Biological Characterization, Mechanistic Investigation and Structure-Activity Relationships of Chemically Stable TLR2 Antagonists

Article abstract of DOI:10.1002/cmdc.202000060

Toll-like receptors (TLRs) build the first barrier in the innate immune response and therefore represent promising targets for the modulation of inflammatory processes. Recently, the pyrogallol-containing TLR2 antagonists CU-CPT22 and MMG-11 were reported; however, their 1,2,3-triphenol motif renders them highly susceptible to oxidation and excludes them from use in extended experiments under aerobic conditions. Therefore, we have developed a set of novel TLR2 antagonists (1–9) based on the systematic variation of substructures, linker elements, and the hydrogen-bonding pattern of the pyrogallol precursors by using chemically robust building blocks. The novel series of chemically stable and synthetically accessible TLR2 antagonists (1–9) was pharmacologically characterized, and the potential binding modes of the active compounds were evaluated structurally. Our results provide new insights into structure-activity relationships and allow rationalization of structural binding characteristics. Moreover, they support the hypothesis that this class of TLR ligands bind solely to TLR2 and do not directly interact with TLR1 or TLR6 of the functional heterodimer. The most active compound from this series (6), is chemically stable, nontoxic, TLR2-selective, and shows a similar activity with regard to the pyrogallol starting points, thus indicating the variability of the hydrogen bonding pattern.

Full text of DOI:10.1002/cmdc.202000060

Accepted Article
Title: Biological characterization, mechanistic investigation and
structure-activity relationship of chemically stable TLR2
antagonists
Authors: Jörg Rademann, Marcel Bermudez, Maria Grabowski,
Manuela S. Murgueitio, Markus Tiemann, Péter Varga,
Thomas Rudolf, Gerhard Wolber, and Günther Weindl
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000060
Link to VoR: https://doi.org/10.1002/cmdc.202000060
01/2020

10.1002/cmdc.202000060
ChemMedChem
FULL PAPER
Biological characterization, mechanistic investigation and
structure-activity relationships of chemically stable TLR2
antagonists
Marcel Bermudez, #[a] Maria Grabowski,#[b] Manuela S. Murgueitio,[a] Markus Tiemann,[a] Péter Varga,[a]
Thomas Rudolf,[a] Gerhard Wolber,*[a] Günther Weindl,*[b,c] and Jörg Rademann*[a]
[a]
Dr. Marcel Bermudez, Dr. Manuela S. Murgueitio, Markus Tiemann, Peter Varga, Thomas Rudolf, Prof. Dr. Gerhard Wolber, Prof. Dr. Jörg Rademann
Institute of Pharmacy (Pharmaceutical and Medicinal Chemistry)
Freie Universität Berlin
Königin-Luise-Str. 2+4, 14195 Berlin, Germany
Email: joerg.rademann@fu-berlin.de, gerhard.wolber@fu-berlin.de
Maria Grabowski,Prof. Dr. Günther Weindl
Institute of Pharmacy (Pharmacology and Toxicology)
Freie Universität Berlin
Königin-Luise-Str. 2+4, 14195 Berlin, Germany
Prof. Dr. Günther Weindl
[b]
[c]
Present address: Section Pharmacology and Toxicology, Pharmaceutical Institute
Universität Bonn
Gerhard-Domagk-Str. 3, 53121 Bonn, Germany
Email: weindl@uni-bonn.de
#
Dr. Marcel Bermudez and Maria Grabowski contributed equally
Supporting information for this article is given via a link at the end of the document.
Abstract: Toll-like receptors (TLRs) build the first barrier in the innate
immune response and therefore represent promising targets for the
modulation of inflammatory processes. Recently, the pyrogallol-
containing TLR2 antagonists CU-CPT22 and MMG-11 have been
reported, however, their 1,2,3-tri-phenol motif renders them highly
susceptible to oxidation and excludes them from use in extended
experiments under aerobic conditions. Therefore, we have developed
a set of novel TLR2 antagonists (1-9) based on the systematic
variation of substructures, linker elements and the hydrogen-bonding
pattern of the pyrogallol precursors by using chemically robust
building blocks. The novel series of chemically stable and
(DAMPs), respectively.[2] TLR2 forms heterodimers with TLR1
and TLR6 and specifically recognizes several components of the
cell wall of gram positive bacteria like di- and tri-acylated
lipoproteins, lipoteichoic acids or lipomannans. After ligand
binding, the preformed dimer undergoes conformational changes
that trigger an intracellular signaling cascade that leads to the
activation of NF-κB and the secretion of pro-inflammatory
cytokines like tumor necrosis factor (TNF) and interleukin (IL)-8.[3]
Under certain circumstances this response is excessive and leads
to severe conditions like sepsis, rheumatoid arthritis, autoimmune
4]
diabetes, asthma and certain types of allergies.[1,
The
synthetically
accessible
TLR2
antagonists
(1-9)
was
modulation of TLR2 function by small molecules has been
postulated as a promising strategy to treat these conditions. To
date only few compounds that modulate TLR2 activity have been
identified and pharmacologically characterized. In 2010 four small
organic molecules with agonistic activity on the receptor were
discovered by high-throughput screening by Guan et al.[5] One of
them was later chemically optimized.[6] In 2012 the first
competitive antagonist CU-CPT22 was discovered by Yin et al.
(Figure 1, left).[7] Virtual screening has successfully been applied
to discover agonists and antagonists for TLR2,[8] but also for other
TLR subtypes.[9] In a previous study, we identified a potent,
competitive and selective TLR2 antagonist MMG-11 [10], however,
it still contained the pyrogallol fragment (Figure 1, right). Since the
pyrogallol scaffold is notorious for its drawbacks including low
chemical stability and poor synthetic accessibility, the
modification of this scaffold to one that is less prone to oxidation
is essential for further optimization steps.
pharmacologically characterized and the potential binding modes of
the active compounds were evaluated structurally. Our results provide
novel insights into structure-activity relationships and allow for
rationalization of structural binding characteristics. Moreover, it
supports the hypothesis this class of TLR ligands bind solely to TLR2
and do not directly interact with TLR1 or TLR6 of the functional
heterodimer. The most active compound from this series (6), is
chemically stable, non-toxic, TLR2-selective and shows a similar
activity with regard to the pyrogallol starting points indicating the
variability of the hydrogen bonding pattern.
Introduction
The first barrier in the innate immune response is formed by the
family of structurally conserved Toll-like receptors (TLRs).[1] In
humans ten functional subtypes (TLR1 – TLR10) have been
identified. TLRs recognize intruding pathogens or endogenous
danger signals released after cell damage or cell death and
activate the innate immune response against them. This happens
through the specific binding of pathogen associated molecular
patterns (PAMPs) and danger associated molecular patterns
1
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Compounds 1, 3, and 6 were synthesized starting from the
corresponding 2,4-, 3,4-, or 3,5-dihydroxy-benzoic acids as
exemplified for compound 6 in Scheme 2. First, both the phenolic
hydroxy groups and the carboxylic acid residues were protected
in one step as O-benzyl-ethers and esters, respectively, using
benzyl bromide with iodide addition and furnishing the tri-O-
benzyl-protected intermediates 12-14.
Figure 1. Previously discovered competitive TLR2 antagonists CU-CPT22 and
MMG-11 both containing the pyrogallol scaffold.[7, 10a]
In this work, we aimed to explore the chemical space around the
pyrogallol-containing antagonists, MMG-11 and CU-CPT22, to
enhance synthetic accessibility and chemical stability, and get
insights into the structure-activity-relationships (SAR) of TLR2
antagonists. We performed synthetic modifications and analog
searches. The synthesized small molecules and selected analogs
were biologically tested for their ability to inhibit TLR2 signaling.
This leads to several novel TLR2 antagonists,
a better
understanding of their SAR and provides a way to rationalize
binding modes of TLR2 antagonists.
Results and Discussion
Compound selection, design and synthesis. A selection of the
compounds that resulted from both chemical synthesis and
similarity-based analog search is shown in Scheme 1. The design
was guided by binding mode investigations of MMG-11 in
complex with TLR2 regarding spatial requirements of the binding
site and potential receptor-ligand interactions. Especially, we
intended to modify the polyphenolic core structure, with the aim of
avoiding the most easily oxidized 1,2-diphenols and 1,2,3-
triphenols or the phenoxy ethers derived from them. Since the
three hydroxyl groups of the pyrogallol scaffold are involved in
hydrogen bonding with the receptor (Figure 3A)[10a], we had to
systematically evaluate these interactions. Therefore, we reduced
the number of hydroxyl groups capable to function as both
hydrogen bond donors and acceptors (1, 3, 5 and 6) and varied
the substitution pattern. For a systematic control, two compounds
still comprising the 1,2,3-trihydroxy motif, 7 and 8, were included
in the study. Furthermore, we introduced methoxy groups, which
can only serve as hydrogen bond acceptors (2 and 4).
Considering the flexibility of the lead structure, we introduced an
amide moiety to rigidify the molecules (1-4 and 6). In order to
enhance synthetic accessibility and the chemical stability, we
exchanged the furan moiety by a phenyl ring in all synthesized
compounds. This resulted in a set of eight synthesis-derived
compounds (1-8, see Scheme 1, 2 and Supporting Information).
In a complementary approach we searched for structural analogs
in the databases which were used for the discovery of MMG-11
by virtual screening.[10a] MMG-11 was used as the query structure
and the databases were searched for similar commercially
available molecules with a Tanimoto coefficient higher than 0.8.
We found three closely related compounds in the Enamine
database (Enamine Ltd, Kiev, Ukraine) which were ordered for
biological evaluation (9-11). Molecular weight and purity (> 95 %)
were confirmed by LC-MS.
Scheme 1. Synthesized and selected compounds. The starting point MMG-
11 is depicted on the top with the different variations highlighted in colors.
Compounds 1 to 8 were rationally designed and synthesized and are shown on
the left side. The compounds selected by analog search (9 to 11) are shown on
the right side.
The tri-O-methyl-protected 3,4-dihydroxy benzoic acid 15 needed
for the synthesis of compound 2 was prepared by an analogous
protocol using methyl iodide for alkylation. Saponification of the
esters 12-15 afforded the free carboxylic acids 16-19 in very good
yields (95%-quantitatively). Next, the prepared carboxylic acids
16-19 or commercially available 3,4-dimethoxy-phenylacetic acid
were activated using O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorphosphate (HATU) in the presence
of ethyl 3-aminobenzoate 20 yielding the protected benzamides 2,
4, and 21-23 as the desired condensation products. Here the
yields were moderate, presumably due to the reduced
nucleophilicity of the aromatic amine in 20. Hydrogenolysis with
palladium on charcoal removed the benzyl ether groups and
The resulting set of eleven derivatives obtained by synthesis and
analog search, provides the possibility to conceive the SAR of
TLR2 antagonists, in particular for the rationalization of the
hydrogen bond pattern of polyphenolic ligands.
2
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furnished the unprotected benzamides 1, 3, and 6 in very good
yields, e.g. 86% for compound 6.
Consequently, compound 11 was excluded from further
investigation. The remaining most active hits, 4, 6, 9 and 10, were
analyzed regarding their potency. For this purpose,
concentration-response curves were assessed and IC50 values
were calculated (Figure 2B). All four compounds showed IC50
values in the µM range, thus confirming their antagonistic effect
as MMG-11 analogs (Table 1Fehler! Verweisquelle konnte
nicht gefunden werden.). Compound 6 turned out to have the
lowest IC50 value of the identified TLR2 inhibitors with similar
results for both heterodimers (TLR2/1: 15.4 µM; TLR2/6: 13.6 µM).
This suggests binding of compound 6 to the TLR2 cavity but no
interaction towards TLR1 or TLR6. While compound 6 effectively
inhibits TLR2 signaling, we could show that it has no influence on
other TLR subtypes (Figure 2C). While keeping the activity of
compound 6 in a comparable range as for CU-CPT22 and MMG-
11, we could replace the highly sensitive 1,2,3-trihydroxyphenyl
structure of pyrogallol in the starting compounds by the chemically
stable 1,3-dihydroxyphenyl substructure of resorcinol. This is
essential for further optimization of TLR2 antagonists, since 6 is
robust, synthetically accessible and no longer susceptibility
toward oxidation.
Scheme 2. Synthesis of inhibitor 6. Reagents and conditions: (a) BnBr, K2CO3,
acetone, reflux, 8 h; (b) NaOH, MeOH, H2O, reflux, 8 h, quant. over 2 steps. (c)
SOCl2, EtOH, reflux, 4 h, 92%. (d) HATU, DIPEA, CH2Cl2, 30 °C, 8 h, 36%; (e)
H2, Pd/C, CH2Cl2, MeOH, 86%. HATU = O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium hexafluorphosphate
Table 1. Table 1: IC50-values of the compounds 4, 6, 9 and 10 by TLR2/1
stimulation (Pam3CSK4, 10 ng/ml) and TLR2/6 stimulation (Pam2CSK4, 1 ng/ml)
in HEK-Blue hTLR2 cells (n=3).
Compound
TLR2/1
TLR2/6
IC50 [µM][a]
IC50 [µM][a]
4
6
44.9 (28.1 – 72.0)
15.4 (8.9 – 26.4)
42.4 (36.9 – 48.7)
44.0 (33.8 – 57.4)
61.9 (18.7 – 204.6)
13.6 (9.9 – 18.6)
42.9 (36.9 – 49.9)
40.7 (30.9 – 53.6)
Aromatic ketone 5, in which the amide linker between two
benzene rings is replaced by a ketomethylene unit, was obtained
via the direct C-acylation of resorcinol (1,3-diphenol) with 3,4-
dimethoxy-phenyl acetic acid using boron trifluoride diethyl
etherate as the activating Lewis acid in 19% yield. Compounds 1-
6 were isolated with >95% purity by column chromatography.
Synthesis of the trihydroxy-derivatives, ethyl 3-(2,3,4-trihydroxy-
9
10
[a] 95% confidence interval shown in brackets
benzamido)-benzoate
7
and
ethyl
3-(3,4,5-trihydroxy-
benzamido)-benzoate 8 was attempted following the same
strategy as in Scheme 2. While the preparation of the tri-O-benzyl-
SAR of novel antagonists. In order to rationalize the bioactivity
and elucidate the potential binding modes of the tested
compounds docking studies were performed. As all characterized
compounds both inhibited TLR2/1 and TLR2/6 signaling, without
showing effects at other TLRs, we hypothesized that they would
bind to the TLR2 monomer and share the same ligand binding
pocket like its pyrogallol-containing precursor, MMG-11.[10a] This
compound has been previously shown to be a competitive
antagonist for the binding of Pam3CSK4 and Pam2CSK4, which
further supports our proposed binding mode. However, an
experimental confirmation of the binding mode is beyond the
scope of this study. Extensive docking analyses were performed
and plausible binding poses selected based on their similarity to
the binding mode of MMG-11 and the amount of performed
interactions between the ligand and the TLR2 binding site. The
resulting proposed binding mode for the most active compound 6
is shown in Figure 3B. Selected docking poses for the remaining
compounds can be found in the Supplementary Figures 2 to 5. 6
is embedded in the front part of the binding pocket with the
dihydroxy-benzamide moiety forming several H-bonds to the
backbone atoms of TLR2. The benzene ring forms hydrophobic
contacts to the Phe325 side chain.
protected precursors of
7
and
8
proceeded smoothly,
debenzylation of the protected intermediates led to the
instantaneous decomposition of these products due to oxidation.
7 and 8 therefore could not be isolated and tested biologically.
Biological validation. To explore the activity of the synthesized
and selected compounds 1 to 6 and 9 to 11 on the TLR2 activity,
reporter cells overexpressing hTLR2 were used. CU-CPT22 was
used as the reference TLR2-antagonist. [7, 10a, 11] Except for 1, all
selected compounds decreased TLR2-mediated NF-κB activation
in a primary screen (Figure 2A). Additionally, 4,6, 9 and 10
reduced Pam3CSK4-induced TLR2/1 responses to less than 40%
at 50 µM and 2-6, 9 and 10 diminished Pam2CSK4-induced
TLR2/6 responses to less than 50% at 50 µM. Compound 6 and
11 abrogated the TLR2 response of both heterodimers almost
completely and therefore showed a similar TLR2 inhibition as CU-
CPT22 (Figure 2A). None of the tested compounds appeared to
have any agonistic TLR2 activity (Figure S1A).
No cytotoxic effects were observed within the activity range
except for 11 which reduced cell viability starting at 50 µM
(Supplementary Figure 1B). Hence, the apparent inhibition of
TLR2-mediated responses by 11 might be due its cytotoxicity.
3
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Figure 2. Inhibition of TLR2-dependent NF-κB activation and IL-8 secretion. (A) HEK-Blue hTLR2 cells were pre-incubated with CU-CPT22 or compound 1-6
and 9-11 for 1 h and then stimulated additionally with TLR2/1 agonist Pam3CSK4 (10 ng/ml) or TLR2/6 agonist Pam2CSK4 (1 ng/ml) for 24 h. (B) HEK-Blue hTLR2
cells were pre-incubated with increasing concentrations of 4, 6, 9, 10 and then stimulated additionally with Pam3CSK4 or Pam2CSK4 for 24 h. Supernatants were
tested for TLR2-mediated NF-κB/AP-1 activity by quantification of SEAP release (OD640). (C) THP-1 macrophages were incubated with CU-CPT22 (25 µM) or
compound 6 for 1 h and afterwards stimulated with Pam3CSK4 (10 ng/ml), Pam2CSK4 (1ng/ml), LPS (10 ng/ml), flagellin (1 µg/ml), CL075 (8 µM) or ODN2006 (5
µM) for 24 h. Supernatants were analyzed for IL-8 secretion by ELISA. Mean +SD or +/-SD (n=3).
Deeper in the pocket further hydrophobic contacts take place
between the benzoate and Leu328, Val313 and Ile314 and the
ethyl moiety and Phe284, Leu317, Leu285, Ile261, Leu266 and
Ile314. H-bond acceptor interactions are formed by the hydroxyl
group on position 5 and the backbone nitrogen atoms of Leu350
and Phe349, in addition to an H-bond donor interaction between
the second hydroxyl group in position 3 and backbone oxygen of
Ser346. These H-bonds are also formed by MMG-11 (Figure 3A)
and have been shown to be important for antagonists binding to
TLR2.[8e] This might explain the lower activity of the other
dihydroxy-benzamido-benzoates (1 and 3, Supplementary Figure
2) and the dimethoxy-benzamido-benzoates (2 and 4,
Supplementary Figure 3). The geometry of the 2,4-dihydroxy-
benzamide 1 causes it to form H-bonds to Ser346 and Lys347 but
not Leu350 and Phe349 leading to a weak activity. The 3,4-
dihydroxy-benzamide 3 is more active than 1 as the mandatory
H-bonds to Leu350 and Phe349 are formed, but less active than
6 as the stabilizing H-bond to Ser326 is missing. In the case of
the dimethoxy-benzamido-benzoates the methoxy groups are
worse and bulkier acceptors than the hydroxyl groups thus
making the formation of the key H-bonds less favorable. For these
compounds we hypothesize a flipped binding mode which allows
the carbonyl-oxygen of the ester to interact with the backbone of
Phe349 and Leu350, without the formation of further stabilizing H-
bonds towards Ser346 the resulting activity still is low. The
dihydroxy-phenyl moiety of compound 5 is surmised to form two
H-bonds towards Phe349 and Leu350, however its scaffold puts
the methoxy groups into proximity of hydrophobic residues, which
is unfavorable for binding and leads to a reduced activity
(Supplementary Figure 4).
4
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Figure 3. Predicted binding pose for MMG-11 and 6. The TLR2 antagonists MMG-11 (A) and 6 (B) bound in the TLR2 ligand binding site are shown. Protein residues
are depicted in ball and stick mode, the compound as sticks. Protein-ligand interactions are color- and shape-coded (yellow sphere – hydrophobic contact area,
green arrow – H-bond donor, red arrow – H-bond acceptor).
In addition to the synthesized compounds we tested three
dihydroxy-phenyl-oxoethyl-furan-carboxylates (9, 10 and 11) of
which one 11 was cytotoxic and therefore omitted in further
analysis. The other two compounds, 9 and 10 show a binding
Experimental Section
mode nearly identical to that of MMG-11 (Supplementary Figure
5). Surprisingly, their activity is much lower, which shows the
importance of the third hydroxyl group for the stabilization of
ligands containing the furan carboxylate scaffold. Interestingly,
this is not the case for the newly developed and rigidified
dihydroxy-benzamido-benzoates like lead structure 6, which
virtually retains the binding affinity despite of having only two
remaining hydroxyl groups.
Chemical synthesis
Chemicals were purchased from Acros, Alfa Aesar, Fluka, Sigma-
Aldrich, and VWR and were used without further purification or
distillation unless otherwise stated. All solvents that were used
during the reactions were obtained from a column-based solvent
system (MBraun, MB-SPS-800). Air or moisture-sensitive
reactions were carried out under a positive pressure of dry
nitrogen or argon in oven-dried glassware. Solution phase
reactions were monitored either by LC-MS techniques or by thin
layer chromatography (TLC) using Analtech silica gel plates (60
F254) and the spots were examined under UV light at 254nm or
stained with developing reagents. LC-MS data were recorded on
an Agilent 1100 series chromatography workstation (Agilent
Conclusion
Taken together, this study reports a set of TLR2 antagonists
derived by chemical synthesis and analog search based on the
previously reported pyrogallol-containing TLR2 antagonists CU-
CPT22 and MMG-11. The series of compounds (1-11) designed
and tested here provides novel insights into the structure-activity
relationships (SAR) of TLR2 antagonists and allows for
rationalization of structural binding characteristics. The findings
prove the possibility to vary the pyrogallol fragment, the hydrogen
bonding pattern, the second aryl aryl fragment and the linker
moiety connecting both fragments. These tolerated variations
have enabled the development of chemically stable drug
candidates without the sensitive pyrogallol scaffold. The non-toxic
and most active compound from this series, inhibitor 6, shows a
comparable activity toward TLR2 as its pyrogallol precursors
without being sensitive toward oxidative degradation as its
pyrogallol derivatives. Furthermore, the biological activity of this
inhibitor supports our hypothesis that this group of TLR
antagonists binds solely to TLR2 and does not directly interact
with the TLR1 or TLR6 part of the functional heterodimer.
Technologies) equipped with
a
single quadruple mass
spectrometer and electrospray ionization (ESI). Eluents A (0.1%
formic acid in Millipore water) and B (0.1% formic acid in
acetonitrile) were used in a linear gradient (5-99% B in 5min).
Removal of solvents was performed using rotary evaporators
from Heidolph. The Biotage®IsoleraTM Spektra One was used for
regular flash purifications with pre-pack flash chromatography
cartridges from Biotage® employing individual gradients derived
from analytical runs or thin layer chromatography (TLC). Non-
HRMS measurements were recorded with an ESI single quad
spectrometer (Agilent) coupled with an analytical HPLC system
(Agilent 1100). HRMS measurements were conducted with an
Agilent 6210 ESI-TOF mass spectrometer. Nuclear magnetic
resonance (NMR) spectra (1H and 13C NMR) were recorded on
a Bruker AVANCE 300MHz, 400MHz and 500MHz instrument
and chemical shifts (δ) were measured in parts per million (ppm).
Coupling constants (J) are expressed in Hertz (Hz). The following
abbreviations are used to describe peak patterns when
appropriate: s (singlet), d (doublet), t (triplet), q (quadruplet), m
(multiplet), and br (broad). The elemental analyzer VARIO EL was
5
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FULL PAPER
used for C-, H-,and N-analysis. Details for compound synthesis
and analytic data are given in the Supporting Information.
General procedure for the preparation of perbenzylated
benzoic acid esters 12-14.
7.72 (d, J = 8.6 Hz, 1H), 7.54 – 7.27 (m, 10H), 6.82 (s, 1H), 6.67
(d, J = 8.8 Hz, 1H), 5.20 (s, 2H), 5.16 (s, 2H).
3,4-Bis-(benzyloxy)-benzoic acid (17)
17 was synthesized according to the general procedure, using 13
(2.30 g, 5.42 mmol) and was obtained as a white solid (1.70 g,
5.09 mmol, 94%). 1H NMR (400 MHz, DMSO-d6) δ: 12.57 (s, 1H),
7.46 (s, 1H), 7.45 (d, J = 7.4 Hz, 1H), 7.39 – 7.19 (m, 10H), 7.06
(d, J = 8.8 Hz, 1H), 5.12 (s, 2H), 5.08 (s, 2H).
Benzyl bromide (6 eq.), sodium iodide (6 eq.) and potassium
carbonate (8 eq.) were added to a solution of the respective
benzoic acid in acetone (40 ml mmol-1) at room temperature. The
mixture was stirred under reflux for 16 h. Then it was cooled to
room temperature and the solvent removed under reduced
pressure. Water was added and the resulting mixture was
extracted with EtOAc. The combined organic layers were dried
over anhydrous Na2SO4 and filtered. EtOAc was evaporated
under reduced pressure and the resulting crude product was
purified by column chromatography over SiO2 (hexane-EtOAc as
eluent) to afford the desired perbenzylated compounds.
Benzyl 2,4-bis-(benzyloxy)-benzoate (12)
3,5-Bis-(benzyloxy)-benzoic acid (18)
18 was synthesized according to the general procedure, using 14
(0.21 g, 0.50 mmol) and was obtained as a white solid comprising
a mixture of acid and sodium salt (0.21 g, quant.). 1H NMR (500
MHz, DMSO-d6) δ: 13.04 (s, 1H) 7.46 –7.32 (m, 10H), 7.16 (s,
2H), 6.93 (s, 1H), 5.14 (s, 4H).
Preparation of 3,4-dimethoxy-benzoic acid (19)
To a suspension of 15 (0.35 g, 1,79 mmol, 1 eq.) in H2O/MeOH
(1:1 v/v, 30 ml) at room temperature was added LiOH
monohydrate (0.23 g, 5.36 mmol, 3 eq.) and the resulting mixture
stirred for 4 h. The reaction mixture was then brought to pH 3
using 1M HCl and extracted with EtOAc. The combined organic
layers were dried over anhydrous Na2SO4 and filtered. The EtOAc
was evaporated under reduced pressure to afford 19 as a white
solid (0.31 g, 1.70 mmol, 95%). 1H NMR (400 MHz, DMSO-d6) δ:
12.67 (s, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.44 (s, 1H), 7.03 (d, J =
8.5 Hz, 1H), 3.82 (s, 3H), 3.79 (s, 3H).
12 was synthesized according to the general procedure using 2,4-
dihydroxybenzoic acid (1.00 g, 6.5 mmol) and was obtained as a
1
pale yellow solid (1.58 g, 3.72 mmol, 57%). H NMR (400 MHz,
CDCl3) δ: 7.91 (d, J = 8.7 Hz, 1H), 7.46 –7.28 (m, 15H), 6.61 (s,
1H), 6.58 (d, J = 8.7 Hz, 1H), 5.32 (s, 2H), 5.11 (s, 2H), 5.06 (s,
2H).
Benzyl 3,4-bis-(benzyloxy)-benzoate (13)
13 was synthesized according to the general procedure using 3,4-
dihydroxybenzoic acid (5.00 g, 32.5 mmol) and was obtained as
a pale yellow solid (7.80 g, 18.53 mmol, 57%). 1H NMR (400 MHz,
CDCl3) δ: 7.71 – 7.65 (m, 2H), 7.49 – 7.31 (m, 15H), 6.93 (d, J =
8.9 Hz, 1H), 5.32 (s, 2H), 5.22 (s, 2H), 5.20 (s, 2H).
Preparation of ethyl 3-amino-benzoate (20)
20 was synthesized according to literature.1 Brown oil, yield:
92%,1H NMR (400 MHz, CDCl3) δ: 7.41 (d, J = 7.7 Hz, 1H), 7.34
(s, 1H), 7.19 (dd, J = 7.8 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 4.33
(q, J = 7.1 Hz, 2H), 3.48 (s, 2H), 1.36 (t, J = 7.1 Hz, 3H).
General procedure for the preparation of benzamides 2, 4,
and 21-23.
Benzyl 3,5-bis-(benzyloxy)-benzoate (14)
14 was synthesized according to the general procedure (without
NaI) using 3,5-dihydroxybenzoic acid (0.77 g, 5 mmol) and was
obtained as a pale yellow solid (0.41 g, 0.95 mmol, 19%). 1H NMR
(500 MHz, DMSO-d6) δ: 7.46 –7.31 (m, 15H), 7.18 (s, 2H), 6.98
(s, 1H), 5.33 (s, 2H), 5.15 (s, 4H).
To a mixture of the appropriate acid (1 eq.) and 20 (1 eq.) in DCM
(5 ml mmol-1) was added HATU (1.2 eq.) and DIPEA (3 eq.) at
0°C and after which it was stirred at 30 °C for 8 h. The solvent
was removed under reduced pressure, water was added and the
resulting mixture was extracted with EtOAc. The combined
organic layers were dried over anhydrous Na2SO4 and filtered.
The EtOAc was evaporated under reduced pressure and the
resulting crude product was purified by column chromatography
over SiO2 (hexane-EtOAc as eluent) to afford the desired amides
2, 4, 21-23.
Methyl 3,4-dimethoxy-benzoate (15)
To a solution of 3,4-dihydroxy-benzoic acid (0.80 g, 5.2 mmol, 1
eq.) in DMF (30 ml) was added methyl iodide (1.50 ml, 25 mmol,
5 eq.) and potassium carbonate (3.59 g, 25 mmol, 5 eq.) at room
temperature. The mixture was stirred at 50°C for 16 h, cooled to
room temperature and concentrated under reduced pressure.
Water was added and the resulting mixture extracted with EtOAc
(3 x 20 ml). The combined organic layers were dried over
anhydrous Na2SO4 and filtered. The EtOAc was evaporated
under reduced pressure and the resulting crude product purified
by column chromatography on SiO2 (hexane-EtOAc as eluent) to
afford 15 as a white solid (0.73 g, 3.74 mmol, 72%). 1H NMR (400
MHz, CDCl3) δ: 7.65 (d, J = 8.4 Hz, 1H), 7.52 (s, 1H), 6.86 (d, J =
8.4 Hz, 1H), 3.91 (s, 6H), 3.87 (s, 3H).
Preparation of benzoic acids 16-18. General procedure
To a suspension of the appropriate benzyl benzoates (12-14) in
EtOH at room temperature was added an excess of 5 M NaOH
solution and the resulting mixture stirred under reflux for 8 h. The
reaction mixture was cooled to room temperature and acidified to
pH 3 by dropwise addition of 12 M HCl. The resulting precipitate
was filtered off, washed with water and dried under reduced
pressure to afford the corresponding acids 16-18.
Ethyl 3-(3,4-dimethoxy-benzamido)-benzoate (2)
2 was synthesized according to the general procedure, using 19
(0.05 g, 0.28 mmol) and was obtained as a yellow solid (0.63 g,
1
0.21 mmol, 76%). H NMR (400 MHz, CDCl3) δ: 8.19 – 8.00 (m,
3H), 7.80 (d, J = 7.5 Hz, 1H), 7.49 (s, 1H), 7.47 – 7.37 (m, 2H),
6.87 (d, J = 8.4 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 3.92 (s, 6H),
1.37 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ: 166.40,
165.59, 152.33, 149.29, 138.46, 131.34, 129.32, 127.21, 125.46,
124.73, 121.10, 119.76, 110.79, 110.45, 61.31, 56.18, 14.43;
HRMS (ESI+) [M + H]+ C18H20NO5 calculated 330.1341 Da, found:
330.1344 m/z.
Ethyl 3-(2-(3,4-dimethoxyphenyl)-acetamido)-benzoate (4)
4 was synthesized according to the general procedure (but
extracted with DCM instead of EtOAc), using 2-(3,4-
dimethoxyphenyl)-acetic acid (0.20 g, 1.02 mmol) and was
obtained as a yellow solid (0.28 g, 0.83 mmol, 81%). 1H NMR (500
MHz, CDCl3) δ: 7.91 – 7.84 (m, 2H), 7.74 (d, J = 7.7 Hz, 1H), 7.49
(d, J = 7.2 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 6.89 – 6.84 (m, 2H),
6.83 (s, 1H), 4.33 (q, J = 7.1 Hz, 2H), 3.88 (s, 3H), 3.87 (s, 3H),
2,4-Bis-(benzyloxy)-benzoic acid (16)
16 was synthesized according to the general procedure, using 12
(1.01 g, 2.36 mmol). The resulting precipitate was recrystallized
from MeOH/DCM (50:1) and afforded 16 as a white solid (0.80 g,
1,89 mmol, 80%). 1H NMR (400 MHz, DMSO-d6) δ: 12.27 (s, 1H),
6
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10.1002/cmdc.202000060
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FULL PAPER
3.68 (s, 2H), 1.35 (t, J = 7.1 Hz, 3H); 13C NMR (126 MHz, CDCl3)
δ: 169.80, 166.30, 149.59, 148.74, 138.00, 131.26, 129.19,
126.66, 125.48, 124.37, 121.84, 120.65, 112.59, 111.84, 61.28,
56.03, 44.43, 14.38; HRMS (ESI+) [M + H]+ C19H22NO5 calculated
344.1498 Da, found: 344.1506 m/z.
Ethyl 3-(2,4-bis-(benzyloxy)-benzamido)-benzoate (21)
21 was synthesized according to the general procedure, using 16
(0.06 g, 0.18 mmol) and was obtained as a brown solid (0.02 g,
0.04 mmol, 20%). 1H NMR (400 MHz, CDCl3) δ: 9.92 (s, 1H), 8.30
(d, J = 8.7 Hz, 1H), 7.94 (s, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.61 –
7.21 (m, 12H), 6.77 (d, J = 9.0 Hz, 1H), 6.73 (s, 1H), 5.19 (s, 2H),
5.14 (s, 2H), 4.37 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H).
Ethyl 3-(3,4-bis-(benzyloxy)-benzamido)-benzoate (22)
22 was synthesized according to the general procedure using 17
(0.10 g, 0.30 mmol), but was not extracted. Instead the solvent of
the reaction mixture was evaporated under reduced pressure and
the residue directly purified by column chromatography. The
6 was synthesized according to the general procedure using 23
(0.09 g, 0.18 mmol) and was obtained as a grey solid (0.05 g, 0.15
mmol, 86%). 1H NMR (500 MHz, DMSO-d6) δ: 10.29 (s, 1H), 9.57
(s, 2H), 8.44 (t, J = 1.9 Hz, 1H), 8.04 (ddd, J = 8.2, 2.2, 1.0 Hz,
1H), 7.67 (dt, J = 7.7, 1.3 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 6.80
(d, J = 2.2 Hz, 2H), 6.43 (t, J = 2.2 Hz, 1H), 4.33 (q, J = 7.1 Hz,
2H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ:
165.93, 165.65, 158.36, 139.64, 136.72, 130.27, 128.94, 124.62,
124.05, 120.72, 105.86, 105.62, 60.75, 14.18; Anal. calcd for
C16H15NO5 (301.3): C, 63.78; H, 5.02; N, 4.65; found: C, 63.83; H,
5.02; N, 4.66%; HRMS (ESI+) [M + H]+ C16H16NO5 calculated
302.1028 Da, found: 302.1033 m/z.
Preparation
of
1-(2,4-dihydroxyphenyl)-2-(3,4-
dimethoxyphenyl)-ethan-1-one (5)
To a solution of 2-(3,4-dimethoxyphenyl)-acetic acid (1.57 g, 8.00
mmol, 1 eq.) in boron trifluoride diethyl etherate (15.84 ml, 56.00
mmol, 7 eq.) at 0 °C resorcinol (1.32 g, 12 mmol, 1.5 eq.) was
desired product was obtained as a yellow solid (0.14 g, 0.28 mmol, added. The mixture was heated for 5 h at 110 °C and then cooled
93%). 1H NMR (400 MHz, CDCl3) δ: 8.09 (s, 1H), 8.03 (d, J = 8.1
Hz, 1H), 7.99 (s, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.55 (d, J = 2.0 Hz,
1H), 7.48 – 7.27 (m, 12H), 6.93 (d, J = 8.4 Hz, 1H), 5.21 (s, 2H),
5.19 (s, 2H), 4.36 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H).
Ethyl 3-(3,5-bis-(benzyloxy)-benzamido)-benzoate (23)
23 was synthesized according to the general procedure (but
extracted with DCM instead of EtOAc), using 18 (0.17 g, 0.50
mmol) and was obtained as a white solid (0.09 g, 0.02 mmol,
36%). m/z (ESI+) [M + H]+ C30H28NO5 calculated 482.2 Da, found:
482.8 m/z.
to 0°C. Cold water (200 ml) was added, the resulting precipitate
collected by filtration, washed with water and recrystallized from
EtOH to afford 5 as a white solid (0.44 g, 1.52 mmol, 19%). H
1
NMR (400 MHz, DMSO-d6) δ: 12.57 (s, 1H), 10.68 (s, 1H), 7.95
(d, J = 8.9 Hz, 1H), 6.90 (d, J = 1.9 Hz, 1H), 6.87 (d, J = 8.2 Hz,
1H), 6.79 (dd, J = 8.2, 2.0 Hz, 1H), 6.39 (dd, J = 8.8, 2.4 Hz, 1H),
6.25 (d, J = 2.4 Hz, 1H), 4.19 (s, 2H), 3.72 (s, 3H), 3.71 (s, 3H);
13C NMR (101 MHz, DMSO-d6) δ: 202.41, 164.95, 164.69, 148.60,
147.63, 133.61, 127.46, 121.53, 113.36, 112.15, 111.85, 108.26,
102.49, 55.50, 55.47, 43.69; HRMS (ESI+) [M + H]+ C16H17O5
calculated 289.1076 Da, found: 289.1074 m/z.
General procedure for the preparation of compounds 1,3, and
6 via O-benzyl-deprotection.
To a mixture of the respective O-benzyl-protected compounds in
THF/MeOH (1:1 v/v, 30 ml mmol-1) was added palladium on active
carbon (20 w%). The resulting suspension was stirred at room
temperature for 8 h under H2 atmosphere (1 bar) and then filtered
over celite. The solvent was evaporated under reduced pressure
and the resulting crude product purified by column
chromatography on SiO2 (hexane-EtOAc as eluent) to afford the
desired compounds 1, 3 and 6.
Biological validation
Cell culture. Human embryonic kidney (HEK)-Blue hTLR2 cells
(InvivoGen, Touluse, France) were used from passage 5 to 20.
Stimulation of the cells with TLR2 agonists leads to production
and secretion of NF-κB-inducible embryonic alkaline phosphatase
(SEAP). The enzyme transforms the substrate QUANTI-Blue
(InvivoGen) into a blue dye that was detected by optical density
(OD) at 640 nm as previously described.[11a,
12]
THP-1 cells
Ethyl 3-(2,4-dihydroxy-benzamido)-benzoate (1)
(DSMZ, Braunschweig, Germany) from passage 10 to 15 were
cultured as previously reported.[13] For the generation of THP-1
macrophages, THP-1 monocytes were first incubated with 25
ng/ml Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for
48h and afterwards rested for 24h. The cell lines were cultured at
37°C in a humidified atmosphere of 5% CO2 and 95% air and were
regularly tested negative for mycoplasma contamination
(VenorGeM Classic Mycoplasma PCR detection kit, Minerva
Biolabs, Berlin, Germany).
1 was synthesized according to the general procedure using 21
(0.05 g, 0.10 mmol) and was obtained as a grey solid (0.03 g, 0.10
1
mmol, 99%). H NMR (400 MHz, DMSO-d6) δ: 12.17 (br s, 1H),
10.35 (s, 1H), 10.25 (br s, 1H), 8.33 (t, J = 1.9 Hz, 1H), 7.95 (ddd,
J = 8.1, 2.1, 0.9 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.70 (dt, J = 7.8,
1.2 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 6.38 (dd, J = 8.7, 2.3 Hz, 1H),
6.33 (d, J = 2.3 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1
Hz, 3H). HRMS (ESI+) [M + H]+ C16H16NO5 calculated 302.1028
Da, found: 302.1027 m/z.
TLR2 ligands.
Ethyl 3-(3,4-dihydroxy-benzamido)-benzoate (3)
The synthetic lipopeptides Pam3CSK4 and Pam2CSK4,
lipopolysaccharide from Escherichia coli O111:B4 (LPS-EB),
flagellin from Salmonella typhimurium (FLA-ST), the
3 was synthesized according to the general procedure using 22
(0.06 g, 0.13 mmol) and was obtained as a white solid (0.03 g,
0.11 mmol, 81%). 1H NMR (500 MHz, DMSO-d6) δ: 10.15 (s, 1H),
9.63 (br s, 1H), 9.27 (br s, 1H), 8.42 (s, 1H), 8.04 (d, J = 8.0 Hz,
1H), 7.65 (d, J = 7.7 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.41 (s, 1H),
7.38 (dd, J = 8.2, 1.9 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 4.32 (q, J
= 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H); 13C NMR (126 MHz, DMSO-
d6) δ: 165.72, 165.44, 149.09, 145.01, 139.92, 130.24, 128.93,
125.51, 124.60, 123.79, 120.68, 119.77, 115.48, 114.92, 60.77,
14.21; HRMS (ESI+) [M + H]+ C16H16NO5 calculated 302.1028 Da,
found: 302.1033 m/z.
thiazoloquinoline compound CL075 and class
B
CpG
oligonucleotide ODN2006 were purchased from Invivogen and
the TLR2 antagonist CU-CPT22 was obtained from Sigma-Aldrich
(Taufkirchen, Germany).
Cell stimulation. HEK-Blue hTLR2 cells (4 x104 cells/well) and
THP-1 macrophages (2 x 105 cells/well) were cultured in 96-well
plates and 24-well plates (TPP, Trasadingen, Switzerland). After
cells were washed with phosphate-buffered saline (PBS, Sigma-
Aldrich), cell stimulation was done in OptiMEM (ThermoFisher
Scientific, Darmstadt, Germany). The tested compounds and CU-
Ethyl 3-(3,5-dihydroxy-benzamido)-benzoate (6)
7
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10.1002/cmdc.202000060
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FULL PAPER
[6]
[7]
[8]
K. Cheng, M. Gao, J. I. Godfroy, P. N. Brown, N. Kastelowitz, H. Yin, Sci
Adv 2015, 1(3).
CPT22 (50 mM) were dissolved in DMSO. Final DMSO
concentrations in cell culture were below 0.2% (v/v). Vehicle
controls showed no significant difference to nonstimulated
controls (data not shown). For co-stimulation of TLR2 agonists
and tested modulators, cells were preincubated with the
compounds or CU-CPT22 for 1 h and afterwards stimulated with
the agonists.
ELISA. Commercially available ELISA kits were used for
detecting human IL-8 levels in cell culture supernatants (ELISA-
Ready Set Go, Invitrogen by Thermo Fisher Scientific).
Cell viability. Cell viability was assessed by the MTT assay in
HEK Blue hTLR2 cells as previously described.[14] 10% (v/v)
DMSO (Carl Roth, Karlsruhe, Germany) served as control and the
viability of untreated cells was defined as 100%.
Statistical analysis. Data of the bar charts are shown as mean
+ SD. Potency (IC50) data are presented as mean with the
confidence interval (95%). Statistical analysis was done by using
GraphPad Prism 6.0 (GraphPad software, San Diego, USA). Non-
linear regression was used to plot and analyze concentration-
response curves and to obtain IC50 values.
K. Cheng, X. Wang, S. Zhang, H. Yin, Angew Chem Int Ed Engl 2012,
51(49), 12246-12249.
aZ. Chen, X. Cen, J. Yang, Z. Lin, M. Liu, K. Cheng, Eur J Med Chem
2019, 169, 42-52; bZ. Chen, X. Cen, J. Yang, X. Tang, K. Cui, K. Cheng,
Chem Commun (Camb) 2018, 54(81), 11411-11414; cP. Durai, H. J.
Shin, A. Achek, H. K. Kwon, R. G. Govindaraj, S. Panneerselvam, D.
Yesudhas, J. Choi, K. T. No, S. Choi, FEBS J 2017, 284(14), 2264-2283;
dM. S. Murgueitio, S. Ebner, P. Hortnagl, C. Rakers, R. Bruckner, P.
Henneke, G. Wolber, S. Santos-Sierra, Biochim Biophys Acta Gen Subj
2017, 1861(11 Pt A), 2680-2689; eM. S. Murgueitio, P. Henneke, H.
Glossmann, S. Santos-Sierra, G. Wolber, ChemMedChem 2014, 9(4),
813-822; fZ. Zhong, L. J. Liu, Z. Q. Dong, L. Lu, M. Wang, C. H. Leung,
D. L. Ma, Y. Wang, Chem Commun (Camb) 2015, 51(56), 11178-11181.
aD. Sribar, M. Grabowski, M. S. Murgueitio, M. Bermudez, G. Weindl, G.
Wolber, Eur J Med Chem 2019, 179, 744-752; bU. Svajger, B. Brus, S.
Turk, M. Sova, V. Hodnik, G. Anderluh, S. Gobec, Eur J Med Chem 2013,
70, 393-399; cH. Yu, H. Jin, L. Sun, L. Zhang, G. Sun, Z. Wang, Y. Yu,
PLoS One 2013, 8(3), e56514.
[9]
[10] aM. Grabowski, M. S. Murgueitio, M. Bermudez, J. Rademann, G.
Wolber, G. Weindl, Biochem Pharmacol 2018, 154, 148-160; bM.
Grabowski, M. S. Murgueitio, M. Bermudez, G. Wolber, G. Weindl,
Biochemical Pharmacology 2020, 171, 113687.
Computational methods. The crystal structure of the
heterodimer of TLR2-TLR1 with bound Pam3CSK4 (PDB-code:
[11] aQ. Su, M. Grabowski, G. Weindl, Int J Med Microbiol 2017, 307(2), 108-
112; bS. Bock, M. S. Murgueitio, G. Wolber, G. Weindl, Pharmacol Res
2016, 105, 44-53.
[15] 24
[16] 25
2Z7X)
was retrieved from the Protein Data Bank
and
used for docking studies with the tested compounds. Prior to
docking the TLR1 monomer, all ligands and water molecules were
removed using Molecular Operating Environment (MOE2019,
Chemical Computing Group, Montreal, QC, Canada). The TLR2
monomer was protonated using the “Protonate 3D” application
[12] Q. Su, G. Weindl, Pharmacol Res 2018, 128, 145-152.
[13] A. Pfalzgraff, L. Heinbockel, Q. Su, K. Brandenburg, G. Weindl, Biochem
Pharmacol 2017, 140, 64-72.
[14] N. Do, G. Weindl, L. Grohmann, M. Salwiczek, B. Koksch, H. C. Korting,
M. Schafer-Korting, Exp Dermatol 2014, 23(5), 326-331.
included in MOE2019. The GOLD Suite v.5.2 (Cambridge
[15] M. S. Jin, S. E. Kim, J. Y. Heo, M. E. Lee, H. M. Kim, S. G. Paik, H. Lee,
J. O. Lee, Cell 2007, 130(6), 1071-1082.
[17]
Crystallographic Data Centre, Cambridge, UK)
was used for
docking with the GoldScore[18] as scoring function with “slow”
parameters. Binding poses were minimized (MMFF94 force
field)[19] and further analyzed in LigandScout 4.2 (Inte:ligand,
Vienna, Austria).[20]
[16] H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig,
I. N. Shindyalov, P. E. Bourne, Nucleic Acids Res 2000, 28(1), 235-242.
[17] G. Jones, P. Willett, R. C. Glen, A. R. Leach, R. Taylor, J Mol Biol 1997,
267(3), 727-748.
[18] M. L. Verdonk, J. C. Cole, M. J. Hartshorn, C. W. Murray, R. D. Taylor,
Proteins 2003, 52(4), 609-623.
[19] T. A. Halgren, J Comput Chem 1996, 17(5-6), 490-519.
[20] aG. Wolber, A. A. Dornhofer, T. Langer, J Comput Aided Mol Des 2006,
20(12), 773-788; bG. Wolber, T. Langer, J Chem Inf Model 2005, 45(1),
160-169; cT. Seidel, G. Ibis, F. Bendix, G. Wolber, Drug Discovery
Today: Technologies 2010, 7(4), e221-e228.
Acknowledgements
We gratefully acknowledge funding by the Deutsche
Forschungsgemeinschaft (DFG, DFG RA 895/16-1)
Keywords: Toll-Like Receptors • Inflammation • Chemical
Synthesis • TLR Selectivity • Molecular Modeling • Structure-
Based Design
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8
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FULL PAPER
Entry for the Table of Contents
The 1,2,3-tri-phenol motif of known TLR2 antagonists is highly susceptible to oxidation and excludes them from use in extended
experiments under aerobic conditions. Here, we report a rationally developed series of novel TLR modulators resulting in compound
6, a novel, chemically stable, non-toxic, TLR2-selective antagonist.
9
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