Structure-activity relationship for the oxadiazole class of antibiotics

Article abstract of DOI:10.1021/jm501661f

The structure-activity relationship (SAR) for the newly discovered oxadiazole class of antibiotics is described with evaluation of 120 derivatives of the lead structure. This class of antibiotics was discovered by in silico docking and scoring against the crystal structure of a penicillin-binding protein. They impair cell-wall biosynthesis and exhibit activities against the Gram-positive bacterium Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant and linezolid-resistant S. aureus. 5-(1H-Indol-5-yl)-3-(4-(4-(trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadiazole (antibiotic 75b) was efficacious in a mouse model of MRSA infection, exhibiting a long half-life, a high volume of distribution, and low clearance. This antibiotic is bactericidal and is orally bioavailable in mice. This class of antibiotics holds great promise in recourse against infections by MRSA.

Full text of DOI:10.1021/jm501661f

Article
pubs.acs.org/jmc
Structure−Activity Relationship for the Oxadiazole Class of
Antibiotics
Edward Spink,†,§ Derong Ding,†,§ Zhihong Peng,† Marc A. Boudreau,† Erika Leemans,†
Elena Lastochkin,† Wei Song,† Katerina Lichtenwalter,† Peter I. O’Daniel,† Sebastian A. Testero,†
Hualiang Pi,† Valerie A. Schroeder,‡ William R. Wolter,‡ Nuno T. Antunes,† Mark A. Suckow,‡
Sergei Vakulenko,† Mayland Chang,*,† and Shahriar Mobashery*,†
†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
S
* Supporting Information
ABSTRACT: The structure−activity relationship (SAR) for the newly
discovered oxadiazole class of antibiotics is described with evaluation of
120 derivatives of the lead structure. This class of antibiotics was
discovered by in silico docking and scoring against the crystal structure of
a penicillin-binding protein. They impair cell-wall biosynthesis and exhibit
activities against the Gram-positive bacterium Staphylococcus aureus,
including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant and linezolid-resistant S. aureus. 5-(1H-Indol-5-yl)-3-
(4-(4-(trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadiazole (antibiotic 75b) was efficacious in a mouse model of MRSA infection,
exhibiting a long half-life, a high volume of distribution, and low clearance. This antibiotic is bactericidal and is orally bioavailable
in mice. This class of antibiotics holds great promise in recourse against infections by MRSA.
ranked compounds for screening with living bacteria.9 We set
INTRODUCTION
■
the bar high from the outset by screening compounds first
Staphylococcus aureus is a leading human bacterial pathogen that
against Escherichia coli and the ESKAPE panel of antibiotics,
is a common source of infections in healthcare and community
instead of against the recombinant protein. The ESKAPE panel
environments. The 2013 Centers for Disease Control and
is composed of Enterococcus faecium, Staphylococcus aureus,
Prevention (CDC) report on antibiotic resistance prioritized
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
methicillin-resistant Staphylococcus aureus (MRSA) as an
aeruginosa, and Enterobacter species, a collection of bacteria that
ongoing serious threat, with 2011 records indicating that
cause the majority of nosocomial infections.15 This strategy for
11 285 of the 23 000 deaths caused by antibiotic-resistant
screening easily eliminates any compound that would not have
bacteria and fungi in the United States were directly attributed
activity against bacteria, so the search was streamlined. The
to MRSA infections.1 Antibiotics that are approved for
treatment of MRSA infections are vancomycin (a glycopep-
discovery produced the lead oxadiazole 1 (Scheme 1).9 We
tide), linezolid (an oxazolidinone), daptomycin (a lipopeptide),
and, more recently, ceftaroline (a β-lactam) and tedizolid (an
Scheme 1. Structure of the Lead Oxadiazole 1
oxazolidinone). Only linezolid and tedizolid are orally
bioavailable among these agents.2 Furthermore, resistance to
each of these antibiotics is known.3−8
We described recently the discovery of the oxadiazole class of
antibiotics.9 The lead in this class came out of an in silico search
for potential inhibitors for penicillin-binding protein 2a
have explored in the present report the structural space for
oxadiazole antibiotics by syntheses of derivatives. These
compounds were in turn screened against the bacterial panel,
from which a number exhibited good anti-MRSA activity. In
another effort in streamlining the discovery process, the
promising compounds went directly into the mouse MRSA
peritonitis model for infection. This model has shown excellent
(PBP2a) of MRSA. PBPs are targets of β-lactam antibiotics.
Inhibition of PBPs by β-lactams is bactericidal, as it interferes
with biosynthesis of cell wall.10 Resistance to β-lactam
antibiotics is widespread,11−14 but the importance of PBPs as
targets for antibiotics has not diminished. We reasoned that
PBPs remain worthy targets for antibiotics, and we sought to
discover a new class of non-β-lactam inhibitors for these
enzymes in this effort.
The in silico search and scoring of 1.2 million compounds
from the ZINC library led to selection and purchase of the top-
Received: October 28, 2014
© XXXX American Chemical Society
A
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Scheme 2. General Synthetic Route To Access the 1,2,4-Oxadiazoles, and the Starting Materials Used for Variations within Ring
a
A
a
Compounds 14, 15, and 28 were prepared from acyl chlorides, compound 40 from a methyl ester, and all others from the corresponding carboxylic
acids. Aromatic substitution: K2CO3, DMF, 60-100 °C. Ullmann coupling: CuI, Cs2CO3, N,N-dimethylglycine·HCl, 1,4-dioxane 90 °C.
correlation between the minimal-inhibitory concentration
(MIC) and ED50 (the effective dose that rescues 50% of the
animals from the infection) for 14 β-lactam antibiotics16 and for
linezolid.17 This is a rapid animal model of infection that results
in 100% fatality within 48 h. The compounds that would show
efficacy would by necessity exhibit reasonable pharmacokinetic
(PK) properties. This approach sped up lead optimization by
identifying compounds with in vivo activity early. The
compounds that resulted in survival of the animals were then
further scrutinized for optimization by additional syntheses
around the structural space and for attributes such as improved
PK, decreased metabolism, and lack of toxicity to mammalian
cells. The ring A of structure 1 (Scheme 1) produced excellent
opportunities for these additional explorations. These efforts
B
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Figure 1. Antibacterial activities of the synthetic 1,2,4-oxadiazole derivatives. The functionality within the green box was altered to generate all the
synthetic compounds in this series, whereas Y was limited to the three entities that are indicated. The MIC values (in μg/mL) measured for S. aureus
ATCC 29213, with the active compounds placed within the shaded blue area (MIC ≤ 8 μg/mL). Compounds in red font underwent in vivo
evaluation. Properties of the lead compounds (57a, 57b, and 58b) have been described previously.9 Compound 57b is identical to compound 1 and
will be referred to as such in the remainder of the text.
led to the SAR for the oxadiazoles as studied by 120 synthetic
derivatives in ring A.
The left-hand portions of the oxadiazoles were accessed by
starting (with a few exceptions) with the corresponding
carboxylic acids, which would ultimately become ring A and
the C5 of the oxadiazole ring (Scheme 2). These were
converted to the corresponding acyl chlorides by reaction with
either oxalyl chloride or thionyl chloride. The starting materials
included several benzoic acid derivatives (7−28) and a variety
of heteroatom-containing carboxylic acids (29−56). Biological
analysis of the lead compound 1 and a few close analogues
established that a hydrogen bond donor at the 4-position of
ring A is generally beneficial for activity against S. aureus.9 Thus,
the phenolic hydroxyl was retained using several different
protected derivatives (7−16), while the 4-amino group (for
anilines) was accessed by starting with the corresponding 4-
nitrobenzoic acid derivatives (17−21), wherein the nitro
functionality was later reduced. We also explored the effect of
several other substituents at the 4-position of the phenyl ring,
as exemplified by precursors 22−28. The heteroatom-
containing starting materials included several pyrazoles (29−
34), pyrroles (35, 36), imidazole 37, triazoles (38, 39), indole
40, indazoles (41, 42), pyrrolopyridine 43, pyridines (44-48),
several aliphatic derivatives (49−53), protected amino acids
RESULTS AND DISCUSSION
■
Synthesis. The focus of this SAR study is the variation of
structure within ring A of the oxadiazole lead. This ring,
attached to position 5 of the 1,2,4-oxadiazole moiety (1),
proved versatile in generating many active antibiotics of this
class. The diphenyl ether portion (rings C and D) was obtained
by reaction of either 4-fluorobenzonitrile (2) or 4-iodobenzoni-
trile (3) with the appropriate counterpart phenol (4a, 4b, or 4c,
Scheme 2). Typically, coupling with 2 was done via
nucleophilic aromatic substitution, and the reaction with 3
was achieved with an Ullmann coupling. Our variations to the
aromatic rings of the diphenyl ether were minimal and involved
only substitution at the 4-position of ring D with a fluoro or
trifluoromethyl group. The para substitution of ring D,
specifically with those two variations, proved beneficial for
improved metabolic stability and lowered clearance. Nitriles 5a,
5b, and 5c were converted into their corresponding N′-
hydroxybenzimidamides 6a, 6b, and 6c, respectively, using
hydroxylamine in refluxing ethanol.
C
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a
Table 1. Minimal-Inhibitory Concentrations (MICs) of Oxadiazoles
MIC (μg/mL)
i
i
i
60b
60c
65b
66b
75b
76b
1
vancomycin
linezolid
b
c
S. aureus ATCC 29213
S. aureus ATCC 27660
1
1
0.5
0.5
0.25
0.5
2
4
4
8
2
2
1
1
4
2
2
0.5
c
S. aureus NRS100 (COL)
>128
1
>128
2
>128
2
>128
32
2
32
4
2
2
2
d
S. aureus NRS119
2
2
2
32
32
2
d
S. aureus NRS120
64
64
>128
1
>128
1
2
16
1
2
2
e
S. aureus VRS1
1−2
0.5−1
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
1
1
2
1−2
64
16
2
f
S. aureus VRS2
0.5
4
0.5
4
16
32
16
>128
128
16
16
16
16
16
16
16
8−16
2
2
S. epidermis ATCC 35547
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
2
1
S. hemolyticus ATCC 29970
S. oralis ATCC 9811
8
2
2
128
64
16
8
32
32
2
0.5
0.6
1
1
S. pyogenes ATCC 49399
B. cereus ATCC 13061
1
1
B. licheniformis ATCC 12759
2
0.5
2
1
b
E. faecalis ATCC 29212
4
2
2
g
E. faecalis 201 (Van S)
8
2
1
2
h
E. faecalis 99 (Van R)
16
8
2
128
0.5
256
0.5
1
g
E. faecium 119-39A (Van S)
1
2
h
E. faecium 106 (Van R)
8
2
1
E. faecium NCTC 7171
2−4
2
2
a
The compounds were screened against E. coli and the ESKAPE panel of bacteria; they exhibited antibacterial activity against Gram-positive bacteria.
b
c
A quality-control strain to monitor accuracy of MIC testing. mecA positive, resistant to methicillin, oxacillin, and tetracycline; susceptible to
d
e
vancomycin and linezolid. mecA positive, resistant to ciprofloxacin, gentamicin, oxacillin, penicillin, and linezolid. Vancomycin-resistant MRSA
f
g
(vanA) clinical isolate from Michigan. Vancomycin-resistant MRSA (vanA) clinical isolate from Pennsylvania. Vancomycin-susceptible clinical
isolate. Vancomycin-resistant clinical isolate. Data from O’Daniel et al.,9 reproduced for the sake of comparison.
h
i
(54, 55), and pyrimidine 56. While most of these precursors
are commercially available, carboxylic acids 9, 12, 13, 20, 27,
and 52, and acyl chlorides 14 and 15, had to be synthesized
(procedures given in the Supporting Information). Once in
hand, the acyl chlorides were allowed to react with 6a, 6b, or 6c
in refluxing pyridine/toluene or 1,4-dioxane to produce the
1,2,4-oxadiazoles.
Many of these immediate oxadiazole products were subjected
to further synthetic manipulation to broaden the structural
diversity of the derivatives (protective group removal, nitro
reduction, metal-catalyzed coupling, substitution on amine,
etc.). These reactions are described in the Experimental
Methods and Supporting Information.
resulted in lower clearance and better metabolic stability, thus
improving the PK properties.
Replacement of the phenol or aniline moieties in ring A with
certain heterocyclic rings improved antibacterial activity.
Introduction of 4-halogen-substituted pyrazoles (60a−c,
61a,b, 62a−c) maintained MIC values of ≤1 μg/mL. The
pyrazolyl compounds also tolerated NO2 (63a−c) and NH2
(64b) substitution in this position; however, introduction of an
isopropyl group on the amine (65a,b) caused the MIC to drop
further to 0.5 μg/mL. The lowest observed MIC value of 0.25
μg/mL came from the ethynyl substituted derivative 66b.
Other sp-hybridized functional group substitutions (67b and
68b) also maintained good activity.
Addition of a 3-hydroxyl group (69b,c) retained activity, and
the addition of fluorine atoms in the 3- and 5-positions on the
phenol (70a−c and 71b,c) and the aniline (72b, 73a,b) was
possible without significant loss of activity, but an additional
methylene spacer between the 1,2,4-oxadiazole and the phenol
ring (74a) increased the MIC to 8 μg/mL. The other
heterocyclic substitutions that retained good activity were the
indolyl compounds (75a−c), the imidazolyl compound (76b),
the substituted pyridinyl compounds (77b,c), and the nitro
substituted pyrrolyl compound (78b).
Replacement of the hydrogen-bond donating phenol and
aniline groups with aryl halogens (80a,b, 81b,c) resulted in loss
of activity, as did replacement with other hydrogen-bond
accepting aryl moieties (82a,b, 84c). Interestingly, the aniline
derivative with a 4-F substitution on the biphenyl ether (58c)
had no antibiotic activity despite the low MIC values for the 4-
H and 4-CF3 substituted compounds (58a,b). All other
substituted aryl systems failed to show activity (87b,c, 88c,
89c, 90c, 91c, 92c, 93b), including changing the hydroxyl
group to the 2- or 3-positions (95a, 96a, 97a). No activity was
seen for unsubstituted pyrazoles (102b,c), and the activity seen
Structure−Activity Relationship (SAR). The SAR for the
synthetic oxadiazole compounds was investigated using
antibacterial screening against the aforementioned ESKAPE
panel of bacteria plus E. coli. The 120 synthetic samples
encompassed modifications in the lead at the 5-position of the
1,2,4-oxadiazole (ring A in Scheme 1), while keeping the 3-
position constant as a 4-substituted diphenyl ether moiety
(Figure 1). The oxadiazoles exhibit activity against Gram-
positive bacteria. We expressly explored the activity against S.
aureus for this study, for reasons that we outlined earlier. The
SAR was evaluated by minimal-inhibitory concentration (MIC)
measurements against S. aureus ATCC 29213, a standard
methicillin-sensitive S. aureus (MSSA) strain for the purpose of
screening. Active compounds (MIC ≤ 8 μg/mL) are enclosed
within the blue shading in Figure 1. The substituents at the 4-
position of ring D were hydrogen, trifluoromethyl, or fluorine
(Figure 1), as indicated earlier. These modifications had little
effect on the in vitro activity of compounds, except for some
specific cases that are discussed below. However, the
substitution with trifluoromethyl or fluorine at this position
D
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Table 2. In Vitro and in Vivo Evaluation of Selected Oxadiazole Analogs
a
PK Parameters
b
antibiotic human plasma protein binding (%) AUC0−8h (μg min/mL) CL (mL/min/kg) XTT HepG2 IC50 (μg/mL) mouse peritonitis (survived/total)
60b
60c
65b
66b
75b
76b
1
97.8 0.3
94.5 2.1
91.6 0.3
96.4 2.7
98.2 3.2
93.5 2.4
99.9 0.1
910
446
22.0
44.9
15.2
2.4
24.1 1.6
18.2 2.9
3.9 0.8
9.8 4.0
75.7 7.3
31.5 0.5
25.8
4/6
2/6
1313
8261
1283
2054
1/6
2/6
15.2
9.7
5/6
3/6
c
c
c
2650
18.9
ED50 = 40 mg/kg
a
b
PK parameters after a single iv dose at 20 mg/kg (n = 2 mice per 5 time points). Mouse peritonitis, S. aureus ATCC27660 given ip at 5 e7 cfu/mL
with 5% mucin. Compounds were given iv at 20 mg/kg at 30 min and 7.5 h after infection. Data from O’Daniel et al.,9 reproduced for the sake of
c
comparison. PK parameters for compound 1 at 50 mg/kg iv.
in the secondary amine series did not extend to larger straight
chain alkyl or cyclic alkyl substitution (104b, 105b, 106b,
107b) or acylation (108b, 109b). Complete replacement of an
aryl moiety with a simple hydroxyl (112a) or methyl (113a)
abolishes activity and underlines the significance for activity of a
spacing group between the 1,2,4-oxadiazole ring and the
hydrogen-bond donating group. No activity was observed for
any of the other heteroaromatic substituents that were
introduced (114a, 115a, 116b, 117b, 118c, 119c, 120b,c,
121b,c, 122b,c, 123b,c, 124a, 125b,c, 78c, 126c, 127c). In a
similar trend observed with the inactive 4-F diphenyl ether
substituted aniline (58c), the 4-F diphenyl ether substituted
imidazole derivative (76c) showed no activity compared to the
active 4-CF3 derivative (76b). The effect of the 4-position
diphenyl ether substitutions on activity in these cases is yet to
be resolved. Several derivatives with saturated cyclic sub-
stitutions were also synthesized; however, activities were poor
(≥32 μg/mL) (128a, 132a, 133b, 134b, 135b, 136a, 137a).
Activity Against Gram-Positive Organisms. Com-
pounds 60b, 60c, 65b, 66b, 75b, and 76b were evaluated
against a panel of Gram-positive organisms. The pyrazoles
showed activity against S. aureus MSSA (ATCC 29213) and
MRSA (ATCC 27660, NRS119, VRS1, and VRS2) strains,
including vancomycin-resistant strains (Table 1). The pyrazoles
were not active against S. aureus NRS120 and other Gram-
positive organisms. Replacement of the pyrazole with an indole
(75b) broadened the spectrum of activity against Gram-positive
organisms. The activity of the indole 75b was similar to that of
the phenol derivative 1 (Table 1).
Plasma-Protein Binding. Protein binding for compounds
60b, 60c, 65b, 66b, 75b, and 76b was determined in human
plasma using equilibrium dialysis. Results are shown in Table 2.
Protein binding of the pyrazoles 60b, 60c, 65b, 66b, and the
imidazole 76b was lower than that of the indole 75b (98.2
3.2%). Although plasma protein binding was high, 43% of the
1500 most frequently prescribed drugs have protein binding
>90%,18 and 12 of the 100 most prescribed drugs have >98%
plasma protein binding.19 Plasma protein binding of many
antibiotics on the market, including daptomycin, oxacillin,
teicoplanin, rifampicin, and clindamycin, is >91%.20−23
Fast Pharmacokinetic (PK) Studies. To rapidly ascertain
the PK properties of the compounds, fast PK studies were
conducted. These studies involve administration of the
compounds using a limited number of animals (n = 2 mice
per time point) for a few time points. This allows us to rapidly
compare the preliminary PK properties of the compounds, so
that full PK studies are conducted only with the most
promising compound(s). All compounds were administered
intravenously (iv) with a single dose at 20 mg/kg. The alkyne
substituted pyrazole 66b had the lowest clearance and the
highest systemic exposure, as measured by area under the curve
(AUC, Table 2). The highest clearance was observed for 60c,
and as a result it had the lowest systemic exposure.
In Vitro Cytotoxicity. We used the XTT assay with HepG2
cells to evaluate the in vitro toxicity of compounds 60b, 60c,
65b, 66b, 75b, and 76b (Table 2). The highest toxicity was
observed for 65b and the lowest for the indole 75b. Compared
to the lead 1, indole 75b was 5-fold less toxic.
In Vivo Efficacy. Compounds 60b, 60c, 65b, 66b, 75b, and
76b were evaluated in the mouse peritonitis model of infection
(Table 2). We used the ICR out-bred strain of mice that
provides a heterogeneous population, similar to the human
situation, thus ensuring the relevance of the antibacterial effect.
This animal model of infection is widely used, it is easy to carry
out, and the end points (death or survival) are rapidly assessed,
making it less resource-intensive compared to other infection
models. In addition, excellent correlation between MIC and
ED50 has been shown for 14 β-lactam antibiotics using this
model.16 The mouse peritonitis infection model continues to
be an important model for evaluation of the efficacy of
antibiotics against human pathogens.17 We use the iv route of
administration in initial efficacy studies, as this allows us to test
the efficacy without knowledge of the oral bioavailability of the
lead. Evaluation was done at 20 mg/kg. The highest efficacy
was observed for indole 75b (Table 2).
Minimal-Bactericidal Concentration (MBC). The MBC
of compound 75b was determined using S. aureus ATCC
29213, S. aureus ATCC 277660, and E. faecium NCTC 7171.
For S. aureus ATCC 29213 (an MSSA strain), the MBC was
the same as the MIC value, while for the two other strains, the
MBC was 2-fold above the MIC values. These data indicated
that compound 75b is bactericidal at concentrations that inhibit
bacterial growth (Table 4).
Full PK Study. A full PK study was conducted with indole
75b after iv and oral (po) administration. This compound had
the lowest in vitro toxicity and the highest efficacy in the mouse
peritonitis infection model. Results are summarized in Figure 2
and Table 3. Antibiotic 75b was characterized by low clearance
of 5.68 mL/min/kg (less than 10% of hepatic blood flow), a
high volume of distribution of 4.73 L/kg, and a terminal half-
life after iv administration of 9.6 h. After oral administration,
maximum concentrations were observed at 6 h, after which
time relatively high concentrations were sustained. The
terminal half-life after oral administration was long (18.6 h).
The oral bioavailability of 75b at 97% was high, and was similar
to that of compound 1.9 Antibiotic 75b had 13-fold higher
E
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Table 3. In Vivo Efficacy of Compound 75b in the Mouse
Peritonitis Model
route of
administration
compd
dose frequency
ED50 (mg/kg)
75b
75b
1
iv
2 doses given at 30 min and 7.5
h after infection
7.6
1.7
po
iv
a
40
75b
1
po
po
po
single dose given at 1 h after
infection
3.1
a
44
linezolid
2.8
a
Data from O’Daniel et al.,9 reproduced for the sake of comparison.
Figure 2. Pharmacokinetics of 75b after single iv and po
administration at 20 mg/kg to mice (n = 3 per time point).
volume of distribution and 3-fold lower clearance than 1 and
was more rapidly absorbed than 1 (t1/2abs of 0.8 h vs 3.3 h9).
Thus, antibiotic 75b has superior PK properties compared to 1.
In Vivo Efficacy of 75b. Antibiotic 75b was evaluated in
the mouse peritonitis infection model using S. aureus ATCC
27660 (MRSA) after iv and po administration (Table 3). The
mean effective dose (ED50) values were 7.6 mg/kg and 1.7 mg/
kg after iv and po doses given at 30 min and 7.5 h after
infection, respectively. The excellent oral efficacy was attributed
to the sustained plasma concentrations of 75b following po
administration. The ED50 value after iv administration of 75b is
6-fold better than that of 1. The ED50 of compound 75b was
also evaluated after a single po dose given at 1 h after infection.
Compound 75b has an excellent ED50 of 3.1 mg/kg,
comparable to that of linezolid of 2.8 mg/kg (Table 3) and
14-fold better than that of 1. These data indicated that
antibiotic 75b is superior than 1 and comparable in efficacy to
linezolid.
such as pyridines (114a, 115a, 120b−122c), triazoles (116b,
117b), and pyroles (78c, 126c, 127c) generally abolish
antibacterial activity, as do aliphatic heterocycles (128a−135b).
Although introducing a pyrazole at ring A generally results in
compounds that are potently active in vitro with living bacteria,
they are also generally cytotoxic. Thus, while pyrazole
derivatives 65b and 66b are among the most active compounds
reported here, they also exhibit the highest toxicity toward
mammalian cells. Replacing the pyrazole with an indole
circumvents these toxicity problems, while retaining antibacte-
rial activity. One compound, antibiotic 75b, shows excellent
efficacy in vivo with a long half-life, a high volume of
distribution, and low clearance. Antibiotic 75b is bactericidal
and is 97% orally bioavailable. This class of antibiotics holds
great promise in treatment of infections by these difficult
human pathogens.
CONCLUSION
EXPERIMENTAL METHODS
■
■
The recent discovery of the oxadiazole class of anti-MRSA
antibiotics provided the opportunity to explore the structural
space for these cell-wall-active antibiotics. We have disclosed in
the present work the SAR for this class by synthesis and
evaluation of 120 structural variants, of which a few dozen
exhibit antibacterial activity against S. aureus and MRSA strains.
Certain heterocycles with the ability to donate hydrogen bonds
are well-tolerated at ring A. Thus, the 4-phenol (57a−59b,
69b−71c) and 4-aniline (72b−73b) analogs are active against
S. aureus, but substituents such as phosphates (84c),
sulfonamides (87b, 87c), amides (88c), and carboxylic acids
(89c) attenuate or abrogate activity. Hydrogen-bond-accepting
substituents on ring A abolish activity (e.g., 80a−82b, 91c, 93b,
94a, 99a−101c). Replacing the phenyl moiety of ring A for an
aromatic heterocyclic ring retains activity in some cases.
Pyrazoles substituted with halogens (60a−62c), a nitro group
(63a−63c), an isopropylamino group (65a, 65b), or sp-
hybridized groups (66b−68b) are all active, as are indoles
(75a−75c) and an imidazole (76b). Pyrazoles containing
amino groups with larger substituents (104b−110b) lead to
abolishment of antibacterial activity. Heteroaromatic systems
In Silico Screening. A library of 1.2 million drug-like compounds
from the ChemDiv subset of the ZINC database24 was prepared for
high-throughput virtual screening against the X-ray structure of PBP2a
(PDB ID: 1VQQ).25 The protein was prepared using the Schrodinger
̈
Preparation Wizard (Schrodinger, LLC, 2009). The top scoring 10%
̈
of the compounds by Schrodinger Glide were cross-docked with
̈
Glide-SP,26−28 Autodock,29 Gold-chemscore, Gold-goldscore, and
Gold-PLP.30 The top-scoring 2000 poses from each were extracted
and refined using Glide-XP mode.26−28 Finally, the best 2500
compounds were clustered according to structural similarity using
hierarchical clustering. Binding poses of these clusters were inspected
visually. From these, 29 compounds were selected and purchased from
ChemDiv for in vitro activity experiments.
Syntheses. The synthetic procedures for the six compounds
chosen for in vivo evaluation are detailed below. These are
representative of the methods that were used for the preparation of
other derivatives. Purity of the final products was generally >95%, as
confirmed by HPLC. Detailed conditions are provided in the HPLC
section.
Methyl 1H-indole-5-carboxylate (40). 1H-Indole-5-carboxylic acid
(0.843 g, 5.23 mmol), methyl iodide (3.21 g, 22.7 mmol), and
NaHCO3 (1.76 g, 20.92 mmol) were stirred in DMF (24 mL) at room
temperature for 3 days at which point water (50 mL) was added to the
Table 4. Pharmacokinetic Parameters of 75b
dose (mg/kg) AUC0−24h (μg min/mL) AUC0‑∞ (μg min/mL) Cmax (μg/mL) Tmax (h) CL (mL/min/kg) Vd (L/kg)
t1/2
F (%)
20 iv
3140
3520
5.68
4.73
t1/2α = 0.54 min
t1/2β = 9.6 h
97
20 po
3060
4060
2.34
6
t1/2abs = 0.83 h
t1/2dist = 15.9 h
t1/2elim = 18.6 h
F
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Journal of Medicinal Chemistry
Article
mixture forming a milky precipitate that was extracted with ethyl
acetate (3 × 30 mL). The combined organic layer was washed with 5%
LiCl (2 × 50 mL) and dried over anhydrous Na2SO4, and the
suspension was filtered. The filtrate was concentrated to dryness in
vacuo to produce an off white solid, which was purified by silica-gel
chromatography (ethyl acetate/hexanes, 1:10) to give the desired
product as a white solid (0.820 g, 90%). 1H NMR (400 MHz, CDCl3)
δ 3.93 (s, 3H), 6.64 (m, 1H), 7.26 (m, 1H), 7.39 (dt, J = 8.6 Hz, 0.8
Hz, 1H), 7.91 (dd J = 8.6 Hz, 1.6 Hz, 1H), 8.42 (m, 1H), 8.48 (s, 1H).
13C NMR (100 MHz, CDCl3) δ 52.1, 104.2, 111.0, 122.2, 123.6, 124.0,
and was concentrated to dryness in vacuo. The residue was purified by
silica-gel chromatography (EtOAc/hexanes, 1:2 to 2:1) to yield the
title compound as a yellow foam (75.4 mg, 80.0%). Mp 184−187 °C.
1H NMR (400 MHz, CDCl3) δ 7.07−7.13 (m, 4H), 7.48 (s, 1H), 7.63
(d, J = 8.4 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 118.3, 119.0, 119.2, 122.3, 124.1, 126.0, 126.3, 127.5 (q, J
= 4.0 Hz), 129.5, 132.4, 158.6, 159.2, 167.3, 170.0. HRMS (ESI): calcd
for C18H13F3N5O2 [M + H]+ 388. 1016, found 388.1010.
N-Isopropyl-3-(3-(4-(4-(trifluoromethyl)phenoxy)phenyl)-1,2,4-
oxadiazol-5-yl)-1H-pyrazol-4-amine (65b). To a solution of
compound 64b (75.4 mg, 0.19 mmol) and acetone (17 μL, 0.23
mmol) in 5 mL of CH2Cl2 were added activated 3 Å molecular sieves
and sodium triacetoxyborohydride (62.0 mg, 0.29 mmol). The mixture
was stirred at room temperature for 7 days. The mixture was filtered
through Celite, which was washed with EtOAc. The filtrate was
concentrated to dryness, and the residue was purified by silica-gel
chromatography (EtOAc/hexanes, 1:3) to afford the desired product
125.7, 127.7, 138.6, 168.5. HRMS (ESI): calcd for C10H10NO2 [M +
H]+ 176.0706, found 176.0710.
5-(4-Chloro-1H-pyrazol-3-yl)-3-(4-(4-(trifluoromethyl)phenoxy)-
phenyl)-1,2,4-oxadiazole (60b). This compound was synthesized
using the same procedure as for 63b and purified by silica-gel
chromatography (EtOAc/hexanes, 1:4) to yield the product as an off-
white powder (58%). Mp 193−195 °C. 1H NMR (400 MHz, CDCl3)
δ 7.16−7.19 (m, 4H), 7.66 (d, J = 8.8 Hz, 2H), 8.02 (s, 1H), 8.17−
8.20 (m, 2H), 13.58 (br, 1H). 13C NMR (100 MHz, DMSO-d6) δ
110.8, 119.8, 120.6, 122.6, 124.8, 125.1, 128.3 (q, J = 3.6 Hz), 130.1,
130.9, 134.1, 158.8, 159.9, 168.0, 170.2. HRMS (ESI): calcd for
C18H11ClF3N4O2 [M + H]+ 407.0517, found 407.0540.
5-(4-Chloro-1H-pyrazol-3-yl)-3-(4-(4-fluorophenoxy)phenyl)-
1,2,4-oxadiazole (60c). This compound was synthesized using the
same procedure as for 63b and was purified by silica-gel
chromatography (EtOAc/hexanes, 1:6) to yield the product as an
off-white powder (61%). Mp 212−214 °C. 1H NMR (400 MHz,
CDCl3) δ 7.07−7.13 (m, 6H), 7.91 (s, 1H), 8.16 (d, J = 9.2 Hz, 2H).
13C NMR (100 MHz, DMSO-d6) δ 110.1, 116.8, 117.0, 117.9, 120.6,
1
as a light green powder (44 mg, 53%). Mp 158−161 °C. H NMR
(400 MHz, CDCl3) δ 1.33 (s, 3H), 1.35 (s, 3H), 3.50−3.56 (m, 1H),
7.15−7.20 (m, 4H), 7.47 (s, 1H), 7.65 (d, J = 8.8 Hz, 2H), 8.18−8.21
(m, 2H). 13C NMR (100 MHz, CDCl3) δ 23.2, 47.8, 116.0, 119.1,
119.6, 122.8, 122.9, 125.9, 126.2, 127.6 (q, J = 3.7 Hz), 129.8, 135.7,
158.9, 159.6, 167.6, 170.6. HRMS (ESI): calcd for C21H19F3N5O2 [M
+ H]+ 430. 1485, found 430.1489.
5-(4-Ethynyl-1H-pyrazol-3-yl)-3-(4-(4-(trifluoromethyl)phenoxy)-
phenyl)-1,2,4-oxadiazole (66b). Compound 103b (0.12 g, 0.26
mmol), KF (31 mg, 0.53 mmol), and 10 mL of MeOH were placed in
a round-bottom flask. The mixture was stirred at room temperature for
17 h. After the completion of the reaction, the solvent was removed in
vacuo, and the residue was purified by silica-gel chromatography
(EtOAc/hexanes, 1:4) to give the compound as an off-white powder
121.8, 121.9, 129.3, 130.3, 133.5, 151.2, 151.3, 157.6, 160.0, 160.2,
167.4, 169.4. HRMS (ESI): calcd for C17H11ClFN4O2 [M + H]+
357.0549, found 357.0544.
1
(84.7 mg, 81%). Mp 206−209 °C. H NMR (500 MHz, CDCl3) δ
5-(4-Iodo-1H-pyrazole-3-yl)-3-(4-(4-trifluoromethyl)phenoxy)-
phenyl)-1,2,4-oxadiazole (62b). This compound was synthesized
according to the procedure for 63b and was purified by silica-gel
chromatography (EtOAc/hexanes, 1:3.5) to afford compound 62b as
3.39 (s, 1H), 7.14−7.19 (m, 4H), 7.64 (d, J = 8.5 Hz, 2H), 8.06 (s,
1H), 8.21−8.23 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 72.8, 83.1,
119.2, 119.8, 122.5, 122.9, 125.6, 126.0, 126.4, 127.6 (q, J = 3.6 Hz),
129.9, 159.1, 159.5, 168.5, 169.6. HRMS (ESI): calcd for
C20H12F3N4O2 [M + H]+ 397.0907, found 397.0914.
1
an off-white powder (64.0%). Mp 222−225 °C. H NMR (400 MHz,
DMSO-d6) δ 7.29−7.36 (m, 4H), 7.80 (d, J = 8.8 Hz, 2H), 8.16 (d, J =
8.4 Hz, 2H), 8.31 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 60.4,
119.1, 120.0, 122.1, 122.8, 124.1, 124.4, 125.5, 127.6 (q, J = 3.5 Hz),
129.4, 137.4, 138.3, 158.1, 159.2, 167.3, 170.4. HRMS (ESI): calcd for
C18H11F3IN4O2 [M + H]+ 498.9873, found 498.9879.
5-(1H-Indol-5-yl)-3-(4-(4-(trifluoromethyl)phenoxy)phenyl)-1,2,4-
oxadiazole (75b). A solution of N′-hydroxy-4-(4-(trifluoromethyl)-
phenoxy)benzimidamide (6b, 1.02 g, 3.44 mmol) in anhydrous THF
(15 mL) was stirred under an argon atmosphere, and sodium hydride
(60% in mineral oil, 0.172 g, 4.30 mmol) was added to the flask. The
mixture was left to stir for 1 h at room temperature, and then a
solution of methyl 1H-indole-5-carboxylate (40, 0.302 g, 1.72 mmol)
in anhydrous THF (15 mL) was added and the mixture heated at
reflux for 3.5 h. Once the solution had cooled to room temperature,
water (50 mL) was added, and the resulting mixture was extracted with
ethyl acetate (3 × 50 mL). The combined organic layer was dried over
anhydrous Na2SO4 and then filtered, and the filtrate was evaporated to
leave an orange residue. This was purified using column chromatog-
raphy on silica gel (dichloromethane/hexanes, 9:1) to give the desired
5-(4-Nitro-1H-pyrazole-3-yl)-3-(4-(4-(trifluoromethyl)phenoxy)-
phenyl)-1,2,4-oxadiazole (63b). 4-Nitropyrazole-3-carboxylic acid
(34, 0.24 g, 1.50 mmol) was dissolved in SOCl2 (2.2 mL, 30.548
mmol), and the solution was stirred at reflux for 2 h. The excess SOCl2
was evaporated to dryness in vacuo, and the residue was taken up in
toluene (15 mL) and pyridine (0.61 mL, 7.0 mmol), followed by the
addition of (Z)-N′-hydroxy-4-(4-(trifluoromethyl)phenoxy)-
benzimidamide (6b, 0.30 g, 1.0 mmol). The resultant mixture was
stirred at reflux overnight. The solvent was evaporated to dryness in
vacuo, and the residue was purified by silica-gel chromatography
(CH2Cl2/MeOH, 100:1) to afford the title compound as a yellow
powder (0.20 g, 48.0%). Mp 204−206 °C. 1H NMR (400 MHz,
CDCl3) δ 7.15−7.18 (m, 4H), 7.65 (d, J = 8.4 Hz, 2H), 8.18−8.20 (m,
2H), 8.55 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 120.0, 120.5,
122.2, 123.5, 125.0, 125.3, 126.2, 128.3 (q, J = 3.5 Hz), 130.2, 133.1,
134.8, 159.2, 159.8, 168.4, 169.3. HRMS (ESI): calcd for C18H10F3N5
NaO4 [M + Na]+ 440.0577, found 440.0579.
3-(3-(4-(4-(Trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadiazol-5-
yl)-1H-pyrazol-4-amine (64b). Anhydrous THF (5 mL) was slowly
added to a mixture of sulfur (0.22 g, 6.86 mmol) and sodium
borohydride (74.4 mg, 1.96 mmol) in a round-bottom flask at room
temperature. After stirring for 10 min, compound 63b (0.10 g, 0.24
mmol) in THF (2.0 mL) was added dropwise to the above mixture
before heating it to 65 °C for 2.5 h. Upon cooling to room
temperature, water (6 mL) and diethyl ether (6 mL) were added, and
the mixture was stirred for 5 min. The layers were separated, and the
aqueous portion was extracted with diethyl ether (3 × 12 mL). The
combined organic layer was washed with brine and dried (Na2SO4)
1
product as a white solid (0.190 g, 26%). Mp 138−141 °C. H NMR
(400 MHz, CDCl3) δ 6.69 (m, 1H), 7.11 (d, J = 9.0 Hz, 2H), 7.14 (d,
J = 8.9 Hz, 2H), 7.29 (m, 1H), 7.48 (dt J = 8.5 Hz, 1.6 Hz 1H), 7.61
(d, J = 9.0 Hz 2H), 8.03 (dd, J = 8.6 Hz, 1 Hz, 1.6 Hz 1H), 8.20 (d, J =
8.9 Hz, 2H), 8.6 (m, 1H), 8.6 (s, 1H). 13C NMR (100 MHz, CDCl3) δ
104.2, 111.9, 116.2, 118.9, 119.8, 122.1, 122.3, 123.5, 124.3 (q, J =
272.2 Hz), 125.9 (q, J = 32.9 Hz), 126.2, 127.5 (q, J = 3.6 Hz), 128.2,
129.7, 138.4, 158.6, 159.7, 168.3, 177.4. 19F NMR (376 MHz, CDCl3)
δ 99.9 (s, 3F). HRMS (ESI): calcd for C23H14F3N3O2 [M + H]+
422.1111, found 422.1078.
5-(1H-Imidazol-4-yl)-3-(4-(4-(trifluoromethyl)phenoxy)phenyl)-
1,2,4-oxadiazole (76b). The compound was synthesized according to
the procedure used for 63b and purified by silica-gel chromatography
(EtOAc/hexanes, 1:4 to 1:1.5) to afford the product as an off-white
powder (72.0%). Mp 243−245 °C. 1H NMR (400 MHz, DMSO-d6) δ
7.29−7.31 (m, 4H), 7.80 (d, J = 8.8 Hz, 2H), 7.99 (s, 1H), 8.12 (d, J =
8.4 Hz, 2H), 8.24 (s, 1H), 13.00 (br, 1H). 13C NMR (100 MHz,
DMSO-d6) δ 119.2, 119.8, 122.4, 122.5, 124.1, 124.4, 126.3, 127.7 (d, J
G
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Article
= 3.0 Hz), 129.4, 138.3, 158.0, 159.2, 167.2, 172.2. HRMS (ESI): calcd
for C18H12 F3N4O2 [M + H]+ 373.0907, found 373.0911.
B% = (Cp − Cf)/Cp × 100
3-(4-(4-Trifluoromethyl)phenoxy)phenyl)-5-(4-((trimethylsilyl)-
ethynyl-1H-pyrazol-3-yl)-1,2,4-oxadiazole (103b). Compound 62b
(0.19 g, 0.38 mmol) was placed in a 10 mL round-bottom flask, and 5
mL of anhydrous THF was added to the flask. Ethynyltrimethylsilane
(0.12 mL, 0.84 mmol), Pd(Ph3P)2Cl2 (21.4 mg, 0.03 mmol), CuI
(9.00 mg, 0.05 mmol), and Et3N (0.14 mL, 0.99 mmol) were added to
the above mixture. The resultant solution was heated in reflux for 5 h.
After completion of the reaction, the solvent was evaporated to
dryness in vacuo, and the residue was purified by silica-gel
chromatography (EtOAc/hexanes, 1:6) to afford the product as a
where Cp and Cf are the total plasma concentration and the free
concentration of compound, respectively.
XTT Cytotoxicity Assay. The XTT cytotoxicity assay was performed
in triplicated using HepG2 cells (ATCC HB-8065), as previously
described.9 The IC50 values were calculated with GraphPad Prism 5
(GraphPad Software, Inc., San Diego, CA).
Animals. Female ICR mice (6−8 weeks old, ∼20-g body weight)
were used for the PK and peritonitis studies. Animals were purchased
from Harlan Laboratories, Inc. (Indianapolis, IN) and given Teklad
2019 Extruded Rodent Diet and water ad libitum. Mice were
1
1
white foam (0.14 g, 79.5%). H NMR (400 MHz, CDCl3) δ 0.32 (s,
maintained in polycarbonate shoebox cages with
/ in. corncob
4
9H), 7.15−7.19 (m, 4H), 7.65 (d, J = 8.8 Hz, 2H), 8.18 (s, 1H), 8.22
(d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 0.1, 93.7, 100.8,
105.5, 119.2, 119.7, 122.6, 122.9, 125.6, 126.0, 126.4, 127.6 (q, J = 3.7
Hz), 129.9, 135.6, 138.5, 159.1, 159.4, 159.5, 168.5, 169.8. HRMS
(ESI): calcd for C23H20F3N4O2Si [M + H]+ 469.1303, found 469.1336.
High Performance Liquid Chromatography (HPLC). The system
used was a PerkinElmer Series 200 Chromatography System
(PerkinElmer, Waltham, MA) equipped with an autosampler, UV−
vis detector, LC pump, NCI 900 Network Chromatography Interface,
and 600 Series Link Chromatography Interface. The samples were
analyzed on a Zorbax RX-C8 analytical column (5.0 μm, 4.6 mm id ×
250 mm, Agilent Technologies, Santa Clara, CA). The mobile phase
consisted of isocratic elution for 10 min with a 1:1 mixture of water/
0.1% trifluoroacetic acid (TFA) and acetonitrile/0.1% TFA at a flow
rate of 1.0 mL/min, with the effluent monitored by UV detection
(detection window set to 250−255 nm).
Microbial Strains. The ESKAPE organisms (E. faecium NCTC
(ATCC 19734), S. aureus ATCC 29213, K. pneumonia ATCC 700603,
A. baumannii ATCC 17961, P. aeruginosa ATCC 17853, E. aerogenes
ATCC 35029) and E. coli ATCC 25922) in the initial screen, S. aureus
ATCC 27660, S. epidermis ATCC 35547, S. hemolyticus ATCC 29970,
S. oralis ATCC 9811, S. pyogenes ATCC 49399, B. cereus ATCC 13061,
B. licheniformis ATCC 12759, and E. faecalis ATCC 29212 were
purchased from the American Type Culture Collection (Manassas,
VA). S. aureus strains NRS100, NRS119, NRS120, VRS1, and VRS2
were obtained from the Network on Antimicrobial Resistance in
Staphylococcus aureus (Chantilly, VA). E. faecalis strains 201 and 99,
and E. faecium strains 119-39A and 106 were collected from Wayne
State University School of Medicine.
(The Andersons Inc., Maumee, OH) and Alpha-dri (Sheperd Specialty
Papers, Inc., Richland, MI) bedding under 12-h light/12-h dark cycle
at 72 2 °F. All procedures involving animals were approved by the
University of Notre Dame Institutional Animal Care and Use
Committee.
Fast Pharmacokinetic (PK) Studies. For fast PK studies, the
compounds were dissolved in 10% DMSO/25% Tween-80/65% water
at a concentration of 5 mg/mL. The dosing formulations were
sterilized by filtration through a 0.2 μm, 13 mm diameter PTFE
membrane attached to an Acrodisc syringe filter (Pall Life Sciences,
Ann Arbor, MI). Mice (n = 2 per time point) were given 100 μL of the
test compound(s) intravenously (iv), equivalent to 20 mg/kg.
Terminal blood was collected at 5 min, 40 min, 2 h, 4 h, and 8 h;
blood was centrifuged at 1200 g for 10 min to harvest plasma.
Full PK Studies. Antibiotic 75b was dissolved in 10% DMSO/25%
Tween-80/65% water at a concentration of 5 mg/mL. Mice (n = 3 per
time point per route of administration) were administered 100 μL of
75b (equivalent to 20 mg/kg) iv. A separate group of mice was given
100 μL of 75b (equivalent to 20 mg/kg) orally (po). Terminal blood
was collected in heparin by cardiac puncture at 2, 5, 10, 20, and 40
min, and at 1, 2, 3, 4, 8, and 24 h after iv administration and at 0.5, 1, 2,
3, 4, 6, 9, 24, and 36 h after po administration. Blood was centrifuged
at 1200 g for 10 min to obtain plasma. Plasma samples were stored at
−80 °C until analysis.
Bioanalytical Method. Plasma (50 μL aliquot) was mixed with 100
μL of acetonitrile containing internal standard (final concentration 8
μg/mL). After centrifugation at 10 000 g for 10 min, the supernatant
was analyzed by ultraperformance liquid chromatography (UPLC)
with UV detection at 285 nm. A Waters Acquity UPLC System
(Waters Corporation, Milford, MA), consisting of a binary pump, an
autosampler, a column heater, and a photodiode array detector, was
used. An Acquity UPLC C18 1.7 μm, 2.1 mm id × 50 mm column was
used. Elution was at 0.5 mL/min with 70% A/30% B for 2 min,
followed by a 10 min linear gradient to 10% A/90% B, and then 70%
A/30% B for 2 min, where A = 0.1% formic acid/water and B = 0.1%
formic acid/acetonitrile. Monitoring was by UV detection at 285 nm.
Calibration curves for each compound were prepared in control
plasma containing internal standard. The concentrations in the PK
samples were obtained using peak area ratio to the internal standard
and the calibration curve regression analysis parameters. The methods
were linear from 0.01 to 100 μg/mL; coefficients of determination R2
range from 0.98 to 0.99.
Pharmacokinetic Parameters. The area under the curve (AUC),
clearance (CL), volume of distribution (Vd), and terminal half-life
were calculated using Phoenix WinNonlin 6.3 (Certara LP, St Louis,
MO) noncompartmental analysis using uniform weighing. Half-lives
were estimated from the linear portion of the initial or terminal phase
of the concentration−time data by linear regression, where the slope of
the line was the rate constant k and t1/2 = ln 2/k.
Mouse Peritonitis Studies. The mouse peritonitis model was used
with S. aureus ATCC 27660, as described previously.9 The final
bacterial inocula contained 5 × 107 cfu/mL and 5% mucin (Sigma-
Aldrich Chemical Co., St Louis, MO). Just prior to inoculation,
bacteria at 108 cfu/mL were mixed 1:1 with 10% mucin. Mice (n = 6
per group) were given 0.5 mL of the bacterial inocula intraperitoneally.
Mice were given two iv doses of the compounds at 30 min and 7.5 h
after infection by tail vein injection. Vehicle and positive control
Minimal-Inhibitory Concentration (MIC) Determination. The
procedure for MIC determination was the same as that previously
reported.9
Minimal-Bactericidal Concentration (MBC) Determination. The
MBC of antibiotic 75b was determined by incubation of 1.5 × 105 cells
of S. aureus ATCC29213, S. aureus ATCC 27660, and E. faecium
NCTC7171 at MIC, 2× MIC, and 4× MIC. Aliquots of 10 μL
(corresponding to 1.5 × 104 cells) were plated on agar plates and
incubated for 48 h, and colonies were counted in the presence and
absence of antibiotic 75c.9 The MBC was the concentration of
antibiotic 75b that resulted in >1000-fold reduction in colonies.
Plasma Protein Binding. Plasma protein binding was determined
using human plasma and a rapid equilibrium dialysis device (Pierce
Biotechnology, Thermo Scientific, Waltham, MA). Human plasma was
thawed and centrifuged at 1200 g for 10 min to remove particulates. A
200 μL aliquot of human plasma was added to the sample chamber
and 350 μL of 0.1 M phosphate buffered saline (pH 7.4) containing
0.15 mM sodium chloride was added to the adjacent chamber. A 2 μL
aliquot of a stock solution of the compounds at a concentration of 1
mM in DMSO was diluted with human plasma to a final drug
concentration of 10 μM and added to the sample chamber. The
compounds were dialyzed at 37 °C in an orbital shaker for 6 h.
Aliquots (50 μL) were taken from the buffer chamber (representing
the free concentration) and from the plasma chamber (representing
the total concentration) and mixed with 100 μL of internal standard in
acetonitrile to a final concentration of 5 μM. Samples were analyzed by
UPLC with UV detection at 285 nm. The plasma protein binding ratio
(B%) was calculated according to the following equation
H
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Journal of Medicinal Chemistry
Article
(vancomycin at 5 mg/kg) groups were included. Mice were monitored
for 48 h, at which time the number of surviving mice were counted.
ED50 Determination. The effective dose that results in survival of
50% of the mice was determined using Probit analysis (XLSTAT, New
York, NY). Groups of six mice per dose level were evaluated in the
mouse peritonitis infection model at iv doses of 2.5, 5, 7.5, 10, 15, and
20 mg/kg and after po doses at 2.5, 5, 10, 20, and 40 mg/kg. The
doses were given at 30 min and 7.5 h after infection. In addition, ED50
values were determined for compound 75b and linezolid after a single
po dose given at 1 h after infection.
faecium during daptomycin therapy. Antimicrob. Agents Chemother.
2005, 49, 1664−1665.
(8) Mendes, R. E.; Tsakris, A.; Sader, H. S.; Jones, R. N.; Biek, D.;
McGhee, P.; Appelbaum, P. C.; Kosowska-Shick, K. Characterization
of methicillin-resistant Staphylococcus aureus displaying increased MICs
of ceftaroline. J. Antimicrob. Chemother. 2012, 67, 1321−1324.
(9) O’Daniel, P. I.; Peng, Z.; Pi, H.; Testero, S. A.; Ding, D.; Spink,
E.; Leemans, E.; Boudreau, M. A.; Yamaguchi, T.; Schroeder, V. A.;
Wolter, W. R.; Llarrull, L. I.; Song, W.; Lastochkin, E.; Kumarasiri, M.;
Antunes, N. T.; Espahbodi, M.; Lichtenwalter, K.; Suckow, M. A.;
Vakulenko, S.; Mobashery, S.; Chang, M. Discovery of a new class of
non-β-lactam inhibitors of penicillin-binding proteins with Gram-
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ASSOCIATED CONTENT
* Supporting Information
■
S
General experimental procedures for MIC determination and
further information on synthetic procedures. Spectral data for
reported compounds and their MIC values against Gram-
positive organisms of the ESKAPE panel. This material is
available free of charge via the Internet at http://pubs.acs.org.
(11) Fisher, J. F.; Meroueh, S. O.; Mobashery, S. Bacterial resistance
to β-lactam antibiotics: compelling opportunism, compelling oppor-
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: mchang@nd.edu. Phone: 574-631-2965.
*E-mail: mobashery@nd.edu. Phone: 574-631-2933.
■
Author Contributions
§E.S. and D.D. contributed equally to this work.
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.).
■
ABBREVIATIONS USED
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■
AUC, area under the curve; DMAP, dimethyl amino pyridine;
DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
Et3N, triethylamine; MIC, minimum-inhibitory concentration;
MRSA, methicillin-resistant Staphylococcus aureus; PK, pharma-
cokinetics; TBS, tert-butyldimethylsilyl; THF, tetrahydrofuran;
TLC, thin layer chromatography; UPLC, ultraperformance
liquid chromatography; UV, ultraviolet
(18) Kratochwil, N. A.; Huber, W.; Muller, F.; Kansy, M.; Gerber, P.
̈
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