Thiazolylaminomannosides as potent antiadhesives of type 1 piliated escherichia coli isolated from crohn's disease patients

Article abstract of DOI:10.1021/jm400723n

Adherent-invasive Escherichia coli (AIEC) have previously been shown to induce gut inflammation in patients with Crohn's disease (CD). We developed a set of mannosides to prevent AIEC attachment to the gut by blocking the FimH bacterial adhesin. The crystal structure of the FimH lectin domain in complex with a lead thiazolylaminomannoside highlighted the preferential position for pharmacomodulations. A small library of analogues showing nanomolar affinity for FimH was then developed. Notably, AIEC attachment to intestinal cells was efficiently prevented by the most active compound and at around 10000-fold and 100-fold lower concentrations than mannose and the potent FimH inhibitor heptylmannoside, respectively. An ex vivo assay performed on the colonic tissue of a transgenic mouse model of CD confirmed this antiadhesive potential. Given the key role of AIEC in the chronic intestinal inflammation of CD patients, these results suggest a potential antiadhesive treatment with the FimH inhibitors developed.

Full text of DOI:10.1021/jm400723n

Article
pubs.acs.org/jmc
Thiazolylaminomannosides As Potent Antiadhesives of Type 1
Piliated Escherichia coli Isolated from Crohn’s Disease Patients
Sami Brument,† Adeline Sivignon,‡,§ Tetiana I. Dumych,⊥ Nicolas Moreau,† Goedele Roos,#
Yann Guerardel,▽ Thibaut Chalopin,† David Deniaud,† Rostyslav O. Bilyy,⊥,*
́
Arlette Darfeuille-Michaud,*,‡,§,∥ Julie Bouckaert,*,▽ and Sebastien G. Gouin*,†
́
†LUNAM Universite,
et des Techniques, 2 rue de la Houssinier
‡Clermont Universite,
UMR 1071 Inserm/Universite
§INRA, Unite
́
CEISAM, Chimie Et Interdisciplinarite,
e, BP 92208, 44322 NANTES Cedex 3, France
d’Auvergne, 63000 Clermont-Ferrand, France
́ ̀ ́
Synthese, Analyse, Modelisation, UMR CNRS 6230, UFR des Sciences
̀
́
́
́
Sous Contrat 2018, 63000 Clermont-Ferrand, France
∥Centre Hospitalier Universitaire, 63000 Clermont-Ferrand, France
⊥Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine
#Algemene Chemie (ALGC), Vrije Universiteit Brussel, Brussels, Belgium
▽Unite
de Glycobiologie Structurale et Fonctionnelle (UGSF), UMR8576 du CNRS, Universite Lille 1, F-59655 Villeneuve d’Ascq
́ ́
Cedex, France
S
* Supporting Information
ABSTRACT: Adherent-invasive Escherichia coli (AIEC) have
previously been shown to induce gut inflammation in patients
with Crohn’s disease (CD). We developed a set of mannosides
to prevent AIEC attachment to the gut by blocking the FimH
bacterial adhesin. The crystal structure of the FimH lectin
domain in complex with a lead thiazolylaminomannoside
highlighted the preferential position for pharmacomodulations.
A small library of analogues showing nanomolar affinity for
FimH was then developed. Notably, AIEC attachment to
intestinal cells was efficiently prevented by the most active
compound and at around 10000-fold and 100-fold lower
concentrations than mannose and the potent FimH inhibitor
heptylmannoside, respectively. An ex vivo assay performed on
the colonic tissue of a transgenic mouse model of CD confirmed this antiadhesive potential. Given the key role of AIEC in the
chronic intestinal inflammation of CD patients, these results suggest a potential antiadhesive treatment with the FimH inhibitors
developed.
was mimicked using a transgenic mouse model expressing
human CEACAM6 receptor. AIEC-infected mice were
abundantly colonized by these bacteria and developed severe
colitis.7
INTRODUCTION
■
Crohn’s disease (CD) is characterized by an aberrant immune
response occurring in a genetically predisposed host in
response to microbes and/or microbial compounds.1,2 In
patients with CD, high concentrations of bacteria forming a
biofilm on the surface of the gut mucosa and increased numbers
of mucosa-associated Escherichia coli are observed.3 These
bacteria, called AIEC for adherent-invasive E. coli, colonize the
ileal mucosa of CD patients.4 They can adhere to intestinal
epithelial cells and replicate rapidly within macrophages and
epithelial cells inducing the secretion of large amounts of TNF-
α.5 For CD patients in whom the ileum is involved in the
disease, an abnormal expression of carcinoembryonic antigen-
related cell adhesion molecules 5 and 6 (CEACAM5 and
CEACAM6) has been reported and studies have shown that
CEACAM6 acts as a receptor for AIEC bacteria.6 The host/
bacteria crosstalk in the context of host susceptibility to CD
As AIEC attachment is mediated by the FimH adhesin
located at the tip of the type 1 pili, which bind to
oligomannosides displayed on intestinal cells, we hypothesized
that the development of FimH antagonists preventing AIEC
binding to the gut could be relevant for CD treatment. Such an
antiadhesive strategy based on FimH inhibition has attracted
much attention during the past few years for the treatment of
urinary tract infections (UTIs). We and others have developed
synthetic mannosides with promising in vitro8,9 and in vivo10,11
antiadhesive potencies. Increased affinity for FimH was
Received: February 21, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
obtained with synthetic O-mannosides bearing hydrophobic
aglycons interacting with two tyrosines (Tyr48 and Tyr137) at
the entrance of the FimH binding site.
conducting the cyclization step with dienes or dienophiles
bearing diverse R groups. We have also previously shown that
the thiomethyl group of the pyrimidine ring can easily be
substituted by nucleophilic displacement,15 which provides
further opportunities to increase the chemical diversity of the
heterocycles.
Our efforts were first focused on the pyrimidine series.
Peracetylated mannosylisothiocyanate 116 was obtained from
the corresponding mannopyranosylchloride17 with potassium
thiocyanate and tetrabutylammonium iodide, as previously
described by Camarasa and co-workers.18 This compound was
engaged in a [4 + 2] cycloaddition reaction with 1,3-
diazadienum iodide19 2 and triethylamine to afford the
pyrimidine 3 in a quantitative yield (Scheme 2). The cyclization
The aim here was to develop a new class of FimH
antagonists bearing N-linked heterocyclic aglycons. N-Manno-
sides have been much less investigated for FimH inhibition.12,13
The aromatic heterocycles should increase the binding affinity
through stacking interactions with Tyr48 and Tyr137 of FimH.
Water solubility issues, generally encountered with O- or C-
mannosides bearing hydrophobic aglycons, may also be
reduced. We selected two heterocycles (pyrimidine and
thiazole) which are accessible by a convenient addition-
cyclization methodology developed in our group.14 In view of
the broad adaptability of the FimH binding site, illustrated by
the numerous FimH antagonists based on O- or C-linked
mannosides described so far,8−11 we hoped for a first hit with
one of the lead heterocyclic mannosides. We then planned to
improve the FimH binding potency by designing a small library
of a second generation of inhibitors with different pharmaco-
phores on the heterocycle.
Scheme 2. Synthesis of the Pyrimidylmannosides 4 and 5
Thiazole was the only heterocycle to be further considered as
1
pyrimidine-based mannosides adopted an unusual C4 config-
uration, unsuitable for FimH inhibition. Co-crystallization of a
lead compound with FimH showed possible pharmacomodu-
lations of the R2 group (Scheme 1) to give supplementary
Scheme 1. Structure of the Mannosides with Pyrimidine and
Thiazole Aglycons
step was followed by a spontaneous deamination and the
1
intermediate presented in Scheme 2 was never isolated. H
NMR analysis revealed a surprisingly high H1−H2 coupling
constant for a mannoside of J1,2 = 9.6 Hz (Supporting
Information Figure S1), not in agreement with the generally
observed 4C1 conformation but coherent with the less
frequently observed 1C4. This assumption was further
confirmed by the crystallographic structure of compound 3
(Figure 1).
The thiomethyl group of 3 was shown to be sensitive to
nucleophilic substitution. An excess of sodium methanolate or
ammonia in methanol led to unprotected compounds 4 and 5
where the thiomethyl group was substituted by a methoxy and
an amino group, respectively. Both compounds retained the
unusual 1C4 conformation during the deprotection steps.
Pyrimidine-based analogues were not further developed
because the 1C4 conformation adopted by the mannose hinders
an optimal fit in the FimH binding site. In fact, compounds 3
and 4 were poor inhibitors of FimH in a cell-based assay
(results not shown). It should be noted that previously
described FimH inhibitors based on triazolylmethyl-C-manno-
stacking interactions with Tyr48. A small library of analogues
was then designed, and the binding potency on FimH evaluated
by a competitive enzyme-linked immunosorbent assay
(ELISA). The ability of the new FimH antagonists to prevent
adhesion of the AIEC reference strain LF82 was assessed in
vitro with guinea pig erythrocytes and the human intestinal cell
line T84 and ex vivo in the colonic loop of a transgenic mouse
model of CD.
1
RESULTS AND DISCUSSION
sides adopting a C4 conformation also displayed a relatively
■
Synthesis. The selected heterocycles would be poorly
grafted on sugars by conventional glycosylation methods due to
the presence of nucleophilic heteroatoms. Thus, the pyrimidine
and thiazole rings were built by an addition-cyclization
methodology developed in our group, performed here on
mannosylisothiocyanate (Scheme 1). Interestingly, this chem-
ical procedure enabled a library of analogues to be generated by
low affinity for FimH.13
The addition-cyclization strategy was then used to build the
thiazole on mannose. Mannosylisothiocyanate 1 was first
reacted with NH3 gas in toluene to form thiourea derivative
6 in 92% yield without deacetylation of the sugar moiety
(Scheme 3). Compound 6 was quantitatively converted into
the thiazadiene 7 with N,N-dimethylformamide dimethyl acetal
B
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
Figure 1. Crystallographic structure of compound 3 showing the unusual C4 conformation.
Scheme 3. Synthesis of Aminothiazole 9
Scheme 4. Structure of the Thiazolylaminomannosides 10−31
(DMFDMA) in dichloromethane. The subsequent addition-
cyclization reaction was first performed with a 2-fold excess of
1-chloropropan-2-one and triethylamine at 60 °C for 16 h.
Under these conditions, compound 8 was obtained in 83%
yield as a pure α-anomer (Scheme 3). Experimental conditions
must be carefully controlled as increasing both the reaction
time to 48 h and the temperature to 70 °C led to the formation
of the β-anomer in a proportion reaching up to 50%. α- and β-
anomers were unambiguously identified with the critical
1
anomeric H−13C 1J coupling constants, which have previously
been shown to be around 170 Hz for α-products and 150 Hz
for β-products.20 The values observed for a mixture of α- and β-
anomers 8, obtained with increased reaction times, were 166
and 149 Hz, respectively (Supporting Information Figure S2).
The deprotection of the α-anomer was carried out with only
C
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. FimH−9 complex showing the ligand 9 bound with its thiazole stacked against the closed tyrosine gate. The same overall structure, with
Tyr48 and Tyr137 and 9 (all shown in ball-and-stick model), is observed in the crystals in both the orthorhombic and the monoclinic space groups
(PDB entry codes 3zl1 and 3zl2). Water molecules are shown as red-dotted spheres expanding to their van der Waals radius (figure created using
Pymol version 0.99). The figure shows hydrous solvation over Tyr137 and hydrophobic shielding of the tyrosine gate. Polar interactions (in Å) are
also displayed and a crown of water rendering Tyr137, Tyr48, and the carboxylate of 9 with the solvent.
0.05 equiv of sodium methanolate in methanol to give the
expected compound 9 quantitatively.
distance of about 3.7 Å. However, these two aromatic groups
are not perfectly stacked for an optimal interaction (Figure 3).
The staggered orientation places the methyl of 9 at the C2 axis
of symmetry of the phenyl ring of Tyr48. Thus, we
hypothesized that the substitution of the methyl group of 9
by an aromatic substituent should enable further stacking
interactions with the side chain of Tyr48. Furthermore, Tyr48
forms part of a nonhydrated ridge which is a hydrophobic
extension of the binding site of FimH in the tip of the protein
(Figure 2). For these reasons, the methyl position was favored
for carrying out pharmacomodulations (i.e., compounds 21−
31) with aromatic or hydrophobic substituents to increase
FimH binding affinity.
Tyr137, the second gate door residue, faces the solvent on
the exterior of the protein and shields the mannose-binding site
from solvent. The electron density of the sulfur atom in 9 is
oriented toward the distant, orthogonally stacked Tyr137
residue (Figure 3A). Although this polarization of the sulfur
atom in 9 toward Tyr137 does not appear to contribute to
binding (positive Eint for 9−Tyr137 in Table 1), it seems to
alleviate significantly the electronic hardness (η (r) in Table 1),
thus favoring the interaction of 9 with both tyrosine gate doors.
Quantum Chemical Calculations. A relaxed potential
surface scan for the rotation of the carbonyl moiety around the
C−C bond connecting the carbonyl and the thiazole in ligand 9
(Figure 4) determined that the carbonyl group of 9 is in its
lowest energy state in the crystal structure of the FimH−9
complex. The methyl group is on the opposite side of the sulfur
of the thiazole and, in this orientation, the acetyl is in an
excellent position to interact with Tyr48 (Figure 3).
The promising binding affinity of 9, and insights into the
interaction with the FimH lectin domain provided by the
crystallographic structure (see following sections), encouraged
us to design a small library of analogues with an aromatic R2
group (Scheme 1). This was achieved by selecting commercial
α-halogenoketones (8 out of 11 with an aromatic group) for
the cyclization step. The second generation of thiazolylamino-
mannosides 21−31 was obtained with excellent overall yields
ranging from 83 to 96% (except for 23) for the addition-
cyclization and deprotection steps (Scheme 4). In each case,
the reaction was over after 16 h and the lower yield observed
for 12 (46%) was attributed to a more difficult purification. The
anomeric substituents of the unprotected compound were
shown to be in dynamic equilibrium in water with a preference
for the α forms (α/β ratio >3/1, except for 28, α/β = 3/2).
Crystal Structure of the FimH Lectin Domain in
Complex with Thiazolylaminomannoside 9. The lead
compound 9 was cocrystallized with the FimH lectin domain to
investigate the influence of the thiazole and to define the
relevant positions for complementation binding in the
hydrophobic tyrosine gate of FimH.
Crystallization was accomplished using the sitting drop vapor
diffusion method in two different space groups, orthorhombic
and monoclinic (Supporting Information Table S1). The
structure in the monoclinic space group was refined to 1.55
́
Å resolution and contains two complexes in the asymmetric
unit; 339 water molecules could be identified in their electron
density. All three FimH−9 complexes are almost identical and
show the ligand 9 bound with its thiazole stacked against the
closed tyrosine gate (Figure 2).
The aromatic side chain of Tyr48, the first gate door residue,
shows a parallel orientation with the thiazole ring of 9 at a
The interaction energy between ligand 9 and Tyr48 and
Tyr137 was then calculated and compared with that of benzene
located at the same position as the heterocycle of ligand 9 as
the reference molecule. The total interaction energy between
D
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
were calculated. For both ligands, the interaction with Tyr48 is
the strongest, while that with Tyr137 is negligible (Table 1).
This can be related to the structure in which Tyr48 is found to
be closer to the ligand, and in parallel, than Tyr137, which is
oriented in a T-shaped stacking position.
In addition, the dispersion and the electrostatic energy
components of the interactions between the ligand and Tyr137
and Tyr48 were determined. From a conceptual point of view,
the dispersion energy can be related to the polarizability (α)
and the electrostatic energy to the local hardness (η (r)).21 The
polarizability α is a global descriptor, giving an estimation of the
dispersion component of the total interaction energy between
ligand 9 or benzene and both Tyr137 and Tyr48. α is the
tendency of an electron cloud to be distorted by an electric field
caused, for example, by the stacking partner. A larger
polarizability is favorable for the interaction. A polarizability
of 99 bohr3 was found for ligand 9, while that for the reference
molecule benzene was 41 bohr3. This result corresponds to the
more favorable interaction energy found for ligand 9.
The local hardness is a measure of negative charge
accumulation and is a local descriptor giving an estimation of
the electrostatic component of the interaction energy at
localized points. A large value of η (r) indicates a high
repulsion energy and thus an unfavorable interaction energy.
Local hardness should only be compared at the same points
(Supporting Information Figure S3). For the interaction with
Tyr48, a local hardness of 58 and 104 kcal/mol was found for
ligand 9 and benzene, respectively. This corresponds to the
more favorable interaction energy of 2.5 kcal/mol calculated
between ligand 9 and Tyr48. For the interaction with Tyr137, a
local hardness of 46 and 95 kcal/mol was found for ligand 9
and benzene, respectively. This large difference in local
hardness is not reflected in the interaction energies calculated
between the ligands and Tyr137. Taken together, the relatively
high polarizability and low local hardness of ligand 9 can
explain its high affinity.
Binding Affinity for FimH. The binding affinity of the
synthetic thiazolylaminomannosides 9 and 21−31 toward
FimH was first evaluated by competitive ELISA. Heptylmanno-
side (HM) was also included in the assay as a reference
displaying a strong nanomolar affinity for FimH.22 The RNaseB
protein, which possesses a complex mixture of oligomannose
glycans (Man5GlcNAc2, Man7GlcNAc2, and Man8GlcNAc2),23
was used as the FimH substrate (Figure 5). The intensity of
FimH binding to the substrate is represented by the optical
density of chromophore absorbance at 450 nm and decreases in
the presence of inhibitors in a dose-dependent manner. The
thiazolylaminomannosides 9 and 21−31 showed a strong
affinity for FimH. Compared to the control (FimH alone),
most of the inhibitors, including HM, already significantly
inhibited FimH binding at 5 nM. The marked signal reduction
obtained with HM at nanomolar concentrations is consistent
with the low dissociation constants of 522 and 7 nM24
previously reported for the HM-FimH complex by surface
plasmon resonance (SPR) and isothermal titration calorimetry
(ITC), respectively. Different inhibitory profiles were observed
depending on the nature of the substituents attached to the
thiazole ring. This clearly shows that improved affinity for
FimH may still be expected with pharmacophores situated at
rather large distances from the mannose binding site. As can be
seen from Figure 5, thiazolylaminomannosides 22 and 30 were
able to block FimH binding toward oligomannose glycans at
the low concentration of 5 nM, while mannosides 21, 24, 26,
Figure 3. Two different views of ligand 9 in the mannose-binding site
of FimH. The hydrophobic stacking of 9 against the closed tyrosine
gate (distances in Å) shows its sulfur atom pointing toward Tyr137
and the parallel but staggered orientation of the thiazole ring of 9
relative to the phenyl group of Tyr48.
Table 1. Interaction Energies Eint, Local Hardness η (r), and
Polarizabilities α, of the Thiazolyl Moiety of 9 and Benzene
thiazolyl of 9
benzene
global
Tyr137 + Tyr48
Tyr137 + Tyr48
Eint (kcal/mol)
α (bohr3)
local
−5.1
99
−3.0
41
Tyr137
0.1
46
Tyr48
Tyr137
Tyr48
Eint (kcal/mol)
−5.1
58
−0.4
95
−2.6
104
η (r) (kcal/mol)
Figure 4. Scan orientation of the carbonyl group.
ligand 9 and both Tyr137 and Tyr48 is −5.1 kcal/mol, while
that of benzene is only −3.0 kcal/mol (Table 1), explaining the
high affinity of ligand 9.
To obtain a more detailed picture, the individual interaction
energies between ligand 9 (or benzene) and Tyr137 or Tyr48
E
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. Affinity testing of thiazolylaminomannosides toward oligomannose glyco-epitopes using enzyme-linked immunosorbent assay (ELISA).
The X-axis indicates the compounds used for testing; the Y-axis represents the optical density of TMB chromophore absorbance at 450 nm. The
controls are (from left to right): FimH alone (positive control, black bar), aFimH (dark-gray bar), aFimH + 2ndAb-HRP (light-gray bar), 2ndAb-
HRP (white bar), and TMB (negligible, not shown). The schematic principle of the assay is shown on the right. Briefly, oligomannose glycans of
RNaseB (as the best suited natural FimH target) and the studied inhibitors compete for binding of FimH lectin, then bound FimH is detected by
antibodies. In the case of the absence of inhibitor or a weak inhibitor, FimH will bind to RNaseB sorbed onto a plate and will produce a high signal of
absorbance, while a strong inhibitor will bind to FimH and prevent it from binding to RNaseB on a plate, resulting in a low optical density signal.
27, and 28 effectively blocked its binding at a higher
concentration of 50 nM. A significantly higher level of
inhibition was observed with 22 and 30 compared to the
HM reference.
Prevention of Bacterial Attachment to Erythrocytes. A
widely used cell-based assay (hemagglutination, HAI) was first
selected to evaluate the antiadhesive potency of the
glycoconjugates 9 and 21−31 toward the CD-associated E.
coli strain AIEC LF82. AIEC adhesion to the gut occurs
through the binding of FimH adhesins to the mannosylated
glycans of intestinal cells. This scenario was first mimicked here
with the highly mannosylated glycocalyx of guinea pig
erythrocytes.
Figure 6. Inhibition of hemagglutination (HAI): inhibition of guinea
pig red blood cell hemagglutination by the type-1 piliated AIEC strain
A 2-fold dilution series of the antagonists was added to wells
containing guinea pig erythrocytes and AIEC LF82 bacteria.
LF82 by the newly synthesized glycoconjugates. The error bars
The formation of the cross-linked network (hemagglutination)
due to the interaction of the E. coli FimH adhesins with the
glycocalyx of the erythrocytes was prevented at a certain
concentration of inhibitors. The lowest concentration at which
hemagglutination is still inhibited is defined as the inhibition
titer of the antagonist. HM was included in the assay to obtain a
relative inhibitory concentration (rIC) for the antagonists by
dividing the inhibitory concentration (IC) of the substance of
interest by the value for HM. In fact, rICs are more accurate
than rough inhibition titers to compare the potency of
antagonists evaluated in different assays.
HAI titers confirmed the good to excellent inhibitory
potencies of the thiazole antagonists. Analogues less potent
than HM in this assay were 9, 21, and 31 (Figure 6).
Antagonist 21, bearing the highly electron-withdrawing group
CF3, was significantly less potent than 9 with the CH3 group.
The same tendency was also observed in the aromatic series.
Compounds 23 and 25 with NO2 and CN groups were less
potent than the unsubstituted phenyl analogue 22. The
electron-withdrawing groups probably hamper interactions
with the Tyr48 side chain. Alkyl-armed thiazoles 9 and 26,
bearing a methyl and a tert-butyl group, showed a similar
inhibitory profile to HM. A significant improvement was
observed with compound 29 bearing a bulky adamantyl group.
The most potent FimH inhibitor 30, identified in the ELISA,
indicate the variation in inhibitory concentrations (ICs, in nM) for the
two assays with variable E. coli LF82 bacterial numbers. rICs relative to
HM are indicated in italic.
was also shown to be the second most potent compound to
prevent AIEC attachment to guinea pig erythrocytes.
Prevention of Bacterial Attachment to Intestinal
Epithelial Cells T84. The capacity of the inhibitors to prevent
adhesion of the AIEC bacteria to intestinal epithelial cells was
then evaluated. This assay is particularly relevant because T84
cells highly express CEACAM6, a receptor for AIEC bacteria,
reflecting the aberrant expression of CEACAM6 in the ileal
mucosa of CD patients.
T84 cells were seeded in a 48-well plate (1.5 × 105 cells per
well) and incubated for 1 h with HM or thiazolylaminomanno-
sides 9 and 21−31 at final concentrations of 0.1, 1, 10, or 100
μM. Intestinal cells were then infected for 3 h with the AIEC
reference strain LF82 at a multiplicity of infection of 10 bacteria
per 1 cell. After washing, infected cells were lysed and samples
were diluted and plated onto agar plates to determine the
number of colony-forming units (CFU). The effect of
mannosides was compared to α-D-mannose (D-Man) treatment
(100, 1000, 10000, and 50000 μM). In the absence of any
treatment, the adhesion level of AIEC LF82 to T84 cells was
F
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
normalized to 100%. The inhibitory ability of mannosides was
expressed as a percentage of residual adhesion.
The residual percentages of AIEC LF82 bacteria associated
with intestinal epithelial cells T84 in the presence of increasing
concentrations of four selected inhibitors (D-Man, HM, 9, 30)
are presented in Figure 7A (for all the results see Supporting
compound HM was therefore 100 times greater than that of α-
D-mannose, indicating that the antiadhesive effect observed on
AIEC bacteria greatly benefits from the anomeric heptyl chain
of HM. Importantly, compound 30 exhibited a dose-dependent
inhibition similar to HM but at a 100-fold lower concentration
(rIC ∼ 0.01). When the residual bacterial adhesion in the
presence of antagonists at 1 μM concentration was compared
(Figure 7B), the much higher potency of 30 was further
evidenced. In fact, no significant decrease in bacterial adhesion
was observed with HM at this concentration, while only 14% of
AIEC remained attached to the cells when 30 was applied (p <
0.001). These results are in good agreement with the previous
assays (competitive ELISA and HAI) and show the very strong
antiadhesive properties of 30 on AIEC.
Prevention of Bacterial Attachment to Colonic Tissue
from CEABAC10 Mice: Ex Vivo Assay. Finally, we explored
ex vivo the relative capacity of D-Man and 30 to prevent the
adhesion of AIEC bacteria to the gut of FVB/N CEABAC10
transgenic mice (transgenic mouse model mimicking AIEC
colonization in CD patients).25 The mice colons were removed
and segmented into four independent loops. A 100 μL solution
containing 2 × 105 AIEC bacteria with or without D-Man (1
μmol and 10 μmol) or compound 30 (0.01 μmol and 0.1
μmol) was injected into the loops and incubated for 1 h within
the tissue. After four washes, the residual bacterial adhesion to
the gut in the presence or absence of inhibitors was determined
after incubation. The adhesion levels normalized to 100% are
presented in Figure 8.
Figure 7. (A) Dose-dependent inhibitory effects on the ability of the
AIEC strain LF82 to adhere to T84 cells obtained with D-Man, HM, 9,
and 30. Horizontal scale: concentration of inhibitors expressed in μM.
Results are expressed in percentage of bacteria adherent to cells
(means SEM); 100% corresponds to adhesion in the absence of any
treatment (NT for nontreated). (B) Comparison of the inhibitory
effects on the AIEC strain LF82 adhesion to T84 cells obtained with
HM, 9, and 21−31 at 1 μM concentration. Results are expressed in
percentage of bacteria adherent to the cells (means SEM); 100%
corresponds to adhesion in the absence of any treatment (NT for
nontreated). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (One-way
ANOVA).
Figure 8. Comparison of the inhibitory effects on AIEC bacterial
adhesion to the colonic tissue of transgenic CEABAC10 mice obtained
with D-Man and 30. Results are expressed as percentage of bacteria
adherent to the colonic mucosa (means SEM, n = 5−8 mice). 100%
corresponds to bacterial adhesion in the absence of treatment (NT for
nontreated). *, p < 0.05; ***, p < 0.001 (one-way ANOVA).
Information Figure S4). All the mannosides tested exerted a
dose-dependent inhibitory effect on AIEC LF82 adhesion. α-D-
Mannose, HM, and all the compounds tested displayed very
large differences when concentration values corresponding to
IC50s were considered. For example, similar residual adhesion
levels (51.6%, 52.4%, and 57.6%) were observed with 1000 μM
of α-D-mannose, 10 μM of HM, and 0.1 μM of compound 30,
respectively (Figure 7A, red bars). The inhibitory potency of
Ten μmol (1.8 mg) of D-Man was shown to decrease the
level of residual adhesion to 41%. In comparison, the minimum
quantity of compound 30 to give a significant inhibitory effect
was much lower, and a residual adhesion of 65% was observed
with 0.1 μmol (0.036 mg) of compound. In this assay,
compound 30 was therefore 10- to 100-fold more potent than
D-Man at preventing AIEC attachment to the colonic mucosa.
Lower inhibitory effects, if any, were observed when 10-fold
G
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
lower quantities of D-Man (1 μmol, 1.8 mg) and 30 (0.01 μmol,
0.0036 mg) were injected into the loops. These results show a
dose-dependent antiadhesive effect and highlight the possibility
of decreasing the residual bacterial adhesion further with higher
doses of FimH inhibitors.
spectrometer, and chemical shifts are reported in parts per
million relative to tetramethylsilane or a residual solvent peak
(CHCl3: H, δ = 7.26; 13C, δ = 77.2; DMSO-d6: H, δ = 2.54,
13C, δ = 40.4). Peak multiplicity is reported as: singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m), and broad
(br). Peak multiplicity and chemical shifts are reported for α
compounds in the case of anomeric mixtures in equilibrium.
High resolution mass spectra HRMS were obtained by
electrospray ionization (ESI) on a Micromass-Waters Q-TOF
Ultima Global or with a Bruker Autoflex III SmartBeam
spectrometer (MALDI). Low-resolution mass spectra (MS)
were recorded with a Thermo Electron DSQ spectrometer. All
reagents were purchased from Acros Organics or Aldrich and
were used without further purification. Column chromatog-
raphy was conducted on silica gel Kieselgel SI60 (40−63 μm)
from Merck. Reactions requiring anhydrous conditions were
performed under argon. Dichloromethane was distilled from
calcium hydride under nitrogen prior to use. Microwave
experiments were conducted in sealed vials in commercial
microwave reactors especially designed for synthetic chemistry
(MultiSYNTH, Milestone). The instrument features a special
shaking system that ensures high homogeneity of the reaction
mixtures. Optical rotations were measured on a 343 Perkin
Elmer at 20 °C in a 1 cm cell in the stated solvent; [α]D values
are given in 10−1 deg·cm2 g−1 (concentration c given as g/100
mL). Compounds tested in vitro were determined to be of
>95% purity by inverse phase HPLC (C-18).
1
1
CONCLUSION
■
We have developed here a synthetic methodology to build
anomeric heterocycles (pyrimidines and thiazoles) on mannose
sugar. This chemical procedure provided access to a new class
of FimH inhibitors not reachable by conventional glycosylation
reactions. As other sugars can be functionalized by such
protocols,26 this cyclization strategy may not be restricted to
the design of FimH inhibitors and is probably applicable to
designing sugar antagonists of other biologically relevant
lectins.27,28 In the present case, the thiazolylaminomannosides
9 and 21−31 were the only compounds adopting the required
1C4 configuration for an optimal fit in the FimH binding
pocket. The pyrimidine derivatives showed a low affinity for the
target (result not shown) and were not further considered. The
lead compound 9 was shown to interact strongly with FimH in
an ELISA assay. The high-resolution crystal structure of the
FimH lectin domain with 9 showed the thiazolyl moieties
interacting with Tyr48 and Tyr137 of the so-called “tyrosine
gate”. Insights into the high affinity of 9 for FimH were
provided by calculating the interaction energies. The results
showed that the thiazole moiety stabilizes the interactions with
the two tyrosines more than the widely used phenyl group
does.
2-[(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranos-1-yl)-
amino]-4-dimethylamino-1,3-thiazabuta-1,3-diene (7).
N,N-Dimethylformamide dimethyl acetal (DMFDMA) (1.3
mL, 9.7 mmol, 1.3 equiv) was added to a solution of 6 (3.11 g,
7.66 mmol, 1 equiv) in dichloromethane (300 mL) under argon
atmosphere. The mixture was stirred at rt for 3 h. The solvent
was evaporated under reduced pressure and the residue purified
by chromatography on a silica gel column with petroleum
spirit−ethyl acetate (3−7 to 4−6) as eluent to afford 7 (3.32 g,
The parallel but staggered orientation of the thiazole with the
aromatic ring of Tyr48 suggested that new pharmacophores
replacing the methyl group of 9 would give additional π-
stacking with Tyr48. Thus, we developed a small library of
analogues mostly based on aromatic compounds. Most of this
second generation of thiazolylaminomannosides showed nano-
molar affinity for FimH. A cell-based assay revealed for the first
time that synthetic mannosides can efficiently prevent the
attachment of AIEC bacteria to human intestinal cells. This
effect was further evidenced ex vivo in the gut of a transgenic
mouse model of CD.25 As AIEC bacteria are known to colonize
the ileal mucosa of patients with CD and to induce gut
inflammation, this finding may open the way to a potential
antiadhesive treatment of CD.
The high capacity of 30 to prevent AIEC adhesion in vitro
and ex vivo compared to HM and α-D-mannose is particularly
promising and justifies further evaluations. Moreover, 30 was
shown to be noncytotoxic and, interestingly, to abolish the
apoptotic effect of FimH for intestinal epithelial cells (Caco2)
at the low concentration of 100 nM (see Supporting
Information.). Future works will focus on the evaluation of
compound 30 in vivo to evaluate whether the strong
antiadhesive effect observed on intestinal epithelial cells and
in the gut correlates with a subsequent improvement in colitis.
This will enable the potency of 30 to be defined for preventing/
treating chronic intestinal inflammation in CD patients in
which AIEC plays a key role.
1
94%) as a mixture of two conformational isomers A and B. H
NMR (300 MHz, CDCl3) δ = 8.82, 8.77 (2 H, 2s, H-9, A + B),
7.69 (1 H, d, J7,1= 7.8 Hz, H-7 B), 7.44 (1 H, d, J = 6.6 Hz, H-7
A), 6.19 (1 H, dd, J1,2 = 2.7 Hz, J1,7 = 8.4 Hz, H-1 A), 5.94 (1 H,
dd, J1,2 = 1.2 Hz, J1,7 = 7.8 Hz, H-1 B), 5.35−5.17 (3 H, m, H-2,
H-3, H-4), 4.31−4.26 (1-H, m, H-6a), 4.12−3.91 (2-H, m, H-5,
H-6b), 3.20, 3.10 (6 H, 2s, H-10, H-10′), 2.15, 2.07, 2.05, 2.00
(12-H, m, CH3CO). 13C NMR (75 MHz, MeOD): δ = 196.3,
194.5 (C-8, A + B), 170.6, 170.5, 170.0, 169.7, 169.5, 169.3
(CH3CO, A + B), 163.3, 162.8 (C-9, A + B), 79.6 et 78.3 (C-1,
A + B), 70.4, 69.9 (C-5, A + B), 70.0, 68.7 (C-3, A + B), 66.4,
65.9 (C-4, A + B), 62.0 (C-6, A + B), 41.6, 41.5, 36.1, 35.8 (C-
10 and C-10′, A + B), 20.9, 20.8 (CH3CO, A + B). HRMS (ES
+): found 489.1354, C19H29N3O9SNa requires 489.1360.
General Procedure for the Addition-Cyclizations.
Example for 8. 1-Chloropropan-2-one (48 μL, 0.709 mmol)
and triethylamine (100 μL, 0.740 mmol) were added to a
solution of 2-[(2,3,4,6-tetra-O-acetyl-α-D-mannopyranos-1-yl)-
amino]-4-dimethylamino-1,3-thiazabuta-1,3-diene 7 (161 mg,
0.349 mmol) in dry THF (5 mL). The mixture was stirred at 60
°C for 16 h then diluted in dichloromethane (20 mL) and
washed with water (3 × 10 mL). The aqueous layer was
extracted with dichloromethane (20 mL). The organic layers
were combined, dried over magnesium sulfate, filtered, and
evaporated under reduced pressure. The residue was purified by
chromatography on a silica gel column with petroleum spirit−
EXPERIMENTAL SECTION
■
■
CHEMISTRY
NMR spectra were recorded at room temperature with a
Bruker Avance 300 Ultra Shield or eBruker Avance III 400
H
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
ethyl acetate (1−1) as eluent to afford 8 as a white solid (83%,
α/β = 9/1).
bicarbonate buffer pH 9.6. Plates were incubated at 4 °C
overnight and then washed (300 μL/well) three times with 10
mM phosphate-buffered saline (PBS) containing 0.15% Tween-
20. All wells were blocked with 250 μL of 3% bovine serum
albumin (BSA) in 10 mM phosphate-buffered saline (PBS)
containing 0.15% Tween-20 (PBST), incubated at 37 °C for 2
h then washed three times with PBST. Thiazole-bearing
mannosides were dissolved in PBST in the concentrations
indicated on graphs and added to microwells. FimH was diluted
in PBST to 0.07 μM and added to each plate well then
incubated for 1 h at room temperature. Wells were washed
three times with PBST before being incubated with 100 μL of
rabbit-anti-FimH antibodies IgG (aFimH), diluted 1:2500 in
PBST, for 1 h at room temperature. Wells were washed 3 times
with PBST and incubated with 100 μL of goat-antirabbit HRP-
labeled secondary antibody, Enzo Life Sciences (2ndAb-HRP),
diluted 1:5000 in PBST, for 1 h at room temperature. They
were then washed three times with PBST and 100 μL of
3,3′,5,5′-tetramethylbenzidine (TMB) was added to each well
and incubated in darkness for 2−5 min. The reaction was
stopped with 100 μL/well of 1N sulfuric acid. Plate absorbance
was analyzed at 450 nm using a BioTek-ELx800 microplate
reader.
5-Acetyl-2-((2,3,4,6-tetra-O-acetyl-α-D-mannopyra-
1
nos-1-yl)amino)thiazole (8). α/β = 4/1. H NMR (300
MHz, CDCl3) δ = 9.00 (1 H, br, NH), 7.98 (1 H, s, H-8α), 7.79
(1 H, s, H-8β), 5.52 (1 H, dd, J2,1 = 1.8 Hz, J2,3 = 2.7 Hz, H-2),
5.34−5.24 (2 H, m, H-3, H-4), 5.14 (1 H, d, J1,2 = 1.8 Hz, H-
1α), 4.32 (1-H, dd, J6b,6a = 12.0 Hz, J6b,5 = 5.4 Hz, H-6b), 4.11−
4.01 (2 H, m, H-5, H-6a), 2.43 (3 H, s, H-11), 2.18, 2.06, 2.01,
2.00 (12 H, 4 s, CH3CO). 13C NMR (75 MHz, CDCl3): δ =
189.8 (C-10), 172.6 (C-7), 170.8, 170.2, 169.7 (CH3CO),
146.7 (C-8), 131.5 (C-9), 82.5 (C-1), 69.4 (C-5 + C-3 or C-4),
69.1 (C-2), 66.0 (C-3 or C-4), 62.2 (C-6), 26.2 (C-11), 20.7
(CH3CO). HRMS (MALDI): found 473.1241, C19H24N2O10S
requires 473.1224.
2-((2,3,4,6-Tetra-O-acetyl-α-D-mannopyranos-1-yl)-
amino)-5-(4-methyl-2-(pyrazin-2-yl)thiazole-5-
1
carbonyl)thiazole (19). [α]D = +27 (c = 0.4, CHCl3). H
NMR (300 MHz, CDCl3) δ = 9.45 (1 H, d, J18,17 = 1.5 Hz, H-
18), 8.65 (1 H, d, J16,17 = 2.5 Hz, H-16), 8.55 (1 H, dd, J17,18
=
2.5 Hz, J17,16 = 1.5 Hz, H-17), 8.12 (1 H, s, H-8), 5.50 (1 H, dd,
J2,1= 1.8 Hz, J2,3 = 2.4 Hz, H-2), 5.40−5.14 (2 H, m, H-1, H-3,
H-4), 4.40 (1 H, dd, J6b,6a = 12.9 Hz, J6b,5 = 5.7 Hz, H-6b),
4.12−3.99 (2 H, m, H-5, H-6a), 2.75 (3 H, s, H-14), 2.16, 2.07,
2.00, 1.90 (12 H, 4 s, CH3CO). 13C NMR (75 MHz, CDCl3): δ
= 177.6 (C-10), 172.5 (C-7), 170.8, 170.5, 170.2, 169.7
(CH3CO), 166.7, 160.5 (Car), 147.7 (C-8), 146.1 (C-16),
144.2 (C-17), 142.0 (C-18), 133.1 (C-11) 129.8 (C-9), 82.3
(C-1), 69.8 (C-3 or C-4), 69.1 (C-2), 68.6 (C-5), 66.0 (C-3 or
C-4), 62.0 (C-6), 20.7 (CH3CO), 18.8 (CH3). HRMS (ES+):
found 634.1263, C26H27N5O10S2 requires 634.1272.
Crystal Structure of the FimH Lectin Domain in
Complex with 9. Crystallization was accomplished in two
different space groups using the sitting drop vapor diffusion
method. The sitting drops were formed by 100 nL of the
precipitant solution (Supporting Information Table S2) layered
on top of 100 nL of 18 mg/mL FimH lectin domain in 20 mM
Na-Hepes at pH 7.4, 150 mM NaCl and complemented with
20 mM 9 just before setup.
Calculations. Ligand 9 was optimized at the B3LYP/6-
31+G(d,p) level using Gaussian 09. A relaxed potential energy
surface scan for the rotation around the C2−C3 bond was
calculated as follows: the starting position of the O−C−C−S
dihedral angle (1−2−3−4 in the figure) was 0°. This angle was
rotated in 36 steps of 10°. For every step, a geometry
optimization was performed at the B3LYP/6-31+G(d,p) level.
For ligand 9 and benzene located at the same position as the
heterocycle of ligand 9, the local hardness was calculated on
two positions (see figure) as the electronic part of the
molecular electrostatic potential divided by twice the number
of electrons.8
General Procedure for the Deprotection. Example for 9.
A solution of sodium methanolate 0.1 M (60 μL, 1 equiv) was
added to a solution of tetra-O-acetylmannopyranosylamino-
thiazole (138 mg, 1 equiv) in methanol (5 mL), and the
mixture was stirred at rt for 3 h. The mixture was diluted with
osmosed water (5 mL) and neutralized with Amberlite IRA-120
(H+) ion-exchange resin, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a C-
18 column with 100−0 to 0−100 water−methanol (linear
gradient) as eluent to afford 9.
5-Acetyl-2-((α-D-mannopyranos-1-yl)amino)thiazole
(9). α/β > 4/1. 1H NMR (300 MHz, D2O) δ = 8.09 (1 H, s, H-
8), 5.34 (1 H, s, J1,2 = 1.7, H-1α), 4.19 (1 H, dd, J2,1 = 1.7 Hz,
J2,3 = 3.3 Hz, H-2), 3.91−3.46 (5 H, m, H-3, H-4, H-5, H-6),
2.54 (3 H, s, H-11). 13C NMR (75 MHz, D2O): δ = 194.1 (C-
10), 174.1 (C-7), 149.3 (C-8), 129.2 (C-9), 83.7 (C-1), 73.3,
70.5, 69.6, 66.7, 60.7 (C-2, C-3, C-4, C-5, C-6), 25.3 (C-11).
HRMS (MALDI): found 327.06049, C11H16N2O6SNa requires
327.06213.
2-(α-D-Mannopyranos-1-yl)amino)-5-(4-methyl-2-(pyr-
azin-2-yl)thiazole-5-carbonyl)thiazole (30). α/β > 3/1. 1H
NMR (400 MHz, CD3OD) δ = 9.35 (1 H, s, H-18), 8.82 (1 H,
d, J16,17 = 2.4 Hz, H-16), 8.76 (1 H, d, H-17), 8.00 (1 H, m, H-
8) 5.17 (1 H, d, J1,2 = 1.8 Hz, H-1), 5.40−5.14 (6 H, m, H-2, H-
3, H-4, H-5, H-6). 13C NMR (100 MHz, DMSO): δ = 176.4
(C-10), 173.6 (C-7), 165.7, 157.5 (Car), 149.8 (C-8), 146.5
(C-16), 145.2 (Car), 144.7 (C-17), 140.7 (C-18), 130.2, 129.1
(C-9, C-11), 83.4 (C-1), 75.0 (C-5), 70.5 (C-3 or C-4), 69.4
(C-2), 67.1 (C-4 or C-3), 60.9 (C-6), 17.3 (CH3). HRMS (ESI
+): found 466.08484, C18H20N5O6S2 requires 466.08495.
ELISA. Immunosorbent microplates were coated with 100
μL of a 10 mg/mL solution of RNaseB in 100 mM carbonate/
V (r)
2N
el
η(r) = −
with N the number of electrons and Vel(r) the electronic part of
the molecular electrostatic potential. The polarizability of
benzene and ligand 9 was analytically calculated at the B3LYP/
6-311+G(d,p) level. Interaction energies between ligand 9,
(benzene) and Tyr137 and Tyr48 were calculated at the MP2/
6-311++G(d,p) level using a basis set superposition error
correction with Gaussian 09 (Figure 9).29
Hemagglutinations. Interaction of E. coli FimH adhesins
with the glycocalyx of guinea pig erythrocytes forms a cross-
linked network in the wells. Glycoconjugates, added in a 2-fold
dilution series, prevent the agglutination reaction. The
inhibition titer is defined as the lowest concentration of the
glycoconjugate at which hemagglutination is still inhibited.
LF82 E. coli were grown statically overnight in LB at 37 °C,
washed three times in ice-cold phosphate-buffered saline, and
redissolved. A 2-fold dilution of glycoconjugates with a starting
concentration of 1 mM was prepared in 25 μL of 20 mM
I
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
ampicillin (100 μg/mL) and erythromycin (20 μg/mL) to
select AIEC LF82 bacteria.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and NMR spectra for new com-
pounds. X-ray data collection, data processing, and refinement
statistics for FimH−9 cocrystals and PDB entries 3zl1 and 3zl2.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
PDB codes 3zl1 and 3zl2
Figure 9. Representation of ligand 9 in the mannose-binding site of
FimH. The orange and purple dots give the position on which the
local hardness was calculated for the interaction with Tyr137 and
Tyr48, respectively.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +0033 2 51 12 54 06. E-mail: r.bilyy@gmail.com
(R.O.B.); Arlette.Darfeuille-Michaud@u-clermont1.fr (A.D-
M.); julie.bouckaert@univ-lille1.fr (J.B.); Sebastien.gouin@
univ-nantes.fr (S.G.G.).
HEPES pH 7.4 with 150 mM NaCl. Importantly, the pipet tip
was changed at every dilution step to avoid carry-over. Next,
the bacterial solution (25 μL) was added to the 2-fold dilution
series of the compound. Finally, 50 μL of guinea pig red blood
cells, washed and diluted in the buffer to 5% of the blood
volume, was added to reach 100 μL. The plates were left on ice
for one night before read-out.
Adhesion Assays of AIEC LF82 Strain on Intestinal
Epithelial Cells T84. E. coli strain LF82 isolated from an ileal
biopsy of a CD patient was used as the AIEC reference strain.3
Bacteria were grown overnight at 37 °C in Luria−Bertani (LB)
broth, and a bacterial suspension was prepared at a
concentration of 1.5 × 108 bacteria/mL in phosphate buffer
saline (PBS) for adhesion assays.
The human intestinal cell line T84, purchased from
American Type Culture Collection (ATCC, CCL-248), was
maintained in an atmosphere containing 5% CO2 at 37 °C in
the culture medium recommended by ATCC. T84 cells were
seeded in 48-well tissue culture plates at a density of 1.5 × 105
cells/well and incubated at 37 °C for 48 h. Before infection,
cells were washed with PBS and incubated for 1 h with HM or
thiazole-bearing mannosides at final concentrations of 0.1, 1,
10, or 100 μM. Effects of mannoside treatment were compared
with α-D-mannose (D-Man) treatment (100, 1000, 10000, and
50000 μM). Epithelial cells were then infected in the presence
of inhibitory compounds with the AIEC reference strain LF82
for 3 h at a multiplicity of infection (MOI) of 10 bacteria per
cell (1.5 × 106 bacteria/well). Monolayers were washed three
times with PBS and lysed with 1% Triton X-100 (Sigma) in
deionized water. Samples were diluted and plated onto LB agar
plates to determine the number of colony-forming units
(CFU).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
J.B. thanks Andrew Thompson at ProximaI of SOLEIL, Saint
Aubin, France, for help with data collection. G.R. acknowledges
the FWO for a postdoctoral fellowship. This work was carried
out with financial support from the French Agence Nationale
de la Recherche (ANR-12-BSV5−0016−01), the Conseil
Reg
National de la Recherche Scientifique, the Minister
l’Enseignement Superieur et de la Recherche in France, the
Institut National de la Sante et de la Recherche Medicale
(UMR1071), the Institut National de la Recherche en
Agronomie (USC INRA 2018), and the Association Francois
́
ional des Pays de La Loire (ERABAC project), the Centre
̀
e de
́
́
́
̧
Aupetit. The work of T.I.D. and R.O.B. was partially supported
by grants from NASU and WUBMRC.
ABBREVIATIONS USED
■
AIEC, adherent-invasive Escherichia coli; BAC, bacterial artificial
chromosome; CD, Crohn’s disease; CEA, carcinoembryonic
antigen; CEABAC, BAC bearing part of the human CEACAM
family gene locus; CEACAM, carcinoembryonic antigen-related
cell adhesion molecules; CFU, colony-forming units;
DMFDMA, N,N-dimethylformamide dimethyl acetal; ELISA,
enzyme-linked immunosorbent assay; FimH, adhesin portion of
type 1 fimbria; HAI, hemagglutination; HM, heptylmannoside;
rIC, relative inhibitory concentration; TNF-α, tumor necrosis
factor- α; Tyr, tyrosine; UTIs, urinary tract infections
Ex Vivo Assay. An adhesion assay of AIEC LF82 bacteria
was performed using colonic tissue from transgenic mice
expressing the human CEACAM6 protein.25 Briefly, 10−12-
week-old FVB/N CEABAC10 transgenic mice were anesthe-
tized and euthanized by cervical dislocation, and colons were
removed. Colons were washed twice in phosphate buffer saline
(PBS) and segmented into four independent loops of ≈ 0.6 cm.
A volume of 100 μL of LF82 bacteria at 2 × 106 bacteria/mL in
PBS or of a mix of LF82 bacteria + D-mannose (1 μmol and 10
μmol) or compound 30 (0.01 μmol and 0.1 μmol) was injected
into the loops. These were incubated for 1 h at 37 °C in an
atmosphere containing 5% CO2 and then opened and washed
four times in PBS. Tissues were homogenized, appropriately
diluted, and plated onto Luria−Bertani agar plates containing
REFERENCES
■
(1) Kaser, A.; Zeissig, S.; Blumberg, R. S. Inflammatory Bowel
Disease. Annu. Rev. Immunol. 2010, 28, 573−621.
(2) Chassaing, B.; Darfeuille-Michaud, A. Commensal microbiota and
enteropathogens in the pathogenesis of IBD. Gastroenterology 2011,
140, 1720−1728.
(3) Darfeuille-Michaud, A.; Neut, C.; Barnich, N.; Lederman, E.; Di
Martino, P.; Desreumaux, P.; Gambiez, L.; Joly, B.; Cortot, A.;
Colombel, J. F. Commensal microbiota and enteropathogens in the
pathogenesis of IBD. Gastroenterology 1998, 115, 1405−1413.
(4) Darfeuille-Michaud, A.; Boudeau, J.; Bulois, P.; Neut, C.; Glasser,
A. L.; Barnich, N.; Bringer, M. A.; Swidsinski, A.; Beaugerie, L.;
Colombel, J. F. High prevalence of invasive Escherichia coli strains in
patients with Crohn’s disease. Gastroenterology 2004, 127, 412−421.
J
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(5) (a) Glasser, A. L.; Boudeau, G.; Barnich, N.; Perruchot, M. H.;
Colombel, J. F.; Darfeuille-Michaud, A. Adherent invasive Escherichia
coli strains from patients with Crohn’s disease survive and replicate
within macrophages without inducing host cell death. Infect. Immun.
2001, 69, 5529−5537. (b) Boudeau, J.; Glasser, A. L.; Masseret, E.;
Joly, B.; Darfeuille-Michaud, A. Invasive Ability of an Escherichia coli
Strain Isolated from the Ileal Mucosa of a Patient with Crohn’s
Disease. Infect. Immun. 1999, 67, 4499−4509.
(6) Barnich, N.; Carvalho, F. A.; Glasser, A. L.; Darcha, C.; Jantscheff,
P.; Allez, M.; Peeters, H.; Bommelaer, G.; Desreumaux, P.; Colombel,
J. F.; Darfeuille-Michaud, A. CEACAM6 a receptor of adherent-
invasive Escherichia coli supports ileal mucosa colonization in Crohn’s
disease patients. J. Clin. Invest. 2007, 117, 1566−1574.
(7) Carvalho, F. A.; Barnich, N.; Sivignon, A.; Darcha, C.; Chan, C.
H.; Stanners, C. P.; Darfeuille-Michaud, A. J. Exp. Med. 2009, 206,
2179−2189.
(8) (a) Gouin, S. G.; Wellens, A.; Bouckaert, J.; Kovensky, J.
Multimeric heptyl mannosides as potent anti-adhesives of uropatho-
genic Escherichia coli. ChemMedChem 2009, 5, 749−755. (b) Almant,
M.; Moreau, V.; Kovensky, J.; Bouckaert, J.; Gouin, S. G. Clustering of
Escherichia coli Type-1 Fimbrial Adhesins by Using Multimeric Heptyl
α-D-Mannoside Probes with a Carbohydrate Core. Chem.Eur. J.
2011, 17, 10029−10038.
Tetrahedron Lett. 1998, 39, 2307−2310. (c) 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 multivalency1. Glycoconjugate J. 1998,
15, 605−613. (d) Kotter, S.; Krallmann-Wenzel, U.; Ehlers, S.;
̈
Lindhorst, T. K. Trivalent α-D-mannoside clusters as inhibitors of type-
1 fimbriae-mediated adhesion of Escherichia coli: structural variation
and biotinylation. J. Chem. Soc., Perkin Trans. 1 1998, 2193−2200.
(13) Schwardt, O.; Rabbani, S.; Hartmann, M.; Abgottspon, D.;
̌
Wittwer, M.; Kleeb, S.; Zalewski, A.; Smiesko, M.; Cutting, B.; Ernst,
B. Design, synthesis and biological evaluation of mannosyl triazoles as
FimH antagonists. Bioorg. Med. Chem. 2011, 19, 6454−6473.
(14) (a) Landreau, C.; Deniaud, D.; Reliquet, A.; Meslin, J. C. A
facile access to imidazo[2,1-b]thiazole and thiazolo[3,2-a]pyrimidine
derivatives. Synthesis 2001, 13, 2015. (b) Landreau, C.; Deniaud, D.;
Evain, M.; Reliquet, A.; Meslin, J. C. Efficient regioselective synthesis
of triheterocyclic compounds: imidazo[2,1-b]benzothiazoles,
pyrimido[2,1-b]benzothiazolones and pyrimido[2,1-b]benzothiazoles.
J. Chem. Soc., Perkin Trans. 1 2002, 6, 741. (c) Landreau, C.; Deniaud,
D.; Reliquet, A.; Meslin, J. C. From thiourea to bicyclic structures: an
original route to imidazo[2,1-b]thiazoles, 5H-thiazolo[3,2-a]-
pyrimidines, 7H-imidazo[2,1-b][1,3]thiazines, and 2H,6H-pyrimido-
[2,1-b][1,3]thiazines. Eur. J. Org. Chem. 2003, 3, 421. (d) Landreau,
C.; Deniaud, D.; Meslin, J. C. From Thiourea to Bicyclic Structures:
An Original Route to Imidazo[2,1-b]thiazoles, 5H-Thiazolo[3,2-
a]pyrimidines, 7H-Imidazo[2,1-b][1,3]thiazines, and 2H,6H-
Pyrimido[2,1-b][1,3]thiazines. J. Org. Chem. 2003, 68, 4912.
(e) Robin, A.; Julienne, K.; Meslin, J. C.; Deniaud, D. Straightforward
Pyrimidine Ring Construction: A Versatile Tool for the Synthesis of
Nucleobase and Nucleoside Analogues. Eur. J. Org. Chem. 2006, 3,
634−643. (f) Kikelj, V.; Setzer, P.; Julienne, K.; Meslin, J. C.; Deniaud,
D. Synthesis of nucleoside derivatives via heterocyclocondensation
reactions. Tetrahedron Lett. 2008, 49, 3273−3275. (g) Kikelj, V.;
Julienne, K.; Meslin, J. C.; Deniaud, D. Original Ring Contraction of
Triazine Derivatives to 1,2,4-Thiadiazoles. Tetrahedron Lett. 2009, 50,
5802−5804.
(9) (a) Firon, N.; Ofek, I.; Sharon, N. Carbohydrate-binding sites of
the mannose-specific fimbrial lectins of enterobacteria. Carbohydr. Res.
1983, 120, 235−249. (b) Firon, N.; Ofek, I.; Sharon, N. Contribution
of hydrophobicity to hemagglutination reactions of Pseudomonas
aeruginosa. Infect. Immun. 1984, 43, 1088−1090. (c) Lindhorst, T. K.;
Kotter, S.; Kubisch, J.; Krallmann-Wenzel, U.; Ehlers, S.; Kren, V.
̈
Effect of p-Substitution of Aryl α-D-mannosides on Inhibiting
Mannose-Sensitive Adhesion of Escherichia coliSyntheses and
Testing. Eur. J. Org. Chem. 1998, 1669−1674. (d) 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. Lead
Optimization Studies on FimH Antagonists: Discovery of Potent and
Orally Bioavailable Ortho-Substituted Biphenyl Mannosides. J. Med.
Chem. 2010, 53, 4779−4792. (e) Sperling, O.; Fuchs, A.; Lindhorst, T.
K. Evaluation of the carbohydrate recognition domain of the bacterial
adhesion FimH: design, synthesis and binding properties of mannoside
ligands. Org. Biomol. Chem. 2006, 4, 3913−3922. (f) Grabosch, C.;
Hartmann, M.; Schmidt-Lassen, J.; Lindhorst, T. K. Squaric Acid
Monoamide Mannosides as Ligands for the Bacterial Lectin FimH:
Covalent Inhibition or Not? ChemBioChem 2011, 12, 1066−1074.
(10) (a) 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, 109−115. (b) Klein,
T.; Abgottspon, D.; Wittwer, M.; Rabbani, S.; Herold, J.; Jiang, X.;
(15) (a) Kikelj, V.; Grosjean, S.; Julienne, K.; Meslin, J. C.; Deniaud,
D. Concerning the Nucleophilic Displacement of a Methylsulfanyl
Group on Substituted Pyrimidinones. Synthesis 2010, 2811−2815.
(b) Grosjean, S.; Julienne, K.; Triki, S.; Meslin, J. C.; Deniaud, D.
Synthesis of nitrogen bicyclic scaffolds: pyrimido [1,2-a] pyrimidine-
2,6-diones. Tetrahedron 2010, 66, 9912−9924.
́
(16) Kuhne, M.; Gyorgydeak, Z.; Lindhorst, T. K. A Simple Method
̈
̈
for the Preparation of Glycosyl Isothiocyanates. Synthesis 2006, 6,
949−951.
(17) Wang, Q.; Fu, J.; Zhang, J. A facile preparation of peracylated α-
aldopyranosyl chlorides with thionyl chloride and tin tetrachloride.
Carbohydr. Res. 2008, 343, 2989−2991.
Kleeb, S.; Luthi, C.; Scharenberg, M.; Bezenco
̧
n, J.; Gubler, E.; Pang,
́ ́
(18) Camarasa, M. J.; Fernandez-Resa, P.; García-Lopez, M. T.; De
̈
L.; Smiesk
̌
o, M.; Cutting, B.; Schwardt, O.; Ernst, B. FimH Antagonists
́ ́
Las Heras, F. G.; Mendez-Castrillon, P. P.; Felix, A. S. A New
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. (c) Jiang, X.; Abgottspon, D.; Kleeb, S.; Rabbani, S.;
Scharenberg, M.; Wittwer, M.; Haug, M.; Schwardt, O.; Ernst, B.
Antiadhesion Therapy for Urinary Tract InfectionsA Balanced PK/
PD Profile Proved To Be Key for Success. J. Med. Chem. 2012, 55,
4700−4713.
Procedure for the Synthesis of Glycosyl Isothiocyanates. Synthesis
1984, 509−510.
(19) (a) Robin, A.; Julienne, K.; Raimbault, S.; Meslin, J. C.;
Deniaud, D. An Efficient Synthesis of 1-Substituted Uracil Ring
Systems and Their Thio Analogues. Synlett. 2005, 18, 2805−2807.
(b) Robin, A.; Julienne, K.; Meslin, J. C.; Deniaud, D. Synthesis of
pyridone and pyridine rings by [4 + 2] hetero-cyclocondensation.
Tetrahedron Lett. 2004, 45, 9557−9559.
(11) Bouckaert, J.; Zhaoli, Li.; Xavier, C.; Almant, M.; Caveliers, V.;
Lahoutte, T.; Weeks, S.; Kovensky, J.; Gouin, S. G. Heptyl α-D-
mannosides grafted on a -cyclodextrin core to interfere with Escherichia
coli adhesion, an in vivo multivalent effect. Chem.Eur. J. 2013, 19,
7847−7855.
(12) (a) Dubber, M.; Lindhorst, T. K. Trehalose-Based Octopus
Glycosides for the Synthesis of Carbohydrate-Centered PAMAM
Dendrimers and Thiourea-Bridged Glycoclusters. Org. Lett. 2001, 3,
(20) Bock, K.; Pedersen, C. A study of 13CH coupling constants in
hexopyranoses. Chem. Soc., Perkin Trans. 2 1974, 293−299.
(21) Mignon, P.; Loverix, S.; Steyaert, J.; Geerlings, P. Influence of
the π−π interaction on the hydrogen bonding capacity of stacked
DNA/RNA bases. Nucleic Acids Res. 2005, 33, 1779−1789.
(22) Bouckaert, J.; Berglund, J.; Schembri, M.; De Genst, E.; Cools,
L.; Wuhrer, M.; Hung, C.-S.; Pinkner, J.; Slattegard, R.; Zavialov, A.;
̈
̊
4019−4022. (b) Konig, B.; Fricke, T.; Wassmann, A.; Krallmann-
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.
̈
Wenzel, U.; Lindhorst, T. K. α-Mannosyl Clusters Scaffolded on
Azamacrocycles: Synthesis and Inhibitory Properties in the Adhesion
of Type 1 Fimbriated Escherichia coli to Guinea Pig Erythrocytes.
K
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(23) Prien, J. M.; Ashline, D. J.; Lapadula, A. J.; Zhang, H.;
Reinholda, V. N. The high mannose glycans from bovine ribonuclease
B isomer characterization by ion trap MS. J. Am. Soc. Mass. Spectr.
2009, 20, 539−556.
(24) Wellens, A.; Lahmann, M.; Touaibia, M.; Vaucher, J.; Oscarson,
S.; Roy, R.; Remaut, H.; Bouckaert, J. The Tyrosine Gate as a Potential
Entropic Lever in the Receptor-Binding Site of the Bacterial Adhesin
FimH. Biochemistry 2012, 51, 4790−4799.
(25) Chan, C. H.; Stanners, C. P. Novel mouse model for
carcinoembryonic antigen-based therapy. Mol. Ther. 2004, 9, 775−785.
(26) Pearson, M. S. M.; Robin, A.; Bourgougnon, N.; Meslin, J.-C.;
Deniaud, D. An Efficient Route to Pyrimidine Nucleoside Analogues
by [4 + 2] Cycloaddition Reaction. J. Org. Chem. 2003, 68, 8583−
8587.
(27) (a) Chen, C.-P.; Song, S.-C.; Gilboa-Garber, N.; Chang, K. S. S.;
Wu, A. M. Studies on the binding site of the galactose-specific
agglutinin PA-IL from Pseudomonas aeruginosa. Glycobiology 1998, 8,
̀
7−16. (b) Chabre, Y. M.; Giguere, D.; Blanchard, B.; Rodrigue, J.;
Rocheleau, S.; Neault, M.; Rauthu, S.; Papadopoulos, A.; Arnold, A. A.;
Imberty, A.; Roy, R. Chem.Eur. J. 2011, 17, 6545−6562.
(28) Giguere, D.; Sato, S.; St-Pierre, C.; Sirois, S.; Roy, R. Aryl O-
̀
and S-galactosides and lactosides as specific inhibitors of human
galectins-1 and -3: role of electrostatic potential at O-3. Bioorg. Med.
Chem. Lett. 2006, 16, 1668−1672.
(29) Frisch, G. W. T. M. J.; Schlegel, H. B.; Scuseria, G. E.; Robb, M.
A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.;
Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.;
Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, Jr. J. A.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,
N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;
Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.;
̈
Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.;
Foresman, J. B.; Ortiz, V. J.; Cioslowski, D. J. Gaussian 09, revision
A.1; Gaussian Inc.: Wallingford CT, 2009.
L
dx.doi.org/10.1021/jm400723n | J. Med. Chem. XXXX, XXX, XXX−XXX