Discovery and characterization of aryl isonitriles as a new class of compounds versus methicillin- and vancomycin-resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.ejmech.2015.06.031

Methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) have emerged as a global health concern. A new class of compounds featuring an aryl isonitrile moiety has been discovered that exhibits potent inhibitory activity against several clinically-relevant MRSA and VRSA isolates. Structure-activity relationship studies have been conducted to identify the aryl isonitrile group as the key functional group responsible for the observed antibacterial activity. The most potent antibacterial aryl isonitrile analogs (MIC 2 μM) did not show any toxicity against mammalian cells up to a concentration of 64 μM.

Full text of DOI:10.1016/j.ejmech.2015.06.031

Accepted Manuscript
Discovery and Characterization of Aryl Isonitriles as A New Class of Compounds
versus Methicillin- and Vancomycin-resistant Staphylococcus aureus
Dexter C. Davis, Haroon Mohammad, Kwaku Kyei-Baffour, Waleed Younis, Cassidy
Noel Creemer, Mohamed N. Seleem, Mingji Dai
PII:
S0223-5234(15)30107-0
10.1016/j.ejmech.2015.06.031
EJMECH 7958
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 March 2015
Revised Date: 9 June 2015
Accepted Date: 13 June 2015
Please cite this article as: D.C. Davis, H. Mohammad, K. Kyei-Baffour, W. Younis, C.N. Creemer, M.N.
Seleem, M. Dai, Discovery and Characterization of Aryl Isonitriles as A New Class of Compounds versus
Methicillin- and Vancomycin-resistant Staphylococcus aureus, European Journal of Medicinal Chemistry
(2015), doi: 10.1016/j.ejmech.2015.06.031.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Insert Table of Contents Graphic and Synopsis Here.
versus
N+
-
C
C
N
13
43 (MIC > 128 µM)
MIC as low as 2 µM agaist MRSA and VRSA
No toxicity at 64 µM against mammalian cells
A novel class of aryl isonitrile compounds has been discovered as potent antimicrobial
compounds against several clinically relevant MRSA and VRSA isolates without any
toxicity against mammalian cells up to a concentration of 64 µM.

ACCEPTED MANUSCRIPT
Discovery and Characterization of Aryl Isonitriles as
A New Class of Compounds versus Methicillin- and
Vancomycin-resistant Staphylococcus aureus
Dexter C. Davis,†,§ Haroon Mohammad,†, ‡ Kwaku Kyei-Baffour,§ Waleed Younis, ‡ Cassidy Noel
,
,
‡
Creemer,§ Mohamed N. Seleem,* and Mingji Dai* §
§Department of Chemistry and Center for Cancer Research, Purdue University, West Lafayette, Indiana
47907, United States
‡Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West
Lafayette, Indiana 47907, United States
†These two authors contributed equally.
*Corresponding authors’ E-mail addresses: mseleem@purdue.edu; mjdai@purdue.edu
KEYWORDS: Antibiotic, Drug Resistance, MRSA, VRSA, Isonitrile
ABSTRACT: Methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and
VRSA) have emerged as a global health concern. A new class of compounds featuring an aryl
isonitrile moiety has been discovered that exhibits potent inhibitory activity against several
clinically-relevant MRSA and VRSA isolates. Structure-activity relationship studies have been
1

ACCEPTED MANUSCRIPT
conducted to identify the aryl isonitrile group as the key functional group responsible for the
observed antibacterial activity. The most potent antibacterial aryl isonitrile analogs (MIC 2 µM)
did not show any toxicity against mammalian cells up to a concentration of 64 µM.
INTRODUCTION
Multidrug-resistant bacterial infections pose a significant global health challenge afflicting more
than 2 million people each year in the United States alone, resulting in over 23,000 fatalities.1
Nearly half of these casualties are due to infections caused by a single pathogen, methicillin-
resistant Staphylococcus aureus (MRSA). Currently prevalent in the community setting, MRSA
is responsible for a wide spectrum of illnesses from superficial skin infections to invasive
diseases including pneumonia, osteomyelitis, and bloodstream infections.2-5 While a robust
arsenal of antibiotics was once capable of treating MRSA infections, strains of this pathogen
have emerged that exhibit resistance to nearly every class of antibiotics, including agents of last
resort such as vancomycin and linezolid.6-11 This underscores the urgent need for the
identification and development of novel therapeutic options capable of treating infections due to
MRSA.12
Recently, we have conducted a whole-cell screening of a small number of in house
generated small molecules (about 250 molecules) against MRSA USA300 with the aim to
identify compounds with novel skeletons to target antibiotic drug resistance. To our delights,
among several hit molecules revealed by this screening effort, compound 1 with an isonitrile
group attached to a stilbene system was shown to be capable of inhibiting bacterial growth at a
concentration of 32 ꢀM (Figure 1). Further analysis revealed this compound is bacteriostatic (the
minimum bactericidal concentration exceeded 128 ꢀM). The presence of an isonitrile moiety in
2

ACCEPTED MANUSCRIPT
this compound is quite unique given that few antimicrobial compounds possessing the isonitrile
moiety in their core structure have been described in literature and all of them are complex
natural products and are difficult to access.13-19 Natural terpene isonitrile-containing molecules
and simplified analogs have been reported to show antimalarial activity as well.20-22 The novel
structural skeleton of compound 1 as an antibacterial compound against drug resistant strains
prompted us to further study of this type of isonitrile compounds. Herein, we report our chemical
synthesis, structure-activity relationship study, and evaluation of the antibacterial performance of
compound 1 and closely related analogs against several clinically-relevant MRSA and VRSA
strains. These efforts have led to the identification of more potent compounds with MIC as low
as 2 ꢀM but do not show any cytotoxicity against mammalian cells up to a concentration of 64
µM. Physiochemical analysis of this potent lead compound has been described to guide the next
stage of developing these promising compounds into the antibiotic drug pipeline.
FIGURE 1 here.
CHEMICAL SYNTHESIS
In general, the stilbene isonitrile analogs were prepared from benzylic bromide 2, which
was converted to phosphonate 3 by Michaelis-Arbuzov reaction.23 The nitro group of 3 was then
converted to an isonitrile group upon a sequence of hydrogenation and Hofmann isonitrile
synthesis using dichlorocarbene.24 Compound 4 then served as a divergent point to synthesize a
collection of analogs with a Horner-Wadsworth-Emmons reaction.25 By treating various ketones
and aldehydes with stabilized phosphonate carbanions derived from phosphonates 4, we obtained
thirty-three stilbene isonitrile analogs (1, 5-25, and 27-37) and one styrene isonitrile analog (26).
This collection also includes compounds with the isonitrile group at different positions on the
3

ACCEPTED MANUSCRIPT
aromatic ring as well as pyridine containing analogs. In order to investigate the importance of the
isonitrile group for the observed biological activity, compound containing a hydrogen atom (42)
or a nitrile group (43) at the isonitrile-substitution position was prepared as well using the
Horner-Wadsworth-Emmons reaction. Additionally, four biary isonitrile analogs (46 and 49-51)
26
were prepared.
Compound 45 was prepared from commercially available amine 33 via
formamide formation followed by dehydration. Compounds 49-51 were synthesized from 2-
bromoaniline derivatives (47) and arylboronic acids. Suzuki cross-coupling converted 47 to
biaryl amines 48 smoothly. The latter was then converted to 49-51 via the aforementioned
formamide formation and dehydration sequence. Lastly, we prepared compound 53 with a
saturated two-carbon chain to investigate the importance of the double bond linker between the
two aromatic moieties. All the newly synthesized compounds were purified using flash
chromatography before entering biological evaluations.
SCHEME 1 here.
BIOLOGICAL RESULTS AND DISCUSSION
Antimicrobial susceptibility analysis of the isonitrile compounds against clinically-
relevant isolates of MRSA and VRSA. The bacterial growth inhibiting activity of these synthetic
analogs of hit compound 1 were subsequently evaluated (Table 2). When these derivatives were
screened against MRSA, via the broth microdilution assay, the results revealed several
interesting structural elements that appear to play an important role in the antimicrobial activity
of these compounds. Initial inspection of the structural moieties of 1 revealed that the presence of
an isonitrile group is essential for its antimicrobial activity. When the isonitrile group of 1 (MIC
4

ACCEPTED MANUSCRIPT
against MRSA ranging from 8-64 ꢀM) was removed (as in compound 42), a complete loss in the
anti-MRSA activity of 42 is observed (MIC > 128 ꢀM). A similar pattern is observed when
reviewing the MIC results for compounds 13 and 43. Compound 13, one of the most potent
derivatives constructed (with MIC values against MRSA as low as 2 ꢀM), contains the isonitrile
group; when the isonitrile group of 13 is replaced with an isosteric nitrile group (resulting in
compound 43), complete loss of antimicrobial activity was observed. Similarly, compound 53
with an isonitrile group is active against several strains evaluated particularly MRSA USA100,
MRSA USA300, MRSA NRS119, and VISA NRS1, while compound 52 without the isonitrile
group lacks antimicrobial activity. These results confirm that the isonitrile group appears
necessary for these compounds to possess activity against MRSA and may play an important role
in binding to the compound’s molecular target.
The presence of a second aromatic substituent (connected to the isonitrile-phenyl group)
also appears critical to the biological activity observed; replacement of this moiety in 1 with a
diethyl phosphonate (as in analog 4a with an ortho-isonitrile group) results in complete loss of
activity against MRSA (MIC > 128 ꢀM). Likewise, substitution of this second aromatic
substituent with a cycloalkane (cf. 26) renders this compound inactive against several MRSA
isolates (including MRSA USA300, MRSA USA500, and MRSA NRS119). The presence of an
alkene bridge between the two aromatic substituents in 1 also appears to be important. When the
alkene bridge between the two aromatic substituents is removed, as in compound 46, this
compound lacks activity against three strains of MRSA (USA300, USA500, and NRS119). A
similar loss in antimicrobial activity is observed with compounds 49 and 50 indicating that the
stilbene isonitrile core of 1 plays an important role in its antimicrobial activity. This notion was
further supported by a direct comparison of compounds 13 and 53. Compound 53 is a saturated
5

ACCEPTED MANUSCRIPT
analog of compound 13 and contains a flexible two-carbon linker between the two aromatic
moieties. In general, compound 53 is less potent than compound 13 against all the strains texted
except for VISA NRS1.
We then evaluated how substituents on the double bond would affect the antimicrobial
activity. Interestingly, removal of the ethyl group of 1 (cf. 13) resulted in a dramatic
improvement in antimicrobial activity (a two-to-eight fold reduction in the MIC against MRSA
was observed). When the ethyl group was replaced by methyl (5), n-propyl (6), n-butyl (7) and
phenyl (8) groups, a noticeable change in the MIC value for these compounds is observed.
We next assessed how substituents on the non-isonitrile-containing aromatic ring would
affect the potency against MRSA. Analogs constructed include substitution of methoxy group
(14-16), fluoride (17-19), trifluoromethyl group (20-22), methyl group (23), n-butyl group (24),
and nitro group (25). Interestingly most of these modifications do not produce a major
improvement in the MIC observed against MRSA, when compared to the activity of 13.
Additionally the positioning of these groups around the benzene ring do not appear to have an
impact on the antimicrobial activity of the compound. While most of these modifications have
little effect on improving the antimicrobial activity of these compounds, one substitution had an
observed deleterious effect. Compound 24, containing a n-butyl group, lacked activity against
most MRSA strains tested (MIC >128 ꢀM); interestingly, 23, with a methyl group is active
against all MRSA strains tested albeit at a higher concentration than 13 (MIC of 23 ranges from
4 to 64 ꢀM against MRSA). This would appear to indicate that the presence of an alkyl group (in
particular one of increased length) is undesirable and can have a negative effect on the activity of
these compounds against MRSA. Analogs containing a pyridine ring were synthesized and tested
as well (27, 30, 33) and reduced antimicrobial activities were observed.
6

ACCEPTED MANUSCRIPT
All the analogs discussed above contain an ortho-substituted isonitrile group. We
wondered how the relative position of the isonitrile group would affect the antimicrobial activity
and prepared eight analogs with the isonitrile group in para- and meta-relationship to the double
bond (cf. 27, 28, 31, 32, 34-37). Different antimicrobial activity patterns are observed. For the
group of 13, 36, and 37, the ortho-substituted compound 13 is still the most potent one against
most of the strains tested and slight improvement was observed for the para-substituted
compound 37 against MSSA (NRS72) and MRSA USA500. Interestingly, for the group of 33,
34, and 35, the para-substituted compound 35 is much more active against all the strains tested
than the ortho- and meta-substituted ones. The groups of 27-29 and 30-31 are less potent than the
aforementioned two groups, which indicate that the position of the nitrogen atom in the pyridine
ring is important for the observed antimicrobial activity as well.
After completing a preliminary examination of the structure-activity relationship of these
compounds, we next moved to assess whether these compounds would retain their activity
against several of the most challenging strains of MRSA (Table 1 and Table 2). When tested
against an array of clinically-relevant MRSA isolates, the most potent compounds (6, 8-18, 20-
21, 25, and 37) did retain their antimicrobial activity. Indeed these compounds possess potent
activity against MRSA isolates prevalent in the healthcare-setting such as MRSA USA100
(responsible for invasive diseases in infected hospitalized patients),27 and MRSA USA200
(associated with more severe morbidity in affected patients due to the production of toxins that
can lead to toxic shock syndrome).28 In addition to this, these compounds exhibit potent activity
against MRSA USA300, a strain that has been linked to the majority of MRSA skin and soft
tissue infections present in the community setting.10,29 Furthermore, these compounds
demonstrate strong antimicrobial activity against MRSA strains exhibiting resistance to
7

ACCEPTED MANUSCRIPT
numerous antibiotic classes including penicillins, aminoglycosides (NRS1, USA200, and
USA500), macrolides (USA100, USA200, USA300, USA500, and USA700), lincosamides
(USA100, USA200, USA500), tetracyclines (NRS1, USA300, and USA500), and
fluoroquinolones (USA100 and USA500). Additionally, compounds 10, 11, 12, 21, 25, 32, and
35 exhibit potent antimicrobial activity (MIC between 4 and 16 ꢀM) against clinical isolates of S.
aureus exhibiting resistance to antibiotics deemed agents of last resort, namely vancomycin
(VRS2). These results indicate cross-resistance between these antibiotics and the aryl isonitrile
compounds is unlikely; this lends further credence to the notion that the aryl isonitrile
compounds have potential to be developed as future alternatives to these antibiotics.
Table 1 and Table 2 here.
Toxicity analysis of most potent aryl isonitrile compounds against mammalian cells.
Identification of compounds exhibiting potent antimicrobial activity is the first step in a lengthy
process for drug development. Many compounds with promising antimicrobial activity fail to
advance further in this process due to concerns about toxicity to mammalian tissues. Selective
toxicity is a critical feature novel antimicrobial compounds must possess. The ability for
antimicrobial agents to exhibit their activity on the target microorganism while not causing harm
to host (mammalian) tissues is important to ascertain early in the drug discovery process. To
determine if compound 1 and its most potent derivatives against MRSA exhibited toxicity to
mammalian tissues, these compounds were screened against a murine macrophage (J774) cell
line utilizing the MTS assay (Figure 2). Initial inspection of the structure-activity relationship
revealed that the isonitrile moiety appeared to be a vital component in the antimicrobial activity
8

ACCEPTED MANUSCRIPT
of these compounds. This was a point of concern given the isonitrile group has been associated
with a high degree of toxicity in certain compounds present in nature.30 However, when the most
potent compound, 13 (containing the isonitrile moiety), and its analog 42 (lacking the isonitrile
moiety) were tested against J774 cells, they produced identical results (neither compound was
toxic up to a concentration of 64 ꢀM). This would indicate that the isonitrile group in these
compounds does not contribute to undesirable toxicity to mammalian cells. This result is similar
to a study conducted at Bayer AG that found compounds, in their discovery pipeline, containing
the isonitrile moiety were not toxic to mice when administered orally or subcutaneously (even at
concentrations in excess of 500 mg/kg).31 In addition to this, at a concentration of 32 ꢀM, all of
the compounds tested, with the exception of 25 with a nitro group, were not toxic. When the
compounds were tested at a concentration of 64 ꢀM, nineteen out of twenty-three compounds
were found to not be toxic to J774 cells (Figure 2). Compounds 11, 12, 19, and 25 were found to
be toxic at 64 ꢀM. When the compounds were tested at 128 ꢀM, all compounds were found to be
toxic with the exception of compounds 15, 30, and 37 (data not presented). For the most active
compounds (such as 13), a 16-to-32 fold difference exists between the concentration at which the
compounds exhibit anti-MRSA activity (MIC) compared to the concentration where toxicity is
observed.
Figure 2 here.
Preliminary study of physicochemical properties of the isonitrole compounds using
kinetic solubility analysis and Caco-2 permeability assay. After confirming that most of the
isonitrile compounds exhibited strong antimicrobial activity against MRSA were not toxic to
9

ACCEPTED MANUSCRIPT
mammalian cells up to a concentration of 64 µM, we next moved to analyze the physicochemical
properties of the most promising compound 13. These properties play an important role in
determining the appropriate route of administration (i.e. systemic vs. local) by which compounds
with biological activity can be delivered to the host.32 Additionally, the physicochemical
properties of a compound will have a direct impact on its pharmacokinetic profile (in particular
absorption and metabolism), and ability to be translated into a viable drug candidate. Indeed, one
study found that 40% of new drug candidates were withdrawn due to issues pertaining to
significant pharmacokinetic problems.33 Compounds possessing a limited physicochemical
profile can have issues pertaining to solubility and permeability which can hinder a compound’s
ability to cross biological membranes, reach the bloodstream, and arrive at the target site of an
infection (thus limiting their use systemically).34
A kinetic solubility screen (using phosphate-buffered saline) and Caco-2 permeability
analysis was performed with compound 13. The solubility screen determined the highest
concentration 13 and three control drugs were capable of being fully dissolved in an aqueous
solvent (PBS). As presented in Table 3, this experiment revealed that compound 13 possessed
partial aqueous solubility (soluble up to 15.6 µM), identical to the control drugs reserpine and
tamoxifen.
Table 3 here.
The Caco-2 permeability assay revealed that compound 13 was not able to permeate
across the Caco-2 bilayer. As presented in Table 4, this compound was unable to cross from the
apical (A) to basolateral (B) surface of the membrane (apparent permeability, Papp = 0.0 cm/sec).
10

ACCEPTED MANUSCRIPT
A similar pattern is observed in the basolateral to apical direction with Papp = 0.0 cm/sec
(indicating this compound is unlikely a substrate for an efflux transporter, like talinolol, which
would be one plausible explanation for the inability of this compound to traverse the membrane).
This is in stark contrast to the control drug warfarin, which is able to effectively permeate across
the membrane from the basolateral to apical surface (Papp = 27.0 x 10-6 cm/sec). This result is a
bit surprising given the size, structure, and calculated partition coefficient (clog P = 4.107) for
13. Thus, in addition to possessing only partial aqueous solubility, 13 also possesses a poor
permeability profile, indicating that, in its present state, this compound would not be suitable for
use systemically.
Table 4 here.
The result from the Caco-2 permeability analysis is in agreement with the overall result
obtained from the kinetic solubility screen indicating that 13, though a promising antimicrobial
candidate, needs to undergo further structural modifications to enhance its physicochemical
profile (in order for it to be used systemically). In addition to modifying the structure of this
compound, formulation technology can be utilized to overcome this compound’s current
limitations. This technology has been used to improve the drug-like properties of promising
compounds with similar kinetic profiles to 13 in order to propel these compounds into further
stages of drug development. By using a spray drying dispersion technique,35 the antisolvent
crystallization method,36 or combining the active compound with an excipient (to create an
amorphous solid dispersion),37 the aqueous solubility, permeability and bioavailability profile of
this compound can be significantly improved. Identifying that 13 has a problematic
11

ACCEPTED MANUSCRIPT
physicochemical profile early in the drug discovery process will permit medicinal chemists and
formulation scientists to invest time and effort to enhancing both the physiochemical and
pharmacokinetic profiles of this promising new antimicrobial compound.
Metabolic stability analysis of 13 via microsomal stability analysis. In addition to
studying the solubility and permeability profile of compound 13, the stability of this compound
to metabolic processes present in the liver was investigated using human liver microsomes
(Table 5). Drugs administered systemically often are subject to various metabolic processes that
can convert the active compound to inactive metabolites. Pharmaceutical compounds that are
slow to be metabolized have multiple advantages including an improved pharmacokinetic
profile, reduced frequency of doses that need to be given to patients (leading to better patient
compliance), while also ensuring the active drug circulates within the patient’s system to assist
with treating and clearing an infection. As the liver is the primary organ for metabolism of drugs
administered systemically in the body, incubating compounds with liver microsomes can shed
valuable insight into the stability of these compounds to metabolic processes.32
When 13 was incubated with human liver microsomes, it was found to be rapidly
metabolized (only 24% of the parent compound remained after one hour) similar to the highly
metabolized control drug, verapamil (13% remained after one hour incubation with liver
microsomes) (Table 5). While verapamil appeared to be metabolized via a NADPH-mediated
process (as 94% of the drug remained after one hour when the co-factor NADPH was removed
from the reaction mixture), 13 does not appear to mimic this result as only 51% of the parent
compound remained after one hour when NADPH was not present. This would appear to suggest
that 13 is metabolized by more than one enzyme system/reaction (one dependent on the co-factor
12

ACCEPTED MANUSCRIPT
NADPH (most likely the cytochrome P450 system), and one independent of NADPH). The
metabolic stability analysis performed lends further credence to the argument that in their present
state, 13, would not be suitable for use in systemic applications to treat MRSA infections.
Table 5 here.
CONCLUSION
In summary, we have discovered a novel class of aryl isonitrile compounds as promising
and potent antimicrobial compounds without apparent toxicity against mammalian cells up to a
concentration of 64 µM. Physicochemical profiling, including solubility, membrane
permeability, and metabolic stability of one of the most potent compounds, 13, has been
conducted as well. These results indicates that modification of the physical structure of
compound 13 is needed to enhance its physicochemical and pharmacokinetic profile so that it
can be developed for systemic use against MRSA infections. In addition, identifying other routes
of administration (such as topical/local administration) is another avenue to pursue to further
develop this promising compound as a novel antimicrobial candidate. Given that S. aureus and
its resistant strains (including MRSA) are a leading cause of uncomplicated skin infections (such
as abscesses, impetigo, and cellulitis),2,10 it is logical to assess if compound 13, and its analogs,
can be used as topical antimicrobial agents for treatment of MRSA skin infections. Topical
agents avoid many concerns relating to solubility, permeability, and systemic toxicity associated
with drugs administered orally or intravenously. Future work with these compounds will look to
pursue two avenues – determining if these compounds can be used as topical antimicrobial
agents for MRSA skin infections and improving the physicochemical profile of these compounds
13

ACCEPTED MANUSCRIPT
(via structural modifications or using formulation technology) so they can potentially be used for
treatment of more invasive MRSA infections.
ACKNOWLEDGMENT
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, VISA, and VRSA strains used in this study and the NIH P30CA023168 for
supporting shared NMR resources to Purdue Center for Cancer Research. M.D. thanks Purdue
University for startup support.
Appendix A. Supplementary data
Supplementary data contain the detailed synthetic procedures, compound characterization data,
and biological test procedures.
REFERENCES
(1) Centers for Disease Control and Prevention. Antibiotic Resistance Threats in the United
States, 2013. http://www.cdc.gov/drugresistance/threat-report-2013/.
(2) Giordano, P.; Weber, K.; Gesin, G.; Kubert, J. Skin and skin structure infections: treatment
with newer generation fluoroquinolones. Ther. Clin. Risk Manag. 2007, 3, 309-317.
(3) Gillet, Y.; Issartel, B.; Vanhems, P.; Fournet, J. C.; Lina, G.; Bes, M.; Vandenesch, F.;
Piémont, Y.; Brousse, N.; Floret, D.; Etienne, J. Association between Staphylococcus aureus
strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in
young immunocompetent patients. Lancet. 2002, 359, 753-759.
14

ACCEPTED MANUSCRIPT
(4) Bocchini, C. E.; Hulten, K. G.; Mason, E. O.; Gonzalez, B. E.; Hammerman, W. A.;
Kaplan, S. L. Panton-Valentine leukocidin genes are associated with enhanced inflammatory
response and local disease in acute hematogenous Staphylococcus aureus osteomyelitis in
children. Pediatrics 2006, 117, 433-440.
(5) David, M. Z.; Daum, R. S. Community-associated methicillin-resistant Staphylococcus
aureus: epidemiology and clinical consequences of an emerging epidemic. Clin. Microbiol. Rev.
2010, 23, 616-687.
(6) Chambers, H. F. Community-associated MRSA-resistance and virulence converge. N. Engl.
J. Med. 2005, 352, 1485-1487.
(7) Frazee, B. W.; Lynn, J.; Charlebois, E. D.; Lambert, L.; Lowery, D.; Perdreau-Remington,
F. High prevalence of methicillin-resistant Staphylococcus aureus in emergency department skin
and soft tissue infections. Ann. Emerg. Med. 2005, 45, 311-320.
(8) Han, L. L.; McDougal, L. K.; Gorwitz, R. J.; Mayer, K. H.; Patel, J. B.; Sennott, J. M.;
Fontana, J. L. High frequencies of clindamycin and tetracycline resistance in methicillin-resistant
Staphylococcus aureus pulsed-field type USA300 isolates collected at a Boston ambulatory
health center. J. Clin. microbiol. 2007, 45, 1350-1352.
(9) Hiramatsu, K. Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic
resistance. Lancet Infect. Dis. 2001, 1, 147-155.
(10) Moran, G. J.; Krishnadasan, A.; Gorwitz, R. J.; Fosheim, G. E.; McDougal, L. K.; Carey,
R. B.; Talan, D. A. Methicillin-resistant S. aureus infections among patients in the emergency
department. N. Engl. J. Med. 2006, 355, 666-674.
15

ACCEPTED MANUSCRIPT
(11) Wilson, P.; Andrews, J. A.; Charlesworth, R.; Walesby, R.; Singer, M.; Farrell, D. J.;
Robbins, M. Linezolid resistance in clinical isolates of Staphylococcus aureus. J. Antimicrob.
Chemother. 2003, 51, 186-187.
(12) Podoll, J. D.; Liu, Y.; Chang, L.; Walls, S.; Wang, W.; Wang, X. Bio-inspired synthesis
yields a tricyclic indoline that selectively resensitizes methicillin-resistant Staphylococcus aureus
(MRSA) to β-lactam antibiotics. Proc. Natl. Acad. Sci. USA 2013, 110, 15573-15578.
(13) Marconi, G. G.; Molloy, B. B.; Nagarajan, R.; Martin, J. W.; Deeter, J. B.; Occolowitz, J.
L. A32390A, a new biologically active metabolite. II. Isolation and structure. J. Antibiot. 1978,
31, 27-32.
(14) Mo, S.; Krunic, A.; Chlipala, G.; Orjala, J. Antimicrobial ambiguine isonitriles from the
cyanobacterium Fischerella ambigua. J. Nat. Prod. 2009, 72, 894-899.
(15) Raveh, A.; Carmeli, S. Antimicrobial ambiguines from the cyanobacterium Fischerella sp.
collected in Israel. J. Nat. Prod. 2007, 70, 196-201.
(16) Sugawara, T.; Tanaka, A.; Imai, H.; Nagai, K.; Suzuki, K. YM-47515, a novel isonitrile
antibiotic from Micromonospora echinospora subsp. echinospora. J. Antibiot. 1997, 50, 944-
948.
(17) Fujiwara, A.; Okuda, T.; Masuda, S.; Shiomi, Y.; Miyamoto, C.; Sekine, Y.; Tazoe, M.;
Fujiwara, M. Fermentation, isolation and characterization of isonitrile antibiotics. Agric. Biol.
Chem. 1982, 46, 1803-1809.
16

ACCEPTED MANUSCRIPT
(18) Marquez, J. A.; Horan, A. C.; Kalyanpur, M.; Lee, B. K.; Loebenberg, D.; Miller, G. H.;
Patel, M.; Waitz, J. A. The hazimicins, a new class of antibiotics taxonomy, fermentation,
isolation, characterization, and biological properties. J. Antibiot. 1983, 36, 1101-1108.
(19) Bettley, F. R. Xanthocillin cream for local treatment. Br. Med. J. 1959, 1, 1226-1227.
(20) Wright, A. D.; Wang, H.; Gurrath, G.; König, G. M.; Kocak, G.; Neumann, G.; Loria, P.;
Foley, M.; Tilley, L. Inhibition of heme detoxification processes underlies the antimalarial
activity of terpene isonitrile compounds from marine sponges. J. Med. Chem. 2001, 44, 873-885.
(21) Schwarz, O.; Brun, R.; Bats, J. W.; Schmalz, H. Synthesis and biological evaluation of
new antimalarial isonitriles related to marine diterpenoids. Tetrahedron Lett. 2002, 43, 1009-
1013.
(22) Singh, C.; Srivastav, N. C.; Puri, S. K. In vivo active antimalarial isonitriles. Bioorg. Med.
Chem. Lett. 2002, 12, 2277-2279.
(23) Arbuzov, B. A. Michaelis–Arbusow- und Perkow-Reaktionen. Pure. Appl. Chem. 1964, 9,
307-353.
(24) Weber, W. P.; Gokel, G. W. An improved procedure for the Hofmann carbylamine
synthesis of isonitriles. Tetrahedron Lett. 1972, 1637-1640.
(25) Zhang, B.; Studer, A. 2-Trifluoromethylated indoles via radical trifluoromethylation of
isonitriles. Org. Lett. 2014, 16, 1216-1219.
17

ACCEPTED MANUSCRIPT
(26) Zhang, B.; Mück-Lichtenfeld, C.; Daniliuc, C. G.; Studer, A. 6-Trifluoromethyl-
phenanthridines through radical trifluoromethylation of isonitriles. Angew. Chem. Int. Ed. 2013,
52, 10792-10795.
(27) Klevens, R. M.; Morrison, M. A.; Nadle, J.; Petit, S.; Gershman, K.; Ray, S.; Harrison, L.
H.; Lynfield, R.; Dumyati, G.; Townes, J. M.; Craig, A. S.; Zell, E. R.; Fosheim, G. E.;
McDougal, L. K.; Carey, R. B.; Fridkin, S. K. Invasive methicillin-resistant Staphylococcus
aureus infections in the United States. JAMA, 2007, 298, 1763-1771.
(28) Lin, Y. C.; Anderson, M. J.; Kohler, P. L.; Strandberg, K. L.; Olson, M. E.; Horswill, A.
R.; Schlievert, P. M.; Peterson, M. L. Proinflammatory exoprotein characterization of toxic
shock syndrome Staphylococcus aureus. Biochemistry, 2011, 50, 7157-7167.
(29) McDougal, L. K.; Steward, C. D.; Killgore, G. E.; Chaitram, J. M.; McAllister, S. K.;
Tenover, F. C. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus
aureus isolates from the United States: Establishing a national database. J. Clin. Microbiol. 2003,
41, 5113-5120.
(30) Goda, M.; Hashimoto, Y.; Shimizu, S.; Kobayashi, M. Discovery of a novel enzyme,
isonitrile hydratase, involved in nitrogen-carbon triple bond cleavage. J. Biol. Chem. 2001, 276,
23480-23485.
(31) Ugi, I.; Fetzer, U.; Eholzer, U.; Knupfer, H.; Offerman, K. Isonitrile Syntheses. Angew.
Chem. Int. Ed. 1965, 4, 472-484.
(32) Kerns, E. H.; Di, L. Drug-like properties: concepts, structure design and methods- from
ADME to toxicity optimization. Amsterdam; Boston: Academic Press; 2008.
18

ACCEPTED MANUSCRIPT
(33) Prentis, R. A.; Lis, Y.; Walker, S. R. Pharmaceutical innovation by the seven UK-owned
pharmaceutical companies (1964-1985). Br. J. Clin. Pharmacol. 1988, 25, 387-396.
(34) Lin, J. H.; Lu, A. Y. Role of pharmacokinetics and metabolism in drug discovery and
development. Pharmacol. Rev. 1997, 49, 403-449.
(35) Kwong, A. D.; Kauffman, R. S.; Hurter, P.; Mueller, P. Discovery and development of
telaprevir: an NS3-4A protease inhibitor for treating genotype 1 chronic hepatitis C virus. Nat.
Biotechnol. 2011, 29, 993-1003.
(36) Lonare, A. A.; Patel, S. R. Antisolvent Crystallization of Poorly Water Soluble Drugs. Int.
J. Chem. Eng. Appl. 2013, 4, 337-341.
(37) Van den Mooter, G. The use of amorphous solid dispersions: A formulation strategy to
overcome poor solubility and dissolution rate. Drug Discov. Today. Technol. 2012, 9, e71-e174.
19

ACCEPTED MANUSCRIPT
Schemes, Figures and Tables (see below)
Me
NC
1
FIGURE 1. Structure of hit compound 1.
1

ACCEPTED MANUSCRIPT
I. Synthesis of compounds 1and 5-37
R1
R2
Br
PO(OEt)2
PO(OEt)2
i) Pd/C, H2
LDA, THF
POEt3, Tol.
120 oC
O2N
CN
O2N
CN
-78 oC to RT
ii) CHCl3, BnEt3NCl
50% KOH, CH2Cl2
R1
R2
2
4
3
1, 5-37
O
Me
13 (X = H)
20 (X = p-CF3)
X
X
R
14 (X = p-OMe) 21 (X = m-CF3)
15 (X = m-OMe) 22 (X = o-CF3)
16 (X = o-OMe) 23 (X = p-Me)
1 (R = Et)
9 (X = p-F)
5 (R = Me)
6 (R = nPr)
7 (R = nBu)
8 (R = Ph)
10 (X = p-CF3)
11 (X = p-OMe)
12 (X = m-CF3)
17 (X = p-F)
18 (X = o-F)
19 (X = m-F)
24 (X = p-nBu)
25 (X = p-NO2)
NC
NC
NC
NC
26
N
N
N
30 (o-NC)
31 (m-NC)
32 (p-NC)
27 (o-NC)
28 (m-NC)
29 (p-NC)
33 (o-NC)
34 (m-NC)
35 (p-NC)
CN
CN
CN
37
36
CN
NC
II. Synthesis of compounds 42 and 43
Y
PO(OEt)2
LDA, THF, -78 oC to RT
Et
or
or
H
Et
38 (Y = H)
CN
39 (Y = CN)
40
41
42
O
O
43
III. Synthesis of compounds 46 and 38-40
Ac2O, HCO2H
73%
POCl3, NEt3
93%
NC
NHCHO
NH2
46
45
44
Br
Ar
Ar
NC
NH2
NH2 i) Ac2O, HCO2H
ii) POCl3, NEt3
PdCl2(PPh3)2
K2CO3, DME
49 (R = Me, Ar = p-tBuPh)
50 (R = Me, Ar = p-CF3Ph)
51 (R = H, Ar = 2-naphthyl)
+
Ar B(OH)2
R
R
R
48
47
49-51
IV. Synthesis of compound 53
PO(OEt)2
CHCl3, BnEt3NCl
50% KOH, CH2Cl2
i) LDA, PhCHO, THF
ii) Pd/C, H2
NO2
52
53
3a
NH2
NC
SCHEME 1. Synthesis of Analogs of Lead Compound 1.
2

ACCEPTED MANUSCRIPT
Table 1: Strains of Staphylococcus aureus utilized in this study.
Strain Name Isolation
Molecular Typing
Antimicrobial Resistance Phenotype
Alternate
Designation
Origin
Source
SCCmec
type
spa type
NARSA
ID1
ATCC700699
VISA
Japan
-
II
TJMBMDMGMK
Resistant to aminoglycosides and
tetracycline (minocycline)
Glycopeptide-intermediate S. aureus
None
NRS1
-
NRS722
NRS119
MSSA 476
-
-
UKJFKBPE
Dialysis-
associated
peritonitis
SA LinR #12
United States
(Massachusetts)
IV
YHGCMBQBLO
Resistant to linezolid
USA100
USA200
United States
(Ohio)
Bloodstream
Bloodstream
Wound
II
II
TJMBMDMGMK
Resistant to erythromycin, clindamycin
and levofloxacin
NRS382
NRS383
NRS384
NRS385
NRS386
VRS2
United States
(North Carolina)
WGKAKAOMQQQ Resistant to erythromycin, clindamycin
and gentamicin
USA300-0114
USA500
United States
(Mississippi)
IV
IV
IV
II
YHGFMBQBLO
YHGCMBQBLO
UJGFMGGM
Resistant to erythromycin, methicillin,
and tetracycline
United States
(Connecticut)
Bloodstream
Bloodstream
Plantar ulcer
Resistant to erythromycin, clindamycin,
trimethoprim/sulfamethoxazole,
levofloxacin, gentamicin and tetracycline
Resistant to erythromycin and methicillin
USA700
United States
(Louisiana)
VRSA
United States
(Pennsylvania)
TJMBMDMGMK
Resistant to vancomycin
1 NARSA = Network on Antimicrobial Resistance in Staphylococcus aureus.
2 NRS72 = Methicillin-sensitive Staphylococcus aureus (MSSA).
3

ACCEPTED MANUSCRIPT
Table 2: Minimum inhibitory concentration (MIC, in M) of isonitrile compounds, linezolid, and
ꢀ
vancomycin against methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus
(MRSA) strains.
Compound
Name
MSSA1
(NRS72) USA100 USA200 USA300 USA500
MRSA
MRSA
MRSA
MRSA
MRSA
MRSA
VISA1
VRSA2
VRS2
USA700 NRS119 NRS1
8
>128
8
16
32
8
8
>128
8
16
32
8
16
128
64
16
32
8
32
>128
>128
32
>128
16
32
>128
>128
16
32
8
8
>128
16
16
16
4
64
>128
>128
32
>128
16
8
128
4
8
16
4
32
>128
>128
32
64
16
1
4a
5
6
7
8
8
8
8
4
16
4
8
16
16
8
4
2
8
8
8
16
16
8
8
4
2
4
8
32
32
16
>128
4
16
16
8
16
16
8
8
32
>128
32
8
16
4
2
4
8
16
8
64
64
>128
8
4
32
32
16
8
8
4
2
2
4
4
4
32
16
8
8
32
32
32
16
9
10
11
12
13
14
15
16
8
8
4
8
2
8
16
8
16
4
32
17
4
8
2
16
16
8
32
4
32
18
8
4
16
4
16
2
32
8
128
16
16
2
128
16
8
2
128
16
19
20
2
4
4
8
8
4
8
2
8
21
8
16
4
-
16
4
>128
4
32
16
>128
8
16
32
>128
8
16
8
-
>128
64
>128
16
8
4
-
4
32
32
>128
8
22
23
24
25
16
>128
2
4
8
8
16
64
32
128
32
32
16
32
32
8
64
64
64
128
32
64
32
32
64
8
>128
16
32
32
16
32
8
32
32
>128
64
64
64
32
64
32
32
64
16
32
64
128
32
64
16
32
64
8
>128
128
128
128
64
64
64
64
32
4
>128
32
128
64
32
64
8
64
64
4
26
27
28
29
30
31
32
33
64
64
64
64
64
16
64
64
2
64
128
64
64
64
32
64
32
8
34
35
4
4
4
16
4
32
4
32
32
>128
>128
16
16
>128
64
32
16
2
16
4
>128
16
16
64
>128
>128
16
16
>128
16
>64
32
2
64
32
>128
>128
>128
8
32
36
37
42
43
46
49
50
51
52
>128
>128
>128
>128
64
>128
32
>128
2
>128
>128
>128
>128
>128
>128
64
>64
64
<1
>128
>128
>128
>128
>128
16
>128
64
2
>128
>128
16
16
>128
16
>128
4
>128
>128
>128
>128
>128
64
>128
4
2
>128
>128
>128
>128
>128
32
>64
128
<1
4
>128
4
8
2
<1
8
53
Linezolid
Vancomycin
<1
2
64
-
<1
-
-
4
-
128
1VISA = Vancomycin-intermediate Staphylococcus aureus.
2VRSA = Vancomycin-resistant Staphylococcus aureus.
4

ACCEPTED MANUSCRIPT
Figure 2: Percent viable mammalian cells (measured as average absorbance ratio (test agent relative to
DMSO)) for cytotoxicity analysis of compounds 1, 11-23, 25, 27, 30, 32, 35, 36, 37, 42, and 51 at 64 µM.
Compounds were tested against J774 cells using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. DMSO was used as a negative control
to determine a baseline measurement for the toxic impact of each compound. The values represent an
average of three samples analyzed for each compound. Error bars represent standard deviation values for
the absorbance values. Asterisks (*) indicate a statistical difference between the values obtained for the
compound relative to the cells treated with DMSO (P < 0.05).
5

ACCEPTED MANUSCRIPT
Table 3: Kinetic solubility assessment of compound 13, reserpine, tamoxifen, and verapamil in
phosphate-buffered saline (PBS).
Compound Tested
Solubility Limit (µM)1
Solubility Analysis
Low solubility
Low solubility
Low solubility
High solubility
15.6
15.6
15.6
>500
13
Reserpine
Tamoxifen
Verapamil
1 Solubility limit corresponds to the highest concentration of test compound where no precipitate was
detected.
6

ACCEPTED MANUSCRIPT
Table 4: Permeability analysis of compound 13, ranitidine, warfarin, and talinolol via the Caco-2
permeability assay.
Compound/Drug
Tested
Mean A
→
B1
Mean B
→
A2
Efflux Ratio3
Permeability
Analysis
Papp
Papp
(10-6 cm/sec)
0.04
(10-6 cm/sec)
0.0
3.1
7.2
8.9
N/A5
13.5
0.3
Not permeable
Low permeability
High permeability
P-gp6 efflux
13
Ranitidine
Warfarin
Talinolol
0.23
27.0
0.05
178
control
1 Mean A
2 Mean B
→
→
B Papp = mean apparent permeability of test compound from apical to basolateral surface
A Papp = mean apparent permeability of test compound from basolateral to apical surface
3 Efflux ratio= ꢀꢀꢁꢁꢂꢂꢂꢂ((ꢄꢃ→→ꢃꢄ))
4 Compound not detected in receiver compartment
5 N/A, not applicable
6 P-gp, P--glycoprotein
7

ACCEPTED MANUSCRIPT
Table 5: Evaluation of metabolic stability of compound 13, verapamil, and warfarin in human liver
microsomes.
Compound/Drug
Tested
Average
remaining after
60 min (%), with
NADPH
Average
remaining after
60 min (%),
without NADPH
51
Notes
24
-
13
Verapamil
13
94
High metabolism
control
Warfarin
93
94
Low metabolism
control
8

ACCEPTED MANUSCRIPT
Highlights
1. Evaluated over forty aryl isonitrile compounds against MRSA and VRSA strains.
2. Identified compounds with MIC as low as 2 µM but no cytotoxicity at 64 µM.
3. Established SAR of these novel isonitrile compounds.
4. Profiled the most potent compound’s physicochemical properties.