Discovery of a new class of non-β-lactam inhibitors of penicillin-binding proteins with gram-positive antibacterial activity

Article abstract of DOI:10.1021/ja500053x

Infections caused by hard-to-treat methicillin-resistant Staphylococcus aureus (MRSA) are a serious global public-health concern, as MRSA has become broadly resistant to many classes of antibiotics. We disclose herein the discovery of a new class of non-β-lactam antibiotics, the oxadiazoles, which inhibit penicillin-binding protein 2a (PBP2a) of MRSA. The oxadiazoles show bactericidal activity against vancomycin-and linezolid-resistant MRSA and other Gram-positive bacterial strains, in vivo efficacy in a mouse model of infection, and have 100% oral bioavailability.

Full text of DOI:10.1021/ja500053x

Article
pubs.acs.org/JACS
Discovery of a New Class of Non-β-lactam Inhibitors of Penicillin-
Binding Proteins with Gram-Positive Antibacterial Activity
Peter I. O’Daniel,† Zhihong Peng,†,¶ Hualiang Pi,†,¶ Sebastian A. Testero,† Derong Ding,† Edward Spink,†
Erika Leemans,† Marc A. Boudreau,† Takao Yamaguchi,† Valerie A. Schroeder,‡ William R. Wolter,‡
Leticia I. Llarrull,† Wei Song,† Elena Lastochkin,† Malika Kumarasiri,† Nuno T. Antunes,†
Mana Espahbodi,† Katerina Lichtenwalter,† Mark A. Suckow,‡ Sergei Vakulenko,† Shahriar Mobashery,*,†
and Mayland Chang*,†
†Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
‡Freimann Life Sciences Center and Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556,
United States
ABSTRACT: Infections caused by hard-to-treat methicillin-resistant
Staphylococcus aureus (MRSA) are a serious global public-health concern,
as MRSA has become broadly resistant to many classes of antibiotics. We
disclose herein the discovery of a new class of non-β-lactam antibiotics,
the oxadiazoles, which inhibit penicillin-binding protein 2a (PBP2a) of
MRSA. The oxadiazoles show bactericidal activity against vancomycin-
and linezolid-resistant MRSA and other Gram-positive bacterial strains, in
vivo efficacy in a mouse model of infection, and have 100% oral
bioavailability.
which is believed to be a reason for the effectiveness of β-
lactams.20,21
INTRODUCTION
■
Methicillin-resistant Staphylococcus aureus (MRSA) is a global
public-health problem that results in 278,000 hospitalizations1
and 19,000 deaths2 annually in the United States alone. MRSA
infections have been increasing from 2% of S. aureus infections
in intensive care units in 1974 to 64% in 2004,3 although more
recent data report stabilization of these cases.4,5 Over the years,
β-lactams were antibiotics of choice for treatment of S. aureus
infections. However, these agents faced obsolescence with the
emergence of MRSA in the early 1960s.6 Presently, the only
effective agents for treatment of MRSA infections are
vancomycin, daptomycin, and linezolid,7 although only line-
zolid can be dosed orally.8 Resistance to all three has
emerged.9−13 Thus, new anti-MRSA therapeutic strategies are
needed, especially agents that are orally bioavailable.14
Clinical resistance to β-lactam antibiotics by MRSA has its
basis in the acquisition of the mecA gene,15 which encodes
penicillin-binding protein 2a (PBP2a), a cell-wall DD-trans-
peptidase.16,17 Staphylococcus aureus normally produces four
PBPs,18 which are susceptible to inhibition by β-lactam
antibiotics. These antibiotics irreversibly acylate the active-site
serine of PBPs, which deprives bacteria of their biosynthetic
functions and results in bacterial death. In contrast, PBP2a is
refractory to inhibition by essentially all commercially available
β-lactams.17 Thus, novel antibiotics that inhibit PBP2a, among
other PBPs, are highly sought. It is worth mentioning that the
structures of essentially all PBPs are highly similar to each other
within the active sites.19 Inhibition by β-lactam antibiotics
results in incapacitation of multiple PBPs in the same organism,
In this study, we report on the oxadiazoles as a new class of
non-β-lactam antibiotics, which was discovered from in silico
screening. Lead optimization, in vitro, and in vivo evaluation
resulted in antibiotics with Gram-positive activity and excellent
oral bioavailability. We investigated the mechanism of action of
the oxadiazoles and found that they inhibit PBP2a of MRSA
and the biosynthesis of cell wall.
RESULTS AND DISCUSSION
■
In Silico Screening and Determination of Minimal-
Inhibitory Concentrations (MICs). We screened in silico 1.2
million compounds from the ZINC database22 individually
complexed to the X-ray structure of PBP2a of MRSA23 as
potential inhibitors. The resulting complexes were scored using
a combination consensus score of four scoring methods, DOCK,
Gold, FlexX, and ChemScore, and 50 top-scoring compounds
were selected for further analysis. Of these compounds, 29 were
either synthesized or purchased and tested for antibacterial
activity against Escherichia coli and a collection of ESKAPE
bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,
and Enterobacter species24), which cause the majority of
nosocomial infections. Compound 1 exhibiting poor but
reproducible MIC against S. aureus and E. faecium emerged
from this screening (Figure 1).
Received: January 3, 2014
Published: February 11, 2014
© 2014 American Chemical Society
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Table 1. Minimal-Inhibitory Concentrations (MICs) of
a
Oxadiazoles
MIC (μg/mL)
4 vancomycin linezolid
microorganism
2
3
b
c
S. aureus ATCC 29213
S. aureus ATCC 27660
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
1
1
4
2
2
2
c
S. aureus NRS100 (COL)
2
2
d
S. aureus NRS119
2
2
32
32
2
d
S. aureus NRS120
2
2
e
S. aureus VRS1
2
512
64
16
2
f
S. aureus VRS2
2
2
S. epidermis ATCC 35547
2
1
S. hemolyticus ATCC 29970
S. oralis ATCC 9811
2
2
32
32
2
32
>32
2
32
32
4
0.5
1
Figure 1. The X-ray structure of the S. aureus PBP 2a (left) is shown as
a solvent-accessible Connolly surface in green. The close up of the
active site (at one o’clock) is depicted in stereo, showing the fitting of
compound 1 in the pose predicted by the program DOCK. Inhibitor
color scheme: oxygen (red), nitrogen (blue), carbon (gray), and
fluorine (aqua).
S. pyogenes ATCC 49399
B. cereus ATCC 13061
0.6
1
1
1
B. licheniformis ATCC 12759
2
2
2
0.5
2
1
b
E. faecalis ATCC 29212
2
2
2
2
g
E. faecalis 201 (Van S)
2
2
>32
2
1
2
h
E. faecalis 99 (Van R)
2
2
128
0.5
1
Syntheses of Leads and In Vitro Evaluation. We
synthesized in solution a library of 370 variants of compound
1 (Scheme 1), which was screened against the same panel of
E. faecium 119−39A
1
1
1
2
g
(Van S)
h
E. faecium 106 (Van R)
2
1
2
2
2
2
256
0.5
1
2
E. faecium NCTC 7171
a
Scheme 1. Synthetic Approach to the Preparation of the
Oxadiazoles
Whereas the compounds were screened against E. coli and the
ESKAPE panel of bacteria, they exhibited antibacterial activity only
against Gram-positive bacteria. A quality-control strain to monitor
accuracy of MIC testing. mecA positive, resistant to methicillin,
b
c
oxacillin, and tetracycline; susceptible to vancomycin and linezolid.
d
mecA positive, resistant to ciprofloxacin, gentamicin, oxacillin,
e
penicillin, and linezolid. Vancomycin-resistant MRSA (vanA) clinical
isolate from Michigan. Vancomycin-resistant MRSA (vanA) clinical
f
g
isolate from Pennsylvania. Vancomycin-susceptible clinical isolate.
h
Vancomycin-resistant clinical isolate.
and phase II metabolism), with 100% of the parent compound
remaining unchanged after a 60-min incubation.
In Vivo Studies. The pharmacokinetic (PK) properties of
compounds 2−4 were evaluated in mice (Figure 2 and Table
2). After a single intravenous (iv) dose of compound 2 at 50
mg/kg, the area under the concentration−time curve (AUC)
was 1,380 μg·min/mL. Compound 2 had moderate clearance of
36.2 mL/min/kg, a large volume of distribution, and a terminal
half-life of 4.4 h. In contrast, compound 3 had 2-fold higher
systemic exposure of 2650 μg·min/mL, lower clearance of 18.9
mL/min/kg, a moderate volume of distribution, and a terminal
half-life of 22 h. Compound 4 had 3.5-fold higher systemic
exposure than compound 3, low clearance of 5.4 mL/min/kg, a
large volume of distribution, and a terminal half-life of 20 h.
The better PK properties for antibiotics 3 and 4 were
anticipated to result in higher in vivo efficacy compared to
antibiotic 2. Furthermore, both antibiotics 3 and 4 exhibited
100% oral bioavailability, indicating that the compounds can be
administered orally.
Antibiotics 3 and 4 were evaluated in the mouse peritonitis
infection model using iv or oral (po) routes of administration.
While both compounds had the same efficacy after iv
administration, antibiotic 3 had superior efficacy following po
dosing. This was attributed to the shorter distribution half-life
of antibiotic 3 compared to that of 4, which suggested that 3
would distribute to the site of infection faster than 4. Since
antibiotic 3 had superior efficacy, a dose response curve against
MRSA peritonitis model was determined. The median effective
clinically important microorganisms. Antibiotics 2−4 emerged
from this screening with excellent antibacterial activities against
S. aureus (including MRSA) and vancomycin-resistant E.
faecium (VRE; Table 1). The MIC values did not change
with increasing bacterial load, indicating that there was no
innoculum effect on the MIC. We also determined the
minimal-bactericidal concentrations (MBCs), which were in
these cases the same as the MIC values, indicating that the
compounds were bactericidal at concentrations at which they
manifested the antibacterial activity. We also note that the
antibacterial activity against the various Gram-positive bacteria
in Table 1 indicates that other PBPs are likely inhibited by
oxadiazoles, as not all express PBP2a, which was used in the in
silico discovery phase of the work.
Compounds 2−4 were evaluated for in vitro toxicity in the
hemolysis and XTT cell viability assays. Compounds 2 and 3
caused 3% hemolysis of red blood cells at 64 μg/mL (32-fold
above the MIC), and compound 4 was not hemolytic at all. The
compounds were metabolically stable in rat liver S9 (phase I
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dose (ED50) of antibiotic 3 after iv administration was 40 mg/
kg and that after po administration was 44 mg/kg, consistent
with the high oral bioavailability of the antibiotic.
Mechanism of Action. The mode of action of compound 3
was investigated using macromolecular synthesis assays,25
which monitor incorporation of radiolabeled precursors[me-
thyl-3H]-thymidine, [5-3H]-uridine, L-[4,5-3H]-leucine, or D-
[2,3-3H]-alanineinto DNA, RNA, protein, or peptidoglycan,
respectively, of S. aureus in the logarithmic phase of growth.
Inhibition of radioactive-precursor incorporation by antibiotic 3
was compared to that by known antibiotics for the given
biosynthetic pathways (ciprofloxacin, rifampicin, tetracycline,
and fosfomycin, respectively, Figure 3). As expected for an
inhibitor of PBPs, antibiotic 3 inhibited peptidoglycan syn-
thesis, but did not inhibit transcription, translation, or
replication (DNA synthesis). The in vivo inhibition studies of
transcription and translation by 3 were followed up by in vitro
assays for the two processes. We utilized a T7 polymerase
transcription assay for the dacB gene encoding E. coli PBP4.
The intensities of the bands for the dacB mRNA were studied
in the presence and in the absence of antibiotic 3 (Figure 4).
There was no effect by antibiotic 3 on this assay. We further
utilized an in vitro assay that evaluated a coupled transcription
and translation system that resulted in the formation of β-
galactosidase, the levels of which could be assayed by a
chromogenic substrate. The presence of compound 3 at
concentrations of up to 160 μg/mL (80-fold above the MIC)
had no effect on this assay (Figure 4). These assays collectively
support the assertion that antibiotic 3 inhibits the biosynthetic
process of the cell wall.
Inhibition of purified recombinant PBP2a by the oxadiazoles
was investigated next. The gene for PBP2a was cloned from a
strain of MRSA and was purified to homogeneity in our
laboratories.26 We utilized a continuous spectrophotometric
assay of PBP2a inhibition, using the chromogenic cephalospor-
in nitrocefin as a reporter molecule. As nitrocefin modifies the
active-site serine of PBP2a, it generates a chromophore, which
can be monitored in inhibition assays. Antibiotics 2 and 3, with
MIC values of 1−2 μg/mL, inhibited PBP2a with IC50 values of
4 μg/mL and 8 μg/mL, respectively. Similar results were
obtained in a noncontinuous PBP2a binding assay using the
fluorescent penicillin BOCILLIN-FL. These data indicated that
Figure 2. Pharmacokinetics of the oxadiazoles after single iv and po
administration at 50 mg/kg to mice (n = 3 per time point); (a)
antibiotic 2, (b) antibiotic 3, and (c) antibiotic 4.
a
Table 2. Pharmacokinetic Parameters of Oxadiazoles
cmpd
dose (mg/kg)
50 iv
AUC0−∞ (μg·min/mL)
Cmax (μg/mL)
Tmax (h)
CL (mL/min/kg)
36.2
Vd (mL/kg)
t1/2
F (%)
2
1380
860
t1/2α = 2.4 min
t1/2β = 4.4 h
3
4
50 iv
2650
2620
18.9
370
t1/2α = 3.5 min
t1/2β = 22 h
50 po
2.5
2
2
t1/2abs = 3.3 h
t1/2dist = 6.0 h
t1/2elim = 40 h
t1/2α = 6.7 min
t1/2β = 20 h
∼100
50 iv
9200
9220
5.4
720
50 po
10.0
t1/2abs = 2.4 h
t1/2dist = 13 h
t1/2elim = 36 h
100
a
n = 3 mice per time point per route of administration; 10−12 time points. AUC0−∞, area under the plasma concentration−time curve from 0 to
infinity; Cmax, maximum concentration observed in plasma; Tmax, time at which maximum plasma concentration is observed; CL, plasma clearance;
Vd, volume of distribution; t1/2α, initial half-life; t1/2β, terminal half-life; t1/2abs, absorption half-life; t1/2dist, distribution half-life; t1/2elim, elimination half-
life; F, oral bioavailability.
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Figure 3. Effect of antibiotic 3 on macromolecular biosynthesis assays, measuring the incorporation of radiolabeled precursors into (a)
peptidoglycan, (b) protein, (c) RNA, and (d) DNA, compared to known antibiotic inhibitors of the same pathways.
biosynthesis. As such, the oxadiazoles hold promise in
treatment of MRSA infections.
EXPERIMENTAL SECTION
■
General. Organic reagents, solvents, penicillin−streptomycin,
nonessential amino acids, L-glutamine, trypan blue, and XTT kits
were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO,
U.S.A.). 1H and 13C NMR spectra were acquired on a Varian INOVA-
500 (Varian, Inc., Palo Alto, CA, U.S.A.). High-resolution mass spectra
were obtained on a JEOL AX505HA mass spectrometer (Tokyo,
Japan) by FAB ionization or on a Bruker micrOTOF/Q2 mass
spectrometer (Bruker Daltonik, Bremen, Germany) by ESI ionization.
Fetal bovine serum (Gibco), Dulbecco’s Modified Eagle’s Medium
(Gibco), and Dulbecco’s phosphate-buffered saline were purchased
from Invitrogen (Grand Island, NY, U.S.A.).
4-(4-(Trifluoromethyl)phenoxy)benzonitrile (5a). A solution
of anhydrous trifluoro-p-cresol (531 mg, 3.27 mmol), copper(I) iodide
(83 mg, 0.4 mmol), cesium carbonate (1.42 g, 4.36 mmol), 4-
iodobenzonitrile (500 mg, 2.18 mmol) and N,N-dimethylglycine
hydrochloride (91 mg, 0.65 mmol) in degassed 1,4-dioxane (10.0 mL)
was heated at 90 °C for 140 h under an atmosphere of nitrogen. The
mixture was cooled, and it was partitioned between ethyl acetate (50
mL) and water (50 mL). The organic layer was separated, and the
aqueous layer was extracted with ethyl acetate (2 × 50 mL). The
combined organic layer was washed with brine (100 mL) and dried
Figure 4. (a) In vitro transcription assay for levels of dacB in the
presence of compound 3. (b) In vitro transcription/translation assay
for β-galactosidase expression levels in the presence of compound 3.
over Na2SO4. The solution was concentrated in vacuo. The resultant
oil was purified by silica gel chromatography (CH2Cl2/hexanes, 1:8 to
the oxadiazoles inhibit the recombinant PBP2a close to the
1:4) The product was recrystallized from hot hexane to give the title
respective values for the MICs. The small difference might be
due to the absence of the membrane environment, where
PBP2a is anchored. Our repeated trials to date in obtaining X-
ray structures of the complexes of the oxadiazoles bound to
PBP2a have not borne fruit.
1
compound as a white solid (286 mg, 50%). H NMR (500 MHz,
CDCl3): δ (ppm): 7.08 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H),
7.66 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H). 13C NMR (125
MHz, CDCl3): δ (ppm): 107.1, 118.7, 119.1 (CH), 120.0 (CH), 124.0
(q, J = 272.0 Hz, C), 127.0 (q, J = 32.5 Hz, C), 127.3, 127.8 (q, J = 3.8
Hz, CH), 134.5 (CH), 158.2, 160.4. 19F NMR (282 MHz, CDCl3): δ
(ppm): −62.05 (s, 3F). MS (m/z): (FAB+) [MH]+ 264. HRMS (m/
z): [MH]+ calcd for C14H8F3NO, 264.0636; found, 264.0621.
4-Phenoxybenzonitrile (5b). A solution of phenol (1.23 g, 13.10
mmol), copper(I) iodide (333 mg, 1.75 mmol), cesium carbonate
(5.69 g, 17.47 mmol), 4-iodobenzonitrile (2.00 g, 8.73 mmol), and
N,N-dimethylglycine hydrochloride (366 mg, 2.62 mmol) in degassed
1,4-dioxane (35 mL) was stirred at 90 °C for 88 h under an
CONCLUSION
■
In summary, we have described here the discovery of a class of
non-β-lactam bactericidal antibiotics active against Gram-
positive bacteria with in vivo efficacy and 100% oral
bioavailability. The oxadiazoles inhibit PBP2a (and likely
other related PBPs), an important enzyme in cell-wall
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atmosphere of nitrogen. The mixture was cooled and then was
partitioned between ethyl acetate (100 mL) and water (100 mL). The
organic layer was separated, and the aqueous layer was washed with
ethyl acetate (2 × 100 mL). The combined organic layers were washed
with brine (100 mL), dried over anhydrous Na2SO4, and concentrated
in vacuo. The crude material was purified by column chromatography
on silica gel (EtOAc/hexanes, 1:12 to 1:8) to give the title compound
column chromatography on silica gel (EtOAc/hexanes, 1:3) to give a
white solid (132 mg, 92%). H NMR (500 MHz, CDCl3): δ (ppm):
1
7.14 (d, J = 9.0 Hz, 2H), 7.16 (d, J = 9.0 Hz, 2H), 7.23−7.27 (m, 2H),
7.63 (d, J = 8.4 Hz, 2H), 8.18 (d, J = 8.8 Hz, 2H) 8.23 (dd, J = 9.0, 5.4
Hz, 2H). 13C NMR (125 MHz, CDCl3): δ (ppm): 116.7 (d, J = 15.2
Hz, CH), 119.0 (CH), 119.8 (CH), 120.8 (d, J = 4.2 Hz, C), 122.9,
124.3 (q, J = 271.6 Hz, C), 126.0 (q, J = 32.5 Hz, C), 127.6 (q, J = 3.8
Hz, CH), 129.7 (CH), 130.8 (d, J = 9.0 Hz, CH), 158.8, 159.6, 165.7
(d, J = 254.1 Hz, C), 168.5, 175.1. 19F NMR (282 MHz, CDCl3): δ
(ppm) 61.87 (s, 3F), 104.90−105.00 (m, 1F). MS (m/z): (FAB+)
[MH]+ 400 (MH+). HRMS (m/z): [MH]+ calcd for C21H12F4N2O2,
400.0835; found, 400.0811.
1
as a white solid (1.70 g, 100%). H NMR (300 MHz, CDCl3): δ
(ppm): 7.00 (d, J = 9.1 Hz, 2H), 7.06 (d, J = 7.6 Hz, 2H), 7.23 (t, J =
7.2 Hz, 1H), 7.41 (t, J = 8.1 Hz, 2H), 7.59 (d, J = 9.1 Hz, 2H). 13C
NMR (75 MHz, CDCl3): δ (ppm): 105.9, 118.0 (CH), 119.0, 120.5
(CH), 125.3 (CH), 130.4 (CH), 134.2 (CH), 154.9, 161.8. MS (m/z):
(FAB+) [MH]+ 196. HRMS (m/z): [MH]+ calcd for C13H9NO,
196.0762; found, 196.0780.
5-(4-(tert-Butyldimethylsilyloxy)phenyl)-3-(4-(4-(trifluoro-
methyl)phenoxy)-phenyl)-1,2,4-oxadiazole (10a). 4-(tert-
Butyldimethylsilyloxy)benzoyl chloride (201 mg, 743 μmol) in toluene
(1.0 mL) was added to (Z)-4-(4-(trifluoromethyl)phenoxy)-N′-
hydroxybenzamidine (128 mg, 254 mmol) in toluene (4.0 mL). The
reaction mixture was refluxed (120 °C) for 27 h. The solution was
then cooled to room temperature, and the organic layer was then
removed under reduced pressure. The crude brown material was
purified by column chromatography (4:1 Hex/EtOAc) to give a yellow
oil, which crystallized upon standing to give yellow crystals (294 mg,
(Z)-4-(4-(Trifluoromethyl)phenoxy)-N′-hydroxybenzamidine
(6a). A solution of 4-(4-(trifluoromethyl)phenoxy)benzonitrile (179
mg, 0.68 mmol) and hydroxylamine (167 μL, 2.72 mmol) in ethanol
(10.0 mL) was refluxed for 1 h. The reaction mixture was cooled to
room temperature and was concentrated in vacuo to give the desired
1
product (201 mg, 100%). H NMR (500 MHz, CDCl3): δ (ppm):
4.94 (bs, 3H), 7.04−7.08 (m, 4H), 7.60 (d, J = 9.0 Hz, 2H), 7.64 (d, J
= 8.8 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ (ppm): 118.6 (CH),
119.7 (CH), 124.3 (q, J = 271.6 Hz, C), 125.7 (q, J = 32.9 Hz, C),
127.4 (q, J = 3.8 Hz, CH), 127.7, 127.9 (CH), 138.0, 152.3, 157.6. 19F
NMR (282 MHz, CDCl3): δ (ppm): −61.88 (s, 3F). MS (m/z):
(FAB+) [MH]+ 297. HRMS (m/z): [MH]+ calcd for C14H11F3N2O2,
297.0851; found, 297.0863.
1
85%). H NMR (500 MHz, CDCl3): δ (ppm): 0.26 (s, 6H), 1.01 (s,
9H), 6.98 (d, J = 9.0 Hz, 2H), 7.13 (d, J = 9.1 Hz, 2H), 7.15 (d, J = 9.0
Hz, 2H), 7.63 (d, J = 9.1 Hz, 2H), 8.11 (d, J = 8.8 Hz, 2H), 8.18 (d, J
= 9.0 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ (ppm): −4.2 (CH3),
18.5 (C), 25.8 (CH3), 117.6 (C), 118.9 (CH), 119.8 (CH), 123.3
(CH), 124.3 (q, J = 271.8 Hz, CF3), 125.9 (q, J = 32.5 Hz, C), 127.5
(q, J = 4.1 Hz, CH), 129.7 (CH), 130.3 (CH), 158.6, 159.6, 159.7,
160.2, 168.3, 175.9. 19F NMR (282 MHz, CDCl3): δ (ppm): 61.9 (s,
3F). MS (m/z): (FAB+) [MH]+ 513. HRMS (m/z): [MH]+ calcd for
C27H27F3N2O3Si, 513.1821; found, 513.1816.
(Z)-N′-Hydroxy-4-phenoxybenzamidine (6b). 4-Phenoxyben-
zonitrile (200 mg, 1.02 mmol) and hydroxylamine (250 μL, 4.09
mmol) in ethanol (5.0 mL) were refluxed for 3 h. The mixture was
cooled and then concentrated in vacuo to give the title compound (233
1
mg, 100%). H NMR (500 MHz, CDCl3): δ (ppm): 4.87 (bs, 2H),
7.01 (d, J = 9.0 Hz, 2H), 7.03 (dd, J = 8.7, 1.1 Hz, 2H), 7.15 (tt, J =
7.4, 1.1 Hz, 2H), 7.36 (dd, J = 8.6, 7.3 Hz, 2H), 7.59 (d, J = 9.0 Hz,
2H). 13C NMR (125 MHz, CDCl3): δ (ppm): 118.6 (CH), 119.6
(CH), 124.3, 127.3, 127.6 (CH), 130.1 (CH), 152.5, 156.6, 159.3. MS
(m/z): (FAB+) [MH]+ 229. HRMS (m/z): [MH]+ calcd for
C13H12N2O2, 229.0977; found, 229.0977.
4-(3-(4-(4-(Trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadia-
zol-5-yl)phenol (3). 5-(4-(tert-Butyldimethylsilyloxy)phenyl)-3-(4-
(4-(trifluoromethyl)phenoxy)-phenyl)-1,2,4-oxadiazole (164 mg, 319
μmol) was dissolved in dry THF (2.0 mL) and TBAF (319 μL, 1.0 M
in THF) was added dropwise at room temperature to the stirred
solution. The reaction was checked for completion by TLC after <1
min. The organic solution was washed with water, 10% HCl, and then
dried with MgSO4. The organic solvents were removed under reduced
pressure to give the crude product which was purified by silica gel
column chromatography (1:4 EtOAc/hexanes) to give the product (88
mg, 70%) as an off white solid. 1H NMR (500 MHz, CDCl3): δ
(ppm): 6.71 (bs, 1H), 6.99 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.8 Hz,
2H), 7.15 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 8.6 Hz, 2H), 8.10 (d, J = 8.6
Hz, 2H), 8.16 (d, J = 8.6 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ
116.4 (CH), 116.7 (C), 119.1 (CH), 119.7 (CH), 122.8 (C), 124.3 (q,
J = 271.6 Hz, C), 126.0 (q, J = 32.5 Hz, C), 127.5 (q, J = 3.8 Hz, CH),
129.8 (CH), 130.6 (C), 158.8 (C), 159.5 (C), 160.3 (C), 168.3 (C),
175.9 (C). 19F NMR (282 MHz, CDCl3): δ (ppm): 61.9 (s, 3F). MS
(m/z): (FAB+) [MH]+ 399. HRMS (m/z): [MH]+ calcd for
C21H13F3N2O3, 399.0957; found, 399.0928.
5-(4-(tert-Butyldimethylsilyloxy)phenyl)-3-(4-phenoxyphen-
yl)-1,2,4-oxadiazole (10b). 4-(tert-Butyldimethylsilyloxy)benzoyl
chloride (1.50 g, 6.6 mmol) in toluene (10.0 mL) was added to
(Z)-N′-hydroxy-4-phenoxybenzamidine (1.96 g, 254 mmol) in toluene
(80.0 mL). The reaction mixture was refluxed (120 °C) for 27 h. The
solution was then cooled to room temperature and the organic solvent
was removed under reduced pressure. The resulting crude brown
material was purified by column chromatography (4:1 hexanes/
EtOAc) to give a yellow oil, which crystallized upon standing to give
yellow crystals (2.25 g 77%). 1H NMR (500 MHz, CDCl3): δ (ppm):
0.26 (s, 6H), 1.01 (s, 9H), 6.97 (d, J = 8.8 Hz, 2H), 7.08−7.11 (m,
4H), 7.18 (t, J = 7.4 Hz, 1H) 7.37−7.41 (m, 2H), 8.10 (d, J = 9.0 Hz,
2H), 8.12 (d, J = 9.0 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ
(ppm): −4.2 (CH3), 18.5 (C), 25.8 (CH3), 117.7 (C), 118.6 (CH),
119.9 (C), 120.9 (CH), 121.9 (C), 124.3 (CH), 129.5 (CH), 130.1
(CH), 130.2 (CH), 156.4 (C), 160.1 (C), 160.2 (C), 168.5 (C), 175.7
(C). MS (m/z): (FAB+) [MH]+ 445. HRMS (m/z): [MH]+ calcd for
C26H28N2O3Si, 445.1947; found, 445.1922.
(Z)-4-(4-(Trifluoromethyl)phenoxy)-O-(4-(4-fluorobenzoyl)-
benzamidoxime (7). A solution of (Z)-4-(4-(trifluoromethyl)-
phenoxy)-N′-hydroxy-benzamidine (130 mg, 0.38 mmol) in methyl-
ene chloride (1.5 mL) and N-ethyldiisopropylamine (133 μL, 0.76
μmol) was cooled in an iced-water bath under an atmosphere of
nitrogen. A solution of 4-fluorobenzoyl chloride (60 μL, 0.51 mmol)
was added dropwise to the previous solution and the mixture was
stirred for 1 h at 0 °C. The solution was allowed to warm to room
temperature and was then stirred for 24 h. Ethyl acetate (25 mL) was
poured into the mixture, and the solution was washed with aq
NaHCO3 (25 mL), water (25 mL), and then brine (25 mL). The
organic layer was then dried over anhydrous MgSO4 and was
concentrated to dryness in vacuo. The solid residue was purified using
column chromatography on silica gel (EtOAc/hexanes, 1:3 to 4:1) to
give the product as a white solid (172 mg, 94%). 1H NMR (500 MHz,
CDCl3): δ (ppm): 5.18 (bs, 1H), 7.07−7.11 (m, 4H), 7.16 (t, J = 8.7
Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 9.0 Hz, 2H), 8.12 (dd, J
= 9.0, 5.4 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ (ppm): 116.0 (d,
J = 22.3 Hz, CH), 118.8 (CH), 119.7 (CH), 124.2 (q, J = 271.6 Hz,
C), 125.9 (d, J = 2.8 Hz, C), 126.0 (q, J = 32.5 Hz, C), 127.1, 127.5 (q,
J = 3.8 Hz, CH), 129.1 (CH), 132.2 (d, J = 9.5 Hz, CH), 157.7 (d, J =
232.5 Hz, C), 159.6, 163.3, 165.0, 167.0. 19F NMR (282 MHz,
CDCl3): δ (ppm): −61.89 (s, 3F), 105.03−105.13 (m, 1F). MS (m/z):
(FAB+) [MH]+ 419. HRMS (m/z): [MH]+ calcd for C21H14F4N2O3,
419.1019; found, 419.1031.
5-(4-Fluorophenyl)-3-(4-(4-(trifluoromethyl)phenoxy)-
phenyl)-1,2,4-oxadiazole (1). (Z)-4-(4-(Trifluoromethyl)phenoxy)-
O-(4-(4-fluorobenzoyl)benzamidoxime (150 mg, 0.358 mmol) was
placed in THF (1.5 mL) under an atmosphere of nitrogen.
Tetrabutylammonium fluoride (1.0 M, 385 μL) was added dropwise,
and the solution was stirred at room temperature for 4 h. The solvent
was removed under reduced pressure, and the product was purified by
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1
4-(3-(4-Phenoxyphenyl)-1,2,4-oxadiazol-5-yl)phenol (2). 5-
(4-(tert-Butyldimethylsilyloxy)phenyl)-3-(4-phenoxyphenyl)-1,2,4-ox-
adiazole (104 mg, 234 μmol) was dissolved in dry THF and then
TBAF (234 μL, 1.0 M in THF) was added dropwise at room
temperature to the stirred solution. The reaction was checked by TLC
after <1 min. The organic solution was washed with water, 10% HCl
and then dried with MgSO4. The organic solvents were removed
under reduced pressure to give the crude product which was purified
by silica gel column chromatography (1:4 EtOAc/hexanes) to give the
mg, 72%) as a white solid. H NMR (500 MHz, CDCl3) δ (ppm):
4.18 (s, 2H), 6.74 (d, J = 8.0 Hz, 2H), 7.13 (t, J = 9.4 Hz, 4H), 7.61 (d,
J = 8.7 Hz, 2H), 7.99 (d, J = 8.0 Hz, 2H), 8.16 (d, J = 8.0 Hz, 2H). 13C
NMR (125 MHz, CDCl3) δ (ppm): 114.0, 114.7 (CH), 118.9 (CH),
119.8 (CH), 123.5, 125.4, 125.8 (C, q, J = 32.5 Hz), 127.5 (CH, q, J =
3.8 Hz), 129.7 (CH), 130.3 (CH), 150.9, 158.5, 159.8, 168.2, 176.3.
19F NMR (282 MHz, CDCl3) δ (ppm): 61.85 (3F, s). MS (m/z):
(FAB+) [MH]+: 398. HRMS (m/z): [MH]+ calcd for C21H14F3N3O2,
397.1038; found 397.1036.
1
title compound (77 mg, 100%) as an off white solid. H NMR (500
Bacterial Strains. E. faecium ATCC 19734, K. pneumonia ATCC
700603, A. baumanni ATCC 17961, P. aeruginosa ATCC 27853, E.
aerogenes ATCC 35029 and E. coli ATCC 25922 were used for the
initial screen and were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, U.S.A.). S. aureus NRS100 (COL),
NRS 119, NRS120, VRS1, and VRS2 were obtained from the Network
on Antimicrobial Resistance in Staphylococcus aureus (Chantilly, VA,
U.S.A.). S. epidermis ATCC 35547, S. hemolyticus ATCC 29970, S.
oralis ATCC 9811, S. pyogenes ATCC 49399, B. cereus ATCC 13061, B.
licheniformis ATCC 12759, E. faecalis ATCC 29212, and E. faecium
NCTC 7171 (ATCC 19434) were obtained from ATCC. E. faecalis
201 and 99, E. faecium 119-39A and 106 were collected from Wayne
State University School of Medicine.
Determination of Minimal-Inhibitory Concentrations (MICs)
and Minimal-Bactericidal Concentrations (MBCs). MICs were
determined by the microdilution procedure in cation-adjusted Mueller
Hinton II Broth (CAMHB II; BBL) in accordance with
recommendations of CLSI.27 The MICs against Streptococcus were
determined in CAMHB II supplemented with 5% lysed horse blood
(Hema Resource & Supply, Inc., Aurora, Oregon, U.S.A.) and the
MICs of oxacillin against S. aureus were determined in the presence of
2% NaCl. The final bacterial innoculum was 5 × 105 CFU/mL, and
the results were read after 16−20 or 20−24 h incubation at 35 °C,
depending on the species. All MIC determinations were performed in
triplicate. We also determined MICs of compound 2 against S. aureus
ATCC 29213 with bacterial innocula of 5 × 106 CFU/mL and 5 × 107
CFU/mL in order to evaluate the innoculum effect on MIC
determination. Inoculated microtiter plates were incubated at 35 °C
for 16−20 h, prior to recording of the results. Three bacterial strains, S.
aureus NRS100 (highly methicillin-resistant strain), S. aureus ATCC
29213 and E. faecalis ATCC 29212 (vancomycin-susceptible strains)
were used to determine MBCs of antibiotics 2 and 3. Determination of
MBCs was performed by the macrodilution method in CAMHB II, by
a variation of a literature method.28 First, 2-fold dilutions of the
antibiotics were made in culture tubes containing 3 mL of CAMHB II,
and the broth was inoculated with 5 × 105 CFU/mL of bacterial
suspension. After incubation for 16−20 h at 35 °C, MICs were
recorded. For MBC determination, broth in the tubes with antibiotics
2 and 3 at concentrations corresponding to the MIC (the lowest
concentration of compound with no visible bacterial growth), 2 ×
MIC and 4 × MIC were mixed, and a 100 μL portion was plated on
antibiotic-free agar plates and incubated for 48 h prior to visualization
and colony counting.
MHz, CDCl3): δ (ppm): 5.83 (bs, 1H), 6.98 (d, J = 8.2 Hz, 2H), 7.09
(m, 4H), 7.18 (t, J = 7.6 Hz, 1H), 8.12 (t, J = 7.7 Hz, 2H), 8.12 (d, J =
6.8 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ (ppm): 116.3 (CH),
117.2, 118.6 (CH), 119.9 (CH), 121.8, 124.4 (CH), 129.5 (CH),
130.2 (CH), 130.6 (CH), 156.4, 159.8, 160.3, 168.5, 175.7. MS (m/z):
(FAB+) [MH]+ 331. HRMS (m/z): [MH]+ calcd for C20H14N2O3,
331.1083; found, 331.1056.
tert-Butyldimethylsilyl 4-(tert-butyldimethylsilyloxy)-
benzoate (8). 4-Hydroxybenzoic acid (1.0 g, 7.24 mmol) and tert-
butyldimethylsilyl chloride (2.4 g, 15.9 mmol) were dissolved in ethyl
acetate (9.0 mL) and then triethylamine was added dropwise. The
reaction mixture was stirred at room temperature for 1 h. The solution
was filtered and then allowed to stand for 1 h to precipitate the
triethylammonium salt. The solution was filtered again, and the filtrate
was removed under reduced pressure to give a clear oil which
crystallized upon standing to give the product as a white solid (2.6 g,
100%), which was contaminated by a small amount of TBDMSCl. 1H
NMR (500 MHz, CDCl3): δ (ppm): 0.23 (s, 6H), 0.36 (s, 6H), 0.99
(s, 9H), 1.02 (s, 9H), 6.86 (d, J = 9.0 Hz, 2H), 7.94 (d, J = 9.0 Hz,
2H). 13C NMR (125 MHz, CDCl3): δ (ppm): −4.5 (CH3), −4.2
(CH3), 18.0 (C), 18.5 (C), 25.8 (CH3), 25.9 (CH3), 120.0 (CH),
124.8 (C), 132.3 (CH), 160.3 (C), 166.7 (C). MS (m/z): (FAB+)
[MH]+ 367. HRMS (m/z): [MH]+ calcd for C19H34O3Si2, 367.2125;
found, 367.2120.
4-(tert-Butyldimethylsilyloxy)benzoyl chloride (9). tert-Butyl-
dimethylsilyl 4-(tert-butyldimethylsilyloxy)benzoate (0.6 g, 1.6 mmol)
was dissolved in dry methylene chloride (15.0 mL), and the solution
was cooled to 0 °C. Oxalyl chloride (160 μL, 1.8 mmol) was added,
followed by dry DMF (4 drops). The reaction mixture was allowed to
warm to room temperature and then stirred for an additional 3.5 h.
The reaction was checked for completion by TLC, and the product
was used immediately in the next step without purification.
3-(4-(4-(Trifluoromethyl)phenoxy)phenyl)-5-(4-nitrophenyl-
1,2,4-oxadiazole (11). To a solution of 4-nitrobenzoylchloride (1.57
g, 8.5 mmol) in toluene (50 mL) was added the (Z)-N′-hydroxy-4-(4-
(trifluoromethyl)phenoxy)benzimidamide (2.51 g, 8.5 mmol). The
reaction mixture was refluxed for 5.5 h. Then the solution was cooled
to room temperature and the product crystallized from the solvent. A
third of the solvent was removed under reduced pressure, and the
solvent was heated to dissolve the product and then cooled to 0 °C
overnight. The solid was collected by suction filtration and washed
with cold toluene to give the pure product (1.89 g, 52%) as a yellow
solid. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.15 (dd, J = 9.1, 0.7 Hz,
2H), 7.18 (d, J = 8.8 Hz, 2H), 7.65 (dd, J = 9.1, 0.7 Hz, 2H), 8.20 (d, J
= 9.0 Hz, 2H), 8.43 (s, 4H). 13C NMR (125 MHz, CDCl3) δ (ppm):
119.2 (CH), 119.8 (CH), 122.3, 124.6 (CH), 126.1 (C, q, J = 32.5
Hz), 127.6 (CH, q, J = 3.8 Hz), 129.4 (CH), 129.7, 129.8, 150.4,
159.4, 169.0, 173.9. 19F NMR (282 MHz, CDCl3) δ (ppm): 61.9 (3F,
s). MS (m/z): (FAB+): 427 (MH+). HRMS (m/z): [MH]+ calcd for
C21H12F3N3O4, 427.0780; found 427.0782.
Hemolysis Assay. A 10-mL aliquot of horse whole blood
(Innovative Research, Burlington, ON, Canada) was centrifuged at
7000 × g for 5 min. The supernatant was removed, and 5 mL of 0.1 M
PBS was added to the red blood cell (RBC) pellet, followed by
centrifugation. This step was repeated three times to afford washed
RBCs, which were resuspended in 5 mL of 0.1 M PBS. Stock solutions
of the compounds were prepared in DMSO. For the assays, a 50-μL
aliquot of the each compound in DMSO was mixed with 750 μL PBS
and 200 μL RBCs suspended in PBS to final concentrations of 2, 4, 8,
16, 32, and 64 μg/mL and incubated at 37 °C for 1 h. The positive
control consisted of 50 μL DMSO, 750 μL of 2% Triton X-100 in 0.1
M PBS, and 200 μL RBCs. The negative control contained 50 μL
DMSO, 750 μL PBS, and 200 μL RBCs. All samples were prepared in
duplicates. After incubation, they were centrifuged at 13,000 × g for 5
min, and the supernatant was collected and analyzed by UV
spectroscopy at 541 nm using an Epoch Microplate Spectropho-
tometer (BioTek Instruments, Inc., Winooski, VT, U.S.A.). Spectro-
4-(3-(4-(4-(Trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadia-
zol-5-yl)aniline (4). Iron powder (132 mg, 2.33 mmol), water (210
μL, 11.8 mmol) and 12 N HCl (10 μL, 0.11 mmol) were added
consecutively to a solution of 3-(4-(4-(trifluoromethyl)phenoxy)-
phenyl)-5-(4-nitrophenyl-1,2,4-oxadiazole (46 mg, 0.11 mmol) in
ethanol (6.0 mL). The mixture was heated at 95 °C for 1 h. The
mixture was filtered while still hot and then washed with additional
ethanol (10 mL). The filtrates were combined, and the solvent was
removed in vacuo. The crude solid product was purified by silica gel
column chromatography (EtOAc/hexanes 1:8) to give the product (31
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scopic data were plotted in Microsoft Excel for calculation of IC50
values.
Liquid Chromatography/Mass Spectrometry. The chromato-
graphic system consisted of a Waters Acquity UPLC System (Waters
Corporation, Milford, MA, U.S.A.) equipped with a binary solvent
manager, an autosampler, a column heater and a photodiode array
detector. All mass spectrometric experiments were performed with a
Waters TQD tandem quadrupole detector (Milford, MA, U.S.A.)
monitored with MassLynx MS software. Mass spectrometry
acquisition was performed in the positive electrospray ionization
mode with MRM. The capillary voltage, cone voltage, extractor
voltage, and RF lens voltage were set at 3.2 kV, 25 V, 3.0 V, and 0.1 V,
respectively. Desolvation gas flow rate was 650 L/h (nitrogen) and the
cone gas flow rate was 50 L/h (nitrogen). The source temperature and
desolvation temperature were held at 150 and 350 °C, respectively.
Samples were analyzed on an Aquity UPLC C18 1.7 μm, 2.1 mm i.d. ×
50 mm column by elution at 0.5 mL/min with 70% A/30% B for 2
min, followed by a 10-min linear gradient to 90% B, then 70% A/30%
B for 2 min (A = 0.1% formic acid/water, B = 0.1% formic acid/
acetonitrile). The MRM transitions used were: 331→121 for
compound 2, 399→121 for compound 3, 398→120 for compound
4, and 401→123 for the internal standard.
Pharmacokinetic Parameters. The area under the mean
concentration−time curve up to the last quantifiable sampling time
(AUC0‑last) was calculated by the trapezoidal rule using the
pharmacokinetic software PK Solutions (version 2.0, Summit Research
Services, Montrose, CO, U.S.A.). AUC0−∞ was calculated as AUC0‑last
+ (Clast/k), where Clast is the concentration at the last quantifiable
sampling time and k is the elimination rate constant. Half-lives (t1/2α
and t1/2β) were estimated from the linear portion of the initial or
terminal linear portion of the concentration−time data by linear
regression, where the slope of the line was the rate constant k and t1/2α
= ln 2/k.
In Vivo Toxicity. A dose tolerance study was conducted with
compound 2. Mice (n = 3 per time per group) were administered a
single iv dose of compound 2 at 25 or 50 mg/kg. Compound 2 was
dissolved in 10% DMSO/25% Tween-80/65% water. A vehicle control
group (n = 3) was included. Mice were clinically monitored for 2 days.
Mouse Peritonitis Model. S. aureus ATCC 27660 (MRSA) was
used for the mouse peritonitis model. Bacteria were grown in brain
heart infusion agar and after overnight incubation they were grown in
brain heart infusion broth (BD Biosciences, Woburn, MA, U.S.A.).
The inoculum was prepared immediately before inoculation by
suspending the colonies in sterile brain heart infusion broth medium,
adjusted to an optical density of 0.5 at 540 nm (equivalent to 108
CFU/mL), and stored on ice until inoculation. A suspension of 10%
mucin (Sigma-Aldrich Chemical Co., St Louis, MO, U.S.A.) in saline
was heated to 105 °C for 45 min and the pH was adjusted to 7.0. Just
prior to inoculation, bacteria at 108 CFU/mL were mixed 1:1 with
mucin. The final inoculum was 5 × 107 CFU/mL and 5% mucin.
Groups of six mice were given each 0.5 mL of 5 × 107 CFU/mL
intraperitoneally. At 30 min after infection, mice were treated with
oxadiazole, vancomycin (at 5 mg/kg), or vehicle given iv by tail vein
injection, with a second dose at 8 h after infection. Mice were clinically
monitored for 2 days, with survival as the primary end point.
Compounds 3 and 4 were also evaluated in the mouse peritonitis
model after po administration. A single dose of the compound was
administered to ICR mice (n = 6 per group) by oral gavage 1 h after
infection. Mice were clinically monitored for 2 days and the number of
mice surviving was recorded. Linezolid (at 10 mg/kg) was used as
positive control.
Metabolic Stability. Compounds (5 μM) were incubated with
male rat liver S9 (0.05 mg protein/mL, BD Biosciences, Woburn, MA,
U.S.A.) and nicotinamide adenine dinucleotide phosphate reduced
(0.5 mM) in potassium phosphate buffer (50 mM, pH 7.4) at 37 °C
for up to 60 min. Aliquots were taken at 0, 10, 30, and 60 min, and the
reaction was terminated by the addition of one and a half volumes of
acetonitrile containing 1 μM internal standard. The reaction mixtures
were centrifuged (13,000 × g, 13 min) and aliquots of the supernatants
were analyzed by ultraperformance liquid chromatography (UPLC)
with multiple-reaction monitoring (MRM) detection. All analyses were
conducted in duplicate.
Cell Lines and Culture. HepG2 cells (ATCC HB-8065) that had
undergone 76 passages and HeLa (ATCC CCL-2) were stored in
liquid nitrogen and were subcultured less than 20 times. Cells were
maintained in monolayer culture at 37 °C and 5% CO2 in Dulbecco’s
Modified Eagle’s medium supplemented with 10% fetal bovine serum,
1% nonessential amino acids, 2 mM L-glutamine, and 1% penicillin−
streptomycin. Cell counts were performed by hemocytometry before
addition of cells to 96-well plates.
In Vitro Cytotoxicity XTT Assay. Cell viability was determined by
the XTT assay, which is based on the mitochondrial dehydrogenase
activity in living cells. Cell density of seeding was optimized in order to
have sufficient cells for analysis, without overgrowth. Compounds were
dissolved in DMSO as stock solutions and diluted to a final
concentration of 1% DMSO. Three controls were used in each 96-
well plate in triplicates: negative control (complete medium and 1%
DMSO without cells), cell growth control (complete medium and
cells), and solvent control (complete medium, cells and 1% DMSO).
After overnight incubation, the cells were treated with the compound
for 16 h at concentrations of 2, 4, 8, 16, 32, 50, 64, and 100 μg/mL.
Experiments were performed in triplicate. After treatment, each well
was washed two times with PBS buffer and 150 μL XTT working
solution (according to the manufacturer’s protocol) was then added to
each well. After an additional 3-h incubation, the absorbance was
measured at two wavelengths: 475 nm (test wavelength) and 660 nm
(reference wavelength for background) using an ELISA reader
(BioTek,Winooski, VT, U.S.A.). All the experiments were repeated
two or three times. IC50 was calculated using GraphPad Prism 5
(GraphPad Software, Inc., San Diego, CA, U.S.A.).
Animals. Female ICR mice (6−8 weeks old, 17−20 g body weight
specific pathogen free,) were obtained from Harlan Laboratories, Inc.
(Indianapolis, IN U.S.A.). All procedures were performed in
accordance with the University of Notre Dame Institutional Animal
Care and Use Committee. Mice were maintained in polycarbonate
shoebox cages with hardwood bedding in a room under a 12:12 h
light/dark cycle and at 72
2 °F and provided with Teklad 2019
Extruded Rodent Diet (Harlan, Madison, WI, U.S.A.) and water ad
libitum.
Animal Dosing and Sample Collection for Pharmacokinetic
Studies. Mice (n = 3 per time point per compound) were
administered a single iv dose of compounds 2 or 3 or 4 at 50 mg/kg.
The compounds were dissolved in 10% DMSO/25% Tween-80/65%
water. Blood samples were collected in heparinized syringes by cardiac
puncture at 2, 5, 10, 20, and 40 min, and at 1, 2, 3, 4, 8, 12, and 24 h
after dose administration. A separate group of female ICR mice (n = 3
per time point) received a single po dose of compound 3 or 4 at 50
mg/kg by gavage. Terminal blood was collected at 0.5, 1, 2, 3, 4, 6, 8,
24, 30, and 48 h. Blood was centrifuged at 1200 × g for 10 min to
collect plasma.
Sample Analysis. A 50-μL aliquot of plasma was mixed with 100
μL of internal standard in acetonitrile to a final concentration of 8 μg/
mL. The sample was centrifuged at 10,000 × g for 10 min, and the
supernatant was collected and analyzed by reversed-phase UPLC-
ESI(+)-MRM of the transitions 331→121 for compound 2, 399→121
for compound 3, 398→120 for compound 4, and 401→123 for the
internal standard. Calibration curves of compounds 2, 3, and 4 were
prepared in blank mouse plasma containing the internal standard from
which unknown concentrations were determined.
A dose response curve for compound 3 was determined. Groups of
six mice were given two iv doses of compound 3 at 10, 20, 30, 40, and
50 mg/kg at 30 min and 8 h after infection. In a separate study, groups
of six mice received two po doses of compound 3 at 30, 40, 50, 60, and
70 mg/kg. Data were plotted in Graph Pad Prism for calculation of
ED50 values.
Macromolecular Synthesis Assays. The effects of compound 3
on DNA, RNA, peptidoglycan, and protein synthesis of S. aureus were
determined by measuring radioactive precursor as reported
previously.29 S. aureus ATCC 29213 was cultured overnight at 37
°C in fresh brain heart infusion medium until midexponential phase.
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Radioactive precursor (1 μCi/mL, [methyl-3H]-thymidine, [5,6-3H]-
uridine, L-[4,5-3H]-leucine, or [2,3-3H]-D-alanine) was added to each
aliquot of cell culture and incubated at 37 °C for 10 min, followed by
addition of 0.25 μg/mL and 0.5 μg/mL of compound 3. Negative and
positive controls (ciprofloxacin, rifampicin, tetracycline, and fosfomy-
cin for inhibition of DNA, RNA, protein synthesis, and peptidoglycan
synthesis, respectively) were included. The mixtures were incubated at
37 °C for up to 2 h. Aliquots (650 μL) were pipetted out every 20 min
for determination of isotope incorporation and viability. The
macromolecules were precipitated in 20% ice-cold trichoroacetic acid
(TCA) on ice for 1 h and filtered through glass fiber filters. The filters
were washed three times with 5% TCA and two times with 95%
ethanol, dried, and placed in vials containing 5 mL of scintillation
cocktail. Radioactivity was measured on a Beckman LS-100 liquid
scintillation counter (Beckman Coulter, Inc., Brea, CA, U.S.A.).
Triplicates were prepared for each assay, and all assays were repeated
at least twice. The effects of compound 3 on radioactive precursor
incorporation over time were expressed as percentage of inhibition
relative to control.
Cloning of E. coli dacB. The dacB gene encoding penicillin-
binding protein PBP4 was amplified from the E. coli K12 chromosome
using the high fidelity PfuUltra II Fusion HS DNA polymerase with
primers PBP4_fw(TTTGTGTCATATGCAAATGTTGATGAG-
TACATTACTC) and PBP4_rv (TTTGTGCTCGAGATTGTTC-
TGATAAATATCTTTATAC), containing NdeI and XhoI restriction
sites, respectively, to facilitate cloning. The resulting PCR fragment,
containing dacB without the first 90 bp encoding the N-terminal signal
peptide, was extracted from 1% agarose gel, purified using the
QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands), digested
with NdeI and XhoI, cleaned with QIAquick PCR purification kit
(Qiagen), and ligated with NdeI and XhoI double digested pET24a
plasmid DNA. The ligation reaction was performed for 3 h at room
temperature. A 5-μL aliquot of the ligation mixture was used to
transform 100 μL of chemically competent E. coli DH5α cells. Colonies
containing the plasmid were selected on LB agar supplemented with
50 μg/mL kanamycin. The construct was verified for the correct insert
using NdeI and XhoI double digestion of plasmids extracted from four
single colonies and further verified by sequencing of both DNA
strands. Plasmid pET24a harboring the dacB gene was purified using
QIAPre spin MiNiprep columns (Qiagen).
compound 3, along with the negative control. The reaction mixtures
were incubated at 37 °C for 2 h, followed by placing the tubes on ice
for 5 min to stop translation. The activity of β-galactosidase was
quantified spectrophotometrically for each reaction using the β-
galactosidase assay kit (Promega, Madison WI) according to the
manufacturer’s instructions. The kit contains the chromogenic
substrate o-nitrophenyl-β-D-galacoside, which is hydrolyzed by β-
galactosidase from the reaction mixtures, resulting in the formation of
o-nitrophenolate. The amount of o-nitrophenolate formed was
measured by absorbance at 420 nm. The amounts of β-galactosidase
from the reaction mixtures were calculated on the basis of a standard
curve.
PBP2a-Binding Assay. PBP2a was cloned in E. coli as previously
described.26 The assay involves the use of BOCILLIN FL, a
commercially available fluorescent penicillin (Molecular Probes, Inc.,
CA), as a labeling reagent for the enzyme active site.30 A typical
reaction mixture (20 μL) contains PBP2a (2.5 μM) and a given
concentration of a putative inhibitor (antibiotic) in 25 mM Hepes, 1
M NaCl (pH 7.0). The mixture is incubated for 5 min at room
temperature. At this point, the amount of uncomplexed (free) PBP is
assayed by the addition of BOCILLIN FL to afford a final
concentration of 30 μM, followed by an additional incubation at 37
°C for 10 min. SDS sample buffer (15 μL) is added to the reaction
mixture, and it is boiled for 3 min. The samples (35 μL in total) are
loaded onto 10% SDS-PAGE, and the gel is developed and scanned
using Storm840 Fluorimager. The fluorescent bands are quantified
using IMAGEQUANT 5.2 software. A control sample is also routinely
prepared in which PBP2a is incubated directly with BOCILLIN FL for
10 min and quenched using the same SDS sample buffer.
Nitrocefin assay. Nitrocefin was synthesized as previously
described,31 following the procedure previously reported.26
AUTHOR INFORMATION
■
Corresponding Authors
mobashery@nd.edu
mchang@nd.edu
Author Contributions
¶Z.P. and H.P. have contributed equally.
In vitro Transcription Assay. In vitro transcription levels of dacB
encoding E. coli PBP4 were evaluated with and without compound 3.
The 6633bp template plasmid DNA, which codes for 1607nt
transcript, was purified using QIAPre spin MiNiprep columns
(Qiagen, Venlo, Netherlands), and linearized with restriction enzyme
XhoI. After linearization, template DNA was purified by phenol/
chloroform extraction and a 2-μL aliquot of the sample (0.5 μg/μL)
was used for each reaction. The dacB gene was transcribed in vitro
using a T7 Transcription kit (Thermo Fisher Scientific Inc., Waltham,
MA). The assay mixtures were prepared according to the
manufacturer’s instructions and supplemented with 0.5, 5, 50, and
160 μg/mL of compound 3, along with the negative control. The assay
mixtures were incubated for 2 h at 37 °C, and then placed on ice to
stop transcription. Template DNA was removed with 2 μL of RNase-
free DNaseI added to each tube, followed by incubation at 37 °C for
15 min, and then 2 μL of 0.5 M EDTA (pH 8.0) was added to each
tube, followed by incubation at 65 °C for 10 min. The reaction
mixtures were diluted 30-fold with DEPC-treated water to a final
concentration of 0.1−0.5 μg/μL RNA transcripts and 8-μL aliquots of
the diluted transcripts were loaded on a 1% denaturing formaldehyde
gel in MOPS buffer (pH 7.0). The gel was visualized by Gel Doc EZ
Imager (Bio-Rad Laboratories, Inc., Hercules, CA).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Grant AI090818 from the
National Institutes of Health (to M.C. and S.M.).
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