Synthesis and biological evaluation of triazole-containing N-acyl homoserine lactones as quorum sensing modulators

Article abstract of DOI:10.1039/c2ob27155a

Many bacterial species are capable of assessing their local population densities through a cell-cell signaling mechanism termed quorum sensing (QS). This intercellular communication process is mediated by small molecule or peptide ligands and their cognate protein receptors. Numerous pathogens use QS to initiate virulence once they achieve a threshold cell number on a host. Consequently, approaches to intercept QS have attracted considerable attention as potential anti-infective therapies. Our interest in the development of small molecule tools to modulate QS pathways motivated us to evaluate triazole-containing analogs of natural N-acyl l-homoserine lactone (AHL) signals as non-native QS agonists and antagonists in Gram-negative bacteria. We synthesized 72 triazole derivatives of five broad structure types in high yields and purities using efficient Cu(i)-catalyzed azide-alkyne couplings. These compounds were evaluated for their ability to activate or inhibit two QS receptors from two prevalent pathogens-LasR from Pseudomonas aeruginosa and AbaR from Acinetobacter baumannii-using bacterial reporter strains. Several triazole derivatives were identified that were capable of strongly modulating the activity of LasR and AbaR. These compounds represent a new and synthetically accessible class of AHL analogs, and could find utility as chemical tools to study QS and its role in bacterial virulence.

Full text of DOI:10.1039/c2ob27155a

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Synthesis and Biological Evaluation of Triazole-Containing
N-Acyl Homoserine Lactones as Quorum Sensing Modulators
Danielle M. Stacy,a Sebastian T. Le Quement,b Casper L. Hansen,b Janie W. Clausen,b Tim
Tolker-Nielsen,c Jacob W. Brummond,a Michael Givskov,c,d Thomas E. Nielsen,b,d,*
and Helen E. Blackwella,*
a Department of Chemistry, University of Wisconsin–Madison, 1101 University Ave., Madison,
WI 53706 USA; b Department of Chemistry, Technical University of Denmark, DK-2800 Kgs.
Lyngby, Denmark; c Department of International Health, Immunology, and Microbiology,
Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark; d
Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological
University, Singapore 637551
Abstract
Many bacterial species are capable of assessing their local population densities through a cell-
cell signaling mechanism termed quorum sensing (QS). This intercellular communication
process is mediated by small molecule or peptide ligands and their cognate protein receptors.
Numerous pathogens use QS to initiate virulence once they achieve a threshold cell number on
a host. Consequently, approaches to intercept QS have attracted considerable attention as
potential anti-infective therapies. Our interest in the development of small molecule tools to
modulate QS pathways motivated us to evaluate triazole-containing analogs of natural N-acyl L-
homoserine lactone (AHL) signals as non-native QS agonists and antagonists in Gram-negative
bacteria. We synthesized 72 triazole derivatives of five broad structure types in high yields and
purities using efficient Cu(I)-catalyzed azide-alkyne couplings. These compounds were
evaluated for their ability to activate or inhibit two QS receptors from two prevalent pathogens
* To whom correspondence should be addressed. blackwell@chem.wisc.edu; ten@kemi.dtu.dk

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– LasR from Pseudomonas aeruginosa and AbaR from Acinetobacter baumannii – using
bacterial reporter strains. Several triazole derivatives were identified that were capable of
strongly modulating the activity of LasR and AbaR. These compounds represent a new and
synthetically accessible class of AHL analogs, and could find utility as chemical tools to study
QS and its role in bacterial virulence.
Introduction
Bacteria can monitor their local population densities using diffusible, small molecule signals
(or autoinducers) in a process called quorum sensing (QS).1-3 As the bacterial population grows
and these signals reach a certain threshold concentration, the bacterial community will alter
gene expression levels to initiate a range of behaviors that will benefit the group.4, 5 These
collective processes are exceptionally diverse, and include luminescence, virulence factor
production, swarming motility, sporulation, and biofilm production.6-8 In pathogens, QS allows
the bacteria to only initiate virulence after reaching a sufficiently high cell density that will
9
overwhelm the host.7, Accordingly, QS is actively being pursued as a new target for the
development of anti-infective strategies, particularly against the antibiotic-resistant and
increasingly prevalent pathogens Pseudomonas aeruginosa and Staphylococcus aureus.10-13
A range of approaches to intercept bacterial QS has been examined over the past ~20 years,
and chemical methods to block QS signals have received significant recent attention from the
chemical biology and drug discovery communities.14-17 Most bacterial QS circuits are mediated
by a low molecular weight ligand binding to its cognate receptor, and this binding event
regulates the transcription of QS genes.18, Productive ligand:receptor binding occurs only
19
when a “quorum” of bacteria is reached (and the ligand concentration is sufficient). In view of
2

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this mechanism, one straightforward chemical approach to intercept QS is to block
signal:receptor binding using non-native small molecules, and our labs and several others have
explored the design and synthesis of such agents.14, 20-25 Compounds that antagonize or agonize
QS receptors can thus result in QS inhibition or activation, respectively. Either class of
compound could hold value as a chemical tool to study QS pathways and phenotypes. In
particular, the QS inhibitors provide a pathway to the development of novel anti-virulence
approaches.
Numerous synthetic QS modulators have been reported to date. The majority of these
compounds are mimics of either natural QS signal molecules or natural products shown to
modulate QS, such as the halogenated furanones produced by certain algae as natural
27
antifoulants.26,
In Gram-negative bacteria, QS signal molecules are typically N-acyl L-
homoserine lactones (AHLs), and considerable recent research has focused on the interception
of AHL signalling via chemical means. Originally studied in the marine symbiont Vibrio
fischeri, AHLs are generated by LuxI-type proteins (the AHL synthases) and are perceived by
intracellular LuxR-type proteins (the AHL receptors).18, 28 Dependent on the synthase, the AHL
products typically have acyl chains of 4–18 carbon atoms, and may have units of unsaturation
and/or altered oxidation states at the 3-position. Once productive AHL:LuxR-type receptor
binding occurs, this complex will most commonly dimerize, bind to specific QS-associated
promoters, and activate transcription of QS target genes.
A range of non-native AHLs have been synthesized and examined as QS agonists and
antagonists in many bacterial species.14, 20-22, 25, 29-44 These signal analogs are believed to act by
competitively binding LuxR-type receptors. Several recent reviews provide comprehensive
descriptions of the known structure-activity relationships (SARs) for LuxR-type receptor
3

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modulation by non-native AHLs;14, 24, 45, 46 we summarize the most salient SAR features here.
The homoserine lactone (HL) head group of native AHLs has been replaced by a range of
heterocycles and cycloalkanes, and several of these analogs are strong QS agonists with
activities comparable to native ligands, while others are strong antagonists. Evaluation of the
HL head group stereochemistry has demonstrated that AHLs containing the natural HL
stereoisomer (i.e., L-HL) are most often markedly more potent than their D-HL analogs, whether
agonists or antagonists. Beyond these head group modifications, the AHL acyl chain has been
the focus of most other AHL derivatization studies, and our laboratories have examined these
analogs extensively over the past decade.30, 32, 33, 35-37, 47-52 A larger proportion of these non-native
AHLs display antagonistic as opposed to agonistic activities, suggesting that the structural
requirements for activation of LuxR-type receptors by AHLs are more stringent. Indeed, most
non-native, AHL-type LuxR-type agonists have acyl chains that closely mimic native AHL
ligands. In turn, the most potent antagonists have largely contained moderately bulky, aromatic
moieties on the acyl group. Such substituents have been postulated to promote hydrophobic
interactions in the LuxR-type receptor ligand-binding pocket (assuming these non-native AHLs
target the same site), which could act, in part, to inactivate the receptor. Lastly, a series of
studies has probed replacement of the AHL amide bond with various isosteres, including
sulfonamides, ureas, and sulfonylureas, and this replacement has largely yielded LuxR-type
antagonists.24
Our laboratory has focused primarily on the development of solid-phase synthetic routes to
non-native AHLs and AHL analogs, and these methods have allowed for the rapid construction
of several focused AHL libraries.30, 33 We now seek to develop complementary solution-phase
methods for AHL synthesis to expand our set of non-native AHLs for study. We have focused
4

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our attention on robust reactions that are tolerant of a range of functional groups and utilize
readily available building blocks. In the current study, we report our development of solution-
phase AHL synthesis methods based on Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC)
reactions (Figure 1).53-55 The azide and alkyne building blocks were either commercially
available or generated from simple precursors via Sonogashira cross-coupling and Cu-catalyzed
aromatic azidation of aromatic halides. Combination of these building blocks with
complementary reagents yielded four sets of novel triazole-containing AHL analogs (types I–
IV, Figure 1A) and varied combinations of complementary building blocks yielded multivalent
AHL analogs (type V, Figure 1B) in a minimal number of synthetic steps. Notably, the type I
analogs had the acyl triazole positioned such that the 2-N atom of the triazole ring overlapped
with the 3-oxo functionality of native AHLs (circled moieties, Fig. 1A); we reasoned that such
overlap could possibly enhance AHL-receptor interactions, if these triazole analogs target the
ligand-binding site of LuxR-type receptors. To our knowledge, triazole HLs of these structure
types are yet to be evaluated for QS activity.56, 57 Screening of the triazole HLs for modulation of
LuxR-type receptors in two prominent pathogens (P. aeruginosa and Acinetobacter baumannii)
revealed several potent QS agonists and antagonists. These results suggest that these triazole
HLs represent a new class of non-native AHL derivatives for use as QS modulators.
Furthermore, the efficient synthetic routes described herein should be readily applicable for the
future generation of expanded sets of triazole HLs to improve their potency and further
elucidate their SAR.
5

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A
Type I
Building Blocks
Complimentary Reagents
N
N
N
O
R
O
O
O
N
N3
H
N
X
Alk
O
H
O
O
O
O
Type II
O
O
O
O
N
H
Ar N3
Alk N3
R1
O
N
H
Analog
Design
Triazole
Formation
N
R
N
H
N
O
N
N
N
O
O
O
O
O
O
Type III
CuAAC
O
O
R
N
Ar N3
Alk N3
R2
N
N
N
H
H
H
O
O
O
O
N
O
Type IV
O
O
R
N
H
X
Alk
Commercially available
N3
N
H
O
N
N
B
Complementary Building Blocks
O
Triazole
Formation
Type V
O
O
O
H
O
N
N
O
N
O
N3
O
H
N
N
N
N
O
H
CuAAC
H
O
O
O
Figure 1. A. Structures of the type I–IV triazole HLs and their corresponding building blocks. The 3-oxo
group of the natural AHL (left) and the corresponding triazole 2-nitrogen in the type I analog are circled.
B. General structure of a dimeric type V triazole HL and corresponding building blocks. Trimeric
derivatives were also synthesized; see text.
Results and Discussion
To access the triazole HLs of types I–IV (Figure 1A) via CuACC reactions, we first
synthesized L-HL building blocks bearing azide or alkyne functionalities. These building blocks
were then used as handles for reaction with complementary alkyne or azide–functionalized
units, respectively. The CuAAC strategy was also used to synthesize triazole type V compounds
containing two or three HL moieties (Figure 1B), as discussed below.
Synthesis of building block type 1: azidoacetyl HL (3). Azidoacetyl HL 3 was readily
available from L-HL, using a procedure involving bromoacetylation and subsequent
displacement of the bromide with N3 (Scheme 1). In our initial two-step procedure to generate
3, bromide 2 was synthesized under Schotten-Baumann conditions and isolated by simple
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extraction. Subsequent reaction of 2 with sodium azide in DMSO, aqueous work-up, and
extraction provided the azide 3 in only moderate overall yield (41% over two steps). We
noticed, however, that both steps proceeded with clean conversion of the starting material as
indicated by TLC. We therefore hypothesized that the observed moderate yield was merely due
to the high solubility of 2 and 3 in water, which was required for the numerous extractions and
troublesome removal of DMSO in the second step. These observations prompted us to attempt a
one-pot reaction, where L-HL was directly converted to the azide building block under
Schotten–Baumann conditions in the presence of both bromoacetylbromide and sodium azide
(Scheme 1). To our satisfaction, this one-pot procedure provided the azidoacetyl HL 3 in
excellent yield (89%) after simple extractive aqueous work-up.
NaHCO3 (3 equiv),
O
BrCH2COBr (1.1 equiv),
O
O
Br
HBr H2N
N
H
CH2Cl2:H2O (1:1), rt, 3 h
(66%)
O
O
2
1
NaN3 (2 equiv)
DMSO, rt, 3h
(62%)
NaHCO3 (2 equiv),
BrCH2COBr (1.1 equiv),
NaN3 (2 equiv)
O
O
N3
N
CH2Cl2:H2O (1:1), rt, 16 h
(89%)
H
O
3
Scheme 1. Synthesis of azidoacetyl L-HL 3.
Synthesis of building block type 2: 4-pentynoyl L-HL (4). A range of standard amide
coupling reagents was surveyed for the reaction of 4-pentynoic acid with L-HL. These studies
revealed PyBOP as the optimal reagent for the synthesis of alkyne derivative 4 (>95% yield;
Scheme 2).
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4-Pentynoic acid (1 equiv),
PyBOP (1 equiv),
O
Et3N (3 equiv),
O
O
HBr H2N
N
H
DMF, rt, 2 h
(>95%)
O
O
4
1
Scheme 2. Synthesis of 4-pentynoyl L-HL 4.
Synthesis of building block types 3 and 4: alkyne- and azidobenzoyl L-HLs. Acylation
of L-HL by 4- and 3-iodobenzoyl chloride, generated in situ by treating the corresponding acids
with oxalyl chloride and catalytic amounts of DMF, provided intermediates 7 and 8 (Scheme 3).
These intermediates were then subjected to Sonogashira cross-coupling with TMS-acetylene to
give coupling products 9 and 10 in excellent yields (91% and 94%, respectively).58 Treatment of
9 and 10 with TBAF afforded the desired alkyne-functionalized benzoyl L-HL building blocks
11 and 12.
Iodides 7 and 8 were also subjected to the azidation conditions reported by Andersen et al.
(Scheme 3).59 Sodium azide, in the presence of copper iodide and N,N’-dimethylethylene
diamine ligand, provided 4- and 3-azidobenzoylated L-HL building blocks 13 and 14 in
satisfactory yields (72% and 78%, respectively).
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O
1) Oxalyl chloride (3 equiv),
DMF (0.05 equiv),
CO2H
O
I
N
CH2Cl2, rt, 5 h
H
I
O
2)
Et3N (3 equiv), DMF, rt, 2 h
L
-HL.HBr (1 equiv),
7 (4-I): 76%
8 (3-I): 90%
5 (4-I)
6 (3-I)
TMS-acetylene (1 equiv),
Pd(PPh3)4 (4 mol %),
CuI (8 mol %),
NaN3 (2 equiv),
CuI (10 mol %),
NaAsc (5 mol %),
NEM:DMF (1:1), rt, 16 h
(MeNHCH2)2 (15 mol %),
DMSO:H2O (5:1), rt, 16 h
O
O
O
O
N
N
H
TMS
H
N3
O
O
9
(4-CCTMS): 91%
13 (4-N3): 72%
14 (3-N3): 78%
10 (3-CCTMS): 94%
TBAF (2.5 equiv),
THF, rt, 16 h
O
O
N
H
O
11 (4-CCH): 58%
12 (3-CCH): 65%
Scheme 3. Synthesis of alkyne- and azide-functionalized benzoyl L-HLs 11–14.
Synthesis of triazole HL libraries (types I–IV). The L-HL building blocks synthesized
above were next subjected to CuACC reactions with a range of complementary, commercially
available alkyne and azide–functionalized units to generate the triazole HL libraries. After
screening a range of reaction conditions, we settled on a straightforward and general CuACC
protocol using CuI and DIPEA in acetonitrile (a representative reaction is shown in Scheme 4).
In all cases, complete conversion of starting material was observed after overnight reaction (~16
h, at room temperature), typically with some precipitation of the desired triazole product. A first
product yield was easily isolated by simple filtration of the reaction mixture. A second quantity
of product was then obtained by evaporation of the filtrate, redissolution of the resulting residue
in hot acetic acid or methanol, subsequent filtration, and precipitation with diethylether. We
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applied this CuACC reaction and isolation protocol (described fully in the Supplementary
Information) to construct the four triazole HL libraries (types I–IV) shown in Figure 2.
R
(1.5 equiv),
CuI (10 mol %),
DIPEA (2 equiv),
N
N
N
O
O
R
O
O
N3
N
N
H
H
MeCN, rt, 16 h
O
O
3
I-A – I-O
Scheme 4. Representative synthesis of type I triazole HLs (I-A–I-O) from building block 3.
Type I
Type IV
O
N
H
N
O
O
N
N
O
R
N
O
O
N
N
N
N
N
N
H
H
n
N
H
O
O
O
R:
I-I
R
n = 2
n = 3
n = 4
n = 5
I-A
4-OPh
3,5-F2
I-J
I-K
I-L
I-B
I-C
I-D
R:
para
meta
3,5-(OCH3)2
n = 2
n = 3
n = 4
n = 5
IV-A
IV-C
IV-E
IV-B
IV-D
IV-F
IV-H
O
N
N
O
N
N
O
O
O
N
N
N
H
N
H
n
IV-G
n
O
O
I-M
H3CO
n = 1
n = 3
n = 4
I-E
I-F
I-G
IV-I
IV-K
IV-M
IV-J
IV-L
IV-N
n = 1
n = 3
n = 4
O
N
N
S
n
N
N
H
IV-O
IV-P
O
I-N
N
N
N
O
O
O
O
N
N
N
H
S
N
O
I-H
N
H
R1:
I-O
O
IV-Q
IV-S
IV-U
IV-W
IV-R
IV-T
IV-V
IV-X
H
R1
3,5-F2
3,5-(OCH3)2
4-OPh
Type II
Type III
O
O
O
N
N
N
R
O
O
H
N
N
O
N
H3CO
H
R
N
N
IV-Y
IV-Z
H
para
meta
R:
N
R:
4-OCH3
4-NH2
4-CH3
3-CHO
4-OH
II-A
II-B
II-C
II-D
II-E
II-F
N
III-A
III-C
III-B
III-D
IV-AA
IV-AC
IV-AE
IV-AB
IV-AD
IV-AF
S
S
3-NH2
R1:
NH2
OH
III-E
III-G
III-F
III-H
III-J
R1
S
OCH3 III-I
Figure 2. Structures of the type I–IV triazole HL libraries.
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In general, the triazole HL library members were isolated in good to excellent yields and
purities after precipitation with diethylether. However, compounds resulting from the reaction
of azidoacetyl L-HL 3 with aliphatic acetylenes were of slightly lower purity (~85%) than all of
the other triazoles synthesized (typically >95%). Reaction yields also varied somewhat within
and between individual libraries. For example, the reaction of azido building blocks 3, 13, and
14 with aromatic acetylenes resulted in higher yields than reactions with aliphatic acetylenes
(see Supplementary Information for full details).
Synthesis of multivalent triazole AHL library (type V). Having developed efficient
methodology for the synthesis of monomeric triazole HL libraries, we reasoned that the above
L-HL building blocks could also serve as precursors for the construction of novel, multivalent
compounds incorporating several L-HL moieties. Multivalent scaffolds remain largely
unexamined in prior studies of AHL analogs,24, 45, 60 and could provide an interesting means to
increase the effective concentration of ligand in a receptor local environment, thus potentially
enhancing ligand activity (agonistic or antagonistic). Receptor geometry also must be
considered, and the relatively imbedded native ligand-binding site of LuxR-type proteins
61
(assuming non-native analogs target this same site) could preclude such a mechanism.14,
Nevertheless, in order to make multivalent compounds for testing, we investigated the reaction
of azido-functionalized L-HL building blocks with di- and triynes, as well as their reaction with
alkyne-functionalized L-HL building blocks.
Starting with azidoacetyl L-HL building block 3, we examined the CuAAC reaction with
diynes, triynes, and L-HL building block 4. Isolation of the desired triazole products from these
reactions, however, proved quite difficult. Even though reaction stoichiometries were tailored to
each specific reaction, purities of the precipitated products typically proved much lower than the
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purities of the above type I–IV AHL analogs. One exception was V-G, stemming from reaction
of 3 with 1,3-diethynylobenzene, which was isolated in satisfactory purity (85%) after
precipitation with diethylether (see Supplementary Information).
Next, we examined similar reactions with azidobenzoyl building blocks 13 and 14. To our
satisfaction, the reaction with these building blocks proved much improved relative to
azidoacetyl building block 3. As was the case for the reactions with 3, reactant stoichiometries
had to be optimized for each specific reaction. In most cases, however, the desired products
could be isolated in good purities and yields using the diethylether precipitation protocol
described above.
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O
O
NH
O
O
N
H
N
O
N
O
O
HN
O
O
N
N
N
N
O
O
N
V-A
V-E
NH
O
N
O
O
N
N
O
O
N
N
O
NH
O
O
NH
O
O
N
H
O
N
H
N
N
N
N
N
O
N
O
V-B
O
V-F
O
O
H
N
H
N
N
N
N
H
N
V-C
N
O
O
N
O
O
NH
N
N
N
O
N
O
O
V-G
O
O
O
H
N
N
H
N
O
O
N
N
O
O
O
O
O
O
HN
V-D
O
O
NH
N
N
O
N
O
N
N
O
O
N
O
NH
N
O
N
HN
N
N
O
N
N
N
NH
O
N
O
N
N
N
V-H
N
O
N
V-I
N
O
HN
O
O
Figure 3. Structures of the type V triazole L-HL library.
LasR antagonism by triazole HLs. We first evaluated the ability of the five triazole HL
libraries to modulate the activity of the major LuxR-type QS receptor in P. aeruginosa, LasR.
P. aeruginosa uses LasR to control virulence factor production (e.g., elastase production),
motility, and biofilm formation, and is an extremely attractive target for small molecule
modulation.61 LasR activity is regulated by its cognate AHL signal, N-(3-oxo-dodecanoyl)-L-
homoserine lactone (OdDHL). We screened the five triazole HL libraries for LasR antagonism
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using an Escherichia coli strain harboring both a LasR expression vector and a LasR β-
galactosidase reporter.62 Compounds were evaluated for receptor antagonism at 100 µM (except
where limited by solubility, see Table 4) against OdDHL at 10 nM. The primary screening data
for each library are listed in Tables 1 and 3–5. Triazole HLs exhibiting >50% LasR antagonism
were subjected to dose response analyses to determine their IC50 values (listed in Table 2).
Table 1. Primary agonism and antagonism assay data
of the type I and type II triazole HL libraries in
LasR.a
P. aeruginosa – LasR
compound
Activation [%]b
Inhibition [%]c
I-A
I-B
I-C
I-D
I-E
0
0
17
39
0
0
1
47
5
0
0
0
0
49
0
3
0
3
42
1
55
75
55
23
52
70
72
14
56
21
71
49
38
7
61
75
77
72
21
65
58
I-F
I-G
I-H
I-I
I-J
I-K
I-L
I-M
I-N
I-O
II-A
II-B
II-C
II-D
II-E
II-F
13
a
See Experimental Section for details of reporter strains
and methods. All assays performed in triplicate. Error
b
≤10%. AHLs evaluated at 100 µM and normalized to
c
OdDHL at 100 µM (100%). AHLs evaluated at 100 µM
against OdDHL at 10 nM.
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Table 2. IC50 values for the most potent LasR
antagonists in this study.a
IC50 value (µM)b
compound
I-B
I-C
I-F
I-G
I-I
I-K
II-A
II-C
II-F
IV-AE
17.6
3.27
16.3
12.4
15.9
13.7
16.5
10.9
4.03
2.64
a
See Supplementary Information for plots of antagonism
b
dose response curves. IC50 values determined by testing
AHLs over a range of concentrations (10 nM – 100 µM)
against OdDHL at 10 nM.
The type I and II triazole HL libraries contained the majority of the active LasR modulators
identified in this study. These two triazole libraries were typified by short acyl chain segments
(one or two carbon atoms) separating the L-HL and functionalized triazole units (Figure 2). In
general, the LasR antagonism trends by the type I and II triazole HL libraries were analogous to
those observed for non-native acyl HLs with similar acyl groups. Notably, these type I and II
triazole HL analogs demonstrated enhanced potency relative to the recently reported AHL
analogs with triazole and tetrazole units in place of the native amide bond.56
In the type I library, the acyl chain segment was a single methylene unit (Figure 2). The
triazole ring, derived from the azidoacetyl building block 3, was oriented differently than it was
in the type II ligands. The 2-N atom of the triazole ring then mimics the 3-oxo functionality
found in OdDHL, the native AHL for LasR. We observed that the type I triazole HLs with alkyl
chains extending from the triazole ring (i.e., I-A–I-D) displayed trends parallel to previously
30
reported 3-oxo acyl-HLs;23,
that is, triazole HLs with short alkyl chains were LasR
antagonists, while triazole HLs with longer chains were LasR agonists (see below). As the alkyl
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chain increased in length from propyl (I-A, 55%) to butyl (I-B, 75%, IC50 value = 17.6 µM),
inhibitory activity increased from moderate to strong (Table 1). Upon further elongation of the
chain from pentyl (I-C, 55%, IC50 value = 3.2 µM)) to hexyl (I-D, 23%), LasR antagonism
diminished and concomitant LasR agonistic activity increased (17 and 39%, respectively; see
below). Interestingly, this transition from antagonist to weak agonist occurred as the entire
length of the acyl chain (counting the shortest distance across the triazole ring) transitioned
from 10 to 11 atoms, a chain length approaching that of the native LasR ligand, OdDHL (with
12 carbon atoms). This result suggests that acyl chain lengths that mimic native AHL ligands
can lend agonistic activity to triazole HLs.
Of the cycloalkyl triazole derivatives in the type I triazole HL library (I-E–I-H), those with
larger ring structures were stronger LasR antagonists than those with smaller rings (Table 1).
For example, the cyclopropyl derivative (I-E) was a moderate LasR inhibitor (52%), but the
cyclopentyl (I-F, 70%, IC50 value = 16.3 µM) and cyclohexyl (I-G, 72%, IC50 value = 12.4 µM)
derivatives were both more potent LasR inhibitors. We note that the inclusion of an additional
methylene unit between the triazole and cyclohexyl group (as in I-H) led to weaker LasR
inhibition (14%) and stronger LasR activation instead; we return to this interesting activity
switch below.
Type I triazole HLs with aryl acyl groups were found to antagonize LasR (Table 1). These
results correlate well with activity trends for phenylacetanoyl HLs (PHLs; e.g., C14, Figure 4),
which we have shown to strongly inhibit many LuxR-type receptors.23, 30 The unsubstituted
phenyl-triazole HL (I-I, 56%, IC50 value = 15.9 µM) was a moderate antagonist of LasR.
Compared to I-I, the 3,5-difluoro derivative (I-K, 71%, IC50 value = 13.7 µM) was a slightly
better LasR antagonist; however, triazole HLs with bulkier substituents, such as the
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methoxynaphthyl derivative (I-M, 38%) and the biphenylether derivative (I-J, 21% inhibition),
were less active. In turn, the thiophene derivative (I-O, 61%) was a moderate LasR antagonist,
and similar inhibitory activity was observed previously with the structurally-related thiophene
AHL D3 (Figure 4).23, 30
O
O
O
O
N
O2N
H2N
N
H
H
O
O
B11
O
O
C14
H3C
HO
O
O
O
O
N
H
N
H
C19
C21
H3CO
O
O
O
O
O
N
H
N
H
O
O
O
C24
C23
O
O
O
N
H
N
H
S
O
D3
O
n
O
R1 n = 1, C2
R2 n = 2, C3
R3 n = 3, C4
R4 n = 4, C5
R5 n = 6, C7
O
N
H
O
S6
Figure 4. Selected non-native AHLs previously evaluated for LasR agonism and antagonism23, 30 with
structures similar to the compounds reported in this study.
In general, the LasR antagonism activity trends for the type II triazole HLs were consistent
with those observed for type I ligands with similar structural scaffolds (Tables 1 and 2). The
para-aniline derivative (II-B, 77%) inhibited LasR somewhat more than meta-aniline (II-F,
58%) in the primary screen; however, dose response analysis revealed that II-F maintained its
inhibitory activity at lower concentrations (IC50 value = 4.03 µM) relative to II-B (IC50 value >
20 µM). Modest inhibition was also observed for phenol derivative II-E (65%), yet an IC50
value could not be obtained due to a large drop off in activity at lower concentrations. In
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contrast, the anisole derivative (II-A, 75%, IC50 value = 16.5 µM) and the toluene derivative
(II-C, 72%, IC50 value = 10.9 µM) continued to inhibit LasR at low concentrations. Our
previous studies had shown that PHLs with similar acyl groups as these triazole HLs (C19,
C21, C23, and C24; Figure 4) were only poor LasR antagonists (< 20%).23, This result
30
indicates that inclusion of triazole functionality into the phenylacetanoyl-HL scaffold can
augment their inhibitory activity against LasR.
Type III triazole HL library. Within the type III triazole HL library, the phenylthioether
derivatives III-C and III-D were the most potent LasR antagonists (Table 3), with the para-
triazole HL III-C slightly more active than the meta-triazole HL III-D. Other members of this
library were only weak LasR antagonists, but the para-triazole HL isomers were still typically
the most potent (e.g., the indole (III-A, 23% vs. III-B, 7%) and phenol derivatives (III-G, 19%
vs. III-H, 11%)).
Table 3. Primary agonism and antagonism assay
data of the type III triazole HL library in LasR.a
P. aeruginosa – LasR
Activation [%]
Inhibition [%]
compound
III-A
III-B
III-C
III-D
III-E
III-F
III-G
III-H
III-I
1
1
0
0
1
0
1
0
0
1
23
7
48
40
14
16
19
11
16
17
III-J
a See Table 1 for footnotes.
Type IV triazole HL library. As a result of their alternate synthesis, the type IV triazole
HLs, synthesized from azidobenzoyl HL building blocks 13 and 14 instead of alkyne-
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functionalized benzoyl HL building blocks 11 and 12, had different triazole linkages relative to
the type III triazole HLs, with the variable substituent installed at the 5-position as opposed to
the 1-position of the triazole (Figure 2). Interestingly, this structural modification yielded
compounds with enhanced antagonistic activities against LasR relative to their type III analogs
(Table 4). Similar to the type III triazole HLs, the para-substituted type IV triazole HLs were
generally stronger LasR antagonists than their meta-substituted analogs. We note that certain
type IV triazole HLs had limited DMSO solubility, so these compounds were evaluated at 10
µM instead of 100 µM in the reporter assays (as indicated in Table 4).
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Table 4. Primary agonism and antagonism assay data of the
type IV triazole HL library in LasR.a
P. aeruginosa – LasR
Activation [%]
Inhibition [%]
100 µM 10 µM
compound
100 µM
10 µMa
IV-A
IV-B
IV-C
IV-D
IV-E
IV-F
IV-G
IV-H
IV-I
1
1
0
0
–
–
–
–
0
0
–
–
–
–
–
–
0
0
–
–
–
2
–
–
–
–
–
–
–
–
0
–
–
–
–
–
0
0
0
0
–
–
1
0
0
0
0
0
–
–
0
0
3
–
1
0
1
0
0
0
6
0
–
0
46
29
60
36
–
–
–
–
54
46
–
–
–
–
–
–
38
21
–
–
–
14
–
–
–
–
–
–
–
–
62
6
12
–
13
–
44
18
50
21
10
14
21
20
50
21
18
17
–
IV-J
IV-K
IV-L
IV-M
IV-N
IV-O
IV-P
IV-Q
IV-R
IV-S
IV-T
IV-U
IV-V
IV-W
IV-X
IV-Y
IV-Z
IV-AA
IV-AB
IV-AC
IV-AD
IV-AE
IV-AF
–
1
1
5
–
17
18
9
21
13
20
11
22
47
–
a
b
See Table 1 for footnotes. Compounds evaluated at 10 µM due to
limited DMSO solubility. OdDHL control remained as listed in Table
1 for all compounds.
The first sub-set of the type IV library (IV-A–IV-H) was designed to evaluate the effects of
alkyl substituents of variable chain lengths (see Figure 2). For the para-substituted series, LasR
antagonism clearly increased with increasing alkyl chain length (from three to six carbons). The
meta-substituted analogs, while less active overall, exhibited a similar inhibitory trend against
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LasR. These results are in accord with activity trends for our previously reported, structurally
related acyl-HLs (R1–R6, Figure 4), where LasR antagonism likewise increased with alkyl
substituent length.34
The second sub-set of type IV triazole HLs (IV-I–IV-P) was investigated to explore the
influence of cycloalkyl substituents on compound activity (Figure 2). These derivatives were
modest to moderate antagonists of LasR (Table 4). Within the sub-set of compounds, the para-
substituted cyclopropyl derivative (IV-I, 54%) was a relatively strong LasR inhibitor at 100
µM. However, when evaluated at the same concentration as the rest of the cycloalkyl series (10
µM), the larger cyclopentyl (IV-K, 21%) and cyclohexyl derivatives (IV-M, 50%) were more
effective LasR inhibitors than the cyclopropyl derivative (IV-I, 10%). A similar inhibition trend
was observed for cycloalkyl-substituted analogs in the type I triazole HL library (as discussed
above). This trend was repeated with the meta-substituted cycloalkyl library members; however,
similar to the other triazoles in this library, the antagonistic activities of the meta-substituted
derivatives were slightly diminished relative to the para-substituted derivatives.
The third sub-set of type IV triazole HLs contained relatively bulky aryl substituents (IV-Q–
IV-Z, Figure 2), and were found to be only modest LasR antagonists (Table 4), suggesting that
there may be a limitation to the size of substituents on the type IV triazole HL scaffolds for
effective LasR inhibition. The difluorophenyl derivatives (IV-S, 1%, and IV-T, 1%) were far
less active here relative to the structurally related (and potent) type I triazole HL (I-K, 71%, IC50
value = 13.7 µM; see above). The unsubstituted phenyl derivatives (IV-Q, 38% at 100 µM, and
IV-R, 21% at 100 µM) were modest LasR inhibitors, but the further modifications of the phenyl
ring tested led to diminished activity. Interestingly, in this sub-set of derivatives, the meta-
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substituted ligands were actually more active than the para-substituted ligands, opposing the
trend observed with the rest of the type IV triazole HL library.
The fourth and final sub-set of type IV triazole HLs contained heterocyclic moieties or
related heteroatom substituents (IV-AA–IV-AF, Figure 2). These derivatives were less active
than their corresponding type I analogs (Table 4), with the one striking exception – the aryl
thioether IV-AE. This type IV triazole HL was a stronger LasR antagonist (62%, IC50 value =
2.64 µM) than its isomeric analog in the type III library (III-C, 48%). Notably, IV-AE was the
strongest LasR antagonist identified in this study, and is comparable in activity to some of the
most potent acyl-HL-derived LasR antagonists we have recently reported.23, 30 Intriguingly, the
structurally related type I ligand (I-N) was a strong LasR agonist instead (see below). Similar to
other derivatives in this library, LasR antagonism was severely diminished for the meta-
substituted thioether analog of IV-AE, IV-AF (6%).
Lastly, the type V triazole HL library contained derivatives with multiple HL rings linked
together by a triazole ring, a phenyl ring, or a tertiary amine (Figure 3). In general, these
multivalent scaffolds were only modestly active as LasR antagonists (Table 5), and the dimeric
compounds (V-A–V-F) were more potent than the two trimeric compounds (V-I and V-H). The
most active compound (V-B, 22%) was derived from meta-alkyne benzoyl HL and a para-
azidobenzoyl HL. Other variants, linked through the opposite alkyne/azide coupling partners
(V-C, 14%) or meta-substituted (V-D, 10%) or para-substituted (V-A, 14%) aromatic rings
were less active. Larger triazole HL dimers linked via a phenyl ring between two triazole-
benzoyl HLs (V-E, 16%, and V-F, 16%) displayed similar modest activities against LasR.
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Table 5. Primary agonism and antagonism assay data
for the type V library in LasR.a
P. aeruginosa–LasR
compound
Activation [%]
Inhibition [%]
V-A
V-B
V-C
V-D
V-E
V-F
V-G
V-H
V-I
0
1
1
1
0
0
8
16
2
14
22
14
10
16
16
6
1
5
a See Table 1 for footnotes.
LasR agonism by triazole HLs. We next examined the ability of the five triazole HL
libraries to agonize LasR using the E. coli reporter strain introduced above. Each compound was
tested for LasR activation at 100 µM (except where limited by solubility, see Tables 1 and 3–5
and Experimental Section). Relative to OdDHL at 100 µM (set to 100%), only seven triazole
HLs were capable of > 10% LasR agonism (I-C, 17%; I-D, 39%; I-H, 47%; I-N, 49%; II-D,
42%; II-F, 13%; and V-H, 16%; see Figures S-1–S-3 in the Supplementary Information for full
primary screening data). Due to their limited activities, none of these synthetic AHLs were
active and soluble over a range of concentrations that enabled the determination of accurate
EC50 values. Still, several of these compounds and their related SAR trends are worthy of
further discussion.
Four of the seven LasR agonists were uncovered in the type I triazole HL library. While
short alkyl-substituted triazoles (propyl or butyl chains) were LasR antagonists (as discussed
above), two with longer alkyl chains had diminishing antagonistic activity and enhanced
agonistic activity in LasR (i.e., pentyl triazole (I-C, 17% agonism) and hexyl triazole (I-D, 39%
agonism)). Again, this activity transition occurred as the entire length of the triazole acyl chain
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approached that of the OdDHL acyl chain (12 atoms). Notably, a third type I agonist (I-H, 47%)
only deviated from triazole I-G, one of the top 10 LasR antagonists (IC50 value = 12.4 µM)
identified herein (see above), by a methylene unit between the cyclohexyl ring and the triazole
unit (Figure 2). Interestingly, acyl HLs structurally similar to I-H and previously studied in our
laboratory (e.g., S6 and B11, Figure 4) do not display LasR agonistic activities, although they
are agonists of other LuxR-type receptors.30, 34 We postulate that the triazole ring in I-H, a 3-oxo
mimic that is absent in both B11 and S6, could play a role in its agonistic activity in LasR. The
fourth type I LasR agonist identified was the aryl thioether derivative (I-N, 49%). This triazole
HL represented the most potent LasR agonist uncovered in this study, and is amongst the most
potent non-native LasR agonists that we have identified to date.20, 23, 30 Two type II triazole HLs
were modest to moderate LasR agonists, meta-anisoyl HL (II-F, 13%) and the meta-
benzaldehyde-substituted HL (II-D, 42%). Lastly, the para-substituted trimeric HL derivative
(V-H) was a modest LasR agonist (16%) – additional studies would be required to determine if
multivalency plays any role in this activation effect. The meta-substituted trimeric HL analog
(V-I, 2%) was inactive, however, corroborating trends observed in the triazole libraries
discussed above.
AbaR antagonism and agonism by triazole HLs. We next examined the ability of the five
triazole HL libraries to modulate the AbaR receptor in A. baumannii. A. baumannii is a human
pathogen that regulates biofilm formation and surface motility through one AHL signal, (R)-N-
(3’-hydroxydodecanoyl)-L-homoserine lactone (OH-dDHL) and one putative LuxR-type
receptor, AbaR.52, 63, 64 Both of these phenotypes are linked to virulence in A. baumannii. We
recently reported a set of non-native AHLs that inhibit motility and biofilm formation in this
pathogen;52 however, to date, few small molecules are known to modulate QS in A. baumannii,
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and we seek additional SAR information to develop optimized AbaR inhibitors. While the LasR
and AbaR receptors recognize structurally similar natural ligands (OdDHL and OH-dDHL,
respectively), we found that the activity trends for identical non-native AHLs in these two
receptors vary greatly.30, 52 Therefore, we hypothesized that screening the triazole AHL libraries
for activity in both LasR and AbaR could reveal additional, unique structure–activity
information for these important QS receptors.
We evaluated the five triazole HL libraries for AbaR antagonism and agonism in an A.
baumannii (ΔabaI) mutant strain harboring an AbaR β-galactosidase reporter (see Experimental
Section for a detailed description of this reporter system and assay protocols).64 Overall, we
identified fewer modulators of AbaR in the triazole HL libraries relative to LasR (23 triazole
HLs displayed > 20% inhibition, and one triazole HL displayed > 10% agonism; listed in Tables
6 and 7). Several of the active compounds merit additional discussion.
Most of the AbaR modulators were type I triazole HLs (12 of the 24 compounds), and
within this structure class, the disparity between their SAR for LasR and AbaR antagonism was
relatively clear. Type I triazole HLs I-A–I-D closely mimic the structure of OdDHL (the natural
ligand of LasR), and these triazoles exhibited SAR in LasR that paralleled that for acyl-HLs (as
discussed above). However, the length of the acyl chain did not influence their ability to inhibit
AbaR (I-A, 47%; I-B, 42%; I-C, 29%; I-D, 41%), and none of these ligands activated this
receptor. These results corroborate finding from our earlier study of AbaR modulators, which
demonstrated that AbaR is very selective with regards to activation by acyl-HLs: only OH-
dDHL strongly activates AbaR and AHLs with other acyl chain lengths and oxidation states at
the 3-position were significantly less active.52 In turn, one sub-set of type I triazole HLs
displayed similar SAR in AbaR as in LasR: compounds with large cycloalkyl substituents (I-F,
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61%; I-G, 68%) were more potent inhibitors than those with smaller ring substituents (I-E,
42%). We highlighted above that the addition of a methylene spacer between the cyclohexyl
group and the triazole group of I-G (to give I-H) had converted a LasR inhibitor (I-G; 72%
antagonism) into a LasR activator (I-H; 47% agonism). In AbaR, however, I-H exhibited
antagonism only (50%).
Table 6. Primary agonism and antagonism assay
data of the most potent triazole HLs in AbaR.a
A. baumannii – AbaR
compound Activation [%]d
Inhibition [%]e
I-A
I-B
I-C
I-D
I-E
0
0
0
0
4
0
0
0
0
0
0
5
9
0
2
0
0
47
42
29
41
42
61
68
50
59
25
32
55
51
21
58
33
22
I-F
I-G
I-H
I-I
I-K
I-N
I-O
II-A
II-B
II-C
II-D
II-F
a
See Experimental Section for details of reporter
strains and methods. All assays performed in
b
triplicate. Error = ±10%. AHLs evaluated at 100
µM and normalized to OH-dDHL at 100 µM (100%).
c
AHLs evaluated at 100 µM against OH-dDHL at
700 nM.
Five type II triazole HLs displayed moderate AbaR antagonism in the reporter gene assay
(Table 6). The toluyl derivative (II-C), harboring a weakly electron–donating substituent, was a
moderate AbaR inhibitor (58%). As the substituents on the phenyl group became more electron
withdrawing, antagonistic activity diminished (e.g., anisole (II-A, 51%) > benzaldehyde (II-D,
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33%) > aniline (II-B, 21%; II-F, 22%) > phenol (II-E, 9%) derivative). Similar to LasR, the
type III triazole HLs were only modest modulators of AbaR (see Figure S-6 in Supplementary
Information for full data). The type IV triazole HL library was more active in AbaR, however,
and contained six antagonists and the only AbaR agonist revealed by the primary screen. The
majority of the AbaR antagonists of this type were alkylated triazoles (IV-A–IV-H; 25-36%).
Interestingly, the lone AbaR agonist identified (aryl thioether IV-AE, 26%) is also the most
potent LasR antagonist identified in this study (62%, IC50 value = 2.64 µM). This divergence in
activity is striking, and suggests that IV-AE could be modulating AbaR and LasR activity by
different mechanisms. We note that the most potent LasR agonist identified in this study was
also an aryl thioether derivative (I-N, 49%), albeit with a type I scaffold. This convergence of
activity for aryl thioester-substituted triazole HLs suggests that further analysis of this
substructure is warranted in new AHL analog design. Finally, while triazole HL IV-AE was
only a moderately weak AbaR agonist, its discovery is significant, as few non-native agonists of
AbaR have been reported to date (only seven including IV-AE).52
Table 7. Primary agonism and antagonism assay data of
the triazole type IV library in AbaR.a
A. baumannii – AbaR
Activation [%]
100 µM 10 µM
Inhibition [%]
100 µM 10 µM
compound
IV-C
IV-D
IV-E
IV-F
IV-I
1
0
–
–
1
–
36
25
–
–
22
–
–
–
30
26
–
–
0
0
–
0
–
IV-Z
IV-AE
–
26
42
–
2
a
b
See Table 6 for footnotes. Compounds evaluated at 10 µM due to
limited DMSO solubility. OH-dDHL control remained as listed in
Table 6 for all compounds.
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Summary and Conclusions
In conclusion, we have developed simple and efficient methodology for the rapid synthesis
of AHL mimics using CuAAC reactions. Efficient protocols were developed for the synthesis of
L-HL building blocks that contain alkyne or azido handles. These building blocks smoothly
underwent cycloaddition reactions with readily–available alkyne and azide counterparts. A
straightforward protocol was developed for the rapid isolation of triazole products via simple
precipitation in generally good to excellent yields and purities. This methodology enabled the
construction of a series of four triazole libraries (type I-IV) where the AHLs were linked to
various hydrophobic groups via a 1,2,3-triazole moiety. In addition, the building blocks were
also used for the construction of a series of multivalent triazole AHL analogs (type V)
incorporating two or three L-HL head groups. The resulting 72 compounds were systematically
evaluated in cell-based, reporter gene assays for their ability to agonize or antagonize two QS
receptors – LasR and AbaR – in the clinically relevant pathogens P. aeruginosa and A.
baumannii, respectively. Several potent and novel agonists and antagonists were identified.
A considerable number of SARs were apparent from the results of the biological screens
(Figure 5). Of the five triazole types, the type I and II triazole HLs were the most active in
general, comprising all but one of the most potent antagonists identified in this study (the outlier
being the type IV triazole HL, IV-AE). The type I and II scaffolds most closely resembled the
AHLs that naturally activate LasR and AbaR (i.e., OdDHL and OH-dDHL, respectively). The
strongest LasR antagonists contained short alkyl chains (I-A–I-D); these triazole derivatives
transitioned from inhibitory to activating in LasR as the chain length approached that of
OdDHL. This trend was not unique to this scaffold, as alkylated type IV ligands (IV-A–IV-H)
displayed parallel activities. Cycloalkyl and heterocyclic type I and II triazole HLs were active
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inhibitors of both receptors: derivatives with large saturated rings (I-G) inhibited both receptors
strongly. Interestingly, the potent inhibitor I-G was converted to a strong LasR agonist upon the
inclusion of a single methylene unit between the triazole and cyclohexyl group (I-H). This
divergence in activity was not observed in the AbaR screen. Unlike previous studies of
structurally related phenylacetanoyl-HLs (i.e., PHLs) in LasR, triazole-derived
phenylacetanoyl-HLs (II-A–II-F) were modest LasR antagonists, suggesting that the inclusion
of triazole functionalities into certain scaffolds heightens inhibitory activity against LasR.
Agonists
Antagonists
N
N
N
N
O
long alkyl chain
phenyl thioether
short alkyl chain
N
N
O
O
O
O
O
O
O
LasR
cycloalkyl
phenyl
N
N
H
H
O
O
O
O
N
H
multiple
substitutents
N
N
N
N
H
saturated linear
or cycloalkyl
N
N
N
N
N
N
O
N
N
O
short alkyl chain
AbaR
phenyl thioether
O
O
cycloalkyl
N
N
N
H
unsubstituted
phenyl
H
O
O
Figure 5. General SAR trends for LasR and AbaR modulators identified in this study.
Of the type III and IV triazole HL libraries, the type IV compounds, with variable
substituents installed at the 5-position as opposed to the 1-position of the triazole (e.g., III-C
and IV-AE), were more active (Figure 5). Within these libraries, alkyl-substituted derivatives
(IV-A–IV-H) displayed activities parallel to the type I triazole HLs (I-A–I-D). Benzoyl
derivatives were typically inhibitory; para-substituted derivatives were generally more active
29