From in vitro to in cellulo: Structure-activity relationship of (2-nitrophenyl)methanol derivatives as inhibitors of PqsD in Pseudomonas aeruginosa

Article abstract of DOI:10.1039/c4ob00707g

Recent studies have shown that compounds based on a (2-nitrophenyl)methanol scaffold are promising inhibitors of PqsD, a key enzyme of signal molecule biosynthesis in the cell-to-cell communication of Pseudomonas aeruginosa. The most promising molecule displayed anti-biofilm activity and a tight-binding mode of action. Herein, we report on the convenient synthesis and biochemical evaluation of a comprehensive series of (2-nitrophenyl)methanol derivatives. The in vitro potency of these inhibitors against recombinant PqsD as well as the effect of selected compounds on the production of the signal molecules HHQ and PQS in P. aeruginosa were examined. The gathered data allowed the establishment of a structure-activity relationship, which was used to design fluorescent inhibitors, and finally, led to the discovery of (2-nitrophenyl)methanol derivatives with improved in cellulo efficacy providing new perspectives towards the application of PqsD inhibitors as anti-infectives. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00707g

Volume 12 Number 32 28 August 2014 Pages 6017–6280
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
ISSN 1477-0520
PAPER
Martin Empting, Rolf W. Hartmann et al.
From in vitro to in cellulo: structure–activity relationship of (2-nitrophenyl)-
methanol derivatives as inhibitors of PqsD in Pseudomonas aeruginosa

Organic &
Biomolecular Chemistry
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From in vitro to in cellulo: structure–activity
relationship of (2-nitrophenyl)methanol derivatives
as inhibitors of PqsD in Pseudomonas aeruginosa†
Cite this: Org. Biomol. Chem., 2014,
12, 6094
Michael P. Storz,a Giuseppe Allegretta,a Benjamin Kirsch,a Martin Empting*a and
Rolf W. Hartmann*a,b
Recent studies have shown that compounds based on a (2-nitrophenyl)methanol scaffold are promising
inhibitors of PqsD, a key enzyme of signal molecule biosynthesis in the cell-to-cell communication of
Pseudomonas aeruginosa. The most promising molecule displayed anti-biofilm activity and a tight-
binding mode of action. Herein, we report on the convenient synthesis and biochemical evaluation of a
comprehensive series of (2-nitrophenyl)methanol derivatives. The in vitro potency of these inhibitors
against recombinant PqsD as well as the effect of selected compounds on the production of the signal
molecules HHQ and PQS in P. aeruginosa were examined. The gathered data allowed the establishment
of a structure–activity relationship, which was used to design fluorescent inhibitors, and finally, led to the
discovery of (2-nitrophenyl)methanol derivatives with improved in cellulo efficacy providing new perspec-
tives towards the application of PqsD inhibitors as anti-infectives.
Received 3rd April 2014,
Accepted 29th May 2014
DOI: 10.1039/c4ob00707g
www.rsc.org/obc
general. In this regard, QS regulates a variety of virulence
factors, which contribute to breaking the first line defences
Introduction
Until recently, bacterial communities were seen as nothing and damaging surrounding tissues leading to dissemination,
more than an accumulation of autonomous single-celled systemic inflammatory-response syndrome, multiple organ
organisms. But today, we are aware that bacteria use cell-to-cell failure, and, finally, death of the host.2 Furthermore, QS con-
communication systems like quorum sensing (QS) to behave col- tributes to the collective coordination of biofilm formation, a
lectively rather than as individuals.1 Small diffusible molecules key reason for bacterial resistance against conventional anti-
are produced by single bacterial cells that can be released into biotics in clinical use.3 Thus, the importance of these regulat-
the environment and detected by surrounding bacteria. Upon ory systems could be exploited for the design of novel anti-
proliferation, the extracellular signal molecule concentrations infectives.
increase along with cell density. Once a certain threshold is
Several groups have successfully targeted QS, which is dis-
reached, receptors are activated by these autoinducers result- cussed as an alternative to the traditional treatment using bac-
ing in population-wide changes in gene expression. This con- tericidal or bacteriostatic agents (for reviews see ref. 4 and 5).
certed switch from low- to high-cell-density mode allows single Novel anti-virulence compounds ideally decrease pathogenicity
bacterial cells to limit their group-beneficial efforts to those without affecting bacterial survival or growth, whereas it is
cell densities which guarantee an effective group outcome. believed that no or less selection pressure is posed on the
Even if QS may not be directly essential for the survival of a bacteria. Hence, a reduced rate of newly occurring resistances,
singular bacterial cell, it is very important for bacteria–host which gradually render existing antibiotics ineffective, is
interactions and pathogenesis upon bacterial infections in expected.6
Among bacteria, very different communication systems
based on distinct autoinducers are utilized. Gram-positive bac-
teria primarily use modified oligopeptides, whereas N-acyl
aHelmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus C23,
66123 Saarbrücken, Germany. E-mail: rolf.hartmann@helmholtz-hzi.de;
homoserine lactones are a major class of signal molecules in
Gram-negative bacteria.1 The opportunistic pathogen P. aerugi-
nosa additionally utilizes a characteristic pqs system, which is
based on the quinolone PQS (Pseudomonas Quinolone Signal)
and its biosynthetic precursor HHQ (2-heptyl-4-quinolone)
(Fig. 1).7
Fax: +49 681 302 70308; Tel: +49 681 302 70300
bPharmaceutical and Medicinal Chemistry, Saarland University, Campus C23, 66123
Saarbrücken, Germany
†Electronic supplementary information (ESI) available: Synthesis and analytics
of all synthetic intermediates and all substrates used in the enzyme inhibition
assay as well as procedure of the mutagenicity test; HHQ and PQS inhibition in
the PA14 wild-type strain. See DOI: 10.1039/c4ob00707g
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Fig. 1 Role of PqsD in HHQ and PQS biosynthesis (top). Structures of previously reported PqsD inhibitors 1 and 2.
Both are able to activate the transcriptional regulator PqsR
leading to the production of various virulence factors like
pyocyanin and hydrogen cyanide (HCN).8 We have shown, that
PqsR antagonists efficiently decrease pyocyanin production
and pathogenicity of P. aeruginosa PA14.9,10 Furthermore, HHQ
and PQS contribute to the formation of biofilms.3
Biosynthesis of HHQ and PQS is accomplished by proteins
Fig. 2 Systematically varied structural features of inhibitor 3.
encoded by the pqsABCD operon. Thereby, experiments using
transposon knockout mutants identified PqsD as key enzyme
in the cellular signal molecule production route.11,12 Recently, PqsD inhibition.16 Indeed, this compound was capable of
Dulcey et al. reported that cytoplasmic PqsD catalyses the con- reducing the HHQ and PQS levels. Furthermore, biofilm for-
mation was significantly inhibited and no antibiotic effects
densation of anthraniloyl-CoA (ACoA) and malonyl-CoA to
2-aminobenzoylacetate (2-ABA, Fig. 1).13 The resulting reactive were observed.
intermediate is then processed to HHQ by PqsC using octanoic
acid as substrate. This second reaction step is supported by
Binding studies of 3 revealed apparent irreversibility and
that binding occurs near the active site residues.17 Both enan-
PqsB by an unknown mechanism. Interestingly, PqsD alone is tiomers showed similar affinity but contrary thermodynamic
also capable of generating HHQ in vitro directly from ACoA profiles. Based on site-directed mutagenesis, isothermal titra-
using β-ketodecanoic acid as secondary substrate.14 This enzy-
tion calorimetry (ITC) analysis, and molecular docking, explicit
matic reaction has been routinely exploited by us to evaluate binding modes were proposed. In these predicted enzyme–
PqsD inhibitors.14–18 Inhibition of PqsD is an attractive strat- inhibitor complexes both enantiomers reside in nearly identi-
cal positions with the main difference being the orientation of
egy to interfere with QS-controlled infection mechanisms,
since it is essential for cellular HHQ/PQS formation. A pqsD the hydroxyl group at the stereogenic center.17
transposon mutant strain of P. aeruginosa PAO1, which is
Herein, we present a target-oriented (in vitro) structure–
activity relationship and optimization of this compound class
deficient in PQS formation, shows decreased levels of pyo-
cyanin and HCN as well as reduced lethality in nematodes.11 based on the (2-nitrophenyl)methanol scaffold by systematic
Furthermore, putative inhibitors of PqsA, an enzyme involved structure variation (Fig. 2) investigating also the time-depen-
dent onset of inhibition. Previously, we reported, that a tetra-
in earlier stages of HHQ biosynthesis, block the cellular
production of the corresponding signal molecules, prevent hedral geometry including an acceptor function is favoured for
systemic dissemination, and attenuate mortality in infected the linker between both phenyl rings.16 However, the intrinsic
mice.19
nitrophenyl moiety bears an increased risk of toxic, mutagenic
Derived from compounds active against FabH, a structurally and carcinogenic side effects.20 Thus, we evaluated the muta-
and functionally related enzyme, we have identified and opti- genicity in Ames Salmonella assays and investigated suitable
chemical replacements. Additionally, the influences of substi-
mized the first PqsD inhibitors demonstrating IC50 values in
the single-digit micromolar range (Fig. 1, 1).14,15 Unfortu- tuents with opposed electronic and hydrophilic properties in
nately, these compounds had no pronounced effect on the 4- and 5-position of the nitrophenyl moiety were studied. Fur-
thermore, a variety of aliphatic and aromatic residues instead
P. aeruginosa PA14 (unpublished data). Recently, in a ligand- of the second phenyl ring were examined.
extracellular signal molecule levels in cell-based assays using
based approach we have identified compound 2 as a novel
inhibitor of PqsD (Fig. 1).16 Ligand efficiency-guided optimi-
The gathered information enabled us to design fluorescent
inhibitors, which may be useful tools to investigate
sation led to compound 3 (Fig. 2), which was used for an enzyme inhibitor interactions and to visualize the target in
initial examination of the effects on PA14 cells mediated by cells.21,22
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Finally, selected compounds were examined regarding their trifluoroacetic acid and basic hydrolysis. This route also
potency to inhibit signal molecule production in P. aeruginosa provided access to the cyclic carbamate 7.
PA14 cells. Thereby, we additionally applied a novel strategy to
The carboxylate 11 was prepared by iso-propylmagnesium
reduce costs and time by the usage of a pqsH-deficient mutant chloride-mediated iodine–magnesium exchange on methyl
which has been selected from a transposon mutant library.23 2-iodobenzoate 9 and subsequent addition to benzaldehyde.
This procedure allows to evaluate the potency of a compound This reaction is followed by spontaneous cyclisation yielding
solely by quantification of HHQ instead of two signal mole- lactone 10, which was hydrolyzed under basic conditions to
cules. In this way we identified compounds with increased yield the desired carboxylate. An analogous method employing
in cellulo activity while a low molecular weight is retained iodine–magnesium exchange was used for synthesis of the
(<250 Da). These optimized fragment-like molecules provide trifluoromethyl and nitril derivatives 14 and 15. In the case
the potential for further improvements by a fragment growing of compounds 21–25 and 28–29, corresponding aldehyde
approach. Additionally, an attempt to correlate in vitro data precursors were commercially available. Hence, the desired
with the effects observed in the cellular assays is made.
products were prepared by direct addition of phenylmagne-
sium chloride.
The synthesis of (2-nitrophenyl)methanol derivatives 3,
33–35, 39, 40, 43–44, and 47–86 followed the general pathways
outlined in Scheme 2. For all compounds, in which Z1 or Z2
were exclusively substituted by hydrogen or methyl, phenyl-
magnesium chloride was added to 36–38 to accomplish
iodine–magnesium exchange in ortho position to NO2 as
described by Knochel and coworkers.24 The generated
Grignard reagents were reacted with the appropriate aldehydes
to form the desired products.
Results and discussion
Chemistry
In order to find alternatives to the nitro group, a variety of
molecules with different chemical functionalities were syn-
thesized (Scheme 1). Assembly of (2-aminophenyl)(phenyl)-
methanol 8 started with the formation of Boc-protected aniline
5. Ortho-lithiation by tert-butyllithium and subsequent reac-
tion with benzaldehyde yielded the alcohol 6. The desired
product 8 was obtained by a two-step deprotection using
For synthesis of the methoxy derivatives 43 (Z1 = OMe) and
44 (Z2 = OMe) from 41 and 42 we utilized 4-methoxyphenyl-
magnesium bromide as novel reagent to accomplish
Scheme 1 Synthesis of compounds 8, 11, 14–15, 21–25, and 28–29. Reagents and conditions: (a) Boc2O, THF, reflux, 38%; (b) tBuLi, THF, −60 °C;
(c) benzaldehyde, THF, −20 °C, 35% (2 steps); (d) TFA, DCM, 0 °C–room temp, 78%; (e) KOH, MeOH–H2O, reflux, 17%; (f) iPrMgCl, THF, −40 °C;
(g) benzaldehyde, THF, −40 °C, 63% (2 steps); (h) NaOH, MeOH–H2O, 50 °C, 39%; (i) iPrMgCl, THF, −40 °C; ( j) benzaldehyde, THF, −40 °C, 47–71%
(2 steps); (k) PhMgCl, THF, 0–50 °C, 36–92%; (l) PhMgCl, THF, −40 °C, 7–12%.
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Scheme 2 Synthesis of compounds 3, 33–35, 39–40 and 43–44 and 47–86. Reagents and conditions: (a) PhMgCl, THF, −40 °C; 20–58%;
(b) PhMgCl, THF, −40 °C; (c) aldehyde, THF, −40 °C, 11–86% (2 steps); (d) 4-methoxyphenylmagnesium bromide, THF, −40 °C; (e) NaBH4, MeOH,
0 °C–room temp, 62%; (f) hydrazine hydrate, EtOH, reflux, 62–89%; (g) NBD chloride, NaHCO3, MeOH, room temp – 50 °C, 40%; (h) HOOC-
(CH2)mNH-NBD (84i, 85i), EDC·HCl, HOBt·H2O, NMM, acetonitrile, room temp, 32–46%; (i) dansyl chloride, TEA, DCM, room temp, 29–45%;
( j) fluorescein iso-thiocyanate, DIEA, DMF, room temp, 36%.
iodine–magnesium exchange in ortho-iodo-nitrobenzenes. ing carboxylic acids. The fluorescein derivative 86 was formed
Application of this method to a broader range of substrates upon reaction with fluorescein iso-thiocyanate.
will be discussed elsewhere.
Essentiality of the nitro-group and Ames test
Fluorescent derivatives were prepared by cleavage of the
phthalimide moiety of 68–70 via the Ing-Manske procedure. After synthesis, compounds were evaluated regarding their
The released amines 77–79 served as a starting point for the inhibitory activity against heterologously expressed and puri-
introduction of fluorophores. Coupling these amines with fied PqsD using ACoA and β-ketodecanoic acid as substrates.14
dansyl chloride yielded derivatives 80–82. Direct attachment of Until recently, β-ketodecanoic acid instead of malonyl-CoA has
NBD to the amine 78 using NBD-chloride afforded 83, whereas been considered as the second substrate in HHQ synthesis,
84 and 85 were synthesized by coupling with the NBD contain- since it has been shown, that addition of β-ketodecanoic acid
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to the anthraniloyl-PqsD complex leads to HHQ formation seems to be more favourable. In contrast, introduction of the
in vitro.14 We have clearly shown that (2-nitrophenyl)methanol electron-donating (EDG) methyl (39, 40) or methoxy (43, 44)
derivatives interfere with the formation of the anthraniloyl- groups led to potent compounds with IC50 values in the range
PqsD complex itself, which allows further usage of β-ketodeca- of the unsubstituted 3. Again, a preference for the introduction
noic acid independently of its function in the bacterial cells.17
of substituents at Z2 was observed (40, 44). These observations
First, a variety of substituents replacing the nitro group in are in accordance with our proposed binding mode reported
ortho position were tested. An amino group (8) in analogy to earlier,17 as the para position to the nitro group provides more
ACoA, which served as template for the inhibitor design,16 led space to accommodate additional substituents. An explanation
to an inactive compound. This was also true for substituents for the general detrimental effect of EWGs on affinity could be
with electron-withdrawing properties similar to the nitro that the nitro group functions as hydrogen bond acceptor (as
group, as trifluoromethyl (14), nitril (15) and halogens (21, 22). in our proposed binding model). This ability might possibly
Furthermore, no activity was observed for molecules bearing be diminished by electron-withdrawing substituents. Unfortu-
potential hydrogen bond acceptors like 7, 10, the carboxylate nately, none of the modifications installed in this part of the
11, 24 and 25. Since the nitro group seems to be essential for scaffold led to an improvement of inhibitor potency. However,
activity, we shifted the position in meta or para position (28, comparing the determined IC50 for 10 min and 30 min, it
29), but inhibitory potency was again completely abolished. An seems that the Z2 position provides the opportunity to modu-
initial toxicity study provided promising results, since no toxic late the binding behaviour. Through the choice of either
effect against human THP-1 macrophages was observed at methyl (EDG) or chloro (EWG) substituents, the onset (see IC50
250 µM of compound 3.16 To assess the mutagenic risk of the at 10 min) can be either slightly accelerated (3 vs. 40) or slowed
compound class, Ames Salmonella assays were performed. down (3 vs. 34).
Compound 3 was tested on Salmonella typhimurium derived
In light of the results gathered so far, we kept the unsubsti-
strains TA100, TA1535 and TA102 with and without metabolic tuted nitrophenyl moiety constant and turned our attention to
activation by liver homogenate (S9 mix). No biologically rele- the second residue R of the methanol moiety. Hydrogen or
vant increase in the number of revertant colonies was observed linear alkyl chains of different length were introduced (45,
at dose levels up to 5000 µg per plate. Thus, the nitro group 47–51). Thereby, shorter residues up to ethyl (45, 47, and 48)
was retained and we focused our efforts on the improvement were favoured over longer variants (49–51). A plausible expla-
of inhibitory activity by introduction of additional substituents nation is the increasing entropic penalty caused by the limit-
into the nitrophenyl moiety.
ation of rotational freedom upon formation of the inhibitor–
enyzme complex.
In vitro SAR
Except iso-butyl-bearing compound 52, branched and cyclic
Recently, we have reported extensive studies on the mode of isomers 53–56 generally inhibited PqsD less efficiently than
action of the (2-nitrophenyl)methanol scaffold.17 In this their linear congeners. This might be due to the narrow
regard, we demonstrated a time-dependent onset of inhibition entrance channel, which hampers the binding of the bulky
based on a slow non-covalent (reversible) interaction. As we residues.
have observed that inhibition onset levels out after 20 min of
Short alkyl linkers were inserted between the tetrahedral
preincubation for compound 3,17 we consider a period of carbon and the phenyl group, but neither compound 57 nor
30 min appropriate for the rapid evaluation of the set of novel 58 showed improved IC50 values compared to 3. Thus, we con-
compounds (n ∼ 50) described herein. Additionally, we cluded that direct attachment to the methanol moiety brings
measured the inhibitory activity using only 10 min of preincu- the aromatic residue in an optimal position and fused a
bation to gain qualitative insight into the effect of inhibitor second benzene ring. But the resulting 1-naphthyl and
modifications on binding behaviour.
2-naphthyl isomers 59 and 60 were less potent PqsD
This examination is relevant, as it has been reported that inhibitors.
time-dependency of enzyme–inhibitor interactions can have
Hence, monocyclic heteroaromatic residues were intro-
significant impact in the efficacy of compounds in the cellular duced. For all the thiophene and pyridine derivatives 61, 62,
system.25
and 65–67 moderate activity without further improvement was
First, we re-evaluated our starting compound 3 applying an observed. The furane derivatives showed conspicuous behav-
optimized protocol for the prolonged pre-treatment period of iour, since the differences in activity between the oxygen in
enzyme with inhibitor (Table 1). As described earlier,17 an 2- or 3-position were tremendous. While the 3-furyl derivative
improvement of potency was observed rendering this com- 64 was almost inactive, the 2-furyl isomer 63 shows improved
pound now a sub-micromolar PqsD inhibitor.
PqsD inhibition. The synthetic route towards fluorescent
In a subsequent step, we investigated the effect of different (2-nitrophenyl)methanol derivatives provided additional
substituents within the nitrophenyl moiety. Compounds inhibitors of PqsD with non-fluorescent residues R as inter-
bearing electron-withdrawing substituents (EWG) as chlorine mediates. The amines 77–79 may be considered as direct
(33, 34) or nitro (35) showed diminished inhibitory activity derivatives of the alkyl compounds 49–51, whereas the term-
(Table 1). However, concerning target affinity (IC50 at 30 min inal methyl was substituted by an amino group, which is
of preincubation) the para substitution pattern (Z2 in Table 1) expected to be protonated under assay conditions. In contrast
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Table 1 PqsD inhibition by (2-nitrophenyl)methanol derivatives
Compounds
Z1
Z2
R
IC50 [µM] (10 min)a,b
IC50 [µM] (30 min)a,c
3
H
Cl
H
NO2
Me
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
Me
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
Me
Et
n-Pr
n-Bu
n-Pentyl
iso-Bu
tert-Pentyl
c-Pentyl
c-Hexyl
1-Adamantyl
CH2Ph
CH2CH2Ph
1-Naphthyl
2-Naphthyl
2-Thienyl
3-Thienyl
2-Furyl
3-Furyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
(CH2)2Phth
(CH2)3Phth
(CH2)4Phth
Et
2-Thienyl
3-Thienyl
2-Furyl
(CH2)2Phth
(CH2)3Phth
(CH2)2NH2
(CH2)3NH2
(CH2)4NH2
3.2 0.1
13.4 1.4
15.0 0.6
15.4 2.0
3.7 0.5
1.9 0.4
2.2 0.5
3.0 0.4
1.6 0.5
1.3 0.3
1.1 0.2
2.8 0.4
5.2 0.8
4.9 0.9
7.9 1.0
15.9 1.1
4.9 1.0
10.1 1.4
11.6 2.2
5.4 0.6
4.6 1.0
10.8 2.5
13.1 1.9
14.3 1.9
5.9 0.9
1.8 0.4
28% @ 50 µM
6.7 1.2
11.7 2.1
6.8 1.4
1.2 0.1
1.9 0.3
1.7 0.5
0.7 0.3
6.2 1.8
3.3 0.5
0.9 0.1
0.9 0.1
0.7 0.1
34.7 4.6
44% @ 50 µM
8.2 2.1
3.5 0.1
3.0 1.3
3.2 0.4
4.3 0.5
46.5 2.1
13.1 1.5
1.3 0.7
0.5 0.1
11.2 0.9
1.6 0.1
5.8 1.0
1.6 0.1
0.6 0.1
1.6 0.3
0.5 0.1
0.7 0.2
0.8 0.1
0.8 0.1
1.0 0.4
2.9 0.1
1.0 0.4
1.2 0.3
6.7 0.1
1.4 0.2
4.5 0.2
2.7 0.1
0.8 0.1
0.9 0.1
5.8 0.2
2.4 0.5
1.5 0.2
6.4 1.9
0.9 0.1
13.1 2.8
1.2 0.1
1.7 0.1
2.2 0.1
0.3 0.1
0.7 0.2
1.6 0.4
0.6 0.1
2.7 0.5
1.6 0.3
1.1 0.1
0.7 0.1
0.7 0.2
40.0 3.7
27.8 1.7
22.4 4.3
5.8 0.2
4.0 0.7
1.4 0.3
3.4 0.2
12.5 4.2
10.0 0.7
1.5 0.3
33
34
35
39
40
43
44
45
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80d
81d
82d
83d
84d
85d
86d
Me
Me
Me
Me
Me
Me
H
H
H
a P. aeruginosa PqsD (recombinantly expressed in Escherichia coli), anthraniloyl-CoA (5 µM), and β-ketodecanoic acid (70 µM). b IC50 values were
determined using a 10 min preincubation period of inhibitor and enzyme followed by a 40 min reaction time. c IC50 values were determined
using a 30 min preincubation period of inhibitor and enzyme followed by a 40 min reaction time. d For the structure of the fluorescent
derivatives 80–86 see Scheme 2.
to the alkyl compounds, the amines 77–79 showed low activity.
The phthalimides 68–70, on the other hand, also differing
This result was expected, as the entrance of the substrate in the length of the alkyl linker, showed potent PqsD inhi-
tunnel is decorated with arginine side chains providing a bition. Compound 68 even demonstrated the lowest IC50 value
repulsive positive surface polarization.
within the investigated set of compounds of around 300 nM.
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So far, novel interesting derivatives with retained potency
Table 2 Comparison of inhibitory potency regarding HHQ production
in a P. aeruginosa pqsH mutant with physicochemical properties
and reduced molecular weight (45, 47, 48, and 63) or even
improved target affinity (68) have been identified. Moreover,
some compounds showed a pronounced difference between
the IC50 values measured via the 10 min and 30 min protocol
(34, 52, 61, and 66) indicating a slow onset of inhibition. As
described above, introduction of a methyl group in para posi-
tion to the nitro group resulted in a reduction of time-depen-
dency while retaining activity for compound 40. These results
encouraged us to synthesize additional selected derivatives
possessing this methyl group (71, 72). Indeed, all of these
methyl-containing compounds showed a fast onset of inhi-
bition (Table 1) while being potent inhibitors of PqsD in the
single-digit micromolar to submicromolar range. However, no
further improvement in target affinity has been gained com-
pared to the most potent compound 68. Nevertheless, together
with the unmethylated congeners (48, 61–63, 68, and 69) inter-
esting pairs of PqsD inhibitors for further evaluation in cellulo
have been yielded.
IC50 (10 min)/IC50 % HHQ
Cmpd MWa
Log Pa,b LEa,c (30 min)a
inhibitiond
3
229.23 2.47
0.52
0.39
0.45
0.37
0.45
0.48
0.43
0.46
0.78
0.71
0.66
0.60
0.52
0.53
0.51
0.45
0.53
6.4
1.2
9.4
2.7
2.3
3.2
1.4
6.0
2.3
1.6
1.4
2.8
1.7
4.9
8.7
0.8
2.0
43
6
33
34
35
39
40
43
44
45
47
48
49
50
51
61
62
63
64
68
69
70
71
72
73
74
75
76
80
81
82
83
84
85
86
263.68 3.16
263.68 3.12
274.23 2.14
243.26 2.93
243.26 2.93
259.26 2.69
259.26 2.55
153.14 0.76
167.16 1.11
181.19 1.64
195.22 2.17
209.24 2.71
223.27 3.24
235.26 2.15
235.26 2.15
219.19 1.63
219.19 1.63
326.30 2.61
340.33 2.84
354.36 2.98
195.22 2.10
249.24 2.61
249.24 2.61
233.22 2.09
340.33 3.07
354.36 3.30
429.49 3.71
443.52 3.94
457.54 4.08
373.32 1.10
444.40 2.15
486.48 2.65
599.61 2.94
n.i.
n.i.
10
20
19
n.i.
n.i.
13
26
61
4
3
7
1
6
2
7
22
n.i.
n.i.
64
74
51 15
73
6
6
0.43 n.d.e
2
0.38
0.34
0.31
0.62
0.46
0.48
0.49
0.34
0.33
0.24
0.24
0.26
0.28
0.21
0.20
0.19
4.0
2.7
1.1
1.2
2.3
2.1
0.8
1.3
1.0
0.6
0.8
2.3
1.3
3.7
1.3
0.9
n.i. @ 125 µM
n.i. @ 125 µM
n.i. @ 125 µM
n.i.
Apparently, various substituents of R are tolerated by PqsD,
which encouraged us to introduce fluorescent groups in this
position. Since promising inhibitory activity was observed for
compound 2, we substituted the pantothenate moiety by
24 0.2
23
20
7
2
dansyl
(5-(dimethylamino)-naphthalene-1-sulfonyl),
NBD
n.i. @ 125 µM
n.i. @ 100 µM
n.i. @ 75 µM
n.i. @ 75 µM
n.i. @ 25 µM
n.i. @ 150 µM
(7-nitrobenz-2-oxa-1,3-diazol-4-yl) and fluorescein fluoro-
phores. The flexible linker of 2 was conserved to provide
sufficient degrees of conformational freedom to adopt to the
sterical requirements of the substrate tunnel. In the case of
the dansyl derivatives 80–82, the chain length of the alkyl
linker was only slightly varied with the intention to position
the hydrophobic fluorophore within the channel. For the more
hydrophilic NBD derivatives 83–85 additional acyl linkers were
introduced as well, thereby shifting the fluorophore towards
the protein surface. Fortunately, an acceptable in vitro activity
was observed for the dansyl derivatives 80–82, and compounds
with NBD (83) or fluorescein (86) directly attached to the
amine 78. However, the NBD fluorophores 84 and 85 contain-
ing an additional acyl-linker were less potent. Unfavourable
interactions and/or entropic penalties might be possible
reasons.
17
27
3
1
n.i.
a Molecular weight (MW), calculated partition coefficient (log P),
ligand efficiency index (LE), and the ratio of IC50 values measured
using different preincubation periods. b Calculated by ACD/Labs 2012
using the ACD/Log P Classic algorithm. c Values calculated as LE = 1.4
(−log IC50)/N using the IC50 value for the prolonged incubation time
(30 min) and N meaning number of non-hydrogen atoms. d Planctonic
P. aeruginosa PA14 pqsH mutant. Inhibitor concentration 250 µM as
not indicated otherwise. Percentage of inhibition was normalized
regarding OD600. n.i. no significant inhibition (<10%). e n.d. means
“not determined”.
Inhibition of signal molecule production in a P. aeruginosa
pqsH mutant
If soluble, all compounds were tested at 250 µM. The
in cellulo results are summarized in Table 2 together with
relevant parameters like molecular weight (MW), calculated
partition coefficient (log P), ligand efficiency index (LE), and
the ratio of IC50 values measured using different preincubation
periods.
In an attempt to elucidate the physicochemical and/or func-
tional requirements for high in cellulo efficacy, we tested
selected compounds regarding their ability to reduce the
signal molecule levels in P. aeruginosa PA14 (Table 2).
Inhibitor 3, which served as a starting point, decreased
HHQ production by 43%. Introduction of methyl groups into
the nitro-phenyl moiety (39 and 40) reduced inhibitory
potency, whereas methoxy substituents (43 and 44) led to inac-
tivity. These results are disappointing, since two of the com-
pounds showed submicromolar in vitro activity. A similar
result was obtained for molecules bearing a substituent in the
nitrophenyl moiety: the chloro compounds 33 and 34 as well
as the dinitrophenyl-derivative 35 showed no or only low
In the wild-type strain, HHQ is converted into PQS by PqsH
and the expression of pqsH depends on the growth period.26
Thus, we quantified HHQ in a pqsH mutant, which is not able
to perform this oxidation, to increase simplicity and reproduci-
bility of this cell-based assay. To ensure the validity of this
novel methodology, we compared the results gathered using
the pqsH mutant with additionally determined PA14 wild-type
data for three selected compounds and observed a good corre-
lation between both data sets (see ESI†).
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activity in the cells. Furthermore, no effects on HHQ pro- features of compounds displaying intracellular activity are all
duction despite of most promising IC50 values were observed the more worthwhile to investigate.
for the phthalimides 68–70, 75, and 76, which were tested at
100–125 µM due to limited solubility.
In this context, time-dependent inhibition was one charac-
teristic of the presented compound class that has first drawn
All congeners of the homologous series 45–51 containing our attention. Reversible target interaction (inhibition) is
unbranched alkyl residues showed good activity in the enzy- characterized by inhibitor binding (kon, “on-rate”) and dis-
matic assay, whereas the in vitro activity slightly decreased for sociation (koff, “off-rate”). Thus, a slow binding behaviour in
the larger alkanes. In the cellular test system, the largest resi- combination with promising (nanomolar) IC50 values would
dues (50 and 51) led to inactivity, while a maximum HHQ inhi- imply long drug-residence times as the “off-rate” should also
bition was observed for ethyl (48). This compound showed an be attenuated along with the inhibition onset (“on-rate”). Such
improved cellular activity compared to the starting point 3. an effective (long-lasting) enzyme blockade can be considered
Interestingly, the variant with methyl in para position to the a favourable scenario in order to shut down cellular signal
nitro group (71) was inactive, although it possesses a slightly molecule synthesis. However, we were not able to establish a
improved IC50 value.
correlation between this phenomenon and improved HHQ
Surprisingly, all four compounds containing hetero- reduction. Some compounds with either high or low ratios of
aromatic pentacycles 61–64 potently reduced the HHQ for- IC50 values measured using 10 min or 30 min of preincubation
mation. These compounds were not among the most potent were effective inhibitors of signal molecule production
PqsD inhibitors in vitro. Moreover, 64 showed only a moderate (compare for example 3 and 61 with 62 and 70). Indeed, we
activity in the double-digit micromolar range against recombi- showed that the methyl modification at the nitrophenyl core
nant PqsD. We can only speculate whether additional alterna- results in compounds with only low time-dependent onset of
tive cellular targets are involved or whether the furanyl residue inhibition along with a decreased or even abolished in cellulo
is converted into a more active compound inside the cell. efficacy (48 vs. 71 and 61 vs. 72). Nevertheless, the most potent
Multiple examples for the instability of furan are reported in inhibitor in our cell assays was 62 showing almost no time-
the literature. However, the thiophene 62 is the most potent dependency of PqsD inhibition.
inhibitor of cellular HHQ formation reported so far. Again, the
methyl modification in Z2 of the nitrophenyl moiety resulting cellular activity, though, was molecular weight. Most com-
in compounds 72–74 was detrimental to in cellulo efficacy. pounds with MW > 300 Da were inactive inside the cells. The
A parameter which seems to have direct influence on intra-
Finally, we examined the effect of fluorophore-labelled only exceptions within the set of tested compounds were NBD-
derivatives 80–86 on P. aeruginosa to evaluate their potential tagged fluorophores 84 and 85 showing only a moderate
for applications in cellular systems. Unfortunately, neither the reduction of HHQ production. This observed mass cut-off is
dansyl- nor fluorescein-labelled inhibitors (80–82 and 86) were much lower than the general value of 600 Da reported for
able to reduce HHQ formation at 250 µM. Consequently, their Gram-negative bacteria and should not be considered a strict
application is restricted to studies using lysed cells or isolated criteria due exceptions mentioned above.30 This finding might
biomolecules. The best results were obtained for NBD deriva- be explained by the described low permeability of outer mem-
tives 84 and 85, which showed at least slight inhibition. This brane of P. aeruginosa compared to other Gram-negative
opens up avenues towards intracellular labelling experiments, bacteria.31
which might be conducted in the future.
Another important physicochemical parameter for cellular
availability is hydrophobicity usually expressed as the octanol–
water partition coefficient log P. None of the fragment-like
compounds reported herein can be considered as strongly
Interpretation of in vitro and in cellulo data
A first conclusion which can be drawn from the detailed lipophilic as none of them exceeds log P ≈ 4 while the majority
experimental data reported herein is that in the case of possesses a value below 3. However, it has been proposed by
(2-nitrophenyl)methanol derivatives in vitro potency expressed others that even lower log P values are beneficial or actually a
either as IC50 or ligand efficiency (LE, Table 2) does not prerequisite for activity against Gram-negative bacteria.30
directly translate into in cellulo activity. However, this obser- Indeed, our most active compound in cellulo possess a log P
vation is not utterly surprising as Gram-negative bacteria, in below 2.5.
general, and P. aeruginosa, in particular, are known to provide
Noteworthy, we have identified compounds within the set
challenging barriers for the effective inhibition of intracellular of tested (2-nitrophenyl)methanol derivatives that are interest-
targets.27–29 The orthogonal sieving ability of the two bacterial ing exceptions to the described MW/log P criteria. Compound
cell membranes blocking larger hydrophobic compounds 71, for example, has low molecular weight (195.22 Da) and log
(outer membrane) as well as smaller hydrophilic entities P (0.62) combined with a substantial in vitro activity (IC50
=
(inner membrane) from entering the cytoplasm is only one 0.6) but no relevant in cellulo activity. We account this finding
obstacle which an anti-infective agent has to overcome.27,28 to the general detrimental effect of the methyl group in para
Additionally, an arsenal of efflux pumps and degrading/meta- position to the nitro group which we have also found for the
bolizing enzymes may hinder a drug from reaching its target.29 other compounds bearing this substitution pattern. On the
Hence, the physicochemical, structural, and functional other hand, developed NDB-tagged inhibitors possess
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increased molecular weight and hydrophobicity, yet show eluent. The purity of compounds used in the biological assays
moderate activity on signal molecule production. Hence, was ≥95% as measured by LC/MS, monitored at 254 nm. The
further optimization of our fragment-like molecules towards methods for LC/MS analysis and a table with analytical data
drug-like compounds via fragment growing approaches may be (including melting points) for all tested compounds are pro-
a rewarding endeavour.
vided in the purity section of the ESI.† All chiral alcohols were
1
isolated as racemates. H and 13C NMR spectra were recorded
on a Bruker DRX-500 instrument at 300 K. Chemical shifts are
reported in δ values (ppm) and the hydrogenated residues of
deuterated solvents were used as internal standard (acetone-
Conclusions
More than fifty derivatives of (2-nitrophenyl)phenylmethanol 3 d6: 2.05, 29.84. CDCl3: δ = 7.26, 77.16. MeOH-d4: δ = 3.31,
have been synthesized and tested for in vitro activity. In this 49.00. DMSO-d6: δ = 2.50, 39.52). Splitting patterns describe
regard, we demonstrated that 4-methoxyphenylmagnesium apparent multiplicities and are designated as s (singlet), d
bromide is a suitable reagent to accomplish efficient I–Mg (doublet), dd (doublet of doublet), t (triplet), td (triplet of
exchange for compound 42. Whereas the nitro group in ortho doublet), q (quartet), m (multiplet). All coupling constants (J)
position turned out to be essential for in vitro PqsD inhibition, are given in hertz (Hz). Mass spectra (ESI) were measured on a
no mutagenicity was observed for compound 3 in an Ames Thermo Scientific Orbitrap. Mass spectra (EI) were measured
test. Improved potency was achieved by the replacement of the on a DSQII instrument (ThermoFisher). Melting points of
eastern phenyl residue. This position turned out to be very tol- samples were determined in open capillaries using a SMP3
erant for various moieties, which allowed the design of fluo- Melting Point Apparatus from Bibby Sterilin and are uncorrected.
rescence-labelled inhibitors.
Infrared spectra were measured on a PerkinElmer Spectrum
For a straightforward evaluation of PqsD inhibitors in the 100 FT-IR spectrometer.
cellular context, signal molecule production was investigated
General method A for synthesis of 3, 39, 40, 48–76
using a pqsH-deficient P. aeruginosa PA14 strain. Some of our
compounds showed significantly improved inhibition of signal A solution of 2-iodo-nitrobenzene 36 or a close derivative
molecule production. An attempt to correlate in vitro data with (1.0 eq.) in THF (10 ml g−1 reagent) was cooled to −40 °C and
in cellulo results has been made identifying low molecular a solution of phenylmagnesium chloride (2 M in THF, 1.1 eq.)
weight and hydrophobicity as important, but not stringent, cri- was added dropwise. After stirring for 30 min at −40 °C, the
teria for intracellular activity. Nevertheless, the presented work aldehyde (1.0 eq.) was added. Then, the reaction mixture was
emphasizes the notion that optimization of intracellular continuously stirred at −40 °C until complete conversion
activity is a challenging multi-parameter problem which (checked by TLC). The reaction was quenched with a saturated
requires further intense research.
solution of NH4Cl (5 ml) and diluted with water (5 ml). The
Finally, novel PqsD inhibitors presented in this work aqueous phase was extracted with ethyl acetate (three times)
possess a fragment-like size and improved efficiency in cellulo. and the combined organic layers were washed with brine,
Together with the structural insight provided by our studies dried, and concentrated under reduced pressure. The residue
regarding the mode of action, we have delivered the basis for a was purified by column chromatography over silica gel to give
fragment-growing optimization process towards PqsD-target- the desired product.
ing anti-infectives.
General method B for synthesis of 10, 14, and 15
A solution of the iodobenzene 9, 12 or 13 (1.00 g, 1.0 eq.) in
30 ml THF was cooled to −40 °C and a solution of iso-propyl-
magnesium chloride (2 M in THF, 1.1 eq.) was added drop-
wise. The solution was stirred for 60 min at −40 °C,
Experimental section
General
(2-Nitrophenyl)methanol 45 was purchased from Sigma- benzaldehyde (1.0 eq.) was added and the reaction was com-
Aldrich and used for biological assays without further purifi- pleted at −40 °C (checked by TLC). The workup was carried
cation. Starting materials were purchased from ABCR, Acros, out as described in method A.
Sigma-Aldrich and Fluka and were used without further purifi-
General method C for synthesis of 21–25
cation. All reactions were conducted under a nitrogen atmos-
phere. During workup drying was achieved by anhydrous To a solution of phenylmagnesium chloride (2 M in THF,
sodium sulfate. Flash chromatography was performed using 1.5 eq.) in 8 ml THF at 0 °C aldehydes 16–20 (1 eq.) were
silica gel 60 (40–63 μm) and the reaction progress was deter- slowly added and the solution was stirred at 50 °C for 30 min.
mined by TLC analysis on ALUGRAM SIL G/UV254 (Macherey- The mixture was cooled to 0 °C, quenched with a saturated
Nagel). Visualization was accomplished through excitation solution of NH4Cl (5 ml), and diluted with water (5 ml). The
using UV light. Purification by semi-preparative HPLC was aqueous phase was extracted with diethyl ether (three times)
carried out on an Agilent 1200 series HPLC system from and the combined organic layers were dried and concentrated
Agilent Technologies, using an Agilent Prep-C18 column (30 × under reduced pressure. The residue was purified by column
100 mm/10 μm) as stationary phase with acetonitrile–water as chromatography over silica gel to give the desired product.
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General method D for synthesis of 28 and 29
Determination of extracellular HHQ levels
To a solution of phenylmagnesium chloride (3.31 ml, 2 M in Extracellular levels of HHQ were determined according to the
THF, 6.62 mmol) in 20 ml THF at −40 °C nitrobenzaldehyde method of Lépine et al. with the following modifications.32,33
26 or 27 (1.00 g, 6.62 mmol) was added slowly. The reaction An aliquot of 500 µl of bacterial cultures were supplemented
mixture was stirred continuously at −40 °C until complete con- with 50 µl of a 10 µM methanolic solution of the internal stan-
version (checked by TLC). The mixture was quenched with a dard (IS) 5,6,7,8-tetradeutero-2-heptyl-4(1H)-quinolone (HHQ-d4)
saturated solution of NH4Cl (5 ml) and diluted with water and extracted with
1 ml of ethyl acetate by vigorous
(5 ml). The workup was carried out as described in method A, shaking. After centrifugation, 400 µl of the organic phase were
followed by purification of the crude product by flash chrom- evaporated to dryness in LC glass vials. The residue was re-dis-
atography (petroleum ether–ethyl acetate 6 : 1).
solved in methanol. UHPLC-MS/MS analysis was performed as
described in detail recently.16 The following ions were moni-
tored (mother ion [m/z], product ion [m/z], scan time [s], scan
width [m/z], collision energy [V], tube lens offset [V]): HHQ:
244, 159, 0.5, 0.01, 30, 106; HHQ-d4 (IS): 248, 163, 0.1, 0.01, 32,
113. Xcalibur software was used for data acquisition and
quantification with the use of a calibration curve relative to the
area of the IS.
General method E for synthesis of 77–79
To a solution of the phthalimides 68–70 (1.53 mmol, 1 eq.) in
ethanol (40 ml) hydrazine hydrate (9.18 mmol, 6 eq.) was
added and the mixture was refluxed for 3 h. Ethanol was
removed in vacuo and ethyl acetate was added. The solution
was washed with water and extracted twice with EtOAc. The
organic phase was dried and the solvent was evaporated to
yield the desired product in sufficient purity.
Calculation of log P values
Experimental values of 67 and 79 were determined by Sirius
T3 titrator from Sirius Analytical. Comparison with values cal-
culated by ACD/Labs 2012 (Build 1996, 31. May 2012) revealed,
that ACD/Log P Classic is the most appropriate algorithm. Con-
sequently, the latter was used for the values given in Table 2.
Expression and purification of recombinant PqsD
Expression and purification of recombinant PqsD was con-
ducted as previously described.16 Briefly, BL21 (λDE3) E. coli
transformed with expression vector pT28b(+)/pqsD were
induced with IPTG overnight. After harvesting and lysis
through sonication, recombinant PqsD possessing a His6-tag
was isolated via immobilized metal ion affinity chromato-
graphy (IMAC) followed by gel filtration. The affinity tag was
removed by thrombin cleavage and a second IMAC step.
Abbreviations
2-ABA
ACoA
2-Aminobenzoylacetate
Anthraniloyl-CoA
Enzyme inhibition assay using recombinant PqsD
dansyl
5-(Dimethylamino)-naphthalene-1-sulfonyl
The standard assay for determination of IC50 values was per-
formed monitoring the enzyme activity by measuring the HHQ
concentration as described recently.14 PqsD was preincubated
with inhibitor for 10 min or 30 min prior to addition of the
substrates ACoA and β-ketodecanoic acid. Quantification of
HHQ was performed analogously, but with some modifi-
cations: The flow rate was set to 750 µl min−1 and an Accucore
RP-MS column, 150 × 2.1 mm, 2.6 µm, (Thermo Scientific) was
used. All test compound reactions were performed in sextupli-
cate. Synthesis of ACoA and β-ketodecanoic acid was per-
formed as described in the ESI.†
EDG/EWG Electron-donating/withdrawing group
HHQ
LE
log P
MW
NBD
PQS
QS
2-Heptyl-4-quinolone
Ligand efficiency
Octanol–water partition coefficient
Molecular weight
7-Nitrobenz-2-oxa-1,3-diazol-4-yl
2-Heptyl-3-hydroxy-4-quinolone
Quorum sensing
Acknowledgements
We thank Carina Scheid, Simone Amann, and Christine
Maurer for performing the PqsD and cellular assays. Further-
more, we thank Prof. Dr Susanne Häußler for kindly supplying
the P. aeruginosa PA14 wild-type strain and PA14 pqsH mutant
and Michael Hoffmann for performing HRMS measurements.
Cultivation of P. aeruginosa PA14 pqsH mutant
For determination of extracellular HHQ levels, cultivation was
performed in the following way: cultures of P. aeruginosa PA14
pqsH transposon mutant23 (initial OD600 = 0.02) were incu-
bated with or without inhibitor (final DMSO concentration
1%, v/v) at 37 °C, 200 rpm and a humidity of 75% for 16 h in
24-well Greiner Bio-One Cellstar plates (Frickenhausen,
Germany) containing 1.5 ml medium per well. Cultures were
generally grown in PPGAS medium (20 mM NH4Cl, 20 mM
KCl, 120 mM Tris-HCl, 1.6 mM MgSO4, 0.5% (w/v) glucose, 1%
(w/v) Bacto™ Tryptone). For each sample, cultivation and
sample work-up were performed in triplicates.
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