Lead optimization studies on FimH antagonists: Discovery of potent and orally bioavailable ortho-substituted biphenyl mannosides

Article abstract of DOI:10.1021/jm300165m

Herein, we describe the X-ray structure-based design and optimization of biaryl mannoside FimH inhibitors. Diverse modifications to the biaryl ring to improve druglike physical and pharmacokinetic properties of mannosides were assessed for FimH binding affinity based on their effects on hemagglutination and biofilm formation along with direct FimH binding assays. Substitution on the mannoside phenyl ring ortho to the glycosidic bond results in large potency enhancements several-fold higher than those of corresponding unsubstituted matched pairs and can be rationalized from increased hydrophobic interactions with the FimH hydrophobic ridge (Ile13) or "tyrosine gate" (Tyr137 and Tyr48) also lined by Ile52. The lead mannosides have increased metabolic stability and oral bioavailability as determined from in vitro PAMPA predictive model of cellular permeability and in vivo pharmacokinetic studies in mice, thereby representing advanced preclinical candidates with promising potential as novel therapeutics for the clinical treatment and prevention of recurring urinary tract infections.

Full text of DOI:10.1021/jm300165m

Article
pubs.acs.org/jmc
Lead Optimization Studies on FimH Antagonists: Discovery of Potent
and Orally Bioavailable Ortho-Substituted Biphenyl Mannosides
Zhenfu Han,† Jerome S. Pinkner,‡ Bradley Ford,§ Erik Chorell,‡ Jan M. Crowley,∥,⊥
Corinne K. Cusumano,‡ Scott Campbell,# Jeffrey P. Henderson,∥,⊥ Scott J. Hultgren,*,‡,∥
and James W. Janetka*,†,∥
‡
§
†Department of Biochemistry and Molecular Biophysics, Department of Molecular Microbiology, Department of Pathology
∥
⊥
#
and Immunology, Center for Women’s Infectious Disease Research, Department of Internal Medicine, and Department of
Anesthesiology, Washington University School of Medicine, 660 S. Euclid Avenue, Saint Louis, Missouri 63110, United States
S
* Supporting Information
ABSTRACT: Herein, we describe the X-ray structure-based
design and optimization of biaryl mannoside FimH inhibitors.
Diverse modifications to the biaryl ring to improve druglike
physical and pharmacokinetic properties of mannosides were
assessed for FimH binding affinity based on their effects on
hemagglutination and biofilm formation along with direct
FimH binding assays. Substitution on the mannoside phenyl
ring ortho to the glycosidic bond results in large potency
enhancements several-fold higher than those of corresponding
unsubstituted matched pairs and can be rationalized from increased hydrophobic interactions with the FimH hydrophobic ridge
(Ile13) or “tyrosine gate” (Tyr137 and Tyr48) also lined by Ile52. The lead mannosides have increased metabolic stability and
oral bioavailability as determined from in vitro PAMPA predictive model of cellular permeability and in vivo pharmacokinetic
studies in mice, thereby representing advanced preclinical candidates with promising potential as novel therapeutics for the
clinical treatment and prevention of recurring urinary tract infections.
mouse models.12 In addition, a recent study concluded that
INTRODUCTION
FimH is a mannose-specific bacterial lectin located at the tip of
■
type 1 pili play an important role in human cystitis13 and it has
been reported that type 1 pili fulfill molecular Koch’s postulates
type 1 pili, an adhesive fiber produced by uropathogenic E. coli
of microbial pathogenesis.14 In agreement with these findings
and in support of a role for FimH in humans, it has been shown
that the fimH gene is under positive selection in human clinical
isolates of UPEC.8a,15 Aspects of the UPEC pathogenic cascade
extensively characterized in a murine model of infection8b,e,10
have been documented in samples from human clinical studies
such as filamentation and IBC formation.16Targeted inhibitors
of FimH adhesion which block both E. coli invasion and biofilm
formation thus hold promising therapeutic potential as new
antibacterials for the treatment of UTI and the prevention of
recurrence.17,18
(UPEC).1 FimH is known to bind to mannosylated human
uroplakins that coat the luminal surface of the bladder2 and has
also been shown to be involved in invasion of human bladder
cells3 and mast cells,4 triggering apoptosis and exfoliation5 and
inducing elevated levels of cAMP.6 Furthermore, FimH
recognizes N-linked oligosaccharides on β1 and α3 integrins,
which are expressed throughout the urothelium.7 Murine
uroplakin is highly homologous to human, and FimH has
been shown to facilitate bacterial colonization and invasion of
the bladder epithelium in murine models.8 Internalized UPEC
is exocytosed in a TLR-4 dependent process;9 however, bacteria
can escape into the host cell cytoplasm, where they are able to
subvert expulsion and innate defenses by aggregating into
biofilm-like intracellular bacterial communities (IBCs) in a
FimH dependent process.8b,c,10 Subsequently, UPEC species
disperse from the IBC, escape into the bladder lumen, and
reinitiate the process by binding and invading naive epithelial
cells where they are able to establish quiescent intracellular
reservoirs that can persist in a dormant state, tolerant to
antibiotic therapy, and subsequently serve as seeds for recurrent
infection.11 In humans, the severity of UTI was increased and
the immunological response was greater in children with
infections caused by type 1 piliated UPEC strains and type 1
pilus expression has been shown to be essential for UTI in
The discovery of simple D-mannose derivatives as inhibitors
of bacterial adherence was first reported almost 3 decades
ago,19 but early mannosides showed only weak inhibition of
bacterial adhesion. Consequently, the vast majority of research
in this area has been focused on multivalent mannosides,20
which have been pursued in an effort to improve binding
avidity to type 1 pili, which can be expressed present in large
numbers on a single bacterium (up to hundreds). While
substantial progress has been made with this approach, these
high molecular weight structures are not suitable for in vivo
Received: February 6, 2012
Published: March 26, 2012
© 2012 American Chemical Society
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Figure 1. (a) Biphenyl mannoside FimH inhibitors with meta-substituted H-bond acceptors. (b) Lead optimization strategy of lead biphenyl
mannoside 3 for increased FimH binding affinity, good druglike physical properties, and improved pharmacokinetics.
evaluation or clinical development as oral drugs. The recent
X-ray crystal structures of D-mannose,21 butyl mannoside,22 and
mannotriose23 bound to FimH have enabled the rational
structure-based design of tighter binding alkyl,22 phenyl,24 and
biphenyl25,26 mannoside FimH inhibitors. The urgency for
developing new orally bioavailable FimH inhibitors26 as a
targeted strategy for the treatment of UTI alternative to broad
spectrum antibiotics is reinforced by the rate of recurrence seen
in these type of infections as well as increasing clinical
resistance of UPEC to first line antibiotic treatments.27
piece of SAR for phenyl mannosides was actually first observed
in 1987 by Firon et al.19 but has gone largely unnoticed until
recently. As shown in Figure 2, Firon reported that p-nitro-o-
chlorophenyl mannoside had a 10-fold increase in potency
relative to p-nitrophenyl mannoside using a yeast hemaggluti-
nation assay, and later in 2006 Sperling et al. reported a 4-fold
increase in activity from o-chlorine substitution on a different
phenyl mannoside.24 We also previously reported an 8-fold and
5-fold increase in activity, respectively, from ortho chlorine and
nitrile substitution on phenyl mannoside but found that larger
groups (e.g., phenyl) decreased potency.25 Intrigued by these
results from us and others, we were interested in elucidating the
molecular basis for this increased potency derived from ortho
substitution on the biphenyl ring.
Structure-Guided Design of Tight-Binding Ortho-
Substituted Biaryl Mannosides. The structure of 1 bound
to FimH suggested that the ortho-position of the ring attached
to mannose is aimed at the Tyr137 residue at the ridge of FimH
binding pocket and that improved hydrophobic contact and
binding affinity to FimH could be achieved by substitution with
small groups but would be sterically encumbered by larger
groups such as an aryl ring.25 Thus, we performed a matched
pair analysis of monoester 1 compared to ortho-substituted
analogues bearing halogen and small alkyl groups shown in
Figure 3. The compounds were evaluated for their potency in
the hemagglutination inhibition (HAI) assay,28 and we
discovered that all biphenyl ring substitutions yielded more
potent inhibitors (Table 1). o-Cl mannoside 4b had a HAI titer
EC90 of 30 nM, which is more than 30-fold better than matched
pair 1, while the Me analogue 4c was 8-fold more active
(HAI titer EC90 = 120 nM). Substitution with CF3 gave the
most potent analogue 4d with an HAI titer EC90 of 30 nM,
whereas the OMe (4e) and F (4a) analogues showed smaller
improvements in activity following the trend CF3 > Cl = Me >
OMe > F. These data suggest that increased hydrophobic
contact with the tyrosine gate and Ile52 or with Ile13 at the
opposite ridge of the mannose binding pocket could explain
RESULTS AND DISCUSSION
■
In an earlier study we reported the discovery of biphenyl
mannosides 1−3 (Figure 1a) which make strong hydrophobic
interactions to residues forming the outer “gate” of the FimH
binding pocket. X-ray crystallographic data of compound 1
bound to FimH revealed both a key π−π interaction of Tyr48
with the second phenyl ring of 1 and a tight H-bond between
Arg98 and the ester carbonyl.25 In this communication we
describe the lead optimization of biphenyl mannoside 3
following the detailed strategy outlined in Figure 1b. Part of
the plan was to improve FimH binding affinity through
increased interactions with FimH using the structure of the
FimH−1 complex to guide our compound design. Directed by
these structure−activity relationships (SARs), the final goal of
optimization was to improve the metabolic stability, bioavail-
ability, bladder tissue exposure, and in general the druglike
properties of the mannosides through a multifaceted approach
including heterocyclic replacements of both phenyl rings,
ortho-substitution off each ring, and amide substitutions with
higher pKa moieties.
Ortho-Substituted Phenyl Mannosides. In addition to
our work on mannoside FimH inhibitors Ernst26 has sub-
sequently described biphenyl mannosides as FimH antagonists
and demonstrated that substitution with chlorine on the ortho-
position of the phenyl ring directly attached to D-mannose
resulted in a substantial increase in FimH inhibition. This key
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Figure 2. Potency enhancement from o-chloro substitution of phenyl mannosides.
Figure 3. Substituted biphenyl mannosides.
this enhanced potency, since better activity correlates well with
increased hydrophobicity as evidenced by the fact that fluoro
analogue 4a shows no improvement in activity relative to
unsubstituted matched pair 1 and the trifluoromethyl analogue
4c, which has the largest hydrophobic surface area, shows
the highest activity. However, it is possible that the orientations
of both phenyl rings are altered slightly26 and are restricted to a
conformation more conducive to improved FimH binding with
Tyr137, Tyr48, Ile52, Ile13, and/or Arg98 residues.
esters such as amides 5a−c (Figure 3 and Table 1). A similar
trend was observed (CF3 > Me > Cl) with CF3 amide 5c having
the best activity, but in this case the Me analogue 5b showed
better activity than the Cl analogue 5a. We also explored
analogues with substitution on the meta position, exemplified
by ester 6, which retains potency relative to 1 but did not lead
to any enhancement. We next developed o-CF3 7 and Me 8
diamide matched pairs to original lead compound 3 which were
exponentially more potent than any previously reported
mannoside FimH inhibitors with an HAI titer EC90 of 8 and
16 nM, respectively. This unprecedented level of cellular
The outcome of this preliminary study directed us to pursue
analogues that were more metabolically stable and soluble than
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Table 1. Potency Enhancement and PAMPA Data from Ortho-Substitution of Biphenyl Mannosides
compd
1 (ester)
HAI titer EC>90 (μM)
biofilm prevention IC50 (μM)
MW (g/mol)
PSA
CLogD7.4
PAMPA log Pe (cm2/s)
−5.42
1.00
0.75
0.03
0.12
0.03
0.19
1.0
0.94
390.4
408.4
424.8
404.4
458.4
420.4
448.4
389.4
423.8
403.4
457.4
446.4
514.5
460.5
126
126
126
126
126
135
152
128
128
128
128
158
158
158
1.17
1.00
1.64
1.82
1.62
0.47
0.93
0.28
0.75
0.93
0.73
0.71
1.15
1.35
4a (F)
4b (Cl)
0.26
0.33
0.17
0.89
−4.29
−3.91
−4.08
4c (Me)
4d (CF3)
4e (OMe)
6 (m-CO2Me)
2 (amide)
5a (Cl)
0.50
0.12
0.06
0.03
0.37
0.01
0.02
1.35
0.52
0.16
0.13
0.74
0.043
0.073
5b (Me)
5c (CF3)
3 (diamide)
7 (CF3)
−3.89
−4.51
−6.27
−8.46
8 (Me)
a
Scheme 1. Synthesis of Ortho-Substituted Biphenyl Mannosides
a
Reagents and conditions: (a) BF3−Et2O, CH2Cl2, reflux; (b) 3-substituted phenylboronic acid derivatives, cat. Pd(PPh3)4, Cs2CO3, dioxane/water
(5/1), 80 °C; (c) cat. MeONa, MeOH, rt; (d) MeNH2/EtOH, rt; (e) bis(pinacolato)diboron, cat. Pd(dppf)Cl2, KOAc, DMSO, 80 °C.
activity corresponds to a 200000-fold improvement over α-D-
mannose and a 15000-fold improvement over an early reported
inhibitor butyl-α-D-mannoside (HAI titer EC90 = 125 μM) and
is 50-fold better than previous lead compound 3.
Synthesis of Ortho-Substituted Biaryl Mannosides.
Mannosides were synthesized by traditional Lewis acid medi-
ated glycosylation of mannose pentaacetate by reaction with
2-substituted 4-bromophenols using BF3 etherate (Scheme 1).25,30
Suzuki cross-coupling with commercially available 3-substituted
phenylboronic acid derivatives gave protected ortho-substituted
4′-biphenyl mannosides in excellent yields, and subsequent
deprotection with NaOMe gave mannosides 4 and 5. 6 was
prepared following the procedure previously described.25
Synthesis of diamides 7 and 8 followed a similar procedure
but required the synthesis of 3,5-di-(N-methylaminocarbonyl)-
phenylboronic acid pinacol ester 9. Shown in Scheme 1, amida-
tion of dimethyl 5-bromoisophthalate by reaction of methyl-
amine in ethanol proceeded in quantitative yield to give
N,N-dimethyl 5-bromoisophthalamide. Installation of the
boronate ester was accomplished by Pd-mediated coupling
with bis(pinacolato)diboron to give 9.31 Suzuki coupling and
deprotection as before yielded compounds 7 and 8.
The biofilm inhibition assay17b,c,29 was utilized to test these
mannosides’ ability to prevent bacteria from forming IBCs, a
critical pathogenic process in the development of UTIs. As
shown in Table 1, the biofilm prevention IC50 values correlate
quite well with the potencies determined by the HAI assay.
Introduction of an ortho-substituent (e.g., methyl) to the
biphenyl mannoside improved the biofilm activity by 8-fold
from mannoside 2 (IC50 = 1.35 μM) to mannoside 5b (IC50
=
0.16 μM). These data confirmed the mannoside’s functional
effect and activity on UPEC derived from FimH inhibition with
a secondary assay. Biofilm IC50 values were utilized in conjunc-
tion with pharmacokinetic parameters as a key measure of the
predicted lowest effective mannoside concentration in the urine
required for efficacy in vivo, to be discussed vide infra.
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Optimization of Mannosides for Increased Tissue
Penetration and Half-Life. To help improve tissue
penetration and identify mannosides with prolonged exposure
in the bladder, we explored amide derivatives containing func-
tional groups with higher pKa (Table 2). Higher pKa
and this new methodology allows for the use of more readily
obtainable heteroaryl bromides as coupling partners. All hetero-
aryl bromides used in Table 3 were commercially available
Table 3. Heterocyclic Modifications to Biphenyl Ring for
Improving Druglike Properties
Table 2. Exploration of Amide Substitution with Increased
pKa
compounds containing basic moieties such as amines tend to
have increased tissue penetration. Therefore, mannosides 10a−f
were prepared via standard 2-(7-aza-1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)
mediated coupling reaction of 4′-(α-D-mannopyranosyloxy)-
biphenyl-3-carboxylic acid25 with various amines. Unexpectedly,
aminoethylamide 10b showed a disappointing 6-fold drop in
activity relative to methylamide 2. However, hydroxyethylamide
10a was equipotent to 2. We also found that more hydrophobic
tertiary amide piperazine analogues 10c and 10d had largely
decreased potency (HAI EC90 = 4 μM). Interestingly, pyridyl-
amides 10e and 10f showed slightly improved activity, most
pronounced with 4-pyridyl derivative 10e. While the reason for
decreased potency of higher pKa substituents is unclear, it is
plausible that this effect could originate from charge−charge
repulsion with Arg98 side chain at the edge of FimH binding
pocket.
Exploration of Heterocyclic Mannosides for Improved
Pharmacokinetics. We next pursued heterocyclic replace-
ments of the terminal biphenyl ring as an alternative approach
to improve the druglike properties of lead mannosides and
potentially decrease the metabolic clearance. In our target
compounds we retained the key H-bond donor to Arg98 of
FimH, so we could directly compare the effects of heterocyclic
ring replacement. In order to synthesize a library of hetero-
cycles in a divergent fashion, we developed a new Suzuki
synthesis using a 4-mannopyranosyloxyphenyl boronate inter-
mediate 1132 in place of 4-bromophenyl α-D-mannoside. Only a
limited number of heteroaryl boronates are commercially available,
with reasonable prices except bromothiophene derivatives 16
and 17 (Scheme 2). 16 was prepared via Curtius reaction
by first treatment of 5-bromothiophene-3-carboxylic acid with
diphenylphosphorylazide (DPPA) to form isocyanate inter-
mediate, then quenching with ammonia. 17 was synthesized
according to previous method.33 As shown in Scheme 2,
Starting from 4-bromophenyl-α-D-mannoside, bis(pinacolato)-
diboron, and Pd(dppf)Cl2 in DMSO, intermediate 11 was
prepared in good yield. Suzuki cross-coupling of 11 with
various aryl bromides followed by acetyl deprotection gave
target compounds. Compounds synthesized, shown in Table 3,
include pyridyl esters 12a and 12b which showed excellent
activity in the HAI titer with improvement over phenyl ester 1.
Furthermore, ring replacement with a thiophene urea
carboxylate led to dramatic advancements in FimH activity as
exemplified by compound 13a which has HAI EC90 = 16 nM.
In order to ascertain whether the carboxylate ester group or
urea was responsible for this large increase in potency, we
synthesized ester 13b and urea 13c to discover that the
enhancement results from a combined effect of both func-
tionals, since 13b and 13c have much decreased activity. It is
unknown why there is a synergistic effect from the compound
with both urea and carboxylate, but from previous work on
thiophene carboxamide ureas33b we have shown that an
intramolecular H-bond exists between the internal NH of the
urea and the carbonyl of the ester. This conformational
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a
Scheme 2. Synthesis of Heterocyclic Mannosides
a
Reagents and conditions: (a) bis(pinacolato)diboron, cat. Pd(dppf)Cl2, KOAc, DMSO, 80 °C; (b) heteroaryl bromide derivatives, cat. Pd(PPh3)4,
i
Cs2CO3, dioxane/water (5/1), 80 °C; (c) cat. MeONa, MeOH, rt; (d) DPPA, Pr2NEt, dioxane, 85 °C; (e) NH3/dioxane (0.5 M), rt.
restriction might enhance binding entropically to FimH, likely
from the urea carbonyl. We also pursued several fused rings
such as isoquinoline derivatives 14 and 15 to examine the
effects of isosteric replacement for the arylcarbonyl H-bond
acceptor,25 where the heterocyclic ring nitrogen is designed to
accept a H-bond from the FimH Arg98 side chain. The
promising HAI assay results for 14a,b to 15a,b clearly provide
much evidence that the orientation of the CN in 15a or
CO in 15b is likely the same as the conformation of CO
of mannoside 1 in the crystal structure of FimH−mannoside
1,25 bringing the potency up by as much as 10-fold over
mannoside 1.
binding studies solidified that the high potencies stemming
from mannosides 5b, 7, 8 are derived directly from extremely
tight binding to the FimH lectin and not other nonspecific
effects from the cell assays.
Although thus far we have not yet been successful in aquiring
an X-ray structure of one of these new ortho-substituted manno-
sides bound to FimH, we have generated an electrostatic
surface with the most potent mannoside 7 docked to FimH
shown in Figure 5. The large boost in binding affinity to FimH
can be attributed to the fact that in our model the ortho
trifluoromethyl group is oriented directly at Ile13, resulting in a
very strong hydrophobic interaction with FimH. The added
hydrophobicity encompassed by the fluorine atoms in manno-
side 7 results in further augmented binding to FimH relative to
ortho methyl mannoside 8. X-ray crystallographic studies are
aggressively being pursued to confirm this proposed hypo-
thetical model and to elucidate potential other reasons for the
improved binding affinity with regard to either compound or
FimH conformation.
Stability and Elimination Kinetics of Mannosides. We
recently reported that mannoside 3 shows efficacy in vivo36 in
the treatment and prevention of established UTIs in mice when
dosed orally, but the compound displayed some metabolic
instability from hydrolysis of the glycosidic bond (Figure 6a)
and very rapid elimination kinetics to the urine partly due to its
low CLogD (Table 1). While renal clearance is an attractive
feature for UTI therapy, we still wanted to develop improved
mannosides encompassing lower clearance rates as well as
increased oral bioavailability and bladder tissue permeability. In
order to have a general idea of the elimination rate of manno-
side 3, PK studies of its urinary clearance were conducted in
mice (Figure 6b). These experiments demonstrated that the
lower oral dosing of 20 mg/kg was unlikely to maintain
effective concentrations (as determined by biofilm IC50)
because of rapid clearance. Maintenance of mannoside 3 levels
above the minimal effective level of 0.74 μM (based on the
biofilm prevention IC50 in Table 1) during in an 8 h period
required a larger, 100 mg/kg dose. During the PK study, we
detected a small amount of a phenolic biphenyl metabolite (R)
(Figure 6a) in the urine, indicating that some glycoside bond
hydrolysis takes place upon oral dosing. Urine levels of the
R group were unchanged from the 100 and 200 mg/kg doses,
Direct FimH Binding Studies and Pharmacophore
Model for Mannosides. In order to better understand how
the excellent potency in cell-based HAI and biofilm assays is
correlated with FimH binding by biaryl mannosides and to
more precisely select the best lead compounds for in vivo
pharmacokinetic (PK) studies, we have developed a biolayer
interferometry method to directly measure the binding affinities
(Kd) of FimH inhibitors. As shown in Table 4, earlier
mannosides 19−3325 showing moderate potency in the HAI
assay had Kd values as low as 1−10 nM. Strikingly, for com-
pounds 1 and 2, Kd values were in the picomolar range. For
mannosides 5, 7, and 8, however, Kd could not be calculated
because the off-rates were too low to measure. In order to
overcome this obstacle, we utilized differential scanning
fluorimetry (DSF) to rank the high-affinity mannosides. DSF
measures the melting temperature change of protein when
binding to small molecules.34 Melting temperature shifts are
proportional to the free energy of binding, and melting tempera-
tures increase even as ligand concentration exceeds the Kd.35 As
illustrated in Table 4 and Figure 4, the melting temperature of
FimH without mannoside was about 60 °C and rose to
between 68 and 74 °C when binding to mannosides 19−33 of
moderate potency. With tight binding mannosides 5b, 7, 8 the
melting temperature of FimH ranged from 74 to 76 °C,
suggesting that improved mannosides likely bind FimH with
low picomolar affinity. Figure 4 illustrates that DSF ranks
mannosides in a similar fashion to the HAI assay except for 5c
and 25, demonstrating that this is a general and reliable method
to qualitatively rank FimH-mannoside binding when Kd values
span many orders of magnitude. Thus, these direct FimH
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a
Table 4. Results of Octet Assay and DSF Assay
Figure 4. Curves of HAI, Octet, and DSF assay results.
Figure 5. Proposed model of mannoside 7 bound to FimH calculated
with APBS (Adaptive Poisson−Boltzmann Solver) software using PDB
code 3MCY.25
mannosides and other carbohydrate-derived compounds37 can
limit their ability to cross cell membranes in the absence of
active transport mechanisms, and so increasing their hydro-
phobicity is one strategy to help improve cell permeability
and oral bioavailability of this class of molecules. The ortho-
substituted mannosides described above were designed for
increased hydrophobic contact with FimH but also in part to
increase the log D and were predicted to improve the solubility,
oral bioavailability, and bladder tissue penetration relative to
starting mannosides 1−3. It was anticipated that the newly
designed inhibitors would also have increased metabolic stabi-
lity via protection of the glycosidic bond from acidic hydrolysis
in the gut and enzymatic hydrolysis by α-mannosidases in
blood and tissues. In order to test these hypotheses experi-
mentally, we used the PAMPA. PAMPA is commonly used as
an in vitro model of passive, transcellular permeability to
predict oral bioavailability for drug candidates.38 We tested the
most potent biphenyl mannosides in this model for prioritiz-
ing compounds to evaluate further in animal PK and efficacy
studies. Shown in Table 1, compound 5b with log Pe of
−3.89 cm2/s proved to have the highest oral absorption, while
the most potent mannosides (determined by HAI assay) 7 and
8 with log Pe of −6.27 and −8.46 cm2/s exhibited significantly
lower oral bioavailability.
a
(∗) N.D. = not determined.
suggesting that metabolism by glycoside bond hydrolysis is
saturated between the 20 and 100 mg/kg doses (Figure 6b).
In Vitro Parallel Artificial Membrane Permeability Assay
(PAMPA). The low log D of polyhydroxylated sugar-based
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Figure 6. (a) Metabolic lability of the mannoside 3 glycosidic bond from hydrolysis to mannose and biphenol. (b) Elimination kinetics and clearance
of mannoside 3 and biphenol (R) hydrolysis product in mouse urine.
Figure 7. (a) Plasma pharmacokinetics of optimized ortho-substituted mannosides 5a−c, 7, 8 and mannoside 3. (b) Elimination clearance kinetics of
optimized ortho-substituted mannosides 5a−c, 7, 8 and mannoside 3 in urine.
In Vivo Pharmacokinetic Studies of Ortho-Substituted
Biphenyl Mannoside Inhibitors. On the basis of these results,
oral PK studies were performed in mice to assess any
improvements in the PK of these ortho-substituted mannosides.
Compounds were dosed at 50 mg/kg, and plasma and urine
samples were taken at 30 min and 1, 2, 3, 4, 6 h after dosing. As
demonstrated in Figure 7, a generally 10-fold higher mannoside
concentration was observed in urine (Figure 7b) compared to
plasma (Figure 7a), indicating a high clearance rate for these
mannosides, which in this case aids in clearing uropathogens on
the bladder surface. We found that compounds 8 and 5b
consistently maintain a high level of concentration in both urine
and plasma, which is well above the predicted minimum
effective concentration within a 6 h period. While 100 mg/kg
dosing of mannoside 3 was required to achieve effective
mannoside concentrations, only 50 mg/kg dosing of mannoside
5b was required, permitting a much larger therapeutic window
for treatment. Taken together with PAMPA results, high oral
bioavailability and in vivo efficacy in recently reported animal
studies36 support mannoside 5b as the most promising
therapeutic candidate for UTI treatment/prevention.
CONCLUSIONS
■
Using a combination of traditional ligand-based and X-ray
structure-guided approaches with SAR driven by cell-based
hemagglutination and biofilm assays in combination with direct
FimH binding assays, we have identified a diverse array of biaryl
mannoside FimH inhibitors that exhibit binding affinities into
the picomolar range. While we found the most potent manno-
side 7 with respect to FimH binding affinity and activity in
cell assays contains an o-trifluoromethyl group off the phenyl
ring adjacent to the mannose ring group, the most promising
inhibitor from in vivo studies is the o-methyl analogue 8
showing prolonged compound exposure in plasma and urine
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Article
collected, dried with Na2SO4, and concentrated. The resulting residue
was purified by silica gel chromatography with hexane/ethyl acetate
combinations as eluent, giving 4-bromo-2-methylphenyl 2,3,4,6-tetra-
O-acetyl-α-D-mannopyranoside (3.22 g) in 77% yield. 1H NMR
(300 MHz, chloroform-d) δ ppm 7.18−7.38 (m, 2H), 6.97 (d, J =
8.79 Hz, 1H), 5.50−5.59 (m, 1H), 5.43−5.50 (m, 2H), 5.32−5.42
(m, 1H), 4.28 (dd, J = 5.63, 12.50 Hz, 1H), 3.99−4.15 (m, 2H), 2.27
(s, 3H), 2.20 (s, 3H), 2.02−2.11 (three singlets, 9H). MS (ESI): found
[M + Na]+, 539.0.
PK studies. We rationalize the large FimH binding affinity
improvement. We have also discovered a variety of heterocyclic
biaryl mannosides that either retain or improve FimH binding
activity. The novel inhibitors of UPEC type 1 mediated
bacterial adhesion reported herein show unprecedented activity
in hemagglutination and biofilm in vitro assays in addition to
desirable PK properties in vivo. Further optimization of lead
mannosides is currently being focused on the identification of
mannose modifications with reduced sugar-like character.39
Biaryl mannosides have high potential as innovative therapeu-
tics for the clinical treatment and prevention of UTIs. This
report describes the most potent mannoside FimH inhibitors to
date that display good oral bioavailability and druglike proper-
ties in vivo. The unique mechanism of action of targeting the
pilus tip adhesin, FimH, circumvents the conventional
requirement for drug penetration of the outer membrane and
the potential for development of resistance by porin mutations,
efflux, or degradative enzymes, all mechanisms that promote
resistance to antibiotics. Current efforts are directed at the
selection of one or more clinical candidate drugs through
rigorous preclinical evaluation in several models of recurrent
UTI with antibiotic resistant forms of UPEC. These preclinical
models will facilitate further optimization of current lead
compounds to clinical candidate drugs which promise to pro-
vide a new and more selective therapy to treat and prevent
chronic and recurrent urinary tract infections for which there is
currently no effective treatment. Furthermore, since mannoside
FimH inhibitors function outside the cell and are not cytotoxic
to bacteria in contrast to all commonly prescribed antibiotics
susceptible to resistance, this innovative therapeutic strategy
could dramatically reduce resistant forms of uropathogenic E. coli.
4′-(α-D-Mannopyranosyloxy)-N,3′-dimethylbiphenyl-3-car-
boxamide (5b). Under nitrogen atmosphere, a mixture of 4-bromo-
2-methylphenyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (0.517 g,
1 mmol), 3-(N-methylaminocarbonyl)phenylboronic acid pinacol ester
(0.392 g, 1.5 mmol), cesium carbonate (0.977 g, 3 mmol), and tetrakis-
(triphenylphosphine)palladium (0.116 g, 0.1 mmol) in dioxane/water
(15 mL/3 mL) was heated at 80 °C with stirring for 1 h under a
nitrogen atmosphere. After cooling to room temperature, the mixture
was filtered through silica gel column to remove the metal catalyst and
salts with hexane/ethyl acetate combinations as eluent. The filtrate was
concentrated and then dried in vacuo. The residue was diluted with
15 mL of methanol containing a catalytic amount of sodium meth-
oxide (0.02 M), and the mixture was stirred at room temperature
overnight. H+ exchange resin (DOWEX 50WX4-100) was added to
neutralize the mixture. The resin was filtered off, and the filtrate was
concentrated. The resulting residue was purified by silica gel
chromatography with CH2Cl2/MeOH combinations as eluent, giving
1
the title compound (0.260 g) in 64% yield for two steps. H NMR
(300 MHz, methanol-d4) δ ppm 7.94 (t, J = 1.65 Hz, 1H), 7.57−7.72
(m, 2H), 7.33−7.50 (m, 3H), 7.23 (d, J = 8.52 Hz, 1H), 5.48 (d, J =
1.92 Hz, 1H), 4.00 (dd, J = 1.79, 3.43 Hz, 1H), 3.83−3.94 (m, 1H),
3.60−3.76 (m, 3H), 3.46−3.58 (m, 1H), 2.87 (s, 3H), 2.24 (s, 3H).
MS (ESI): found [M + H]+, 404.2.
Methyl 3′-Fluoro-4′-(α-D-mannopyranosyloxy)biphenyl-3-
carboxylate (4a). 4-Bromo-2-fluorophenyl 2,3,4,6-Tetra-O-
acetyl-α-D-mannopyranoside. 4-Bromo-2-fluorophenyl 2,3,4,6-
tetra-O-acetyl-α-D-mannopyranoside was prepared using the same
procedure as for 4-bromo-2-methylphenyl 2,3,4,6-tetra-O-acetyl-α-D-
mannopyranoside in the synthesis of 5b. Yield: 25%. 1H NMR
(300 MHz, chloroform-d) δ ppm 7.30 (dd, J = 2.34, 10.03 Hz, 1H),
7.21 (td, J = 1.79, 8.79 Hz, 1H), 7.08 (t, J = 8.52 Hz, 1H), 5.48−5.58
(m, 2H), 5.46 (d, J = 1.65 Hz, 1H), 5.31−5.41 (m, 1H), 4.23−4.31
(m, 1H), 4.13−4.22 (m, 1H), 4.05−4.13 (m, 1H), 2.20 (s, 3H), 2.02−
2.08 (three singlets, 9H). MS (ESI): found [M + Na]+, 543.0.
Methyl 3′-Fluoro-4′-(α-D-mannopyranosyloxy)biphenyl-3-
carboxylate (4a). 4a was prepared using the same procedure as for
5b with 4-bromo-2-fluorophenyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyr-
anoside and 3-methoxycarbonylphenyl boronic acid as the reactants.
Yield: 66%. 1H NMR (300 MHz, methanol-d4) δ ppm 8.21 (t, J = 1.65
Hz, 1H), 7.99 (td, J = 1.44, 7.83 Hz, 1H), 7.84 (ddd, J = 1.10, 1.92,
7.69 Hz, 1H), 7.35−7.62 (m, 4H), 5.55 (d, J = 1.92 Hz, 1H), 4.10 (dd,
J = 1.79, 3.43 Hz, 1H), 3.94 (s, 3H), 3.86−4.00 (m, 1H), 3.59−3.86
(m, 4H). MS (ESI): found [M + Na]+, 431.1.
Methyl 3′-Chloro-4′-(α-D-mannopyranosyloxy)biphenyl-3-
carboxylate (4b). 4-Bromo-2-chlorophenyl 2,3,4,6-Tetra-O-
acetyl-α-D-mannopyranoside. 4-Bromo-2-chlorophenyl 2,3,4,6-
tetra-O-acetyl-α-D-mannopyranoside was prepared using the same
procedure as for 4-bromo-2-methylphenyl 2,3,4,6-tetra-O-acetyl-α-D-
mannopyranoside in the synthesis of 5b. Yield: 46%. 1H NMR
(300 MHz, chloroform-d) δ ppm 7.55 (d, J = 2.47 Hz, 1H), 7.33 (dd,
J = 2.47, 8.79 Hz, 1H), 7.06 (d, J = 8.79 Hz, 1H), 5.58 (dd, J = 3.02,
10.16 Hz, 1H), 5.52 (s, 1H), 5.49−5.54 (m, 1H), 5.33−5.42 (m, 1H),
4.22−4.32 (m, 1H), 4.04−4.17 (m, 2H), 2.21 (s, 3H), 2.07 (s, 3H),
2.05 (s, 3H), 2.04 (s, 3H). MS (ESI): found [M + Na]+, 561.0 .
Methyl 3′-Chloro-4′-(α-D-mannopyranosyloxy)biphenyl-3-
carboxylate (4b). 4b was prepared using the same procedure as
for 5b with 4-bromo-2-chlorophenyl 2,3,4,6-tetra-O-acetyl-α-D-manno-
pyranoside and 3-methoxycarbonylphenyl boronic acid as the reactants. It
was further purified by HPLC (C18, 15 mm × 150 mm column; eluent,
acetonitrile/water (0.1% TFA)). Yield: 43%. 1H NMR (300 MHz,
methanol-d4) δ ppm 3.59−3.71 (m, 1 H), 3.71−3.85 (m, 3 H), 3.94 (s, 3 H),
4.01 (dd, J = 9.34, 3.30 Hz, 1 H), 4.12 (dd, J = 3.30, 1.92 Hz, 1 H),
EXPERIMENTAL SECTION
General Synthesis, Purification, and Analytical Chemistry
Procedures. Starting materials, reagents, and solvents were purchased
■
1
from commercial vendors unless otherwise noted. H NMR spectra
were measured on a Varian 300 MHz NMR instrument. The chemical
shifts were reported as δ ppm relative to TMS using residual solvent
peak as the reference unless otherwise noted. The following abbrevia-
tions were used to express the multiplicities: s = singlet; d = doublet;
t = triplet; q = quartet; m = multiplet; br = broad. High-performance
liquid chromatography (HPLC) was carried out on Gilson GX-281
using Waters C18 5 μm, 4.6mm × 50 mm and Waters Prep C18 5 μm,
19 mm × 150 mm reverse phase columns, eluting with a gradient
system of 5:95 to 95:5 acetonitrile/water with a buffer consisting of
0.05% TFA. Mass spectrometry (MS) was performed on an HPLC/
MSD instrument using electrospray ionization (ESI) for detection. All
reactions were monitored by thin layer chromatography (TLC) carried
out on Merck silica gel plates (0.25 mm thick, 60F254), visualized by
using UV (254 nm) or dyes such as KMnO4, p-anisaldehyde, and
CAM. Silica gel chromatography was carried out on a Teledyne ISCO
CombiFlash purification system using prepacked silica gel columns
(12−330 g sizes). All compounds used for biological assays are greater
than 95% pure based on NMR and HPLC by absorbance at 220 and
254 nm wavelengths.
Procedures for the Preparation of Biphenyl Mannoside
Derivatives through Suzuki Coupling Reaction with Bromo-
phenyl Mannoside Derivatives as Intermediates: 4′-(α-D-
Mannopyranosyloxy)-N,3′-dimethylbiphenyl-3-carboxamide
(5b). 4-Bromo-2-methylphenyl 2,3,4,6-Tetra-O-acetyl-α-D-
mannopyranoside. Under nitrogen atmosphere and at room tem-
perature, boron trifluoride diethyl etherate (3.41 g, 24 mmol) was
added dropwise into a solution of α-D-mannose pentaacetate (3.12 g,
8 mmol) and 4-bromo-2-methylphenol (2.99 g, 16 mmol) in 100 mL
of anhydrous CH2Cl2. After a few minutes the mixture was heated to
reflux and kept stirring for 45 h. The reaction was then quenched with
water, and extraction was with CH2Cl2. The CH2Cl2 layer was
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5.61 (d, J = 1.65 Hz, 1 H), 7.40−7.49 (m, 1 H), 7.49−7.62 (m, 2 H),
7.68 (d, J = 2.20 Hz, 1 H), 7.82 (ddd, J = 7.76, 1.85, 1.10 Hz, 1 H),
7.98 (dt, J = 7.83, 1.30 Hz, 1 H), 8.14−8.25 (m, 1 H). MS (ESI):
found [M + H]+, 425.1.
and 20 min in a sealed vial by microwave. Then the mixture was parti-
tioned between AcOEt and 1 N HCl aqueous solution. The organic
layer was collected, dried with Na2SO4, then concentrated. The result-
ing residue was purified by silica gel chromatography with AcOEt/Hex
combinations as eluent, giving the title compound (0.240 g) in 84%
Methyl 4′-(α-D-Mannopyranosyloxy)-3′-methylbiphenyl-3-
carboxylate (4c). 4c was prepared using the same procedure as for
5b with 4-bromo-2-methylphenyl 2,3,4,6-tetra-O-acetyl-α-D-manno-
pyranoside and 3-methoxycarbonylphenyl boronic acid as the
reactants. Yield: 54%. 1H NMR (300 MHz, methanol-d4) δ ppm
8.20 (t, J = 1.51 Hz, 1H), 7.94 (td, J = 1.41, 7.90 Hz, 1H), 7.77−7.87
(m, 1H), 7.52 (t, J = 7.55 Hz, 1H), 7.39−7.48 (m, 2H), 7.27−7.38
(m, 1H), 5.56 (d, J = 1.65 Hz, 1H), 4.08 (dd, J = 1.92, 3.30 Hz, 1H),
3.94−4.01 (m, 1H), 3.90−3.94 (m, 3H), 3.68−3.83 (m, 3H), 3.55−
3.65 (m, 1H), 2.31 (s, 3H). MS (ESI): found [M + Na]+, 427.1.
Methyl 4′-(α-D-Mannopyranosyloxy)-3′-(trifluoromethyl)-
biphenyl-3-carboxylate (4d). 4-Bromo-2-(trifluoromethyl)-
phenyl 2,3,4,6-Tetra-O-acetyl-α-D-mannopyranoside. 4-Bromo-
2-(trifluoromethyl)phenyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside
was prepared using the same procedure as for 4-bromo-2-methyl-
phenyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside in the synthesis of
5b. Yield: 54%. 1H NMR (300 MHz, chloroform-d) δ ppm 7.74 (d, J =
2.20 Hz, 1H), 7.61 (dd, J = 2.33, 8.93 Hz, 1H), 7.15 (d, J = 8.79 Hz,
1H), 5.61 (d, J = 1.92 Hz, 1H), 5.48−5.56 (m, 1H), 5.33−5.48 (m,
2H), 4.21−4.34 (m, 1H), 3.97−4.14 (m, 2H), 2.21 (s, 3H), 1.99−2.12
(three singlets, 9H). MS (ESI): found [M + Na]+, 593.0.
Methyl 4′-(α-D-Mannopyranosyloxy)-3′-(trifluoromethyl)-
biphenyl-3-carboxylate (4d). 4d was prepared using the same
procedure as for 5b with 4-bromo-2-(trifluoromethyl)phenyl 2,3,4,6-
tetra-O-acetyl-α-D-mannopyranoside and 3-methoxycarbonylphenyl
boronic acid as the reactants. Yield: 53%. 1H NMR (300 MHz,
methanol-d4) δ ppm 8.23 (t, J = 1.51 Hz, 1H), 8.01 (td, J = 1.37, 7.69
Hz, 1H), 7.80−7.93 (m, 3H), 7.52−7.67 (m, 2H), 5.66 (d, J = 1.65
Hz, 1H), 4.07 (dd, J = 1.79, 3.43 Hz, 1H), 3.95 (s, 3H), 3.90−4.00
(m, 1H), 3.66−3.87 (m, 3H), 3.52−3.66 (m, 1H). MS (ESI): found
[M + Na]+, 481.0.
1
yield. H NMR (300 MHz, DMSO-d6) δ ppm 10.02 (s, 1H), 7.90
(td, J = 2.03, 6.66 Hz, 1H), 7.72−7.81 (m, 1H), 7.45−7.60 (m, 2H),
7.28 (d, J = 8.52 Hz, 1H), 7.16 (d, J = 2.47 Hz, 1H), 7.03 (dd, J = 2.61,
8.38 Hz, 1H), 3.86 (s, 3H), 3.56 (s, 3H). MS (ESI): found [M + Na]+,
309.2.
Dimethyl 4-(α-D-Mannopyranosyloxy)biphenyl-2,3′-dicar-
boxylate (6). 6 was prepared via glycosidation between α-D-mannose
pentaacetate and methyl 5-hydroxy-2-(3-methoxycarbonylphenyl)-
benzoate following the procedure previously described.25 Yield: 73%.
1H NMR (300 MHz, methanol-d4) δ ppm 3.56−3.68 (m, 4 H), 3.68−
3.84 (m, 3 H), 3.86−3.98 (m, 4 H), 4.06 (dd, J = 3.30, 1.92 Hz, 1 H),
5.60 (d, J = 1.92 Hz, 1 H), 7.29−7.43 (m, 2 H), 7.45−7.53 (m, 2 H),
7.56 (d, J = 2.47 Hz, 1 H), 7.84−7.92 (m, 1 H), 7.93−8.04 (m, 1 H).
MS (ESI): found [M + H]+, 449.0.
4′-(α-D-Mannopyranosyloxy)-N,N′-dimethyl-3′-(trifluoro-
methyl)biphenyl-3,5-dicarboxamide (7) and N1,N3-Dimethyl-
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1,3-di-
carboxamide (9). Dimethyl 5-bromobenzene-1,3-dicarboxylate (10.6 g,
36.8 mmol) was dissolved in a 33 wt % solution of methylamine in
EtOH (30 mL), and the mixture was stirred for 6 h at room tempera-
ture. The precipitate that formed during the reaction was filtered to
give 5.3 g (53%) of the intermediate 5-bromo-N1,N3-dimethyl-
benzene-1,3-dicarboxamide as a white solid. Concentration of the remain-
ing filtrate yielded an additional 4.6 g (46%) of product. 5-Bromo-
N1,N3-dimethylbenzene-1,3-dicarboxamide (5.3 g, 19.5 mmol),
Pd(dppf)Cl2 (0.87 g, 1.2 mmol), bis(pinacolato)diboron (6.1 g,
24 mmol), and potassium acetate (7.8 g, 80 mmol) were dissolved in
DMSO (100 mL). The solution was stirred under vacuum and then
repressurized with nitrogen. This process was repeated 3 times, and
then the resultant mixture was stirred at 80 °C for 5 h under a nitrogen
atmosphere. After removal of the solvent under high vacuum, the
crude material was purified by silica gel chromatography to give 9 as a
Methyl 4′-(α-D-Mannopyranosyloxy)-3′-methoxybiphenyl-3-
carboxylate (4e). 4e was prepared using the same procedure as for
5b. In the first step of glycosidation reaction 4-bromo-2-
methoxyphenol was used as glycosidation acceptor, and in the second
step of Suzuki coupling reaction 3-methoxycarbonylphenylboronic
acid was used instead. All intermediates were directly taken to the next
step reaction without further purification. 4e was further purified by
HPLC (C18, 15 mm × 150 mm column; eluent, acetonitrile/water
1
light tan solid (2.2 g, 35%). H NMR (300 MHz, DMSO-d6) δ ppm
8.63 (m, 2H), 8.41 (t, J = 1.51 Hz, 1H), 8.23 (d, J = 1.65 Hz, 2H), 2.79
(d, J = 4.40 Hz, 6H), 1.33 (s, 12H). MS (ESI): found [M + H]+, 319.2.
4′-(α-D-Mannopyranosyloxy)-N,N′-dimethyl-3′-(trifluoro-
methyl)biphenyl-3,5-dicarboxamide (7). With the procedure
outlined in the synthesis of 5b with 4-bromo-2-(trifluoromethyl)phenyl
2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (0.57 g) and 9 (0.48 g), the
title compound (0.340 g) was obtained in 69% yield for the two steps.
1H NMR (300 MHz, methanol-d4) δ ppm 8.17−8.24 (m, 1H), 8.14
(d, J = 1.65 Hz, 2H), 7.92 (d, J = 1.92 Hz, 1H), 7.87 (dd, J = 2.20, 8.79
Hz, 1H), 7.57 (d, J = 8.79 Hz, 1H), 5.64 (d, J = 1.65 Hz, 1H), 4.04
(dd, J = 1.65, 3.30 Hz, 1H), 3.87−3.96 (dd, 1H), 3.64−3.83 (m, 3H),
3.48−3.63 (m, 1H), 2.93 (s, 6H). MS (ESI): found [M + H]+, 515.1.
4′-(α-D-Mannopyranosyloxy)-N,N′,3′-trimethylbiphenyl-3,5-
dicarboxamide (8). 8 was prepared using the same procedure as for
5b with 9 as the Suzuki coupling partner. Yield: 56%. 1H NMR
(300 MHz, methanol-d4) δ ppm 8.09−8.23 (m, 3H), 7.46−7.59
(m, 2H), 7.33 (d, J = 8.52 Hz, 1H), 5.57 (d, J = 1.92 Hz, 1H), 4.08
(dd, J = 1.92, 3.30 Hz, 1H), 3.97 (dd, J = 3.43, 9.48 Hz, 1H), 3.67−
3.85 (m, 3H), 3.60 (ddd, J = 2.47, 5.01, 7.35 Hz, 1H), 2.96 (s, 6H),
2.32 (s, 3H). MS (ESI): found [M + H]+, 461.2.
1
(0.1% TFA)). Yield: 3%. H NMR (300 MHz, methanol-d4) δ ppm
8.21 (t, J = 1.51 Hz, 1H), 7.96 (td, J = 1.44, 7.83 Hz, 1H), 7.84 (ddd,
J = 1.24, 1.92, 7.83 Hz, 1H), 7.49−7.61 (m, 1H), 7.30 (d, J = 8.24 Hz,
1H), 7.24 (d, J = 1.92 Hz, 1H), 7.13−7.21 (m, 1H), 5.47 (d, J =
1.92 Hz, 1H), 4.11 (dd, J = 1.79, 3.43 Hz, 1H), 3.94 (s, 3H), 3.92
(s, 3H), 3.86−4.03 (m, 1H), 3.67−3.86 (m, 4H). MS (ESI): found
[M + Na]+, 443.1.
3′-Chloro-4′-(α-D-mannopyranosyloxy)-N-methylbiphenyl-
3-carboxamide (5a). 5a was prepared using the same procedure as
1
for 5b. Yield: 59%. H NMR (300 MHz, methanol-d4) δ ppm 8.03
(t, J = 1.51 Hz, 1H), 7.69−7.83 (m, 3H), 7.32−7.67 (m, 3H), 5.60
(d, J = 1.65 Hz, 1H), 4.12 (dd, J = 1.79, 3.43 Hz, 1H), 4.01 (dd, J =
3.57, 9.34 Hz, 1H), 3.69−3.84 (m, 3H), 3.61−3.69 (m, 1H), 2.95
(s, 3H). MS (ESI): found [M + Na]+, 424.1.
4′-(α-D-Mannopyranosyloxy)-N-methyl-3′-(trifluoromethyl)-
biphenyl-3-carboxamide (5c). 5c was prepared using the same
procedure as for 5b. Yield: 65%. 1H NMR (300 MHz, methanol-d4) δ
ppm 7.98 (t, J = 1.65 Hz, 1H), 7.77−7.87 (m, 2H), 7.65−7.77
(m, 2H), 7.42−7.60 (m, 2H), 5.58 (d, J = 1.65 Hz, 1H), 3.99 (dd, J =
1.79, 3.43 Hz, 1H), 3.81−3.91 (m, 1H), 3.59−3.80 (m, 3H), 3.45−
3.58 (m, 1H), 2.87 (s, 3H). MS (ESI): found [M + H]+, 458.1.
Dimethyl 4-(α-D-Mannopyranosyloxy)biphenyl-2,3′-di-
carboxylate (6). Methyl 5-Hydroxy-2-(3-methoxycarbonyl-
phenyl)benzoate. The reactants of methyl 2-bromo-5-hydroxy-
benzoate (0.231 g, 1 mmol), 3-methoxycarbonylphenylboronic acid (0.214 g,
1.2 mmol), palladium acetate (0.022 g, 0.1 mmol), potassium carbo-
nate (0.346 g, 2.5 mmol), and tetrabutylammonium bromide (0.322 g,
1 mmol) in 1.2 mL of water were heated with stirring at 70 °C for 1 h
Procedure for the Preparation of Mannosides via Amide
Coupling Reaction: N-(2-Hydroxyethyl)-4′-(α-D -
mannopyranosyloxy)biphenyl-3-carboxamide (10a). Under
nitrogen atmosphere at 0 °C anhydrous DMF (5 mL) was added into
a round-bottom flask containing 4′-(α-D-mannopyranosyloxy)biphenyl-
3-carboxylic acid25 (0.038 g, 0.1 mmol) and HATU (0.046 g, 0.12 mmol).
After the mixture was stirred for 10 min, aminoethanol (0.007 g, 0.12 mmol)
and then N,N-diisopropylethylamine (0.039 g, 0.3 mmol) were added. The
mixture was stirred overnight while being warmed to room temperature
naturally. The solvent was removed, and the residue was purified by HPLC
(C18, 15 mm × 150 mm column; eluent, acetonitrile/water (0.1% TFA)) to
1
give the title compound (0.032 g) in 76% yield. H NMR (300 MHz,
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deuterium oxide) δ ppm 3.53−3.63 (m, 2 H), 3.68−3.94 (m, 6 H), 4.11
(dd, J = 9.20, 3.43 Hz, 1 H), 4.22 (dd, J = 3.30, 1.92 Hz, 1 H), 5.66 (d, J =
1.65 Hz, 1 H), 7.21 (d, J = 8.79 Hz, 2 H), 7.47−7.62 (m, 3 H), 7.66−7.78
(m, 2 H), 7.87 (t, J = 1.65 Hz, 1 H). MS (ESI): found [M + H]+, 420.1.
N-(2-Aminoethyl)-4′-(α-D-mannopyranosyloxy)biphenyl-3-
carboxamide (10b). 10b was prepared using the same procedure as
as eluent. The filtrate was concentrated, then dried in vacuo. Into the
residue, 6 mL of methanol with a catalytic amount of sodium meth-
oxide (0.02 M) was added, and the mixture was stirred at room tem-
perature overnight. The solvent was removed. The resulting residue
was purified by silica gel chromatography with CH2Cl2/MeOH
combinations containing 2% NH3/H2O as eluent, giving rise to 12b
1
1
for 10a. Yield: 60%. H NMR (300 MHz, methanol-d4) δ ppm 3.14−
(0.031 g) in 40% yield. H NMR (300 MHz, CD3OD) δ ppm 3.53−
3.26 (m, 2 H), 3.57−3.66 (m, 1 H), 3.66−3.83 (m, 5 H), 3.87−4.00
(m, 1 H), 4.03 (dd, J = 3.30, 1.92 Hz, 1 H), 5.49−5.62 (m, 1 H),
7.19−7.31 (m, 2 H), 7.49−7.59 (m, 1 H), 7.59−7.72 (m, 2 H), 7.75−
7.89 (m, 2 H), 8.03−8.21 (m, 1 H). MS (ESI): found [M + H]+,
419.2.
3.65 (m, 1 H), 3.67−3.83 (m, 3 H), 3.89−3.96 (m, 1 H), 3.99 (s, 3 H),
4.04 (dd, J = 3.43, 1.79 Hz, 1 H), 5.57 (d, J = 1.92 Hz, 1 H), 7.22−7.37
(m, 2 H), 7.58−7.73 (m, 2 H), 8.54 (t, J = 2.06 Hz, 1 H), 8.97 (d, J =
2.20 Hz, 1 H), 9.04 (d, J = 1.92 Hz, 1 H). MS (ESI): found [M + H]+,
392.1.
3′-(Piperazin-1-ylcarbonyl)biphenyl-4-yl α-D-mannopyrano-
side (10c). 10c was prepared using the same procedure as for 10a.
Yield: 65%. 1H NMR (300 MHz, methanol-d4) δ ppm 3.54−3.67
(m, 1 H), 3.67−3.85 (m, 4 H), 3.85−3.99 (m, 4 H), 4.03 (dd, J = 3.30,
1.92 Hz, 1 H), 5.54 (d, J = 1.65 Hz, 1 H), 7.19−7.29 (m, 2 H), 7.42
(dt, J = 7.69, 1.24 Hz, 1 H), 7.50−7.64 (m, 3 H), 7.67−7.79 (m, 2 H).
MS (ESI): found [M + H]+, 445.3.
Methyl 4-[4-(α-D-Mannopyranosyloxy)phenyl]pyridine-2-
carboxylate (12a). 12a was prepared using the same procedure as
for 12b and was purified by HPLC (C18, 15 mm × 150 mm column;
eluent, acetonitrile/water (0.1% TFA)). Yield: 15%. 1H NMR (300 MHz,
methanol-d4) δ ppm 3.51−3.63 (m, 1 H), 3.65−3.84 (m, 3 H), 3.88−
3.95 (m, 1 H), 4.00−4.13 (m, 4 H), 5.62 (d, J = 1.65 Hz, 1 H), 7.28−
7.40 (m, 2 H), 7.82−7.95 (m, 2 H), 8.13 (dd, J = 5.49, 1.92 Hz, 1 H),
8.55 (d, J = 1.65 Hz, 1 H), 8.73 (d, J = 5.49 Hz, 1 H). MS (ESI): found
[M + H]+, 392.2.
3′-[(4-Methylpiperazin-1-yl)carbonyl]biphenyl-4-yl α-D-
mannopyranoside (10d). 10d was prepared using the same
1
procedure as for 10a. Yield: 87%. H NMR (300 MHz, methanol-
Methyl 3-(Carbamoylamino)-5-[4-(α-D-mannopyranosyloxy)-
phenyl]thiophene-2-carboxylate (13a). 13a was prepared using
the same procedure as for 12b and was purified by HPLC (C18,
15 mm × 150 mm column; eluent, acetonitrile/water (0.1% TFA)).
d4) δ ppm 2.96 (s, 3 H), 3.05−3.65 (m, 9 H), 3.68−3.85 (m, 3 H),
3.87−3.97 (m, 1 H), 4.03 (dd, J = 3.30, 1.92 Hz, 1 H), 5.54 (d, J =
1.65 Hz, 1 H), 7.17−7.30 (m, 2 H), 7.42 (dt, J = 7.62, 1.27 Hz, 1 H),
7.50−7.66 (m, 3 H), 7.67−7.80 (m, 2 H). MS (ESI): found [M + H]+,
459.0.
4′-(α-D-Mannopyranosyloxy)-N-(pyridin-4-yl)biphenyl-3-car-
boxamide (10e). 10e was prepared using the same procedure as for
10a. Yield: 93%. 1H NMR (300 MHz, methanol-d4) δ ppm 3.57−3.69
(m, 1 H), 3.69−3.84 (m, 3 H), 3.93 (dd, J = 9.34, 3.30 Hz, 1 H),
4.04 (dd, J = 3.30, 1.92 Hz, 1 H), 5.56 (d, J = 1.65 Hz, 1 H), 7.18−7.37
(m, 2 H), 7.57−7.78 (m, 3 H), 7.82−8.06 (m, 2 H), 8.24 (t, J = 1.65
Hz, 1 H), 8.36−8.51 (m, 2 H), 8.68 (d, J = 7.42 Hz, 2 H). MS (ESI):
found [M + H]+, 453.1.
4′-(α-D-Mannopyranosyloxy)-N-(pyridin-3-yl)biphenyl-3-car-
boxamide (10f). 10f was prepared using the same procedure as for
10a. Yield: 75%. 1H NMR (300 MHz, methanol-d4) δ ppm 3.55−3.68
(m, 1 H), 3.68−3.85 (m, 3 H), 3.88−3.98 (m, 1 H), 4.04 (dd, J = 3.43,
1.79 Hz, 1 H), 5.56 (d, J = 1.92 Hz, 1 H), 7.20−7.32 (m, 2 H), 7.52−
7.73 (m, 3 H), 7.80−7.91 (m, 1 H), 7.91−7.99 (m, 1 H), 8.04 (dd, J =
8.65, 5.63 Hz, 1 H), 8.23 (t, J = 1.65 Hz, 1 H), 8.59 (d, J = 5.49 Hz,
1 H), 8.67−8.79 (m, 1 H), 9.55 (s, 1 H). MS (ESI): found [M + H]+,
453.2.
1
Yield: 10%. H NMR (300 MHz, methanol-d4) δ ppm 3.58 (ddd, J =
7.21, 4.88, 2.47 Hz, 1 H), 3.66−3.83 (m, 3 H), 3.83−3.96 (m, 4 H),
4.02 (dd, J = 3.30, 1.92 Hz, 1 H), 5.54 (d, J = 1.65 Hz, 1 H), 7.12−7.27
(m, 2 H), 7.56−7.69 (m, 2 H), 8.12 (s, 1 H). MS (ESI): found
[M + H]+, 455.1.
Methyl 5-[4-(α-D-Mannopyranosyloxy)phenyl]thiophene-2-
carboxylate (13b). 13b was prepared using the same procedure as
for 12b and was purified by silica gel chromatography with CH2Cl2/
1
MeOH combinations. Yield: 23%. H NMR (300 MHz, methanol-d4)
δ ppm 3.53−3.62 (m, 1 H), 3.68−3.81 (m, 3 H), 3.85−3.94 (m, 4 H),
4.02 (dd, J = 3.60, 2.1 Hz, 1 H), 5.54 (d, J = 1.8 Hz, 1 H), 7.19
(m, 2 H), 7.34 (d, J = 3.9 Hz, 1 H), 7.64 (m, 2 H), 7.75 (d, J = 3.9 Hz,
1 H). MS (ESI): found [M + Na]+, 419.1.
(5-Bromo-3-thienyl)urea (16). Under nitrogen atmosphere N,N-
diisopropylethylamine (0.390 g, 3 mmol) was added to a solution of
5-bromothiophene-3-carboxylic acid (0.207 g, 1 mmol) and DPPA
(0.330 g, 1.2 mmol) in dioxane (5 mL) at room temperature. After
being stirred for 30 min, the mixture was heated at 85 °C for 1.5 h. After
the mixture cooled to room temperature, 0.5 M ammonia solution in
dioxane (12 mL) was added. After 30 min, the solvents were removed
and the resulting residue was purified by silica gel chromatography
with CH2Cl2/MeOH combinations to give (5-bromo-3-thienyl)urea
Procedure for the Preparation of Biphenyl Mannoside Deri-
vatives through Suzuki Coupling Reaction with 4-(4,4,5,5-
Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl 2,3,4,6-Tetra-O-
acetyl-α-D-mannopyranoside (11) as Intermediates: Methyl
5-[4-(α-D-Mannopyranosyloxy)phenyl]pyridine-3-carboxylate
(12b). 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl
2,3,4,6-Tetra-O-acetyl-α-D-mannopyranoside (11). Under nitro-
gen atmosphere, a mixture of 4-bromophenyl 2,3,4,6-tetra-O-acetyl-
α-D-mannopyranoside (2.791 g, 5.55 mmol), bis(pinacolato)diboron
(1.690 g, 6.66 mmol), potassium acetate (2.177 g, 22.18 mmol), and
(1.1′-bis(diphenylphosohino)ferrocene)dichloropalladium(II) (0.244 g,
0.33 mmol) in DMSO (50 mL) was heated at 80 °C with stirring for
2.5 h. The solvent was removed and the resulting residue was purified
by silica gel chromatography with hexane/ethyl acetate combinations
1
(0.072 g) in 32% yield. H NMR (300 MHz, DMSO-d6) δ ppm 8.80
(s, 1H), 7.09 (s, 2H), 5.87 (s, 2H). MS (ESI): found [M + H]+, 223.0.
1-{5-[4-(α-D-Mannopyranosyloxy)phenyl]thiophen-3-yl}urea
(13c). 13c was prepared using the same procedure as for 12b and
was purified by HPLC (C18, 15 mm × 150 mm column; eluent,
1
acetonitrile/water (0.1% TFA)). Yield: 80%. H NMR (300 MHz,
methanol-d4/acetonitrile-d3(3/1)) δ 7.50−7.62 (m, 2H), 7.11−7.21
(m, 3H), 7.08 (d, J = 1.37 Hz, 1H), 5.53 (d, J = 1.65 Hz, 1H), 4.02
(dd, J = 1.92, 3.30 Hz, 1H), 3.82−3.95 (m, 1H), 3.66−3.81 (m, 3H),
3.51−3.64 (m, 1H). MS (ESI): found [M + H]+, 397.1.
1
Methyl 5-[4-(α-D-Mannopyranosyloxy)phenyl]thiophene-3-
carboxylate (13d). 13d was prepared using the same procedure as
for 12b and was purified by silica gel chromatography with CH2Cl2/
as eluent to give 11 (2.48 g) in 81% yield. H NMR (300 MHz,
chloroform-d) δ ppm 1.33 (s, 12 H), 1.98−2.12 (m, 9 H), 2.20 (s,
3 H), 3.93−4.19 (m, 2 H), 4.21−4.36 (m, 1 H), 5.32−5.42 (m, 1 H),
5.45 (dd, J = 3.57, 1.92 Hz, 1 H), 5.51−5.62 (m, 2 H), 7.00−7.15
(m, 2 H), 7.67−7.84 (m, 2 H). MS (ESI): found [M + Na]+, 573.2.
Methyl 5-[4-(α-D-Mannopyranosyloxy)phenyl]pyridine-3-
carboxylate (12b). Under nitrogen atmosphere, a mixture of 11
(0.132 g, 0.24 mmol), methyl 5-bromonicotinate (0.043 g, 0.2 mmol),
cesium carbonate (0.196 g, 0.6 mmol), and tetrakis(triphenylphos-
phine)palladium (0.023 g, 0.02 mmol) in dioxane/water (5 mL/1 mL)
was heated at 80 °C with stirring for 1 h. After cooling, the mixture
was filtered through a silica gel column to remove the metal catalyst
and salts with hexane/ethyl acetate (2/1) containing 2% triethylamine
1
MeOH combinations. Yield: 33%. H NMR (300 MHz, methanol-d4)
δ ppm 3.55−3.63 (m, 1 H), 3.68−3.81 (m, 3 H), 3.84−3.94 (m, 4 H),
4.02 (dd, J = 3.30, 1.8 Hz, 1 H), 5.53 (d, J = 1.8 Hz, 1 H), 7.18 (m,
2 H), 7.59 (m, 2 H), 7.64 (d, J = 1.5 Hz, 1 H), 8.11 (d, J = 1.5 Hz,
1 H). MS (ESI): found [M + Na]+, 419.1.
4-(Isoquinolin-7-yl)phenyl α-D-Mannopyranoside (14a). 14a
was prepared using the same procedure as for 12b and was purified by
HPLC (C18, 15 mm × 150 mm column; eluent, acetonitrile/water
1
(0.1% TFA)). Yield: 73%. H NMR (300 MHz, methanol-d4) δ 9.73
(s, 1H), 8.59−8.71 (m, 1H), 8.47−8.58 (m, 2H), 8.42 (d, J = 6.59 Hz, 1H),
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8.33 (d, J = 8.79 Hz, 1H), 7.76−7.89 (m, 2H), 7.25−7.41 (m, 2H),
5.60 (d, J = 1.92 Hz, 1H), 4.06 (dd, J = 1.92, 3.30 Hz, 1H), 3.87−4.00
(m, 1H), 3.68−3.87 (m, 3H), 3.55−3.68 (m, 1H). MS (ESI): found
[M + H]+, 384.2.
Melt curves were fitted to the Boltzmann equation to determine the
melting temperature (Tm) (y = A2 + (A1 − A2)/(1 + exp((x − x0)/dx))
where x0 is the Tm) using OriginPro 8 (OriginLab, Northampton
MA). Melting temperatures are represented as the mean of three
replicates plus or minus 1 standard deviation in Figure 4.
PK Studies. Compound levels in mouse urine and plasma were
made using an AB Sciex API-4000 QTrap (AB Sciex, Foster City, CA)
as previously described.36 Selected reaction monitoring (SRM) mode
quantification was performed using the following MS/MS transitions
[precursor mass/charge ratio (m/z)/product m/z]: compound 3, 447/
285; compound 5a, 424/262; compound 5b, 404/242; compound 5c,
548/296; compound 7, 515/353; compound 8, 461/299; compound 3 R
group, 285/254.
PAMPA Method. Materials. The assay was carried out using
Multiscreen PVDF 96-well plates (Millipore, Billerica, MA) using the
company’s transporter receiver plate. The lipid (dioleyoylphosphoti-
dylcholine (DOPC)/stearic acid (80:20, wt %) in dodecane was
obtained from avanti polar lipids (Alabaster, AL). Hank’s buffered
salt solution (HBSS), pH 7.4, was obtained from MediaTech
(Manassas, VA).
4-(Quinazolin-6-yl)phenyl α-D-Mannopyranoside (14b). 14b
1
was prepared using the same procedure as for 12b. Yield: 28%. H
NMR (300 MHz, methanol-d4) δ 9.51 (s, 1H), 9.15 (s, 1H), 8.21−
8.35 (m, 2H), 8.02 (d, J = 8.52 Hz, 1H), 7.63−7.80 (m, J = 8.79 Hz,
2H), 7.14−7.32 (m, J = 8.79 Hz, 2H), 5.50 (d, J = 1.37 Hz, 1H), 3.94−
4.03 (m, 1H), 3.80−3.94 (m, 1H), 3.62−3.80 (m, 3H), 3.48−3.62
(m, 1H). MS (ESI): found [M + H]+, 385.1.
4-(Isoquinolin-5-yl)phenyl α-D-Mannopyranoside (15a). 15a
was prepared using the same procedure as for 12b and was purified by
HPLC (C18, 15 mm × 150 mm column; eluent, acetonitrile/water
1
(0.1% TFA)). Yield: 90%. H NMR (300 MHz, methanol-d4) δ 9.80
(s, 1H), 8.45−8.60 (m, 2H), 8.35 (d, J = 6.59 Hz, 1H), 8.01−8.21
(m, 2H), 7.45−7.55 (m, 2H), 7.31−7.42 (m, 2H), 5.62 (d, J =
1.92 Hz, 1H), 4.07 (dd, J = 1.92, 3.30 Hz, 1H), 3.89−3.99 (m, 1H),
3.70−3.86 (m, 3H), 3.60−3.69 (m, 1H). MS (ESI): found [M + H]+,
384.2.
4-(1-Oxo-1,2-dihydroisoquinolin-7-yl)phenyl α-D-Mannopyr-
anoside (15b). 15b was prepared using the same procedure as for
12b and was purified by HPLC (C18, 15 mm × 150 mm column;
eluent, acetonitrile/water (0.1% TFA)). Yield: 75%. 1H NMR
(300 MHz, methanol-d4) δ 8.51 (d, J = 2.20 Hz, 1H), 7.93−8.05
(m, 1H), 7.62−7.77 (m, 3H), 7.21−7.31 (m, 2H), 7.18 (d, J = 7.14
Hz, 1H), 6.71 (d, J = 7.14 Hz, 1H), 5.56 (d, J = 1.92 Hz, 1H), 4.04
(dd, J = 1.92, 3.57 Hz, 1H), 3.88−4.00 (m, 1H), 3.69−3.87 (m, 3H),
3.57−3.69 (m, 1H). MS (ESI): found [M + H]+, 400.2.
Affinity Measurement by Biolayer Interferometry. Samples or
buffer (200 μL per well) was dispensed into 96-well microtiter plates
(Greiner Bio-One, Monroe, NC) and maintained at 30 °C with 1000 rpm
shaking. Premanufactured pins for individual assays were made by
biotinylating FimH lectin domain25 at a 1:1 molar ratio with NHS-
PEO4-biotin (Thermo Fisher, Rockford, IL), diluting it to 50 μg/mL
in 20 mM HEPES, pH 7.5, 150 mM NaCl (HBS), immobilizing it on
high-binding streptavidin-coated biosensor tips (Super Streptavidin,
Methods. Each of the test compounds was diluted to 2.5 mM in
DMSO (Sigma, St. Louis, MO) and further diluted prior to testing to
2.5 μM in HBSS, pH 7.4. The assay was performed using a Millipore
96-well multiscreen-IP PAMPA plate, and 5 μL of the lipid suspension
was directly added to the PVDF membrane of the filter plate. Immedi-
ately following the addition of the lipid to the membrane, 200 μL of
HBSS solution containing the test compound was added to the donor
(upper) chamber. HBSS (300 μL) was also added to the receiver plate,
and the filter and receiver plates were assembled and incubated
overnight at room temperature in a moistened sealed bag to prevent
evaporation. The concentration in the receiver plate as well as an
equilibrium plate, which represents a theoretical, partition-free sample,
was determined by HPLC−tandem mass spectrometry. Analysis of
each compound was performed in triplicate.
Analysis. Analyses of both the acceptor and equilibrium samples
were performed using an AB 3200 triple quadrupole mass spectro-
meter linked to a Shimadzu DGU-20A HPLC instrument with a
Prevail C18 column (3 μm, 2.1 mm × 10 mm) with a flow rate of
0.35 mL/min. The mobile phase used was A (0.1% formic acid in
water) and B (0.1% formic acid in methanol). The elution gradient
method is shown in the Table 5. Data acquisition and peak height
determination were performed using Analyst, version 1.4.2.
́
ForteBio, Inc., Menlo Park, CA) for 10 min at 30 °C, blocking the pins
with 10 μg/mL biocytin for 2 min, washing in HBS for 1 h, and rinsing
in 15% sucrose in HBS. Pins were then air-dried for 30 min and stored
in their original packaging with a desiccant packet. Assays were
performed by rewetting premade pins with HBS for 15 min, then
storing them in fresh HBS until use. Individual affinity assays were
́
performed on an Octet Red instrument (ForteBio, Inc., Menlo Park,
Table 5
CA) and consisted of a short baseline measurement followed by
incubation of pins for 10 min with seven 2-fold dilutions (in HBS) of
compound in a concentration range experimentally determined to give
well-measured association and dissociation kinetics, then a 30 min
dissociation phase in HBS. Each experimental pin was referenced to a
biocytin-blocked pin to control for instrument drift and a second
biocytin-blocked pin that was passed through a duplicate experiment
in the same 96-well plate to control for nonspecific binding of the
compound to the pin. Kinetics data and affinity constants were
generated automatically by the global fitting protocol in ForteBio Data
Analysis, version 6.3. Typical signal for compound binding was
approximately 0.2 “nm shift” units, while the noise level of the instru-
ment is around 0.0025 nm.
time, min
solvent A, %
solvent B, %
0.01
0.50
1.00
2.00
2.01
6.01
95
95
5
5
5
95
95
5
5
95
stop
Calculations. The log10 of the effective permeability (log Pe) was
DSF Method. FimH lectin domain (residues 1−158 of UPEC J96
FimH, without an affinity tag)22 was purified as described previously.
An amount of 5 μg of FimH in 5 μL of HBS was mixed with HBS to
yield a final volume of 50 μL containing compound at a final
concentration of 100 μM and a “5×” final concentration of SYPRO
Orange (sold as a “5000×” stock in DMSO and mixed with HBS to a
working stock of 50× immediately before use, Life Technologies Inc.;
Grand Island, NY) . Compounds were diluted to 100 μM from stocks
in DMSO and compared to matched control wells with FimH alone
plus 0.2% DMSO. Then 50 μL mixtures were placed in 96-well clear-
bottom PCR plates and subjected to a melt curve from 20 to 90 °C in
0.5 °C increments of 15 s each followed by a fluorescence read of the
“HEX” channel in a Bio-Rad CFX96 thermocycler (Bio-Rad, Hercules, CA).
calculated using the following equation
⎧
⎪
⎫
⎪
⎭
⎛
⎜
⎞
⎪
[drug]acceptor
⎟
⎟
⎠
⎨
⎬
log P = log C × −ln 1 −
e
⎜
⎝
⎪
⎩
[drug]equilibrium
where
C =
VDVA
(VD + VA)(area)(time)
where the drug concentration is the peak areas for the analyte and
VD and VA are the volume of the donor and acceptor compart-
ments, respectively. The area is the surface area of the PVDF membrane
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Journal of Medicinal Chemistry
Article
(0.11 cm2), and time is the incubation in seconds (64 800 s). Each
value is the mean of triplicates performed on the same day.
(6) Bishop, B. L.; Duncan, M. J.; Song, J.; Li, G.; Zaas, D.; Abraham,
S. N. Cyclic AMP-regulated exocytosis of Escherichia coli from infected
bladder epithelial cells. Nat. Med. 2007, 13, 625−630.
(7) Eto, D. S.; Jones, T. A.; Sundsbak, J. L.; Mulvey, M. A. Integrin-
mediated host cell invasion by type 1-piliated uropathogenic
Escherichia coli. PLoS Pathog. 2007, 3, e100.
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR spectra of compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
(8) (a) Chen, S. L.; Hung, C. S.; Pinkner, J. S.; Walker, J. N.;
Cusumano, C. K.; Li, Z.; Bouckaert, J.; Gordon, J. I.; Hultgren, S. J.
Positive selection identifies an in vivo role for FimH during urinary
tract infection in addition to mannose binding. Proc. Natl. Acad. Sci.
U.S.A. 2009, 106, 22439−22444. (b) Justice, S. S.; Hung, C.; Theriot,
J. A.; Fletcher, D. A.; Anderson, G. G.; Footer, M. J.; Hultgren, S. J.
Differentiation and developmental pathways of uropathogenic
Escherichia coli in urinary tract pathogenesis. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 1333−1338. (c) Wright, K. J.; Seed, P. C.; Hultgren,
S. J. Development of intracellular bacterial communities of
uropathogenic Escherichia coli depends on type 1 pili. Cell. Microbiol.
2007, 9, 2230−2241. (d) Nicholson, T. F.; Watts, K. M.; Hunstad, D.
A. OmpA of uropathogenic Escherichia coli promotes postinvasion
pathogenesis of cystitis. Infect. Immun. 2009, 77, 5245−5251.
(e) Justice, S. S.; Lauer, S. R.; Hultgren, S. J.; Hunstad, D. A.
Maturation of intracellular Escherichia coli communities requires SurA.
Infect. Immun. 2006, 74, 4793−4800. (f) Anderson, G. G.; Goller, C.
C.; Justice, S. S.; Hultgren, S. J.; Seed, P. C. Polysaccharide capsule and
sialic acid-mediated regulation promote biofilm-like intracellular
bacterial communities during cystitis. Infect. Immun. 2010, 78, 963−
975.
AUTHOR INFORMATION
■
Corresponding Author
*For S.J.H.: phone, 314-362-7059; e-mail, hultgren@borcim.
wustl.edu. For J.W.J.: phone, 314-362-0509; e-mail, janetkaj@
biochem.wustl.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was completed in part by funding from the National
Institutes of Health (NIH) American Recovery and Reinvest-
ment Act (ARRA) Challenge Grant 1RC1DK086378 and a
Washington University OTM Bear Cub Grant.
ABBREVIATIONS USED
■
UTI, urinary tract infection; UPEC, uropathogenic E. coli;
PAMPA, parallel artificial membrane permeability assay; IBC,
intracellular bacterial community; HAI, hemagglutination
inhibition; SAR, structure−activity relationship; PK, pharma-
cokinetic; PSA, polar surface area; DSF, differential scanning
(9) Song, J.; Bishop, B. L.; Li, G.; Grady, R.; Stapleton, A.; Abraham,
S. N. TLR4-mediated expulsion of bacteria from infected bladder
epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 14966−14971.
(10) Anderson, G. G.; Palermo, J. J.; Schilling, J. D.; Roth, R.; Heuser,
J.; Hultgren, S. J. Intracellular bacterial biofilm-like pods in urinary
tract infections. Science 2003, 301, 105−107.
fluorimetry; IC50, half maximal inhibitory concentration; EC>90
,
(11) (a) Shilling, J. D.; Lorenz, R. G.; Hultgren, S. J. Effect of
trimethoprim-sulfamethoxazole on recurrent bacteriuria and bacterial
persistence in mice infected with uropathogenic Escherichia coli. Infect.
Immun. 2002, 70, 7042−7049. (b) Mulvey, M. A.; Schilling, J. D.;
Hultgren, S. J. Establishment of a persistent Escherichia coli reservoir
during the acute phase of a bladder infection. Infect. Immun. 2001, 69,
452−459. (c) Mysorekar, I. U.; Hultgren, S. J. Mechanisms of
uropathogenic Escherichia coli persistence and eradication from the
urinary tract. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 14170−14175.
(12) Connell, H.; Agace, W.; Klemm, P.; Schembri, M.; Marild, S.;
Svanborg, C. Type 1 fimbrial expression enhances Escherichia coli
virulence for the urinary tract. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
9827−9832.
over 90% of maximal effective concentration; po, oral; HATU,
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; DPPA, diphenylphosphorylazide; methyl
man., methyl α-D-mannoside; butyl man., butyl α-D-mannoside;
phenyl αD man., phenyl α-D-mannoside
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