Synthesis and bacterial biofilm inhibition studies of ethyl N-(2-phenethyl) carbamate derivatives

Article abstract of DOI:10.1039/c0ob00063a

An 88 member library based upon the marine bacterial metabolite ethyl N-(2-phenethyl) carbamate was evaluated for bacterial biofilm inhibition against a panel of medically relevant strains. These studies culminated in the discovery of a new class of molec

Full text of DOI:10.1039/c0ob00063a

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Synthesis and bacterial biofilm inhibition studies of ethyl N-(2-phenethyl)
carbamate derivatives†
Steven A. Rogers, Daniel C. Whitehead, Trey Mullikin and Christian Melander*
Received 29th April 2010, Accepted 9th June 2010
First published as an Advance Article on the web 8th July 2010
DOI: 10.1039/c0ob00063a
An 88 member library based upon the marine bacterial
metabolite ethyl N-(2-phenethyl) carbamate was evaluated
for bacterial biofilm inhibition against a panel of medically
relevant strains. These studies culminated in the discovery of
a new class of molecules capable of inhibiting the formation
of S. aureus biofilms with low micromolar IC50 values.
handful of aliphatic subunits. None of the analogues demonstrated
improved activity in comparison to 2d.13 Based on these results
and our success with 2-AI derivatives, we raised the question as
to whether or not this metabolite (2d) would display anti-biofilm
properties against more medically relevant bacteria. Furthermore,
if this was the case and compound 2d was active against medically
relevant bacteria, would the synthesis and screening of a more
structurally diverse library of 2d analogues provide potent anti-
biofilm compounds? We were particularly eager to investigate a
library of analogues based on 2d, due to their relative structural
simplicity and ease of synthesis and purification as compared to
our 2-aminoimidazole-based modulators. Presented herein is an
account of the results of this pilot study.
Introduction
Bacterial biofilms are defined as surface-adhered communities of
bacteria encased in an extracellular matrix of biomolecules.1 This
particular phenotype of bacterial growth is incredibly resilient
to conventional antibiotics, antiseptics, and host defences.2 In
fact, biofilms of medically relevant bacteria constitute more than
80% of all bacterial infections.3 Additionally, biofilms have been
implicated in the persistence of infections of indwelling medical
devices,4 and are responsible for the mortality and morbidity of
all cystic fibrosis (CF) patients.5
Despite the preponderance of severe medical conditions that
are influenced by bacterial biofilms, there exists a relative dearth
of small, drug-like molecular scaffolds that affect their forma-
tion and maintenance.6 Examples of various compound classes
known to possess anti-biofilm activity include homoserine lactone
derivatives,7 brominated furanones,8 and ursine triterpenes.9 Ad-
ditionally, high throughput screening approaches10 and computer
aided drug design methods11 have also resulted in the discovery of
a few novel scaffolds that possess anti-biofilm activity.
Results and discussion
Ethyl N-(2-phenethyl) carbamate 2d was synthesized from com-
mercially available materials by routine acylation methodology
(ethyl chloroformate/TEA in DCM) (Scheme 1). Compound 2d
was isolated in 96% yield without recourse to chromatographic
purification.
Our group has had marked success in the development of
novel molecular scaffolds that can both inhibit and disperse
bacterial biofilms across order, class, and phylum. Our unifying
strategy towards the development of these molecules has been
through the systematic design and optimization of structural
motifs embedded within the core structure of the marine natural
product bromoageliferin.12
Scheme 1 Preparation of metabolite 2d and library design.
With the aim of introducing a new class of molecules possessing
potent anti-biofilm activity, we sought to evaluate a library
of analogues based upon the bacterial metabolite ethyl N-(2-
phenethyl) carbamate (2d), isolated from the marine bacteria
SCRC3P79 (Cytophaga sp.).13 A. Yamada et al. reported that
2d exhibited moderate antibiofilm activity against the marine
a-proteobacteria Rhodospirillum salexigens. Yamada performed
preliminary analogue synthesis by varying the aromatic appendage
with substituted benzene rings and the ethyl appendage with a
Similar to Yamada et al., we found that 2d displayed mediocre
antibiofilm activity against R. salexigens, giving a 59.7% in-
hibition at a 200 mM concentration as judged by a crystal
violet reporter assay.14 Interestingly, a 200 mM concentration of
2d also displayed activities against various medically relevant
bacterial strains, inhibiting 63.1%, 68.1%, 80.2%, 52.0% and
40.8% of biofilm formation for S. epidermidis, methicillin-resistant
S. aureus (MRSA), vancomycin-resistant Enterococcus faecium
(VRE), multi-drug resistant Acinetobacter baumannii (MDRAB),
and E. coli respectively (Table 1).
Department of Chemistry, North Carolina State University, 2620 Yarbrough
Dr., Raleigh, NC, 27695-8204, USA. E-mail: christian_melander@
ncsu.edu; Fax: +1 919-515-5079; Tel: +1 919-513-2960
† Electronic supplementary information (ESI) available: Experimental
details for synthesis and biology and analytical data for all compounds.
See DOI: 10.1039/c0ob00063a
After successfully obtaining antibiofilm activity for 2d against
medically relevant bacteria, a three part structure–activity analysis
was designed (see Scheme 1), which entailed a systematic modula-
tion of the metabolite’s aromatic head region, carbamate linkage,
and tail group.
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Table 1 Biofilm inhibition activity of 2d against various bacteria
Table 2 Biofilm inhibition (IC50) against MRSA and E. coli
Strain
% Inhibition (200 mM 2d)
Compound
MRSA IC50/mM
E. coli IC50/mM
S. epidermidis
MRSA
VRE
R. salexigens
MDRAB
E. coli
63.1
68.1
80.2
59.7
52.0
40.8
5a
8a
9a
10a
10c
—
28.3
—
34.6
—
49.8
4.87
4.70
—
66.4
R. salexigens, MDRAB and E. coli at a 200 mM concentration.
None of the compounds displayed notable antibiofilm activity
against S. epidermidis, VRE, R. salexigens, or MDRAB. Never-
theless, compounds 4c, 8a, 9a, and 10a displayed greater than
90% inhibition of MRSA biofilms at 200 mM concentration.
Furthermore, compounds 5a, 9a, and 10c exhibited greater than
80% inhibition of E. coli biofilms at 200 mM concentration.
Dose-response curves were generated for the lead compounds
for the inhibition of MRSA and E. coli biofilms (Table 2).
Non-bactericidal antibiofilm activity was verified through colony
count analysis of the planktonic viability in the presence and
in the absence of each compound at their IC50 value (i.e. the
concentration that inhibits 50% of biofilm formation, see ESI
for details†). Against MRSA, IC50 values were determined to be
49.8 mM, 4.87 mM and 4.70 mM for 8a, 9a, and 10a respectively.
Against E. coli, IC50 values were determined to be 28.3 mM,
34.6 mM and 66.4 mM for 5a, 9a, and 10c respectively.
The natural product analogues were synthesized using the same
method used to prepare 2d. Specifically, the respective amine
was reacted with 0.9 equivalents of the requisite chloroformate,
isocyanate, dicarbonate, or isothiocyanate in the presence of 2.0
equivalents of triethylamine in dichloromethane (see ESI for
details†). Each of the listed amines was reacted independently
with each acylating reagent to produce an 88 member pilot library
in yields ranging from 76–98%. Various aromatic head groups
were used, incorporating the indole, triazole, indane, tetrahydro-
quinoline, indoline, and pyridine, as well as para-amino, para-
methoxy, and para-bromo substituted phenyl rings. The carbamate
heteroatomic core was varied through the substitution with a
thiocarbamate, urea, and thiourea linkages. Tail modifications
were made through the incorporation of the (-)-menthyl, benzyl,
t-butyl and cholesteryl groups (Scheme 2).
Given the potency of our lead compounds toward inhibiting
MRSA biofilms, we next explored their activity against various
other S. aureus strains. Specifically, we screened 8a, 9a, and 10a
against three additional S. aureus strains (ATCC #’s 29213, 29740,
and 25923). IC50 values were determined for each compound
against each of the S. aureus strains; in some cases, the compounds
were found to be more potent than they were against MRSA.
IC50 values for compound 8a were found to be 21.2 mM, 24.3 mM
and 71.9 mM against 29213, 29740 and 25923 respectively. For 9a
they were found to be 124 mM, 82.2 mM and 19.7 mM for 29213,
29740 and 25923 respectively. Lastly, for compound 10a, which
was found to be the most active compound overall, IC50 values
were determined to be 4.70 mM, 2.84 mM and 37.4 mM for 29213,
29740 and 25923 respectively (Table 3). Again, planktonic viability
in the presence of the test compounds was verified through colony
count analysis.
Interestingly, our most potent inhibitors of the S. aureus strains
including MRSA contained (-)-menthyl carbamates. Indeed, (-)-
menthol and its derivatives have long been shown to have various
antimicrobial and antiplasmid effects on bacteria.15 Along with
(-)-menthol (13), the related natural products thymol (14) and
carvacrol (15) (Scheme 3, dashed box) are also known to possess
antimicrobial activity.16 In light of this observation, we prepared
the thymyl and carvacryl carbamate analogues of 9a and 10a.
We chose these two compounds for analogue design because
they had the lowest IC50 values against MRSA and both worked
well against 29213, 29740, and 25923 (see Tables 2 and 3).
Additionally, we prepared the stereochemical antipodes of 9a and
10a by employing (+)-menthyl carbamate. Finally, we prepared
the cyclohexyl carbamate derivatives of 9a and 10a as a control
(Scheme 3).
Scheme 2 Analogue library.
Once 2a–12h had been synthesized, they were screened for their
ability to inhibit biofilm formation of S. epidermidis, MRSA, VRE,
With compounds 9i–10l in hand, they were then screened
for biofilm inhibition activity along with (-)-menthol and
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Table 3 Biofilm inhibition (IC50) against other S. aureus strains
as well as marrying this novel menthyl carbamate motif with our
2-aminoimidazole compounds. The results of these studies will be
reported in due course.
Compound
29213 IC50/mM
29740 IC50/mM
25923 IC50/mM
8a
9a
10a
21.2
124
4.70
24.3
82.2
2.84
71.9
19.7
37.4
Acknowledgements
The authors would like to thank the University of North Carolina
General Administration Competitive Research Fund, the V Foun-
dation for a Predoctoral Jimmy V Scholar award (S.A.R.) and the
NCSU Office of Undergraduate Research for an undergraduate
research fellowship (T.M.).
Notes and references
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Scheme 3 Analogues of compounds 9a and 10a.
(-)-menthol methyl ether against MRSA and 29213. Interestingly,
none of the analogues depicted in Scheme 3 displayed any notable
biofilm inhibition activity against either MRSA or 29213 at a
200 mM concentration, with the exception of 9k and 10k. These
compounds were found to have identical antibiofilm properties as
their enantiomers, 9a and 10a. Importantly, both (-)-menthol and
(-)-menthol methyl ether were found to be completely inactive.
Lastly, 2d, 8a, 9a, and 10a were preliminarily screened for
cytotoxicity. This was assessed using a red blood cell hemolysis
assay using difibrinated sheep blood. In each case, the carbamates
were found to show no red blood cell lysis up to the highest
concentration tested (1.2 mM, see ESI for details†).
In summary, by targeting analogues of the bacterial metabolite
2d, we have discovered a novel class of biofilm inhibitors based
upon a menthyl carbamate scaffold. The culmination of this
study resulted in two potent compounds (9a and 10a) that
display low micromolar IC50 values for the inhibition of various
S. aureus biofilms including those from the medically relevant
MRSA. This scaffold represents a unique new class of compounds
for combating bacterial biofilms. Although they currently lack the
ability to disperse preformed biofilms, this disadvantage is off-
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agents that have low IC50 values were needed to completely
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equilibrium of the biofilm development cycle in that planktonic
cells will always continue to form films as long as they are viable.
We have recently demonstrated that employing a combination
therapy of antibiofilm agents with antibiotics is more effective at
completely alleviating the biofilm source since the planktonic cells
are constantly being eliminated from the equilibrium.[17] Current
efforts in our labs are focused on further tuning this new scaffold
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The Royal Society of Chemistry 2010
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