Gram-negative uropathogenic Escherichia coli (UPEC) bacteria are a causative pathogen of urinary tract infections (UTIs). Previously developed antivirulence inhibitors of the type 1 pilus adhesin, FimH, demonstrated oral activity in animal models of UTI but were found to have limited compound exposure due to the metabolic instability of the O-glycosidic bond (O-mannosides). Herein, we disclose that compounds having the O-glycosidic bond replaced with carbon linkages had improved stability and inhibitory activity against FimH. We report on the design, synthesis, and in vivo evaluation of this promising new class of carbon-linked C-mannosides that show improved pharmacokinetic (PK) properties relative to O-mannosides. Interestingly, we found that FimH binding is stereospecifically modulated by hydroxyl substitution on the methylene linker, where the R-hydroxy isomer has a 60-fold increase in potency. This new class of C-mannoside antagonists have significantly increased compound exposure and, as a result, enhanced efficacy in mouse models of acute and chronic UTI.
Gram-negative uropathogenic Escherichia coli (UPEC) bacteria are a causative pathogen of urinary tract infections (UTIs). Previously developed antivirulence inhibitors of the type 1 pilus adhesin, FimH, demonstrated oral activity in animal models of UTI but were found to have limited compound exposure due to the metabolic instability of the O-glycosidic bond (O-mannosides). Herein, we disclose that compounds having the O-glycosidic bond replaced with carbon linkages had improved stability and inhibitory activity against FimH. We report on the design, synthesis, and in vivo evaluation of this promising new class of carbon-linked C-mannosides that show improved pharmacokinetic (PK) properties relative to O-mannosides. Interestingly, we found that FimH binding is stereospecifically modulated by hydroxyl substitution on the methylene linker, where the R-hydroxy isomer has a 60-fold increase in potency. This new class of C-mannoside antagonists have significantly increased compound exposure and, as a result, enhanced efficacy in mouse models of acute and chronic UTI.
Since the 1970s, antibiotic
resistance has been steadily increasing
and now multidrug resistant pathogens are an imminent threat. Antibiotics
target processes essential for bacterial replication and thus induce
strong pressure to evolve resistance to these drugs. An alternate
therapeutic strategy is to disarm the pathogen by inhibiting critical
host–pathogen interactions necessary for persistence in a host.
Herein, we describe a promising antibiotic-sparing strategy using
small molecule C-mannosides that specifically block
the ability of uropathogenic Escherichia coli (UPEC) to colonize the lower urinary tract by neutralizing the FimH
adhesin. In mouse models, we show that C-mannosides
are oral drugs that are effective in both preventing and treating
urinary tract infections (UTIs). These compounds represent advanced
preclinical candidates for UTI therapy.Microbial adherence
to host cells is a critical initial step in
most infectious diseases.[1] The ability
of bacteria to bind and invade epithelial tissues allows them to establish
a niche within the host and evade host immune responses.[2] While antimicrobial therapy has traditionally
been effective in treating bacterial infections, resistance to the
most commonly used antimicrobials is on the rise,[3] highlighting the need for new therapeutic strategies.[4] The study of critical host–pathogen interactions
during bacterial infection has revealed promising novel targets for
therapeutic intervention.[5] Monoclonal antibodies,
vaccines, glycoconjugates, and small molecules have all been developed
to disrupt bacterial adherence by competitively blocking the bacterial
protein(s) involved in the recognition of various host receptors.[6] This process and molecular mechanism has been
most thoroughly studied in urinary tract infection (UTI) pathogenesis.A key step in UTI pathogenesis is the initial colonization of the
bladder epithelium by uropathogenic E. coli (UPEC). This binding is mediated by hair-like fibers, termed type
1 pili, that are tipped with the virulence factor FimH adhesin.[7] The lectin domain of FimH binds mannosylated
glycoproteins expressed on the luminal surface of human and murine
bladder cells. The terminal FimH residue is a prototypical two-domain
adhesin, which is joined to the distal end of the linear tip fibrillum
of type 1 pili by a donor strand exchange reaction with subunit FimG.[7,8] FimG then undergoes its own donor strand exchange reaction with
subunit FimF, which adapts the fibrillum tip to the long pilus rod,
which consists of a homopolymer of ∼1000 FimA subunits coiled
into a rigid right-handed helical structure that is capable of unwinding
into a linear fiber.[9] The FimH lectin domain
contains a deep acidic pocket that recognizes α-d-mannose
with stereochemical specificity.[8] FimH-mediated
binding to mannosylated uroplakins[10] or
α1,β3 integrins[11] facilitates
bacterial colonization and invasion of bladder epithelial cells.[12]UTIs present a significant burden for
women, with nearly 20 million
cases reported annually.[13] Despite antibiotic
treatment, 20–40% of these women will have at least one recurrence
within 6 months of their initial diagnosis.[14] This results in a significant economic impact, approximately two
billion dollars in the U.S. alone,[13] associated
with these common and painful infections. The majority of UTIs (85–95%)
are caused by members of the Enterobacteriaceae family;
UPEC are isolated in approximately 80–85% of community-acquired
UTI, and other Enterobacteriaceae account for 5–10%
of infections.[15] Because of the increasing
prevalence of recurrent infections, as well as the increasing emergence
of antibiotic resistant strains,[16] including
multidrug resistant UPEC such as the ESBL (extended spectrum β-lactamase)
strain ST131,[17] the desire for new UTI
therapeutics has escalated rapidly in recent years. The requirement
for FimH to cause disease has led to its classification as a promising
and validated therapeutic target[18] for
UTI and, more recently, for Crohn’s disease.[19] Inhibition of FimH function and activity circumvents bacterial
bladder cell adhesion, invasion, and subsequent intracellular biofilm
formation, making the bacteria unable to cause or propagate an existing
infection.We have previously developed[20] small
molecule glycosides based on α-d-mannose (O-mannosides), such as 1 and 4 (Figure ), as tight-binding
ligands and potent antagonists of FimH both in vitro and in animal
models of UTI. Although, phenyl α-d-mannosides were
first reported by Firon and Sharon in the 1980s as inhibitors of yeast
agglutination by mannose-specific enterobacteria,[21] the true viability of synthetic mannosides as oral therapeutics
for treatment of UTI was not validated in vivo until 2010,[20a,22] where the oral activity of several biphenyl α-d-mannosides
in animal models of acute and chronic UTI was demonstrated. Since
then, other related mannosides have also been reported[23] and glycodendrimer[24] FimH antagonists. X-ray crystallographic data of mannosides binding
to the FimH lectin domain has aided in the rational design of higher
affinity ligands, with improvement gained through interactions to
the “tyrosine gate” at the periphery of the deep mannose
binding pocket[6c,8,20c] and to a small hydrophobic pocket[20b,23a,25] near the mannoside glycosidic bond. While O-mannosides have good efficacy in animal models of UTI,
they have low bioavailability and half-life, which can possibly be
attributed to the metabolic instability of the O-glycosidic
bond[20b] to either hydrolysis in the stomach
or intestinal tract or, alternately, from the enzymatic action of
mannosidases.
Figure 1
Examples of simple glycosidic bond replacement of early
lead O-mannosides 1 and 4, with alternate
linkers to the biphenyl aglycone.
Examples of simple glycosidic bond replacement of early
lead n class="Chemical">O-mannosides 1 and 4, with alternate
linkers to the biphenyl aglycone.
In this article, we specifically address and eliminate this
potential
metabolic liability of O-mannosides with new C-mannosides, which have the labile O-glycosidic
bond replaced with stable carbon-based linkers to the aglycone portion
of the mannoside. Herein, we report on a novel, stereoselective, synthetic
route to construct this new class of biaryl, C-glycosideFimH antagonists, which encompass a unique R-hydroxy
methylene bond to the aglycone. We have employed X-ray crystallography
and computational docking studies of mannosides to develop a pharmacophore
model of stereospecific C-mannoside binding to FimH.
Furthermore, we show that relative to O-mannosides,
these new C-mannosides have greatly increased compound
exposure, which we postulate to result from an increase in metabolic
stability of the glycosidic bond. Exactingly, C-mannosides also have
significantly improved in vitro FimH activity and in vivo efficacy
in animal models of UTI.
Results and Discussion
N-, S-Linked Mannosides
At the onset of this work,
we synthesized two compounds, one containing
a nitrogen (2) and the other a sulfur atom (3) in place of the oxygen glycosidic bond (1) (Figure ). As shown in Scheme A, these S- and N-mannosides were prepared using
a traditional glycosylation methodology with slight modification,
first coupling α-d-mannose with methyl 4′-aminobiphenyl-3-carboxylate
to give N-linked mannoside 2, then glycosylating mannose-pentaacetate
with 4-bromobenzenethiol to give 3a, followed by a Suzuki
reaction with 3-methoxycarbonylphenyl boronic acid and deprotection
to give S-linked mannoside 3. We found that both analogues
had slightly lower activity (HAI = 4 μM) to that of O-mannoside 1 when tested in a hemagglutination
(HA) inhibition assay using the clinical E. coli strainUTI89. To help guide our SAR, we also tested 2 and 3 in an isothermal shift melting point assay and
found that they had equivalent binding affinity to FimH, relative
to 1. Next, we synthesized an N-linked heterocycle, triazolomannoside,
via “click” chemistry methodology. Shown in Scheme B, the reaction of
azido mannoside 7(26) with phenylacetylene
and copper sulfate, followed by sodium methoxide deacetylation, gave
phenyl triazole mannoside 8 in good yield. However, mannoside 8 lost substantial potency relative to 1, only
exhibiting an HAI of 32 μM.[27] On
the other hand, it was shown by another group that 8 still
retains good FimH binding affinity (IC50 = 0.25 μM)
as determined in a competitive binding assay.[28] Hoping to build from these initial results with N- and S-mannosides, we aimed to construct potentially
more metabolically stable, carbon-based linkers, or C-mannosides.
Scheme 1
Synthesis of N-, S-, and Triazole-Linked
Biphenyl Mannosides
Reagents and conditions: (a)
methyl 4′-aminobiphenyl-3-carboxylate, EtOH, 55 °C; (b)
4-bromobenzenethiol, BF3–OEt2, DCM, 0
°C to rt; (c) 3-methoxycarbonylphenyl boronic acid, Pd(PPh3)4, Cs2CO3, dioxane/water
(5/1), 80 °C; (d) NaOMe/MeOH, 0 °C to rt; (e) phenylacetylene,
CuSO4, Na ascorbate, EtOH/H2O (4/1), rt.
Synthesis of N-, S-, and Triazole-Linked
Biphenyl Mannosides
Reagents and conditions: (a)
methyl 4′-aminobiphenyl-3-carboxylate, EtOH, 55 °C; (b)
4-bromobenzenethiol, BF3–OEt2, DCM, 0
°C to rt; (c) 3-methoxycarbonylphenyl boronic acid, Pd(PPh3)4, Cs2CO3, dioxane/water
(5/1), 80 °C; (d) NaOMe/MeOH, 0 °C to rt; (e) phenylacetylene,
CuSO4, Na ascorbate, EtOH/H2O (4/1), rt.
C-Linked Amide Mannosides
Because
of ease of synthesis, we first targeted a series of simple C-linked amide mannosides. Shown in Scheme , cyano mannoside 9(29) was obtained by reaction of mannose pentaacetate
with trimethylsilyl cyanide and boron trifluoride diethyl etherate
to give 9 as a 2:1 mixture of α and β isomers.
Compound 9 was then hydrolyzed to carboxyl mannoside 10 by heating in 25% aq HCl for 2 days. 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid
hexafluorophosphate (HATU) mediated coupling of 10 with
methyl 4′-aminobiphenyl-3-carboxylate yielded the target C-linked amide mannoside 11. In parallel, we
pursued methylene spaced amides in order to investigate the effect
of length and flexibility of the linker. Thus, starting from cyano
mannoside 9, we synthesized aminomethyl mannoside intermediate 12.[30] Acylation with 4-bromo-benzoyl
chloride gave amide mannoside 13, which was coupled to
methoxycarbonylphenylboronic acid via Suzuki reaction, to give methylene-spaced
amide 14 as a matched pair of 1. Testing
of 11, 13, and 14 in the HA
assay revealed that all analogues had reduced potency, relative to 1, with HAIs of 16 μM. We rationalized that these, and
the triazole linker 8, do not allow sufficient flexibility
to mimic the conformation, or key FimH binding interactions, observed
for O-mannoside 1. Hence, we next pursued
direct methylene (CH2) spaced C-mannosides,
which provide a closer structural match to O-, N-, and S-mannosides 1–3.
Scheme 2
Synthesis of C-Linked Amide Mannosides
Reagents and conditions: (a)
25% HCl aq, 50 °C; (b) HATU, DIPEA, 0 °C to rt, DMF; (c)
4-bromobenzoyl chloride, pyridine, rt; (d) Pd(PPh3)4, Cs2CO3, dioxane/water(5/1), 80 °C.
Synthesis of C-Linked Amide Mannosides
Reagents and conditions: (a)
25% HCln class="Chemical">aq, 50 °C; (b) HATU, DIPEA, 0 °C to rt, DMF; (c)
4-bromobenzoyl chloride, pyridine, rt; (d) Pd(PPh3)4, Cs2CO3, dioxane/water(5/1), 80 °C.
Direct Methylene C-Linked
Mannosides
Methylene-spaced, C-mannoside
analogues of O-mannoside 4 and ortho-methylated O-mannoside 23 were obtained using a newly
developed, multistep synthetic procedure, starting from nitrile 15 (Scheme ). The non-ortho substituted analogues were synthesized
first. Bromide intermediates 17R and 17S were constructed in a one-pot, two-step sequence, first reducing
nitrile 15 to aldehyde 16 with DIBAL, followed
by the addition of an organolithium reagent (formed by lithiation
of 1,4-dibromobenzene (R = H, step b). The organolithium addition
yielded a mixture of alcohol isomers (17R and 17S), which were separable by silica gel chromatography. Following a
palladium-mediated cross-coupling of 17R and 17S with 3-(N-methylaminocarbonyl)phenyl boronate, 18R and 18S were obtained in good yield, respectively.
Subsequent benzyl deprotection produced methylene-linked C-mannosides 5, 6R and 6S.
Following testing in the HA assay, we discovered that methylene-spaced
analogue 5 (HAI = 1 μM) was equipotent to its matched
pair O-mannoside 4 (HAI = 0.5 μM)
(Figure ). Surprisingly,
we found the secondary alcohol isomers 6S and 6R showed differential potency, with a pronounced stereochemical preference
for the R-hydroxyl group for FimH binding affinity.
The isomer 6S had 25-fold less potency in the HA assay
(HAI = 24 μM) than 5, whereas isomer 6R gained 4-fold activity (HAI = 0.25 μM).
Scheme 3
Synthesis of C-Linked Methylene and Hydroxy-Methylene
Mannosides
Synthesis of C-Linked Methylene and Hydroxy-Methylene
Mannosides
Reagents and conditions: (a)
DIBAL, CH2Cl2, −78 °C; (b) diethyl
ether, −78 °C to −20 °C; (c) Dess–Martin
periodinane, pyridine, CH2Cl2, 0 °C; (d)
Li(tOBu)3AlH, THF, −40 to 0 °C;
(e) 3-(N-methyaminocarbonyl)phenylboronic acid pinacol
ester, Pd(PPh3)4, Cs2CO3, dioxane/water (5/1), 80 °C; (f) 10% Pd/C, H2, MeOH,
rt.On the basis of these key results, we
synthesized the ortho-methyl analogues 22 and 21 based on our
lead O-glycoside 23 (Figure ).[20a] Shown in Scheme , C-mannosides 21 and 22 were constructed in a similar fashion as for 5 and 6, only differing in the organolithium reagent (4-bromo-2-methyl-iodobenzene
[R = Me, step b]) used for the addition to aldehyde 16. We discovered that methylene C-mannoside 22 (HAI = 2 μM) had a 30-fold decrease in activity relative
to 23, which is in contrast to the results obtained from 5, which retained activity relative to its matched pair, 4. However, we did discover the same R-stereospecificity
of the benzylic alcohols observed with 6R and 6S also exists in the more potent ortho-methyl series,
with C-mannoside 21R having an HAI of
30 nM (relative to 62 nM for 23) and S-isomer 21S having an HAI of only 24 μM. This striking increase in the
FimH activity of R-isomers 6R and 21R was attributed exclusively to a change in their relative
FimH binding affinities when compared to 6S and 4 and 21S and 23, respectively (Figure ). It should be noted
that although the R and S stereochemical
assignment of the hydroxymethylene linker in 6 and 21 were only speculation at this time, we later confirmed
the stereochemistry through small molecule X-ray crystal structure
of a derivative of a key precursor, 19R (Figure ). From the vast potency difference
seen between 21R and 21S, we can also presuppose
that our potency-based assignments are correct for the less effective 6R and 6S compounds.
Figure 2
Direct comparison of
the potencies of C-linked
methylene mannosides 21R, 21S, and 22 to those of similar O-mannoside analogues 23, 24, and 25.
Figure 3
In vitro analysis of mannoside potency. The difference in the conformational
stability of the FimH lectin domain in the presence of mannoside compared
to that in the absence of mannoside, as determined by differential
scanning fluorimetry, is presented on the left axis. Measurement of
the melting temperature is described in the methods. The hemagglutination
inhibition data is presented relative to d-mannose on the
right axis and is described in the methods.
Figure 6
(A) Derivatization of key building block 19R (precursor
to 21R). (B) Small molecule X-ray structure of acetylated
intermediate 27, confirming the R-stereochemistry
of the 21R benzylic hydroxyl group.
Direct comparison of
the potencies of C-linked
methylene mannosides 21R, 21S, and 22 to those of similar O-mannoside analogues 23, 24, and 25.In vitro analysis of mannoside potency. The difference in the conformational
stability of the FimH lectin domain in the presence of mannoside compared
to that in the absence of mannoside, as determined by differential
scanning fluorimetry, is presented on the left axis. Measurement of
the melting temperature is described in the methods. The hemagglutination
inhibition data is presented relative to d-mannose on the
right axis and is described in the methods.
Stereoselective Methodology for Recycling the Undesired 19S-Isomer into the 19R-Isomer
To improve
upon our yield of R-hydroxybenzyl C-mannosides, we explored alternate synthetic strategies. In this
study, we sought to maximize the yield of 19R in step
d, depicted in Scheme , which generates a mixture of diastereomers, yielding 19R in only 16%, and the 19S isomer in 20%. We first investigated
modified reaction conditions, including a systematic evaluation of
solvents. However, these changes resulted in a lower reaction yield
and also gave rise to an inseparable 4-iodo-3-methyl byproduct. Variation
of other reaction parameters, such as temperature, concentration,
additives, and order of addition, were also attempted, but these unproductively
increased the 19S isomer ratio. As a possible solution,
we envisioned a transformation to convert 19S into the
desired 19R isomer. Therefore, we implemented a two-step
oxidation and selective reduction protocol, allowing us to efficiently
recycle the undesired 19S isomer into 19R. This stereoselective recycling method improved the R:S ratios from 1:1.2 (steps c,d) to 28:1 (steps
c–f), also increasing the overall yield of 19R from 16% to 28% (calculated from compound 15). The
oxidation of 19S (or unresolved mixtures of 19S and 19R) was carried out using Dess–Martin periodinane
in pyridine to give the aryl ketone intermediate 19a (not
shown) in 74%. Stereoselective reduction using the bulky lithium tri-tert-butoxyaluminum hydride reagent in THF gives 19R with a respectable 17:1 (R:S)
diastereoselectivity and an excellent yield of 84%. Upon further investigation,
we elucidated that the corresponding bulky potassium tri-sec-butylborohydride (K-selectride), the unhindered lithium aluminum
hydride (LAH), and sodium borohydride (NaBH4) reagents
all favored stereoselectivity for the undesired 19S isomer.
This strongly suggests that the stereoselectivity for the hydride
delivery, and reduction comes from a combination of both steric and
electronic interactions between the chirality of the α-d-mannoside and the chosen reducing agent. A careful review of the
literature reveals that this particular use of mannose, or any other
sugar, as a chiral director for the stereoselective reduction of ketones
has not been reported to date and could be precedent for further investigation.
We surmise that this methodology could be applied to other glycoside
systems to effect a large array of diastereoselective or possibly
even enantioselective ketone reductions.
Prodrugs of Mannosides
In a parallel strategy to improve
the oral bioavailability and half-life (compound exposure) of O-mannosides, we designed and synthesized various prodrugs[31] of lead O-mannoside 23. The prodrugs include tetraacetate 23a, the 6-position
phosphate 23b, and dimethylglycine acetate 23c (Scheme ). Although 23a is the precursor in the synthesis of 23,[20b] it was synthesized by reacetylating mannoside 23. To selectively phosphorylate the 6-position, as in prodrug 23b, mannoside 23 was reacted with phosphoryl
chloride (POCl3) in water and trimethyl phosphate. Finally,
prodrug 23c was constructed via silylation of 23, followed by selective deprotection of the primary trimethylsilyl
group. Next, coupling of the free 6-hydroxy group with N,N-dimethylaminoglycine was achieved using N,N-diisopropylcarbodiimide (DIC) to give
prodrug 23c upon protecting group removal with TFA.
Scheme 4
Structures and Synthesis of Prodrug Analogues of O-Mannoside 23
Reagents and conditions:
(a)
Ac2O, pyridine, rt; (b) POCl3 trimethylphosphate,
H2O, 0 °C; (c) TMSCl, Et3N, DMF, 0 °C;
(d) AcOH, acetone/MeOH, 0 °C to rt; (e) N,N-dimethylglycine hydrochloride, DMAP, DIC, CH2Cl2/DIPEA, rt; (f) TFA, CH3CN, 0 °C.
Structures and Synthesis of Prodrug Analogues of O-Mannoside 23
Reagents and conditions:
(a)
Ac2O, n class="Chemical">pyridine, rt; (b) POCl3 trimethylphosphate,
H2O, 0 °C; (c) TMSCl, Et3N, DMF, 0 °C;
(d) AcOH, acetone/MeOH, 0 °C to rt; (e) N,N-dimethylglycine hydrochloride, DMAP, DIC, CH2Cl2/DIPEA, rt; (f) TFA, CH3CN, 0 °C.
X-ray Crystallography and Computational Modeling
To
better understand the molecular interactions of O-mannosides 23 and its matched disubstituted analogue 24 (Figure )[20b] with FimH, and to help us design
improved mannosides based on C-mannoside 21R, we obtained co-crystal structures of 23 and 24 bound to FimH. A high-resolution 1.67 Å X-ray crystal
structure of 24 (PDB 5F2F)[25] and a 1.75
Å structure of 23 (PDB 5F3F) bound to the FimH lectin domain were
obtained (Figure A) and used to develop a pharmacophore model
for mannoside FimH ligands. The O-linked aglycone
projects toward the “lid” of the binding pocket, which
engages in favorable hydrophobic and H-bonding interactions. The ortho-methyl substituent on the biphenyl A-ring makes hydrophobic
interactions in a small pocket formed by I52, Y137, and N138. The
biphenyl B-ring forms favorable π–π stacking interactions
with Y48, and the meta-substituted amide also makes
polar contacts with FimH. For example, the carbonyl of the amide forms
a predicted electrostatic or H-bonding interaction with the neighboring
salt bridge, R98, and/or Y137 residues. In Figure B, comparison of mannosides 23 and 24 to non-ortho-substituted 1 (PDB 3MCY),[20c] highlights a marked difference in
the biphenyl group B-ring conformation upon installation of the ortho-methyl group. On the basis of this new X-ray structure
of 23 and previously reported structures,[23a,25,32] it is apparent that the hydrophobic
contacts of the ortho-substituents within the small
pocket, and the resultant twisted B-ring conformation, improves π–π
stacking in the “tyrosine gate” with Y48 and Y137.
Figure 4
(A) X-ray
structure of 23 bound to FimH (PDB 5F3F). Water-mediated
H-bond of amide carbonyl to Y48. (B) Overlay of 23 with 24 (PDB 5F2F) and 1 (PBD 3MCY). Altered biphenyl ring conformation and amide carbonyl
orientation of ortho-substituted mannosides 23 and 24 compared to 1.
(A) X-ray
structure of 23 bound to FimH (PDB 5F3F). Water-mediated
H-bond of amidecarbonyl to Y48. (B) Overlay of 23 with 24 (PDB 5F2F) and 1 (PBD 3MCY). Altered biphenyl ring conformation and amidecarbonyl
orientation of ortho-substituted mannosides 23 and 24 compared to 1.
Computational Docking of C-Mannosides to FimH
Because we have not been able to obtain a co-crystal structure
of C-mannoside 21R, we have formulated
a computational binding model, shown in Figure . As seen in the
structure of 23 (Figure A), there is a crystallized water molecule between
the glycoside anomeric oxygen and the D140 and N135FimH side chains.
The location of the water suggests it is involved in a H-bonding interaction
between the anomeric oxygen and FimH. Our computational docking model
of the diastereomers 21S and 21R indicates
that the R-isomer (21R, Figure B) is favored energetically
over the S-isomer (21S, Figure A) due to a productive water-mediated
H-bond in the former. This same water molecule, positioned between
the hydroxyl group of the R-isomer and residue(s)
D140 and N135, is not accessible to the S-isomer.
This preference is also predicted from thermodynamic calculations,
as the calculated ΔG of binding for the R-isomer is favored by 2.8 kcal/mol over that of the S-isomer. These observations can explain the strong stereospecific
preference for FimH binding the R-isomer, relative
to the S-isomer, and why methylene spaced mannoside 22 loses activity[33] compared to O-mannoside 23, as the CH2 is not
able to participate in H-bonding to this water molecule. In the absence
of X-ray crystallography data of FimH binding, we determined the stereochemistry
of the 21R benzylic hydroxyl by obtaining a small molecule
X-ray crystal structure of crystalline C-mannoside
aryl bromide intermediate, 27 (Figure ). The high resolution X-ray of 27 unequivocally
confirms that the benzylic hydroxyl group of intermediate 19R. By logical deduction, 19R and its final product 21R, both have the R-stereochemistry, reinforcing
our computational docking model invoking a stereospecific water-mediated
H-bond to FimH.
Figure 5
Computational docking models of isomeric C-mannosides:
(A) S-hydroxy (21S) and (B) R-hydroxy (21R) bound to the FimH mannose-binding
pocket. The accepted model of 21R is bound to FimH through
water-mediated H-bond to D140 and N135.
Computational docking models of isomeric C-mannosides:
(A) n class="Chemical">S-hydroxy (21S) and (B) R-hydroxy (21R) bound to the FimHmannose-binding
pocket. The accepted model of 21R is bound to FimH through
water-mediated H-bond to D140 and N135.
(A) Derivatization of key building block 19R (precursor
to 21R). (B) Small molecule X-ray structure of acetylated
intermediate 27, confirming the R-stereochemistry
of the 21R benzylic hydroxyl group.
Pharmacokinetics (PK) of Mannosides
To further examine
the therapeutic potential of the new C-mannosides
and O-mannoside prodrugs, we evaluated their pharmacokinetic
(PK) behavior in vivo. Mannosides were delivered orally in a suspension
of 10% cyclodextrin. Previous administration utilized DMSO as a vehicle.
Our decision to administer in cyclodextrin was 2-fold: (1) cyclodextrin
is commonly used for increasing the aqueous solubility and bioavailability
of drugs[34] and (2) DMSO has been demonstrated
to modulate the host immune response, obfuscating any observed results.[35] The concentration of mannoside detected in the
urine of mice was monitored at multiple time points following oral
delivery over a period of 8 h and quantitatively measured by HPLC
and mass spectrometry. As previously published, O-linked mannosides, including both 23 and 24, were found to accumulate at high concentrations in the urine as
early as 1 h post dosing,[20b] followed by
a steady almost linear decrease throughout the collection time frame
(Figure A). Despite this continual decrease in concentration,
the relative concentration of each mannoside remained well above the
HAI EC90 (0.062 μM) and biofilm IC50 (0.15
μM) of 23 throughout this period. In contrast to 23 and 24, the concentrations of 21R and 23a (the acetate prodrug of 23) remained
substantially higher throughout the period measured, resulting in
a significantly increased AUC. Shown in Figure A, the concentration of both 21R and 23a in the urine 8 h following oral delivery was
significantly higher than 23. Compound levels of 21R reached 50 μM at 8 h, which is 1600 times
higher than its HAI potency. Acetate prodrug 23a also showed enhanced concentration, presumably from higher gut permeability,
resulting in a 20-fold higher concentration (20 μM) of 23 relative to direct oral dosing of 23 (1 μM)
at 8 h.
Figure 7
(A) Mannoside levels were quantified in the urines of mice over
a period of 8 h following oral gavage of drug as described in the
methods. Data is presented as the mean and standard deviation from
at least three independent experiments. Differences in concentration
at the 8 h time point were tested for significance using Mann–Whitney
U test. (* P < 0.05). (B) Measurement of mannoside
in the plasma of rats following either a 10 mg/kg oral dose (dashed
lines) or a 3 mg/kg intravenous dose (solid lines). Details regarding
the measurement of mannoside are described in the methods.
(A) Mannoside levels were quantified in the urines of mice over
a period of 8 h following oral gavage of drug as described in the
methods. Data is presented as the mean and standard deviation from
at least three independent experiments. Differences in concentration
at the 8 h time point were tested for significance using Mann–Whitney
U test. (* P < 0.05). (B) Measurement of mannoside
in the plasma of rats following either a 10 mg/kg oral dose (dashed
lines) or a 3 mg/kg intravenous dose (solid lines). Details regarding
the measurement of mannoside are described in the methods.To further expand on this result, we conducted
a more thorough
examination of the PK properties of 23 and 21R as well as the analogous ring-constrained isoquinolone O-mannoside 25(25) (Figure ) in rats (Figure B). Recent examination
of the PK behavior and efficacy of 25 in mice identified
it as a leading therapeutic candidate in the O-mannosides
series.[25] In contrast to the previous study
(Figure A), here we
monitored the concentration of mannoside in the plasma of rats following
either a 10 mg/kg oral dose (dashed lines) or a 3 mg/kg intravenous
dose (solid lines) (Figure B). Analysis of the rat PK data revealed that, while C-mannoside 21R encompassed the highest level
of compound exposure (as assessed by Cmax and AUCall) and the lowest clearance rate, at 34.9 mL/min/kg
(23 clearance rate = 98.4 mL/min/kg) O-mannoside 25 shows the highest clearance rate (408
mL/min/kg) and volume of distribution (Vdss = 6.7 L) but the highest bioavailability (7%) of all compounds tested.
This suggests that the biaryl B-ring isoquinolone aglycone of 25 significantly enhances permeability in the gut, while on
the other hand, C-mannoside 21R and
the O-mannoside prodrug 23a have a longer
half-life and extended compound exposure, presumably from a combination
of increased bioavailability from the prodrugs and metabolic stability
of the C-mannoside. Because O-mannosides 24 and 25 were stable (t1/2 > 2h) in plasma, simulated gut fluid (pH 1.2), simulated
intestinal fluid (pH 6.8), and liver microsomes, we hypothesize that
the increased exposure of C-mannoside 21R is likely due
to evading the enzymatic action of mannosidases. This combined data
constitutes key structure–property relationships (SPR) and
was useful for guiding further optimization of the C-mannosides described below.
Synthesis of Optimized C-Linked Isoquinolone
Mannosides
Inspired by the enhanced potency and increased
compound exposure of C-mannoside 21R, coupled with the improved oral bioavailability of 25, we synthesized its C-mannoside matched pair analogue, 28R. Suzuki cross-coupling between 27 and 1-hydroxyisoquinoline-7-boronate
ester, followed by acetyl deprotection, generated biaryl isoquinolone C-mannoside 28R (Table ). In an alternate synthesis, 19R was used in place of 27 in the Suzuki reaction, but
attempts to remove the benzyl protecting groups via hydrogenation
resulted in formation of the saturated dihydroisoquinolone analogue 29R. For direct comparison, we repeated this hydrogenation
on O-glycoside 25 to give corresponding
analogue 30. We found that both isoquinolone 28R and dihydroisoquinolone 29R have excellent potency,
with HAI titers of 8 and 6 nM, respectively. This equates to a ∼4.5-fold
increase in potency over the O-mannoside matched
pairs, 25 and 30 (HAI = 31 and 32 nM, respectively).
Following these exciting results, we evaluated fused ring systems
like 25 that would still retain their H-bonding and FimH
binding attributes. Using similar procedures as described above, we
synthesized aminoisoquinoline 31R and isoquinoline 32R, both possessing a nitrogen atom that occupies a position
equivalent to the carbonyl oxygen on the isoquinolone ring of 25. Isoquinoline 32R has a different ring fusion,
being attached to the aglycone A-ring at the 5-position (versus the
7-position in 31R, 28R, and 29R). As expected, the C-mannoside 31R was 3-fold more potent than its matched O-mannoside
pair 33, with an HAI of 6 nM. However, the 5-position
isoquinolineC-mannoside 32R had lower
potency relative to its O-mannoside pair 34(24) (HAI = 62 nM), with an HAI of 125 nM
(Table ). This unexpected
result can be rationalized from docking studies (not shown), wherein
we found that due to a dramatic shift in ring conformation of the C-mannosides relative to the O-mannosides
(Figure ), a steric
clash exists between FimH and this 5-position ring substitution of 32R in the C-mannoside series, which does
not exist in 34 of the O-mannoside series.
When comparing the matched pair C- and O-mannosides shown in Table , it is apparent that the cLogP values are approximately 1
Log unit lower in the C-mannoside series. For example,
comparing 33 to 31R, we see a drop in cLogP
from 2.28 to 1.2, respectively, suggesting that 31R has
respectable oral drug-like physical properties like 25 (cLogP 1.4) but with enhanced glycosidic bond stability.
Table 1
Compound Structures and Comparison
of Biological Activity and Physical Properties Between O- and C-Mannoside Matched Pairs (ND = not determined)
In Vivo Efficacy of Mannosides
in Acute and Chronic Cystitis
Given the improved in vivo
PK behavior of both the C-mannoside 21R and the prodrug 23a, we
next investigated whether these enhancements improved their potency
in animal models of UTI.[20a,36] The distinct pathophysiology
of UTI that has been identified in these murine models has also been
observed in humans, demonstrating their relevance.[37] To measure efficacy in these models, we examined the ability
of mannosides to prevent the initial pathogenic steps
of UPEC infection in our prophylactic model of acute UTI as well as
their ability to treat an established infection in
our model of chronic UTI. Despite the disparity in potency and PK
behavior, the efficacy of all the compounds, in either model, were
nearly identical (Figure ). When tested as a prophylaxis therapy (Figure A), where the mannoside
is dosed 30 min prior to UPEC infection and bacterial titers enumerated
6 h postinfection, tetraacetate prodrug 23a and phosphate
prodrug 23b (see Supporting Information data) each showed improved efficacy compared to 23.
In contrast, the glycine acetate prodrug 23c (see Supporting Information data) did not show enhanced
efficacy, perhaps due to its increased stability in conversion to
drug or decreased oral absorption in the gut relative to 23a and 23b.
Figure 8
(A) Prophylactic treatment of an acute UTI.
Mannoside was dosed
orally at 25 mg/kg in 10% cyclodextrin, 30 min prior to infection
with 107 CFUs UPEC. Bladders were harvested 6 h postinfection
and bacterial burdens enumerated. (B) Mannoside treatment of chronic
UTI. Mice were treated orally with 50 mg/kg of mannoside in 10% cyclodextrin,
after 14 days of chronic UPEC infection. Bladders were harvested 6
h following oral dosing and bacterial burdens enumerated. Data from
at least two independent experiments are presented in each panel;
bars indicate geometric means. Differences between treated groups
and vehicle were tested for significance using Mann–Whitney
U test (*** P = 0.0001, **** P <
0.0001).
(A) Prophylactic treatment of an acute UTI.
Mannoside was dosed
orally at 25 mg/kg in 10% cyclodextrin, 30 min prior to infection
with 107 CFUs UPEC. Bladders were harvested 6 h postinfection
and bacterial burdens enumerated. (B) Mannoside treatment of chronic
UTI. Mice were treated orally with 50 mg/kg of mannoside in 10% cyclodextrin,
after 14 days of chronic UPEC infection. Bladders were harvested 6
h following oral dosing and bacterial burdens enumerated. Data from
at least two independent experiments are presented in each panel;
bars indicate geometric means. Differences between treated groups
and vehicle were tested for significance using Mann–Whitney
U test (*** P = 0.0001, **** P <
0.0001).Both the O-linked
and C-linked
mannosides accumulated in the urine at concentrations over 10-fold
higher than their HAI EC90 and biofilm IC50 (Figure ) up to 8 h following
oral dosing. Thus, we hypothesized that 6 h post-treatment was too
early to discriminate relative mannoside potency in the chronic UTI
model (Figure B).
To investigate this possibility, we first monitored the pharmacodynamics
(PD) of O-linked mannoside 24 in our
treatment model of chronic UTI to evaluate its therapeutic window.
Diamide mannoside 24 has similar PK to monoamide mannoside 23 but was found to be 4-fold more potent in our HA assay
(HAI = 16 nM), identifying it as one of our most effective O-linked mannosides in vitro. Following oral delivery of 24 in the chronic UTI model, we found a significant decrease
in bladder burden from 107 CFUs to approximately 105 CFUs 2 h postdosing (Figure ). Bladder burdens
continued to decrease to 104 CFUs at 6 h but leveled off
at 12 hours, when the geometric mean of the bacterial burden increased
to 105. Interestingly, the 12-hour time point appears to
be comprised of a bimodal distribution, consisting of mice that have
either high or low bacterial burdens in the bladder (Figure ). Taken together, this data
demonstrates a rapid reduction of bladder burdens following oral delivery
of O-linked mannoside 24 and an increase
of burdens in a subpopulation of mice 12 hours after treatment that
may represent bladder repopulation once mannoside levels fall below
the HAI.
Figure 9
Pharmacodynamics of O-linked mannoside. Chronically
infected mice were treated orally with 50 mg/kg of mannoside 24. Bladders were harvested at the designated time points
following oral dosing and bacterial burden enumerated. Bars indicate
geometric means. Differences between the designated time point and
the 0 h time point were tested for significance using Mann–Whitney
U test (* P < 0.05, ** P <
0.001).
Pharmacodynamics of O-linked mannoside. Chronically
infected mice were treated orally with 50 mg/kg of mannoside 24. Bladders were harvested at the designated time points
following oral dosing and bacterial burden enumerated. Bars indicate
geometric means. Differences between the designated time point and
the 0 h time point were tested for significance using Mann–Whitney
U test (* P < 0.05, ** P <
0.001).
C-Mannosides
Displays Significantly Improved
and Prolonged Efficacy in Vivo
Given the observed bimodal
distribution of bladder burdens 12 hours after treatment with mannoside 24, we next decided to investigate the influence of the improved
pharmacokinetic behavior of C-mannoside 21R on its ability to treat chronic cystitis at this time point following
oral delivery of the mannoside (Figure A). Additionally,
to accentuate any differences in efficacy at this time point, we reduced
the mannoside dosages by half, treating only with a 25 mg/kg dose.
At 12 h post treatment, the O-linked mannoside 24 resulted in a noticeable, but not significant, decrease
in bladder burdens compared to vehicle. Treatment with prodrug 23a resulted in a similar, but statistically significant,
decrease in bladder burdens. In contrast, it was found that treatment
with C-mannoside 21R at 25 mg/kg resulted
in a dramatic and significant decrease in CFUs as compared to both
vehicle and O-linked mannosides 24 and 23a. Indeed, bladder burdens of mice treated with 21R were uniformly three logs lower than untreated mice, clearly demonstrating
the improved efficacy of the C-linked mannoside.
Figure 10
Improved
efficacy of C-linked mannosides. (A)
Chronically infected mice were treated orally with 25 mg/kg of mannoside.
Bladders were harvested 12 hours following oral dosing and bacterial
burdens enumerated. (B) Mice were treated orally with 25 mg/kg of
mannoside 30 min prior to infection. Bladders were harvested 6 h following
infection and bacterial burdens enumerated. Data from at least two
independent experiments are presented; bars indicate geometric means.
Differences between treated groups and vehicle were tested for significance
using Mann–Whitney U test (**P < 0.01,
*** P = 0.0001, **** P < 0.0001).
Improved
efficacy of C-linked mannosides. (A)
Chronically infected mice were treated orally with 25 mg/kg of mannoside.
Bladders were harvested 12 hours following oral dosing and bacterial
burdens enumerated. (B) Mice were treated orally with 25 mg/kg of
mannoside 30 min prior to infection. Bladders were harvested 6 h following
infection and bacterial burdens enumerated. Data from at least two
independent experiments are presented; bars indicate geometric means.
Differences between treated groups and vehicle were tested for significance
using Mann–Whitney U test (**P < 0.01,
*** P = 0.0001, **** P < 0.0001).Furthermore, using the newly optimized
heterocyclic C-mannosides, 28R, 29R and 31R, we found significantly augmented
activity relative to O-mannoside 25 when
tested in the prophylactic model
of UTI (Figure B),
described above, where compound was dosed orally (25 mg/kg) 30 min
before infection. The in vivo activity seen with 28R, 29R, and 31R is similar to 21R,
but it can be argued that 29R and 31R perform
slightly better in preventing an infection in this model of acute
UTI. However, there is a clear difference in efficacy displayed between
the C-mannosides and O-mannoside 25. This is nicely illustrated by direct comparison of O-mannoside 25 and its matched C-mannoside 28R, which decreases bacterial titers 2 Logs
lower (100-fold). Taken together with the fact that O-mannoside 25 and C-mannoside 21R are equivalent in both FimH binding (DSF = 75.4 °C)
and target inhibition (HAI = 31 nM), this result suggests that the C-mannosides have superior metabolic stability.
Conclusion
Carbohydrates, and in particular small glycosides, are typically
unstable in vivo due to their inherent susceptibility to hydrolysis
in the stomach or intestine and to enzymatic hydrolysis by various
glycosidases present in the gut, plasma, and some tissues. This instability
can critically limit the oral bioavailability, half-life. and overall
therapeutic potential of glycoside-based drugs[38] like FimHmannoside antagonists. The replacement of O-glycosidic bonds with sulfur, carbon, or nitrogen-based
linkers is one strategy that can be utilized to increase the metabolic
stability of glycosides while also theoretically helping to increase
their bioavailability. However, the synthesis of unnatural glycoside
linkers is challenging and the influence of these changes on activity
are not always predictable or straightforward. In the current manuscript,
we implemented a rational strategy to improve mannoside PK and efficacy
by replacing the glycosidic bond of the O-mannosides
with a number of unique carbon-based linkers that are likely stable
to metabolism. To access these novel C-mannosides,
we have designed a new stereoselective synthetic route for the assembly
of hydroxymethyl C-mannosides. While increased stability
to mannosidases is the most reasonable explanation for the improved
efficacy in vivo, other properties may be contributing to this behavior.
Current investigations are confirming this hypothesis through both
in vitro and in vivo studies which are beyond the scope of this initial
communication.In summary, mannoside FimH antagonists represent
a first-in-class
antivirulence drug in the treatment of chronic UTI and the prevention
of recurrent cystitis. Herein, we have rationally designed a novel C-mannoside FimH antagonist 21R, which we subsequently
modified, based on O-mannosideSAR and isoquinolone
derivative 25,[25] to generate
several optimized analogues, 28R, 29R, and 31R. These C-mannosides have become preclinical
candidates for use as novel antivirulence drugs in UTI therapy.[18] Along with other lead compounds, they are currently
being profiled in advanced in vivo PK, efficacy, and toxicologuey
studies in preparation for selecting a lead candidate drug for clinical
trials. Upon successful clinical development, a mannoside would not
only represent the first small molecule FimH antagonist drug for UTI
but also the first antibiotic-sparing therapeutic for the treatment
of Gram-negative infections. This could also pave the way for the
new development of other antibiotic-sparing drugs targeting other
lectins or virulence factors, which are desperately needed for other
infectious diseases.
Experimental Section
General
Synthesis, Purification, and Chemistry Procedures
Starting
materials, reagents, and solvents were purchased from
commercial vendors unless otherwise noted. In general, anhydrous solvents
are used for carrying out all reactions. 1HNMR spectra
were measured on a Varian 300 MHz NMR instrument or Varian 400 MHz
NMR instrument equipped with an auto sampler. The chemical shifts
were reported as δ ppm relative to TMS using residual solvent
peak as the reference unless otherwise noted. The following abbreviations
were used to express the peak multiplicities: s = singlet; d = doublet;
t = triplet; q = quartet; m = multiplet; br = broad. Melting points
were determined on a Kofler micro hot stage and were uncorrected.
High-performance liquid chromatography (HPLC) was carried out on GILSON
GX-281 using Waters C18 5 μM 4.6 mm × 50 mm and Waters
Prep C18 5 μM 19 mm × 150 mm reverse phase columns, eluted
with a gradient system of 5:95 to 95:5 acetonitrile:water with a buffer
consisting of 0.05–0.1% TFA. Mass spectroscopy (MS) was performed
on HPLC/MSD using a gradient system of 5:95 to 95:5 acetonitrile:water
with a buffer consisting of 0.05–0.1% TFA on a C18 or C8 reversed
phased column and electrospray ionization (ESI) for detection. All
reactions were monitored by thin layer chromatography (TLC) carried
out on either Merck silica gel plates (0.25 mm thick, 60F254) or Millipore
Silica gel aluminum sheets (60F254) and visualized by using UV (254
nm) or dyes such as KMnO4, p-anisaldehyde,
and CAM (Hannesian’s Stain). 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% purity based on NMR
and HPLC by absorbance at 220 and 254 nm wavelengths.
General Procedure
for the Suzuki Coupling Reactions (Amounts
and Volumes Are Specified within Individual Procedures)
Cesium
carbonate (Cs2CO3) was activated by adding it
to a round-bottom flask, which was then heated to 250 °C under
vacuum for 2 min and then allowed to cool to rt under vacuum for an
additional 10 min, after which time a nitrogen atmosphere was continuously
maintained. Next, the desired mannosyl bromide (or mannosyl boronate
ester) derivative was dissolved into dioxane and the solution was
added dropwise, followed by the addition of the desired boronate-aglycone
(or bromide-aglycon if mannosyl boronate was used) and, finally a
small amount of water. After allowing the reaction contents to stir
for 5 min at rt, a catalytic amount of tetrakis(triphenylphosphine)palladium(0)
[Pd(Ph3)4] was added and the reaction flask
was evacuated under high vacuum and backfilled with N2 three
times and then placed in an oil bath preheated to 80 °C and allowed
to stir for the time specified (typically 1.5 h). Upon completion,
the reaction was cooled to rt and solvents were evaporated under reduced
pressure. The crude reaction residue was then purified and deprotected
as specified.
Detailed Procedures for the Synthesis of
Final Mannosides and
Intermediates
α-d-Mannose
(0.360 g, 2 mmol) and methyl 4′-aminobiphenyl-3-carboxylate
(0.454 g, 2 mmol) were dissolved into ethanol (5 mL), and the reaction
was heated to 55 °C for 17 h. After cooling down to rt, the white
precipitate that formed was collected by filtration. The precipitate
was washed with ethanol (2 × 3 mL) and then dried in vacuo to
afford pure 2 in 77% yield. Analytical data for 2: 1HNMR (300 MHz, acetonitrile-d3 and D2O) δ ppm 3.28–3.38 (m,
1H), 3.54–3.59 (m, 2H), 3.64–3.71 (m, 2H), 3.86 (s,
3H), 3.88–3.92 (m, 1H), 4.89 (d, J = 1.1 Hz,
1H), 6.80–6.91 (m, 2H), 7.44–7.58 (m, 3H), 7.75–7.90
(m, 2H), 8.11–8.20 (m, 1H). 13CNMR (100 MHz, methanol-d4/dimethyl sulfoxide-d6; 10/1) δ ppm 52.8, 62.8, 68.6, 72.9, 76.2, 78.9, 83.3,
115.6 (×2), 127.8, 128.1, 128.7 (×2), 130.2, 130.8, 131.7,
131.9, 142.8, 147.5, 168.4. ESI-MS found: [M + H]+, 390.1.
Step 1: Under nitrogen
atmosphere at 0 °C, boron trifluoride diethyl etherate (0.427
g, 3.0 mmol) was added dropwise into a solution of α-d-mannose pentaacetate (0.390 g, 1.0 mmol) and 4-bromobenzenethiol
(0.378 g, 2.0 mmol) in 6 mL of CH2Cl2. After
5 min, the mixture was warmed to rt and allowed to stir for 48 h.
The reaction was then quenched with water and extracted with CH2Cl2. The CH2Cl2 layer was
collected, dried over Na2SO4, and concentrated
in vacuo. The resulting residue was purified by silica gel chromatography
(ethyl acetate–hexanes gradient elution), giving rise to the
intermediate 4-bromophenyl 2,3,4,6-tetra-O-acetyl-1-thio-α-d-mannopyranoside (3a) (0.40 g) in 77% yield. Analytical
data for 3a: 1HNMR (300 MHz, chloroform-d) δ ppm 2.01–2.10 (m, 9H), 2.16 (s, 3H), 4.10
(dd, J = 12.1, 2.5 Hz, 1H), 4.30 (dd, J = 12.1, 6.0 Hz, 1H), 4.43–4.61 (m, 1H), 5.24–5.40
(m, 2H), 5.43–5.52 (m, 2H), 7.32–7.39 (m, 2H), 7.42–7.49
(m, 2H). ESI-MS found: [2 M + H+] 1039.1.Step 2:
Following the general Suzuki-coupling procedure, mannosyl bromide 3a (0.20 g, 0.39 mmol) from step 1, commercially available
3-methoxycarbonylphenylboronic acid (0.106 g, 0.59 mmol), cesium carbonate
(0.381 g, 1.17 mmol), and tetrakis(triphenylphosphine)palladium (0.045
g, 0.04 mmol) in dioxane/water (5 mL/1 mL) were reacted under N2 at 80 °C for 1 h. Upon completion, the reaction was
cooled to rt, and the mixture was filtered through a silica gel column
(ethyl acetate–hexanes, 2/1 isocratic elution) to remove the
metal catalyst and salts. The filtrate was concentrated then dried
in vacuo. To the crude residue was added MeOH (6 mL) and a catalytic
amount of sodium methoxide (0.02 M), and the mixture was stirred at
rt overnight. H+ exchange resin (DOWEX 50WX4-100) was added
to neutralize the mixture, the resin was filtered off, and the filtrate
concentrated. The resulting residue was purified by HPLC [C18, 15
mm × 150 mm column; eluent, acetonitrile/water (0.1% TFA)] to
give 3 (0.095 g) in 63% yield. Analytical data for 3: 1HNMR (300 MHz, methanol-d4) δ ppm 3.66–3.91 (m, 4H), 3.94 (s, 3H),
4.01–4.16 (m, 2H), 5.51 (d, J = 1.4 Hz, 1H),
7.51–7.69 (m, 5H), 7.81–7.93 (m, 1H), 8.00 (dt, J = 7.8, 1.4 Hz, 1H), 8.24 (t, J = 1.7
Hz, 1H). 13CNMR (100 MHz, methanol-d4 /dimethyl sulfoxide-d6; 20/1)
δ ppm 52.8, 62.6, 68.7, 73.2, 73.6, 76.0, 90.2, 128.6 (×2),
128.6, 129.5, 130.4, 132.0, 132.5, 133.2 (×2), 135.9, 140.0,
141.8, 168.1. ESI-MS found: [M + H+] 407.1.
The solution of 2,3,4,6-tetra-O-acetyl-α-d-mannosyl cyanide (9)[29,30] (0.107g, 0.3 mmol) in 25% hydrochloric acid
was heated at 50 °C for 48 h. The solvent was removed. Water
(10 mL) and H+ exchange resin (DOWEX 50WX4-100) was added and kept
stirring for 5 min. The resin was filtered off, and the filtrate was
concentrated and then dried in vacuo to give crude α-d-mannopyranosyl carboxylic acid (10). To the crude product,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (0.274 g,
0.72 mmol) and anhydrous DMF (6 mL) were added at 0 °C. After
stirring for 10 min, methyl 4′-aminobiphenyl-3-carboxylate
(0.164 g, 0.72 mmol), and then N,N-diisopropylethylamine (0.233 g, 1.80 mmol) were added. The mixture
was stirred overnight while being warmed to rt naturally. The solvent
was removed and the residue was purified by silica gel chromatography
(methanol–dichloromethane gradient elution) to give the title
compound (0.066 g) in 52% yield. Analytical data for 11: 1HNMR (300 MHz, methanol-d4) δ ppm 3.50 (dt, J = 4.67, 2.33 Hz, 1H),
3.58–3.65 (m, 2H), 3.76 (dd, J = 11.81, 7.42
Hz, 1H), 3.86–4.03 (m, 4H), 4.52 (t, J = 2.61
Hz, 1H), 4.57 (d, J = 2.75 Hz, 1H), 7.55 (t, J = 7.69 Hz, 1H), 7.60–7.68 (m, 2H), 7.68–7.76
(m, 2H), 7.82–7.91 (m, 1H), 7.97 (dt, J =
7.69, 1.37 Hz, 1H), 8.21–8.28 (m, 1H). 13CNMR (100
MHz, methanol-d4) δ ppm 52.7, 63.3,
68.5, 70.1, 73.2, 79.6, 80.4, 121.9 (×2), 128.4 (×2), 128.6,
129.1, 130.2, 132.0, 132.4, 137.4, 138.9, 142.2, 168.5, 170.1. ESI-MS
found: [M + H+] 418.1.
To a solution of [(α-d-mannopyranosyl)methyl]amine
(12) (0.58 mmol) (obtained from 2,3,4,6-tetra-O-acetyl-α-d-mannosyl cyanide 9(29,30)) in pyridine (10 mL) was added 4-bromo-benzoyl chloride
(2.2 g, 10 mmol), and the reaction was stirred at RT overnight. Excess
acid chloride was quenched by the addition of MeOH (2 mL). After 30
min, the reaction was concentrated in vacuo and dried. To the crude
residue was added 25 mL of [0.5 M] sodium methoxide in MeOH (pH ∼
10), and the reaction was stirred for 3 h at RT. H+ exchange
resin (DOWEX 50WX4-100) was washed with MeOH and then added while
stirring for 15 min. After filtration, the crude product was obtained
by concentrating the filtrate and then was purified by silica gel
chromatography (methanol–dichloromethane gradient elution).
It was found that pyridine-HCl was a large contaminant, so the crude
product was redissolved in MeOH (10 mL) and charged with 0.5 mL of
3 M NaOH and stirred for 5 min. The reaction was then neutralized
with H+ exchange resin (DOWEX 50WX4-100). After filtration
and concentrating the filtrate, the title product was isolated (120
mg, 0.32 mmol; 55% over 2 steps). Analytical data for 13: 1HNMR (400 MHz, methanol-d4) δ ppm 3.63–3.68 (m, 4H), 3.75–3.85 (m, 4H),
4.03–4.07 (m, 1H), 7.63 (d, J = 8.8 Hz, 1H),
7.75 (d, J = 8.4 Hz, 1H). 13CNMR (100
MHz, methanol-d4) δ ppm 40.2, 62.6,
70.0, 70.3, 72.7, 76.3, 77.4, 127.1, 130.3 (×2), 132.7 (×2),
134.6. ESI-MS found: [M + H+] 376.0 (100%), 378.0 (97.3%).
Similar to the synthesis of compound 9, acetyl 2,3,4,6-tetra-O-benzyl-α-d-mannopyranoside (56.97 g, 97.84 mmol) was dissolved into dry
acetonitrile (800 mL) under N2, and the reaction was cooled
to 0 °C. Trimethylsilyl cyanide (36.86 mL, 293.53 mmol) was added,
followed by the dropwise addition of boron trifluoride diethyl etherate
(2.46 mL, 19.57 mmol). After 5 min, the reaction was brought to rt
and stirred for an additional 30 min. Upon completion, brine (400
mL) and ethyl acetate (400 mL) were added and the reaction was stirred
vigorously for 5 min. The layers were then partitioned, and the aqueous
layer was extracted with ethyl acetate (3 × 100 mL). The organic
portions were then combined and washed with 1 M aq HCl (2 × 200
mL) and brine (200 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude residue contained
a 2:1 mixture of α- and β-anomers, which were easily separated
by silica gel chromatography (ethyl acetate–hexanes gradient),
the more nonpolar product being the desired α-mannoside 15, obtained in 51% yield (the β-mannoside byproduct
was obtained in 27% yield). Analytical data for 15:[31b]1HNMR (300 MHz, chloroform-d) δ ppm 3.72–3.76 (m, 1H), 3.80–3.91
(m, 3H), 3.93–3.97 (m, 1H), 4.01–4.08 (m, 1H), 4.51–4.71
(m, 7H), 4.81 (d, J = 2.3 Hz, 1H), 4.88 (d, J = 11.0 Hz, 1H), 7.17–7.22 (m, 2H), 7.27–7.40
(m, 18H). ESI-MS found: [M + Na+] 572.2.
Similarly to previously reported
protocols,[31b,39] at −78 °C, DIBAL/hexane
(1.0 M, 11.3 mL) was added dropwise into the solution of 2,3,4,6-tetra-O-benzyl-α-d-mannopyranosyl cyanide (15) (4.97 g, 9.04 mmol) in CH2Cl2 (150
mL) under N2. The mixture was stirred for 30 min, maintaining
a temperature of −78 °C. Then, the reaction was diluted
with CH2Cl2 (150 mL) and then acidified with
the addition of 0.2 M aq HCl (400 mL), stirring for 10 min at rt,
and then filtered through Celite (to help break up emulsion) into
a separatory funnel. The distinct layers were separated, and the aqueous
layer was then extracted an additional time with CH2Cl2. The two organic fractions were combined and washed 2×
with brine (100 mL). The organic layer was dried over Na2SO4, which also cleared up any remaining emulsion, and
then concentrated to give intermediate carbaldehyde 16 as the crude product. Because of its instability, this intermediate
was used without further purification, after drying 30 min to 1 h
under high vacuum. Compound 16 was confirmed by ESI-MS,
found [M + Na+] 575.5.
Into a flask containing 1,4-dibromobenzene
(0.354 g, 1.5 mmol) in ether (5 mL) was added BuLi/hexanes (2.5 M,
0.5 mL) at −78 °C. One hour later, crude aldehyde 16 (synthesized from 0.56 mmol of carbonitrile 15) was added. The mixture was stirred at −78 °C for 1
h and then was warmed slowly to −40 °C over 1 h. Then
0.5 Naq HCl was used to quench the reaction, and ethyl acetate was
use for extraction. The organic layer was collected, dried with Na2SO4, and concentrated, and the resulting residue
was partially purified by silica gel chromatography (ethyl acetate–hexane
gradient elution). The diastereomers, (2,3,4,6-tetra-O-benzyl-α-d-mannopyranosyl)-(4-bromophenyl)-methan-1(R)-ol (17R) and (2,3,4,6-tetra-O-benzyl-α-d-mannopyranosyl)-(4-bromophenyl)-methan-1(S)-ol (17S) were separated and collected, the
more nonpolar fractions containing intermediate 17R.
After separation, the impure compounds 17R (0.075 g,
0.1 mmol) and 17S (0.034 g, 0.048 mmol) were used without
further purification in the synthesis of 18R and 18S, respectively. Both compounds 17R and 17S were separately confirmed by ESI-MS, found [M + Na+] 731.1.
Into a flask containing 4-bromo-2-methyl-iodobenzene (9.04
mL, 63.29 mmol) in anhydrous Et2O (150 mL) under N2 was added BuLi/hexanes (2.5 M, 21.7 mL) dropwise at −78
°C. After 1 h, the freshly prepared crude 16 [from
starting material 15 (4.97 g, 9.04 mmol)] was dissolved
into Et2O (50 mL) and added via cannula over a period of
5 min. The mixture was stirred at −78 °C for 30 min and
then slowly warmed to 0 °C over 1.5 h. Saturatedaq NH4Cl was used to quench the reaction, and the reaction was extracted
with ethyl acetate (2 × 100 mL). The organic fractions were then
combined and washed with brine (100 mL), dried over Na2SO4, and concentrated in vacuo. The resulting residue
was mixture of diastereomers, which were purified and separated by
silica gel chromatography (1/9, v/v, ethyl acetate–hexanes,
isocratic elution) to give (2,3,4,6-tetra-O-benzyl-α-d-mannopyranosyl)-(4-bromo-2-methylphenyl)-methan-1(R)-ol (19R) as a syrup in 16% yield (1.05g,
1.45 mmol) and (2,3,4,6-tetra-O-benzyl-α-d-mannopyranosyl)-(4-bromo-2-methylphenyl)-methan-1(S)-ol (19S) as a syrup in 20% yield (1.30 g,
1.80 mmol); the fractions coming out of the column earlier containing
isomer 19R. Analytical data for 19R: 1HNMR (400 MHz, chloroform-d) δ ppm
2.29 (s, 3H); 3.49 (br s, 1H), 3.70–3.83 (m, 2H), 3.89 (t, J = 5.9 Hz, 1H), 3.94–3.99 (m, 1H), 4.10 (t, J = 5.1 Hz, 1H), 4.13–4.18 (m, 1H), 4.21–4.28
(m, 1H), 4.40 (s, 2H), 4.49 (s, 2H), 4.56–4.64 (m, 3H), 4.71
(d, J = 11.7 Hz, 1H), 5.08 (d, J = 5.1 Hz, 1H), 7.13–7.18 (m, 2H), 7.28–7.41 (m, 21H).
ESI-MS found: [M + Na+] 745.5 (100%), 747.5 (97.3%). Analytical
data for 19S: 1HNMR (400 MHz, chloroform-d) δ ppm 2.18 (s, 3H), 3.19 (br s, 1H), 3.67–3.73
(m, 2H), 3.76–3.85 (m, 3H), 4.03–4.11 (m, 2H), 4.44–4.62
(m, 7H), 4.67–4.73 (m, 1H), 5.06 (d, J = 5.5
Hz, 1H), 7.16–7.37 (m, 23H). ESI-MS found: [M + Na+] 745.5 (100%), 747.5 (97.3%).
Two-Step Oxidation and
Reduction Protocol to Convert 19S into 19R (Step 1 19S to 19a)
The 19S product was converted
into the 19R isomer via a two-step oxidation reduction
procedure. First, oxidation to the ketone intermediate (19a) was achieved by dissolving 19S (0.58 g, 0.80 mmol)
in dry CH2Cl2 (50 mL) and dry pyridine (0.16
mL, 2.01 mmol) under N2 at 0 °C. Dess–Martin
periodinane (DMP) (0.68 g, 1.61 mmol) was added portionwise, and the
reaction mixture was kept at 0 °C for 1 h and then allowed to
warm to 15 °C over an additional 1.5 h. Upon completion, the
reaction flask was cooled in an ice bath and a 1:1 mixture of 10%
aqNa2S2O3 (6 mL) and saturatedaqNaHCO3 (6 mL) was added, and the reaction was stirred for
5 min at rt. The layers were then separated, and the aqueous layer
was extracted an additional time with CH2Cl2 (5 mL). The organic fractions were combined and washed sequentially
with saturatedaqNaHCO3 (10 mL) and brine (10 mL), dried
over Na2SO4, and concentrated in vacuo without
heating. The residue was quickly purified by silica gel chromatography
(ethyl acetate–hexane gradient elution), and pure compound
eluent was again concentrated in vacuo in the absence of heat to afford
the desired ketone intermediate (2,3,4,6-tetra-O-benzyl-α-d-mannopyranosyl)-(4-bromo-2-methylphenyl)-methanone (19a) in 74% yield (0.43 g, 0.59 mmol). Analytical data for 19a: 1HNMR (400 MHz, chloroform-d) δ ppm 2.40 (s, 3H), 3.50–3.56 (m, 1H), 3.61 (d, J = 10.6 Hz, 1H), 3.67–3.77 (m, 2H), 4.05 (t, J = 9.0 Hz, 1H), 4.43 (d, J = 12.1 Hz,
1H), 4.49–4.61 (m, 3H), 4.64–4.74 (m, 2H), 4.76–4.85
(m, 2H), 4.89 (d, J = 11.0 Hz, 1H), 5.12 (d, J = 2.7 Hz, 1H), 7.19–7.24 (m, 2H), 7.31–7.45
(m, 20H), 7.60 (d, J = 8.6 Hz, 1H). ESI-MS found:
[M + Na+] 743.5 (100%), 745.5 (97.3%).
Two-Step Oxidation
and Reduction Protocol to Convert 19S into 19R (Step 2 19a to 19R)
Next, selective reduction
to the (R)-alcohol was achieved by reacting ketone
(19a) (0.31 g, 0.43 mmol) in dry THF (30 mL) under N2 at −40 °C, with the dropwise addition of lithium
tri-tert-butoxyaluminum hydride (1 M in hex; 0.87
mL, 0.87 mmol). The reaction was warmed to 0 °C over 1 h and
then stirred an additional 1 h at 0 °C. Upon completion, the
reaction was diluted with ethyl acetate (60 mL). Saturatedaq potassium
sodium tartrate (30 mL) was added, and the reaction was vigorously
stirred 1 h at rt. At this time, the layers were separated and the
aqueous layer was additionally extracted with ethyl acetate (2 ×
15 mL), using 1 M aq HCl to break up any remaining emulsion. The organic
fractions were then combined, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel
chromatography (ethyl acetate–hexane gradient elution) to afford
the desired R-isomer 19R (0.26 g, 0.36
mmol) in 84% yield (with 16% of the 19S (0.050 g, 0.069
mmol) isomer also generated). Analytical data as reported above.
The title compound was
synthesized following the general Suzuki-coupling procedure. Although
separable, for this reaction the purified 19R/S (0.130 g, 0.18 mmol, generated from the reaction of aldehyde 16) was taken as a mixture of isomers and was reacted with
commercially available N-methyl-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzamide
(0.071g, 0.27 mmol), cesium carbonate (0.176 g, 0.54 mmol), and tetrakis(triphenylphosphine)palladium
(0.021 g, 0.018 mmol) in dioxane/water (5 mL/1 mL), and the reaction
was stirred under N2, at 80 °C for 1 h. Upon completion,
the solvent was removed and the resulting products were separated
and purified by silica gel chromatography (methanol–dichloromethane
gradient elution). 20R (0.046 g, 0.059 mmol) was obtained
in 33% yield as the more nonpolar compound, and the more polar 20S (0.055 g, 0.071 mmol) was obtained in 39% yield. Analytical
data for 20R: 1HNMR (400 MHz, chloroform-d) δ ppm 2.38 (s, 3H), 2.88–3.17 (m, 4H), 3.65
(dd, J = 10.6, 4.3 Hz, 1H), 3.71–3.84 (m,
2H), 4.02 (d, J = 5.5 Hz, 2H), 4.09–4.21 (m,
2H), 4.30–4.44 (m, 4H), 4.51–4.68 (m, 4H), 5.17 (dd, J = 5.9, 3.1 Hz, 1H), 6.15 (d, J = 4.7
Hz, 1H), 7.08–7.39 (m, 22H), 7.43–7.54 (m, 2H), 7.62–7.67
(m, 1H), 7.68–7.73 (m, 1H), 7.87–7.90 (m, 1H). MS (ESI)
found: [M + Na+] 800.6. Analytical data for 20S: 1HNMR (400 MHz, chloroform-d) δ
ppm 2.27 (s, 3H), 3.03 (d, J = 4.8 Hz, 3H), 3.13
(br s, 1H), 3.61–3.89 (m, 5H), 3.98–4.10 (m, 1H), 4.12–4.20
(m, 1H), 4.33–4.61 (m, 7H), 4.69 (d, J = 11.4
Hz, 1H), 5.14 (d, J = 5.1 Hz, 1H), 6.19 (d, J = 4.3 Hz, 1H), 7.04–7.59 (m, 24H), 7.61–7.76
(m, 2H), 7.91–8.01 (m, 1H). ESI-MS found: [M + Na+] 800.6.
4′-(-α-d-Mannopyranosyloxy)-N,3′-dimethyl-[1,1′biphenyl]-3-carboxamide[20b] (0.20 g, 0.5 mmol) was dissolved in trimethyl
phosphate (5 mL) and water (9 uL, 0.5 mmol). The reaction was cooled
to 0 °C, and then phosphoryl trichloride (142 μL, 1.5 mmol)
was slowly added and then stirred for 3 h at 0 °C. The reaction
was neutralized by adding crushed ice and then conc ammonia. The solvent
was removed in vacuo, and the residue was purified by HPLC [C18, 15
mm × 150 mm column; eluent, acetonitrile/water (0.1% TFA)]. Pure
fractions were combined and lyophilized to give the title compound
as a white powder in 29% yield (0.070 g, 0.145 mmol). Analytical data
for 23b: 1HNMR (400 MHz, dimethyl sulfoxide-d6) δ ppm 2.26 (s, 3H), 2.81 (d, J = 4.70 Hz, 3H), 3.42–3.68 (m, 3H), 3.75 (dd, J = 9.00, 3.13 Hz, 1H), 3.86–3.97 (m, 2H), 4.03 (dd, J = 9.78, 5.87 Hz, 1H), 5.45 (d, J = 1.96
Hz, 1H), 7.24 (d, J = 8.61 Hz, 1H), 7.43–7.60
(m, 3H), 7.76 (dd, J = 7.43, 1.57 Hz, 2H), 8.06 (s,
1H), 8.56 (d, J = 4.30 Hz, 1H). 13CNMR
(100 MHz, dimethyl sulfoxide-d6) δ
ppm 16.1, 26.3, 66.3, 70.1, 70.6, 73.3, 73.3, 98.7, 115.1, 124.8,
125.4, 125.6, 127.3, 128.8, 128.9, 129.1, 133.0, 135.1, 139.9, 154.3,
166.6. ESI-MS found: [M + H+] 484.3
Step 1: 4′-(-α-d-Mannopyranosyloxy)-N,3′-dimethyl-[1,1′biphenyl]-3-carboxamide[20b] (0.202 g, 0.50 mmol) was stirred in a flask
with triethylamine (0.38 mL, 2.75 mmol) and anhydrous DMF (2 mL) at
under N2 at 0 °C. Trimethylsilyl chloride (0.35 mL,
2.75 mmol) was added dropwise, and the reaction was brought to rt
and stirred an additional 3.5 h. The reaction was then quenched with
ice water, and the reaction mixture was extracted twice with ethyl
acetate. The organic extractions were then combined and concentrated
under reduced pressure to give the crude 4′-[2,3,4,6-tetra-O-trimethylsilyl-α-d-mannopyranosyloxy]-N,3′-dimethyl-[1,1′biphenyl]-3-carboxamide
intermediate, which was then redissolved in acetone/methanol (1/1.5
mL). The reaction was cooled to 0 °C, and acetic acid (0.055
mL, 0.96 mmol) was added. The reaction was allowed to slowly warm
to rt and was monitored for progress by TLC (ethyl acetate–hexanes,
1/1). After 9 h, NaHCO3 (0.16 g, 1.90 mmol) was added and
the solvents were removed in vacuo. Purification by silica gel chromatography
(ethyl acetate–hexanes gradient elution) gave the corresponding
4′-[2,3,4,-tri-O-trimethylsilyl-α-d-mannopyranosyloxy]-N,3′-dimethyl-[1,1′biphenyl]-3-carboxamide
in 61% yield (0.19 g, 0.36 mmol). Analytical data: 1HNMR
(400 MHz, chloroform-d) δ ppm 0.18 (d, J = 4.3 Hz, 18H), 0.23 (s, 9H), 1.90 (dd, J = 7.0, 5.9 Hz, 1H), 2.30 (s, 3H), 3.05 (d, J =
4.7 Hz, 3H), 3.62–3.68 (m, 1H), 3.69–3.77 (m, 2H), 4.01–4.05
(m, 3H), 5.39 (d, J = 1.6 Hz, 1H), 6.20 (br s, 1H),
7.17 (d, J = 8.6 Hz, 1H), 7.36–7.44 (m, 2H),
7.45–7.50 (m, 1H), 7.67 (dt, J = 7.7, 1.8
Hz, 2H), 7.95 (t, J = 1.8 Hz, 1H). ESI-MS found:
[M + H+] 489.4;Step 2: N,N-Dimethylglycine hydrochloride (0.0154 g, 0.11 mmol) was
dissolved into CH2Cl2/diisopropylethylamine
(DIPEA) (5/0.2 mL). Dimethylaminopyridine (DMAP) (0.0024 g, 0.02 mmol)
was added, followed by the 4′-[2,3,4,-tri-O-trimethylsilyl-α-d-mannopyranosyloxy]-N,3′-dimethyl-[1,1′biphenyl]-3-carboxamide (0.062 g,
0.10 mmol) from step 1 and finally N,N-diisopropylcarbodiimide (DIC) (0.020 mL, 0.13 mmol). The reaction
was stirred for 16 h and then solvent was removed under reduced pressure
and the residue was redissolved into acetonitrile (3 mL) and the reaction
was cooled to 0 °C. Trifluoroacetic acid (0.08 mL) was added,
and the reaction was stirred for 2 h at 0 °C. The solvent was
removed in vacuo, and the residue was purified by HPLC [C18, 15 mm
× 150 mm column; eluent, acetonitrile/water (0.1% TFA)]. Pure
fractions were combined and lyophilized to give the title compound
as a white powder (0.015 g, 0.031 mmol) in 31% yield. Analytical data
for 23c: 1HNMR (400 MHz, methanol-d4) δ ppm 2.32 (s, 3H), 2.89 (s, 6H), 2.95
(s, 3H), 3.71–3.85 (m, 2H), 3.94–4.00 (m, 1H), 4.06
(d, J = 5.48 Hz, 2H), 4.11 (t, J = 2.54 Hz, 1H), 4.42 (m, 1H), 4.61 (dd, J = 11.74,
1.56 Hz, 1H), 5.57 (d, J = 1.57 Hz, 1H), 7.23 (d, J = 8.61 Hz, 1H), 7.47–7.53 (m, 3H), 7.73–7.78
(m, 2H), 8.05 (m, 1H). 13CNMR (100 MHz, methanol-d4) δ ppm 16.5, 27.0, 44.4 (×2), 57.9,
66.5, 68.3, 71.9, 72.5, 72.8, 99.9, 115.6, 126.4, 126.5, 126.5, 129.0,
130.1, 130.6, 130.6, 135.3, 136.1, 142.3, 155.5, 166.7, 170.7. ESI-MS
found: [M + H+] 489.4;
Dimethylaminopyridine
(8.9 mg, 0.07 mmol) and (2,3,4,6-tetra-O-benzyl-α-d-mannopyranosyl)-(4-bromo-2-methylphenyl)-methan-1(R)-ol (19R) (1.06 g, 1.47 mmol) were dissolved
in dry pyridine (5 mL) under N2, and the reaction was cooled
to 0 °C. Acetic anhydride (0.21 mL, 2.21 mmol) was added dropwise,
and after 5 min the ice bath was removed. After stirring 1 h at rt,
the reaction was cooled to 0 °C and quenched with MeOH (0.5 mL)
and the pyridine was removed in vacuo. The residue was redissolved
in CH2Cl2, (25 mL) and washed successively with
water (10 mL), 1 M aq HCl (2 × 10 mL), and water (10 mL), dried
over Na2SO4, and concentrated in vacuo. The
residue was purified by silica gel chromatography (ethyl acetate–hexane
gradient elution) to afford compound 26 (1.11 g, 1.46
mmol) in 99% yield. Analytical data for 26: 1HNMR (400 MHz, chloroform-d) δ ppm 1.91 (s,
3H), 2.38 (s, 3H), 3.60–3.69 (m, 1H), 3.69–3.78 (m,
1H), 3.78–3.87 (m, 2H), 3.91–4.04 (m, 2H), 4.34 (dd, J = 6.7, 3.5 Hz, 1H), 4.39–4.46 (m, 1H), 4.50–4.69
(m, 6H), 4.81 (d, J = 11.3 Hz, 1H), 6.15 (d, J = 6.7 Hz, 1H), 7.07–7.13 (m, 1H), 7.23–7.38
(m, 22H). ESI-MS found: [M + Na+] 787.5 (100%), 789.5 (97.3%).
Following a modified
literature protocol for benzyl ether removal,[40] (2,3,4,6-tetra-O-benzyl-α-d-mannopyranosyl)-(4-bromo-2-methylphenyl)-methan-1(R)-ol acetate (26) (1.0 g, 1.30 mmol) was dissolved
in dry CH2Cl2 (50 mL) under N2, and
the reaction was cooled to −78 °C. Boron trichloride (1
M in CH2Cl2; 10.4 mL, 10.4 mmol) was added dropwise,
and the reaction was stirred for 30 min. Once removal of the benzyl
ethers was complete, the excess reagent was quenched by the addition
of MeOH (1 mL). At this time, it was observed (via TLC and LCMS) that
upon quenching, a minor product corresponding to benzylic acetate
cleavage was produced. The reaction mixture containing both the tetraacetate
and pentaacetate intermediates was concentrated under reduced pressure
and purified by silica gel chromatography (methanol–dichloromethane
gradient elution). Both intermediates were collected and combined
and then redissolved in dry pyridine (10 mL) under N2,
and the reaction was cooled to 0 °C. Dimethylaminopyridine (7.0
mg, 0.06 mmol) was added, followed by acetic anhydride (0.77 mL, 8.16
mmol), and the reaction was stirred for 5 min at 0 °C, then brought
to rt. After 1 h, the reaction was cooled to 0 °C and quenched
with MeOH (0.25 mL). The pyridine was removed in vacuo, and the residue
was then redissolved in CH2Cl2, (25 mL) and
washed successively with water (10 mL), 1 M aq HCl (2 × 10 mL),
and water (10 mL), dried over Na2SO4, and concentrated
in vacuo. Purification by silica gel chromatography (ethyl acetate–hexanes
gradient) gave the desired compound 27 (0.51 g, 0.88
mmol) in 68% yield. Analytical data for 27: mp 115–118
°C (diethyl ether–hexanes). 1HNMR (400 MHz,
chloroform-d) δ ppm 1.90 (s, 3H), 1.97 (s,
3H), 2.01 (s, 6H), 2.08 (s, 3H), 2.37 (s, 3H), 3.84–3.90 (m,
1H), 3.94 (dd, J = 12.1, 2.0 Hz, 1H), 4.14–4.23
(m, 2H), 5.10 (t, J = 8.6 Hz, 1H), 5.30 (dd, J = 9.0, 3.1 Hz, 1H), 5.47 (t, J = 2.9
Hz, 1H), 6.12 (d, J = 7.0 Hz, 1H), 7.15–7.20
(m, 1H), 7.23–7.30 (m, 2H). ESI-MS found: [M + Na+] 595.2 (100%), 597.2 (97.3%).
Following the general Suzuki-coupling
procedure, the acetylated mannosyl bromide 27 (0.10 g,
0.174 mmol), commercially available1-hydroxyisoquinoline-7-boronate
ester (0.095 g, 0.35 mmol), cesium carbonate (0.17 g, 0.052 mmol),
and tetrakis(triphenylphosphine)palladium (0.03 g, 0.026 mmol) in
dioxane/water (5 mL/1 mL) were reacted under N2 at 80 °C
for 1.5 h. Upon completion, the reaction was then cooled to rt, and
solvents were evaporated under reduced pressure. The crude reaction
residue was then redissolved into CH2Cl2 (typically
a colorless solid byproduct remains insoluble) and partially purified
by column chromatography to remove metal catalysts and salts to give
the impure acetate-protected intermediate. The mannoside intermediate
was then redissolved in MeOH (3 mL) and cooled to 0 °C. [1 M]
Sodium methoxide in MeOH was added dropwise until a pH of 9–10
was achieved. After 5 min, the ice bath was removed and the reaction
was stirred for 2 h. Upon completion, the reaction was neutralized
with H+ exchange resin (DOWEX 50WX4-100). The resin was
filtered, and the filtrate was concentrated in vacuo. The resulting
residue was purified by HPLC [(C18, 15 mm × 150 mm column; eluent,
acetonitrile/water (0.05% TFA)] to give compound 28R (0.035
g, 0.082 mmol) in 47% yield. Analytical data for 28R: 1HNMR (400 MHz, methanol-d4) δ
ppm 2.52 (s, 3H), 3.64–3.75 (m, 4H), 4.05 (br s, 1H), 4.09–4.15
(m, 1H), 4.26 (br s, 1H), 5.25 (d, J = 6.7 Hz, 1H),
6.70 (d, J = 7.0 Hz, 1H), 7.18 (d, J = 7.0 Hz, 1H), 7.52–7.61 (m, 2H), 7.62–7.67 (m, 1H),
7.72 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 8.6 Hz, 1H), 8.54 (s, 1H). 13CNMR (100 MHz, methanol-d4) δ ppm 19.6, 63.0, 69.0, 69.9, 71.0,
73.2, 78.3, 81.6, 107.7, 125.5, 125.5, 127.3, 128.2, 128.8, 129.0,
130.0, 132.6, 137.8, 138.8, 140.1, 140.7, 141.3, 165.1. ESI-MS found:
[M + H+] 428.4, [M – 18 + H+] 410.3,
[2M + H+] 855.6.
Following the general Suzuki-coupling
procedure, mannosyl bromide 19R (0.220 g, 0.304 mmol),
commercially available1-hydroxyisoquinoline-7-boronate ester (0.165
g, 0.608 mmol), cesium carbonate (0.297 g, 0.912 mmol), and tetrakis(triphenylphosphine)palladium
(0.046 g, 0.053 mmol) in dioxane/water (10 mL/2 mL) were reacted under
N2, at 80 °C for 1.5 h. Upon completion, the reaction
was then cooled to rt and solvents were evaporated under reduced pressure.
The crude reaction residue was then redissolved into CH2Cl2 (typically a colorless solid byproduct remains insoluble)
and partially purified by column chromatography to remove metal catalysts
and salts to give the impure benzyl-protected intermediate. The mannoside
intermediate was then redissolved in MeOH (5 mL), and 10% wt Pd/C
(0.150 g, 0.14 mmol) was added. The reaction was stirred under 1 atm
of H2 for 16 h. Upon completion, the Pd/C was filtered
off and the filtrate was concentrated and dried in vacuo. The resulting
residue was purified by HPLC [(C18, 15 mm × 150 mm column; eluent,
acetonitrile/water (0.05% TFA)] to give compound 29R (0.06
mg, 0.140 mmol) in 46% yield. Analytical data for 29R: 1HNMR (400 MHz, methanol-d4) δ ppm 2.49 (s, 3H), 3.01 (t, J = 6.7 Hz,
2H), 3.52 (t, J = 6.7 Hz, 2H), 3.63–3.74 (m,
4H), 4.05 (br s, 1H), 4.10 (dd, J = 6.7, 2.0 Hz,
1H), 4.25 (t, J = 2.5 Hz, 1H), 5.24 (d, J = 7.0 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.44–7.53
(m, 2H), 7.61 (d, J = 7.8 Hz, 1H), 7.75 (dd, J = 7.8, 2.0 Hz, 1H), 8.18 (d, J = 1.6
Hz, 1H). 13CNMR (100 MHz, methanol-d4) δ ppm 19.6, 28.7, 40.8, 63.0, 69.0, 69.9, 71.0, 73.2,
78.2, 81.6, 125.3, 126.6, 128.9, 129.2, 129.8, 130.2, 131.7, 137.6,
139.7, 140.2, 141.0, 141.1, 168.3. ESI-MS found: [M + H+] 430.4, [M – 18 + H+] 412.4, [2M + H+] 859.6.
The previously
reported compound 7-[4-(α-d-mannopyranosyloxy)-3-methylphenyl]-isoquinolin-1-one[20b] (25) (30 mg, 0.073 mmol) was dissolved
into MeOH (3 mL), and 10% wt Pd/C (30 mg) was added. The reaction
was stirred under 1 atm of H2 for 16 h, after which the
reaction was filtered and the filtrate was concentrated in vacuo.
The resulting residue was purified by HPLC [(C18, 15 mm × 150
mm column; eluent, acetonitrile/water (0.05% TFA)] to give compound 30 (0.30 mg, 0.072 mmol) in 99% yield. Analytical data for 30: 1HNMR (400 MHz, methanol-d4) δ ppm 2.30 (s, 3H), 2.96–3.03 (m, 2H),
3.49–3.55 (m, 2H), 3.57–3.65 (m, 1H), 3.72–3.81
(m, 3H), 3.97 (d, J = 9.4 Hz, 1H), 4.08 (br s, 1H),
5.55 (s, 1H), 7.31 (dd, J = 19.4, 8.0 Hz, 2H), 7.40–7.47
(m, 2H), 7.70 (d, J = 7.8 Hz, 1H), 8.14 (s, 1H). 13CNMR (100 MHz, methanol-d4)
δ ppm 16.6, 28.7, 40.9, 62.7, 68.4, 72.1, 72.7, 75.5, 99.9,
115.9, 126.3, 126.3, 128.9, 129.1, 130.2, 131.5, 135.1, 139.2, 141.0,
155.7, 168.4. ESI-MS found: [M + H+] 416.4, [2M + H+] 831.6.
7-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinolin-1-amine
(31a) and Isoquinolin-1-amine-7 Boronic Acid (31b)
Potassium acetate (264 mg, 2.69 mmol) was activated by
adding it to a round-bottom flask, which was then heated to 250 °C
under vacuum for 2 min and then allowed to cool to rt under vacuum
for an additional 10 min, after which time a nitrogen atmosphere was
continuously maintained. Dry DMSO (2 mL) was added, followed by the
addition of commercially available 7-bromoisoquinolin-1-amine (150
mg, 0.67 mmol) and bis(pinacolato)diboron (256 mg, 1.0 mmol). Pd(dppf)Cl2 (49.2 mg, 0.067 mmol) was added, and the reaction flask was
evacuated under high vacuum and then backfilled with N2 three times. The flask was then placed in an oil bath preheated
to 80 °C and allowed to stir for 2.5 h. The reaction was cooled
to rt, and solvents were evaporated under reduced pressure. The crude
reaction residue was then redissolved into CH2Cl2 and allowed to sit for 5 min to allow for byproducts to precipitate.
The precipitate was filtered off. Evaporation of the CH2Cl2 in vacuo resulted in more byproduct precipitation,
and so the residue was redissolved in CH2Cl2 and the process was repeated until no further precipitation was
observed. The crude brown residue was then diluted with water (1 mL)
and lyophilized to removed trace DMSO, resulting in a brown solid,
which was comosed of a mix of the boronate ester and the boronic acid
(31a/b), as determined by LCMS. This crude
mixture was used without further purification. Analytical data for 31a: ESI-MS found, [M + H+] 271.3. Analytical data
for 31b: ESI-MS found, [M + H+] 189.2
Following the general Suzuki-coupling
procedure, mannosyl
bromide 27 (0.092 g, 0.160 mmol), isoquinolin-1-amine-7-boronate
ester/acid 31a/b (0.086, 0.32 mmol), cesium
carbonate (0.16 g, 0.48 mmol), and tetrakis(triphenylphosphine)palladium
(0.028 g, 0.024 mmol) in dioxane/water (5 mL/1 mL) were reacted under
N2 at 80 °C for 1.5 h. Upon completion, the solvent
was removed under reduced pressure and mixture was filtered through
a silica gel column (ethyl acetate–hexanes, 2/1 isocratic elution)
to remove the metal catalyst and salts. The filtrate was concentrated
then dried in vacuo. The crude compound was then redissolved into
MeOH (3 mL) and cooled to 0 °C. Sodium methoxide [1M] in MeOH
was added dropwise until a pH of 9–10 was achieved. After 5
min, the ice bath was removed and the reaction was stirred for 30
min. Upon completion, the reaction was neutralized with H+ exchange resin (DOWEX 50WX4-100). The resin was filtered, and the
filtrate was concentrated in vacuo. The resulting residue was purified
by HPLC [(C18, 15 mm × 150 mm column; eluent, acetonitrile/water
(0.05% TFA)] to give compound 31R (0.018 g, 0.042 mmol)
in 26% yield. Analytical data for 31R: 1HNMR (400 MHz, methanol-d4) δ ppm
8.71 (s, 1H), 8.26 (d, J = 8.2 Hz, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.62–7.72 (m, 3H), 7.54 (d, J = 7.0 Hz, 1H), 7.24 (d, J = 7.0 Hz, 1H),
5.27 (d, J = 7.0 Hz, 1H), 4.27 (t, J = 2.9 Hz, 1H), 4.13 (dd, J = 6.8, 2.2 Hz, 1H),
4.01–4.07 (m, 1H), 3.63–3.73 (m, 4H), 2.55 (s, 3H). 13CNMR (100 MHz, methanol-d4)
δ ppm 19.7, 63.0, 69.0, 69.9, 70.8, 73.1, 78.4, 81.5, 113.0,
119.6, 123.6, 125.8, 127.8, 129.3, 129.6, 130.3, 135.0, 137.7, 138.1,
138.9, 142.3, 143.0, 156.1. ESI-MS found: [M + H+] 427.4.
Following the general Suzuki-coupling
procedure, mannosyl bromide 27 (0.092 g, 0.160 mmol),
commercially purchased 5-isoquinolinylboronic
acid (0.056 g, 0.32 mmol), cesium carbonate (0.16 g, 0.48 mmol), and
tetrakis(triphenylphosphine)palladium (0.028 g, 0.024 mmol) in dioxane/water
(5 mL/1 mL) were reacted under N2 at 80 °C for 1.5
h. Upon completion, the solvent was removed under reduced pressure
and mixture was filtered through a silica gel column (ethyl acetate–hexanes,
2/1 isocratic elution) to remove the metal catalyst and salts. The
filtrate was concentrated then dried in vacuo. The crude compound
was then redissolved into MeOH (5 mL) and cooled to 0 °C. Sodium
methoxide [1M] in MeOH was added dropwise until a pH of 9–10
was achieved. After 5 min, the ice bath was removed and the reaction
was stirred for 30 min. Upon completion, the reaction was neutralized
with H+ exchange resin (DOWEX 50WX4-100). The resin was
filtered, and the filtrate was concentrated in vacuo. The resulting
residue was purified by HPLC [(C18, 15 mm × 150 mm column; eluent,
acetonitrile/water (0.05% TFA)] to give compound 32R (0.025
g, 0.060 mmol) in 38% yield. Analytical data for 32R: 1HNMR (400 MHz, methanol-d4) δ
ppm 9.75 (s, 1H), 8.48 (dd, J = 15.8, 7.2 Hz, 2H),
8.28 (d, J = 6.7 Hz, 1H), 8.02–8.13 (m, 2H),
7.74 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.34 (s, 1H), 5.30 (d, J = 7.0 Hz,
1H), 4.28 (br s, 1H), 4.14–4.19 (m, 1H), 4.04 (br s, 1H), 3.69
(br s, 4H), 2.54 (s, 3H). 13CNMR (100 MHz, methanol-d4) δ ppm 19.6, 63.1, 69.1, 70.1, 70.8,
73.2, 78.4, 81.4, 124.2, 128.5, 129.1, 129.6, 129.6, 130.7, 131.5,
132.9, 134.2, 137.4, 137.6, 138.1, 141.6, 142.3, 149.6. ESI-MS found:
[M + H+] 412.3.
Step 1: Under nitrogen
atmosphere, the mixture of 4-bromo-2-methylphenyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside[20b] (2.869 g, 5.55 mmol), bis(pinacolato)diboron
(1.690g, 6.66 mmol), potassium acetate (2.177 g, 22.18 mmol), and
(1.1′-bis(diphenylphosohino)ferrocene)dichloropalladium(II)
(Pd(dppf)Cl2) (0.244 g, 0.33 mmol) in DMSO (50 mL) was
heated at 80 °C with stirring for 2 h. The solvent was removed,
and the resulting residue was purified by silica gel chromatography
(ethyl acetate–dichloromethane gradient elution) to give the
intermediate boronate ester, 2-methyl-4-[4,4,5,5-tetramethyl-(1,3,2)dioxaborolan-2-yl]phenyl
2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside
(33a),[25] (2.50 g, 4.43 mmol)
in 79% yield. Analytical data: 1HNMR (400 MHz, chloroform-d) δ ppm 1.34 (s, 12H), 2.05 (d, J = 2.3 Hz, 6H), 2.06 (s, 3H), 2.21 (s, 3H), 2.30 (s, 3H), 4.03–4.09
(m, 2H), 4.27–4.33 (m, 1H), 5.39 (t, J = 12.0
Hz, 1H), 5.48 (dd, J = 3.3, 1.8 Hz, 1H), 5.54–5.60
(m, 2H), 7.09 (d, J = 8.2 Hz, 1H), 7.55–7.67
(m, 2H). ESI-MS found: [M + Na+] 587.4.Step 2: Following
the general Suzuki-coupling procedure, 33a (0.150, 0.27
mmol) from step 1, 7-bromoisoquinolin-1-amine (0.060, 0.27 mmol),
cesium carbonate (0.26 g, 0.80 mmol), and tetrakis(triphenylphosphine)palladium
(0.031 g, 0.027 mmol) in dioxane/water (5 mL/1 mL) were reacted under
N2, at 80 °C for 1.5 h. Upon completion, the solvent
was removed under reduced pressure, and mixture was filtered through
a silica gel column (ethyl acetate–hexanes, 2/1 isocratic elution)
to remove the metal catalyst and salts. The filtrate was concentrated
then dried in vacuo. The crude compound was then redissolved into
MeOH (3 mL) and cooled to 0 °C. Sodium methoxide [1M] in MeOH
was added dropwise until a pH of 9–10 was achieved. After 5
min, the ice bath was removed and the reaction was stirred for 2 h.
Upon completion, the reaction was neutralized with H+ exchange
resin (DOWEX 50WX4-100). The resin was filtered, and the filtrate
was concentrated in vacuo. The resulting residue was purified by HPLC
[(C18, 15 mm × 150 mm column; eluent, acetonitrile/water (0.05%
TFA)] to give compound 33 (0.040 g, 0.097 mmol) in 36%
yield. Analytical data for 33: 1HNMR (400
MHz, methanol-d4) δ ppm 2.34 (s,
3H), 3.57–3.64 (m, 1H), 3.72–3.83 (m, 3H), 3.98 (dd, J = 9.4, 3.1 Hz, 1H), 4.10 (br s, 1H), 5.60 (s, 1H), 7.21
(d, J = 6.7 Hz, 1H), 7.36 (d, J =
8.2 Hz, 1H), 7.52 (d, J = 7.0 Hz, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.64 (s, 1H), 7.93 (d, J = 8.6 Hz, 1H), 8.21 (d, J = 8.6 Hz, 1H), 8.64 (s,
1H). 13CNMR (100 MHz, methanol-d4) δ ppm 16.6, 62.7, 68.4, 72.1, 72.7, 75.6, 99.8, 113.0,
116.0, 119.6, 123.0, 127.0, 127.6, 129.2, 129.5, 130.6, 133.6, 134.8,
137.3, 142.9, 156.0, 156.4. ESI-MS found: [M + H+] 413.4.
Hemagglutination Assay
The hemagglutination inhibition
(HAI) assay was performed with n class="Species">UTI89 bacteria and guinea pig red blood
cells, as previously described.[25]
Biofilm
Assay
The biofilm assay was performed with
UTI89 bacteria as previously described.[25]
Differential Scanning Fluorimetry (DSF)
The DSF assay
was performed with FimHL (10 μM) in the absence or
presence of 100 μM n class="Chemical">mannoside, as previously described.[25]
Computational Molecular Modeling
Computer-generated
docking was performed with AutoDock Vina. Ligand coordinates were
generated in eLBOW within the Phenix program suite. Simple optimization
of mannosides 21R and 21S, as described
by its SMILES sequence, was manually inspected and modified to ensure
the correct chair conformation of the mannose pucker. These pdb coordinates
were then converted to pdbqt format using Babel. The X-ray coordinates
of FimH from the 23 co-crystal structure was converted
to its topology file using AutoDock Tools. The grid box was centered
at the mannose binding pocket of FimH, and its dimensions (40 ×
40 × 40 Å3) were chosen to accommodate multiple
potential binding modes at or near the binding pocket. The exhaustiveness
of the search was set to a value of 20. The top binding modes and
scores within this grid space were generated by AutoDock Vina and
visualized in PyMOL.
Animal Infections
The bacterial
inoculum and infection
of mice was performed as previously described.[20a] Briefly, the bacterial strain UTI89 with a kanamycin antibiotic
cassette inserted in the chromosome (UTI89KAN) was grown
under type 1-inducing conditions, 2 × 24 static growth in LB
at 37 °C. Bacteria were harvested, washed, and resuspended in
sterile PBS to the appropriate concentration. Six to eight week C3H/HeN
female mice were purchased from Envigo (formerly Harlan Laboratories).
Mice were anesthetized by inhalation of isoflurane and infected by
transurethral inoculation of 50 μL of bacterial suspension at
the appropriate density. At the designated time point, mice were killed
by cervical dislocation under anesthesia and bladders harvested and
processed by mechanical disruption. Processed bladders were serially
diluted and plated on LB with 50 μg/mL of kanamycin to enumerate
bacterial burden.
Treatment of Chronic Urinary Tract Infection
in Mice
Mice were infected with 1–2 × 108 UTI89KAN, and the infection was allowed to continue
for 2 weeks.
Twelve days post inoculation, the urine from mice was collected and
titered to identify chronically infected mice (urine titers ≥105). At 2 weeks post inoculation, chronically infected mice
were treated orally with 100 μL of mannoside resuspended in
10% cyclodextrin to the appropriate concentration to generate the
designated dosage. Bladders were harvested and titered to determine
the bacterial burden at the designated time points following treatment.
Prophylactic Treatment of Acute Urinary Tract Infection in Mice
Prophylactic treatment of mice involved oral delivery of mannoside
at 25 mg/kg 30 min prior to infection with UTI89KAN. Mice
were infected with 1–2 × 107 UTI89KAN, and bladders were harvested and titered to determine the bacterial
burden 6 h following inoculation.
Mouse Pharmacokinetic (PK)
Studies
For dosing in mice,
100 μL of a solution of mannoside in 10% cyclodextrin [10 mg/mL
(50 mg/kg)] was inoculated with a gavage needle into the mouse stomach.
Urine was collected at 1, 3, 6, and 8 h after dosing and spiked with
an internal standard. Analysis for test article levels by LC–MS/MS
was performed using a C18 reversed phase column and an AB Sciex API-4000
QTrap instrument (a gradient of acetonitrile and water in 0.1% formic
acid), with ion spray detection (+) ESI. Selected reaction monitoring
(SRM) mode quantification was performed for the following MS/MS transitions
[precursor mass/charge ratio (m/z)/product m/z]: compound 21R, 418.20/238.20 amu; compound 23, 404.20/242.20
amu (Levels of 23 were quantitated after dosing of prodrug, 23a); compound 24, 461.20/299.20 amu.
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Authors: Lijuan Pang; Simon Kleeb; Katrin Lemme; Said Rabbani; Meike Scharenberg; Adam Zalewski; Florentina Schädler; Oliver Schwardt; Beat Ernst Journal: ChemMedChem Date: 2012-05-29 Impact factor: 3.466
Authors: Julie Bouckaert; Jenny Berglund; Mark Schembri; Erwin De Genst; Lieve Cools; Manfred Wuhrer; Chia-Suei Hung; Jerome Pinkner; Rikard Slättegård; Anton Zavialov; Devapriya Choudhury; Solomon Langermann; Scott J Hultgren; Lode Wyns; Per Klemm; Stefan Oscarson; Stefan D Knight; Henri De Greve Journal: Mol Microbiol Date: 2005-01 Impact factor: 3.501
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Authors: Hyun Koo; Raymond N Allan; Robert P Howlin; Paul Stoodley; Luanne Hall-Stoodley Journal: Nat Rev Microbiol Date: 2017-09-25 Impact factor: 60.633
Authors: Amarendar Reddy Maddirala; Roger Klein; Jerome S Pinkner; Vasilios Kalas; Scott J Hultgren; James W Janetka Journal: J Med Chem Date: 2019-01-02 Impact factor: 7.446
Authors: Bernadette Jones-Freeman; Michelle Chonwerawong; Vanessa R Marcelino; Aniruddh V Deshpande; Samuel C Forster; Malcolm R Starkey Journal: Mucosal Immunol Date: 2021-02-04 Impact factor: 7.313
Authors: Xiao Li; Kaichen Zhou; Jingyu Wang; Jiahe Guo; Yang Cao; Jie Ren; Tao Guan; Wenchao Sheng; Mingyao Zhang; Zhi Yao; Quan Wang Journal: Front Med (Lausanne) Date: 2021-06-18
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Figure 1
Scheme 1
Scheme 2
Scheme 3
Figure 2
Figure 3
Figure 6
Scheme 4
Figure 4
Figure 5
Figure 7
Figure 8
Figure 9
Figure 10