Antivirulence Isoquinolone Mannosides: Optimization of the Biaryl Aglycone for FimH Lectin Binding Affinity and Efficacy in the Treatment of Chronic UTI

Article abstract of DOI:10.1002/cmdc.201600006

Uropathogenic E. coli (UPEC) employ the mannose-binding adhesin FimH to colonize the bladder epithelium during urinary tract infection (UTI). Previously reported FimH antagonists exhibit good potency and efficacy, but low bioavailability and a short half-life in vivo. In a rational design strategy, we obtained an X-ray structure of lead mannosides and then designed mannosides with improved drug-like properties. We show that cyclizing the carboxamide onto the biphenyl B-ring aglycone of biphenyl mannosides into a fused heterocyclic ring, generates new biaryl mannosides such as isoquinolone 22 (2-methyl-4-(1-oxo-1,2-dihydroisoquinolin-7-yl)phenyl α-d-mannopyranoside) with enhanced potency and in vivo efficacy resulting from increased oral bioavailability. N-Substitution of the isoquinolone aglycone with various functionalities produced a new potent subseries of FimH antagonists. All analogues of the subseries have higher FimH binding affinity than unsubstituted lead 22, as determined by thermal shift differential scanning fluorimetry assay. Mannosides with pyridyl substitution on the isoquinolone group inhibit bacteria-mediated hemagglutination and prevent biofilm formation by UPEC with single-digit nanomolar potency, which is unprecedented for any FimH antagonists or any other antivirulence compounds reported to date.

Full text of DOI:10.1002/cmdc.201600006

DOI: 10.1002/cmdc.201600006
Communications
Antivirulence Isoquinolone Mannosides: Optimization of
the Biaryl Aglycone for FimH Lectin Binding Affinity and
Efficacy in the Treatment of Chronic UTI
Cassie Jarvis,[a] Zhenfu Han,[a] Vasilios Kalas,[b] Roger Klein,[b] Jerome S. Pinkner,[b]
Bradley Ford,[b] Jana Binkley,[b] Corinne K. Cusumano,[b] Zachary Cusumano,[d] Laurel Mydock-
McGrane,[d] Scott J. Hultgren,*[b, c] and James W. Janetka*[a, c]
Uropathogenic E. coli (UPEC) employ the mannose-binding ad-
hesin FimH to colonize the bladder epithelium during urinary
tract infection (UTI). Previously reported FimH antagonists ex-
hibit good potency and efficacy, but low bioavailability and
a short half-life in vivo. In a rational design strategy, we ob-
tained an X-ray structure of lead mannosides and then de-
signed mannosides with improved drug-like properties. We
show that cyclizing the carboxamide onto the biphenyl B-ring
aglycone of biphenyl mannosides into a fused heterocyclic
ring, generates new biaryl mannosides such as isoquinolone
22 (2-methyl-4-(1-oxo-1,2-dihydroisoquinolin-7-yl)phenyl a-d-
mannopyranoside) with enhanced potency and in vivo efficacy
resulting from increased oral bioavailability. N-Substitution of
the isoquinolone aglycone with various functionalities pro-
duced a new potent subseries of FimH antagonists. All ana-
logues of the subseries have higher FimH binding affinity than
unsubstituted lead 22, as determined by thermal shift differen-
tial scanning fluorimetry assay. Mannosides with pyridyl substi-
tution on the isoquinolone group inhibit bacteria-mediated he-
magglutination and prevent biofilm formation by UPEC with
single-digit nanomolar potency, which is unprecedented for
any FimH antagonists or any other antivirulence compounds
reported to date.
ic and healthcare burdens worldwide. UTIs predominantly af-
flict women, who have a lifetime risk of 50% for developing an
acute infection and 20% for experiencing multiple, recurrent
infections.[1] Antibiotic resistance[2] among uropathogenic
E. coli (UPEC), which account for the overwhelming majority of
UTIs, is a rising problem. In addition, the ability of UPEC to es-
tablish quiescent reservoirs may lead to the observed recalci-
trance to antibiotic therapy, as chronic bladder reservoirs may
serve as seeds for recurrent UTI and contribute to the trou-
bling nature of this disease.[3] Furthermore, a history of UTI sig-
nificantly predisposes one to recurrent UTI.[4] Thus, antibiotic-
sparing approaches, such as antivirulence strategies[5] that pre-
vent UPEC colonization, represent viable therapeutics to ad-
dress this emerging threat. UPEC express long fibrillar appen-
dages at their cell surface termed type 1 pili,[6] which are
tipped with FimH,[7] to mediate attachment to superficial facet
cells of the bladder epithelium. FimH is a two-domain adhe-
sin[8] comprised of an N-terminal lectin domain, which medi-
ates specific recognition of mannosylated receptors on the
bladder epithelium, and a C-terminal pilin domain, which an-
chors FimH to the tip of the pilus. Upon binding, FimH triggers
host cell invasion and intracellular colonization by UPEC. A pro-
portion of the UPEC population evades expulsion by escaping
into the cytoplasm and replicating to form intracellular bacteri-
al communities (IBCs), from which infection may propagate to
neighboring cells. Formation of IBCs is part of a mechanism
that allows UPEC to gain a foothold in the bladder and build
up in numbers. Targeting FimH with small-molecule[9] and mul-
tivalent carbohydrate mimetics[10] to interfere with bacterial ad-
hesion, invasion, and IBC and biofilm formation represents
a promising therapeutic strategy for combatting recurrent and
antibiotic-resistant UTIs.
Urinary tract infections (UTIs) represent the second most
common infectious disease, imposing enormous socioeconom-
[a] C. Jarvis, Dr. Z. Han, Prof. J. W. Janetka
Washington University School of Medicine
Department of Biochemistry and Molecular Biophysics
660 S. Euclid Ave., St. Louis, MO 63110 (USA)
E-mail: janetka@biochem.wustl.edu
In preliminary studies we identified biphenyl mannosides 2
and 3 (Figure 1) as promising antagonists of FimH by X-ray-
structure-based design.[11] Compound 3 exhibits good inhibi-
tion of FimH function using UPEC strain UTI89 as determined
by a hemagglutination assay (HAI=0.5 mm) and biofilm assay
(IC50 =1.35 mm). This study revealed that meta substitution on
the biphenyl B-ring with a hydrogen bond acceptor, such as
the methyl amide of 3, was optimal. Further structure–activity
relationship (SAR) studies from us and others[12] produced man-
nosides such as 5 and 6, indicating that addition of a small
substituent at the ortho position of the biphenyl A-ring leads
to marked enhancements in potency. For example, ortho-
methyl and chloro groups as with 5 and 6 were found to in-
[b] V. Kalas, R. Klein, J. S. Pinkner, Dr. B. Ford, J. Binkley, Dr. C. K. Cusumano,
Prof. S. J. Hultgren
Washington University School of Medicine
Department of Molecular Microbiology
660 S. Euclid Ave., St. Louis, MO 63110 (USA)
E-mail: hultgren@wusm.wustl.edu
[c] Prof. S. J. Hultgren, Prof. J. W. Janetka
Washington University School of Medicine
Center for Women’s Infectious Disease Research (cWIDR)
660 S. Euclid Ave., St. Louis, MO 63110 (USA)
[d] Dr. Z. Cusumano, Dr. L. Mydock-McGrane
Fimbrion Therapeutics Inc.
4041 Forest Park Ave., St. Louis, MO 63108 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201600006.
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Figure 1. Progression of biphenyl a-d-mannoside FimH inhibitors.
crease HAI titers (60 and 125 nm) relative to 3 (500 nm). The
addition of another B-ring substituent also improved activity
(compounds 7 and 8). This increase in potency was attributa-
ble to an increase in relative binding affinity for the FimH
lectin domain as measured by differential scanning fluorimetry
(DSF). In vivo investigation of mannoside 5 demonstrated that
the compound is orally bioavailable and efficacious against
acute,[13] chronic,[14] and antibiotic-resistant[15] UTI in mice. How-
ever, despite this efficacy, the oral bioavailability of 5 is low,
and compound exposure in the urine was significantly de-
creased by eight hours. Herein we report on newly optimized
mannosides with significantly enhanced FimH potency, oral
bioavailability, and efficacy in treating chronic UTIs.
the C2, C3, C4, and C6 hydroxy groups of mannose and the
main chain of F1 and side chains of N46, D54, Q133, N135, and
D140. The O-linked aglycone projects toward the lid of the
binding pocket and positions functional groups for favorable
interactions. We discovered that the ortho-methyl substituent
of the A-ring promotes hydrophobic interactions, as it packs
snugly against the surface formed by I52, Y137, and N138. The
B-ring phenyl forms favorable p–p stacking interactions with
Y48 and the amide carbonyl of 7 indirectly interacts with the
phenol of Y48 through a water-mediated hydrogen bond net-
work, as evidenced by the ideal bent geometry and proximal
distances (3–3.2 Š) of hydrogen bond donor–acceptor pairs. In
addition, the secondary nitrogen atom of the two amides form
electrostatic and potential H-bonding interactions with the
neighboring R98 and Y137 residues, but these groups lie 4.1
and 4.6 Š away, respectively. However, the electron density of
the second amide near Y137 reveals two different orientations
of the amide carbonyl in which the carbonyl could be acting
as an H-bond acceptor to the phenol. Comparison of manno-
side 7 with 2 (Figure 2B) by structural overlay highlights a un-
ambiguous difference in the orientation of the biphenyl group.
Based on the structure of 7 presented here and a related ana-
logue,[12a] the improvement in activity of mannosides with
ortho-substituted groups on the A-ring (5–8) relative to unsub-
stituted 2 and 3 can be explained by: 1) increased hydropho-
bic contact of the ortho substituent encompassed by the mo-
lecular surface formed by I52, Y137, and N138; 2) improved p–
p stacking with Y48; and 3) decreased entropic cost of binding
due to restriction in ring rotation. Altogether, this structure
highlights the essential interactions that ortho-substituted
mannosides form with the rim of the mannose-binding pocket,
or the “tyrosine gate” of Y48 and Y137, responsible for in-
creased potency. Despite the good activity of 5 and 7 from in
vitro assays, in vivo pharmacokinetic (PK) studies show that
both mannosides have low oral bioavailability and half-life. We
hypothesize that they are metabolically unstable from hydroly-
sis of the methyl amide and/or hydrolysis of the O-glycosidic
To better understand the improved interactions of 5 and 7
with FimH, a high-resolution (1.67 Š) X-ray crystal structure of
7 (PDB ID: 5F2F) bound to the FimH lectin domain was ob-
tained (Figure 2A). As noted in prior co-crystal structures,[11a,16]
the mannose ring is bound tightly in a deep hydrophilic
pocket by an intricate network of hydrogen bonds between
Figure 2. Structural basis of FimH–mannoside interactions. A) Crystal struc-
ture of compound 7 bound to FimH lectin domain (PDB ID: 5F2F). Unambig-
uous electron density from the FoÀFc map (black mesh) and contoured at
1s reveals compound 7 (green sticks) bound in the mannose binding
pocket of FimH (blue ribbon and surface). B) Comparison of mannoside
binding poses. Overlay of compound 2 (yellow sticks; PDB ID: 3MCY) on 7
bound to the FimH lectin domain.
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bond via the action of a-mannosidases. In an effort to improve
the metabolic stability of these compounds, we examined the
effects of replacing the amide with non-hydrolyzable bioisos-
teres.
hydrolysis. Indeed, we found that 16 shows a fivefold improve-
ment in HA potency (HAI=0.1 mm) over 3. This result suggests
that the isoquinolone amide carbonyl is oriented in a way that
mimics the more energetically favorable binding conformation
to FimH retaining the interaction with R98. This rationale can
also be used to explain the increased activity of 7 over 5. The
addition of the second meta-substituted methyl amide in 7
proposes that the conformation where the amide is in proximi-
ty to the salt bridge (and Y48) is statistically higher than 5.
Based on these exciting results, we synthesized new manno-
sides 22 and 17b, the matched pairs of 16 and 17, by intro-
ducing an ortho-methyl group on the A-ring of the biaryl agly-
cone to increase potency. Mannoside 22 was synthesized using
two different synthetic routes, as shown in Scheme 1A and 1B.
In route 1A, aryl boronate mannoside 18[11b] was coupled to 7-
bromo-1(2H)-isoquinolone via a palladium-mediated cross-cou-
pling to give protected tetraacetate 21. In route 1B, the bro-
moisoquinolone was first converted into the boronate ester
19. Suzuki cross-coupling of 19 to bromophenyl mannoside
20[11b] gave 21. In both routes, mannose acetate deprotection
was performed with sodium methoxide to give 22 in good
yields. Isoquinoline 17b was synthesized in a similar fashion
from 18 and 5-bromoisoquinoline. When tested in the HA
assay, both isoquinolone 22 and isoquinoline 17b had a signifi-
cant fourfold increase in activity (Table 1) relative to 16 and 17,
respectively.
Our structure of 7 shows an electrostatic or hydrogen bond-
ing interaction of the meta-substituted amide carbonyl with
R98, similar to the ester carbonyl of 2 (Figure 2B; PDB ID:
3MCY).[11a] The importance of this interaction can be demon-
strated by 1, in which the ester is located in the para position,
resulting in a marked decrease in potency (Figure 1). To deter-
mine whether we could mimic the amide of 3 and retain this
interaction with FimH, we designed analogues with non-hydro-
lyzable isoelectronic and isosteric groups (Figure 3). In our ear-
lier studies, we evaluated a small set of mannosides containing
simple amide isosteres in the B-ring meta position, such as
nitro 9, nitrile 10, and sulfonamide 14. All isosteres retained
similar potency, while analogues incapable of the same hydro-
gen bonding—phenol 11, fluoride 12, and methyl alcohol
13—all lost potency relative to that of 3. This data provided
additional evidence that the salt bridge interaction with R98
provides the basis for increased FimH binding and inhibition of
adhesion by the HA assay. In another set of compounds, we
used nitrogen-containing heterocycles to mimic the spatial ori-
entation and electronics of the amide carbonyl of compound
7. Isoquinoline 15 displays similar activity as observed with 3,
while the isomeric isoquinoline analogue 17 had slightly im-
proved activity, with HAI=0.25 mm. This discrepancy in activity
is most likely explained by the different orientations of the ni-
trogen, predicted to interact with R98 which mimics the car-
bonyl oxygen in 3. Interestingly, the analogue with improved
activity, 17, directs the carbonyl mimic in an ‘endo-like’ confor-
mation pointing in the direction of mannose, in contrast to 15,
which assumes an ‘exo-like’ orientation pointing away from the
mannose ring. To maintain this interaction, we proposed that
cyclizing or ‘fusing’ the amide of 3 onto the B-ring phenyl
group as with isoquinolone 16 would maintain this hydrogen
bonding interaction and also aid in protecting the amide from
Given the excellent potency displayed by 22, we wanted to
more closely examine its biological behavior in both in vitro
and in vivo assays. We first evaluated 5, 7, and 22 in a biofilm
prevention assay followed by a thermal shift assay by differen-
tial scanning fluorimetry (DSF) to quantitatively calculate rela-
tive binding strength to the FimH lectin domain (Table 1). Early
mannosides 2 and 3 prevent biofilm formation with IC50 values
of 0.94 and 1.35 mm, respectively, whereas 5 and 7 display
~10-fold higher potency (Table 1). We now report that 22 is
equipotent to 5 and 7 in the biofilm and melting point shift
assays. Upon the identification of 22, we next turned our at-
Figure 3. Potency and SAR of meta-position amide bioisostere analogues of biphenyl mannoside 3.
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Scheme 1. Synthesis of A-ring o-methyl B-ring isoquinolone mannoside 22. Reagents and conditions: a) bis(pinacolato)diboron, cat. Pd(dppf)Cl2, KOAc, DMSO;
b) cat. Pd(PPh3)4, Cs2CO3, dioxane/H2O, 808C; c) NaOMe/MeOH.
Table 1. In vitro biological and PK profile of initial and optimized mannosides.
[a]
[a]
Compd
HAI [mm]
Biofilm IC50 [mm]
DSF Tm [8C]
cLogD
Rat CL [mLminÀ1 kgÀ1
]
Rat Vdss [LkgÀ1
]
Rat F [%][a]
Rat t1/2 [h][a]
2
3
5
7
17b
22
1.0
0.50
0.062
0.016
0.062
0.031
0.94
1.35
0.16
0.12
ND
73.4
73.1
75.5
75.8
ND
1.17
0.28
0.93
1.35
1.21
1.4
ND
ND
98
ND
ND
408
ND
ND
1.3
ND
ND
6.7
ND
ND
1.4
ND
ND
7.0
ND
ND
2.8
ND
ND
<3
0.13
75.5
[a] Calculated from a 10 mgkgÀ1 oral (p.o.) dose, 10% cyclodextrin formulation; ND: not determined.
tention to assessing the drug-like properties and pharmacoki-
netics of our compounds in order to prioritize them for in vivo
studies. cLogD is often used as a good indicator of solubility.
While mannosides are sugar-based, and are therefore predict-
ed to have relatively good water solubility, our compounds
have hydrophobic biaryl aglycones which encompass most of
the molecular weight of the mannoside, causing the solubility
to be variable. We calculated the LogD values of the five ana-
logues in Table 1 and found that the change from a methyl
ester 2 to amide 3 leads to a large drop in LogD from 1.17 to
0.28. Interestingly, introduction of the ortho-methyl group in
amide 5 results in a large increase in LogD (0.93), almost reca-
pitulating the solubility of ester 3. More surprising is that the
addition of the second methyl amide in 7 further increases the
LogD to 1.35, which is identical to that of cyclic amide/quino-
lone 22 (LogD 1.4). Therefore, we conclude that LogD is not
well correlated to the improved activity of 7 and 22. Further-
more, evaluation in plasma and liver microsomes (Supporting
Information) suggests that all compounds are metabolically
stable in both mouse and human species.
eral time points (1, 3, and 6 h), and mannoside concentration
was determined by mass spectrometry. As illustrated in Fig-
ure 4A, the initial urine concentration of 7 and 22 were elevat-
ed relative to 5 at 1 h post-doing, but level off and then dis-
play lower clearance for the remainder of the time course. A
follow-up i.v./p.o. crossover study of 5 and 22 in rat (Table 1;
Supporting Information), revealed that 22 (p.o. dose at
10 mgkgÀ1) has higher bioavailability (F=7.0%) than 5, but
also higher clearance (CL=408) and a larger volume of distri-
bution (Vdss =6.7 LkgÀ1). Finally, we tested the lead mannosides
in a murine model of chronic UTI, which was described previ-
ously.[14] Briefly, introduction of bacteria into the urinary tract
results in an acute infection, which in a subpopulation of mice
can progress to a chronic infection as a result of an over-exu-
berant host response. Chronic infection is defined by severe
pyuria and persistent bacteriuria. Elimination of bacteria in
mice with chronic UTI requires antibiotic treatment. Shown in
Figure 4B, all three compounds showed excellent efficacy rela-
tive to the control group, significantly dropping the bacterial
burden (CFUs per bladder) of chronically infected mice by sev-
eral log units. Excitingly, lead 22 displayed the best efficacy
with a substantial 10-fold additional drop in CFUs per bladder
relative to 5, 7, and isoquinoline analogue 17b (Supporting In-
formation). The enhanced efficacy over 5 and 7 can be ration-
alized from the increased bioavailability of 22 and possibly the
high volume of distribution (Vdss), indicating substantial tissue
Subsequently, we performed an in vivo pharmacokinetic (PK)
study of the three lead mannosides in mice. In past studies, we
have correlated compound levels in the urine at 6 h post-
dosing as a good predictor of pharmacodynamic (PD) efficacy.
Therefore, compounds were dosed via oral gavage (formulated
in 10% cyclodextrin) at 50 mgkgÀ1. Urine was collected at sev-
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tional restrictions of the isoquinolone ring and the increased
basicity of the ester- and amine-bearing substituents, in an at-
tempt to improve both affinity and PK. To verify that the rigidi-
ty afforded by the isoquinolone moiety in compounds 23–25
was responsible for the increase in binding observed, we syn-
thesized compounds 28a–b to maintain similar interactions,
while affording greater flexibility in the FimH binding pocket.
We used a similar route involving a Suzuki cross-coupling of 4-
methoxycarbonylphenylboronic acid to bromophenyl manno-
side 20, followed by saponification of compound 26 to 27,
and subsequent HATU-mediated amine coupling (Scheme 3).
Subsequent to synthesis, the focused set of substituted quino-
lones were tested in the HA, biofilm, and DSF assays described
above. Shown in Figure 5, almost all analogues of 22 had im-
proved activity in the HA and biofilm assays at a level correlat-
ing well with the relative increased binding strength by DSF.
The pyridyl acetamides 25b and 25c were identified as the
most potent analogues, with nearly identical activity (HAI=
1 nm, biofilm IC50 =20 nm, and DSF Tm =76.38C). While the two
acyclic amide derivatives, 28a and 28b, display potency (31
and 62 nm) in the HA assay, their matched quinolone pairs,
23b and 23c, have better HAI titers of 4 and 8 nm. As elucidat-
ed by DSF melting point data, this notable eightfold enhance-
ment in activity is derived largely from increased FimH binding
as a result of the conformational restriction of the amide in
quinolones 23b and 23c. Notably, the intermediate acid 24,
used to synthesize 25b and 25c, showed a lower DSF melting
temperature (Tm =74.88C) and biofilm inhibition with an IC50
value of 120 nm, whereas the HA inhibition assay showed an
improvement, with HAI=16 nm. Interstingly, when evaluated
for oral PK in rats (Supporting Information), we discovered that
the two analogues 23b and 25d showed only minor evidence
of bioavailability and when tested in the animal model of
chronic UTI, analogues 23a and 23c showed no significant
effect in decreasing the bacterial burden in mice when dosed
orally (50 mgkgÀ1). On the other hand, 28b displayed similar
activity to that of 5 and 7 in the animal model (Supporting In-
formation). The basis for the lack of oral bioavailability or effi-
cacy in mice or rats remains unclear. All substituents of 22
have a basic, high pKa and create a tertiary amide on the man-
noside, so it is possible that either change prompts alternate
metabolism in the stomach, gut, and/or plasma, resulting in
the lack of oral exposure. Nonetheless, this new class of quino-
lone mannosides has the highest FimH affinity and potency of
any antagonists reported to date (e.g., 25b HAI=1 nm). They
are also capable of preventing bacterial biofilm formation of
UPEC at activity levels well below that of any previously report-
ed small molecule (e.g., 25c biofilm IC50 =18 nm).
Figure 4. Mannoside PK/PD. A) In vivo mouse PK of 5, 7, and 22 following
oral delivery at 50 mgkgÀ1. Urine mannoside concentrations over time indi-
cate equivalent concentrations of 7 and 22. Data are the mean ÆSEM from
at least three animals. B) In vivo mouse PD model of UTI89 chronically infect-
ed mice dosed with 5, 7, and 22 at 50 mgkgÀ1 p.o. (10% cyclodextrin formu-
lation). Compound 22 shows significantly 10-fold enhanced efficacy relative
to 5 and 7 at 6 h post-dosing. Differences were tested for significance within
the designated treatments using Mann–Whitney U-test (****p<0.0001, *p<
0.05).
exposure, which is also supported by its rapid clearance (CL).
The new lead mannoside 22 has elevated potency, better
drug-like physical properties, bioavailability, and improved
in vivo efficacy. To further improve the drug oral bioavailability
and half-life, we synthesized a series of N-substituted manno-
sides based on 22, designed to explore structure–property re-
lationships (SPR), specifically the effects of pKa on half-life,
clearance, tissue exposure, and oral bioavailability.
To access this library based on 22, we used a synthetically
feasible route for regioselectively substituting the isoquinolone
nitrogen versus the oxygen. Thus, substitution reactions with
isoquinolone 21 were performed with various alkyl bromides
as shown in Scheme 2. Subsequent acetyl deprotection with
NaOMe/MeOH was completed to obtain target mannosides
23a–d in respectable yields. Acetamide ester 23d was used as
a key intermediate to access further diversity in substitution of
the isoquinolone through installation of a terminal amide. To
this end, saponification of 23d to carboxylic acid 24, followed
by standard peptide coupling to various amines with HATU,
produced isoquinolone mannosides 25a–d in excellent yields.
These compounds were designed to combine the conforma-
The development of small-molecule antagonists of the
type 1 pilus adhesin FimH for the clinical treatment and pre-
vention of UTI holds promise as an antibiotic-sparing strategy.
Our current pursuit in identifying a compound for clinical de-
velopment has involved a number of lead optimization studies
on improving interactions with FimH, as well as understanding
structure–property relationships from these modifications
in vivo. In this study we pursued a structure-based approach in
which we obtained a high-resolution X-ray structure of 7
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Scheme 2. Synthesis of isoquinolone mannoside analogues with varied pKa substituents. Reagents and conditions: a) Br-R, NaH, DMF, 08C; b) NaOMe/MeOH
(0.02m), RT; c) NaOH (0.2m), MeOH, RT; d) HATU and DMF added at 08C, DIEA and 18/28-amine-R’ added, brought to RT; e) NaOMe/MeOH (0.02m), RT.
isoquinolone-based mannosides ever reported. In
summary, we have discovered new lead biaryl man-
noside 22, which is a promising preclinical candidate
drug for the treatment and prevention of urinary
tract infections.
Abbreviations: UTI, urinary tract infection; UPEC, uropa-
thogenic E. coli; SAR, structure–activity relationship; HA,
hemagglutination; HAI, HA inhibition titer; IBCs, intracel-
lular bacterial communities; PD, pharmacodynamics; PK,
pharmacokinetics.
All animal studies using mice were approved by the
Animal Studies Committee of Washington University
(Animal Protocol Number 20150226).
Acknowledgements
Scheme 3. Synthesis of acyclic mannoside amides as matched pairs to pyridyl isoquino-
lones 23b–c and 25b–c. Reagents and conditions: a) cat. Pd(PPh3)4, Cs2CO3, dioxane/H2O,
808C; b) NaOMe/MeOH (0.02m), RT; c) NaOH (0.2m), MeOH, RT; d) HATU and DMF added
at 08C, DIEA and 18-amine added and brought to RT.
We thank Jan Crowley and Jeff Henderson (NIH MS fa-
cility, cWIDR) for generating bioanalytical data from
the PK studies, Rick Stegeman (X-ray facility, Biochem-
istry Department) for help with X-ray crystallography,
bound to FimH for identifying key interactions aimed at im-
and Scott Wildman (Biochemistry Department) for help with mo-
proved potency. Mannosides containing simple isosteric or iso-
electronic replacements of the biaryl B-ring amide[11–12] have
lecular modeling and docking of FimH ligands. This project was
funded in part by US National Institutes of Health (NIH) grants
been previously studied and have similar potency and proper-
1RC1K086378 (Washington University) from the National Institute
ties to those having the amide. In contrast, the conformational-
of Diabetes and Digestive and Kidney Diseases (NIDDK), and
ly restricted cyclic amide isoquinolone and isoquinoline iso-
1R43AI106112-01A1 (Fimbrion Therapeutics, Inc.) from the Na-
steres reported herein produce improved mannosides with sig-
tional Institute of Allergy and Infectious Diseases (NIAID).
nificantly elevated potency, better drug-like properties, and
in vivo efficacy. Consequently, quinolone mannoside 22 has
Keywords: biofilms
infections · uropathogenic E. coli
· FimH · mannosides · urinary tract
substantially improved bioavailability and a 10-fold increased
efficacy in the treatment of chronic UTI in mice. Furthermore,
substitution of 22 has led to a new class of the most potent
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