Amide isosteres of oroidin: Assessment of antibiofilm activity and C. elegans toxicity

Article abstract of DOI:10.1021/jm900378s

The synthesis and antibiofilm activities of sulfonamide, urea, and thiourea oroidin analogues are described. The most active derivative was able to selectively inhibit P. aeruginosa biofilm development and is also shown to be nontoxic upward of 1 mM to th

Full text of DOI:10.1021/jm900378s

4582 J. Med. Chem. 2009, 52, 4582–4585
DOI: 10.1021/jm900378s
Amide Isosteres of Oroidin: Assessment of
Antibiofilm Activity and C. elegans Toxicity
Justin J. Richards,† Samuel Reyes,† Sean D. Stowe,‡
Ashley T. Tucker,‡ T. Eric Ballard,†,§ Laura D. Mathies,
John Cavanagh,‡ and Christian Melander*,†
†Department of Chemistry, Department of Genetics, and
‡Department of Molecular and Structural Biochemistry,
North Carolina State University, Raleigh, North Carolina 27695.
§Current address: Department of Chemistry, University of Virginia,
Charlottesville, VA 22904.
Received March 25, 2009
Abstract: The synthesis and antibiofilm activities of sulfonamide,
urea, and thiourea oroidin analogues are described. The most active
derivative was able to selectively inhibit P. aeruginosa biofilm
development and is also shown to be nontoxic upward of 1 mM
to the development of C. elegans in comparison to other similar
isosteric analogues and the natural product oroidin.
Figure 1. Nonmicrobicidal biofilm modulators.
be a moderate inhibitor of Pseudomonas aeruginosa PAO1/-
=
PA14 biofilm growth (PAO1 IC50 =190 μM, PA14 IC50
166 μM).14
We have recently focused on the development of methodol-
ogies to access oroidin analogues for antibiofilm screening.15-17
Of these approaches, development of conditions for a generic
reductive acylation reaction has allowed access to previously
unattainable oroidin derivatives through the use of acid
chlorides, anhydrides, succinimide esters, and trichloro-
methylketone pyrroles.18 In a continued effort, we sought to
further apply this approach in generating isosteric analogues
possessing sulfonamide, urea, and thiourea functionalities to
further probe the structure-activity relationships (SAR) of
the oroidin family in the context of antibiofilm activity and
preliminary toxicity.
The Gram-negative γ-proteobacteria Pseudomonas aerugi-
nosa PA14 and Acinetobacter baumannii were selected as the
biofilm-forming bacteria employed in the study. Pseudomonas
aeruginosa is one of the most well studied organisms with
respect to biofilm behavior.19 It is the second most commonly
isolated pathogen in cases of nosocomial acquired pneu-
monia.20 Additionally, the inability to treat cystic fibrosis
patients with chronic P. aeruginosa infections has been di-
rectly correlated with the emergence and pathogenicity of
P. aeruginosa biofilms.21 Acinetobacter baumannii has been
identified in recent hospital outbreaks throughout healthcare
systems in both Europe and the U.S.22,23 The speed and
prevalence with which multidrug resistance (MDR) is occur-
ring in this bacterium has posed as a significant impediment to
A. baumannii remediation therapies. In addition to examining
the antibiofilm properties of these new derivatives, it was also
a major goal of this study to further delineate the toxicity
associated with oroidin-derived compounds. This was accom-
plished by investigating how the most active member of the
newly formed library and previously synthesized isosteric
analogues affected larvae development in the eukaryote
Caenorhabditis elegans in comparison to the natural product
oroidin.
Bacterial biofilm formation is often described as a devel-
opmental process initiated when free floating (planktonic)
bacteria adhere toasurface suitablefor growthand initiate the
formation of a microcolony.1 Occupation of a biofilm growth
state confers to the bacteria a unique set of phenotypic traits
that include resistance to microbicides and antibiotics that
would often lead to eradication.2-4 In a medical setting,
biofilms pose a serious threat to individuals who suffer from
a myriad of diseases. Recent estimates have attributed bio-
film-associated infections as being responsible for upward of
75% of microbial infections in the human body.5 This pro-
blem is further exacerbated by the increased spread of anti-
biotic resistance. Additionally, biofilms are known to infect
patients with in-dwelling medical devices (IMDsa) such as
catheters and heart stents.6-8 Remediation of biofilm infected
IMDs is traditionally accomplished by device removal be-
cause of the lack of antibiotic efficacy.
As the medical community works toward new approaches
aimed at combating the deleterious effects of biofilms, one
area that has garnered significant attention is the identifica-
tion of nonmicrobicidal modulators of biofilm growth and
maintenance (Figure 1).9-13 By not directly killing bacteria, it
ispostulated that developmentof resistance tothesemolecules
would be mitigated or significantly impaired. Implementation
of remediation therapies that focus on the co-dosing of an
antibiofilm modulator with an antibiotic also provides an
attractive avenue for treatment. One of the few naturally
occurring scaffolds shown to possess nonmicrobicidal anti-
biofilm properties are compounds derived from the oroidin
class of natural products. Oroidin 5 has been reported to
*To whom correspondence should be addressed. Phone: 919-513-
2960. Fax: 919-515-5369. E-mail: christian_melander@ncsu.edu.
a Abbreviations: IMD, in-dwelling medical device; IC50, biofilm
inhibitory concentration of 50%; SAR, structure-activity relationship;
MDR, multidrug resistance; Pd/C, palladium on carbon; THF, tetra-
hydrofuran; rt, room temperature; TEA, triethylamine; TFA, trifluor-
oacetic acid; SE, standard error; 2-AI, 2-aminoimidazole; NIEHS,
National Institute of Environmental Health and Safety; DHS, dihy-
drooroidin.
A total of 19 analogues (9 prepared from sulfonyl chlorides,
8 prepared from isocyanates, 2 prepared from isothiocyanates)
were synthesized by slightly varying the reaction conditions
previously reported (Schemes 1 and 2).18 While no base was
used under the initial acylation conditions, it was found that
r
pubs.acs.org/jmc
Published on Web 07/09/2009
2009 American Chemical Society

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4583
Table 1. Antibiofilm Activity of Select Analogues
Scheme 1. Synthesis of Sulfonamide Analogues
PA14
IC50 ( SE (μM)
PA14 dispersion at
100 μM ( SE (%)
analogue
8a
10.1 ( 1.96
25.7 ( 2.19
22.6 ( 3.24
42 ( 4
55 ( 3
25 ( 5
22a
26a
consistent (33-92%). The commercial availability of a wide
array of these acylating reagents also made it possible to probe
the effect that larger ring systems (15, 16, and 24) had on
antibiofilm activity. Coupled products generated through the
application of the methodology were then exposed to TFA-
mediated deprotection in dichloromethane. The resulting TFA
salts were exchanged for the corresponding hydrochloride salts
which were then used for biological assessment.
All newly synthesized derivatives were first subjected to
static biofilm inhibition assays performed at 100 μM utilizing
a crystal violet reporter assay (Supporting Information).15,24
Compounds whose inhibition values exceeded 80% in the
initial screen were subsequently assayed for IC50 (biofilm
inhibition) values. The most active compound in each class
of molecules (sulfonamide, urea, thiourea) was then analyzed
for biofilm dispersion activity. Surprisingly, none of the newly
developed sulfonamides or ureas/thioureas were able to effec-
tively inhibit the formation of A. baumannii biofilms greater
than 30% at 100 μM. This is in contrast to previous studies
where the biological activity of most analogues could be
conserved over γ-proteobacteria.25,26 However, many of the
derivatives were determined to be potent inhibitors of
P. aeruginosa PA14 biofilm development with 11 out of the
19 analogues exhibiting PA14 IC50 under 50 μM. In general,
the sulfonamides were the most active (IC50 =10-46 μM),
followed closely by the two thioureas (IC50=22-26 μM) and
last the ureas (IC50=25-50 μM). The most active analogues
from each class are summarized in Table 1. Nonmicrobicidal
behavior of 8a, 22a, and 26a against PA14 was validated by
performing growth curve experiments at the calculated IC50.
No change in bacterial cell density was observed for all
compounds throughout a 24 h time period (Supporting
Information).
Scheme 2. Synthesis of Urea and Thiourea Analogues
In parallel with previous observations of other oroidin
derivatives, there was a decrease in the activities observed in
the dispersion experiments.14,15,18,27 Analogues 8a, 22a, and
26a were only modest dispersal agents against preformed
PA14 biofilms at 100 μM (42%, 55%, and 25%, respectively).
Despite the lack of biofilm dispersal activity, the inhibition
results further strengthened the argument that inhibition
activityofthe oroidin scaffoldagainst PA14 can bemodulated
to deliver molecules even more potent than those found with
an amide bond in a similar arrangement to the natural
products.15,25 Also, activity can be tuned to display selectivity
against biofilm forming proteobacteria which reside in the
same class through the incorporation of sulfonamide or urea/
thiourea functionalities.
addition of a single equivalent of triethylamine prior to
addition of the acylating reagent and keeping the solution at
-78 °C throughout the course of the reaction led to optimum
yields. In general, the generation of sulfonamide derivatives
proceeded smoothly (57-88%) while the corresponding
yields for the formation of the ureas and thioureas were less

4584 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Richards et al.
Figure 2. C. elegans toxicity thresholds of selected hexyl chain based isosteric oroidin analogues.
The continued investigation into the development of anti-
biofilm analogues derived from the oroidin scaffold has
inevitably led us to further question the possible utilization
of these molecules as therapeutic treatments in biofilm reme-
diation efforts. Ultimately, the molecules must retain their
noncytotoxicbiofilm-modulatingproperties withoutaffecting
the surrounding cellular environments. Preliminary cytotoxi-
city experiments involving GH4C1 rat pituitary cells and N2A
mouse neuroblastoma cells have shown that select 2-amino-
imidazole (2-AI) analogues possessed nontoxic activity at
concentrations up to 600 μM.27,28 To further investigate the
toxicity profiles of a specific class of 2-AI derivatives, we
analyzed the effects they had on the development of the
multicellular eukaryote Caenorhabditis elegans.
The overall goal of the assay was to examine what effect
various concentrations of 2-AI molecules would have on late
larval stage C. elegans development (Supporting Information
for detailed assay). The National Institute of Environmental
Health and Safety (NIEHS) has validated preliminary screens
for eukaryotic toxicity in model organisms such as C. elegans
as a means for the strategic testing of possible toxic sub-
stances.29,30 Briefly, in 96-well microtiter plates, the nema-
todes were monitored for their ability to develop into egg-
laying adults, for the eggs to hatch into early stage larvae, and
fortheselarvaetosubsequently feedona bacterialsuspension.
Observations were made through visualization under a dis-
secting microscope, with the results being interpreted in a
binary manner. Either the wells remained turbid and opaque
signaling that the worms had not fed on the bacterial suspen-
sion at the given concentration of the 2-AI compound (toxic)
or the wells became transparent indicating that the 2-AI
derivative had not interfered with developmental activities
at that particular concentration (nontoxic). Ivermectin, a
potent nematocide, was employed as a positive control. Using
this experimental design, thresholds of toxicity toward
C. elegans development were then determined.
examine a number of other hexyl aliphatic chain 2-AI analo-
gues in which the amide bond was modified in an analogous
fashion (Figure 2).25 The results from the toxicity screens are
summarized in Figure 2. Oroidin 5 was found to display a
threshold of 650 μM, meaning that any concentration over
that mark was found to be toxic to the worms. DHS 27 was
slightly more toxic, exhibiting a detrimental effect at concen-
trations greater than 480 μM. However, many of the aliphatic
derivatives chosen for analysis were found to be nontoxic at
much higher concentrations (8a, 29, 30, 31), with most even
exceeding the maximum concentration of 1 mM used in the
study. Analogue 28 was close to the 1 mM mark, eliciting a
toxic effect at concentrations greater than 950 μM. These
results are the first to demonstrate the associated toxicity of
oroidin and related analogues to the development of
C. elegans. The relative lack of toxicity at such high concen-
trations undoubtedly provides a suitable foundation for the
continued exploration of the affects of these compounds in
other model organisms.
In conclusion, employment of a reductive acylation strategy
to incorporate sulfonamide, urea, or thiourea functionalities
into the oroidin backbone has led to the discovery that other
amide isosteres have the potential to be potent and selective
modulators of P. aeruginosa biofilm development. Further-
more, related isosteric analogues that bear aliphatic side chains
are reported to be highly nontoxic in nature to the develop-
ment of C. elegans in comparison to the natural product
oroidin. Ultimately, it is envisioned that the reductive acyla-
tion reaction will prove amenable for the synthesis of a variety
of biotinylated conjugates to help further probe the mechanism
of action of these biofilm modulating nontoxic compounds.
Acknowledgment. Financial support for this work was
graciously provided by the NIH (Grant R01 GM55769), the
Jimmy V. NCSU Cancer Therapeutics Training Program, the
UNCGA Competiveness Research Fund, and the North
Carolina Biotechnology Center.
A logical starting point for the assay was to first assess the
toxicity of oroidin and dihydrosventrin (DHS) 27, which was
one of the first 2-AI analogues assembled from the oroidin
skeleton that was found to possess biofilm modulating prop-
erties.14,26 In addition to assaying the most active member of
the present isosteric library (8a), it was decided to also
Supporting Information Available: Antibiofilm activity
charts, growth curves, C. elegans toxicity assay procedures,
characterization data, and representative spectra from each
class of compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4585
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