Antivirulence C-Mannosides as Antibiotic-Sparing, Oral Therapeutics for Urinary Tract Infections

Article

pubs.acs.org/jmc

Antivirulence C‑Mannosides as Antibiotic-Sparing, Oral Therapeutics

for Urinary Tract Infections

Laurel Mydock-McGrane,^†,#Zachary Cusumano,^†,#Zhenfu Han,^‡Jana Binkley,^§Maria Kostakioti,^§

Thomas Hannan,^†Jerome S. Pinkner,^§Roger Klein,^§Vasilios Kalas,^§Jan Crowley,^∥Nigam P. Rath,^⊥

Scott J. Hultgren,^†,§,∥and James W. Janetka*^,†,‡,∥

^†Fimbrion Therapeutics, Inc., Saint Louis, Missouri 63108 United States

§

∥

^‡Departments of Biochemistry and Molecular Biophysics, and Chemistry, Department of Molecular Microbiology, Center for

Women’s Infectious Disease Research, Washington University School of Medicine, Saint Louis, Missouri 63110 United States

^⊥Department of Chemistry and Biochemistry, University of Missouri, Saint Louis, Missouri 63121 United States

S

* Supporting Information

ABSTRACT: 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 pharmacoki-

netic (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.

therapeutic strategies.⁴The study of critical host−pathogen

INTRODUCTION

■

interactions during bacterial infection has revealed promising

Since the 1970s, antibiotic resistance has been steadily

novel targets for therapeutic intervention.⁵Monoclonal anti-

increasing and now multidrug resistant pathogens are an

bodies, vaccines, glycoconjugates, and small molecules have all

imminent threat. Antibiotics target processes essential for

been developed to disrupt bacterial adherence by competitively

bacterial replication and thus induce strong pressure to evolve

blocking the bacterial protein(s) involved in the recognition of

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

various host receptors.⁶This 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

uropathogenic Escherichia coli (UPEC) to colonize the lower

binding is mediated by hair-like ﬁbers, termed type 1 pili, that

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

are tipped with the virulence factor FimH adhesin.⁷The lectin

domain of FimH binds mannosylated glycoproteins expressed

on the luminal surface of human and murine bladder cells. The

(UTIs). These compounds represent advanced preclinical

terminal FimH residue is a prototypical two-domain adhesin,

candidates for UTI therapy.

Microbial adherence to host cells is a critical initial step in

which is joined to the distal end of the linear tip ﬁbrillum of

most infectious diseases.¹The ability of bacteria to bind and

type 1 pili by a donor strand exchange reaction with subunit

FimG.^7,8FimG then undergoes its own donor strand exchange

invade epithelial tissues allows them to establish a niche within

the host and evade host immune responses.²While

reaction with subunit FimF, which adapts the ﬁbrillum tip to

antimicrobial therapy has traditionally been eﬀective in treating

bacterial infections, resistance to the most commonly used

Received: June 28, 2016

antimicrobials is on the rise,³highlighting the need for new

A

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Figure 1. Examples of simple glycosidic bond replacement of early lead O-mannosides 1 and 4, with alternate linkers to the biphenyl aglycone.

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.⁹The FimH

lectin domain contains a deep acidic pocket that recognizes α-

D-mannose with stereochemical speciﬁcity.⁸FimH-mediated

binding to mannosylated uroplakins¹⁰or α1,β3 integrins¹¹

facilitates bacterial colonization and invasion of bladder

epithelial cells.¹²

glycosidic bond^20bto either hydrolysis in the stomach or

intestinal tract or, alternately, from the enzymatic action of

mannosidases.

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-glycoside

FimH antagonists, which encompass a unique R-hydroxy

methylene bond to the aglycone. We have employed X-ray

crystallography and computational docking studies of manno-

sides 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.

UTIs present a signiﬁcant burden for women, with nearly 20

million cases reported annually.¹³Despite antibiotic treatment,

20−40% of these women will have at least one recurrence

within 6 months of their initial diagnosis.¹⁴This results in a

signiﬁcant economic impact, approximately two billion dollars

in the U.S. alone,¹³associated 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.¹⁵Because

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 ST131,¹⁷the 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 target¹⁸

for UTI and, more recently, for Crohn’s disease.¹⁹Inhibition 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.

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 1). As shown in Scheme 1A, these S- and N-

Scheme 1. Synthesis of N-, S-, and Triazole-Linked Biphenyl

a

Mannosides

We have previously developed²⁰small molecule glycosides

based on α-^D-mannose (O-mannosides), such as 1 and 4

(Figure 1), 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,²¹the true viability of synthetic manno-

sides as oral therapeutics for treatment of UTI was not

validated in vivo until 2010,^20a,22where 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 reported²³and glycodendrimer²⁴

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,8,20cand to a small

hydrophobic pocket^20b,23a,25near 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-

a

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.

B

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a

Scheme 2. Synthesis of C-Linked Amide Mannosides

a

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.

a

Scheme 3. Synthesis of C-Linked Methylene and Hydroxy-Methylene Mannosides

a

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.

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-pentaace-

tate 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 strain UTI89. 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 method-

ology. Shown in Scheme 1B, the reaction of azido mannoside

7²⁶with phenylacetylene and copper sulfate, followed by

sodium methoxide deacetylation, gave phenyl triazole manno-

side 8 in good yield. However, mannoside 8 lost substantial

potency relative to 1, only exhibiting an HAI of 32 μM.²⁷On

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.²⁸Hoping 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.

C-Linked Amide Mannosides. Because of ease of

synthesis, we ﬁrst targeted a series of simple C-linked amide

mannosides. Shown in Scheme 2, cyano mannoside 9²⁹was

obtained by reaction of mannose pentaacetate with trimethyl-

silyl 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)

C

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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.

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 12.³⁰Acylation 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.

15 (Scheme 3). 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 1).

Surprisingly, we found the secondary alcohol isomers 6S and

6R showed diﬀerential potency, with a pronounced stereo-

chemical 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).

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

D

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a

Scheme 4. Structures and Synthesis of Prodrug Analogues of O-Mannoside 23

a

Reagents and conditions: (a) Ac₂O, pyridine, rt; (b) POCl₃trimethylphosphate, 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.

On the basis of these key results, we synthesized the ortho-

methyl analogues 22 and 21 based on our lead O-glycoside 23

(Figure 2).^20aShown in Scheme 3, 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 3). 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 6). 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.

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 3,

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 prodrugs³¹of

lead O-mannoside 23. The prodrugs include tetraacetate 23a,

the 6-position phosphate 23b, and dimethylglycine acetate 23c

(Scheme 4). Although 23a is the precursor in the synthesis of

23,^20bit 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

E

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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.

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.

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.

bonding interaction with the neighboring salt bridge, R98, and/

or Y137 residues. In Figure 4B, comparison of mannosides 23

and 24 to non-ortho-substituted 1 (PDB 3MCY),^20chighlights

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 structur-

es,^23a,25,32it 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.

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 5. As seen in the structure of

23 (Figure 4A), there is a crystallized water molecule between

the glycoside anomeric oxygen and the D140 and N135 FimH

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 5B) is

favored energetically over the S-isomer (21S, Figure 5A) due to

a productive water-mediated H-bond in the former. This same

X-ray Crystallography and Computational Modeling.

To better understand the molecular interactions of O-

mannosides 23 and its matched disubstituted analogue 24

(Figure 2)^20bwith 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)²⁵and a 1.75 Å

structure of 23 (PDB 5F3F) bound to the FimH lectin domain

were obtained (Figure 4A) 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-

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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.

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.

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

activity³³compared to O-mannoside 23, as the CH₂is 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

6). 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.

Pharmacokinetics (PK) of Mannosides. To further

examine the therapeutic potential of the new C-mannosides

and O-mannoside prodrugs, we evaluated their pharmacoki-

netic (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 drugs³⁴

and (2) DMSO has been demonstrated to modulate the host

immune response, obfuscating any observed results.³⁵The

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 7A). 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 7A, 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

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Table 1. Compound Structures and Comparison of Biological Activity and Physical Properties Between O- and C-Mannoside

Matched Pairs (ND = not determined)

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.

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 (t_1/2> 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 Man-

nosides. 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 1). In an alternate synthesis, 19R was

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 25²⁵(Figure 2) in rats (Figure 7B). Recent

examination of the PK behavior and eﬃcacy of 25 in mice

identiﬁed it as a leading therapeutic candidate in the O-

mannosides series.²⁵In contrast to the previous study (Figure

7A), 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 7B).

Analysis of the rat PK data revealed that, while C-mannoside

21R encompassed the highest level of compound exposure (as

assessed by C_maxand AUC_all) 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 (V_dss= 6.7 L) but the highest

bioavailability (7%) of all compounds tested. This suggests that

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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

10⁷CFUs 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).

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 isoquinoline C-mannoside 32R

had lower potency relative to its O-mannoside pair 34²⁴(HAI =

62 nM), with an HAI of 125 nM (Table 1). 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-manno-

sides (Figure 5), 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 1, 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.

has been identiﬁed in these murine models has also been

observed in humans, demonstrating their relevance.³⁷To

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 8). When tested as a prophylaxis therapy

(Figure 8A), where the mannoside is dosed 30 min prior to

UPEC infection and bacterial titers enumerated 6 h post-

infection, 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.

Both the O-linked and C-linked mannosides accumulated in

the urine at concentrations over 10-fold higher than their HAI

EC₉₀and bioﬁlm IC₅₀(Figure 7) 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 8B). To investigate this possibility, we ﬁrst

monitored the pharmacodynamics (PD) of O-linked manno-

side 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 10⁷CFUs to approximately

10⁵CFUs 2 h postdosing (Figure 9). Bladder burdens

continued to decrease to 10⁴CFUs 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 9). 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

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,36The distinct pathophysiology of UTI that

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in the prophylactic model of UTI (Figure 10B), 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.

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).

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 drugs³⁸like FimH mannoside 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.

subpopulation of mice 12 hours after treatment that may

represent bladder repopulation once mannoside levels fall

below the HAI.

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 10A). 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.

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

Furthermore, using the newly optimized heterocyclic C-

mannosides, 28R, 29R and 31R, we found signiﬁcantly

augmented activity relative to O-mannoside 25 when tested

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).

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yl]-3-carboxylic Acid Methyl Ester (2). 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: ¹H NMR (300 MHz, acetonitrile-d₃and 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).

¹³C NMR (100 MHz, methanol-d₄/dimethyl sulfoxide-d₆; 10/1) δ

designed a novel C-mannoside FimH antagonist 21R, which we

subsequently modiﬁed, based on O-mannoside SAR and

isoquinolone derivative 25,²⁵to generate several optimized

analogues, 28R, 29R, and 31R. These C-mannosides have

become preclinical candidates for use as novel antivirulence

drugs in UTI therapy.¹⁸Along 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.

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.

4′-(α-^D-Mannopyranosylthio)-[1,1′biphenyl]-3-carboxylic Acid

Methyl Ester (3). 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 CH₂Cl₂layer 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:

¹H NMR (300 MHz, chloroform-d) δ ppm 2.01−2.10 (m, 9H), 2.16

EXPERIMENTAL SECTION

General Synthesis, Puriﬁcation, and Chemistry Procedures.

Starting materials, reagents, and solvents were purchased from

commercial vendors unless otherwise noted. In general, anhydrous

■

1

solvents are used for carrying out all reactions. H NMR 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 chromatog-

raphy 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 N₂three 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.

(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 N₂at 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

1

give 3 (0.095 g) in 63% yield. Analytical data for 3: H NMR (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). ¹³C

NMR (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.

4′-[(α-^D-Mannopyranosyl)methyl]-N-methyl-[1,1′biphenyl]-3-car-

boxamide (5). The title compound was obtained from the preparation

of 6S as a side product in 35% yield (0.005 g, 0.013 mmol). Analytical

1

data for 5: H NMR (400 MHz, methanol-d₄) δ ppm 2.93−3.00 (m,

4H), 3.04−3.13 (m, 1H), 3.62−3.88 (m, 6H), 4.09−4.17 (m, 1H),

7.39 (d, J = 8.2 Hz, 2H), 7.48−7.57 (m, 1H), 7.62 (d, J = 7.8 Hz, 2H),

7.74−7.79 (m, 2H), 8.06 (br s, 1H). ¹³C NMR (100 MHz, methanol-

d₄) δ ppm 26.9, 36.1, 63.1, 69.4, 72.0, 72.7, 76.4, 80.4, 126.6, 126.9,

128.1 (×2), 130.1, 130.9 (×2), 131.0, 136.2, 139.6, 139.7, 142.6, 170.7.

ESI-MS found: [M + H⁺] 388.2.

4′-[(α-^D-Mannopyranosyl)-(R)-hydroxymethyl]-N-methyl-[1,1′bi-

phenyl]-3-carboxamide (6R). A mixture of 18R (0.039 g, 0.051

mmol) and 10% wt Pd/C (0.044 g, 0.02 mmol) in MeOH (10 mL)

was stirred under 1 atm of H₂for 16 h. The Pd/C was ﬁltered oﬀ, and

the ﬁltrate was concentrated and dried in vacuo to give title compound

Detailed Procedures for the Synthesis of Final Mannosides

and Intermediates. 4′-(α-^D-Mannopyranosylamino)-[1,1′biphen-

1

6R (0.020 g, 0.050 mmol) in 99% yield. Analytical data for 6R: H

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NMR (300 MHz, methanol-d₄) δ ppm 2.95 (s, 3H), 3.54−3.81 (m,

4H), 3.86−4.05 (m, 2H), 4.25 (t, J = 3.0 Hz, 1H), 5.01 (d, J = 8.0 Hz,

1H), 7.46−7.61 (m, 3H), 7.67 (d, J = 8.2 Hz, 2H), 7.74−7.88 (m,

2H), 8.08 (t, J = 1.7 Hz, 1H). ¹³C NMR (100 MHz, methanol-d₄) δ

ppm 27.0, 62.8, 69.0, 69.9, 72.6, 72.9, 77.8, 82.4, 126.7, 127.0, 127.8

(×2), 128.7 (×2), 130.1, 131.0, 136.2, 140.8, 142.6, 143.5, 170.7. ESI-

MS found: [M + Na⁺] 426.2.

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). ¹³C NMR (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.

4′-[(α-^D-Mannopyranosyl)-(S)-hydroxymethyl]-N-methyl-[1,1′bi-

phenyl]-3-carboxamide (6S). A mixture of 18S (0.028 g, 0.037

mmol) and 10% wt Pd/C (0.032 g, 0.015 mmol) in MeOH (10 mL)

was stirred under 1 atm of H₂for 16 h. 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 6S (0.010 g, 0.025 mmol) in

67% yield [compound 5 was also isolated as a product in 35% yield

4-Bromo-N-[(α-^D-mannopyranosyl)methyl]-benzamide (13). 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: ¹H NMR (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). ¹³C

NMR (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%).

1

(0.005 g, 0.013 mmol)]. Analytical data for 6S: H NMR (300 MHz,

methanol-d₄) δ ppm 2.95 (s, 3H), 3.49−3.58 (m, 1H), 3.59−3.84 (m,

4H), 3.86−4.02 (m, 2H), 5.04 (d, J = 9.0 Hz, 1H), 7.48−7.60 (m,

3H), 7.71 (d, J = 8.2 Hz, 2H), 7.76−7.84 (m, 2H), 8.08 (s, 1H). ¹³C

NMR (100 MHz, methanol-d₄) δ ppm 27.0, 63.4, 69.1, 69.9, 71.2,

72.9, 77.1, 84.0, 126.8, 127.1, 128.3 (×2), 128.7 (×2), 130.2, 131.0,

136.2, 141.4, 142.3, 142.3, 170.6. ESI-MS found: [M + Na⁺] 426.2.

1-α-^D-Mannopyranosyl-4-phenyl-1H-1,2,3-triazole (8).^27,28Step

1: Under a nitrogen atmosphere, ethanol/water (4 mL/1 mL) was

added into the RB ﬂask containing 2,3,4,6-tetra-O-acetyl-α-^D-

mannopyranosyl azide (7) (0.075 g, 0.20 mmol) (obtained from ^D-

mannose pentaacetate²⁷), phenyl acetylene (0.026 g, 0.24 mmol),

copper(II) sulfate pentahydrate (0.01 g, 0.04 mmol), and sodium

ascorbate (0.016 g, 0.08 mmol). The mixture was stirred at room

temperature overnight. The solvent was removed and the residue was

puriﬁed by silica gel chromatography (ethyl acetate−hexanes gradient

elution), giving rise to the tetraacetate protected intermediate 1-

(2,3,4,6-tetra-O-acetyl-α-^D-mannopyranosyl)-4-phenyl-1H-1,2,3-tria-

zole (8a) (0.030 g, 0.062 mmol) in 31% yield. Analytical data for 8a:

¹H NMR (300 MHz, chloroform-d) δ ppm 2.05−2.12 (m, 9H), 2.20

4′-[([(α-^D-Mannopyranosyl)methyl]amino)carbonyl]-[1,1′biphen-

yl]-3-carboxylic Acid Methyl Ester (14). To a stirred solution of 4-

bromo-N-[(α-^D-mannopyranosyl)methyl]-benzamide (13) (0.38 g, 0.1

mmol), 3-methoxycarbonylphenylboronic acid (0.027 g, 0.15 mmol),

and cesium carbonate (0.098 g, 0.3 mmol) in dioxane/water (5 mL/1

mL) under nitrogen atmosphere was added tetrakis-

(triphenylphosphine)palladium (0.012 g, 0.01 mmol) was heated at

80 °C for 1 h. The reaction was concentrated to dryness and dissolved

in DMSO and was puriﬁed by HPLC [C18, 15 mm × 150 mm

column; eluent, acetonitrile/water (0.1% TFA)]. After lyopholiztion of

pure fractions, the title compound (24 mg, 0.056 mmol) was obtained

(s, 3H), 3.89−3.99 (m, 1H), 4.08 (dd, J = 12.50, 2.61 Hz, 1H), 4.40

(dd, J = 12.50, 5.36 Hz, 1H), 5.40 (t, J = 8.79 Hz, 1H), 5.95−6.04 (m,

2H), 6.07 (d, J = 2.75 Hz, 1H), 7.34−7.53 (m, 3H), 7.79−7.92 (m,

2H), 7.96 (s, 1H). ESI-MS found: [M + Na⁺] 498.1.

Step 2: Tetraacetate 8a (0.028 g, 0.059 mmol) from step 1 was

stirred in 6 mL of MeOH with a catalytic amount of sodium

methoxide (0.02 M) at room temperature overnight. H⁺exchange

resin (DOWEX 50WX4-100) was added to neutralize the mixture pH.

The resin was ﬁltered oﬀ, and the ﬁltrate was concentrated and then

dried in vacuo, giving rise to the title compound (0.015 g, 0.049

mmol) in 83% yield. Analytical data for 8: ¹H NMR (300 MHz,

methanol-d₄) δ ppm 3.34−3.44 (m, 1H), 3.70−3.92 (m, 3H), 4.12

(dd, J = 8.52, 3.57 Hz, 1H), 4.71−4.80 (m, 1H), 6.08 (d, J = 2.75 Hz,

1H), 7.30−7.41 (m, 1H), 7.41−7.51 (m, 2H), 7.77−7.90 (m, 2H),

8.51 (s, 1H). ¹³C NMR (100 MHz, methanol-d₄) δ ppm 62.6, 68.7,

70.1, 72.6, 78.7, 88.5, 122.1, 126.8 (×2), 129.5, 130.0 (×2), 131.5,

149.1. MS-ESI found: [M + Na⁺] 330.2.

1

in 55% yield. Analytical data for 14: H NMR (400 MHz, methanol-

d₄) δ ppm 3.68−3.71 (m, 4H), 3.78−3.83 (m, 3H), 3.87 (t, J = 3.2 Hz,

1H), 3.93 (s, 3H), 4.07−4.15 (m, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.73

(d, J = 7.6 Hz, 2H), 7.89 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.0 Hz, 2H),

8.01 (dd, J = 1.6, 8.0 Hz, 1H), 8.26 (s, 1H). ¹³C NMR (100 MHz,

methanol-d₄) δ ppm 40.2, 52.8, 62.7, 69.9, 70.4, 72.7, 76.5, 77.5, 128.1

(×2), 129.0, 129.2 (×2), 129.9, 130.4, 132.1, 132.7, 134.8, 141.7,

144.4, 168.3, 170.0. ESI-MS found: [M + H⁺] 432.2.

2,3,4,6-Tetra-O-benzyl-α-^D-mannopyranosyl Cyanide (15). Sim-

ilar 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

Na₂SO₄and 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:^31b¹H NMR (300 MHz, chloroform-d) δ ppm 3.72−3.76 (m, 1H),

4′-[(α-^D-Mannopyranosylcarbonyl)amino]-[1,1′-biphenyl]-3-car-

boxylic Acid Methyl Ester (11). The solution of 2,3,4,6-tetra-O-acetyl-

α-^D-mannosyl cyanide (9)^29,30(0.107g, 0.3 mmol) in 25% hydro-

chloric 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

1

data for 11: H NMR (300 MHz, methanol-d₄) δ ppm 3.50 (dt, J =

L

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Journal of Medicinal Chemistry

Article

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.

4.51 (m, 6H), 4.52−4.76 (m, 2H), 4.85−4.99 (m, 1H), 6.98−7.45 (m,

22H), 7.47−7.68 (m, 4H), 7.71−7.84 (m, 2H), 8.03 (t, J = 1.7 Hz,

1H). ESI-MS found: [M + Na⁺] 786.2.

(2,3,4,6-Tetra-O-benzyl-α-^D-mannopyranosyl)-(4-bromo-2-meth-

ylphenyl)-methan-1(R/S)-ol (19R/S). Into a ﬂask containing 4-bromo-

2-methyl-iodobenzene (9.04 mL, 63.29 mmol) in anhydrous Et₂O

(150 mL) under N₂was 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. Saturated aq 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-mannopyr-

anosyl)-(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

2,3,4,6-Tetra-O-benzyl-α-^D-mannopyranosyl Carbaldehyde (16).

Similarly to previously reported protocols,^31b,39at −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.

1

(2,3,4,6-Tetra-O-benzyl-α-^D-mannopyranosyl)-(4-bromophenyl)-

methan-1(R/S)-ol (17R/S). Into a ﬂask containing 1,4-dibromoben-

zene (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 N aq 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-bromo-

phenyl)-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.

4′-[(2,3,4,6-Tetra-O-benzyl-α-^D-mannopyranosyl)-(R)-hydroxy-

methyl]-N-methyl-[1,1′biphenyl]-3-carboxamide (18R). Following

the general Suzuki-coupling procedure, the impure mannosyl bromide

17R (0.075 g, 0.1 mmol), commercially available N-methyl-3-(4,4,5,5-

tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzamide (0.039g, 0.15

mmol), cesium carbonate (0.098 g, 0.3 mmol), and tetrakis-

(triphenylphosphine)palladium (0.012 g, 0.01 mmol) in dioxane/

water (5 mL/1 mL) were stirred under N₂, at 80 °C for 1 h. Upon

completion, the solvent was removed, and the resulting residue was

puriﬁed by silica gel chromatography (methanol−dichloromethane

gradient elution) to give 4′-[(2,3,4,6-tetra-O-benzyl-α-^D-mannopyr-

anosyl)-(R)-hydroxymethyl]-N-methyl-[1,1′biphenyl]-3-carboxamide

(18R) in 9% total yield (0.039 g, 0.051 mmol) (3 steps; based on 0.56

column earlier containing isomer 19R. Analytical data for 19R: H

NMR (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: ¹H NMR (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). (2,3,4,6-Tetra-O-benzyl-α-^D-

mannopyranosyl)-(4-bromo-2-methylphenyl)-methanone (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 N₂at 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% aq Na₂S₂O₃(6 mL) and saturated aq

NaHCO₃(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 saturated aq NaHCO₃(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)

1

mmol carbonitrile 15). Analytical data for 18R: H NMR (300 MHz,

acetonitrile-d₃) δ ppm 2.89 (d, J = 4.7 Hz, 3H), 3.49−3.69 (m, 2H),

3.72−3.83 (m, 2H), 3.85−3.96 (m, 1H), 4.06 (dd, J = 8.0, 3.0 Hz,

2H), 4.13−4.22 (m, 1H), 4.27−4.46 (m, 2H), 4.48−4.68 (m, 5H),

4.76 (d, J = 11.3 Hz, 1H), 4.90−5.00 (m, 1H), 7.03−7.15 (m, 1H),

7.18−7.41 (m, 20H), 7.43−7.56 (m, 3H), 7.57−7.65 (m, 2H), 7.69−

7.80 (m, 2H), 8.00 (t, J = 1.6 Hz, 1H). ESI-MS found: [M + Na⁺]

786.2.

1

in 74% yield (0.43 g, 0.59 mmol). Analytical data for 19a: H NMR

(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). (2,3,4,6-Tetra-O-benzyl-α-^D-

mannopyranosyl)-(4-bromo-2-methylphenyl)-methan-1(R)-ol

(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

N₂at −40 °C, with the dropwise addition of lithium tri-tert-

butoxyaluminum hydride (1 M in hex; 0.87 mL, 0.87 mmol). The

4′-[(2,3,4,6-Tetra-O-benzyl-α-^D-mannopyranosyl)-(S)-hydroxy-

methyl]-N-methyl-[1,1′biphenyl]-3-carboxamide (18S). The title

compound was synthesized from 17S (0.034 g, 0.048 mmol) following

the same procedure described for 18R. 18S was obtained in 6.5%

(0.028 g, 0.037 mmol) (3 steps, based on 0.56 mmol carbonitrile 15).

1

Analytical data for 18S: H NMR (300 MHz, acetonitrile-d₃) δ ppm

2.90 (d, J = 4.7 Hz, 3H), 3.48 (d, J = 4.4 Hz, 1H), 3.57−3.62 (m, 1H),

3.62−3.85 (m, 3H), 3.86−3.94 (m, 2H), 3.95−4.03 (m, 1H), 4.33−

M

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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). Saturated aq 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.

preparation of 21R as a side-product in 12% yield (0.003 g, 0.0072

mmol). Analytical data for 22: H NMR (400 MHz, methanol-d₄) δ

1

ppm 2.44 (s, 3H), 2.95 (s, 3H), 3.04 (d, J = 7.4 Hz, 2H), 3.69 (m,

3H), 3.83 (m, 2H), 3.86−3.92 (m, 1H), 4.04−4.21 (m, 1H), 7.31 (d, J

= 7.8 Hz, 1H), 7.42−7.47 (m, 1H), 7.50 (m, 2H), 7.75 (m, 2H), 8.05

(s, 1H). ¹³C NMR (100 MHz, methanol-d₄) δ ppm 19.8, 26.9, 33.7,

63.1, 69.3, 72.1, 72.7, 76.5, 79.4, 125.6, 126.6, 126.8, 130.0, 130.1,

130.9, 131.7, 136.1, 137.8, 138.1, 139.8, 142.7, 170.7. ESI-MS found:

[M + H⁺] 402.3.

4′-(2,3,4,6-Tetra-O-acetyl-α-^D-mannopyranosyloxy)-N,3′-dimeth-

yl-[1,1′biphenyl]-3-carboxamide (23a). 4′-(-α-^D-Mannopyranosy-

loxy)-N,3′-dimethyl-[1,1′biphenyl]-3-carboxamide^20b(0.072 g, 0.178

mmol) was dissolved in anhydrous pyridine (1 mL) and acetic

anhydride (1 mL). 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 62% yield

4′-[(2,3,4,6-Tetra-O-benzyl-α-^D-mannopyranosyl)-(R/S)-hydroxy-

methyl]-N,3′-dimethyl-[1,1′biphenyl]-3-carboxamide (20R/S). 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)

1

(0.063 g, 0.11 mmol). Analytical data for 23a: H NMR (400 MHz,

dimethyl sulfoxide-d₆) δ ppm 1.94 (s, 3H), 2.00 (s, 3H), 2.05 (s, 3H),

2.16 (s, 3H), 2.32 (s, 3H), 2.81 (d, J = 4.30 Hz, 3H), 3.93−4.11 (m,

2H), 4.19 (dd, J = 12.13, 5.09 Hz, 1H), 5.22 (t, J = 9.98 Hz, 1H),

5.33−5.45 (m, 2H), 5.80 (s, 1H), 7.23 (d, J = 8.61 Hz, 1H), 7.46−7.65

(m, 3H), 7.77 (d, J = 7.83 Hz, 2H), 8.07 (s, 1H), 8.54 (d, J = 4.30 Hz,

1H). ¹³C NMR (100 MHz, chloroform-d) δ ppm 16.4, 20.8 (×3),

21.0, 27.0, 62.3, 66.0, 69.2, 69.5, 69.6, 96.0, 114.7, 125.3, 125.7, 125.7,

128.1, 129.0, 129.7, 130.0, 135.0, 135.3, 141.1, 153.9, 168.4, 169.9,

170.1, 170.2, 170.7. ESI-MS found: [M + Na⁺] 594.3.

1

4′-(6-Dihydrogen Phosphate-α-^D-mannopyranosyloxy)-N,3′-di-

methyl-[1,1′biphenyl]-3-carboxamide (23b). 4′-(-α-^D-Mannopyrano-

syloxy)-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

was obtained in 39% yield. Analytical data for 20R: H NMR (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: ¹H NMR (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.

1

(0.070 g, 0.145 mmol). Analytical data for 23b: H NMR (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). ¹³C NMR (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

4′-[(α-^D-Mannopyranosyl)-(R)-hydroxymethyl]-N,3′-dimethyl-

[1,1′biphenyl]-3-carboxamide (21R). Compound 20R (0.046 g,

0.059 mmol) and 10% wt Pd/C (0.050 g, 0.024 mmol) were stirred

in MeOH (5 mL) under 1 atm of H₂for 16 h. The Pd/C was then

ﬁltered oﬀ, 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 21R (0.020 g, 0.048

mmol) in 81% yield. [Compound 22 was also isolated as a product in

4′-[6-O-(N,N-Dimethylaminoacetyl)-α-^D-mannopyranosyloxy]-

N,3′-dimethyl-[1,1′biphenyl]-3-carboxamide (23c). 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 N₂at 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]-

1

12% yield (0.003 g, 0.0072 mmol)]. H NMR (400 MHz, methanol-

d₄) δ ppm 2.51 (s, 3H), 2.95 (s, 3H), 3.57−3.78 (m, 4H), 4.00−4.07

(m, 1H), 4.10 (dd, J = 6.9, 2.4 Hz, 1H), 4.25 (t, J = 2.9 Hz, 1H), 5.24

(d, J = 6.7 Hz, 1H), 7.45−7.57 (m, 3H), 7.62 (d, J = 8.2 Hz, 1H),

7.71−7.83 (m, 2H), 8.07 (t, J = 1.6 Hz, 1H). ¹³C NMR (100 MHz,

methanol-d₄) δ ppm 19.6, 26.9, 63.0, 69.0, 69.9, 71.0, 73.3, 78.2, 81.6,

125.6, 126.7, 126.9, 128.9, 130.1, 130.1, 130.9, 136.1, 137.7, 140.5,

141.2, 142.6, 170.7. ESI-MS found: [M + Na⁺] 440.3.

4′-[(α-^D-Mannopyranosyl)-(S)-hydroxymethyl]-N,3′-dimethyl-

[1,1′biphenyl]-3-carboxamide (21S). The title compound was

synthesized from 20S (0.055 g, 0.071 mmol), following the same

procedure as for 21R. Compound 21S (0.025 g, 0.062 mmol) was

1

obtained in 88% yield. Analytical data for 21S: H NMR (400 MHz,

methanol-d₄) δ ppm 2.51 (s, 3H), 2.95 (s, 3H), 3.56 (dd, J = 1.0 Hz,

1H), 3.67 (d, J = 8.2 Hz, 1H), 3.70−3.82 (m, 3H), 3.91 (d, J = 9.4 Hz,

1H), 4.10 (dd, J = 9.0, 2.0 Hz, 1H), 5.28 (d, J = 8.6 Hz, 1H), 7.34−

7.63 (m, 4H), 7.69−7.90 (m, 2H), 8.07 (s, 1H). ¹³C NMR (100 MHz,

methanol-d₄) δ ppm 19.9, 27.0, 63.4, 67.5, 69.0, 69.9, 73.6, 77.3, 83.4,

126.0, 126.7, 127.0, 129.2, 130.1, 130.3, 130.9, 136.1, 137.4, 140.2,

140.9, 142.3, 170.6. ESI-MS found: [M + Na⁺] 440.3.

1

3-carboxamide in 61% yield (0.19 g, 0.36 mmol). Analytical data: H

NMR (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

4′-[(α-^D-Mannopyranosyl)methyl]-N,3′-dimethyl-[1,1′biphenyl]-

3-carboxamide (22). The title compound was obtained from the

N

DOI: 10.1021/acs.jmedchem.6b00948

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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;

× 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−

1

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-mannopyr-

anosyloxy]-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,

hexanes). H NMR (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%).

7-(4-[(α-^D-Mannopyranosyl)-(R)-hydroxymethyl)-3-methylphen-

yl]-1(2H)-isoquinolinone (28R). 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 N₂at 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.

1

0.031 mmol) in 31% yield. Analytical data for 23c: H NMR (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). ¹³C NMR (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;

(2,3,4,6-Tetra-O-benzyl-α-^D-mannopyranosyl)-(4-bromo-2-meth-

ylphenyl)-methan-1(R)-ol Acetate (26). 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:

¹H NMR (400 MHz, chloroform-d) δ ppm 1.91 (s, 3H), 2.38 (s, 3H),

1

Analytical data for 28R: H NMR (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). ¹³C NMR (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.

3,4-Dihydro-7-(4-[(α-^D-mannopyranosyl)-(R)-hydroxymethyl]-3-

methylphenyl)-1(2H)-isoquinolinone (29R). 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 H₂for

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%

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%).

(2,3,4,6-Tetra-O-acetyl-α-^D-mannopyranosyl)-(4-bromo-2-meth-

ylphenyl)-methan-1(R)-ol Acetate (27). 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

1

yield. Analytical data for 29R: H NMR (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). ¹³C NMR (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.

O

DOI: 10.1021/acs.jmedchem.6b00948

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Journal of Medicinal Chemistry

Article

3,4-Dihydro-7-[4-(α-D-mannopyranosyloxy)-3-methylphenyl]-

1(2H)-isoquinolinone (30). 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: ¹H NMR (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). ¹³C NMR (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 N₂three 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 CH₂Cl₂and allowed to sit

for 5 min to allow for byproducts to precipitate. The precipitate was

ﬁltered oﬀ. Evaporation of the CH₂Cl₂in 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

137.7, 138.1, 138.9, 142.3, 143.0, 156.1. ESI-MS found: [M + H⁺]

427.4.

5-(4-[(α-^D-Mannopyranosyl)-(R)-hydroxymethyl]-3-methylphen-

yl)-isoquinoline (32R). 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 N₂at 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: ¹H NMR

(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). ¹³C NMR (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.

7-[4-(α-^D-Mannopyranosyloxy)-3-methylphenyl]- isoquinolin-1-

amine (33). 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−dichloro-

methane 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: ¹H NMR (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

7-(4-[(α-^D-Mannopyranosyl)-(R)-hydroxymethyl]-3-methylphen-

yl)-isoquinolin-1-amine (31R). 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 N₂at 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.

1

Analytical data for 31R: H NMR (400 MHz, methanol-d₄) δ ppm

1

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). ¹³C NMR

(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,

g, 0.097 mmol) in 36% yield. Analytical data for 33: H NMR (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). ¹³C NMR (100 MHz,

P

DOI: 10.1021/acs.jmedchem.6b00948

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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 UTI89 bacteria and guinea pig red

blood cells, as previously described.²⁵

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b00948.

■

S

¹H and ¹³C NMR, HPLC, and MS analytical data for new

compounds and the details of the crystallography

experiments, including the X-ray crystal determinants

for both for the small molecule 27 and FimH co-crystal

structure with 23; details of the prophylactic acute UTI

mouse model with 23 and prodrugs 23a−c, as well as the

rat pharmacokinetic study of 21R, 23, and 25 (PDF)

Molecular formula strings (CSV)

Bioﬁlm Assay. The bioﬁlm assay was performed with UTI89

bacteria as previously described.²⁵

Diﬀerential Scanning Fluorimetry (DSF). The DSF assay was

performed with FimH_L(10 μM) in the absence or presence of 100 μM

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.

AUTHOR INFORMATION

Corresponding Author

*Phone: 314-362-0509. Fax: 314-362-7183. E-mail: janetkaj@

biochem.wustl.edu. Address: 660 South Euclid Avenue, Box

8231, St. Louis, Missouri 63110, United States.

■

Author Contributions

^#L.M.-M. and Z.C. contributed equally to the work

Notes

The authors declare the following competing ﬁnancial

interest(s): Scott Hultgren and James Janetka are co-founders

and stockholders in Fimbrion Therapeutics, Inc.

Animal Infections. The bacterial inoculum and infection of mice

was performed as previously described.^20aBrieﬂy, the bacterial strain

UTI89 with a kanamycin antibiotic cassette inserted in the

chromosome (UTI89_KAN) 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 × 10⁸UTI89_KAN, 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.

ACKNOWLEDGMENTS

■

We thank Rick Stegeman (X-ray facility, Biochemistry Depart-

ment) for help with X-ray crystallography studies and Scott

Wildman (Biochemistry Department) for help with molecular

modeling and docking of FimH ligands. We also thank Drs.

Rasesh Shah and Jay Wendling at Seventh Wave for conducting

the rat PK study. This work was funded in part by the NIH

grants 1RC1DK086378 (Washington University) from

NIDDK, and 5R43AI106112-02 (Fimbrion Therapeutics,

Inc.) from NIAID. Support for the Biomedical Mass

Spectrometry Resource to do the mouse urine PK bioanalytical

studies is provided by the NIH/NIGMS grant no.

P41GM103422. For the small molecule X-ray structure of

compound 27, we acknowledge funding from the NSF (MRI,

CHE-0420497) for the purchase of the ApexII diﬀractometer.

ABBREVIATIONS USED

■

UTI, urinary tract infection; UPEC, uropathogenic E. coli;

CFUs, colony forming units; SAR, structure−activity relation-

ships; SPR, structure−property relationships; HA, hemaggluti-

nation; HAI, hemagglutination Inhibition; IBC, intracellular

bacterial community; PD, pharmacodynamics; PK, pharmaco-

kinetics; C_max, concentration maximum

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 UTI89_KAN

.

Mice were infected with 1−2 × 10⁷UTI89_KAN, 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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