Design, synthesis and biological evaluation of non-natural modulators of quorum sensing in Pseudomonas aeruginosa

Article abstract of DOI:10.1039/c2ob25198a

Many species of bacteria employ a mechanism of intercellular communication known as quorum sensing which is mediated by small diffusible signalling molecules termed autoinducers. The most common class of autoinducer used by Gram-negative bacteria are N-acylated-l-homoserine lactones (AHLs). Pseudomonas aeruginosa is a clinically important bacterium which is known to use AHL-mediated quorum sensing systems to regulate a variety of processes associated with virulence. Thus the selective disruption of AHL-based quorum sensing represents a strategy to attenuate the pathogenicity of this bacterium. Herein we describe the design, synthesis and biological evaluation of a collection of structurally novel AHL mimics. A number of new compounds capable of modulating the LasR-dependent quorum sensing system of P. aeruginosa were identified, which could have value as molecular tools to study and manipulate this signalling pathway. Worthy of particular note, this research has delivered novel potent quorum sensing antagonists, which strongly inhibit the production of virulence factors in a wild type strain of this pathogenic bacterium.

Full text of DOI:10.1039/c2ob25198a

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PAPER
Design, synthesis and biological evaluation of non-natural modulators of
quorum sensing in Pseudomonas aeruginosa†‡
James T. Hodgkinson,a Warren R. J. D. Galloway,a Megan Wright,a Ioulia K. Mati,a Rebecca L. Nicholson,a
Martin Welchb and David R. Spring*a
Received 26th January 2012, Accepted 6th March 2012
DOI: 10.1039/c2ob25198a
Many species of bacteria employ a mechanism of intercellular communication known as quorum sensing
which is mediated by small diffusible signalling molecules termed autoinducers. The most common class
of autoinducer used by Gram-negative bacteria are N-acylated-L-homoserine lactones (AHLs).
Pseudomonas aeruginosa is a clinically important bacterium which is known to use AHL-mediated
quorum sensing systems to regulate a variety of processes associated with virulence. Thus the selective
disruption of AHL-based quorum sensing represents a strategy to attenuate the pathogenicity of this
bacterium. Herein we describe the design, synthesis and biological evaluation of a collection of
structurally novel AHL mimics. A number of new compounds capable of modulating the LasR-dependent
quorum sensing system of P. aeruginosa were identified, which could have value as molecular tools to
study and manipulate this signalling pathway. Worthy of particular note, this research has delivered novel
potent quorum sensing antagonists, which strongly inhibit the production of virulence factors in a wild
type strain of this pathogenic bacterium.
Quorum sensing is mediated by small diffusible signalling
Introduction
molecules termed autoinducers that are synthesized intracellu-
larly are released into the surrounding milieu.1 The reliance of
Quorum sensing is a mechanism of intercellular communication
employed by numerous species of bacteria.1–3 This process
quorum sensing upon this ‘language’ of small molecules pro-
allows the cells comprising a bacterial colony to coordinate their
genome expression in a cell-density dependent manner, thus
facilitating population-dependent adaptive activity.1,4–7 The
behaviours regulated by quorum sensing are extremely diverse in
nature, with many playing critical roles in the mediation of both
pathogenic and symbiotic bacteria–host interactions.1,8,9 For
example, root nodulation is a significant quorum sensing-depen-
dent phenotype in symbiots and several clinically relevant patho-
gens use quorum sensing to control processes associated with
virulence.1,10–14 Given the large variety of behaviours regulated
by quorum sensing and the widespread impact of these upon
healthcare, the environment and agriculture, it is unsurprising
that there has been significant interest in further understanding
this form of intercellular communication.1,15,16
vides chemical opportunity to investigate these signalling path-
ways at a molecular level.1,15,17 Indeed, recent years have
witnessed significant efforts directed towards the discovery of
non-native compounds that can modulate quorum sensing
systems, with either agonist or antagonist activity.1,15
The most common class of autoinducer used by Gram-
negative bacteria are N-acylated-L-homoserine lactones
(AHLs).1–3,15,18 Most natural AHLs share conserved structural
characteristics, namely a homoserine lactone ring (the ‘head
group’) unsubstituted at the β and γ positions which is N-acy-
lated at the α position with an acyl group (the ‘tail group’).1,19 In
the majority of Gram-negative bacterial species, AHL-based
quorum sensing typically involves the same series of events. The
AHL is generated by an enzyme (a LuxI-type synthase) where-
upon it freely diffuses out of the cell. Above a certain threshold
concentration, the AHL binds to its cognate cytoplasmic receptor
(a LuxR-type transcriptional regulator). The resulting AHL–
LuxR-type protein complex then modulates the expression of
genes associated with bacterial group processes.1,3,15,20 Homo-
logues of LuxI and LuxR have been identified in a large number
of bacterial genomes; in general each bacterial species responds
specifically to its own unique AHL(s), with different LuxI-type
synthase and LuxR-type receptors employed.1,9
aDepartment of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, UK. E-mail: spring@ch.cam.ac.uk; Fax: +44 (0) 1223-
336362; Tel: +44 (0) 1223-336498
bDepartment of Biochemistry, University of Cambridge, Cambridge, UK
†This article is part of the Organic & Biomolecular Chemistry 10th
Anniversary issue.
‡Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra for most active antagonists. Biological screening data. See
DOI: 10.1039/c2ob25198a
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Fig. 1 (A) Natural AHL molecules employed by P. aeruginosa in quorum sensing. (B) Some examples of AHL-mimics capable of modulating
quorum sensing in P. aeruginosa which contain modifications in the head and tail groups. 1 (Smith et al.42) and 2 (Marsden et al.43) are inhibitors of
LasR mediated quorum sensing. 3 and 4 are agonists of RhlR-mediated signalling (Lee et al.44 and Glansdorp et al.45). 5–9 modulate the activity of
LuxR homologue mediated quorum sensing in P. aeruginosa: 5 and 6 are LasR antagonists, 7 and 8 are LasR agonists and 9 is a RhlR agonist (Smith
et al.27,42). 10 and 11 are potent antagonists against LasR (Geske et al.46).
The Gram-negative bacterium Pseudomonas aeruginosa is a
clinically important pathogen, associated with a range of life-
threatening hospital-acquired infections and a common cause of
mortality in cystic fibrosis patients.21–26 P. aeruginosa uses (at
least) three different types of quorum sensing systems, two of
which are AHL-based.1,27–29 Each AHL system involves a dis-
tinct AHL molecule, LuxI-type synthase LuxR-type receptor. N-
(3-Oxododecanoyl)-L-homoserine lactone (OdDHL) is generated
by LasI with LasR being the cognate receptor. N-Butanoyl-L-
homoserine lactone (BHL) is generated by RhlI and is detected
by the RhlR protein.1,27–29 These AHL-dependent systems are
interlinked with a third system employing a chemically distinct
autoinducer (termed the Pseudomonas quinolone signal,
PQS23,24), forming an intricate hierarchical signaling
network.1,13,29 The Las system is considered to stand at the top,
with LasR-OdDHL positively regulating the Rhl and PQS sig-
nalling systems;1,30–35
P. aeruginosa is known to use quorum sensing to control a
variety of processes associated with virulence.1,21,29,36 For
example the Las system regulates the production of the virulence
factors elastase, alkaline protease and exotoxin A37 and the Rhl
system regulates rhamnolipid production (a rhamnose-based bio-
surfactant) and the virulence factors hydrogen cyanide and pyo-
cyanin. The Rhl system is also required for optimal production
of elastase and alkaline protease.38 Thus the selective disruption
of AHL-based quorum sensing using non-native small molecules
represents a strategy to attenuate the virulence of the bacterium,
allowing the host immune system a better chance of clearing the
infection before the bacteria cause too much tissue
damage.1,15,23,24,29 Indeed, there is proof-of-concept from animal
studies that the virulence of P. aeruginosa can be partially
attenuated in vivo by the inhibition of quorum sensing.1,39,40
P. aeruginosa is well known for its low susceptibility to anti-
biotics and antibiotic resistant strains of P. aeruginosa are widely
reported; thus novel methods to inhibit its virulence would be of
significant clinical value.41
Most work on the small molecule modulation of AHL-based
quorum sensing in Gram negative bacteria has focused upon the
identification of compounds that can interact with the LuxR-type
receptor proteins.1 Given the fact that the Las system is generally
considered to be located at the top of the quorum sensing hierar-
chy in P. aeruginosa (vide supra), it is unsurprising that the
LasR receptor is usually the main target for activator or inhibitor
development in this bacterium.1,17 Indeed, in recent years a
range of non-natural compounds capable of modulating LasR-
mediated quorum sensing via interaction with this receptor
protein have been reported.1,15 Synthetic agents targeting the
BHL-RhlR signalling system are also known.1,15 Many of these
Las- and Rhl-system modulators are based upon the structure of
the natural AHL signalling molecules, OdDHL and BHL, with
modifications in either the ‘head’ or ‘tail’ portions1,15,29 (Fig. 1).
For example, the Suga group has identified several non-natural
agonists and antagonists of natural AHL binding to LasR in
which the head group of OdDHL had been replaced with an aro-
matic group (e.g. 1, Fig. 1).42 Recently, our own group has
reported the discovery of a novel chloro-pyridine pharmacophore
that can be used in the place of the native homoserine lactone
moiety.43 For example, compound 2 was found to be an inhibitor
of a LasR mediated quorum sensing phenotype in P. aeruginosa
(Fig. 1). OdDHL and BHL analogues bearing cyclic carbocycles
(e.g. cyclopentanone, cyclohexanol) as head group replacements
have also been reported to be capable of modulating LasR and
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RhlR mediated quorum sensing in this bacterium (3–9,
Fig. 1).27,28,42,44 The Blackwell group have identified a range of
non-native AHL analogues containing modifications in the tail
region which were capable of modulating the activity of the
LasR receptor in P. aeruginosa. Of particular interest, the incor-
poration of aromatic functionalities in the tail section (e.g. 4-
bromo benzene in 10 and indole in 11, Fig. 1) generally resulted
in analogues with inhibitor activity.16,46 It has also been shown
that the central amide connective function of AHLs (the ‘amide
linker’, Fig. 1) represents a suitable target for chemical modifi-
cation.3 For example, Doutheau and co-workers have reported a
range of synthetic modulators of LuxR-based quorum sensing in
Vibrio fischeri in which the amide function has been replaced
with various non-native moieties.47–49
either the natural head or tail portions of the native AHL auto-
inducer. We were therefore interested in carrying out exploratory
studies into the quorum sensing modulatory activity of a small
set of ‘chimeric’ compounds in which both the natural head and
tail sections had been replaced with non-native structural moi-
eties of proven biological relevance (that is non-native head or
tail sections from molecules which are known to modulate
quorum sensing).50 Specifically, the exploratory studies
described in this report focused upon a small collection of com-
pounds of the general form 13 that contained a (hetero)aromatic
or carbocyclic head group together with a bromo benzene or
indole tail. Through this work a series of structurally novel
modulators of LasR-mediated quorum sensing in P. aeruginosa
were identified. In particular, new potent antagonists which were
capable of strongly inhibiting the formation of virulence factors
were discovered. Overall, the data obtained from these studies
provide further insights into the molecular features required for
small molecule modulation of LasR-mediated quorum sensing in
P. aeruginosa which should provide valuable information for the
rational design of next-generation chemical tools to study and
manipulate this system.1,3,51
Inspired by our results in this field,43–45 and those obtained by
the research groups of Blackwell46 and Suga,27,42 we sought to
design new AHL-mimics incorporating non-native head and tail
structural moieties which were capable of modulating quorum
sensing in P. aeruginosa. This would expand the set of chemical
tools available for the study and manipulation of this form of
intercellular communication in this bacterium. In particular,
structurally novel antagonists would be of interest from a thera-
peutic perspective. Herein we describe the design, synthesis and
biological evaluation of novel AHL mimics based upon two
general structural frameworks (Fig. 2). Guided by previous
results from Suga and our own research group, one set of com-
pounds (of the general form 12) was based around the structures
of OdDHL and BHL in which the natural acyl chains were
retained but the homoserine lactone head group was substituted
with non-natural aromatic and heteroaromatic moieties.
Results and discussion
Synthesis of AHL-mimics with (hetero)aromatic head groups
Mimics of BHL (14–17) were readily prepared by the direct
coupling of butyryl chloride (18) with a range of (hetero)aro-
matic amines 19 (Scheme 1). OdDHL-mimics which incorpor-
ated an (hetero)aromatic head group (compounds 20–23) were
generated by modification of or previously reported synthetic
route towards natural beta-ketoamide AHLs (Scheme 2).52 Reac-
tion of Meldrum’s acid (24) with decanoyl chloride (25) gener-
ated adduct 26. Treatment of this crude material with methanol
furnished methyl 3-oxododecanoate (27). The ketone group was
then protected as an acetal to form 28 and subsequent ester
hydrolysis yielded acid derivative 29. DCC-mediated coupling
of this key intermediate with a range of aromatic amines (30)
furnished ketone-protected amide derivatives 31. Subsequent
Most studies on the small molecule modulation of AHL-based
quorum sensing have employed compounds which retained
Fig. 2 General structures of target AHL mimics with head and tail
modifications.
Scheme 1 Synthesis of BHL mimics with a (hetero)aromatic head
group.
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Scheme 2 Synthesis of OdDHL mimics with an aromatic head group. Note that 23 has previously been reported by the Suga group.40
Scheme 3 Synthesis of chimeric AHL mimics. DMP = Dess–Martin periodinane.
TFA-mediated deprotection yielded the final OdDHL-mimics
20–23.
phenylacetyl tail group and a (hetero)aromatic head group, were
prepared in two steps by the coupling of readily synthesised 4-
bromophenylacetyl chloride (34) with the appropriate (hetero)-
aromatic amine (35 and 36 respectively, Scheme 3) under Schot-
ten–Baumann conditions. Alternatively, reaction of chloride 34
with the amino alcohols 37 or 38 furnished compounds 39 and
40 respectively which contained a bromophenylacetyl tail group
with a carbocyclic head group. Oxidation using Dess–Martin
periodinane then generated the corresponding keto-derivatives
41 and 42. Compound 43, which incorporates an indole-functio-
nalised tail group and an aromatic head group, could be readily
Synthesis of ‘chimeric’ AHL mimics with head and tail
modifications
We were interested in carrying out some preliminary exploratory
studies on the quorum sensory modulatory activity of a small set
of ‘chimeric’ AHL mimics which incorporated a (hetero)aro-
matic or carbocyclic head group together with a bromo
benzene or indole tail. Compounds 32–33 containing a bromo
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accessed by the DCC-mediated coupling of indole-3-butyric acid
(44) with the aromatic amine 45 (Scheme 4).
PAO-JP1 was grown for 12 h in the presence of one of the syn-
thetic AHL mimics (10 μM concentration) added at the start of
the growth curve in the place of OdDHL. Agonists of the LasR
receptor would be expected to increase the amount of pyocyanin
present at the end of the growth period relative to that observed
in the negative control where no OdDHL was present. None of
the compounds examined were found to have significant agon-
istic activity, though there was evidence that some (23, 16, 14
and 32) could be partial agonists (partial efficacy relative to the
OdDHL, the full agonist).
Biological screening
Evaluation of compounds for LasR agonistic activity. Com-
pounds were evaluated for LasR agonism by measuring their
effect upon the production of the virulence factor pyocyanin by a
lasI mutant of P. aeruginosa (PAO-JP1).53 This strain does not
contain a functional LasI synthase and thus cannot produce
endogenous OdDHL. It has been shown that lasI mutants are
compromised with regards to pyocyanin production.54,55 The
lasI mutant retains the LasR receptor and therefore pyocyanin
production would be expected to be stimulated by the addition
of exogenous OdDHL.
PAO-JP1 cells were grown for 13 h in the presence or absence
of exogenously-supplied OdDHL (10 μM) added at the start of
the growth period (positive and negative controls respectively)
and the amount of pyocyanin produced measured (Fig. 3). As
expected, a much larger quantity of pyocyanin was present in
cells grown in the presence of OdDHL, the natural agonist of
LasR. These values were compared with those obtained when
Evaluation of compounds for LasR antagonistic activity.
Small molecules that antagonize quorum sensing are commonly
screened using tailored bacterial reporter strains. Typically in
such systems, expression of an easily-assayable output (e.g.,
lacZ-encoded β-galactosidase) is driven from a LuxR-dependent
promoter. Usually, elsewhere on the same construct, luxR is
engineered to be expressed constitutively. When such a reporter
construct is introduced into a luxI-genetic background, transcrip-
tion of the reporter gene (lacZ) becomes entirely dependent on
the addition of exogenous AHLs. In antagonist screening trials,
the cognate AHL is added at a fixed concentration (usually, just
enough to stimulate robust expression of the reporter gene) and
antagonist molecules are identified by their ability to compete
with the cognate AHL and reduce reporter gene expression.
Such a system offers the clear advantage of being defined.
However, the disadvantage is that it does not faithfully mimic
the situation in wild-type cells, where the endogenous AHL is
continually produced and can therefore more effectively “out-
compete” any added antagonist. This is why many antagonists
that appear extremely potent when identified using engineered
reporter systems often fail to elicit the anticipated response when
Scheme 4 Synthesis of AHL mimic 43 with an indole head group.
Fig. 3 Stimulation of pyocyanin activity by AHL analogues. Cultures of the P. aeruginosa strain PAO-JP1 were grown in Luria broth (LB) medium
in the presence of OdDHL (10 µM) or the indicated AHL analogues (10 µM) with good aeration at 37 °C for 13 h (initial OD600 of 0.05 t = 0). After
growth pyocyanin production was quantified as previously described.56 The amount of pyocyanin produced by each culture in the presence of the
AHL analogues was compared directly to the pyocyanin produced in the presence of OdDHL. No effect on growth was observed for any of the ana-
logues (see ESI†). DMSO (−) was added as a control. The data represent the averages and standard deviations from the results of 3 independent bio-
logical repeats.
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tested in wild-type cells. In the current work, we therefore chose
to screen our small molecules by looking for inhibition of
quorum sensing-dependent phenotypes in wild-type cells.
Specifically, the compounds were initially evaluated for LasR
antagonism by measuring their effects upon pyocyanin pro-
duction by the wild type P. aeruginosa strain PAO1. Cells were
grown for 13 h in the presence of a synthetic AHL mimic (at a
concentration of 200 μM) added at the start of the growth period.
The amount of pyocyanin present was measured and compared
with that obtained when the cells were grown for 13 h in the
absence of any synthetic AHL mimic; antagonists of the LasR
receptor would be expected to reduce the quantity of pyocyanin
produced compared (Fig. 4).
The ‘chimeric’ compounds (which contained both non-native
head and tail regions) were found to be largely inactive (32,
39–43) or only moderately active (33). Compounds which
retained the acyl chain (tail) of the native signalling molecule of
the Rhl-system, BHL, were typically found to display only slight
antagonistic effects (14–17). In contrast, the majority of com-
pounds which contained the tail region found in OdDHL
(specifically compounds 20, 21 and 22) were found to have
strong antagonistic activities without affecting cell growth. In
particular, compounds 22 and 20 inhibited the production of
pyocyanin by 73 and 93% respectively. These findings corrobo-
rate those of the Greenberg,57 Suga27,42 and Blackwell9 groups
in which structurally related OdDHL analogues bearing aromatic
head groups were reported to be LasR antagonists.58 Taken
together, our data strongly suggest that the natural 3-oxo-dodeca-
noyl tail group of OdDHL is important for the LasR modulatory
activity of compounds which are based around the AHL scaffold
which is in agreement with other studies.1,9 A comparison with
literature data suggests that the presence of the native OdDHL
acyl chain may be especially important for inducing strong LasR
antagonism when non-native head groups are present.59 For
example, compound 10, which has a non-native tail region and
native head group has been reported to be a potent LasR antag-
onist.46 On the other hand the chimeric compounds examined in
this study which contained the same tail group (32–33, 39–42)
displayed only moderate levels of activity.60 Indeed, the rela-
tively poor results obtained with such compounds containing
both abiotic head and tail moieties imply that one cannot gener-
ate potent antagonists simply by making chimeras of previously
identified active agents without consideration of other factors.
There are some interesting additional observations. Compound
23 was found to be much less active than 20 despite being very
similar in molecular structure. This is in agreement with previous
work by the Suga group, in which 23 was reported to have no
antagonist activity upon P. aeruginosa (in a quorum sensing con-
trolled reporter gene assay).42 The authors postulated that, in the
case of OdDHL mimics containing the native acyl chain and aro-
matic head groups, a hydrogen bond acceptor is required in
either the meta or para position for antagonistic activity, with the
exact position depending on the nature of the substituent; in the
case of a hydroxyl group, ortho placement is required. Suga and
co-workers have suggested that this substituent is involved in a
key hydrogen bonding interaction within the binding pocket of
the LasR protein, a role which is presumably fulfilled by the car-
bonyl group of the homoserine lactone moiety in the native auto-
inducer OdDHL.42 The high antagonistic activities of methoxy
analogues 22 and 20 are broadly consistent with this model;
Fig. 4 Antagonist activity of AHL analogues on pyocyanin production in PA01. Cultures of PA01 were grown in Luria broth (LB) medium in the
presence of AHL analogues (200 µM) with good aeration at 37 °C for 13 h (initial OD600 of 0.05 t = 0). After growth pyocyanin production was quan-
tified as previously described.56 No effect on growth was observed for any of the analogues (see ESI†). DMSO (−) was added as a control. The data
represent the averages and standard deviations from the results of 3 independent biological repeats.
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methoxy groups are capable of acting as hydrogen bond accep-
tors and the higher antagonism activity of 22 could possibly be
attributed to a more favourable positioning of the methoxy group
in the LasR binding pocket for formation of the hydrogen
bonding interaction. However, the relatively high antagonistic
activity of compound 21 would not be predicted on the basis of
these binding requirements; 21 contains an iodine atom in the
meta position of the aromatic ring which would not be expected
to be capable of forming hydrogen bonding interactions. If one
assumes that 20–22 act as antagonists by targeting the same site
of the LasR receptor (and that the binding mode of these com-
pounds is the same as the native ligand OdDHL), than the antag-
onistic activity of 21 corroborates recent observations by the
Blackwell group which question the proposed critical nature of a
hydrogen bonding interaction between the head group of AHLs
and AHL mimics and the LasR receptor protein for binding.9 In
addition, it has been suggested that π–π stacking or hydrophobic
interactions could play key roles in the binding of aromatic-type
AHL mimics to the LasR receptor.9
Cells grown in the presence of the two most active antagonists
(20 and 22) were also assayed for the amount of elastase present
(Fig. 5). Elastase production is regulated by the las system (and
to a lesser extent, also by the rhl system); antagonists of LasR-
mediated quorum sensing would therefore be expected to
decrease the amount of elastase produced in a given time period.
Both 20 and 22 were found to be antagonists by this assay, with
20 identified as the most active. These results mirror the relative
LasR-antagonistic activities identified by the pyocyanin assay.
Fig. 5 Antagonist activity of AHL analogues 20 and 22 on elastase pro-
duction in PA01. Cultures of PA01 were grown in the presence of the
AHL analogues (200 µM) in LB medium with good aeration at 37 °C for
7 h (initial OD600 of 0.05 t = 0). No effect on growth was observed for
any of the analogues (see ESI†). DMSO (−) was added as a control. The
amount of secreted elastase present in the culture supernatant was quan-
tified as previously described61 with minor modifications (see ESI†).
DMSO (−) was added as a control. The data represent the averages and
standard deviations from the results of 3 independent biological repeats.
Conclusions
Herein we have described the design and synthesis of a collec-
tion of structurally novel AHL mimics incorporating various
non-native head and tail moieties. Biological evaluation of these
compounds led to the identification of a number of analogues
capable of modulating LasR-based quorum sensing in the patho-
genic bacterium P. aeruginosa. Thus, through this work the set
of chemical tools available for the study and manipulation of this
method of intercellular communication has been expanded. In
terms of agonism, only weakly active compounds were ident-
ified; arguably of more interest are the results obtained in the
context of antagonism. The inhibition of quorum sensing in P.
aeruginosa using non-natural small molecules has been ident-
ified as an attractive alternative approach to the use of traditional
antibiotics for the treatment of infections caused by this organ-
ism.1 Three compounds with significant LasR-antagonistic
activity in P. aeruginosa were discovered (20–22), none of
which were found to be toxic to the host bacterium (Table 1).
More specifically, these compounds were able to strongly inhibit
the production of virulence factors that contribute to the patho-
genicity of this clinically-relevant pathogen in a wild type strain
of the organism.
Table 1 Summary of the activities of three most active antagonists
% Inhibitiona
Compound
Pyocyanin
Elastase
93
67
73
2
2
4
63
3
ND
34
4
In addition, some general SAR trends could be delineated in
the context of LasR antagonism. Compounds containing both
non-native head and tail regions were found to be largely inac-
tive and the highest levels of antagonist activity were observed
in compounds which contained the tail region found in OdDHL,
the native signalling molecule of the LasR-system. In addition,
a % Inhibition of virulence factor production as determined from the
data illustrated in Fig. 4 and 5. ND = not determined.
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our data corroborate recent observations which question the criti-
cal requirement of a hydrogen bonding interaction to the head
group of AHLs and AHL-mimics head group for strong LasR
binding.9 SAR data of this sort may facilitate the rational design
of de novo design of next-generation agents with improved
activities.1,62 Further analogue syntheses and SAR studies which
build on the results presented in this report are ongoing and the
results of this work will be reported in due course.
Bruker Avance 500 BB ATM (125 MHz) and Bruker Avance
500 Cryo Ultrashield (125 MHz). Chemical shifts (δC) are
quoted in ppm, to the nearest 0.1 ppm, and are referenced to the
residual non-deuterated solvent peak. Where appropriate, coup-
ling constants are reported in Hertz to the nearest 0.5 Hz and
data are reported as for proton magnetic resonance spectra
without integration. Assignments were supported by DEPT
editing and determined either on the basis of unambiguous
chemical shift or coupling pattern, by patterns observed in 2D
experiments (HMBC and HMQC) or by analogy to fully inter-
preted spectra for related compounds. LCMS spectra were
recorded on an HP/Agilent LCMS APCI 120-1000 full gradient
machine.
Experimental
General information
Reactions were performed using oven-dried glassware apparatus
under an atmosphere of nitrogen with anhydrous, freshly distilled
solvents unless otherwise stated. Dichloromethane, ethyl acetate,
methanol, n-hexane, acetonitrile and toluene were distilled from
calcium hydride. Diethyl ether was distilled over a mixture of
lithium aluminium hydride and calcium hydride. Petroleum ether
was distilled before use and refers to the fraction between
40–60 °C. All other reagents were used as obtained from com-
mercial sources. Room temperature refers to ambient tempera-
ture. Yields refer to chromatographically and spectroscopically
pure compounds unless otherwise stated. All flash chromato-
graphy was carried out using slurry-packed Merck 9325 Keisel-
gel 60 silica gel. Where possible, reactions were monitored by
thin layer chromatography (TLC) performed on commercially
prepared glass plates precoated with Merck silica gel 60 F254 or
aluminium oxide 60 F254. Visualisation was by the quenching
of UV fluorescence (νmax = 254 nm) or by staining with ceric
ammonium molybdate, potassium permanganate or Dragendorff ’s
reagent (0.08% w/v bismuth subnitrate and 2% w/v KI in 3 M
aq. AcOH). Infrared spectra were recorded neat or as a solution
in the designated solvent on a Perkin-Elmer Spectrum One spec-
trometer with internal referencing. Selected absorption maxima
(vmax) are reported in wavenumbers (cm−1). The abbreviation
“br” indicates a broad peak. Melting points were obtained using
a Büchi® melting point apparatus (model B-545) and are uncor-
rected. Proton magnetic resonance spectra were recorded using
an internal deuterium lock at ambient probe temperatures (unless
otherwise stated) on the following instruments: Bruker DPX-400
(400 MHz), Bruker Avance 400 QNP (400 MHz) Bruker Avance
500 BB ATM (500 MHz) and Bruker Avance 500 Cryo Ultra-
shield (500 MHz). Chemical shifts (δH) are quoted in ppm, to
the nearest 0.01 ppm, and are referenced to the residual non-
deuterated solvent peak. Coupling constants (J) are reported in
Hertz to the nearest 0.5 Hz. Data are reported as follows: chemi-
cal shift, integration, multiplicity [br, broad; s, singlet; d,
doublet; t, triplet; q, quartet; quint, quintet; sextet; sept, septet;
m, multiplet; or as a combination of these (e.g. dd, dt, etc.)],
coupling constant(s) and assignment. Proton assignments were
determined either on the basis of unambiguous chemical shift or
coupling pattern, by patterns observed in 2D experiments
(1H–1H COSY, HMBC and HMQC) or by analogy to fully inter-
preted spectra for related compounds. Carbon magnetic reson-
ance spectra were recorded by broadband proton spin decoupling
at ambient probe temperatures (unless otherwise stated) using an
internal deuterium lock on the following instruments: Bruker
DPX-400 (100 MHz), Bruker Avance 400 QNP (100 MHz) and
General procedure 1: coupling of acid chlorides with
(hetero)aromatic amines
Pyridine (1.1 equiv.) and butyryl chloride (1 equiv.) were added
to a solution of the appropriate (hetero)aromatic amine (1 equiv.)
in CH2Cl2 (∼1 mL per mmol amine) at room temperature. The
reaction mixture was stirred until TLC analysis indicated com-
plete consumption of the amine (∼18 h). The mixture was
diluted with CH2Cl2, washed with aqueous HCl (∼3 N solution),
dried (MgSO4) and the solvent removed under reduced pressure.
The crude product material was purified by column chromato-
graphy if required.
N-(2-Bromopyridin-4-yl)butyramide
(14). Prepared
by
general procedure 1 using 4-amino-2-bromopyridine (200 mg,
1.16 mmol), pyridine (0.10 mL, 1.24 mmol) and butyryl chlor-
ide (0.12 mL, 1.16 mmol). The crude product material was
purified by column chromatography (SiO2, Et2O) to yield the
title compound as an orange oil (55 mg, 20%). Rf 0.3 (SiO2;
diethyl ether); vmax (neat)/cm−1 1468, 1504, 1575, 1687 (amide),
2874, 2963, 3048, 3142, 3240; δH (400 MHz, CDCl3) 8.47 (1H,
s, NH), 8.12 (1H, d, J = 5.5 Hz, CHNCBr), 7.82 (1H, d, J = 1.5
Hz, NHCCHCBr), 7.42 (1H, dd, J = 5.5 Hz, 1.5 Hz,
NHCCHCHN), 2.33 (2H, t, J = 7.5 Hz, CH2CO), 1.67 (2H,
sextet, J = 7.5 Hz, CH2CH2CO), 0.91 (3H, t, J = 7.5 Hz, CH3);
δC (100 MHz; CDCl3) 172.2 (CvO (amide)), 150.3 (CHNCBr),
147.0 (NHC), 142.7 (CBr), 117.1 (CHCBr), 112.8 (CHCHN),
39.5 (CH2CO), 18.6 (CH2CH2CO), 13.6 (CH3); LCMS (ES+)
(MeCN) 243 (M+).
N-(3-Methoxyphenyl)butyramide (16). Prepared by general
procedure 1 using meta-anisidine (0.13 mL, 1.16 mol), pyridine
(0.10 mL, 1.24 mmol) and butyryl chloride (0.12 mL,
1.16 mmol). The crude product material was purified by column
chromatography (SiO2, 2 : 1 Et2O–pet. ether) to yield the title
compound as a brown oil (224 mg, 100%). Rf 0.3 (SiO2; 2 : 1
Et2O–pet. ether); vmax (neat)/cm−1 1427, 1454, 1492, 1543,
1598, 1661 (amide), 2963, 3301; δH (400 MHz, CDCl3) 7.13
(1H, s, NH), 7.36 (1H, s, NHCCHCOMe), 7.22 (1H, t, J = 8.0
Hz, NHCCHCH), 6.97 (1H, d, J = 8.0 Hz, NHCCHCH), 6.67
(1H, dd, J = 8.0 Hz, 2.0 Hz, NHCCHCHCH), 3.83 (3H, s,
OCH3), 2.35 (2H, t, J = 7.5 Hz, CH2CO), 1.79 (2H, sextet, J =
7.5 Hz, CH2CH2CO), 1.03 (3H, t, J = 7.5 Hz, CH3); δC
(100 MHz; CDCl3) 171.5 (CvO (amide)), 160.1 (COMe),
139.2 (NHC), 129.6 (NHCCHCH), 111.9 (NHCCHCHCH),
This journal is © The Royal Society of Chemistry 2012
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General procedure 3: acetal group removal
110.1 (NHCCHCOMe), 105.5 (NHCCHCH), 55.3 (OCH3), 39.7
(CH2CO), 19.0 (CH2CH2CO), 13.7 (CH3); LCMS (ES+)
(MeCN) 194 (M + H)+.
TFA (∼20 equiv.) was added to a solution of the protected dicar-
bonyl (1 equiv.) in CH2Cl2 (∼20 mL mmol−1) at room tempera-
ture. The reaction mixture was stirred at room temperature until
TLC analysis indicated complete consumption of starting
material (∼18 h). The solvent was removed under reduced
pressure and the crude product material purified by column
chromatography.
N-(3-Iodophenyl)butyramide (15). Prepared by general pro-
cedure 1 using 3-iodoaniline (0.14 mL, 1.16 mol), pyridine
(0.10 mL, 1.24 mmol) and butyryl chloride (0.12 mL,
1.16 mmol). The crude product material was purified by column
chromatography (SiO2, 1 : 1 Et2O–pet. ether) to yield the title
compound as a brown oil (335 mg, 100%). Rf 0.25 (SiO2; 1 : 1
Et2O–pet. ether); vmax (neat)/cm−1 1414, 1474, 1529, 1580,
1662 (amide), 2872, 2931, 2962, 3071, 3106, 3175, 3291; δH
(400 MHz, CDCl3) 7.14 (1H, s, NH), 7.73 (1H, s, NHCCHCI),
7.28 (1H, d, J = 7.5 Hz, NHCCHCHCH), 7.22 (1H, d, J = 7.5
Hz, NHCCHCH), 6.82 (1H, t, J = 8.0 Hz, NHCCHCH), 2.12
(2H, t, J = 7.0 Hz, CH2CO), 1.54 (2H, sextet, J = 7.5 Hz,
CH2CH2CO), 0.8 (3H, t, J = 7.5 Hz, CH3); δC (100 MHz;
CDCl3) 171.4 (CvO (amide)), 139.1 (NHC), 133.2
(NHCCHCHCH), 130.4 (NHCCHCH), 128.5 (NHCCHCI),
119.0 (NHCCHCH), 94.1 (CI), 39.6 (CH2CO), 18.9
(CH2CH2CO), 13.7 (CH3); LCMS (ES+) (MeCN) 290 (M +
H)+; m.p. 62–64 °C (1 : 1 Et2O–pet. ether).
N-(3-Methoxyphenyl)-3-oxododecanamide (20). The protected
analogue of the final product was prepared by general method 2
using 2-(2-nonyl-1,3-dioxolan-2-yl)acetic acid (29, 200 mg,
0.78 mmol), CH2Cl2 (5 mL), DCC (240 mg, 1.16 mmol),
DMAP (57 mg, 0.47 mmol) and meta-anisidine (0.09 mL,
0.78 mmol). The crude material was purified by column chrom-
atography (SiO2, 1 : 2 EtOAc–pet. ether) to yield a brown oil
(155 mg, 55%). A sample of this material (133 mg) was depro-
tected by general method 3 using 0.5 mL TFA and 5 mL
CH2Cl2. The crude material was purified by column chromato-
graphy (SiO2, 1 : 1 EtOAc–pet. ether) to give the title compound
as a white solid (63 mg, 54%). Rf 0.6 (SiO2; 1 : 1 EtOAc–pet.
ether); vmax (neat)/cm−1 1429, 1456, 1494, 1548, 1596, 1659
(amide), 1717 (ketone), 2854, 2924, 3293; δH (400 MHz;
CDCl3) 9.16 (1H, s, NH), 7.27 (1H, t, J = 2.0 Hz,
NHCCHCOMe), 7.21 (1H, t, J = 8.0 Hz, NHCCHCH), 7.03
(1H, dd, J = 8.0 Hz, 1.0 Hz, NHCCHCH), 6.66 (1H, dd, J = 8.0
Hz, 2.0 Hz, NHCCHCHCH), 3.80 (3H, s, OCH3), 3.54 (2H, s,
C(O)CH2CO), 2.56 (2H, t, J = 7.5 Hz, CH2CO), 1.69–1.53 (2H,
m, J = 7.0, CH2CH2CO), 1.31–1.25 (12H, br m, alkyl CH2),
0.87 (3H, t, J 7.0, CH3); δC (100 MHz; CDCl3) 208.0 (CvO
(ketone)), 163.4 (CvO (amide)), 160.1 (COMe), 138.7 (NHC),
N-(4-Methoxyphenyl)butyramide (17). Prepared by general
procedure 1 using para-anisidine (143 mg, 1.16 mol), pyridine
(0.10 mL, 1.24 mmol) and butyryl chloride (0.12 mL,
1.16 mmol). The crude product material was purified by column
chromatography (SiO2, 2 : 1 Et2O–pet. ether) to yield the title
compound as a brown oil (185 mg, 83%).
Rf 0.3 (SiO2; 2 : 1 Et2O–pet. ether); vmax (neat)/cm−1 1411,
1466, 1510, 1530, 1609, 1648 (amide), 2838, 2872, 2967, 3280;
δH (400 MHz, CDCl3) 7.19 (1H, s, NH), 7.42 (2H, dd, J = 6.5
Hz, 2.0 Hz, NHCCH), 6.87 (2H, dd, J = 6.5 Hz, 2.0 Hz,
NHCCHCH), 3.8 (3H, s, OCH3), 2.33 (2H, t, J = 7.5 Hz,
CH2CO), 1.77 (2H, sextet, J = 7.5 Hz, CH2CH2CO), 1.02 (3H, t,
J = 7.5 Hz, CH3); δC (100 MHz; CDCl3) 171.2 (CvO (amide)),
156.3 (COMe), 131.1 (NHC), 121.8 (NHCCHCH), 114.1
(NHCCHCH), 55.5 (OCH3), 39.4 (CH2CO), 19.1 (CH2CH2CO),
129.6
(NHCCHCH),
112.2
(NHCCHCHCH),
110.4
(NHCCHCOMe), 105.7 (NHCCHCH), 55.3 (OCH3), 48.8
(C(O)CH2CO), 44.2 (CH2C(O)CH2CO), 31.8 (CH2), 29.34
(CH2), 29.30 (CH2), 29.20 (CH2), 28.9 (CH2), 23.3 (CH2), 22.6
(CH2), 14.1 (CH3); LCMS (ES+) (MeCN) 320 (M + H)+.
N-(3-Iodophenyl)-3-oxododecanamide (21). The protected
analogue of the final product was prepared by general method 2
using 2-(2-nonyl-1,3-dioxolan-2-yl)acetic acid (29, 200 mg,
0.78 mmol), CH2Cl2 (5 mL), DCC (240 mg, 1.16 mmol),
DMAP (57 mg, 0.47 mmol) and 3-iodoaniline (0.09 mL,
0.78 mmol). The crude material was purified by column chrom-
atography (SiO2, 2 : 1 Et2O–pet. ether) to yield an orange oil
(257 mg, 72%). A sample of this material (237 mg) was depro-
tected by general method 3 using 0.7 mL TFA and 5 mL
CH2Cl2. The crude material was purified by column chromato-
graphy (SiO2, 1 : 2 Et2O–pet. ether) to give the title compound
as a white solid (168 mg, 78%). Rf 0.17 (SiO2; 1 : 2 Et2O–pet.
ether (30–40)); vmax (neat)/cm−1 1474, 1542, 1586, 1654
(amide), 1715 (ketone), 2850, 2916, 3284; δH (500 MHz,
CDCl3) 9.27 (1H, s, NH), 7.97 (1H, t, J = 2.0 Hz, NHCCHCI),
7.53–7.50 (1H, m, NHCCHCH), 7.45 (1H, ddd, J = 8.0 Hz, 1.5
Hz, 1.0 Hz, NHCCHCHCH), 7.04 (1H, t, J = 8.0 Hz,
NHCCHCH), 3.55 (2H, s, C(O)CH2CO), 2.57 (2H, t, J = 7.5
Hz, CH2CO), 1.65–1.59 (2H, m, CH2CH2CO), 1.29–1.21 (12H,
br m, alkyl CH2), 0.88 (3H, t, J = 7.0 Hz, CH3); δC (125 MHz;
CDCl3) 208.1 (CvO (ketone)), 163.5 (CvO (amide)), 138.6
13.7 (CH3); LCMS (ES+) (MeCN) 194 (M
m.p. 87–89 °C (2 : 1 Et2O–pet. ether).
+
H)+;
General procedure 2: coupling of 2-(2-nonyl-1,3-dioxolan-2-yl)-
acetic acid with (hetero)aromatic amines
DCC (1.5 equiv.) and DMAP (0.6 equiv.) were added to a sol-
ution of 2-(2-nonyl-1,3-dioxolan-2-yl)acetic acid (29, 1.0 equiv.)
in CH2Cl2 (∼5 mL per mmol 29) at room temperature. The reac-
tion mixture was stirred at room temperature for 20 min. The
appropriate (hetero)aromatic amine (1 equiv.) was added and the
reaction mixture stirred at room temperature until TLC analysis
indicated complete consumption of the starting acid (∼18 h).
The reaction mixture was filtered through a pad of Celite®
washing with CH2Cl2, the filtrate washed with aqueous HCl
(∼3 N solution), dried (MgSO4) and the solvent removed under
reduced pressure. The crude product material was purified by
column chromatography if required.
(NHC), 133.5 (NHCCHCHC
̲
6040 | Org. Biomol. Chem., 2012, 10, 6032–6044
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N-(3-Chlorophenyl)-4-(1H-indol-3-yl)butanamide (43). DCC
(303 mg) and DMAP (72 mg) were added to a solution of
indole-3-butyric acid (200 mg, 0.98 mmol) in anhydrous
CH2Cl2 (5 ml) under nitrogen at room temperature. The mixture
was stirred for 20 min at room temperature. 3-Chloroaniline
(0.1 ml, 0.98 mmol) was added and the reaction mixture stirred
for 12 h at rt. The reaction mixture was filtered through a pad of
Celite® washing with CH2Cl2, the filtrate washed with aqueous
HCl (∼3 N solution), dried (MgSO4) and the solvent removed
under reduced pressure. The crude product material was purified
by column chromatography (SiO2, Et2O) to yield the title com-
pound as a yellow oil (225 mg, 0.72 mmol, 73%). Rf 0.46 (SiO2,
Et2O); vmax (CDCl3)/cm−1 3403 (NH indole), 3301 (NH amide),
2927 (CH), 1666 (CvO amide), 1593 (CvC aromatic), 1530
(CvC aromatic); δH (400 MHz, CDCl3) 8.04 (1H, br s. indole
NH), 7.58 (1H, d, J = 7.0 Hz, aryl CH), 7.53 (1H, s, aryl CH),
7.33 (1H, d, J = 8.0 Hz, aryl CH), 7.27 (1H, br s, NH amide),
7.25 (1H, partially obscured by solvent, aryl CH), 7.21–7.16
(2H, m, aryl CH), 7.11 (1H, ddd, J = 8.0 Hz, 8.0 Hz, 1.0 Hz,
aryl CH), 7.05 (1H, ddd, J = 8.0 Hz, 2.0 Hz, 1.0 Hz, aryl CH),
6.94 (1H, d, J = 2.0 Hz, aryl CH), 2.86–2.81 (2H, m, CH2C-
(vO)NH), 2.37–2.33 (2H, m, NHC(vO)CH2CH2CH2),
2.16–2.11 (2H, m, NHC(vO)CH2CH2CH2); δC (100 MHz;
CDCl3) 171.6 (CvO amide), 139.0 (C), 136.4 (C), 134.5 (C),
129.9 (CH), 127.4 (C), 124.1 (CH), 122.0 (CH), 121.7 (CH),
119.9 (CH), 119.3 (CH), 118.8 (CH), 117.7 (CH), 115.3 (C)
111.2 (CH), 36.9 (CH2), 25.7 (CH2), 24.4 (CH2); LCMS (ES−)
(MeCN) 313.2 (M − H)+ for 37Cl, 311.2 (M − H)+ for 36Cl.
(NHCCHCI), 119.2 (NHCCHCH), 94.1 (NHCCHCI), 48.4 (C
(O)CH2CO), 44.3 (CH2C(O)CH2CO), 31.8 (CH2), 29.4 (CH2),
29.3 (CH2), 29.2 (CH2), 29.0 (CH2), 23.3 (CH2), 22.7 (CH2),
14.1 (CH3); LCMS (ES+) (MeCN) 416 (M + H)+; m.p.
73–75 °C (1 : 2 Et2O–pet. ether).
N-(4-Methoxyphenyl)-3-oxododecanamide (22). The protected
analogue of the final product was prepared by general method 2
using 2-(2-nonyl-1,3-dioxolan-2-yl)acetic acid (29, 200 mg,
0.78 mmol), CH2Cl2 (5 mL), DCC (240 mg, 1.16 mmol),
DMAP (57 mg, 0.47 mmol) and para-anisidine (96 mg,
0.78 mmol). The crude material was purified by column chrom-
atography (SiO2, 2 : 1 Et2O–pet. ether) to yield a white solid
(236 mg, 83%). A sample of this material (214 mg) was depro-
tected by general method 3 using 0.7 mL TFA and 5 mL
CH2Cl2. The crude material was purified by column chromato-
graphy (SiO2, 1 : 1 Et2O–pet. ether) to give the title compound
as a white solid (126 mg, 67%). Rf 0.18 (SiO2; 1 : 1 Et2O–pet.
ether); vmax (neat)/cm−1 1418, 1468, 1517, 1552, 1654 (amide),
1711 (ketone), 1727, 2849, 2918, 3301; δH (500 MHz, CDCl3)
9.00 (1H, s, NH), 7.46–7.43 (2H, m, NHCCH), 6.88–6.85 (2H,
m, NHCCHCH), 3.79 (3H, s, OCH3), 3.55 (2H, s, C(O)
CH2CO), 2.57 (2H, t, J = 7.5 Hz, CH2CO), 1.65–1.59 (2H, m,
CH2CH2CO), 1.30–1.26 (12H, br m, alkyl CH2), 0.88 (3H, t, J
= 7.0 Hz, CH3); δC (125 MHz; CDCl3) 208.1 (CvO (ketone)),
163.2 (CvO (amide)), 156.5 (COMe), 130.7 (NHC), 121.9
(NHCCH), 114.1 (NHCCHCH), 55.5 (OCH3), 48.7 (C(O)
CH2CO), 44.2 (CH2C(O)CH2CO), 31.8 (CH2), 29.4 (CH2), 29.3
(CH2), 29.2 (CH2), 29.0 (CH2), 23.4 (CH2), 22.7 (CH2), 14.1
(CH3); LCMS (ES+) (MeCN) 320 (M + H)+; m.p. 91–94 °C
(1 : 1 Et2O–pet. ether).
4-Bromophenylacetyl chloride (34). Oxalyl chloride (0.30 ml,
3.5 mmol, 1.5 equiv.) was added to a stirred solution of 4-bro-
mophenyl acetic acid (500 mg, 2.33 mmol, 1.0 equiv.) in anhy-
drous CH2Cl2 (20 ml) at 0 °C under a nitrogen atmosphere. One
drop of DMF was then added and the reaction mixture stirred for
30 min at 0 °C. The flask was warmed to room temperature and
stirred for at least 80 min. The solvent was removed under
reduced pressure to yield the title compound as a crude yellow
oil which was used immediately and without purification in the
next steps.
N-(3-Hydroxyphenyl)-3-oxododecanamide (23). The protected
analogue of the final product was prepared by general method 2
using 2-(2-nonyl-1,3-dioxolan-2-yl)acetic acid (29, 200 mg,
0.78 mmol), CH2Cl2 (5 mL), DCC (240 mg, 1.16 mmol),
DMAP (57 mg, 0.47 mmol) and 3-aminophenol (85 mg,
0.78 mmol). The crude material was purified by column chrom-
atography (SiO2, 1 : 1 EtOAc–pet. ether) to yield a yellow solid
(68 mg, 25%). A sample of this material (58 mg) was depro-
tected by general method 3 using 0.25 mL TFA and 5 mL
CH2Cl2. The crude material was purified by column chromato-
graphy (SiO2, 4 : 1 Et2O–pet. ether) to give the title compound
as a white solid (11 mg, 23%). Rf 0.3 (SiO2; 4 : 1 Et2O–pet.
ether); vmax (neat)/cm−1 1446, 1491, 1506, 1601, 1641 (amide),
1698 (ketone), 2384, 2851, 2915, 3201; δH (400 MHz; CDCl3)
9.43 (1H, NH), 7.79 (1H, s, NHCCHCOH), 7.18 (1H, t, J = 8.0
Hz, NHCCHCH), 6.74 (1H, d, J = 8.0 Hz, NHCCHCH), 6.66
(1H, dd, J = 8.0 Hz, 2.0 Hz, NHCCHCHCH), 3.61 (2H, s, C(O)
CH2CO), 2.59 (2H, t, J = 7.0 Hz, CH2CO), 1.6–1.66 (2H, m,
CH2CH2CO), 1.23–1.3 (12H, br m, alkyl CH2), 0.9 (3H, t, J =
7.0 Hz, CH3); δC (100 MHz; MeOD) 207.8 (CvO (ketone)),
168.5 (CvO (amide)), 160.0 (COH), 141.6 (NHC), 131.6
(NHCCHCH), 113.5, 113.3 (NHCCHCH and NHCCHCHCH),
109.4 (NHCCHCOH), 44.9 (C(O)CH2CO), 34.1 (CH2C(O)
CH2CO), 31.17–31.59, 25.5, 24.7 (seven signals, CH2), 15.4
(CH3); LCMS (ES+) (MeCN) 306 (M + H)+; m.p. 105–110 °C
(4 : 1 Et2O–pet. ether). This compound has been reported pre-
viously but spectroscopic data is not available.
General procedure 4: coupling of 4-bromophenylacetyl chloride
(34) with (hetero)aromatic amines
A solution of 4-bromophenylacetyl chloride (1 equiv.) in
CH2Cl2 (∼7 mL per mmol susbtrate) was added dropwise to a
vigorously stirred mixture of the appropriate (hetero)aromatic
amine (1 equiv.) and Na2CO3 (2 equiv.) in water (∼7 mL per
mmol substrate) at room temperature. The reaction was stirred
vigorously at room temperature open to the air for 12 h. The
reaction mixture was diluted with CH2Cl2 and the aqueous and
organic layers separated. The aqueous layer was washed with
CH2Cl2 and the combined organic layers dried (MgSO4) and the
solvent removed under reduced pressure. The crude product
material was purified by column chromatography if required.
2-(4-Bromophenyl)-N-(3-chlorophenyl)acetamide (32). Pre-
pared by general procedure 4 using 3-chloroaniline (0.1 mL,
0.86 mmol), Na2CO3 (273 mg), water (6 mL) and 4-bromophe-
nylacetyl chloride (200 mg, 0.86 mmol) dissolved in CH2Cl2
This journal is © The Royal Society of Chemistry 2012
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2-(4-Bromophenyl)-N-((1S*,2S*)-2-hydroxycyclopentyl)aceta-
mide (40). Prepared by general procedure 4 using trans-2-ami-
nocyclopentanol hydrochloride (161 mg, 1.17 mmol), Na2CO3
(372 mg), water (6 mL) and 4-bromophenylacetyl chloride
(273 mg, 1.17 mmol) dissolved in CH2Cl2 (6 mL). The crude
product material (pale yellow solid) was purified by column
chromatography (SiO2, 4 : 1 EtOAc–CH2Cl2) to yield the title
compound as a white solid (266 mg, 76%).
(6 mL). The crude product material (white solid) was purified by
column chromatography (SiO2, 1 : 1 Et2O–pet. ether) to yield the
title compound as an off-white solid (154 mg, 55%). Rf 0.29
(SiO2, 1 : 1 Et2O–pet. ether); vmax (MeOD)/cm−1 3332 br (OH
solvent), 3276 (NH amide), 2395, 1654 (CvO amide), 1590
(CvC aromatic), 1482 (CvC aromatic); δH (400 MHz, MeOD)
7.74 (1H, dd, J = 2.0 Hz, NHCCHCCl), 7.50 (2H, d, J = 8.5 Hz,
CBrCH), 7.43 (1H, ddd, J = 8.0 Hz, 1.0 Hz, 1.0 Hz,
NHCCHCH), 7.29 (2H, d, J = 8.5 Hz, CBrCHCH), 7.29 (1H,
dd, J = 8.0 Hz, 8.0 Hz, NHCCHCH), 7.11 (1H, ddd, J = 8.0 Hz,
2.0 Hz, 1.0 Hz, NHCCHCHCH), 3.68 (2H, s, CH2); δC
(100 MHz; MeOD) 171.7 (CvO), 141.2 (NHC), 135.7 (C),
135.4 (C), 132.6 (CH), 132.1 (CH), 131.0 (NHCCHCH), 125.0
(CClCHCH), 121.8 (CBr), 120.9 (NHCCHCCl), 119.1
(NHCCHCH), 43.8 (CH2); LCMS (ES+) (MeCN) 325.8
(M + H)+ for 35Cl and 81Br, 327.8 (M + H)+ for 37Cl and 81Br;
m.p. 136–138 °C (1 : 1 Et2O–pet. ether).
Rf 0.20 (SiO2, 4 : 1 EtOAc–CH2Cl2); vmax (CDCl3)/cm−1
3350 br (OH), 3281 (NH amide), 2952 (CH), 1631 (CvO
amide), 1553 (CvC amide); δH (400 MHz, CDCl3) 7.52 (2H, d,
J = 8.5 Hz, CBrCH), 7.16 (2H, d, J = 8.5 Hz, CBrCHCH), 5.52
(1H, br s, OH), 4.27 (1H, br d, J = 1.5 Hz, NH), 3.95 (1H, dq, J
= 6.0 Hz, 1.5 Hz, CHOH), 3.86–3.79 (1H, m, NHCH), 3.55
(2H, s, CH2C(vO)), 2.14–1.98 (2H, m), 1.85–1.75 (1H, m),
1.72–1.62 (2H, m), 1.37–1.29 (1H, m); δC (100 MHz; CDCl3)
172.4 (CvO), 133.4 (CBrCHCHC), 132.1 (CBrCH), 131.0
(CBrCHCH), 121.5 (CBr), 79.6 (CHOH), 61.1 (NHCH), 42.7
(CH2C(vO)), 32.6 (COHCH2), 30.5 (NHCHCH2), 21.3
(CH2CH2CH2CHOH); LCMS (ES+) (MeCN) 299.9 (M + H)+
for 81Br; m.p. 110–114 °C (4 : 1 EtOAc–CH2Cl2).
2-(4-Bromophenyl)-N-(2-chloropyridin-4-yl)acetamide
(33).
Prepared by general procedure 4 using 4-amino-2-chloropyridine
(111 mg, 0.86 mmol), Na2CO3 (273 mg), water (6 mL) and 4-
bromophenylacetyl chloride (200 mg, 0.86 mmol) dissolved in
CH2Cl2 (6 mL). The crude product material (yellow oil) was
purified by column chromatography (SiO2, 20 : 1 Et2O–pet.
ether) followed by washing with HCl (to remove unreacted start-
ing amine running with the same Rf) to yield the title compound
as an amorphous white solid (39 mg, 14%). Rf 0.22 (SiO2, 20 : 1
Et2O–pet. ether); vmax (CDCl3)/cm−1 3246 (amide NH), 3053
(CH aromatic), 2927 (CH), 1681 (amide CvO), 1578 (CvN),
1504 (CvC aromatic); δH (400 MHz, CDCl3) 8.17 (1H, br d, J
= 5.0 Hz, NHCCHCHN), 7.55 (1H, s, CHCCl), 7.47 (2H, d J =
8.5 Hz, CBrCH), 7.31 (1H, br d, J = 4.5 Hz, NHCCHCHN),
7.14 (2H, d, J = 8.0 Hz, CBrCHCH), 3.67 (2H, s, CH2); δC
(100 MHz; CDCl3) 169.4 (CvO), 151.7 (C), 149.4 (CH), 147.6
(C), 132.3 (CH), 132.3 (C), 131.0 (CH), 122.0 (C), 113.6 (CH),
112.6 (CH), 44.0 (CH2); LCMS (ES+) (MeCN) 324.9 (M + H)+
for 35Cl and 79Br, 326.9 (M + H)+ for 35Cl and 81Br and/or 37Cl
and 79Br, 328.8 (M + H)+ for 37Cl and 81Br.
General procedure 5: oxidation using Dess–Martin periodinane
Dess–Martin periodinane (1.1 equiv.) was added to a solution of
the amide (1 equiv.) in anhydrous CH2Cl2 under nitrogen at
room temperature. The reaction mixture was stirred at room
temperature for 12 h and the solvent removed under reduced
pressure. The crude product material was purified by column
chromatography if required.
2-(4-Bromophenyl)-N-(2-oxocyclopentyl)acetamide (42). Pre-
pared by general procedure 5 using amide 40 (266 mg,
0.89 mmol), anhydrous CH2Cl2 (10 mL). The crude product
material (white solid) was purified by column chromatography
(SiO2, 1 : 1 CH2Cl2–EtOAc) to yield the title compound as a
white solid (175 mg, 66%). Rf 0.35 (SiO2, 1 : 1 EtOAc–
CH2Cl2); vmax (CDCl3)/cm−1 3251 (NH amide), 3063 (CH aro-
matic), 2962 (CH), 2906 (CH), 1745 (CvO ketone), 1641
(CvO amide), 1547 (CvC aromatic); δH (400 MHz, CDCl3)
7.40 (2H, d, J = 8.5 Hz, CBrCH), 7.09 (2H, d, J = 8.5 Hz,
CBrCHCH), 5.76 (1H, br s, NH), 4.03 (1H, ddd, J = 13.5 Hz,
7.5 Hz, 7.5 Hz, NHCH), 3.47 (2H, s, CH2C(vO)), 2.57–2.51
(1H, m, C(vO)CH), 2.33 (1H, dd, J = 9.0 Hz, 1.5 Hz, C(vO)
CH′), 2.14–2.05 (1H, m), 2.02–1.94 (1H, m), 1.84–1.71 (1H,
m), 1.54–1.43 (1H, dddd, J = 12.5 Hz, 12.5 Hz, 12.5 Hz, 7.0
Hz); δC (100 MHz; CDCl3) 215.2 (CvO ketone), 171.0 (CvO
amide), 134.4 (CBrCHCHC), 132.5 (CBrCH), 131.5
(CBrCHCH), 121.8 (CBr), 58.6 (NHCH), 43.1 (CH2C(vO)),
35.3 (C(vO)CH2), 30.3 (CH2), 18.4 (CH2); LCMS (ES+)
(MeCN) 297.9 (M + H)+ for 81Br; m.p. 124–127 °C (1 : 1
EtOAc–CH2Cl2).
2-(4-Bromophenyl)-N-((1S*,2S*)-2-hydroxycyclohexyl)aceta-
mide (39). Prepared by general procedure 4 using trans-2-ami-
nocyclohexanol hydrochloride (177 mg, 1.17 mmol), Na2CO3
(372 mg), water (6 mL) and 4-bromophenylacetyl chloride
(273 mg, 1.17 mmol) dissolved in CH2Cl2 (6 mL). The crude
product material (white solid) was purified by column chromato-
graphy (SiO2, 20 : 1 EtOAc–CH2Cl2) to yield the title compound
as a white solid (179 mg, 49%). Rf 0.20 (SiO2, 4 : 1 EtOAc–
CH2Cl2); vmax (CDCl3)/cm−1 3300 br (OH), 3286 (NH amide),
2932 (CH), 2851 (CH), 1644 (CvO amide), 1618, 1550 (CvC
aromatic); δH (400 MHz, CDCl3) 7.47 (2H, d, J = 8.5 Hz,
CBrCH), 7.14 (2H, d, J = 8.5 Hz, CBrCHCH), 5.34 (1H, br s,
NH), 3.65–3.57 (1H, m, NHCHCHOH), 3.54 (2H, s, CH2C
(vO)), 2.05–1.99 (1H, m), 1.89–1.82 (1H, m), 1.73–1.62 (2H,
m), 1.35–1.14 (1H, m), 1.12–1.02 (1H, m); δC (100 MHz;
CDCl3) 172.1 (CvO), 133.6 (CBrCHCHC), 132.1 (CBrCH),
131.0 (CBrCHCH), 121.5 (CBr), 75.4 (CHOH), 56.0 (NHCH),
43.0 (CH2), 34.5 (CH2), 31.3 (CH2), 24.5 (CH2), 23.9 (CH2);
2-(4-Bromophenyl)-N-(2-oxocyclohexyl)acetamide (41). Pre-
pared by general procedure 5 using amide 39 (179 mg,
0.58 mmol), anhydrous CH2Cl2 (10 mL). The crude product
material (white solid) was purified by column chromatography
(SiO2, 3 : 1 CH2Cl2–EtOAc) to yield the title compound as a
white solid (102 mg, 57%). Rf 0.38 (SiO2, 1 : 3 EtOAc–
LCMS (ES+) (MeCN) 313.9 (M
+
H)+ for 81Br;
m.p. 110–114 °C (4 : 1 EtOAc–CH2Cl2).
6042 | Org. Biomol. Chem., 2012, 10, 6032–6044
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CH2Cl2); vmax (CDCl3)/cm−1 3256 (NH amide), 3068 (CH aro-
matic), 2942 (CH), 2861 (CH), 1719 (CvO ketone), 1638
(CvO amide), 1553 (CvC aromatic); δH (400 MHz, CDCl3)
7.40 (2H, d, J = 8.0 Hz, CBrCH), 7.09 (2H, d, J = 8.5 Hz,
CBrCHCH), 6.35 (1H, br s, NH amide), 4.38 (1H, ddd, J = 12.5
Hz, 6.5 Hz, 6.5 Hz, NHCH), 3.45 (2H, s, CH2C(vO)), 2.55
(1H, ddddd, J = 6.0 Hz, 6.0 Hz, 3.0 Hz, 3.0 Hz, 3.0 Hz),
2.46–2.40 (1H, m), 2.34–2.25 (1H, m), 2.10–2.02 (1H, m),
1.83–1.75 (1H, m), 1.73–1.65 (1H, m), 1.54 (1H, qt, J = 13.5
Hz, 4,5 Hz), 1.23 (1H, qd, J = 12.5 Hz, 4.5 Hz); δC (100 MHz;
CDCl3) 208.0 (CvO ketone), 170.0 (CvO amide), 134.0
(CBrCHCHC), 132.4 (CBrCH), 131.4 (CBrCHCH), 121 (CBr),
58.6 (NHCH), 43.4 (CH2C(vO)), 41.5 (NHCHC(vO)CH2),
35.7 (CH2), 28.4 (CH2), 24.4 (CH2); LCMS (ES+) (MeCN)
312.0 (M + H)+ for 81Br, 313.03; m.p. 116–118 °C (1 : 3
EtOAc–CH2Cl2).
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Acknowledgements
This work was supported by grants from the Engineering and
Physical Sciences Research Council, Biotechnology and Biologi-
cal Sciences Research Council, Medical Research Council,
Royal Society, Frances and Augustus Newman Foundation, and
Wellcome Trust. J.T.H is supported by an Medical Research
Council strategic priority studentship awarded to MW and DRS.
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ligand). There is a relative lack of work pertaining to the de novo rational
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were selected from molecules which are known to modulate LuxR-type
quorum sensing.
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