Expanded Structure-Activity Studies of Lipoxazolidinone Antibiotics

Article abstract of DOI:10.1021/acsmedchemlett.9b00015

The lipoxazolidinone family of marine natural products, which contains an unusual 4-oxazolidinone core, was found to possess potent antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA). Herein, we expanded our previous synthet

Full text of DOI:10.1021/acsmedchemlett.9b00015

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Expanded Structure−Activity Studies of Lipoxazolidinone
Antibiotics
Kaylib R. Robinson, Jonathan J. Mills,† and Joshua G. Pierce*
Department of Chemistry and Comparative Medicine Institute, NC State University, Raleigh, North Carolina 27695, United States
S
* Supporting Information
ABSTRACT: The lipoxazolidinone family of marine natural
products, which contains an unusual 4-oxazolidinone core,
was found to possess potent antimicrobial activity against
methicillin resistant Staphylococcus aureus (MRSA). Herein,
we expanded our previous synthetic efforts by preparing
selected aryl derivatives of the lipoxazolidinones and further
evaluating the potential to expand the activity of this class of
molecules to Gram-negative pathogens. With these analogs, we explored the effect of varying the substitution pattern around the
aromatic ring, increasing the chain length between the oxazolidinone core and the aryl system, and how altering the position of
more polar functional groups affected the antimicrobial activity. Finally, we utilized a TolC knockout strain of E. coli to
demonstrate that our compounds are subject to efflux in Gram-negative pathogens, and activity is restored in these knockouts.
Together these results provide additional data for the further development of 4-oxazolidinone analogs 5, 20, and 21 for the
treatment of infectious disease.
KEYWORDS: Oxazolidinones, antibiotics, MRSA, heterocycles
products, the synoxazolidinones, which have demonstrated
antibiofouling activity, and 2,2-dimethyl-2-(4-hydroxyphenyl)-
4-oxazolidinone.8−13 Both the lipoxazolidinones and synox-
azolidinones are structurally related to the 2-oxazolidinone
antibiotic linezolid, and its derivatives yet possess quite distinct
three-dimensional structures (Figure 1B). Linezolid was FDA
approved in 2000 for the treatment of methicillin resistant
Staphylococcus aureus (MRSA) and vancomycin resistant
Enterococcus (VRE), two multidrug resistant organisms of
high clinical relevance.14 Like all antibiotics, growing resistance
threatens the long-term longevity of these compounds in the
clinic.15
Previously, our group has synthesized analogs of the
lipoxazolidinones that are potent against methicillin susceptible
S. aureus (MSSA) and MRSA; however, these analogs have
potential liabilities, including high lipophilicity.16,17 Since little
was known regarding the SAR of the right-hand domain of the
lipoxazolidinones, we drew inspiration from one of the analogs
that we previously synthesized (6, Figure 2), which had similar
antimicrobial activity against MSSA and MRSA to that of the
natural product, and a cLogP of 3.84, compared to a cLogP of
6.66 for 1 and 5.26 for simplified analog 5 (Figure 2).16
Inspired to further explore this initial result, the goal of this
study was 2-fold: first, we planned to synthesize a panel of aryl
derivatives aiming to maintain high levels of antimicrobial
activity while exploring the effect of aryl substitution patterns;
second, we sought to vary chain lengths and install more polar
ultidrug resistant (MDR) bacteria pose a significant
Mthreat to human and animal health, and there is a
critical need for antibiotics with activity against MDR strains.1
The marine environment provides a plethora of novel
compounds with unique chemical scaffolds and biological
activities, which can serve as valuable starting points for
antibiotic development.2−5 Previously, our group developed
the first total synthesis of lipoxazolidinone A (1), a 4-
oxazolidinone containing antimicrobial natural product iso-
lated from marine sediment off the coast of Guam (Figure
1A).6,7 The lipoxazolidinone family of natural products
contains an unusual 4-oxazolidinone moiety at its core, and
this heterocycle is only found in two other families of natural
Received: January 10, 2019
Accepted: February 26, 2019
Figure 1. (A) Biologically active 4-oxazolidinones. (B) Clinically
relevant 2-oxazolidinones.
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00015
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ACS Medicinal Chemistry Letters
Letter
Table 1. Initial Evaluation of Antimicrobial Activity of Aryl
a
Lipoxazolidinone Analogs
b
c
d
compd
MSSA
MRSA
A. baumannii
6
1
4
0.5
2
64
nt
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
linezolid
2
4
4
4
4
64
8
>128
0.25
2
16
16
16
16
16
8
8
16
2
2
2
0.5
2
2
2
2
64
128
nt
nt
128
nt
nt
nt
>128
nt
nt
nt
nt
nt
nt
nt
128
nt
nt
nt
Figure 2. Comparison of the cLogP and MIC values for previously
synthesized 4-oxazolidinone analogs.
functionality around the aromatic ring to further elucidate the
structure−activity relationship for these aryl derivatives and the
oxazolidinones more broadly.
Utilizing a synthetic approach that was previously developed
and optimized to a one-pot procedure by our group, we were
able to synthesize a panel of 26 oxazolidinone analogs (Figure
3).9 In short, the TBS-protected α-hydroxyamides were
8
4
64
0.125
0.5
8
2
8
4
8
4
4
8
0.25
0.5
1
0.5
8
8
2
0.5
128
128
nt
>128
128
2
16
4
4
1
64
a
b
c
nt = not tested. All MIC values in μg/mL. ATCC 29213. ATCC
d
33591. ATCC 19606.
or weak electron-donating groups (13−15, 20) at the 4-
position were found to have the most potent antimicrobial
activity against MSSA and MRSA, while analogs with strong
electron-withdrawing (17) or strong electron-donating sub-
stituents (19) at the 4-position had significantly less activity.
Relocation of the electron-withdrawing substituent to the 2- or
3-position (22, 23) or incorporating di- and trisubstitution
(24−27) around the aryl ring also resulted in a decrease in
activity. Similarly, lengthening the chain by an additional
methylene resulted in analogs following a similar antimicrobial
trend, with those having electron-withdrawing substituents
(31−33) possessing an increase in antimicrobial activity and
disubstitution (35) resulting in a decrease in activity. One of
the most promising analogs was biphenyl compound 20 which
possessed potent activity against MSSA and MRSA.
Additional derivatives with extended alkyl chains between
the exocyclic ketone and the aryl ring (28, 36) were also
synthesized to determine if the length of the alkyl chain would
affect the antimicrobial activity. With these compounds in
hand it was demonstrated that extending the alkyl chain by two
to four methylene units did not result in a significant decrease
in antimicrobial activity.
Figure 3. (A) One-pot strategy to synthesize 4-oxazolidinones. (B)
Novel aryl analogs of lipoxazolidinone A.
synthesized via known procedures and then heated to reflux
with acylated Meldrum’s acid derivatives in toluene for 1 h.
After removal of the solvent the mixture was resuspended in
dichloromethane, and trifluoroacetic acid was added to induce
cyclization/dehydration over 24 h. Through this procedure an
array of electron-rich and electron-poor aryl derivatives were
prepared in moderate to good yields (see the Supporting
Information for full experimental protocols and character-
ization data/spectra).
The lipoxazolidinone derivatives were then tested in MIC
assays against strains of MSSA and MRSA to compare their
activity (Table 1). In addition, select analogs were tested
against A. baumannii to determine if they possessed any activity
against this Gram-negative pathogen. All of the analogs tested
were found to have good to moderate activity against MSSA
and MRSA. Analogs with weak electron-withdrawing (11, 12)
A few of the analogs displayed modest activity against A.
baumannii, suggesting that the structures could be modified to
further increase activity against Gram-negative organisms.
Specifically, the bromo- and chloro-substituted derivatives (6
and 12, respectively) had MICs of 64 μg/mL against A.
baumannii, demonstrating that modifications could potentially
B
DOI: 10.1021/acsmedchemlett.9b00015
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ACS Medicinal Chemistry Letters
Letter
be made to further increase activity against additional bacteria.
To further explore the potential for Gram-negative bacteria to
be subject to the 4-oxazolidinone antibiotics, we employed the
use of a series of E. coli knockouts to evaluate the role of efflux
and influx on the activity of our lead compounds (Table
2).18−20 These experiments revealed that the knockout of the
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: jgpierce@ncsu.edu. Homepage: http://piercegroup.
wordpress.ncsu.edu.
ORCID
Kaylib R. Robinson: 0000-0003-2967-540X
Joshua G. Pierce: 0000-0001-9194-3765
Present Address
Table 2. Initial Evaluation of Antimicrobial Activity of Lead
a
Oxazolidinones against E. coli Mutants s
†Department of Biochemistry, Vanderbilt University School of
Medicine, 2215 Garland Avenue, 607 Light Hall, Nashville,
Tennessee 37232 (USA). E-mail: jonathan.mills@vanderbilt.
edu.
compd
SQ110
SQ110 DTC
SQ110 LPTD
5
14
20
21
>128
>128
>128
>128
>128
1−2
2
0.25
0.06
2
32
32
32
32
32
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
linezolid
a
All MIC values in μg/mL.
Funding
We are grateful to the NIH (1R01GM110154) for generous
support of this work and to NC State University for support of
our program. Partial funding for this work was also obtained
through the NC State University Chancellor’s Innovation
Fund.
TolC efflux mechanism sensitized the E. coli to 5, 14, 20, and
21, suggesting that efflux, and not simply influx, is likely
responsible for the diminished activity in Gram-negative
organisms.21 We are optimistic that further chemical
modification of the antibiotic scaffold can further overcome
these efflux mechanisms.22−24
Notes
The authors declare no competing financial interest.
Finally, we evaluated the toxicity of compounds 5 and 21 in
red blood cell hemolysis assays and also A549 cell toxicity
assays. We observed <1% hemolysis at up to 40 μM
concentrations for both 5 and 21. In the mammalian cellular
toxicity assay, compound 5 was shown to have IC50 against
A549 cells of 10 μg/mL, and for 21, an IC50 of 14 μg/mL (see
Table S1 in the Supporting Information for details) after an
extended 72 h exposure. These compounds are classified as
mildly toxic, and current studies are underway to further
increase the therapeutic window of these oxazolidinones in the
next phase of optimization.
In summary, we have utilized a previously developed
synthetic route to access aryl-substituted 4-oxazolidinones.
These new analogs allowed us to further probe the structure−
activity relationship of the right-hand side of the lip-
oxazolidinones, as well as indicating whether an increase in
chain length between the core pharmacophore and the aryl
system affected biological activity. Further, we have gained
additional insight into the mechanism of resistance in Gram-
negative organisms. Taken together, these results in con-
junction with ongoing mechanism of action studies will be
utilized to further drive structure−activity relationship studies
toward analogs with additional heteroatoms and/or hetero-
cycles in hopes of expanding biological activity to additional
high-priority pathogens.
ACKNOWLEDGMENTS
■
Mass spectrometry data, NMR data, and X-ray data were
obtained at the NC State Molecular, Education, Technology
and Research Innovation Center (METRIC). The authors
thank Prof. Christian Melander and Dr. Roberta Melander
(University of Notre Dame) for helpful advice throughout the
progression of this project. We also acknowledge the receipt of
the E. coli knockouts from Dr. Alexander Mankin (University
of Illinois Chicago) and helpful advice from the Mankin/
Vazquez lab. The S. aureus strains in Table 1 were provided by
the Network on Antimicrobial Resistance in Staphylococcus
aureus (NARSA) for distribution by BEI Resources, NIAID,
NIH. We also acknowledge Dr. Melanie Cushion’s lab
(University of Cincinnati College of Medicine) for conducting
toxicity studies under a NIAID Preclinical Services contract.
ABBREVIATIONS
■
MDR, multidrug resistant; MIC, minimum inhibitory concen-
tration; MRSA, methicillin resistant Staphylococcus aureus;
MSSA, methicillin susceptible Staphylococcus aureus
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The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.9b00015.
Experimental procedures and analytical data (1H and
13C NMR) for all new compounds and bioassay
procedures (PDF)
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ACS Medicinal Chemistry Letters
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DOI: 10.1021/acsmedchemlett.9b00015
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX