Target selectivity of FimH antagonists

Article abstract of DOI:10.1021/jm3010338

Mannose-based FimH antagonists are considered new therapeutics for the treatment of urinary tract infections (UTIs). They prevent the adhesion of uropathogenic Escherichia coli (UPEC) to urothelial cell surfaces triggered by the lectin FimH, which is located at the tip of bacterial type 1 pili. Because all reported FimH antagonists are α-d-mannosides, they are also potential ligands of mannose receptors of the human host system. We therefore investigated the selectivity range of five FimH antagonists belonging to different compound families by comparing their affinities for FimH and eight human mannose receptors. On the basis of the detected selectivity range of approximately 5 orders of magnitude, no adverse side effects resulting from nonselective binding to the human receptors have to be expected. FimH antagonists can therefore be further considered as potential therapeutics for the treatment of UTI.

Full text of DOI:10.1021/jm3010338

Article
pubs.acs.org/jmc
Target Selectivity of FimH Antagonists
Meike Scharenberg, Oliver Schwardt, Said Rabbani, and Beat Ernst*
Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
S
* Supporting Information
ABSTRACT: Mannose-based FimH antagonists are consid-
ered new therapeutics for the treatment of urinary tract
infections (UTIs). They prevent the adhesion of uropatho-
genic Escherichia coli (UPEC) to urothelial cell surfaces
triggered by the lectin FimH, which is located at the tip of
bacterial type 1 pili. Because all reported FimH antagonists are
α-D-mannosides, they are also potential ligands of mannose
receptors of the human host system. We therefore investigated
the selectivity range of five FimH antagonists belonging to
different compound families by comparing their affinities for FimH and eight human mannose receptors. On the basis of the
detected selectivity range of approximately 5 orders of magnitude, no adverse side effects resulting from nonselective binding to
the human receptors have to be expected. FimH antagonists can therefore be further considered as potential therapeutics for the
treatment of UTI.
with these various mannose receptors would have a profound
impact on these processes and could cause severe side effects. A
high selectivity of FimH antagonists is therefore of importance
for a clinical application and should be evaluated in the early
stages of preclinical development.
INTRODUCTION
■
Urinary tract infections (UTIs) are primarily caused by
uropathogenic Escherichia coli (UPEC) (70−95% of cases)
expressing type 1 pili.1 At the tip of these pili, the lectin FimH
is located. It enables the bacteria to adhere to oligomannosides
of the glycoprotein uroplakin Ia (UPIa), which is located on
uroepithelial cells.2 This initial adhesion is a prerequisite for the
infection to take place, because it prevents the rapid clearance
of E. coli from the urinary tract by the bulk flow of urine and at
the same time enables the invasion of the host cells.2a,b FimH
antagonists, such as α-D-mannopyranosides, have been shown
to interfere with the attachment of UPEC to their host cells,
thus providing a novel therapeutic opportunity for prevention
and treatment of UTIs as an alternative to antibiotics.3 To date,
several FimH antagonists have been investigated in vitro.4
Furthermore, in vivo studies with methyl α-D-mannopyranosi-
de,5a n-heptyl α-D-mannopyranoside (1, Figure 1),5b,d biphenyl
α-D-mannopyranosides such as 2 and 3,5c−f and indolinylphenyl
α-D-mannopyranosides like 55g exhibited a considerable
potential to reduce bacterial infections.
Target selectivity is of great concern in drug development
and should be evaluated in the early stages of preclinical
development.6 Because all reported FimH antagonists are α-D-
mannopyranosides and therefore also potential ligands for
mannose receptors of the human host system, target selectivity
of these FimH antagonists is a pivotal concern. Although
various antagonists were already tested in vivo,5 their target
selectivity was not verified so far. Mammalian mannose
receptors are present on many tissues throughout the whole
body and are involved in numerous biological processes, such
as cell−cell adhesion7 and serum glycoprotein homeostasis.8
They also intervene in the innate and the adaptive immune
response by recognizing molecular patterns on pathogens.7,9
Consequently, nonselective interactions of FimH antagonists
The majority of human mannose-binding lectins belong to
the group of pathogen-recognition receptors (PRRs). Most
PRRs are members of the C-type lectin superfamily.10 They are
either secreted as soluble plasma proteins or expressed as
membrane-bound proteins on the surface of cells of the
immune system such as macrophages, dendritic cells, or
Langerhans cells. Secreted PRRs, such as the mannose binding
protein (MBL)11 and the lung surfactant protein D (SP-D),12
bind to pathogens and simultaneously associate with cell
surface receptors, triggering signaling pathways such as the
lectin complement activation pathway, which results in
enhanced phagocytosis of the pathogens as well as activation
of the host defense system.13 MBL and SP-D belong to the
collectin family and share a similar collagen-like domain
connected to the C-terminal C-type lectin domain, which
contains the carbohydrate recognition domain (CRD). They
consist of homotrimers, which oligomerize with 2−6 other
trimers, forming high molecular weight complexes.14 Trans-
membrane PRRs, which are classified into type I and type II C-
type lectins, are also involved in the phagocytosis of pathogens,
leading to their elimination or their processing for antigen
presentation.15 The type I C-type lectins such as the
macrophage mannose receptor (MMR)9 contain multiple
CRDs within a single polypeptide backbone. In contrast, the
type II C-type lectins such as langerin,16 DC-specific ICAM-3-
grabbing nonintegrin (DC-SIGN),17 DC-specific ICAM-3-
Received: July 14, 2012
Published: October 22, 2012
© 2012 American Chemical Society
9810
dx.doi.org/10.1021/jm3010338 | J. Med. Chem. 2012, 55, 9810−9816

Journal of Medicinal Chemistry
Article
Figure 1. FimH antagonists tested for their selectivities for various human mannose-binding lectins: n-heptyl α-D-mannopyranoside (1),4d biphenyl
α-D-mannopyranoside derivatives 2 and 3,5d squaric acid derivative 4, and indolylphenyl derivative 5.5g IC50 values for FimH were obtained by a
competitive binding assay.4d
grabbing nonintegrin related (DC-SIGNR),18 dectin-2,19 and
a
Scheme 1
dendritic cell lectin (DLEC)20 exhibit only a single CRD.
However, by formation of homomultimers, type II C-type
lectins can greatly enhance their binding affinity. This was
shown for the trimeric langerin21 and tetrameric DC-SIGN and
DC-SIGNR.19 The multimeric arrangement of the CRDs
further supports the discrimination between innate and
extrinsic carbohydrate epitopes.22
For defense mechanisms against a broad range of micro-
organisms, human mannose-binding receptors require highly
specific binding. Thus, MMR exhibits a preference for branched
sugars with terminal D-mannose, L-fucose, or N-acetyl-D-
glucosamine moieties that are specifically expressed on
mycobacteria and fungi.9a Dectin-2 selectively binds to high
mannose structures predominantly expressed on the surfaces of
yeast and fungi,23 whereas DC-SIGN recognizes high mannose
oligosaccharides and Lewis blood group antigens such as Lewisx
or Lewisa, found on mycobacteria, some viruses (e.g., HIV), and
fungi.18,24
a
Reagents and conditions: (a) H2 (1 atm), cat. PtO2, morpholine,
MeOH, 45 min. (b) Diethyl squarate, MeOH, 1 day, 61% (2 steps).4c
(c) N-Methylpiperazine, DIPEA, MeOH, 18 h, 90%.
To ensure that FimH antagonists do not cause any adverse
side effects due to nonselective binding to human mannose
receptors, their selectivity profile to eight different PRRs was
established. Nonselective binding may also have a profound
impact on the serum half-life of FimH antagonists, since
binding to PRRs often triggers endocytosis and would result in
their elimination from circulation. To address this selectivity
issue, we tested five mannose-based FimH antagonists with
diverse aglycones (alkyl, biphenyl, squaric acid, and indolinyl-
phenyl derivatives) for their binding affinity to various
mannose-binding lectins (MBL, SP-D, MMR, DC-SIGN, DC-
SIGNR, langerin, dectin-2, and DLEC).
easily available from peracetylated D-mannose.4c,26 Because the
reported procedure4c for the hydrogenation to aniline 7 using
palladium on charcoal as a catalyst resulted in a substantial loss
of the chloro substituent, platinum dioxide in the presence of
morpholine was applied.27 The mannosylated ethyl squarate 8
was then obtained in analogy to Sperling et al.4c Finally,
treatment of ester 8 with N-methylpiperazine yielded amide 4
in 90%, which was ready for biological testing.
Binding Assays. The cell-free competitive binding assay4d
is based on the interactions of a biotinylated polyacrylamide
(PAA) glycopolymer [Manα1−3(Manα1−6)Manβ1−
4GlcNAcβ1−4GlcNAcβ-PAA, TM-PAA] with the mannose
receptors. Complexation of the biotinylated glycopolymer with
streptavidin coupled to horseradish peroxidase allows for the
quantification of the binding potencies of the tested FimH
antagonists.
For our selectivity study, two parameters, the protein
concentration and the TM-PAA concentration, were optimized
to obtain comparable optical densities (ODs) for the different
lectins in the competitive binding assay (Figure 2 and Table 1).
Because of distinctive coating properties of the proteins,
different protein concentrations (2.5−20 μg/mL) were
RESULTS AND DISCUSSION
■
With a competitive binding assay,4d five high-affinity FimH
antagonists belonging to different compound families (Figure 1,
1−54d,5d,g) were tested for their selectivity for eight human
mannose receptors.
Synthesis of FimH Antagonists. n-Heptyl α-D-mannopyr-
anoside (1),25 the biphenyl α-D-mannopyranosides 25d and 3,5d
and the indolylphenyl α-D-mannopyranoside 55g were synthe-
sized as previously reported. The synthesis of FimH antagonist
4 (Scheme 1) started from nitrophenyl mannoside 6, which is
9811
dx.doi.org/10.1021/jm3010338 | J. Med. Chem. 2012, 55, 9810−9816

Journal of Medicinal Chemistry
Article
nanomolar FimH affinities of the five investigated antagonists
(Figure 1), the affinities for the human lectins are at least 5
orders of magnitude lower, indicating an excellent selectivity
margin for a therapeutic application of these FimH antagonists.
Infection studies in a mouse disease model, using compounds
1−3, were previously reported.5d In these studies, a high dose
of 50 mg/kg was applied, resulting in a substantial reduction of
the bacterial infection caused by UPEC UTI89 [reduction of
the colony-forming units (CFU) in the urine by 2 orders of
magnitude and in the bladder by 4 orders of magnitude].
Furthermore, the in vivo pharmacokinetic parameters were
determined, including the maximal plasma concentrations
(Cmax) of the FimH antagonists after iv application. Maximal
concentrations of 39 μg/mL for 1, 35 μg/mL for 2, and 39 μg/
mL for 3 were detected in blood samples, which correspond to
Figure 2. Assay development and optimization on the example of the
human lectin SP-D. Different concentrations of coated SP-D (1−10
μg/mL) in combination with various TM-PAA concentrations (0.5−5
μg/mL) were tested.
C
max values of 144, 80, and 97 μM, respectively. Low binding to
mammalian mannose receptors is expected even at these
concentrations, since the IC50 values of the antagonists 1−3 for
the eight tested human mannose receptors are approximately
10-fold higher than the detected maximal blood concentrations
in treated mice. Furthermore, with improved antagonists like
indolylphenyl derivative 5,5g the dose of 50 mg/kg could be
reduced to 1 mg/kg, thus additionally increasing the selectivity
margin.
Binding affinities of various human mannose receptors to
monosaccharides, such as D-mannose, L-fucose, and D-galactose,
have already been characterized in previous studies. Mono-
valent sugars showed only weak binding affinities in the
millimolar range toward DC-SIGN,18 DC-SIGNR,28 dectin-2,23
langerin,21 or MMR.29 The functional affinity to carbohydrates
necessary for pathogen capturing is predominantly an effect of
avidity, caused by the combined strength of multiple
interactions with ligands. The presentation of multivalent
carbohydrates on the pathogen surface and the multimerization
and/or clustering of the receptors on the host cells greatly
support binding between the interaction partners. Therefore,
multivalent presentations of α-D-mannosidic antagonists4a,30
might be prone to cause severe side effects due to strong
binding to human mannose receptors.
Table 1. Optimized Protein and TM-PAA Concentrations
Used in the Competition Assays for Each Individual Lectin
μg/mL
protein
FimH
[protein]
[TM-PAA]
OD415 nm
20
10
5
0.25
5
2.20
1.82
1.88
2.03
2.12
1.92
1.93
2.15
2.10
MBP
SP-D
5
MMR
5
2
langerin
dectin-2
DLEC
10
10
10
2.5
10
2
5
5
DC-SIGN
DC-SIGNR
1
5
necessary to obtain comparable levels of immobilization. The
TM-PAA concentration in turn required adaptation due to
different affinities of the various lectins. Because of the
multivalent oligosaccharide presentation, the affinity of the
polymer is expected to be higher than the affinity of the
corresponding free oligosaccharide. We therefore used low TM-
PAA concentrations between 0.5 and 5 μg/mL, which
correspond to 16−160 μM TM-PAA assuming a molecular
mass of approximately 30 kDa. As a representative example, the
results of this optimization process for the lectin SP-D are
summarized in Figure 2. Briefly, when 5 μg/mL SP-D was used
for the immobilization step, an OD415nm of approximately 2 was
obtained with 5 μg/mL TM-PAA. The protein and polymer
concentrations leading to comparable ODs for the other
investigated lectins are summarized in Table 1.
For the competitive binding assays, concentrations of 1 mM
antagonists and 50 mM D-mannose (positive control) were
used. The results are summarized in Figure 3. The ODs
obtained in the absence of an antagonist were set to 100% TM-
PAA binding, the background in the absence of the polymer to
0% TM-PAA binding. D-Mannose showed a strong inhibition of
binding for all proteins at a concentration of 50 mM (more
than 90% inhibition). As expected, at a concentration of 1 mM,
the antagonists 1−5 strongly inhibited binding of the polymer
to FimH, whereas none of the antagonists showed relevant
inhibition potencies for the tested human lectins. The highest
inhibition of TM-PAA binding was observed for the antagonists
3 (54%) and 5 (58%) to MMR, for compound 3 (63%) to
langerin, and for compound 2 (50%) to DLEC (indicated by
asterisks, Figure 3). On the basis of their ODs, the IC50 value
(concentration at 50% inhibition) of these antagonists can be
estimated to be in the order of 1 mM. As compared to the low
CONCLUSION
■
On the basis of the presented data, adverse side effects resulting
from nonselective binding of monovalent FimH antagonists to
the investigated mannose-binding lectins are not considered to
be a critical issue for their potential therapeutic application to
treat UTI. Although this selection does not cover the entire
mammalian mannose-binding proteins, it represents the most
abundant and best-characterized receptors expressed in various
tissues. The 105-fold lower affinity for the tested human
receptors as compared to the bacterial FimH lectin confirms a
high selectivity safety range. This primarily results from the fact
that the investigated FimH antagonists were optimized by
introducing hydrophobic substituents at their reducing end,
enabling the interaction with the tyrosine gate, the entrance to
the ligand-binding site, which is a unique feature of FimH.31
Furthermore, because of the importance of multivalent ligand
presentation in nature, monovalent α-D-mannopyranosides in
general can be considered to exhibit only low affinities to
human mannose receptors.
EXPERIMENTAL SECTION
General Methods. Commercially available reagents were
purchased from Sigma-Aldrich or Acros. Methanol (MeOH) was
■
9812
dx.doi.org/10.1021/jm3010338 | J. Med. Chem. 2012, 55, 9810−9816

Journal of Medicinal Chemistry
Article
Figure 3. Selectivity profile of FimH antagonists 1−5. Competitive binding assays with FimH, MBP, SP-D, MMR, langerin, dectin-2, DLEC, DC-
SIGN, and DC-SIGNR to evaluate the selectivity of compounds 1−5. Inhibitory capacities of the compounds were tested at a concentration of 1
mM. D-Mannose (Man) served as a positive control (50 mM). The binding signals of TM-PAA to the proteins in absence of antagonists were set to
100%, and background signals were set to 0%. Asterisks indicate inhibition of TM-PAA binding by 50% or more. The assays were performed in
triplicate.
dried by distillation from sodium methoxide. Optical rotation was
measured at 20 °C on a Perkin-Elmer 341 polarimeter. Nuclear
magnetic resonance (NMR) spectra were obtained on a Bruker
Avance 500 UltraShield spectrometer at 500.13 MHz (1H) or 125.76
MHz (13C). Chemical shifts are given in ppm and were calibrated on
was achieved using 2D methods (COSY and HSQC). Electron spray
ionization mass spectra (ESI-MS) were recorded on a Waters
micromass ZQ instrument. High-resolution mass spectra (HR-MS)
were obtained on an ESI Bruker Daltonics micrOTOF spectrometer
equipped with a TOF hexapole detector. Reactions were monitored by
TLC using glass plates coated with silica gel 60 F254 and visualized by
1
residual solvent peaks. Assignment of the H and 13C NMR spectra
9813
dx.doi.org/10.1021/jm3010338 | J. Med. Chem. 2012, 55, 9810−9816

Journal of Medicinal Chemistry
Article
using UV light and/or by charring with a molybdate solution (a 0.02
M solution of ammonium cerium sulfate dihydrate and ammonium
molybdate tetrahydrate in aqueous 10% H2SO4) with heating to 150
°C for 5 min. MPLC separations were carried out on a CombiFlash Rf
from Teledyne Isco equipped with RP-18 reversed-phase flash
columns. LC-MS separations were done on a Waters system equipped
with a Waters SunFire C18 OBD (5 μm, 19 mm × 150 mm) column,
sample manager 2767, pump 2525, PDA 2525, and micromass ZQ
mass spectrometer.
powder after a final lyophilization from H2O/dioxane. [α]D +74.5 (c
1.00, MeOH). 1H NMR (500 MHz, CD3OD): δ 2.47 (s, 3H, NCH3),
2.76 (m, 4H, 2 CH2), 3.66 (ddd, J = 2.1, 5.6, 9.8 Hz, 1H, H-5), 3.71
(dd, J = 5.6, 11.8 Hz, 2H, H-6a), 3.74 (t, J = 9.7 Hz, 1H, H-4), 3.79
(dd, J = 2.1, 11.8 Hz, 2H, H-6b), 3.88 (m, 4H, 2 CH2), 3.96 (dd, J =
3.4, 9.4 Hz, 1H, H-3), 4.09 (dd, J = 1.8, 3.3 Hz, 1H, H-2), 5.48 (d, J =
1.4 Hz, 1H, H-1), 7.15 (dd, J = 2.7, 8.9 Hz, 1H, C6H3), 7.33 (d, J = 8.9
Hz, 1H, C6H3), 7.35 (d, J = 2.7 Hz, 1H, C6H3). 13C NMR (125 MHz,
CD3OD): δ 45.4 (NCH3), 47.4 (2C, 2 CH2), 55.0 (2C, 2 CH2), 62.7
(C-6), 68.2 (C-4), 71.8 (C-2), 72.3 (C-3), 76.0 (C-5), 101.2 (C-1),
119.0, 121.6, 123.8, 125.0, 135.1, 150.2 (C6H3), 164.9, 169.2 (CC),
183.3, 185.9 (2 CO). HR-MS: m/z calcd for C21H26ClN2NaO8 [M +
Na]+, 506.1306; found, 506.1303.
Expression and Purification of DC-SIGN CRD-Fc and DC-
SIGNR CRD-Fc. Plasmids containing the full-length cDNA of DC-
SIGN and DC-SIGNR were kindly provided by Daniel A. Mitchell,
Clinical Sciences Research Institute, Warwick Medical School
(Coventry, United Kingdom). Standard molecular techniques32 were
used for the cloning of the CRD of DC-SIGN (DC-SIGN CRD; aa
residues 250−404, GenBank accession no. M98457) and DC-SIGNR
(DC-SIGNR CRD; aa residue 262−398, GenBank accession no.
Q9H2x3). The DC-SIGN/DC-SIGNR CRD encoding inserts were
amplified by PCR using primers containing the restriction sites EcoRI
and NcoI, respectively. The insert was ligated into the corresponding
cloning site of the pFUSE-hIgG2-Fc2 expression vector (Invivogen,
Toulouse, France). The constructs were amplified in chemocompetent
DH5α E. coli (Novagen, Lucerne, Switzerland), and their correctness
was confirmed by DNA sequencing.
Compound Purity. The test compounds 1−3 and 5 were purified
by reversed-phase chromatography (RP-18 column, gradient of MeOH
in H2O, compound 1−35d,25) or chromatography on silica (DCM/
MeOH/H2O, compound 55g) followed by Bio-Gel P2 (exclusion limit
1800 Da, Bio-Rad Laboratories) size exclusion chromatography
(elution with water containing up to 20% MeOH at 0.25 mL/min)
prior to HPLC, HR-MS, NMR, and activity testing. Compound 4 was
purified by preparative LC-MS (Waters SunFire C18 OBD column,
H2O/MeCN + 0.2% HCO2H). The purity of all test compounds was
determined by NMR and HPLC to be ≥95% [method A (compounds
2, 3, and 5): Beckman Coulter Gold, consisting of pump 126, DAD
168 (190−410 nm), and autosampler 508; column, Waters Atlantis T3
(3 μm, 2.1 mm × 100 mm); A, H2O + 0.1% TFA; B, MeCN + 0.1%
TFA; detection, 254 or 270 nm; gradient, 5% B → 95% B (22 min);
and flow rate, 0.5 mL/min. Method B (compounds 1 and 4): Agilent
1100/1200 with UV detector (190−410 nm) and ELSD; column,
Waters Atlantis T3 (3 μm, 2.1 mm × 100 mm); A, H2O + 0.1% TFA;
B, H2O/MeCN (90:10) + 0.1% TFA; gradient, 5% B (1 min), 5% B →
70% B (15 min), 70% B (1 min), 70% B → 5% B (3 min); flow rate,
1
CHO-K1 cells (American Type Culture Collection No. CCL-61)
were cultivated in Ham's Nutrient Mixture F-12 supplemented with 2
mM L-glutamate (Invitrogen, Paisley, United Kingdom), 10% fetal calf
serum (FCS, Invitrogen), 100 U/mL penicillin, and 100 μg/mL
streptomycin (both Sigma-Aldrich, Basel, Switzerland). The CHO-K1
cells were transfected with the DC-SIGN CRD or DC-SIGNR CRD
expression vector using the FuGENE HD transfection reagent (Roche
Applied Science, Rotkreuz, Switzerland). Stably transfected CHO-K1
cells were selected by treatment with Zeocin (0.5 μg/mL, Invitrogen),
and single clones were obtained by limiting dilution. For protein
production, the cells were cultivated as described above, and the
culture medium containing the secreted DC-SIGN CRD-Fc and DC-
SIGN CRD-Fc chimera was harvested weekly. Purification of the
recombinant protein was achieved by applying conditioned medium
on a protein A-sepharose column (BioVision, Mountain View, CA)
attached to a fast protein liquid chromatography apparatus [BioLogic
(FPLC) system, BioRad, Reinach BL, Switzerland], with loading buffer
I [20 mM Tris/HCl, pH 7.6, 150 mM NaCl, and 0.05% (v/v) Tween-
20]. The protein was eluted with elution buffer I (0.5 M acetic acid/
ammonium acetate, pH 3.4). The collected protein was further
purified on a L-fucose-sepharose column (prepared in house) using
loading buffer II (20 mM Tris/HCl, pH 7.8, 0.5 M NaCl, and 25 mM
CaCl2) and elution buffer II (20 mM Tris/HCl, pH 7.8, 0.5 M NaCl,
and 2 mM EDTA). For long-term storage, the protein was stored at
−80 °C.
FimH, Human Langerin, DLEC, SP-D, Mannose Binding
Protein (MBP), Dectin-2, and MMR. The FimH CRD linked with a
thrombin cleavage site to a 6His-tag (FimH-CRD-Th-6His) was
expressed in E. coli strain HM125 and purified by affinity
chromatography as described in Rabbani et al.4d Human langerin,
DLEC, SP-D, MBP, dectin-2, and MMR were purchased from R&D
systems (Minneapolis, MN).
Competitive Binding Assay. Biotinylated trimannose (TM)-PAA
polymer (20 μL, 1 mg/mL, Lectinity, Moscow) was mixed with 80 μL
of assay buffer (20 mM HEPES, 150 mM NaCl, and 10 mM CaCl2,
pH 7.4), 20 μL of FCS, and 80 μL of streptavidin−horseradish
peroxidase conjugate (500 U/mL, Roche, Mannheim, Germany) and
incubated for 2 h at 37 °C. The complex was stable for several weeks
when stored at 4 °C.
0.5 mL/min; and detection, 254 nm or ELSD]. For the H NMR
spectrum and HPLC trace of compound 4, see the Supporting
Information.
4-Amino-2-chlorophenyl α-D-Mannopyranoside (7). A sus-
pension of 64c,26 (430 mg, 1.28 mmol), morpholine (30 μL), and PtO2
(50 mg) in MeOH (20 mL) was hydrogenated (1 atm H2) for 45 min.
Then, the mixture was filtered and concentrated in vacuo to give crude
7 (443 mg) as a colorless oil, which contained approximately 15%
morpholine and was used in the next step without further purification.
1H NMR (500 MHz, CD3OD): δ 3.71−3.81 (m, 3H, H-4, H-5, H-6a),
3.83 (dd, J = 1.7, 12.7 Hz, 1H, H-6b), 3.95 (dd, J = 3.4, 9.1 Hz, 1H, H-
3), 4.11 (dd, J = 1.8, 3.4 Hz, 1H, H-2), 5.27 (d, J = 1.7 Hz, 1H, H-1),
6.61 (dd, J = 2.7, 8.7 Hz, 1H, C6H3), 6.78 (d, J = 2.7 Hz, 1H, C6H3),
7.09 (d, J = 8.7 Hz, 1H, C6H3). 13C NMR (125 MHz, CD3OD): δ 62.6
(C-6), 68.4 (C-4), 71.9 (C-2), 72.3 (C-3), 75.7 (C-5), 102.1 (C-1),
115.7, 117.4, 121.2, 153.2 (6C, C6H3). ESI-MS: m/z calcd for
C12H17ClNO6 [M + H]+, 306.1; found, 306.0.
2-Chloro-4-[(2-ethoxy-3,4-dioxocyclobuten-1-yl)amino]-
phenyl α-D-Mannopyranoside (8).4c To a solution of 7 (443 mg)
in MeOH (15 mL) was added diethyl squarate (379 μL, 2.56 mmol)
under argon, and the reaction mixture was stirred at rt for 1 d. Then,
the solvent was removed in vacuo, and the residue was purified by
MPLC on RP-18 (H2O/MeOH) to yield 8 (337 mg, 61% from 6) as a
yellow solid. 1H NMR (500 MHz, CD3OD): δ 1.47 (t, J = 7.1 Hz, 3H,
OCH2CH3), 3.66 (dt, J = 3.7, 9.9 Hz, 1H, H-5), 3.75 (d, J = 3.7 Hz,
2H, H-6), 3.79 (t, J = 9.8 Hz, 1H, H-4), 4.00 (dd, J = 3.4, 9.6 Hz, 1H,
H-3), 4.16 (dd, J = 1.8, 3.3 Hz, 1H, H-2), 4.81 (q, J = 7.1 Hz, 2H,
OCH2CH3), 5.56 (d, J = 1.5 Hz, 1H, H-1), 7.23 (m, 1H, C6H3), 7.32
(d, J = 9.0 Hz, 1H, C6H3), 7.47 (s, 1H, C6H3). 13C NMR (125 MHz,
CD3OD): δ 15.7 (OCH2CH3), 61.4 (C-6), 67.2 (C-4), 70.8 (C-2),
71.4 (C-3), 71.6 (OCH2CH3), 74.9 (C-5), 100.0 (C-1), 119.2, 120.2,
122.4, 124.7, 134.6, 152.5 (C6H3), 168.9, 176.2 (CC), 183.3, 186.9
(2 CO). ESI-MS: m/z calcd for C18H21ClNO9 [M + H]+, 430.1;
found, 430.1.
2-Chloro-4-[(2-(4-methylpiperazin-1-yl)-3,4-dioxocyclobut-
en-1-yl)amino]phenyl α-D-Mannopyranoside (4). Compound 8
(72.5 mg, 0.169 mmol) was dissolved in MeOH (7.5 mL) at 50 °C.
After it was cooled to rt, N-methylpiperazine (28.0 μL, 0.252 mmol)
and diisopropyl-ethylamine (DIPEA) (145 μL) were added, and the
reaction mixture was stirred for 18 h at rt. Then, the solvent was
removed in vacuo, and the residue was purified by LC-MS (RP-18,
H2O/MeCN + 0.2% HCO2H) to give 4 (73.9 mg, 90%) as a white
For assay development FimH, DC-SIGN, DC-SIGNR, MBP,
langerin, DLEC, SP-D, dectin-2, and MMR were each diluted in
assay buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, and 10 mM
9814
dx.doi.org/10.1021/jm3010338 | J. Med. Chem. 2012, 55, 9810−9816

Journal of Medicinal Chemistry
Article
CaCl2) at concentrations of 1, 3, 5, and 10 μg/mL and were coated on
microtiter plates (F96 MaxiSorp, Nunc, Langenselbold, Germany)
with 100 μL/well overnight at 4 °C. The coating solution was
discarded, and the wells were blocked with 200 μL/well of a 3% bovine
serum albumin (BSA) solution in assay buffer for 2 h at 4 °C. After
three washing steps with assay buffer, the streptavidin−peroxidase-
coupled TM-PAA polymer (0.5−5 μg/mL) in assay buffer (50 μL/
well) was added to the wells. The plates were incubated for 3 h at 25
°C and 350 rpm and then washed four times with assay buffer. After
the addition of 100 μL/well of 2,2′-azino-bis-(3-ethylbenzthiazoline-6-
sulfonic acid) (ABTS) substrate (Invitrogen), the colorimetric reaction
was allowed to develop for 4 min and then stopped by adding 100 μL/
well of 2% aqueous oxalic acid. The OD was measured at 415 nm on a
microplate reader (Spectramax 190, Molecular Devices, CA).
For measuring the binding properties of FimH antagonists to the
mannose binding receptors, a mix (total volume 100 μL/well) of the
test compounds 1−5 (final concentration 1 mM) or α-D-mannose
(final concentration 50 mM) and the streptavidin−peroxidase-coupled
TM-PAA polymer in assay buffer (final concentration see Table 1) was
added to the protein-coated wells and developed as described above.
and IbStructural basis for the selective binding of FimH adhesin to
uroplakin Ia. J. Biol. Chem. 2006, 281, 14644−14653. (d) Zhou, G.;
Mo, W. J.; Sebbel, P.; Min, G. W.; Neubert, T. A.; Glockshuber, R.;
Wu, X. R.; Sun, T. T.; Kong, X. P. Uroplakin Ia is the urothelial
receptor for uropathogenic Escherichia coli: evidence from in vitro
FimH binding. J. Cell Sci. 2001, 114, 4095−4103.
(3) (a) Ofek, I.; Hasy, D. L.; Sharon, N. Anti-adhesion therapy of
bacterial diseases: Prospects and problems. FEMS Immunol. Med.
Microbiol. 2003, 38, 181−191. (b) Sharon, N. Carbohydrates as future
anti-adhesion drugs for infectious diseases. Biochim. Biophys. Acta, Gen.
Subj. 2006, 1760, 527−537. (c) Ernst, B.; Magnani, J. L. From
carbohydrate leads to glycomimetic drugs. Nat. Rev. Drug Discovery
2009, 8, 661−677.
(4) (a) Lindhorst, T. K.; Kieburg, C.; Krallmann-Wenzel, U.
Inhibition of the type 1 fimbriae-mediated adhesion of Escherichia
coli to erythrocytes by multiantennary alpha-mannosyl clusters: The
effect of multivalency. Glycoconjugate J. 1998, 15, 605−613.
(b) Bouckaert, J.; Berglund, J.; Schembri, M.; De Genst, E.; Cools,
L.; Wuhrer, M.; Hung, C. S.; Pinkner, J.; Slattegard, R.; Zavialov, A.;
Choudhury, D.; Langermann, S.; Hultgren, S. J.; Wyns, L.; Klemm, P.;
Oscarson, S.; Knight, S. D.; De Greve, H. Receptor binding studies
disclose a novel class of high-affinity inhibitors of the Escherichia coli
FimH adhesin. Mol. Microbiol. 2005, 55, 441−455. (c) Sperling, O.;
Fuchs, A.; Lindhorst, T. K. Evaluation of the carbohydrate recognition
domain of the bacterial adhesin FimH: design, synthesis and binding
properties of mannoside ligands. Org. Biomol. Chem. 2006, 4, 3913−
3922. (d) Rabbani, S.; Jiang, X.; Schwardt, O.; Ernst, B. Expression of
the carbohydrate recognition domain of FimH and development of a
competitive binding assay. Anal. Biochem. 2010, 407, 188−195.
(e) Pang, L.; Kleeb, S.; Lemme, K.; Rabbani, S.; Scharenberg, M.;
ASSOCIATED CONTENT
■
S
* Supporting Information
1
HPLC trace and H NMR spectrum for compound 4. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +41 61 267 1551. Fax: +41 61 267 1552. E-mail: beat.
ernst@unibas.ch.
■
Zalewski, A.; Schadler, F.; Schwardt, O.; Ernst, B. FimH antagonists:
̈
Structure-activity and structure-property relationships for biphenyl α-
D-mannopyranosides. ChemMedChem 2012, 7, 1404−1422.
Notes
The authors declare no competing financial interest.
(5) (a) Aronson, M.; Medalia, O.; Schori, L.; Mirelman, D.; Sharon,
N.; Ofek, I. Preventing of colonization of the urinary tract of mice with
Escherichia−coli by blocking of bacterial adherance with methyl alpha-
D-mannoypyranoside. J. Infect. Dis. 1979, 139, 329−332. (b) Wellens,
A.; Garofalo, C.; Nguyen, H.; Van Gerven, N.; Slattegard, R.;
Hernalsteens, J.-P.; Wyns, L.; Oscarson, S.; De Greve, H.; Hultgren, S.;
Bouckaert, J. Intervening with urinary tract infections using anti-
adhesives based on the crystal structure of the FimH-oligomannose-3
complex. PLoS One 2008, 30;3, e2040. (c) Han, Z.; Pinkner, J. S.;
Ford, B.; Obermann, R.; Nolan, W.; Wildman, S. A.; Hobbs, D.;
Ellenberger, T.; Cusumano, C. K.; Hultgren, S. J.; Janetka, J. W.
Structure-based drug design and optimization of mannoside bacterial
FimH antagonists. J. Med. Chem. 2010, 53, 4779−4792. (d) Klein, T.;
Abgottspon, D.; Wittwer, M.; Rabbani, S.; Herold, J.; Jiang, X.; Kleeb,
ACKNOWLEDGMENTS
■
Plasmids containing the full-length cDNA of DC-SIGN and
DC-SIGNR were kindly provided by Dr. Daniel Mitchell,
Clinical Sciences Research Institute, Warwick Medical School
(Coventry, United Kingdom). The financial support by the
Swiss National Science Foundation (SNF interdisciplinary
grant K-32K1-120904) is gratefully acknowledged.
ABBREVIATIONS USED
■
ABTS, 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid);
BSA, bovine serum albumin; CFU, colony-forming units;
CRD, carbohydrate recognition domain; DC-SIGN, DC-
specific ICAM-3-grabbing nonintegrin; DC-SIGNR, DC-
specific ICAM-3-grabbing nonintegrin related; DLEC, dendritic
cell lectin; DIPEA, diisopropyl-ethylamine; FCS, fetal calf
serum; MBP, mannose binding protein; MMR, macrophage
mannose receptor; OD, optical density; PAA, polyacrylamide;
TM-PAA, Manα1−3(Manα1−6)Manβ1−4GlcNAcβ1−
4GlcNAcβ-PAA; PRR, pathogen-recognition receptors; SP-D,
lung surfactant protein D; UPEC, uropathogenic Escherichia
coli; UPIa, uroplakin Ia; UTI, urinary tract infection
S.; Luethi, C.; Scharenberg, M.; Bezenco̧ n, J.; Gubler, E.; Pang, L.;
Smiesko, M.; Cutting, B.; Schwardt, O.; Ernst, B. FimH antagonists for
the oral treatment of urinary tract infections: From design and
synthesis to in vitro and in vivo evaluation. J. Med. Chem. 2010, 53,
8627−8641. (e) Han, Z.; Pinkner, J. S.; Ford, B.; Chorell, E.; Crowley,
J. M.; Cusumano, C. K.; Campbell, S.; Henderson, J. P.; Hultgren, S. J.;
Janetka, J. W. Lead optimization studies on FimH antagonists:
Discovery of potent and orally bioavailable ortho-substituted biphenyl
mannosides. J. Med. Chem. 2012, 55, 3945−3959. (f) Cusumano, C.
K.; Pinkner, J. S.; Han, Z.; Greene, S. E.; Ford, B. A.; Crowley, J. R.;
Henderson, J. P.; Janetka, J. W.; Hultgren, S. J. Treatment and
prevention of urinary tract infection with orally active FimH inhibitors.
Sci. Transl. Med. 2011, 3, 1−10. (g) Jiang, X.; Abgottspon, D.; Kleeb,
S.; Rabbani, S.; Scharenberg, M.; Wittwer, M.; Haug, M.; Schwardt, O.;
Ernst, B. Anti-adhesion therapy for urinary tract infectionsA
balanced PK/PD-profile proved to be key for success. J. Med. Chem.
2012, 55, 4700−4713.
REFERENCES
■
(1) Ronald, A. The etiology of urinary tract infection: Traditional and
emerging pathogens. Am. J. Med. 2002, 113, 14S−19S.
(2) (a) Mulvey, M. A. Adhesion and entry of uropathogenic
Escherichia coli. Cell. Microbiol. 2002, 4, 257−271. (b) Wiles, T. J.;
Kulesus, R. R.; Mulvey, M. A. Origins and virulence mechanisms of
uropathogenic Escherichia coli. Exp. Mol. Pathol. 2008, 85, 11−19.
(c) Xie, B.; Zhou, G.; Chan, S. Y.; Shapiro, E.; Kong, X. P.; Wu, X. R.;
Sun, T. T.; Costello, C. E. Distinct glycan structures of uroplakins Ia
(6) Huggins, D. J.; Sherman, W.; Tidor, B. Rational approaches to
improving selectivity in drug design. J. Med. Chem. 2012, 55, 1424−
1444.
9815
dx.doi.org/10.1021/jm3010338 | J. Med. Chem. 2012, 55, 9810−9816

Journal of Medicinal Chemistry
Article
(7) Khoo, U. S.; Chan, K. Y. K.; Chan, V. S. F.; Lin, C. L. S. DC-
SIGN and L-SIGN: The SIGNs for infection. J. Mol. Med. 2008, 86,
861−874.
(8) Lee, S. J.; Evers, S.; Roeder, D.; Parlow, A. F.; Risteli, J.; Risteli,
L.; Lee, Y. C.; Feizi, T.; Langen, H.; Nussenzweig, M. C. Mannose
receptor-mediated regulation of serum glycoprotein homeostasis.
Science 2002, 295, 1898−1901.
(9) (a) East, L.; Isacke, C. M. The mannose receptor family. Biochim.
Biophys. Acta, Gen. Subj. 2002, 1572, 364−386. (b) Dommett, R. M.;
Klein, N.; Turner, M. W. Mannose-binding lectin in innate immunity:
Past, present and future. Tissue Antigens 2006, 68, 193−209.
(10) Weis, W. I.; Taylor, M. E.; Drickamer, K. The C-type lectin
superfamily in the immune system. Immunol. Rev. 1998, 163, 19−34.
(11) Mori, K.; Kawasaki, T.; Yamashina, I. Isolation and character-
ization of endogenous ligands for liver mannan-binding protein. Arch.
Biochem. Biophys. 1988, 264, 647−656.
(23) McGreal, E. P.; Rosas, M.; Brown, G. D.; Zamze, S.; Wong, S. Y.
C.; Gordon, S.; Martinez-Pomares, L.; Taylor, P. R. The carbohydrate-
recognition domain of Dectin-2 is a C-type lectin with specificity for
high mannose. Glycobiology 2006, 16, 422−430.
(24) Appelmelk, B. J.; van Die, I.; van Vliet, S. J.; Vandenbroucke-
Grauls, C.; Geijtenbeek, T. B. H.; van Kooyk, Y. Cutting edge:
Carbohydrate profiling identifies new pathogens that interact with
dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells.
J. Immunol. 2003, 170, 1635−1639.
(25) Abgottspon, D.; Roelli, G.; Hosch, L.; Steinhuber, A.; Jiang, X.;
Schwardt, O.; Cutting, B.; Smiesko, M.; Jenal, U.; Ernst, B.; Trampuz,
A. Development of an aggregation assay to screen FimH antagonists. J.
Microbiol. Methods 2010, 82, 249−255.
(26) (a) Vervoort, A.; De Bruyne, C. K. Enzymatic hydrolysis of
substituted phenyl-alpha-D-mannopyranosides. Carbohydr. Res. 1970,
12, 277−280. (b) De Prijcker, J.; De Bock, A.; De Bruyne, C. K. α-D-
mannosidase-catalyzed hydrolysis of substituted phenyl α-D-manno-
pyranosides. Carbohydr. Res. 1978, 60, 141−153.
(27) Kosak, J. R. Catalytic hydrogenation of aromatic halonitro
compounds. Ann. N.Y. Acad. Sci. 1970, 172, 175−185.
(28) Guo, P.; Feinberg, H.; Conroy, E.; Mitchell, D. A.; Alvarez, R.;
Blixt, O.; Taylor, M. E.; Weis, W. I.; Drickamer, K. Structural basis for
distinct ligand-binding and targeting properties of the receptors DC-
SIGN and DC-SIGNR. Nat. Struct. Mol. Biol. 2004, 11, 591−598.
(29) Linehan, S. A.; Martinez-Pomares, L.; da Silva, R. P.; Gordon, S.
Endogenous ligands of carbohydrate recognition domains of the
mannose receptor in murine macrophages, endothelial cells and
secretory cells; potential relevance to inflammation and immunity. Eur.
J. Immunol. 2001, 31, 1857−1866.
(30) (a) Imberty, A.; Chabre, Y. M.; Roy, R. Glycomimetics and
glycodendrimers as high affinity microbial anti-adhesins. Chemistry
2008, 14, 7490−7499. (b) Hartmann, M.; Lindhorst, T. K. The
bacterial lectin FimH, a target for drug discoveryCarbohydrate
inhibitors of type 1 fimbriae-mediated bacterial adhesion. Eur. J. Org.
Chem. 2011, 3583−3609.
(12) Kishore, U.; Greenhough, T. J.; Waters, P.; Shrive, A. K.; Ghai,
R.; Kamran, M. F.; Bernal, A. L.; Reid, K. B. M.; Madan, T.;
Chakraborty, T. Surfactant proteins SP-A and SP-D: Structure,
function and receptors. Mol. Immunol. 2006, 43, 1293−1315.
(13) Wallis, R.; Mitchell, D. A.; Schmid, R.; Schwaeble, W. J.; Keeble,
A. H. Paths reunited: Initiation of the classical and lectin pathways of
complement activation. Immunobiology 2010, 215, 1−11.
(14) (a) Jensen, P. H.; Weilguny, D.; Matthiesen, F.; McGuire, K. A.;
Shi, L.; Hojrup, P. Characterization of the oligomer structure of
recombinant human mannan-binding lectin. J. Biol. Chem. 2005, 280,
11043−11051. (b) Teillet, F.; Dublet, B.; Andrieu, J. P.; Gaboriaud,
C.; Arland, G. J.; Thielens, N. M. The two major oligomeric forms of
human mannan-binding lectin: Chemical characterization, carbohy-
drate-binding properties, and interaction with MBL-associated serine
proteases. J. Immunol. 2005, 174, 2870−2877. (c) Sim, R. B.; Clark,
H.; Hajela, K.; Mayilyan, K. R. Collectins and host defence. Novartis
Found. Symp. 2006, 279, 170−181. (d) Ohya, M.; Nishitani, C.; Sano,
H.; Yamada, C.; Mitsuzawa, H.; Shimizu, T.; Saito, T.; Smith, K.;
Crouch, E.; Kuroki, Y. Human pulmonary surfactant protein D binds
the extracellular domains of toll-like receptors 2 and 4 through the
carbohydrate recognition domain by a mechanism different from its
binding to phosphatidylinositol and lipopolysaccharide. Biochemistry
2006, 45, 8657−8664.
(31) Hung, C. S.; Bouckaert, J.; Hung, D.; Pinkner, J.; Widberg, C.;
DeFusco, A.; Auguste, C. G.; Strouse, R.; Langermann, S.; Waksman,
G.; Hultgren, S. J. Structural basis of tropism of Escherichia coli to the
bladder during urinary tract infection. Mol. Microbiol. 2002, 44, 903−
915.
(32) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning. A
Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press:
Cold Spring Harbor, NY, 1989; Vols. 1−3.
(15) Kerrigan, A. M.; Brown, G. D. C-type lectins and phagocytosis.
Immunobiology 2009, 214, 562−575.
(16) Valladeau, J.; Ravel, O.; Dezutter-Dambuyant, C.; Moore, K.;
Kleijmeer, M.; Liu, Y.; Duvert-Frances, V.; Vincent, C.; Schmitt, D.;
Davoust, J.; Caux, C.; Lebecque, S.; Saeland, S. Langerin, a novel C-
type lectin specific to Langerhans cells, is an endocytic receptor that
induces the formation of Birbeck granules. Immunity 2000, 12, 71−81.
(17) Geijtenbeek, T. B. H.; Torensma, R.; van Vliet, S. J.; van
Duijnhoven, G. C. F.; Adema, G. J.; van Kooyk, Y.; Figdor, C. G.
Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3
receptor that supports primary immune responses. Cell 2000, 100,
575−585.
(18) Feinberg, H.; Mitchell, D. A.; Drickamer, K.; Weis, W. I.
Structural basis for selective recognition of oligosaccharides by DC-
SIGN and DC-SIGNR. Science 2001, 294, 2163−2166.
(19) Kanazawa, N. Dendritic cell immunoreceptors: C-type lectin
receptors for pattern-recognition and signaling on antigen-presenting
cells. J. Dermatol. Sci. 2007, 45, 77−86.
(20) Kanazawa, N.; Tashiro, K.; Miyachi, Y. Signaling and immune
regulatory role of the dendritic cell immunoreceptor (DCIR) family
lectins: DCIR, DCAR, dectin-2 and BDCA-2. Immunobiology 2004,
209, 179−190.
(21) Stambach, N. S.; Taylor, M. E. Characterization of carbohydrate
recognition by langerin, a C-type lectin of Langerhans cells.
Glycobiology 2003, 13, 401−410.
(22) Crouch, E. C.; Smith, K.; McDonald, B.; Briner, D.; Linders, B.;
McDonald, J.; Holmskov, U.; Head, J.; Hartshorn, K. Species
differences in the carbohydrate binding preferences of surfactant
protein D. J. Dermatol. Sci. 2006, 35, 84−94.
9816
dx.doi.org/10.1021/jm3010338 | J. Med. Chem. 2012, 55, 9810−9816