Second-generation aryl isonitrile compounds targeting multidrug-resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmc.2019.03.034

Antibiotic resistance remains a major global public health threat that requires sustained discovery of novel antibacterial agents with unexploited scaffolds. Structure-activity relationship of the first-generation aryl isonitrile compounds we synthesized led to an initial lead molecule that informed the synthesis of a second-generation of aryl isonitriles. From this new series of 20 compounds, three analogues inhibited growth of methicillin-resistant Staphylococcus aureus (MRSA) (from 1 to 4 μM) and were safe to human keratinocytes. Compound 19, with an additional isonitrile group exhibited improved activity against MRSA compared to the first-generation lead compound. This compound emerged as a candidate worthy of further investigation and further reinforced the importance of the isonitrile functionality in the compounds’ anti-MRSA activity. In a murine skin wound model, 19 significantly reduced the burden of MRSA, similar to the antibiotic fusidic acid. In summary, 19 was identified as a new lead aryl isonitrile compound effective against MRSA.

Full text of DOI:10.1016/j.bmc.2019.03.034

Accepted Manuscript
Second-generation aryl isonitrile compounds targeting multidrug-resistant
Staphylococcus aureus
Kwaku Kyei-Baffour, Haroon Mohammad, Mohamed N. Seleem, Mingji Dai
PII:
S0968-0896(19)30262-7
https://doi.org/10.1016/j.bmc.2019.03.034
BMC 14826
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 February 2019
9 March 2019
19 March 2019
Please cite this article as: Kyei-Baffour, K., Mohammad, H., Seleem, M.N., Dai, M., Second-generation aryl
isonitrile compounds targeting multidrug-resistant Staphylococcus aureus, Bioorganic & Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.bmc.2019.03.034
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Second-generation aryl isonitrile compounds targeting multidrug-resistant
Staphylococcus aureus
Kwaku Kyei-Baffoura,1, Haroon Mohammadb,1, Mohamed N. Seleemb,c*, and Mingji Daia,c*
a Department of Chemistry, Center for Cancer Research and Institute for Drug Discovery, Purdue
University, 720 Clinic Drive, West Lafayette, Indiana, 47907, United States of America
b Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University,
625 Harrison Street, West Lafayette, Indiana, 47907, United States of America
c Purdue Institute of Inflammation, Immunology and Infectious Disease, 610 Purdue Mall, West
Lafayette, Indiana, 47907, United States of America
*Corresponding Authors:
Mingji Dai
Department of Chemistry and Center for Cancer Research
720 Clinic Drive, West Lafayette, IN 47907
Phone: 765-496-7898 Fax: 765- 496-2592
Email: mjdai@purdue.edu
Mohamed N. Seleem
Department of Comparative Pathobiology
Purdue University College of Veterinary Medicine
625 Harrison St., West Lafayette, IN, 47907
Phone: 765-494-0763 Fax: 765-496-2627
Email: mseleem@purdue.edu
1 These authors contributed equally.

ABSTRACT
Antibiotic resistance remains a major global public health threat that requires sustained
discovery of novel antibacterial agents with unexploited scaffolds. Structure-activity relationship
of the first-generation aryl isonitrile compounds we synthesized led to an initial lead molecule
that informed the synthesis of a second-generation of aryl isonitriles. From this new series of 20
compounds, three analogues inhibited growth of methicillin-resistant Staphylococcus aureus
(MRSA) (from 1 – 4 µM) and were safe to human keratinocytes. Compound 19, with an
additional isonitrile group exhibited improved activity against MRSA compared to the first-
generation lead compound. This compound emerged as a candidate worthy of further
investigation and further reinforced the importance of the isonitrile functionality in the
compounds’ anti-MRSA activity. In a murine skin wound model, 19 significantly reduced the
burden of MRSA, similar to the antibiotic fusidic acid. In summary, 19 was identified as a new
lead aryl isonitrile compound effective against MRSA.

INTRODUCTION
Bacterial infections resistant to currently available antibiotics continue to pose a major
global public health threat that requires the constant identification and development of new
antibacterial agents. Though extensive research efforts and funding have recently been focused
to identify new agents to treat Gram-negative bacterial infections, particularly those caused by
carbapenem-resistant Enterobacteriaceae (CRE), the reality remains that Gram-positive bacteria
(particularly methicillin-resistant S. aureus (MRSA)) are more prevalent sources of community-
acquired and nosocomial infections, particularly in the United States of America.1-3 In its
landmark report in 2013, the US Centers for Disease Control and Prevention noted that serious
infections caused by MRSA (>80,000) alone outnumbered infections attributed to extended-
spectrum β-lactamase producing Enterobacteriaceae (26,000), CRE (9,000), multidrug-resistant
Acinetobacter (7,300), and multidrug-resistant Pseudomonas aeruginosa (6,700) combined.4
Furthermore, significantly more fatalities were attributed to MRSA infections (11,285 deaths)
relative to infections caused by the aforementioned drug-resistant Gram-negative pathogens
(3,240 deaths).4 Given MRSA infections continue to persist both in healthcare and community
settings, and resistant isolates to key antibiotics (vancomycin and linezolid) used to treat MRSA
infections have emerged,3, 5-8 new antibacterial agents are still needed.
Current antibacterial discovery programs often focus on optimizing existing antibiotic
scaffolds (i.e. β-lactams, quinolones, glycopeptides, oxazolidinones) to develop new
therapeutics. This approach has yielded regulatory approval for several new antibacterial agents
to treat MRSA infections, including delafloxacin, dalbavancin, oritavancin, and tedizolid
phosphate with improved potency relative to other antibiotics in the same drug class.9 However,
bacterial resistance to these newer agents is most probably inevitable, particularly given the

similarity in structure to existing antibiotics, which will further hinder clinicians’ ability to
effectively treat drug-resistant bacterial infections. Identifying novel antibacterial agents with
unique scaffolds or mechanisms of action is critical to circumvent the growing challenge of
treating drug-resistant bacterial infections.
As part of efforts to identify compounds with unique functionalities and novel structural
moieties capable of targeting multidrug-resistant bacterial infections, we recently identified aryl
isonitriles as a unique class of compounds with anti-MRSA activity.10 Though extensive efforts
in isolating naturally occurring isonitriles from marine sponges and cyanobacteria have been
fruitful, these and other naturally occurring isonitriles with antimicrobial activity have been
precluded from extensive medicinal chemistry and structure-activity-relationship (SAR)
studies.11-14 This is primarily because their complex structural motifs make them very difficult to
access. Therefore, the isonitrile functionality and the aryl isonitrile scaffold remain one of the
least extensively studied scaffolds. Previous SAR studies on the first series of over 40 aryl
isonitriles revealed the isonitrile functionality as the most essential structural component that
contributed to the antibacterial activity of the compounds. The presence of a second non-
isonitrile-bearing aromatic ring and an alkene bridge/linker were also shown to be important.
However, substituents incorporated on this bridge did not result in improved antibacterial
activity (Figure 1).
Figure 1. First generation SAR.

With this in mind, the current work aims to further explore the structural-anti-MRSA
activity of a second generation of aryl isonitriles, characterize their spectrum of antibacterial
activity, and evaluate the most promising analogue’s activity in a mouse model of MRSA
infection. This exploration takes into consideration a novel group of stilbene bis-isonitriles. New
stilbene aryl isonitriles with medicinally-relevant structural molecules and heterocyclic moieties,
and those with a saturated linker have also been evaluated (Figure 2). From this effort,
compound 19 emerged as a new lead compound with antibacterial activity in vitro and in vivo
against MRSA (Figure 3).
Figure 2. Design strategy for the second-generation aryl isonitrile compounds.

Figure 3. Structural evolution leading to current lead aryl isonitrile compound and minimum
inhibitory concentration (MIC) values against methicillin-resistant S. aureus (MRSA).
RESULTS
Synthesis of new aryl isonitrile analogues
Following the identification of 1 as the initial hit aryl isonitrile and subsequent synthetic
exploration resulting in 2 as the lead molecule (Figure 3), our initial synthetic efforts were
focused on incorporating medicinally-relevant groups to the aryl isonitrile core. These groups
included electron donating and electron withdrawing small molecules such as fluoro, chloro,
trifluoromethyl, and methoxy groups.15-17 To access these novel stilbene isonitriles, Michaelis-
Arbuzov reaction involving the commercially available nitrobenzyl bromide starting material (3)
was used to form nitrobenzyl phosphonate (4).18 Subsequent reduction of the nitro group,
followed by Hofmann isonitrile conversion of the resulting amine, led to the formation of the
isonitrile phosphonate (5).19 Serving as the divergent point, the isonitrile phosphonate was then
subjected to Horner-Wadsworth-Emmons (HWE)20 reaction to produce isonitrile compounds 6,
7, 8, 9, 10, 11, and 12 from the corresponding aldehydes (Scheme 1). The aforementioned

synthetic procedure was also used to synthesize aryl isonitriles to explore the importance of
incorporating heterocyclic moieties as the second aromatic ring. These analogues included furan
(12) and quinoline (13 and 14) groups.
As part of SAR studies on the first-generation molecules, the isonitrile functionality was
identified as the integral component of the stilbene aryl isonitriles (Figure 1). Bis-isonitriles 17,
18, and 19 were therefore synthesized to explore the influence of an additional isonitrile group
on the antibacterial activity of the lead molecule 2. Similarly, using 4 as the divergent point, the
stilbene bis-isonitriles 17, 18, and 19 were synthesized (Scheme 2). HWE reaction involving 4
and the corresponding nitro substituted benzaldehydes was employed as the first step in the
formation of dinitrostilbenes. The selective reduction of the nitro group followed by Hoffmann
isonitrile synthesis led to compounds 17 and 18. Bis-isonitrile 19 was prepared by a stepwise
formylation and POCl3-promoted dehydration of the corresponding diamine.
Finally, after the synthesis of the stilbene isonitriles and the bis-isonitriles, the question of
the impact of an alkane bridge on the biological activity arose. This led to the synthesis of
compounds 21-28 (Scheme 3). In the synthesis of aryl isonitriles with an alkane linker, HWE
reaction involving 4 and various aldehydes was first employed to form the stilbene compounds.
A sequence of reduction and Hofmann isonitrile synthesis afforded the corresponding aryl
isonitriles
in
good
yields.

Scheme 1. Synthesis of stilbene aryl isonitrile compounds.
Scheme 2. Synthesis of stilbene bis-isonitriles.
Scheme 3. Synthesis of isonitriles with an alkane bridge.

Initial screening and structure-activity relationship of new aryl isonitrile analogues against
MRSA NRS123 (USA400)
In order to investigate the antibacterial activity of the newly synthesized isonitrile
analogues, the minimum inhibitory concentration (MIC) against MRSA NRS123 was determined
using the broth microdilution assay (Table 1). The newly synthesized stilbene bis-isonitriles 17
(MIC = 16 µM), 18 (MIC = 4 µM), and 19 (MIC = 8 µM) all exhibited moderate to good anti-
MRSA activity. The most potent analogues were compounds where the isonitrile moiety was
located in the meta- (18) or para- position (19). The moderate to good activity exhibited by the
stilbene bis-isonitriles further emphasized the importance of the isonitrile group for antibacterial
activity against MRSA. An eight-fold reduction in activity was observed when the most potent
stilbene bis-isonitrile 18 (MIC = 4 µM) was transformed to its saturated bis-isonitrile analogue
22 (MIC = 32 µM). The observance of a similar pattern for bis-isonitriles 17 (MIC =16 µM) and
19 (MIC = 8 µM) with respect to the saturated bis-isonitrile analogues 21 and 23 (MIC > 64 µM)
further emphasized the importance of the alkene linker.
Intrigued by the importance of the non-isonitrile-bearing aromatic ring resulting from the
previous SAR studies, heterocyclic moieties with established therapeutic properties and widely
known in medicinal chemistry to improve the physicochemical and pharmacokinetic properties
of lead molecules were investigated.21, 22 While none of these heterocyclic-containing aryl
isonitriles positively improved the activity of the lead compound, the quinoline-containing
compound 13 (MIC = 8 µM) was more potent than the quinoline analogue 14 (MIC > 64 µM)
and furan analogue 12 (MIC = 32 µM).

With the knowledge of the medicinal importance of F, -OCH3, Cl and CF3 and their value
in the enhancement of the pharmacokinetic and physiochemical activity of several therapeutic
small molecule candidates, installing these species on the aryl isonitrile compounds was
investigated.15-17 Of the methoxy substituted derivatives, the meta-substituted methoxy 7 (MIC =
2 µM) was the most potent analogue, but adding the second methoxy group (8) diminished the
compound’s anti-MRSA activity (MIC = 64 µM). Interestingly, none of the analogues with
electron-withdrawing fluoro, chloro, and trifluoromethyl groups exhibited a major improvement
in anti-MRSA activity. This reveals that having the right electronic environment around the
second aromatic ring is important.
Table 1: Minimum inhibitory concentration (MIC, in µM) of second-generation aryl
isonitrile analogues and control antibiotics against methicillin-resistant Staphylococcus
aureus (MRSA) NRS123 (USA400).
Compound/Drug MRSA NRS123
Name
(USA 400)
MIC
32
2
6
7
64
32
64
>64
32
8
>64
16
4
8
9
10
11
12
13
14
17
18
19
21
22
23
24
8
>64
32
>64
64

>64
>64
16
32
1
25
26
27
28
Linezolid
Vancomycin
1
1N.D. = Not determined
Evaluation of most potent analogues against additional strains of S. aureus
Based upon the initial screening results, the four most potent analogues from this series
(7, 13, 18, and 19) were further evaluated against additional multidrug-resistant S. aureus clinical
isolates (Table 2). The S. aureus clinical isolates included strains resistant to linezolid (NRS119)
and vancomycin (VRS10, VRS11a), two antibiotics frequently prescribed to treat MRSA
infections. Against methicillin-sensitive S. aureus, the compounds inhibited growth from 2 – 16
µM. When evaluated against MRSA NRS384 (USA300), all four aryl isonitrile compounds
inhibited growth at concentrations ranging from 1 – 4 µM. Methoxy analog 7 (MIC = 1 µM) and
bis-isonitrile 19 (MIC = 2 µM) were more potent than first generation lead 2 (MIC = 4 µM)
against MRSA NRS384. However, quinoline isonitrile 13 (MIC = 4 µM) and bis-isonitrile 18
(MIC = 4 µM) exhibited similar potency to 2. Against MRSA NRS119, 7 and 19 were the most
potent analogues (MIC = 1 µM), followed by 13 (MIC = 4 µM), and 18 (MIC = 4 µM). Once
again, the second-generation analogues 7 and 19 were more potent than the first-generation lead
2 (MIC = 4 µM). All four compounds were more potent than linezolid (MIC = 32 µM) against
MRSA NRS119. The MIC results obtained against MRSA NRS384 (USA300) and MRSA
NRS119 (linezolid-resistant S. aureus) demonstrate that incorporating a second isonitrile
functionality (as in compound 19) improved the antibacterial activity of the first-generation lead
molecule 2.

Against two S. aureus strains (VRS10 and VRS11a) resistant to vancomycin (MIC > 64
µM), compounds 7 and 19 retained their antibacterial activity (MIC ranged from 2 – 8 µM).
Interestingly, 13 (MIC = 32 µM) exhibited very poor antibacterial activity against VRS10. Thus,
this compound was eliminated from further biological evaluation.
Table 2: Minimum inhibitory concentration (MIC, in µM) of the four most potent aryl
isonitrile compounds and control antibiotics against methicillin-sensitive (MSSA),
methicillin-resistant (MRSA), linezolid-resistant (LRSA), and vancomycin-resistant S.
aureus (VRSA).
Compound S. aureus
MRSA
NRS119
MRSA
NRS384 NRS385
MRSA
VRS10
(VRSA)
VRS11a
(VRSA)
Name
NRS107
(MSSA)
(LRSA)
(USA300) (USA500)
2
1
4
4
1
32
1
1
4
4
2
2
1
4
2
32
8
8
2
4
7
13
18
19
N.D.1
N.D.
16
8
4
1
N.D.
16
8
2
>64
16
4
2
Linezolid
Vancomycin
1
>64
1N.D. = Not determined
Figure 4: Time-kill analysis of aryl isonitrile compounds 7, 18, 19, and linezolid (all at 4 ×
MIC) against methicillin-resistant Staphylococcus aureus (MRSA NRS123) over a 24-hour

incubation period at 37C. DMSO served as a negative control. The error bars represent
standard deviation values obtained from triplicate samples used for each compound/antibiotic
studied.
Investigation of the antibacterial spectrum of activity of the aryl isonitrile compounds
To examine their spectrum of antibacterial activity, compounds 7, 18, and 19 were
examined against clinical isolates from the ESKAPE pathogens (Enterococcus faecium (E),
Klebsiella pneumoniae (K), Acinetobacter baumannii (A), Pseudomonas aeruginosa (P), and
Enterobacter species (E)) (Table 3). Collectively, these six pathogens are a significant source of
hospital-acquired bacterial infections and exhibit resistance to most clinically available
antibiotics.23, 24 All four compounds were significantly less potent or inactive (MIC ≥ 64 µM)
against vancomycin-resistant E. faecium (VRE), K. pneumoniae, A. baumannii, P. aeruginosa,
and E. cloacae, similar to the antibiotics erythromycin, linezolid, and vancomycin. The
compounds were also evaluated against two additional clinically-relevant Gram-positive
bacterial pathogens, Staphylococcus epidermidis and Streptococcus pneumoniae. All three
compounds exhibited potent activity against methicillin-resistant S. epidermidis (MIC = 1 µM).
Against, penicillin-resistant S. pneumoniae, 7 was the most potent compound (MIC = 4 µM)
followed by 19 (MIC = 8 µM). Compound 18 was the least potent analogue against S.
pneumoniae (MIC = 16 µM).
Table 3: Minimum inhibitory concentration (MIC, in µM) of the three most potent aryl
isonitrile compounds and control antibiotics against clinically-relevant Gram-positive and
Gram-negative bacterial pathogens.
Bacterial Strain

S.
S.
E. faecium
A.
E.
K.
Pseudomon
as
epidermi pneumoni ATCC7002 bauman cloac pneumoni
Test Agent
dis
NRS101
ae ATCC
700677
21
nii
BAA-
1605
>64
>64
>64
16
ae
BAA-
1134
>64
>64
>64
ae ATCC aeruginosa
BAA-
1705
>64
ATCC
15442
>64
1
1
4
16
8
>64
>64
>64
N.D.
7
18
19
>64
>64
1
>64
>64
Erythromy
cin
N.D.1
N.D.
>64
>64
>64
Linezolid
Vancomyci
n
2
4
2
1
0.50
>64
>64
32
>64
>64
>64
>64
>64
>64
1 N.D. = Not determined
Effect of the outer membrane and efflux pump on antibacterial activity against Gram-
negative bacterial pathogens
We hypothesized that the lack of antibacterial activity against Gram-negative bacteria
observed for the aryl isonitrile compounds was due either to the presence of the outer membrane
(OM) or due to expulsion via efflux pumps, two common resistance mechanisms utilized by
Gram-negative bacterial pathogens.25 The OM has been known to prevent numerous antibiotics
(including erythromycin) from gaining entry into the bacterial cell at sufficient concentrations to
bind to/inhibit the molecular target. To examine if the OM was impeding the antibacterial
activity of the aryl isonitrile compounds, compound 19 and control antibiotics were incubated
with the same Gram-negative bacterial species presented in Table 2 in the presence of a
subinhibitory concentration of the membrane-permeabilizing agent colistin. As presented in
Table 4, a noticeable improvement in the antibacterial activity of compound 19 was observed
against A. baumannii (MIC = 16 µM) and E. cloacae (MIC = 4 µM) indicating the OM was
having a deleterious effect on the antibacterial activity of this compound. Interestingly, no
improvement in antibacterial activity for 19, in the presence of colistin, was observed against K.

pneumoniae and P. aeruginosa, (similar to linezolid) suggesting the presence of the outer
membrane was not solely responsible for the lack of activity observed in these particular
pathogens. The antibacterial activity of the antibiotic erythromycin, in the presence of colistin,
improved against A. baumannii (MIC = 4 µM), E. cloacae (MIC = 1 µM), K. pneumoniae (MIC
= 16 µM), and P. aeruginosa (MIC = 4 µM). Erythromycin is thought to be capable of diffusing
across the OM but at a very slow rate.26 Thus permeabilizing the OM is expected to enhance
penetration of the antibiotic into bacterial cells.
We next examined if the presence of efflux pumps may be responsible for the lack of
antibacterial activity observed for the aryl isonitrile compounds against Gram-negative bacteria
(Table 4). These pathogens express different efflux pumps that confer resistance to numerous
antibiotics including the AdeIJK efflux pump in A. baumannii, AcrAB-TolC efflux pump
(present in E. coli E. cloacae, and K. pneumoniae) and its homologue MexAB-OprM (expressed
by P. aeruginosa).26, 27 We evaluated the antibacterial activity of 19 against an Escherichia coli
strain (JW25113) deficient in the AcrAB-TolC efflux pump responsible for excluding many
xenobiotics from accumulating inside E. coli cells.25 No discernible improvement in antibacterial
activity of 19 was observed against the mutant E. coli strain relative to the wild-type strain
(BW25113). In contrast, there was noticeable improvement in the antibacterial activity of
erythromycin (MIC = 2 µM) and linezolid (MIC = 16 µM), two substrates of the AcrAB-TolC
efflux pump.26, 28 This suggests that the presence of efflux pumps alone on the surface of the OM
on Gram-negative bacteria may not be responsible for conferring resistance to the aryl isonitrile
compounds. We next investigated if the combination of the outer membrane and efflux pumps
may impede the antibacterial activity of the aryl isonitrile compounds. The compound and
control antibiotics were incubated with a subinhibitory concentration of colistin against E. coli

BW25113 and E. coli JW25113. No improvement in the antibacterial activity of 19 (MIC > 64
µM) was observed in the presence of colistin against E. coli JW25113. Similarly, no
improvement in the MIC of linezolid was observed against E. coli JW25113 in the absence and
presence of colistin, indicating the lack of antibacterial activity of linezolid observed against E.
coli is due primarily to efflux. The MIC of erythromycin against E. coli JW25113, in contrast,
improved one-fold in the presence of colistin indicating both the OM and efflux pumps interfere
with the effect of this antibiotic against Gram-negative bacteria, in agreement with previous
reports.26, 29 Due to the lack of antibacterial activity observed against VRE and Gram-negative
bacterial pathogens, we moved to evaluate the aryl isonitrile compounds antibacterial activity in
vivo against MRSA.
Table 4: Minimum inhibitory concentration (MIC, in µM) of 19 and control antibiotics
against Gram-negative bacterial pathogens in the presence of a subinhibitory concentration
of colistin (COL).
Bacterial Strain
Test Agent
A.
E.
K.
P.
E. coli
BW25113
E. coli
JW25113
(ΔtolC)
baumanni cloaca pneumonia aeruginos
i
e
e ATCC
a ATCC
BAA-
1605
BAA- BAA-1705
1134
15442
(-
(+COL
(-
(+COL
COL)
)
COL
)
)
1
16
4
4
1
>64
16
>64
4
>64
64
>64
1
>64
2
>64
0.50
19
Erythromyci
n
Linezolid
>64
16
>64
>64
>64
>64
16
16
1 No colistin added to the media
In silico pharmacokinetic evaluation of compound 19

MRSA is a source of infection for both superficial skin lesions and complicated systemic
infections. To determine a suitable animal model of MRSA infection to evaluate the aryl
isonitrile compounds, the pharmacokinetic profile of compound 19 and linezolid were simulated
utilizing a dose of 600 mg (as is commonly administered for linezolid in adult human patients).
As shown in Table 5, the results indicate that compound 19 would not be suitable for oral
administration for the treatment of systemic MRSA infections as it is not expected to attain a
concentration in plasma/blood sufficient to inhibit bacterial growth. The maximum plasma
concentration (Cmax) predicted for compound 19 is 0.17 µg/mL (0.73 µM), whereas the MIC
against MRSA ranges from 1 to 8 µM. Intravenous administration of compound 19 is predicted
to result in a slow rate of clearance (12.58 mL/min-kg) correlating with a long half-life (22.90
hours) which could alleviate the need for multiple daily dosing. The high values obtained for the
volume of distribution at steady-state for 19 (24.94 L/kg compared to 1.11 L/kg for linezolid)
indicate this compound is expected to distribute extensively into tissues. This may be due to the
high degree of lipophilicity (cLogP = 3.88) present with the compound. These pharmacokinetic
simulations suggest that though intravenous administration of 19 may be possible for treatment
of systemic MRSA infections, the extensive distribution of the compound into tissues would
necessitate a higher dose be administered/continuous infusion of compound (to ensure the
concentration remained above the MIC to inhibit MRSA growth). The values obtained for
linezolid via the in silico pharmacokinetic simulation overall were in close proximity to
experimental values obtained by Stalker, et al. from healthy human subjects administered a
single 625 mg dose of linezolid either orally or intravenously.30 However, the simulation
underestimated the Cmax for linezolid and overestimated the half-life and volume of distribution
compared to the experimental values. Based upon the in silico pharmacokinetic simulation, 19

appears most suitable for evaluation topically to treat localized/uncomplicated MRSA skin
infections.
Table 5: In silico pharmacokinetic analysis for compound 19 and linezolid (simulated at 600
mg).
Oral
Linezolid
Intravenous
Linezolid
19
Linezolid
Linezolid
19
(Simulated) (Simulated) (Experiment (Simulated) (Simulated) (Experiment
al)1
12.7
1.33
1.03
al)
13.4
0.5
-
Cmax2 (µg/mL)
tmax3 (hours)
0.17
3
0.80
5.33
2.75
0.92
-
-
-
-
-
-
Fraction absorbed
(FAlast)
CL4 (mL/min-kg)
t1/25 (hours)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12.58
22.90
12.82
24.94
9.68
1.09
12.31
17.04
1.11
1.749
4.40
-
MRT6 (hours)
7
Vd (L/kg)
0.5810
-
Vss8 (L/kg)
1.12
1(adapted from Stalker DJ et al. J Antimicrob Chemother 2003, 51: 1239–46 (Table 2, 625 mg
dose))
2Cmax = maximum concentration of drug in plasma/blood
3tmax = time required to reach Cmax
4CL = rate of clearance
5t1/2 = half-life
6MRT = mean residence time
7Vd = volume of distribution
8Vss = volume of distribution at steady-state
7Clearance for linezolid (experimental) obtained by dividing the mean clearance value by the
mean weight of patients
8 Volume of distribution for linezolid (experimental) obtained by dividing the mean Vd by the
mean weight of patients
The active aryl isonitrile compounds are safe to mammalian keratinocytes
S. aureus is a leading source of skin and soft tissue infections, both uncomplicated and
invasive, globally.31-34 As such, based upon the in silico pharmacokinetic data, we decided to
investigate the antibacterial activity of the second-generation aryl isonitriles as topical
antibacterial agents in a MRSA murine skin infection mouse model. Before exposing mice to the

compounds, we evaluated the safety profile of 7, 18, and 19 against keratinocytes (HaCaT). All
three compounds were safe up to the maximum tested concentration of 128 µM (more than 90%
of HaCaT cells remained viable after 24 hours of exposure to the compounds) (Figure 5).
Figure 5: Toxicity analysis of aryl isonitrile compounds against human keratinocytes
(HaCaT). Percent viable mammalian cells (measured as average absorbance ratio (test agent
relative to DMSO)) for cytotoxicity analysis of compounds 7, 18, and 19 (tested in triplicate) at
64 and 128 µM against HaCaT cells over a 24 hour period using the MTS 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
assay.
Dimethyl sulfoxide (DMSO) was used as a negative control to determine a baseline
measurement for the cytotoxic impact of each compound. The absorbance values represent an
average of a minimum of three samples analyzed for each compound. Error bars represent
standard deviation values. A one-way ANOVA, with post hoc Dunnet’s multiple comparisons
test, determined statistical difference between the values obtained for each compound and
DMSO (P < 0.05).
Compound 19 significantly reduces the burden of MRSA in a skin infection mouse model

After confirming the aryl isonitrile compounds are safe to keratinocytes, we moved to
investigate the antibacterial activity of the newly synthesized analogues in a mouse model of
MRSA skin infection. Given the safety profile against keratinocytes was identical for all three
aryl isonitrile compounds, 19 was selected for this experiment. Given 18 and 19 are structurally
similar, the more potent compound in vitro against MRSA USA300 was selected for evaluation.
After the formation of an abscess at the site of infection, mice were treated twice daily for five
days. As the skin wounds were uncomplicated and localized, treatment was administered
topically directly onto the surface of the abscesses. Mice were euthanized 12 hours after the last
dose was administered and the abscesses were harvested to enumerate MRSA CFU. As presented
in Figure 6, 19 (79.02% reduction) was as effective as the control antibiotic fusidic acid (77.78%
reduction) in reducing the burden of MRSA in the wounds of infected mice after five days of
treatment. Importantly, no excess inflammation or toxicity was observed in wounds after
exposure to the compounds or fusidic acid.

Figure 6: Reduction of MRSA USA300 in infected lesions of mice. Average percent reduction
of MRSA CFU/mL in murine skin lesions after treatment with 19 or fusidic acid. A one-way
ANOVA with post-hoc Dunnet’s multiple comparisons found no statistical difference between
mice treated with fusidic acid and mice treated with compound 19.
DISCUSSION
Antibiotics have played a critical role in resolving bacterial infections throughout the
world since their initial discovery. However, bacteria have proven to be shrewd microorganisms
capable of acquiring resistance to multiple antibiotics utilizing an array of clever mechanisms
(e.g. through modification of the drug target, degradation of the antibiotic, and efflux of the
antibiotic). As such there is a continuous need to identify and develop novel antibacterial agents
capable of treating drug-resistant bacterial infections. MRSA remains a significant cause of
superficial and invasive antibiotic-resistant infections globally. Though several antibacterial
agents effective against MRSA are currently in clinical trials, the remarkable ability of this
organism to develop resistance to different antibiotics necessitates new antibacterial agents,
particularly from unexploited scaffolds, be identified and developed.
We previously synthesized and evaluated over 40 aryl isonitrile compounds for
antibacterial activity against MRSA.10 The first-generation compounds inhibited MRSA growth
at concentrations ranging from 2 – 64 µM10 but none of the compounds possessed a suitable
physicochemical profile to evaluate their effectiveness in an animal model of MRSA infection.
The present study aimed to expand the library of aryl isonitrile compounds to better understand
these limitations and further characterize the compounds’ structure-antibacterial activity
relationship. To this end, 20 second-generation aryl isonitrile compounds consisting of stilbene

bis-isonitriles, stilbene aryl isonitriles with medicinally-relevant molecules and heterocyclic
moieties, and those with a saturated linker were synthesized and evaluated for antibacterial
activity.
The newly synthesized analogues were initially evaluated against a clinical isolate of
MRSA USA400. From the initial screening, inclusion of an additional isonitrile moiety, the
stilbene core, and the non-isonitrile-bearing aromatic ring were all found to be key for anti-
MRSA activity. Four compounds (7, 13, 18, and 19) inhibited growth of MRSA at a
concentration ranging from 2 – 8 µM, similar to the lead compound (2) from the first-generation
aryl isonitriles. Bis-isonitrile 19 was more potent than the first-generation lead 2 against MRSA
NRS384 (USA300) and MRSA NRS119 (LRSA) which further confirmed the importance of the
isonitrile functionality.
Importantly, three compounds retained potent antibacterial activity against MRSA
isolates exhibiting resistance to vancomycin and linezolid, antibiotics frequently used as agents
of last resort for treatment of MRSA infections.35,36 Though not prevalent clinically, the
identification of S. aureus strains exhibiting resistance to vancomycin and linezolid, two key
therapeutic options for treatment of MRSA infections, is a noteworthy problem. The lack of
cross-resistance observed between the aryl isonitrile compounds and vancomycin and linezolid is
important therapeutically as it provides a potential alternative source of treatment against
linezolid-resistant and vancomycin-resistant S. aureus infections.
Previously, the first-generation aryl isonitrile analogues were only evaluated against
clinical isolates of S. aureus. We were curious to investigate the spectrum of antibacterial
activity of the second-generation aryl isonitrile compounds. Thus, three of the most potent
analogues against MRSA (7, 18, and 19) were evaluated against a panel of clinically-relevant

pathogens, including members of the ESKAPE group, S. epidermidis, and S. pneumoniae. The
compounds exhibited potent antibacterial activity against the Gram-positive pathogens S.
epidermidis and S. pneumoniae. Interestingly, the compounds were inactive against vancomycin-
resistant E. faecium. The lack of antibacterial activity against VRE (a Gram-positive pathogen
similar to MRSA) is not entirely surprising given certain antibiotics that are active against S.
aureus (such as ampicillin and vancomycin) are inactive against vancomycin-resistant E.
faecium.37 The aryl isonitrile compounds also lacked antibacterial activity against five Gram-
negative bacterial pathogens tested (A. baumannii, K. pneumoniae, E. cloacae, E. coli, and P.
aeruginosa). The lack of antibacterial activity against Gram-negative pathogens is not surprising
given most small molecules are unable to permeate the complex outer membrane (OM) present
in Gram-negative bacteria. Those that are capable of diffusing across the OM may be susceptible
to expulsion (decreasing the intracellular concentration of compound/drug) by a number of
different efflux pumps expressed by Gram-negative bacteria, including the AcrAB-TolC pump
present in E. coli.25 Thus we evaluated the antibacterial activity of the aryl isonitrile compounds
after permeabilizing the OM (using a subinhibitory concentration of colistin) and against an E.
coli strain deficient in the AcrAB-TolC efflux pump. Interestingly, there was improvement in the
MIC for compound 19 observed in the presence of colistin against A. baumannii and E. cloacae
but not against E. coli, K. pneumoniae, and P. aeruginosa. Against a mutant E. coli strain
deficient in AcrAB-TolC, no improvement in the MIC of 19 was observed indicating that this
compound may not be a substrate for efflux. This suggests that though the outer membrane may
contribute to the lack of antibacterial activity for the aryl isonitrile compounds observed against
specific Gram-negative bacterial pathogens, additional resistance mechanism(s) may play a role
as well. Alternatively, the compounds may have a weaker affinity for the molecular target in

Gram-negative bacteria as opposed to against MRSA, S. epidermidis, and S. pneumoniae.
Though the molecular target of the aryl isonitrile compounds is currently unknown, this question
is being intensely investigated using both genetic and phenotypic approaches. Identification of
the molecular target may provide key insight into the difference in potency observed for the aryl
isonitrile compounds against S. aureus relative to other bacterial pathogens.
The aryl isonitrile compounds possessed good in vitro activity against drug-resistant S.
aureus isolates and were safe to mammalian cells (non-toxic to human keratinocytes at 128 µM,
more than 30-fold higher than the concentration where they inhibited MRSA growth in vitro).
However, an in silico pharmacokinetic evaluation of 19 revealed this compound may not be
effective in treatment of systemic MRSA infections (as the maximum predicted concentration of
the compound in plasma was lower than the MIC of the compound against MRSA). As a means
to address this problem while still retaining antibacterial activity, the alkene core of the current
lead 19 is undergoing further structural diversification with molecular entities we hypothesize
will help improve the physicochemical profile of the lead compound.
Based upon the in vitro antibacterial activity assay results and in silico pharmacokinetic
simulation, we proceeded to evaluate the effectiveness of 19 as a topical antibacterial to treat
MRSA skin infection. MRSA remains a major source of skin and soft tissue infections (SSTIs)
throughout the world.31-34, 38, 39 Treatment of SSTIs can be challenging given the emergence of
resistance to several antibiotics frequently used in the clinic. One example is the antibiotic
fusidic acid, which is prescribed for use topically in Europe for the treatment of SSTIs. Extensive
use of fusidic acid has resulted in the emergence of resistant isolates40 that necessitates new
therapeutic agents to treat uncomplicated, localized S. aureus SSTIs. In the United States of
America, MRSA USA300 is the predominant strain linked to community-acquired SSTIs.41 As

such, we moved to evaluate the efficacy of compound 19 administered topically in a MRSA
USA300 skin wound mouse model. Compound 19 significantly reduced the burden of MRSA
USA300 in infected abscesses by over 70% (after only five days of treatment) similar to fusidic
acid.42
CONCLUSIONS
In conclusion, the present study identified four new aryl isonitrile compounds bearing
potent antibacterial activity against MRSA in vitro. The analogues were inactive against
important Gram-negative bacterial pathogens, and their activity appeared to be negatively
impacted by the presence of the outer membrane. The newly synthesized analogues were safe to
keratinocytes at concentrations up to 128 µM and significantly reduced the burden of MRSA in
infected wounds in a mouse model of MRSA skin infection. However, in silico pharmacokinetic
simulation revealed compound 19 would not effectively permeate across the GI tract and may
extensively distribute into tissues thus precluding its evaluation in a systemic animal model of
MRSA infection. Addressing these limitations and deducing the molecular target are necessary
questions to resolve to further develop aryl isonitrile compounds as a novel class of antibacterial
agents to treat drug-resistant S. aureus infections.
EXPERIMENTAL SECTION
Biological Methods
Bacterial strains and reagents
Relevant information pertaining to all bacterial isolates used in this study are presented in
Supplementary Table 1. Clinical isolates of S. aureus and S. epidermidis were obtained through
the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA) program. Isolates
of S. pneumoniae, E. faecium, A. baumannii, K. pneumoniae, E. cloacae, and P. aeruginosa were

obtained from the American Type Culture Collection (ATCC). E. coli strains BW25113 and
JW25113 were obtained from The Coli Genetic Stock Center (CGSC), Yale University.
Antibiotics were purchased commercially and dissolved in DMSO (for linezolid), ethanol (for
erythromycin), or sterile deionized water (for colistin and vancomycin). Stock 10 mM solutions
were prepared for all antibiotics. Brain heart infusion broth (BHI), Tryptic soy broth (TSB),
Tryptic soy agar (TSA), phosphate-buffered saline (PBS), Dulbeco’s modified Eagle’s medium
(DMEM), fetal bovine serum (FBS), and 96-well plates were all purchased from commercial
vendors.
Evaluation of antibacterial activity of compounds and control antibiotics
The minimum inhibitory concentration of the aryl isonitrile analogues and control
antibiotics was determined using the broth microdilution assay following the Clinical and
Laboratory Standards Institute guidelines, with the following modifications.43 Bacteria were
cultured either in TSB or BHI (for E. faecium) and exposed to either compounds or control
antibiotics, in triplicate, in 96-well plates. For permeabilization of the outer membrane, Gram-
negative bacterial pathogens were exposed to a subinhibitory concentration of colistin equivalent
to either ¼ × MIC (for A. baumannii, E. cloacae, E. coli, and P. aeruginosa) or ½ × MIC (for K.
pneumoniae). Plates were incubated aerobically at 37 ºC for 18 – 24 hours before the MIC values
were recorded. S. pneumoniae was incubated with compounds at 37 ºC + 5% CO2. The MICs
reported represent the lowest concentration of each compound/drug necessary to inhibit visual
bacterial growth.
In silico pharmacokinetic evaluation of 19
Compound 19’s pharmacokinetic profile was examined in silico, using chemPK version
2.0 (Cyprotex, Inc.) simulating a dose of 600 mg administered both orally and intravenously.

Toxicity assessment of aryl isonitrile analogues against human keratinocytes
Compounds 7, 18, and 19 were assayed (at concentrations of 16, 32, 64, and 128 µM)
against a human keratinocyte (HaCaT) cell line (AddexBio, San Diego, CA, USA) to determine
the potential toxic effect to mammalian skin cells in vitro, as previously described.44-48 In brief,
cells were cultured in DMEM supplemented with 10% FBS at 37 C with CO2 (5%). Control
cells received DMSO alone at a concentration equal to that in drug-treated cell samples. The
cells were incubated with the compounds (in triplicate) in a 96-well plate at 37 ºC with CO2 (5%)
for 24 hours. The assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) was subsequently added and
the plate was incubated for four hours. Absorbance readings (at OD490) were taken using a
kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable
cells after treatment with each compound was expressed as a percentage of the viability of
DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity data
was analyzed via a one-way ANOVA, with post hoc Dunnet’s multiple comparisons test (P <
0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).
Evaluation of 19 in a murine model of MRSA skin infection
The mice study was conducted under the guidelines of the Purdue University Animal
Care and Use Committee (PACUC) and carried out in strict accordance with the
recommendations in the Guide for the Care and Use of Laboratory Animals of the National
Institutes of Health. The method used for this study was similar to one described in previous
reports, with slight modifications.44, 49-53 Three groups (n = 5) of eight-week old female Balb/c
mice (obtained from Envigo, Indianapolis, IN, USA) received an intradermal injection (40 μL)
containing 1.32 × 109 CFU/mL MRSA USA300. Following formation of an abscess at the

injection site (~48 hours post-infection), topical treatment was initiated subsequently with each
group of mice receiving a 2% suspension (formulated in petroleum jelly) of fusidic acid or 19.
One group of mice received the vehicle alone (negative control). Each group of mice was housed
separately in a ventilated cage with appropriate bedding, food, and water. Mice were checked at
least four times daily during infection and treatment to ensure no adverse reactions were
observed. Mice were treated twice daily for five days, before they were humanely euthanized via
CO2 asphyxiation 12 hours after the last dose was administered. Wounds were aseptically
extracted and subsequently homogenized in PBS (2 mL). The homogenized tissue was then
serially diluted in PBS before plating onto mannitol salt agar plates. Plates were incubated for at
least 16 hours at 37 C before viable CFU were counted and MRSA reduction in the skin wound
(relative to the negative control) post-treatment was determined for each group.
Acknowledgements:
The authors would like to thank the Network of Antimicrobial Resistance in Staphylococcus
aureus (NARSA) program supported under NIAID/NIH Contract # HHSN272200700055C for
providing the MRSA and VRSA strains used in this study. MD thanks financial support from
NIH (R35 GM128570) and unrestricted grants from Eli Lilly and Amgen. The NIH P30
CA023168 is acknowledged for supporting shared NMR resources to Purdue Center for Cancer
Research. The authors would like to thank Dr. Waleed Younis (South Valley University, Qena,
Egypt) and Nader S. Abutaleb (Purdue University) for assistance with biological experiments.

REFERENCES
1.
Blaskovich, M. A. T.; Hansford, K. A.; Butler, M. S.; Jia, Z.; Mark, A. E.; Cooper, M. A.
Developments in Glycopeptide Antibiotics. ACS Infect Dis 2018, 4, 715-735.
2.
Sievert, D. M.; Ricks, P.; Edwards, J. R.; Schneider, A.; Patel, J.; Srinivasan, A.; Kallen,
A.; Limbago, B.; Fridkin, S.; National Healthcare Safety Network, T.; Participating, N. F.
Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of
data reported to the National Healthcare Safety Network at the Centers for Disease Control and
Prevention, 2009-2010. Infect Control Hosp Epidemiol 2013, 34, 1-14.
3.
Mohammad, H.; Thangamani, S.; Seleem, M. N. Antimicrobial peptides and
peptidomimetics - potent therapeutic allies for staphylococcal infections. Curr Pharm Des 2015,
21, 2073-88.
4.
United States, 2013; 2013; pp 1-114.
5. de Dios Caballero, J.; Pastor, M. D.; Vindel, A.; Maiz, L.; Yague, G.; Salvador, C.; Cobo,
Centers for Disease Control and Prevention (CDC). Antibiotic Resistance Threats in the
M.; Morosini, M. I.; del Campo, R.; Canton, R.; Group, G. S. Emergence of cfr-Mediated
Linezolid Resistance in a Methicillin-Resistant Staphylococcus aureus Epidemic Clone Isolated
from Patients with Cystic Fibrosis. Antimicrob Agents Chemother 2015, 60, 1878-82.
6.
of linezolid-resistant Staphylococcus. J Antimicrob Chemother 2013, 68, 4-11.
7. Smith, T. L.; Pearson, M. L.; Wilcox, K. R.; Cruz, C.; Lancaster, M. V.; Robinson-Dunn,
Gu, B.; Kelesidis, T.; Tsiodras, S.; Hindler, J.; Humphries, R. M. The emerging problem
B.; Tenover, F. C.; Zervos, M. J.; Band, J. D.; White, E.; Jarvis, W. R. Emergence of
vancomycin resistance in Staphylococcus aureus. Glycopeptide-Intermediate Staphylococcus
aureus Working Group. N Engl J Med 1999, 340, 493-501.