Comparison of affinity ranking by target-directed dynamic combinatorial chemistry and surface plasmon resonance

Article abstract of DOI:10.24820/ark.5550190.p010.913

Target-directed dynamic combinatorial chemistry (tdDCC) is a powerful method to screen ligands for pharmacologically relevant targets. Generating a dynamic library from reversibly reacting building blocks in the presence of a target protein leads to the a

Full text of DOI:10.24820/ark.5550190.p010.913

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Comparison of affinity ranking by target-directed dynamic combinatorial
chemistry and surface plasmon resonance
Priska Frei, Marleen Silbermann, Tobias Mühlethaler, Xiaohua Jiang,
Oliver Schwardt, Rachel Hevey, and Beat Ernst*
Department of Pharmaceutical Sciences, University of Basel, Klingelbergstr. 50, CH-4056 Basel, Switzerland
E-mail: Beat.Ernst@unibas.ch
Dedicated to Steven Hanessian, for his friendship and support over many years
Received 02-20-2019
Accepted 04-21-2019
Published on line 05-02-2019
Abstract
Target-directed dynamic combinatorial chemistry (tdDCC) is a powerful method to screen ligands for
pharmacologically relevant targets. Generating a dynamic library from reversibly reacting building blocks in
the presence of a target protein leads to the amplification of the most potent library constituents. In previous
studies on tdDCC, these compounds were identified in a qualitative “hit/no-hit”-manner. However, the precise
relationship between the degree of amplification and the affinity of the library constituent has not yet been
evaluated. To study the amplification–affinity relationship, we compared tdDCC experiments, employing
reversible acylhydrazone formation and the bacterial adhesin FimH as a target, with affinities of the library
constituents as determined by surface plasmon resonance.
Keywords: Acylhydrazone, FimH antagonists, dynamic combinatorial chemistry, supramolecular chemistry,
drug discovery
DOI: https://doi.org/10.24820/ark.5550190.p010.913
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Introduction
Dynamic combinatorial chemistry (DCC) describes the generation of dynamic compound libraries from
reversibly reacting building blocks. These libraries, which are under thermodynamic control, remain adaptive
by continuous interconversion of building blocks and products. Therefore, addition of a protein target alters
their equilibrium composition by binding, thereby stabilizing, and ultimately amplifying specific library
constituents. Target-directed DCC (tdDCC) exhibits a self-screening ability, leading to the amplification of those
members of the library with the highest affinity for the protein target, as depicted for an acylhydrazone library
in Figure 1. This makes tdDCC a valuable tool for drug discovery.1-6
Figure 1. Schematic illustration of tdDCC. Reversibly reacting hydrazide and aldehyde building blocks generate
a dynamic acylhydrazone library, which, when challenged with the target FimH, responds with a shift in
its equilibrium composition including amplification of selected high-affinity ligands.
Whereas earlier reports focused on small libraries in a qualitative “hit/no-hit”-manner,3-6 a more precise
affinity ranking is required to increase the value of tdDCC. So far, the relationship between the extent of
amplification and affinity was addressed in detail only by Nasr et al.7 Furthermore, it has been noted that if
several library constituents exhibit high affinity, the identification of the best binders might be difficult.8
Finally, theoretical considerations regarding the relationship between host-ligand interactions and extent of
amplification have been reported.9
In this communication, we have examined in greater detail the relationship between the amplification of
library members in tdDCC experiments and their dissociation constants (KD). As target protein we selected the
bacterial adhesin FimH, located at the distal tip of type 1 pili of uropathogenic E. coli (UPEC) strains, which are
the cause of the majority of urinary tract infections (UTI).10-12 In the initial step of infection, FimH binds to the
highly mannosylated surface protein uroplakin 1a on urothelial host cells,13 a process which can be prevented
with FimH antagonists such as aryl mannosides.14-20
We have reported previously that in addition to ligand affinity, also the ratio of scaffold to fragment
building blocks, sample preparation, analysis, and method of data processing can be crucial factors in dictating
experiment success.21 Here, we extend the reported protocol for tdDCC using the bacterial adhesin FimH as
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target protein to a larger scale. When aldehyde scaffold 121 reacts reversibly with hydrazide fragments
2a-u (→ 3a-u; Scheme 1), the acylhydrazone library 3a-u is obtained. At neutral pH in aqueous media,
equilibrium formation is facilitated by aniline as a nucleophilic catalyst.22
OH
OH
OH
OH
NH2
O
O
O
HO
HO
HO
HO
F
F
H2N
+
N
R
O
O
H
pH 7.0
O
N
3a-u
1
2a-u
CHO
N
H
R
O
R:
S
S
Me
Me
N
N
N
N
N
Cl
Me
Me
e
a
b
c
d
g
f
S
S
Me
HN
O
OMe
Me
Me
HN
h
o
i
j
k
l
m
n
u
S
S
S
Cl
CF3
tBu
Cl
Cl
Cl
Me
p
q
r
s
t
Scheme 1. Aldehyde scaffold 1 and hydrazide fragments 2a-u used for the reversible generation of
acylhydrazones 3a-u at neutral pH with aniline promotion.
Equilibration was carried out in the absence (blank library) or presence (template library) of biotinylated
full-length FimH (FimHFL-B)23 for three days.21 The reversibility of the acylhydrazone exchange reaction was
then blocked by an adjustment in pH, which effectively locks in the library composition and renders the library
suitable for analysis.24 Prior to UV-HPLC analysis, the protein–ligand complex was captured using commercially
available streptavidin agarose, and any unbound ligand removed from the sample (Figure 2A and 2B). The
protein-bound ligand was then released by a further increase in pH together with addition of the FimH
antagonist n-heptyl α-D-mannopyranoside25 (4, Figure 2C). The supernatant was then analyzed with HPLC and
the chromatograms of template libraries compared to those of equally treated blank samples.
On account of toxicological and stability concerns over the acylhydrazone moiety, we subsequently
explored its potential for bioisosteric replacement.
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Figure 2. Schematic representation of the experimental tdDCC set-up.21
A. After sufficient equilibration, the library composition is made static through a pH increase (pH 8.5), and
then the biotinylated target protein – including bound ligands – is captured with commercially available
streptavidin agarose.
B. After centrifugation, the supernatant (containing unbound ligands) is discarded and the agarose-
protein-ligand complex resuspended.
C. Bound ligands are released from the captured FimH into bulk solvent by the addition of excess of
n-heptyl α-D-mannopyranoside (4) 25 and a further pH increase (pH 12). After a final centrifugation step,
the supernatant is removed and analyzed by HPLC.
Results and Discussion
Target-directed dynamic combinatorial chemistry (tdDCC)
Because the outcomes of tdDCC experiments are influenced not only by the ratio of scaffolds to fragments,21
but also by the ratio of library constituents to target protein,26 we studied this issue in more detail by
employing different amounts of building blocks, while keeping the protein concentration constant. Dynamic
libraries were generated with varying concentrations of aldehyde scaffold 1 (600 µM, 400 µM, and 200 µM)
together with hydrazide fragments 2a-u each at 100 µM. All libraries were equilibrated in the presence and
absence of 100 µM FimHFL-B (measured in triplicates). In this experimental design, the amount of scaffold 1 is
determining the maximum attainable acylhydrazone concentration. Assuming full conversion of the aldehyde,
facilitated by the addition of excess hydrazides, concentration ratios of 6:1, 4:1, and 2:1 between
acylhydrazones and target protein should be reached. With a 1:1 ratio, accurate detection was not possible
due to an insufficient amount of acylhydrazones, while unsatisfactory solubility became an issue at higher
protein concentrations. The libraries were analyzed by UV-HPLC, detecting optical density at 310 nm.
Conveniently, only acylhydrazones absorb UV light at this particular wavelength, but not other library
constituents such as aniline, unreacted hydrazides, or n-heptyl α-D-mannopyranoside (4). Peaks in the
resulting chromatograms were assigned using reference samples. Because both 3f and 3g, as well as 3m and
3n, gave overlapping signals which could not be resolved, we treated these signals as containing equal
amounts of both constituents.
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Composition of each library was determined based on the relative peak area (RPA), where the summated
peak areas in each chromatogram were set to 100% and each peak assigned its fraction. Based on this
information, the normalized change of RPA21 between template and blank samples could be calculated and
the influence of FimHFL-B on the library composition assessed (Figure 3). A positive value indicates
amplification of a compound in the presence of FimHFL-B, whereas a negative value indicates depletion.
Figure 3. Normalized change of RPA21 between template and blank libraries. Bars from light to dark grey
indicate 6:1, 4:1, and 2:1 concentration ratios of total acylhydrazones to FimHFL-B. Triplicates of all
libraries were generated in the presence and absence of 100 µM FimHFL-B. Error bars indicate error
propagation of standard deviations of RPAs over three measurements.21
The most pronounced influence of FimHFL-B on the library composition was observed when core aldehyde 1
was present at 200 µM, resulting in a 2:1 ratio of acylhydrazones to target protein. When the scaffold was
employed at higher concentrations (400 µM and 600 µM), changes in composition of the libraries were less
pronounced. To evaluate if the normalized changes of RPA correlated with the affinities of the corresponding
acylhydrazones, KD values for 3a-u were determined by surface plasmon resonance (SPR) using a previously
established procedure.21 Owing to the structural similarity of the compounds, differences in observed KD
values were not drastic (267 nM to 760 nM; Table 1). Obviously, this narrow distribution of affinities places
highest demands on the applied analytical tools.
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Table 1. Affinities measured by surface plasmon resonance.
Compound
KD [nM]
Compoun
KD [nM]
d
3k
3l
3m
3n
3o
3p
3q
3r
1
316021
359
427
508
484
390
440
377
286
337
536
376
76021
3a
3b
3c
3d
3e
3f
3g
3h
3i
52021
358
267
492
55021
461
642
33021
462
3s
3t
3u
3j
Results of tdDCC experiments are commonly reported in a “hit/no-hit”-manner. Amplification (“hit”) and
depletion (“no-hit”) were only considered significant when the propagated error did not cross the baseline
(Figure 3). Otherwise, compound concentrations were regarded as unchanged. Ligands 3d, 3i, 3p and 3q were
amplified by the target in each of the three described experimental designs, whereas 3a and 3t were only
amplified in the replicates with a 2:1 acylhydrazones to FimHFL-B ratio. The investigation of compounds with a
negative normalized change of RPA revealed that signals 3l, 3u, and overlapping 3f and 3g were decreased, in
accordance with lower affinities. For acylhydrazones with intermediate affinities, the combined signal of 3m
and 3n stayed unchanged with 200 µM and 400 µM, but was slightly increased with 600 µM of core aldehyde 1.
Finally, 3j (KD of 462 nM) and 3o (KD of 440 nM) remained unchanged in all libraries.
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Figure 4. Quantitative relationship between the normalized change of relative peak area (RPA) and the
dissociation constant (KD) for different acylhydrazones. Pearson’s correlation coefficient r is given as a
measure of alignment.
Even though the majority of normalized changes of RPA correlated with the affinity data obtained by SPR,
deviations were found for some acylhydrazones: albeit exhibiting rather high affinities of 358 nM and 337 nM,
the normalized changes of RPA for 3c and 3r remained unchanged, while 3e with a KD of only 492 nM was
amplified. Furthermore, 3b, 3h, and 3s with rather low affinities of 523 nM, 642 nM, and 536 nM, respectively,
remained unchanged, whereas 3k with an affinity of 427 nM was decreased. Some of these aberrations could
potentially result from the fast binding kinetics (both on- and off-rates) between acylhydrazones and FimHFL-B
,
as was observed in the SPR experiments (see Supporting Information). Since the kinetic constants could not be
uniquely determined in all cases, some of the reported KD values may be erroneous. Apart from the fast
binding kinetics21 which may have impeded the SPR measurements, we currently have no explanation for
these outliers. However, it is important to keep in mind that when different techniques for affinity
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measurements are utilized, deviations resulting from some inherent errors of measurements are often
unavoidable.
To assess the quantitative relationship between tdDCC and SPR results, the normalized change of RPA for
each acylhydrazone was plotted against its KD value. In the case of overlapping signals in the tdDCC
experiment, the KD value used was the average of the two acylhydrazones. Linear regression of the
experimental data obtained using a 6:1 substrate ratio showed a moderate Pearson’s correlation coefficient (r)
of -0.502 (Figure 4A). For the 4:1 substrate ratio, the correlation coefficient slightly increased to -0.574 (Figure
4B), and for the 2:1 ratio, the highest correlation with an r-value of -0.655 was obtained (Figure 4C).
Overall, the tdDCC experiments delivered results comparable to SPR. Given the narrow range of KD-values
in the compound series, this clearly highlights the sensitivity of tdDCC. Furthermore, the tdDCC approach
offers great economy of time: while SPR often requires independent synthesis and purification prior to affinity
measurement, tdDCC combines the two steps into a single assay, thus clearly accelerating the process of hit
identification. An additional benefit is that the described tdDCC protocol requires only standard laboratory
equipment, while SPR requires an elaborate and costly instrument.
Bioisosteric replacement
Besides their use in tdDCC,21,24,27-31 several acylhydrazones have been reported to exhibit therapeutic
properties32 in areas such as cancer,33-34 viral35-36 and bacterial37-38 infection, and pain and inflammation.39
Furthermore, hydrazone linkages have been exploited for pH-responsive drug delivery.40 However, both the
cytotoxic activity inherently linked to anti-cancer drugs and the instability of the hydrazone moiety which
affords its pH-responsiveness give rise to general concerns towards inclusion of the acylhydrazone group in
potential FimH antagonists. Jumde et al. recently reported on bioisosteric replacement of the acylhydrazone
moiety.41 Therefore, in an effort to improve on stability and reduce toxicity, we generated a small library of
bioisosteric analogues.
To explore possible bioisosteres of acylhydrazone, six alternatives to 3f were synthesized (→ 5-10;
Table 2). Conveniently, reduction of 3f with NaBH3CN yielded hydrazide 5. Ureas 6 and 7 were synthesized
from the corresponding anilines and amines, which were coupled via intermediates formed from 4-
nitrophenyl chloroformate. Thioureas 8 and 9 were generated from the same aniline and amine starting
materials through activation with 1,1’-thiocarbonyldiimidazole. Lastly, amide 10 was obtained by first
assembling the aglycone from acid and amine precursors using standard peptide coupling, followed by
mannosylation.
The affinities of compounds 5-10 were evaluated in a competitive fluorescence polarization assay
(FPA),14,17 using a non-biotinylated version of the FimH full-length protein (FimHFL).23 In type 1 pili of UPECs,
the FimH subunit is stabilized by the N-terminal donor strand of the adjacent FimG subunit. Because isolated
FimH turned out to be unstable, FimHFL-B and FimHFL required stabilization by a peptide consisting of the 15
terminal amino acids of FimG which mimics the donor strand. In the case of FimHFL-B, biotin was linked to the
pentadecapeptide, which does not alter FimHFL-B’s binding properties as compared to FimHFL. Hence, affinities
determined with either of the constructs should be comparable. In the competitive FPA, the compounds under
investigation displace a fluorescently labeled FimH antagonist (see 11 in the Supporting Information)14 whose
fluorescence polarization depends on target binding. By running a ligand dilution series, the dissociation
constants could be determined and are summarized in Table 2. For 3f, a KD value of 515 nM was obtained,
which is in excellent agreement with the affinity measured by SPR (550 nM; Table 1). All bioisosteres except for
amide 10 exhibited a diminished affinity for FimHFL. Noteworthy, only when the benzoyl moiety of the
acylhydrazone was preserved as in hydrazide 5 and amide 10, a strong loss of affinity could be avoided.
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In summary, as evidenced by antagonist 10, and as described by Jumde et al.41 replacement by an amide
provides a good starting point for further optimization of acylhydrazones.
Table 2. Dissociation constant KD and relative KD (rKD) as determined with the fluorescece polarization assay.
KD
KD
Cpd.
Structure
rKD
Cpd.
Structure
rKD
[nM]
[nM]
OH
OH
O
HO
HO
OH
OH
F
6.7
9
O
O
HO
HO
4
3600 17
515.0
-
7
3498
2828
H
N
H
N
O
O
OH
OH
O
OH
OH
O
HO
HO
HO
HO
F
F
1.0
0
5.4
9
O
O
S
3f
8
O
N
N
H
N
H
N
H
OH
OH
OH
O
OH
O
HO
HO
HO
HO
F
F
1.5
2
3.2
4
O
O
5
6
783.6
1097
9
1673
O
H
N
H
N
H
N
N
H
S
OH
OH
O
OH
OH
O
HO
HO
HO
HO
F
F
2.1
3
0.8
7
O
O
10
447.5
O
O
H
N
N
N
H
H
O
Conclusions
In tdDCC experiments, the resulting composition is generally only qualitatively ranked in a “hit/no-hit”-
manner. One goal of this communication was to explore whether a tdDCC ranking correlated to an affinity
ranking that had been determined using surface plasmon resonance (SPR) experiments. We therefore
established a 21-membered acylhydrazone library using aldehyde scaffold 1 and the commercially available
hydrazide fragments 2a-u (→ 3a-u). TdDCC acylhydrazone libraries were generated both in the absence and
presence of biotinylated target protein FimHFL-B, and then analyzed using UV-HPLC. By calculating the
normalized changes of relative peak area (RPA) between template and blank libraries, the influence of FimHFL-B
could be assessed. Surprisingly, the library composition observed post-equilibration was influenced by the
acylhydrazones/FimHFL-B ratio (6:1, 4:1, and 2:1), where differences between components became more
enhanced with greater relative amounts of FimHFL-B (i.e. up- or down-regulation was more pronounced). When
the tdDCC results were qualitatively ranked in a “hit/no-hit”-manner, the majority of amplified acylhydrazones
indeed also exhibited high affinities in SPR experiments, whereas lower KDs correlated with down-regulated
compounds. Next, when the normalized changes of RPA were plotted against KD values, a linear correlation
was observed. The best alignment was obtained from the libraries with a 2:1 ratio of acylhydrazone to protein
target, but the correlation was diminished when libraries with the 4:1 and 6:1 ratios were evaluated. These
results suggest that a stoichiometric ratio between library constituents and target protein would be ideal for
the generation of libraries in which all members exhibit affinity. However, different ratios are conceivable for
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libraries, which cover a wider range of affinities. In a situation where only a few good binders are present,
their formation would more efficiently outcompete the others.
In subsequent efforts, replacement of the potentially hazardous acylhydrazone moiety with various
bioisosteres was investigated. Whereas urea (→ 6 and 7) and thiourea (→ 8 and 9) analogues of parent
compound 3f exhibited decreased affinities, hydrazide derivative 5 retained affinity and amide analogue 10
mildly enhanced the affinity, indicating that the latter two bioisosteres could represent a good starting point
for further optimization of acylhydrazone hits from tdDCC.
In summary, applying tdDCC to FimHFL-B using acylhydrazone libraries of structurally related mannosides
successfully confirmed the high sensitivity of this approach. Most importantly, a linear association between
the normalized change of RPA and the KD values determined by SPR could be observed.
Experimental Section
General. Affinity values were determined using a Biacore T200 system (GE Healthcare). UV-HPLC
measurements were made using an Agilent 1100/1200 system (Agilent). FPA was measured on a Synergy H1
hybrid multimode microplate reader (BioTek Instruments Inc., Winooski, VT, USA).
FPA,14,17 protein production,21 SPR measurements,21 and tdDCC experiments21 were conducted as previously
described. UV-HPLC analysis of libraries: Column: Waters Altlantis T3, 3 µm, 2.1 x 150 mm (Waters
Corporation), solvent A: H2O + 0.01% TFA (trifluoroacetic acid); solvent B: MeCN + 0.01% TFA. Detection: UV
absorption at 310 nm. Gradient: 5% → 25% B (35 min) → 50% B (65 min), flow rate: 0.3 mL/min, injection
volume: 20 µL.
Synthesis
General. NMR spectra were recorded on a Bruker Avance DMX-500 (500.1 MHz) spectrometer. Assignment of
1H NMR and 13C NMR spectra was achieved using 2D methods (COSY, HSQC). Chemical shifts are expressed in
ppm using residual CHCl3, MeOH, or DMSO as references. Optical rotations were measured with a PerkinElmer
Polarimeter 341 and infrared spectra were measured on a PerkinElmer Spectrum One FT-IR Spectrometer.
Electrospray ionization mass spectrometry (ESI-MS) data were obtained on a Waters Micromass ZQ
instrument. High resolution (HR)MS analysis were carried out using an Agilent 1100 LC equipped with a
photodiode array detector and a Micromass QTOF I equipped with a 4 GHz digital-time converter. Reactions
were monitored by thin layer chromatography (TLC) using glass plates coated with silica gel 60 F254 (Merck)
and visualized by UV light and/or by charring with a molybdate solution (0.02 M solution of ammonium cerium
sulfate dihydrate and ammonium molybdate tetrahydrate in aqueous 10% H2SO4). Medium pressure liquid
chromatography (MPLC) separations were carried out on a CombiFlash Rf
with RediSep
(Teledyne ISCO, Inc.)
disposable normal-phase or RP-18 (LiChroprepRP18, Merck) reversed-phase flash columns. Commercially
available reagents were purchased from Fluka, Aldrich, Alfa Aesar, Fluorochem, and Apollo. Solvents were
purchased from Sigma-Aldrich, Acros Organics, or VWR.
Synthesis of the acylhydrazone library
General procedure A for acylhydrazone formation. A flask was charged with a magnetic stirrer, then aldehyde
121 and hydrazide 2a,c-e,g-h,j-t were dissolved in H2O/MeCN (2 mL, 7:3). AcOH (100 µL) was added and the
mixture was stirred at r.t. for 5-22 h until only product was detected by MS. Then, the mixture was neutralized
with 1 M aq. NaOH and the solvents were removed under reduced pressure. The residue was purified by MPLC
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on RP-18 (H2O/MeCN, 5:95 to 20:80) to give the desired products 3a,c-e,g-h,j-t. For synthesis of 3b, 3f, 3i and
3u, please see reference 21.
Note: Compounds 3a, 3d, and 3l were obtained as inseparable mixtures of E- and Z-isomers, with the E-isomer
most likely representing the bigger fraction due to its sterically more favorable conformation. For further
evaluation, we conducted a high temperature NMR measurement (CD3OD, 60 °C) with compound 3l, which
clearly showed a decreased resolution, suggesting faster conversion of the two isomers. Further, HPLC traces
at concentrations similar to the DCC experiments showed only one peak. When the reaction was catalyzed by
aniline instead of AcOH, the same E/Z-ratio was obtained. Thus, the ratio of isomers is expected to be similar
in tdDCC and SPR experiments.
N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]nicotinohydrazide (3a). Prepared according to general
procedure A from aldehyde 1 (10 mg, 33.1 µmol) and nicotinic hydrazide (2a, 4.5 mg, 33.1 µmol). Yield: 8.1 mg
(58%) as an inseparable mixture of E- and Z-isomers (approx. 5:1). NMR data are given for the E-conformer.
[]2D0
1
+129.0 (c 0.35, MeOH); H NMR (500 MHz, (CD3)2SO):  = 3.39–3.54 (m, 4H, H-4, H-5, H-6a, H-6b), 3.60
(dd, J 3.8, 10.6 Hz, 1H), 3.69 (d, J 7.8 Hz, 1H, H-3), 3.88 (s, 1H, H-2), 4.47 (s, 1H, OH-4), 4.89 (m, 2H, OH-3, OH-
6), 5.14 (s, 1H, OH-2), 5.50 (s, 1H, H-1), 7.45 (t, J 8.3 Hz, 1H, Ar-H), 7.50 (d, J 8.6 Hz, 1H, Ar-H), 7.52–7.58 (m,
1H, Ar-H), 7.61 (d, J 11.9 Hz, 1H, Ar-H), 8.25 (d, J 7.7 Hz, 1H, Ar-H), 8.38 (s, 1H, HC=N), 8.75 (d, J 4.3 Hz, 1H, Ar-
H), 9.06 (s, 1H, Ar-H), 12.05 (s, 1H, NH); 13C NMR (125 MHz, (CD3)2SO):  60.9 (C-6), 66.5 (C-4), 69.8 (C-2), 70.5
(C-3), 75.5 (C-5), 99.7 (C-1), 113.9 (d, JC,F 20 Hz), 118.6, 123.6, 124.3, 129.1 (d, JC,F 6 Hz), 129.4, 135.4, 145.4
(d, JC,F 11 Hz; 8C, Ar-C), 146.9 (d, JC,F 2 Hz, HC=N), 148.6, 152.1 (2C, Ar-C), 152.5 (d, JC,F 245 Hz, Ar-C), 161.8
(C=O); IR (KBr):  3429 (vs, OH, NH), 1652 (vs, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C19H20FN3NaO7
[M+Na]+: 444.1183, found: 444.1181.
(E)-N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-2-methylthiazole-5-carbohydrazide (3c). Prepared
according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 2-methyl-thiazole-4-carboxylic acid
[]2D0
1
hydrazide (2c, 5.2 mg, 33.1 µmol). Yield: 8.4 mg (57%).
+117.7 (c 0.24, MeOH); H NMR (500 MHz,
(CD3)2SO):  2.75 (s, 3H, CH3), 3.41 (m, 1H, H-5), 3.44–3.54 (m, 2H, H-6a, H-4), 3.60 (dd, J 5.7, 11.2 Hz, 1H, H-
6b), 3.88 (s, 1H, H-3), 3.69 (s, 1H, H-2), 4.46 (t, J 5.7 Hz, 1H, OH-6), 4.82 (d, J 5.4 Hz, 1H, OH-3), 4.87 (d, J 5.5 Hz,
1H, OH-4), 5.11 (d, J 3.3 Hz, 1H, OH-2), 5.49 (s, 1H, H-1), 7.38–7.49 (m, 2H, Ar-H), 7.55 (d, J 11.7 Hz, 1H, Ar-H),
8.29 (s, 1H, Ar-H), 8.51 (s, 1H, HC=N), 11.79 (s, 1H, NH); 13C NMR (125 MHz, (CD3)2SO):  18.8 (CH3), 60.9 (C-6),
66.5 (C-4), 69.8 (C-2), 70.6 (C-3), 75.5 (C-5), 99.7 (C-1), 113.7 (d, JC,F 20 Hz), 118.6, 124.4 (d, JC,F 2 Hz), 125.4,
129.3 (d, J 7 Hz), 145.3 (d, JC,F 11 Hz; 6C, Ar-C), 147.1 (HC=N), 148.3, 152.5 (d, JC,F 245 Hz), 156.9 (3C, Ar-C),
166.5 (C=O); IR (KBr):  3413 (vs, OH, NH), 1659 (s, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C18H20FN3NaO7S
[M+Na]+: 464.0904, found: 464.0905.
N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-2,4-dimethylthiazole-5-carbohydrazide (3d). Prepared
according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 2,4-dimethyl-thiazole-5-carboxylic
acid hydrazide (2d, 5.7 mg, 33.1 µmol). Yield: 3.2 mg (21%) as a 5:1 mixture of E- and Z-conformer. NMR data
[]20
are given for the E-conformer.
+110.5 (c 0.11, MeOH); 1H NMR (500 MHz, (CD3)2SO):  2.51, 2.66 (2 s, 6H,
D
2 CH3), 3.41 (d, J 7.8 Hz, 1H, H-5), 3.43–3.55 (m, 2H, H-4, H-6a), 3.60 (dd, J 5.3, 11.1 Hz, 1H, H-6b), 3.68 (d, J 8.4
Hz, 1H, H-3), 3.88 (s, 1H, H-2), 4.46 (t, J 5.7 Hz, 1H, OH-6), 4.85 (s, 1H, OH-3), 4.88 (d, J 12.0 Hz, 1H, OH-4), 5.13
(s, 1H, OH-2), 5.49 (s, 1H, H-1), 7.45 (m, 1H, Ar-H), 7.50 (d, J 7.2 Hz, 1H, Ar-H), 7.57 (d, J 11.7 Hz, 1H, Ar-H), 8.02
(s, 1H, HC=N), 11.72 (s, 1H, NH); 13C NMR (125 MHz, (CD3)2SO): 18.5 (2C, 2 CH3), 60.9 (C-6), 66.5 (C-4), 69.8 (C-
2), 70.6 (C-3), 75.5 (C-5), 99.7 (C-1), 114.2 (d, JC,F 19 Hz), 118.8, 124.2 (d, JC,F 3 Hz), 128.9, 142.3, 145.2 (d, JC,F
11 Hz; 6C, Ar-C), 150.5 (HC=N), 152.4 (d, JC,F 245 Hz), 160.3, 162.2 (3C, Ar-C), 170.2 (C=O); IR (KBr):  3436
(OH, NH, vs), 1646 (s, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C19H22FN3NaO7S [M+Na]+: 478.1060, found:
478.1061.
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(E)-6-Chloro-N’-[3-fluoro-4-(α-D-mannopyranosyloxy)benzylidene]nicotinohydrazide
(3e).
Prepared
according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 6-chloropyridine-3-carbohydrazide
[]2D0
1
(2e, 5.7 mg, 33.1 µmol). Yield: 3.8 mg (25%).
+74.1 (c 0.30, MeOH); H NMR (500 MHz, (CD3)2SO): 
3.38–3.60 (m, 4H, H-6a, H-6b, H-5, H-4), 3.68 (d, J 9.1 Hz, 1H, H-3), 3.88 (s, 1H, H-2), 5.47 (s, 1H, H-1), 7.41 (m,
1H, Ar-H), 7.45 (d, J 8.6 Hz, 1H, Ar-H), 7.60 (s, 1H, Ar-H), 7.62 (d, J 3.0 Hz, 1H, Ar-H), 8.35 (d, J 8.3 Hz, 1H,
13
HC=N), 8.37 (d, J 9.6 Hz, 1H, Ar-H), 8.93 (s, 1H, Ar-H); C NMR (125 MHz, (CD3)2SO):  60.9 (C-6), 66.4 (C-4),
69.8 (C-2), 70.5 (C-3), 75.5 (C-5), 99.8 (C-1), 113.7 (d, JC,F 20 Hz), 118.50, 123.8, 124.1, 138.9 (8C, Ar-C), 147.0
(HC=N), 149.3, 151.9, 152.4 (d, JC,F 244 Hz; 3C, Ar-C), 174.0 (C=O); IR (KBr):  3436 (vs, OH, NH), 1634 (s, C=N-
NH-C=O) cm-1; HRMS: m/z: Calcd for C19H19ClFN3NaO7 [M+Na]+: 478.0793, found: 478.0799.
(E)-N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-1-methyl-1H-pyrrole-2-carbohydrazide
(3g).
Prepared according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 1-methyl-1H-pyrrole-2-
[]20
1
carboxylic acid hydrazide (2g, 4.6 mg, 33.1 µmol). Yield: 9.5 mg (68%).
+132.5 (c 0.61, MeOH); H NMR
D
(500 MHz, CD3OD):  3.65 (m, 1H, H-5), 3.69–3.81 (m, 3H, H-4, H-6a, H-6b), 3.93 (dd, J 3.1, 9.6 Hz, 1H, H-3),
3.96 (s, 3H, CH3), 4.08 (s, 1H, H-2), 5.56 (s, 1H, H-1), 6.13 (s, 1H, Ar-H), 6.93 (s, 2H, Ar-H), 7.41 (t, J 8.1 Hz, 1H,
Ar-H), 7.45 (d, J 8.6 Hz, 1H, Ar-H), 7.71 (d, J 11.9 Hz, 1H, Ar-H), 8.15 (s, 1H, HC=N); 13C NMR (125 MHz, CD3OD):
 37.0 (CH3), 62.6 (C-6), 68.2 (C-4), 71.8 (C-2), 72.3 (C-3), 76.0 (C-4), 101.2 (C-1), 108.7, 115.2 (d, JC,F 20 Hz),
119.5, 124.8, 125.7 (d, J 4 Hz), 130.7, 131.4 (d, JC,F 7 Hz; 7C, Ar-C), 146.7 (d, JC,F 6 Hz, HC=N), 147.0 (d, JC,F 11
Hz, Ar-C), 152.4 (C=O), 154.6 (d, JC,F 246 Hz, Ar-C); IR (KBr):  3436 (vs, OH, NH), 1634 (s, C=N-NH-C=O) cm-1;
HRMS: m/z: Calcd for C19H22FN3NaO7 [M+Na]+: 446.1339, found: 446.1341.
(E)-N’-(3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene)-2-methylfuran-3-carbohydrazide (3h). Prepared
according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 2-methyl-furan-carboxylic acid
[]2D0
1
hydrazide (2h, 4.6 mg, 33.1 µmol). Yield: 9.1 mg (65%).
+98.9 (c 0.56, MeOH); H NMR (500 MHz,
CD3OD):  2.63 (s, 3H, CH3), 3.67 (d, J 3.6 Hz, 1H, H-5), 3.72–3.83 (m, 3H, H-4, H-6a, H-6b), 3.95 (dd, J 2.8, 9.4
Hz, 1H, H-3), 4.10 (s, 1H, H-2), 5.59 (s, 1H, H-1), 6.85 (s, 1H, Ar-H), 7.42–7.52 (m, 3H, Ar-H), 7.76 (d, J 11.9 Hz,
1H, Ar-H), 8.22 (s, 1H, HC=N); 13C NMR (125 MHz, CD3OD):  13.7 (CH3), 62.6 (C-6), 68.2 (C-4), 71.7 (C-2), 72.3
(C-3), 76.0 (C-5), 101.2 (C-1), 109.4, 115.0, 115.3 (d, JC,F = 20 Hz), 119.4, 126.0, 142.1, 147.3 (d, JC,F 11 Hz; 8C,
Ar-C), 147.9 (d, JC,F 1 Hz, HC=N), 154.6 (d, JC,F 246 Hz), 160.0 (2C, Ar-C), 163.1 (C=O); IR (KBr):  3414 (vs, OH,
NH), 1619 (vs, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C19H21FN2Na O8 [M+Na]+: 447.1182, found: 447.1182.
(E)-N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-4-methoxybenzhydrazide (3j). Prepared according
to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 4-methoxybenzhydrazide (2j, 5.5 mg, 33.1
[]2D0
1
µmol). Yield: 4.0 mg (27%).
+106.6 (c 0.26, MeOH); H NMR (500 MHz, CD3OD):  3.64 (m, 1H, H-5),
3.69–3.81 (m, 3H, H-4, H-6a, H-6b), 3.88 (s, 3H, CH3), 3.93 (dd, J 3.0, 9.4 Hz, 1H, H-3), 4.08 (s, 1H, H-2), 5.57 (s,
1H, H-1), 7.05 (d, J 8.5 Hz, 2H, Ar-H), 7.43 (t, J 8.2 Hz, 1H, Ar-H), 7.49 (d, J 8.5 Hz, 1H, Ar-H), 7.77 (d, J 11.9 Hz,
1H, Ar-H), 7.92 (d, J 8.6 Hz, 2H, Ar-H), 8.25 (s, 1H, HC=N); 13C NMR (125 MHz, CD3OD):  56.0 (CH3), 62.6 (C-6),
68.2 (C-4), 71.7 (C-2), 72.3 (C-3), 76.0 (C-5), 101.2 (C-1), 115.0 (2C), 115.4 (d, JC,F 20 Hz), 119.5, 125.96, 126.07
(d, JC,F 2 Hz; 6C, Ar-C), 130.7 (2C), 131.1 (d, JC,F 7 Hz), 147.3 (d, JC,F 10 Hz; 4C, Ar-C), 148.4 (HC=N), 154.6 (d, JC,F
246 Hz, Ar-C), 164.5 (C=O), 166.7 (Ar-C); IR (KBr):  3439 (vs, OH, NH), 1648 (s, C=N-NH-C=O) cm-1; HRMS: m/z:
Calcd for C21H23FN2NaO8 [M+Na]+: 473.1336, found: 473.1336.
(E)-N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-1H-indole-2-carbohydrazide
(3k).
Prepared
according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 1H-indole-2-carboxylic acid
[]2D0
1
hydrazide (2k, 5.8 mg, 33.1 µmol). Yield: 7.7 mg (51%).
+100.9 (c 0.47,MeOH); H NMR (500 MHz,
CD3OD):  3.65 (m, 1H, H-5), 3.70–3.82 (m, 3H, H-4, H-6a, H-6b), 3.93 (dd, J 3.0, 9.4 Hz, 1H, H-3), 4.09 (s, 1H, H-
2), 5.56 (s, 1H, H-1), 7.15–7.27 (m, 2H, Ar-H), 7.38–7.50 (m, 3H, Ar-H), 7.73 (s, 1H, Ar-H), 8.06 (d, J 6.4 Hz, 1H,
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Ar-H), 8.17 (s, 1H, HC=N), 8.22 (d, J 7.1 Hz, 1H, Ar-H); 13C NMR (125 MHz, CD3OD):  62.6 (C-6), 68.2 (C-4), 71.8
(C-2), 72.3 (C-3), 76.0 (C-5), 101.2 (C-1), 112.8, 115.2 (d, JC,F 19 Hz), 119.5, 122.25, 122.31, 123.9, 125.7, 127.6,
127.7, 129.6, 131.5 (d, JC,F 7 Hz), 138.0 (12C, Ar-C), 146.2 (HC=N), 147.0 (d, JC,F 11 Hz), 154.6 (d, JC,F 246 Hz; 2C,
Ar-C), 157.3 (C=O); IR (KBr):  3415 (vs, OH, NH), 1619 (s, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for
C22H22FN3NaO7 [M+Na]+: 482.1339, found: 482.1340.
N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-2-(1H-indol-2-yl)acetohydrazide
according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and indole-3-acetic hydrazide (2l, 6.3
[]20
(3l).
Prepared
mg, 33.1 µmol). Yield: 12.8 mg (82%) as a 2:1 mixture of E- and Z-conformation.
+99.1 (c 0.39, MeOH); 1H
D
NMR (500 MHz, CD3OD; E:Z = 2:1, normalized to E conformation):  3.61–3.68 (m, 1.5H, H-5 E+Z), 3.70–3.81
(m, 6.5H, CH2 E, H-6a E+Z, H-6b E+Z, H-4 E+Z), 3.91–3.96 (m, 1.5H, H-3 E+Z), 4.08 (dd, J 1.9, 3.3 Hz, 1H, H-2 E),
4.10 (dd, J 1.9, 3.4 Hz, 0.5H, H-3 Z), 4.20 (s, 1H, CH2 Z), 5.57 (d, J 1.6 Hz, 1H, H-1 E), 5.58 (d, J 1.6 Hz, 0.5H, H-1
Z), 6.99–7.07 (m, 2H, Ar-H E+Z), 7.08–7.15 (m, 1.5H, Ar-H E+Z), 7.21 (s, 0.5H, Ar-H Z), 7.25 (s, 1H, Ar-H E), 7.47–
7.33 (m, 4.5H, 3 Ar-H E, 3 Ar-H Z), 7.57 (d, J 12.6 Hz, 0.5H, Ar-H Z), 7.61 (d, J 7.9 Hz, 1H, Ar-H E), 7.65 (d, J 8.0
13
Hz, 0.5H, Ar-H Z), 7.70 (dd, J 1.6, 12.0 Hz, 1H, Ar-H E), 7.91 (s, 0.5H, CH=N Z), 8.06 (s, 1H, CH=N E); C NMR
(125 MHz, CD3OD; only E conformer):  32.7 (CH2), 62.6 (C-6), 68.2 (C-4), 71.8 (C-2), 72.3 (C-3), 76.0 (C-5),
101.2 (C-1), 126.1, 124.9, 122.6, 122.4, 120.0, 119.4, 115.5, 112.4, 101.4, 148.1 (16 C, 14 Ar-C, HC=N, C=O); IR
(KBr):  3429 (vs, OH, NH), 1651 (s, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C23H24FN3NaO7 [M+Na]+:
496.1496 found: 496.1496.
(E)-N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-4-methylbenzohydrazide (3m). Prepared according
to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and p-toluic hydrazide (2m, 5.0 mg, 33.1 µmol).
[]20
Yield: 7.7 mg (54%).
+104.2 (c 0.47, MeOH); 1H NMR (500 MHz, CD3OD):  2.42 (s, 3H, CH3), 3.65 (d, J 5.4
D
Hz, 1H, H-5), 3.70–3.81 (m, 3H, H-4, H-6a, H-6b), 3.93 (d, J 9.3 Hz, 1H, H-3), 4.08 (s, 1H, H-2), 5.57 (s, 1H, H-1),
7.34 (d, J 7.8 Hz, 2H, Ar-H), 7.43 (t, J 8.2 Hz, 1H, Ar-H), 7.49 (d, J 8.5 Hz, 1H, Ar-H), 7.76 (d, J 11.9 Hz, 1H, Ar-H),
7.83 (d, J 7.7 Hz, 2H, Ar-H), 8.25 (s, 1H, HC=N); 13C NMR (125 MHz, CD3OD):  21.5 (CH3), 62.6 (C-6), 68.2 (C-4),
71.7 (C-2), 72.3 (C-3), 76.0 (C-5), 101.2 (C-1), 115.4 (d, JC,F 21 Hz), 119.4, 126.1, 128.8 (2C), 130.4 (2C), 131.0 (d,
JC,F 7 Hz), 131.2, 144.3, 147.4 (d, JC,F 12 Hz; 11C, Ar-C), 148.8 (HC=N), 154.6 (d, JC,F 246 Hz, Ar-C), 167.1 (C=O); IR
(KBr):  3421 (vs, OH, NH), 1651 (s, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C21H23FN2NaO7 [M+Na]+:
457.1387, found: 457.1387.
(E)-N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-5-methylthiophene-2-carbohydrazide
(3n).
Prepared according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 5-methyl-thiophene-2-
[]20
1
carboxylic acid hydrazide (2n, 5.2 mg, 33.1 µmol). Yield: 7.8 mg (54%).
+156.0 (c 0.47, MeOH); H NMR
D
(500 MHz, (CD3)2SO):  2.52 (s, 3H, CH3), 3.39–3.55 (m, 3H, H-4, H-5, H-6a), 3.60 (dd, J 4.9, 10.9 Hz, 1H, H-6b),
3.69 (d, J 8.9 Hz, 1H, H-3), 3.88 (s, 1H, H-2), 4.46 (t, J 5.8 Hz, 1H, OH-6), 4.83 (d, J 3.6 Hz, 1H, OH-3), 4.87 (t, J
8.4 Hz, 1H, OH-4), 5.12 (d, J 3.0 Hz, 1H, OH-2), 5.50 (s, 1H, H-1), 6.92 (s, 1H, Ar-H), 7.39–7.66 (m, 3H, Ar-H),
7.78 (m, 1H, Ar-H), 8.18 (s, 1H, HC=N), 11.75 (s, 1H, NH); 13C NMR (125 MHz, (CD3)2SO):  15.1 (CH3), 60.9 (C-6),
66.5 (C-5), 69.9 (C-2), 70.6 (C-3), 75.5 (C-4), 99.7 (C-1), 114.03 (d, JC,F 38 Hz), 118.8, 124.2, 125.4, 129.2, 135.1,
142.5, 145.49 (d, JC,F 79 Hz), 152.5 (d, JC,F 245 Hz), 161.2 (11C, 10 Ar-C, HC=N), 166.4 (C=O); IR (KBr):  3413
(vs, OH, NH), 1619 (vs, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C19H21FN2NaO7S [M+Na]+: 463.0951, found:
463.0954.
(E)-4-Chloro-N’-[3-fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-carbohydrazide (3o). Prepared according
to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 4-chlorobenzhydrazide (2o, 5.6 mg, 33.1
[]20
1
µmol). Yield: 4.5 mg (30%).
+112.4 (c 0.24, MeOH); H NMR (500 MHz, CD3OD):  = 3.63 (ddd, J 2.2, 5.3,
D
9.5 Hz, 1H, H-5), 3.69–3.82 (m, 3H, H-4, H6a, H-6b), 3.93 (dd, J 3.4, 9.4 Hz, 1H, H-3), 4.08 (dd, J 1.7, 3.1 Hz, 1H,
H-2), 5.57 (s, 1H, H-1), 7.44 (t, J 8.3 Hz, 1H, Ar-H), 7.50 (d, J 9.2 Hz, 1H, Ar-H), 7.54 (d, J 8.5 Hz, 2H, Ar-H), 7.76
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(m, 1H, Ar-H), 7.92 (d, J 8.5 Hz, 2H, Ar-H), 8.26 (s, 1H, HC=N); 13C NMR (125 MHz, CD3OD):  62.6 (C-6), 68.2 (C-
4), 71.7 (C-2), 72.3 (C-3), 76.0 (C-5), 101.1 (C-1), 115.5 (d, JC,F 20 Hz), 119.4, 126.2, 130.0 (2C), 130.5 (2C), 130.8
(d, JC,F 7 Hz), 132.8, 139.5, 147.5 (d, JC,F 11 Hz; 11C, Ar-C), 149.3 (HC=N), 154.6 (d, JC,F 246 Hz, Ar-C), 165.9
(C=O); IR (KBr):  3436 (vs, OH, NH), 1651 (s, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C20H20ClFN2NaO7
[M+Na]+: 477.0841, found: 477.0841.
(E)-3-Chloro-N’-[(3-fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-4-methylthiophene-2-carbohydrazide
(3p). Prepared according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 3-chloro-4-methyl-2-
[]20
thiophenecarboxylic acid hydrazide (2p, 6.3 mg, 33.1 µmol). Yield: 5.4 mg (34%).
+90.7 (c 0.15, MeOH);
D
1H NMR (500 MHz, (CD3)2SO):  2.20 (s, 3H, CH3), 3.37–3.55 (m, 3H, H-6a, H-4, H-5), 3.60 (dd, J 3.6, 11.2 Hz, 1H,
H-6b), 3.68 (dd, J 2.9, 9.0 Hz, 1H, H-3), 3.87 (s, 1H, H-2), 4.47 (s, 1H, OH-6), 4.90 (s, 2H, OH-3, OH-4), 5.13 (s,
1H, OH-2), 5.49 (s, 1H, H-1), 7.40–7.48 (m, 2H, Ar-H), 7.56 (d, J 11.9 Hz, 1H, Ar-H), 7.65 (m, 1H, Ar-H), 8.06 (m,
1H, Ar-H), 11.77 (s, 1H, NH); 13C NMR (125 MHz, (CD3)2SO):  14.3 (CH3), 60.9 (C-6), 66.5 (C-4), 69.8 (C-2), 70.5
(C-3), 75.5 (C-5), 99.7 (C-1), 113.9 (d, JC,F 20 Hz), 118.6 (d, JC,F 6 Hz), 124.3, 129.1, 131.2, 136.6, 143.6, 143.8 (d,
JC,F 9 Hz), 145.3 (9C, Ar-C), 146.8 (HC=N), 152.4 (d, JC,F = 245 Hz, Ar-C), 171.2 (C=O); IR (KBr):  3430 (vs, OH,
NH), 1642 (s, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C19H20ClFN2NaO7S [M+Na]+: 497.0561, found:
497.0561.
(E)-5-Chloro-N’-[3-fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-thiophene-2-carbohydrazide
(3q).
Prepared according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 5-chlorothiophene-2-
[]20
1
carboxylic acid hydrazide (2q, 5.6 mg, 33.1 µmol). Yield: 3.9 mg (26%).
+138.3 (c 0.15, MeOH); H NMR
D
(500 MHz, (CD3)2SO):  3.40–3.56 (m, 3H, H-4, H-5, H-6a), 3.62 (dd, J 4.1, 11.2 Hz, 1H, H-6b), 3.70 (dd, J 3.0, 9.1
Hz, 1H, H-3), 3.90 (s, 1H, H-2), 4.49 (t, J 5.6 Hz, 1H, OH-4), 4.89 (m, 2H, OH-3, OH-6), 5.15 (s, 1H, OH-2), 5.53 (s,
1H, H-1), 7.29 (d, J 4.1 Hz, 1H, Ar-H), 7.43–7.69 (m, 3H, Ar-H), 8.39 (m, 2H, Ar-H, HC=N), 12.00 (s, 1H, NH); 13C
NMR (125 MHz, (CD3)2SO):  61.0 (C-6), 66.5 (C-5), 69.8 (C-2), 70.5 (C-3), 75.5 (C-4), 99.7 (C-1), 114.4 (d, JC,F 19
Hz), 118.8, 124.4, 126.6, 128.6 (d, JC,F 6 Hz), 130.4, 134.5, 137.3 (8C, Ar-C), 143.5 (HC=N), 145.4 (d, JC,F 11 Hz),
152.4 (d, JC,F 245 Hz; 2C, Ar-C), 160.0 (C=O); IR (KBr):  3436 (vs, OH, NH), 1651 (s, C=N-NH-C=O) cm-1; HRMS:
m/z: Calcd for C18H18ClFN2NaO7S [M+Na]+: 483.0405, found: 483.0406.
(E)-N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-2-naphthohydrazide (3r). Prepared according to
general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 2-naphthhydrazide (2r, 6.2 mg, 33.1 µmol).
[]20
Yield: 3.9 mg (25%).
+81.6 (c 0.16, MeOH); 1H NMR (500 MHz, CD3OD):  3.65 (m, 1H, H-5), 3.69–3.82 (m,
D
3H, H-4, H-6a, H-6b), 3.93 (dd, J 2.1, 9.4 Hz, 1H, H-3), 4.09 (s, 1H, H-2), 5.58 (s, 1H, H-1), 7.45 (t, J 8.2 Hz, 1H,
Ar-H), 7.53 (d, J 8.5 Hz, 1H, Ar-H), 7.57–7.66 (m, 2H, Ar-H), 7.80 (d, J 11.9 Hz, 1H, Ar-H), 7.94–8.06 (m, 4H, Ar-
H), 8.32 (s, 1H, HC=N), 8.50 (s, 1H, Ar-H); 13C NMR (125 MHz, CD3OD):  62.6 (C-6), 68.2 (C-4), 71.8 (C-2), 72.3
(C-3), 76.1 (C-5), 101.2 (C-1), 115.5 (d, JC,F = 20 Hz), 119.5, 125.0, 126.2, 128.1, 128.9, 129.2, 129.5, 129.6,
130.1, 131.0 (d, JC,F 7 Hz), 131.3, 134.0, 136.6, 147.5 (d, JC,F 11 Hz; 15C, Ar-C), 149.0 (HC=N), 154.6 (d, JC,F 246
Hz, Ar-C), 167.1 (C=O); IR (KBr):  3422 (vs, OH, NH), 1651 (s, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for
C24H23FN2NaO7 [M+Na]+: 493.1387 found: 493.1388.
(E)-N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]-4-(trifluoromethyl)benzo-hydrazide (3s). Prepared
according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 4-(trifluoromethyl)benzhydrazide
[]20
(2s, 6.8 mg, 33.1 µmol). Yield: 8.6 mg (53%).
+98.8 (c 0.39, MeOH); 1H NMR (500 MHz, CD3OD):  3.64 (d,
D
J 6.2 Hz, 1H, H-5), 3.69–3.81 (m, 3H, H-4, H-6a, H-6b), 3.93 (dd, J 2.2, 9.3 Hz, 1H, H-3), 4.08 (s, 1H, H-2), 5.58 (s,
1H, H-1), 7.45 (t, J 8.2 Hz, 1H, Ar-H), 7.51 (d, J 8.5 Hz, 1H, Ar-H), 7.77 (d, J 11.8 Hz, 1H, Ar-H), 7.84 (d, J 8.0 Hz,
2H, Ar-H), 8.10 (d, J 8.0 Hz, 2H, Ar-H), 8.28 (s, 1H, HC=N); 13C NMR (125 MHz, CD3OD):  62.6 (C-6), 68.2 (C-4),
71.7 (C-2), 72.3 (C-3), 76.1 (C-5), 101.1 (C-1), 115.5 (d, JC,F 20 Hz), 119.4 (2C, Ar-C), 125.2 (q, JC,F 267 Hz, CF3),
126.3 (d, J 3 Hz), 126.7 (q, J 3 Hz, 2C), 129.6 (2C), 130.7 (d, JC,F 7 Hz), 134.6 (q, JC,F 33 Hz), 137.9, 147.6 (d, JC,F 11
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Hz; 9C, Ar-C), 149.7 (HC=N), 154.6 (d, JC,F 246 Hz, Ar-C), 165.6 (C=O); IR (KBr):  3430 (vs, OH, NH), 1663 (vs,
C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C21H20F4N2NaO7 [M+Na]+: 511.1104 found: 511.1107.
(E)-4-N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzylidene]benzo[b]thiophene-2-carbohydrazide
Prepared according to general procedure A from aldehyde 1 (10 mg, 33.1 µmol) and 3-chloro-
[]20
(3t).
benzo[b]thiophene acid hydrazide (2t, 7.5 mg, 33.1 µmol). Yield: 4.2 mg (25%).
+82.4 (c 0.18, MeOH); 1H
D
NMR (500 MHz, (CD3)2SO):  3.38–3.51 (m, 3H, H-6a, H-6b, H-5), 3.59 (s, 1H, H-4), 3.68 (s, 1H, H-3), 3.88 (s, 1H,
H-2), 4.47 (s, 1H, OH-4), 4.71–4.96 (m, 2H, OH-3, OH-6), 5.11 (s, 1H, OH-2), 5.49 (s, 1H, H-1), 7.35–7.69 (m, 5H,
Ar-H), 8.44–8.84 (m, 3H, 2 Ar-H, HC=N), 12.09 (s, 1H, NH); 13C NMR (125 MHz, (CD3)2SO):  60.9 (C-6), 66.5 (C-
4), 69.8 (C-2), 70.5 (C-3), 75.5 (C-5), 99.8 (C-1), 114.2 (d, JC,F 20 Hz), 118.6, 122.4, 122.6, 123.4, 124.4, 126.0,
127.4, 127.7, 132.6, 143.5, 145.4, 147.5, 152.5 (d, JC,F 245 Hz; 15C, Ar-C, HC=N), 160.2 (C=O); IR (KBr):  3460
(vs, OH, NH), 1656 (vs, C=N-NH-C=O) cm-1; HRMS: m/z: Calcd for C22H20ClFN2NaO7S [M+Na]+: 533.0561 found:
533.0562.
Synthesis of bioisosteres
General procedure B for deprotection of acetylated mannosides. Protected mannosides were dissolved in dry
MeOH (2 mL) under argon atmosphere and freshly prepared 1 M NaOMe (100 µL) was added. The mixtures
were stirred at r.t. for 30-45 min, until TLC (CH2Cl2/MeOH, 9:1) showed no remaining starting material. The
mixtures were neutralized with amberlite ion-exchange resin (H+-form, IR120, Sigma Aldrich), filtered, and
concentrated in vacuo. The residues were purified by MPLC (RP-18; H2O/MeCN, 95:5 to 20:80) to yield 60-82%
of the desired products.
OH
OH
O
OH
OH
O
HO
HO
HO
HO
F
F
a)
O
O
O
O
H
N
N
N
N
H
H
3f
5
Scheme 2. Reagents and conditions: a) NaBH3CN, aq. HCl., MeOH, r.t., 23 h, 95%.
N’-[3-Fluoro-4-(α-D-mannopyranosyloxy)benzyl]benzohydrazide (5). Compound 3f (4.5 mg, 10.7 µmol, 1 eq.)
was dissolved in MeOH (3 mL) and NaBH3CN (6.7 mg, 107 µmol, 10 eq.) and five drops of 36% aq. HCl. were
added. The mixture was flushed with argon for 5 min and stirred at r.t. After 23 h the mixture was neutralized
with solid NaOH and concentrated. The residue was purified by MPLC on RP-18 (H2O/MeCN, 95:5 to 20:80) to
[]20
give 5 (4.3 mg, 95%).
+66.3 (c 0.22, MeOH); 1H NMR (500 MHz, CD3OD):  3.64–3.79 (m, 4H, H-4, H-5, H-
D
6a, H-6b), 3.91 (dd, J 3.4, 9.3 Hz, 1H, H-3), 4.00 (s, 2H, CH2), 4.06 (dd, J 1.8, 3.3 Hz, 1H, H-2), 5.46 (d, J 1.5 Hz,
1H, H-1), 7.13 (d, J 8.4 Hz, 1H, Ar-H), 7.24 (dd, J 1.9, 11.9 Hz, 1H, Ar-H), 7.31 (t, J 8.4 Hz, 1H, Ar-H), 7.42 (t, J 7.5
Hz, 2H, Ar-H), 7.50 (m, 1H, Ar-H), 7.69–7.75 (m, 2H, Ar-H); 13C NMR (125 MHz, CD3OD):  = 55.5 (CH2), 62.6 (C-
6), 68.2 (C-4), 71.9 (C-2), 72.3 (C-3), 75.8 (C-5), 101.6 (C-1), 117.9 (d, JC,F = 19 Hz), 120.0, 126.2 (d, JC,F 3 Hz),
128.2, 129.5, 132.6, 134.5, 134.8 (d, JC,F 17 Hz), 144.8 (d, JC,F = 11 Hz), 154.5 (d, JC,F 245 Hz; 12C, Ar-C), 169.0
(C=O); IR (KBr):  3438 (vs, OH), 1646 (s, C=O) cm-1; HRMS: m/z: Calcd for C20H24FN2NaO7 [M+Na]+: 445.1387,
found: 445.1386.
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OAc
OAc
O
OAc
OAc
O
F
13
HO
OAc
OAc
O
AcO
AcO
AcO
AcO
b)
F
F
NO2
AcO
AcO
O
O
a)
OAc
NO2
NH2
12
14
15
OR
OR
O
OAc
OAc
O
RO
RO
AcO
AcO
F
F
c)
d)
O
O
NO2
O
O
N
N
N
O
H
H
H
16
R = Ac: 17
R = H: 6
e)
Scheme 3. Reagents and conditions: a) BF3∙Et2O, CH2Cl2, MS 4Å, 50 °C, 21 h, 39%; b) H2, Pd(OH)2, THF/ MeOH
(2:1), r.t., 2h, 75%; c) 4-nitrophenyl chloroformate, DIPEA, CH2Cl2, 2 h; d) benzylamine, DIPEA, THF, 2.5 h,
29% (over two steps); e) NaOMe, MeOH, r.t., 45 min, 70%.
2-Fluoro-4-nitrophenyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (14). In a two-necked flask, activated MS
4Å (ca. 500 mg), peracetylated D-mannose (12, 500 mg, 1.28 mmol, 1.2 eq.), 3-fluoro-4-nitrophenol (13, 168
mg, 1.07 mmol, 1.0 eq.), and dry CH2Cl2 (10 mL) were mixed and cooled down in an ice bath. Under argon
atmosphere, BF3∙Et2O (395 µL, 3.20 mmol, 3.0 eq.) was added dropwise and the reaction heated to 50 °C. The
mixture was refluxed for 21 h. Then it was cooled down to r.t., diluted with EtOAc, and filtered over celite. The
filtrate was subsequently washed with satd. aq. NaHCO3 and brine. The organic layer was dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified by MPLC on silica (petroleum ether/EtOAc, 1:0 to
[]20
4:6) to give 14 (241 mg, 39%).
+92.8 (c 1.00, CHCl3); 1H NMR (500 MHz, CDCl3):  2.03, 2.05, 2.07, 2.22 (4
D
s, 12H, 4Ac-CH3), 4.06–4.15 (m, 2H, H-5, H-6a), 4.27 (dd, J 5.9, 12.8 Hz, 1H, H-6b), 5.39 (t, J 9.9 Hz, 1H, H-4),
5.52 (dd, J 1.8, 3.5 Hz, 1H, H-2), 5.55 (dd, J 3.5, 9.9 Hz, 1H, H-3), 5.65 (d, J 1.5 Hz, 1H, H-1), 7.36 (m, 1H, Ar-H),
8.02–8.08 (m, 2H, Ar-H); 13C NMR (125 MHz, CDCl3):  20.63, 20.64, 20.65, 20.8 (4 Ac-CH3), 61.9 (C-6), 65.6 (C-
4), 68.4 (C-3), 68.9 (C-2), 70.2 (C-5), 96.9 (C-1), 113.0 (d, JC,F 23 Hz), 117.1 (d, JC,F 1 Hz), 120.5 (d, JC,F 4 Hz), 143.0
(d, JC,F 7 Hz), 148.8 (d, JC,F 11 Hz), 151.9 (d, JC,F 254 Hz; 6C, Ar-C), 169.6, 169.8, 169.9, 170.4 (4 C=O); ESI-MS:
m/z: Calcd for C20H22FNNaO12 [M+Na]+: 510.10, found: 510.14.
4-Amino-2-fluorophenyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (15). A two-necked flask equipped with
a magnetic stirrer was charged with 14 (241 mg, 0.527 mmol). Under argon atmosphere, THF/MeOH (2:1, 15
mL) and Pd(OH)2/C (25 mg) were added. The flask was evacuated five times and filled with H2. Under hydrogen
atmosphere, the mixture was stirred at r.t. until TLC (CH2Cl2/MeOH, 8:2) indicated completion of the reaction.
The mixture was filtered over celite and concentrated. Purification by MPLC (petroleum ether/EtOAc, 1:0 to
[]20
0:1) gave 15 (181 mg, 75%).
+74.5 (c 2.01, CHCl3); 1H NMR (500 MHz, CDCl3):  1.97, 2.00, 2.01, 2.12 (4 s,
D
12H, 4 Ac-CH3), 3.71 (s, 2H, NH2), 4.07 (dd, J 1.8, 12.1 Hz, 1H, H-6a), 4.18–4.28 (m, 2H, H-5, H-6b), 5.22 (s, 1H,
H-1), 5.29 (t, J 9.9 Hz, 1H, H-4), 5.44 (dd, J 1.7, 3.3 Hz, 1H, H-2), 5.47 (dd, J 3.4, 9.9 Hz, 1H, H-3), 6.29 (m, 1H, Ar-
13
H), 6.37 (dd, J 2.6, 12.5 Hz, 1H, Ar-H), 6.86 (t, J 8.8 Hz, 1H, Ar-H); C NMR (125 MHz, CDCl3):  20.46, 20.49,
20.6 (4C, 4 Ac-CH3), 62.1 (C-6), 65.8 (C-4), 68.7 (C-3), 69.2 (2C, C-3, C-5), 98.6 (d, JC,F 1 Hz, C-1), 103.3 (d, JC,F 22
Hz), 110.2 (d, JC,F 3 Hz), 121.4 (d, JC,F 2 Hz), 134.9 (d, JC,F 12 Hz), 144.1 (d, JC,F 10 Hz), 154.3 (d, JC,F 246 Hz; 6C, Ar-
C), 169.6, 169.7, 169.8, 170.4 (4 C=O); ESI-MS: m/z: Calcd for C20H24FNNaO10 [M+Na]+: 480.13, found: 480.13.
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4-Nitrophenyl (4’-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyloxy)-3’-fluorophenyl)carbamate (16). In a two-
necked flask, 4-nitrophenyl chloroformate (20.7 mg, 0.103 mmol, 1 eq.) was dissolved in dry CH2Cl2 (2 mL).
Under argon atmosphere, a solution of 15 (47 mg, 0.103 mmol, 1 eq.) and DIPEA (17.6 µL, 0.103 mmol, 1 eq.)
in dry CH2Cl2 (2 mL) was added dropwise. The mixture was stirred at r.t. for 2 h, until TLC (petroleum
ether/EtOAc, 1:1) showed completion of the reaction. The mixture was diluted with EtOAc and washed with 1
M aq. HCl. The organic layer was dried over Na2SO4, filtered, and concentrated to give 16 (quant.) which was
used without further purification. ESI-MS: m/z: Calcd for C27H27FN2NaO14 [M+Na]+: 645.13, found: 645.16.
1-Benzyl-3-[4’-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyloxy)-3’-fluorophenyl]urea (17). Crude 16 was
dissolved in dry THF (2 mL) and benzylamine (11.3 mg, 0.103 mmol, 1 eq.) and DIPEA (17.6 µL, 0.103 mmol, 1
eq.) were added. The reaction was stirred for 1.5 h, until TLC (CH2Cl2/MeOH, 9:1) indicated full consumption of
the starting material. The mixture was concentrated, dissolved in EtOAc, washed with 1 M aq. HCl and the
aqueous layer re-extracted with EtOAc (3x). The organic layer was dried over Na2SO4, filtered and the solvent
removed. Purification by MPLC (petroleum ether/EtOAc, 1:0 to 0:1) gave 17 (17.1 mg, 29% over two steps).
[]2D0
1
+35.8 (c 0.89, CHCl3); H NMR (500 MHz, CDCl3):  2.01, 2.02, 2.06, 2.18 (4 s, 12H, 4 Ac-CH3), 4.08 (dd, J
3.4, 13.2 Hz, 1H, H-6a), 4.21–4.28 (m, 2H, H-6b, H-5), 4.37 (d, J 5.7 Hz, 2H, CH2), 5.32–5.38 (m, 2H, H-4, H-1),
5.41 (t, J 5.6 Hz, 1H, NH), 5.49 (dd, J 1.8, 3.4 Hz, 1H, H-2), 5.52 (dd, J 3.5, 9.9 Hz, 1H, H-3), 6.91 (dd, J 1.2, 8.9
Hz, 1H, Ar-H), 6.94 (s, 1H, NH), 7.00 (t, J 8.8 Hz, 1H, Ar-H), 7.21–7.27 (m, 4H, Ar-H), 7.27–7.32 (m, 2H, Ar-H); 13C
NMR (125 MHz, CDCl3):  20.65, 20.66, 20.8 (4C, 4 Ac-CH3), 44.2 (CH2), 62.1 (C-6), 65.8 (C-3), 68.9 (C-2), 69.3
(C-4), 69.5 (C-5), 98.1 (C-1), 109.1 (d, JC,F 23 Hz), 115.6 (d, JC,F 3 Hz), 120.1 (d, JC,F 2 Hz), 127.4, 127.5, 128.7,
135.5 (d, JC,F 9 Hz), 138.6, 138.8 (d, JC,F 12 Hz), 153.5 (d, JC,F 247 Hz; 12C, Ar-C), 155.4 (NC=O), 169.8, 170.0,
170.7 (4C, 4 C=O); ESI-MS: m/z: Calcd for C28H31FN2Na O11 [M+Na]+: 613.18, found: 613.22.
1-Benzyl-3-[3’-fluoro-4’-(α-D-mannopyranosyloxy)phenyl]urea (6). Prepared according to general procedure
[]20
B from 17 (17.7 mg, 30.0 µmol). Yield: 8.8 mg (70%).
+109.9 (c 0.44, MeOH); 1H NMR (500 MHz, CD3OD):
D
 3.70–3.81 (m, 4H, H-4, H-5, H-6a, H-6b), 3.90 (m, 1H, H-3), 4.06 (dd, J 1.8, 3.3 Hz, 1H, H-2), 4.37 (s, 2H, CH2),
5.35 (d, J 1.7 Hz, 1H, H-1), 6.95 (ddd, J 1.3, 2.4, 8.9 Hz, 1H, Ar-H), 7.19–7.27 (m, 2H, Ar-H), 7.29–7.34 (m, 4H,
Ar-H), 7.38 (dd, J 2.5, 13.3 Hz, 1H, Ar-H); 13C NMR (125 MHz, CD3OD):  44.5 (CH2), 62.7 (C-6), 68.3 (C-4), 71.9
(C-2), 72.3 (C-3), 75.7 (C-5), 102.3 (C-1), 108.8 (d, JC,F 24 Hz), 115.7 (d, JC,F 3 Hz), 121.2 (d, JC,F 2 Hz), 128.1,
128.3, 129.6, 137.1 (d, JC,F 10 Hz), 140.4 (d, JC,F 11 Hz), 140.9, 154.7 (d, JC,F 243 Hz; 12C, Ar-C), 158.0 (C=O); IR
(KBr):  3347 (vs, OH), 1635 (s, C=O), 1515 (vs, NH) cm-1; HRMS: m/z: Calcd for C20H24FN2NaO7 [M+Na]+:
445.1387, found: 445.1392.
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F
OAc
OAc
O
OAc
OAc
O
HO
18
OAc
OAc
O
AcO
AcO
AcO
AcO
CN
b)
F
F
AcO
AcO
a)
O
O
OAc
NH2
12
19
20
CN
OR
OR
O
RO
RO
H
O
F
H2N
N
c)
20
O2N
O
d)
O
H
H
N
N
21
22
R = Ac: 23
R = H: 7
O
e)
Scheme 4. Reagents and conditions: a) BF3∙Et2O, CH2Cl2, MS 4Å, 50 °C, 29 h, 60%; b) H2, Pd(OH)2, THF/MeOH
(2:1), r.t., 2 h, 64%; c) 4-nitrophenyl chloroformate, DIPEA, CH2Cl2, r.t., 22 h, 53%; d) DIPEA, THF, r.t., 2 h,
19%; e)NaOMe/MeOH, r.t., 45 min, 83%.
4’-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyloxy)-3’-fluorobenzonitrile (19). In a two-necked flask,
activated MS 4Å (500 mg), peracetylated D-mannose (12, 500 mg, 1.28 mmol, 1.2 eq.), 3-fluoro-4-
hydroxybenzonitrile (18, 146 mg, 1.07 mmol, 1.0 eq.), and dry CH2Cl2 (10 mL) were mixed and cooled down in
an ice bath. Under argon atmosphere, BF3∙Et2O (395 µL, 3.20 mmol, 3 eq.) was added slowly and the reaction
heated to 50 °C. The mixture was refluxed for 29 h. Then, it was diluted with EtOAc, filtered over celite, and
subsequently washed with satd. aq. NaHCO3 and brine. The organic layer was dried over Na2SO4, filtered, and
[]20
concentrated. The residue was purified by MPLC (toluene/EtOAc, 1:0 to 6:4) to give 19 (356 mg, 60%).
D
+88.6 (c 1.00, CHCl3); 1H NMR (500 MHz, CDCl3):  2.03, 2.04, 2.07, 2.21 (4 s, 12H, 4 Ac-CH3), 4.06–4.14 (m, 2H,
H-5, H-6a), 4.27 (dd, J 6.0, 12.9 Hz, 1H, H-6b), 5.38 (t, J 9.9 Hz, 1H, H-4), 5.51 (dd, J 1.8, 3.4 Hz, 1H, H-2), 5.53
13
(dd, J 3.5, 9.9 Hz, 1H, H-3), 5.63 (d, J 1.4 Hz, 1H, H-1), 7.34 (t, J 8.3 Hz, 1H, Ar-H), 7.42–7.48 (m, 2H, Ar-H); C
NMR (125 MHz, CDCl3):  20.37, 20.39, 20.41, 20.5 (4 Ac-CH3), 61.7 (C-6), 65.4 (C-4), 68.2 (C-3), 68.7 (C-2), 69.9
(C-5), 96.6 (C-1), 107.0 (d, JC,F 8 Hz), 117.1 (d, JC,F 2 Hz), 120.3 (d, JC,F 22 Hz), 129.2 (d, JC,F 4 Hz), 147.2 (d, JC,F 11
Hz), 152.2 (d, JC,F 252 Hz; 7C, Ar-C, CN), 169.4, 169.5, 169.6, 170.1 (4 C=O); IR (KBr):  2232 (w, CN), 1751 (vs,
C=O) cm-1; ESI-MS: m/z: Calcd for C21H22FNNaO10 [M+Na]+: 490.11, found: 490.06.
4’-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyloxy)-3’-fluorophenylmethanamine (20). A two-neckeds flask
was charged with 19 (50 mg, 0.107 mmol) and a magnetic stirrer. Under argon atmosphere, THF/MeOH (6 mL,
2:1) and Pd(OH)2/C (15 mg) were added. The flask was evacuated five times and filled with H2. Under hydrogen
atmosphere, the mixture was stirred at r.t. until TLC (CH2Cl2/MeOH, 8:2) indicated completion of the reaction.
The mixture was filtered over celite, and concentrated. Purification by MPLC (CH2Cl2/MeOH, 1:0 to 8:2) gave
[]20
1
20 (32.2 mg, 64%).
+93.4 (c 1.00, CHCl3); H NMR (500 MHz, CDCl3):  2.03, 2.04, 2.07, 2.20 (4 s, 12H, 4
D
Ac-CH3), 3.83 (s, 2H, CH2), 4.10 (d, J 10.3 Hz, 1H, H-6a), 4.22–4.30 (m, 2H, H-5, H-6b), 5.37 (t, J 10.0 Hz, 1H, H-
4), 5.46 (d, J 1.6 Hz, 1H, H-1), 5.52 (dd, J 1.8, 3.4 Hz, 1H, H-2), 5.57 (dd, J 3.5, 10.0 Hz, 1H, H-3), 7.02 (d, J 8.3 Hz,
1H, Ar-H), 7.09–7.16 (m, 2H, Ar-H); 13C NMR (125 MHz, CDCl3):  20.60, 20.63, 20.8 (4C, 4 Ac-CH3), 45.3 (CH2),
62.1 (C-6), 65.8 (C-4), 68.7 (C-3), 69.3 (C-2), 69.5 (C-5), 97.7 (C-1), 115.5 (d, JC,F 19 Hz), 119.4 (d, JC,F 1 Hz), 122.9
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(d, JC,F 3 Hz), 139.7 (d, JC,F 6 Hz), 141.9 (d, JC,F 11 Hz), 153.4 (d, JC,F 248 Hz; 6C, Ar-C), 160.7, 169.8, 169.8, 170.5
(4 C=O); ESI-MS: m/z: Calcd for C21H27FNO10 [M+H]+: 472.16, found: 472.13.
4-Nitrophenyl phenylcarbamate (22). To a mixture of aniline (21, 49.0 µL, 0.537 mmol) and 4-nitrophenyl
chloroformate (108 mg, 0.537 mmol) in THF (2 mL) was added DIPEA (91.9 µL, 0.537 mmol). The mixture was
stirred at r.t. After 22 h, TLC (toluene/EtOAc, 1:1) indicated no remaining starting materials. The mixture was
diluted with EtOAc and washed with 1 M aq. HCl. The aqueous layer was extracted with EtOAc, the combined
organic layers were dried over Na2SO4, filtered and concentrated. The residue was purified by MPLC
(toluene/EtOAc, 1:0 to 1:1) to give 22 (72.8 mg, 52%). 1H NMR (500 MHz, CDCl3):  7.06 (s, 1H, NH), 7.16 (t, J
7.4 Hz, 1H, Ar-H), 7.34–7.41 (m, 4H, Ar-H), 7.45 (d, J 7.8 Hz, 2H, Ar-H), 8.25–8.31 (m, 2H, Ar-H); 13C NMR (125
MHz, CDCl3):  119.0 (2C), 122.1 (2C), 124.6, 125.2 (2C), 129.3 (2C), 136.6, 145.1 (12C, Ar-C), 150.1 (C=O),
155.4 (Ar-C).
1-[4’-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyloxy)-3’-fluoro]-3-phenylurea (23). A mixture of 20 (50 mg,
107 µmol), DIPEA (18.2 µL, 107 µmol) and 22 (27.4 mg, 107 µmol) in dry THF (2 mL) was stirred at r.t. for 2 h,
until TLC (CH2Cl2/MeOH, 9:1) showed no remaining starting material. The mixture was diluted with EtOAc and
washed with 1 M aq. HCl. The aqueous layer was extracted three times with EtOAc, and the combined organic
layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by MPLC
[]20
1
(CH2Cl2/MeOH, 1:0 to 9:1) to give 23 (12.1 mg, 19%).
+51.2 (c 0.61, CHCl3); H NMR (500 MHz, CDCl3): 
D
= 2.02, 2.03, 2.06, 2.19 (4 s, 12H, 4 Ac-CH3), 4.08 (dd, J 2.0, 12.1 Hz, 1H, H-6a), 4.20 (ddd, J 2.0, 5.2, 10.0 Hz, 1H,
H-5), 4.25 (dd, J 5.2, 12.1 Hz, 1H, H-6b), 4.32 (d, J 5.9 Hz, 2H, CH2), 5.33–5.39 (m, 2H, H-4, NH), 5.42 (d, J 1.5 Hz,
1H, H-1), 5.49 (dd, J 1.8, 3.4 Hz, 1H, H-2), 5.54 (dd, J 3.5, 10.0 Hz, 1H, H-3), 6.80 (s, 1H, NH), 6.96 (d, J 8.4 Hz,
13
1H, Ar-H), 7.03 (dd, J 1.8, 11.4 Hz, 1H, Ar-H), 7.05–7.11 (m, 2H, Ar-H), 7.24–7.30 (m, 4H, Ar-H); C NMR (125
MHz, CDCl3):  20.6, 20.7, 20.8 (4C, 4 Ac-CH3), 43.2 (CH2), 62.1 (C-6), 65.8 (C-4), 68.8 (C-3), 69.2 (C-2), 69.6 (C-
5), 97.6 (C-1), 115.8 (d, JC,F 19 Hz), 119.4, 121.1, 123.3 (d, JC,F 4 Hz), 124.1, 129.3, 135.9 (d, JC,F 6 Hz), 138.2,
142.3 (d, JC,F 11 Hz), 153.4 (d, JC,F 249 Hz; 12C, Ar-C), 155.8 (C=O(NH)2), 169.7, 169.9, 170.0, 170.6 (4 C=O); ESI-
MS: m/z: Calcd for C28H31FN2NaO11 [M+Na]+: 613.18, found: 613.31.
1-(3’-Fluoro-4’-α-D-mannopyranosyloxy)-3-phenylurea (7). Prepared according to general procedure B from
[]20
23 (12.1 mg, 20.5 µmol). Yield: 7.2 mg (83%).
+88.1 (c 0.36, MeOH); 1H NMR (500 MHz, CD3OD):  3.66–
D
3.80 (m, 4H, H-4, H-5, H6a, H-6b), 3.91 (dd, J 3.4, 9.2 Hz, 1H, H-3), 4.06 (dd, J 1.8, 3.3 Hz, 1H, H-2), 5.44 (d, J 1.6
Hz, 1H, H-1), 6.97 (t, J 7.4 Hz, 1H, Ar-H), 7.07 (d, J 8.4 Hz, 1H, Ar-H), 7.11 (dd, J 1.9, 11.9 Hz, 1H, Ar-H), 7.21–
7.28 (m, 2H, Ar-H), 7.31 (t, J 8.4 Hz, 1H, Ar-H), 7.35 (dd, J 1.0, 8.5 Hz, 2H, Ar-H); 13C NMR (125 MHz, CD3OD): 
43.6 (CH2), 62.6 (C-6), 68.2 (C-4), 71.9 (C-2), 72.3 (C-3), 75.8 (C-5), 101.7 (C-1), 116.2 (d, JC,F 19 Hz), 120.3,
123.6, 124.3 (d, JC,F 3 Hz), 129.8, 137.0 (d, JC,F 6 Hz), 140.8, 144.4 (d, JC,F 11 Hz), 154.6 (d, JC,F 246 Hz; 12 C, Ar-
C), 158.2 (C=O); IR (KBr):  3371 (vs, OH), 1651 (s, C=O) cm-1; HRMS: m/z: Calcd for C20H24FN2NaO7 [M+Na]+:
445.1387, found: 445.1385.
OAc
OAc
O
OH
OH
O
OAc
OAc
O
AcO
AcO
HO
HO
AcO
AcO
F
F
F
a)
b)
O
O
O
S
S
N
N
N
N
NH2
H
H
H
H
15
24
8
Scheme 5. Reagents and conditions: a) i. TCDI, CH2Cl2, r.t., 17 h; ii. benzylamine, CH2Cl2, r.t., 3 h, 52% (over two
steps); b) NaOMe, MeOH, 0 °C, 45 min, 71%.
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1-Benzyl-3-[4’-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyloxy)-3’-fluorophenyl]thiourea (24). Compound 15
(47.0 mg, 0.103 mmol) and 1,1’-thiocarbonyldiimidazole (TCDI; 18.3 mg, 0.103 mmol.) were dissolved in dry
CH2Cl2 (2 mL). The mixture was stirred at r.t. for 17 h. Then benzylamine (11.2 µL, 0.103 mmol) was added and
the mixture was stirred for 3 h until TLC (petroleum ether/EtOAc, 1:1) showed no remaining starting material.
The mixture was diluted with CH2Cl2, and washed with 1 M aq. HCl, satd. aq. NaHCO3 and brine. The organic
layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by MPLC
[]20
(petroleum ether/EtOAc, 1:0 to 0:1) to give 24 (32.3 mg, 52%).
+51.6 (c 1.07, CHCl3); 1H NMR (500 MHz,
D
CDCl3):  1.96, 1.99, 2.02, 2.15 (4 s, 12H, 4 Ac-CH3), 4.04 (dd, J 2.4, 12.1 Hz, 1H, H-6a), 4.12 (ddd, J 2.3, 5.2,
10.1 Hz, 1H, H-5), 4.20 (dd, J 5.2, 12.2 Hz, 1H, H-6b), 4.81 (d, J 5.2 Hz, 2H, CH2), 5.32 (t, J 10.1 Hz, 1H, H-4), 5.41
(s, 1H, H-1), 5.43 (dd, J 1.8, 3.3 Hz, 1H, H-2), 5.48 (dd, J 3.4, 10.0 Hz, 1H, H-3), 6.23 (s, 1H, NH), 6.92 (d, J 8.7 Hz,
1H, Ar-H), 7.02 (dd, J 2.2, 11.0 Hz, 1H, Ar-H), 7.16 (t, J 8.7 Hz, 1H, Ar-H), 7.22–7.27 (m, 3H, Ar-H), 7.27–7.32 (m,
2H, Ar-H), 7.96 (s, 1H, NH); 13C NMR (125 MHz, CDCl3):  20.59, 20.61, 20.62, 20.8 (4 Ac-CH3), 49.4 (CH2), 62.1
(C-6), 65.7 (C-4), 68.5 (C-3), 69.1 (C-2), 69.7 (C-5), 97.5 (C-1), 114.4 (d, JC,F 20 Hz), 119.9 (d, JC,F 2 Hz), 121.6 (d,
JC,F 4 Hz), 127.7, 127.9, 128.8, 137.0, 142.4 (d, JC,F 11 Hz), 153.4 (d, JC,F 252 Hz; 12C, Ar-C), 169.7, 169.8, 169.9,
170.4 (4 C=O), 181.1 (C=S); ESI-MS: m/z: Calcd for C28H32FN2O10S [M+H]+: 607.18, found: 607.30.
1-Benzyl-3-[3-fluoro-4-(α-D-mannopyranosyloxy)phenyl]thiourea (8). Prepared according to general
[]2D0
1
procedure B from 24 (32.2 mg, 53.1 µmol). Yield: 16.7 mg (71%).
+95.6 (c 0.84, MeOH); H NMR (500
MHz, CD3OD):  3.65–3.81 (m, 4H, H-4, H-5, H-6a, H-6b), 3.90 (dd, J 3.4, 9.1 Hz, 1H, H-3), 4.06 (dd, J 1.8, 3.3
Hz, 1H, H-2), 4.80 (s, 2H, CH2), 5.45 (d, J 1.6 Hz, 1H, H-1), 7.03 (ddd, J 1.4, 2.3, 8.8 Hz, 1H, Ar-H), 7.25 (m, 1H,
Ar-H), 7.27–7.36 (m, 6H, Ar-H); 13C NMR (125 MHz, CD3OD):  49.0 (CH2), 62.7 (C-6), 68.2 (C-4), 71.8 (C-2), 72.3
(C-3), 75.9 (C-5), 101.7 (C-1), 114.5 (d, JC,F 21 Hz), 120.4, 121.9 (d, JC,F 5 Hz), 128.2, 128.6, 129.5, 135.1 (d, JC,F
9
Hz), 139.9, 143.2 (d, JC,F 11.0 Hz), 154.3 (d, JC,F 246 Hz; 12C, Ar-C), 182.9 (C=S); IR (KBr):  3295 (vs, OH), 1563
(vs), 1509 (vs) cm-1; HRMS: m/z: Calcd for C20H24FN2NaO6S [M+Na]+: 461.1159 found: 461.1161.
OAc
OAc
O
OAc
OAc
O
OH
OH
O
AcO
AcO
F
O
AcO
AcO
HO
HO
F
F
NH2
20
NH2
b)
O
O
a)
H
H
H
H
N
N
N
N
21
25
9
S
S
Scheme 6. Reagents and conditions: a) i. TCDI, CH2Cl2, r.t., 18 h; ii. 20, CH2Cl2, r.t., 6 h, 7% (over two steps); b)
NaOMe, MeOH, r.t., 45 min, 66%.
1-[4’-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyloxy)-3’-fluorobenzyl]-3-phenylthiourea (25). 1,1’-Thio-
carbonyldiimidazole (18.9 mg, 0.106 mmol) was dissolved in dry CH2Cl2 (2 mL) and 21 (9.7 µL, 0.106 mmol) was
added. The mixture was stirred at r.t. for 18 h until TLC (toluene/EtOAc, 1:1) showed no remaining aniline.
Then, 20 (50 mg, 0.106 mmol) in dry CH2Cl2 (2 mL) was added and the mixture stirred for another 6 h. Then, it
was diluted with CH2Cl2 and subsequently washed with 1 M aq. HCl, satd. aq. NaHCO3, and brine. The organic
layer was dried over Na2SO4, filtered, and the solvent removed. The residue was purified by MPLC
[]20
1
(toluene/EtOAc, 1:0 to 1:1) to give 25 (4.8 mg, 7 %).
+49.8 (c 0.24, CHCl3); H NMR (500 MHz, CDCl3): 
D
2.03, 2.06, 2.19 (3 s, 12H, 4 Ac-CH3), 4.09 (dd, J 1.9, 12.2 Hz, 1H, H-6a), 4.20 (ddd, J 1.8, 5.2, 9.9 Hz, 1H, H-5),
4.26 (dd, J 5.3, 12.1 Hz, 1H, H-6b), 4.83 (d, J 5.7 Hz, 2H, CH2), 5.36 (t, J 10.0 Hz, 1H, H-4), 5.45 (s, 1H, H-1), 5.49
(m, 1H, H-2), 5.55 (dd, J 3.5, 10.0 Hz, 1H, H-3), 6.22 (s, 1H, NH), 7.01 (d, J 8.4 Hz, 1H, Ar-H), 7.09 (dd, J 1.5, 11.3
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Hz, 1H, Ar-H), 7.12 (t, J 8.2 Hz, 1H, Ar-H), 7.15–7.26 (m, 2H, Ar-H), 7.34 (m, 1H, Ar-H), 7.44 (t, J 7.8 Hz, 2H, Ar-
13
H), 7.72 (s, 1H, NH); C NMR (125 MHz, CDCl3):  20.66, 20.67, 20.68, 20.8 (4 Ac-CH3), 48.3 (CH2), 62.1 (C-6),
65.8 (C-4), 68.7 (C-3), 69.3 (C-2), 69.6 (C-5), 97.6 (C-1), 116.1 (d, JC,F 19 Hz), 119.3, 123.6 (d, JC,F 4 Hz), 125.6,
127.8, 128.8, 130.4, 134.0 (d, J 6 Hz), 142.8 (d, JC,F 11 Hz), 153.4 (d, JC,F 249 Hz; 12C, Ar-C), 169.7, 169.8, 169.9,
170.5 (4 C=O), 181.3 (C=S); ESI-MS: m/z: Calcd for C28H31FN2NaO10S [M+H]+: 629.16, found: 629.15.
1-(3-Fluoro-4-α-D-mannopyranosyloxybenzyl)-3-phenylthiourea (9). Prepared according to general procedure
[]20
B from 25 (4.6 mg, 7.9 µmol). Yield: 2.3 mg (66%).
+61.4 (c 0.12, MeOH); 1H NMR (500 MHz, CD3OD):  =
D
3.66–3.79 (m, 4H, H-4, H-5, H-6a, H-6b), 3.91 (dd, J 3.4, 9.1 Hz, 1H, H-3), 4.06 (dd, J 1.8, 3.4 Hz, 1H, H-2), 4.76
(s, 2H, CH2), 5.45 (d, J 1.7 Hz, 1H, H-1), 7.09 (d, J 8.4 Hz, 1H, Ar-H), 7.15 (dd, J 2.0, 11.9 Hz, 1H, Ar-H), 7.21 (m,
1H, Ar-H), 7.28–7.39 (m, 5H, Ar-H); 13C NMR (125 MHz, CD3OD):  48.2 (CH2), 62.6 (C-6), 68.2 (C-4), 71.9 (C-2),
72.3 (C-3), 75.8 (C-5), 101.7 (C-1), 116.6 (d, JC,F 19 Hz), 120.2 (d, JC,F 1 Hz), 124.7 (d, JC,F 3 Hz), 125.9 (d, JC,F
3
Hz), 127.0, 130.3, 139.5, 144.4, 154.5 (d, JC,F 246 Hz; 12C, Ar-C), 182.8 (C=S); IR (KBr):  3422 (vs, OH), 1514
(m, NH) cm-1; HRMS: m/z: Calcd for C20H24FN2NaO6S [M+Na]+: 461.1159 found: 461.1160.
OR
O
OR
F
NH2
O
RO
RO
F
HO
F
27
a)
O
b)
HO
H
O
N
O
H
COOH
N
O
O
26
28
R = Ac: 29
R = H: 10
c)
Scheme 7. Reagents and conditions: a) HBTU, HOBt, DIPEA, DMF, r.t., 2.5 h, 18%; b) 11, BF3∙Et2O, CH2Cl2/
MeCN, MS 4Å, 50-75 °C, 48 h, 12%; c) NaOMe, MeOH, r.t., 45 min, 60%.
3-Fluoro-4-hydroxy-N-(2-oxo-2-phenylethyl)benzamide (28). 3-Fluoro-4-hydroxybenzoic acid (26, 50.0 mg,
0.641 mmol, 1 eq.), HBTU (243 mg, 1.28 mmol, 2 eq.), HOBt hydrate (12% water; 98.4 mg, 0.205 mmol, 2 eq.),
and 2-aminoacetophenone hydrochloride (27, 35.2 mg, 1.28 mmol, 2 eq.) were dissolved in anhydrous DMF
(1.5 mL). Then, DIPEA (110 µL, 2.56 mmol, 4 eq.) was added and the mixture was stirred at r.t. for 2.5 h. Then,
it was diluted with EtOAc and subsequently washed with 1 M aq. HCl and brine. The organic layer was dried
over Na2SO4, filtered and the solvents were removed in vacuo. The residue was purified by MPLC
(toluene/EtOAc, 1:0 to 1:1) to give 28 (31.8 mg, 18%). 1H NMR (500 MHz, CD3OD):  4.86 (s, 2H, CH2), 6.99 (t, J
8.5 Hz, 1H, Ar-H), 7.53 (t, J 7.7 Hz, 2H, Ar-H), 7.60 (dd, J 2.1, 8.4 Hz, 1H, Ar-H), 7.62–7.67 (m, 2H, Ar-H), 8.02–
8.08 (m, 2H, Ar-H); 13C NMR (125 MHz, CD3OD):  47.7 (CH2), 116.5 (d, JC,F 20 Hz), 118.5 (d, JC,F 3 Hz), 125.3 (d,
JC,F 3 Hz), 126.7 (d, JC,F 6 Hz), 129.0, 129.9, 134.8, 136.5, 150.0 (d, JC,F 13 Hz), 152.4 (d, JC,F 242 Hz; 12C, Ar-C),
169.1 (CONH), 196.4 (C=O); ESI-MS: m/z: Calcd for C15H12FNNaO3 [M+Na]+: 296.07, found: 295.49.
4-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyloxy)-3-fluoro-N-(2-oxo-2-phenylethyl)benzamide (29). A two-
necked flask was charged with activated MS 4Å (50 mg), peracetylated D-mannose (12, 54.5 mg, 0.140 mmol,
1.2 eq.) and dry CH2Cl2 (2 mL). Under argon atmosphere, 28 (31.8 mg, 0.116 mmol, 1 eq.) in dry MeCN (2 mL)
was added. The mixture was refluxed at 50 °C for 24 h, and another 24 h at 75°C. When TLC (petroleum
ether/EtOAc, 1:1) showed no remaining mannose precursor, the mixture was diluted with EtOAc, filtered over
celite, and washed with satd. aq. NaHCO3 and brine. The organic layer was dried over Na2SO4, filtered and
concentrated. The residue was purified by MPLC (petroleum ether/EtOAc, 1:0 to 1:1) to yield 29 (8.3 mg, 12%).
[]2D0
1
Unreacted 28 (15.5 mg, 49%) could be recovered.
+60.1 (c 0.42, CHCl3); H NMR (500 MHz, CDCl3): 
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2.04, 2.04, 2.07, 2.21 (4 s, 12H, 4 Ac-CH3), 4.09 (dd, J 2.1, 12.2 Hz, 1H, H-6a), 4.16 (ddd, J 1.9, 5.3, 9.9 Hz, 1H, H-
5), 4.28 (dd, J 5.4, 12.3 Hz, 1H, H-6b), 4.94 (d, J 4.1 Hz, 2H, CH2), 5.38 (t, J 10.1 Hz, 1H, H-4), 5.53 (dd, J 1.8, 3.3
Hz, 1H, H-2), 5.55–5.60 (m, 2H, H-3, H-1), 7.22 (s, 1H, NH), 7.28 (t, J 8.2 Hz, 1H, Ar-H), 7.53 (t, J 7.7 Hz, 2H, Ar-
H), 7.62 (d, J 8.6 Hz, 1H, Ar-H), 7.65 (t, J 7.4 Hz, 1H, Ar-H), 7.70 (dd, J 1.9, 11.1 Hz, 1H, Ar-H), 8.03 (d, J 7.5 Hz,
2H, Ar-H); 13C NMR (125 MHz, CDCl3):  20.64, 20.65, 20.66, 20.8 (4 Ac-CH3), 46.9 (CH2), 62.0 (C-6), 65.7 (C-4),
68.6 (C-3), 69.1 (C-2), 69.8 (C-5), 97.0 (C-1), 116.2 (d, JC,F 20 Hz), 118.1, 123.4 (d, JC,F 4 Hz), 128.0, 129.0, 129.9
(d, JC,F 6 Hz), 134.2, 134.4, 146.1 (d, JC,F 11 Hz), 152.8 (d, JC,F 250 Hz; 12C, Ar-C), 165.4, 169.7, 169.8, 169.9,
170.5, 194.0 (6 C=O); ESI-MS: m/z: Calcd for C29H30FNNaO12 [M+Na]+: 626.17, found: 626.24.
3-Fluoro-4-α-D-mannopyranosyloxy-N-(2-oxo-2-phenylethyl)benzamide (10). Prepared according to general
[]20
procedure B from 29. Yield: 3.6 mg (60%).
+88.3 (c 0.18, MeOH); 1H NMR (500 MHz, CD3OD):  3.62 (ddd,
D
J 2.4, 5.6, 9.8 Hz, 1H, H-5), 3.68–3.81 (m, 3H, H-4, H-6a, H-6b), 3.93 (dd, J 3.4, 9.5 Hz, 1H, H-3), 4.09 (dd, J 1.8,
3.4 Hz, 1H, H-2), 4.87 (s, 2H, CH2), 5.62 (d, J 1.5 Hz, 1H, H-1), 7.50 (t, J 8.5 Hz, 1H, Ar-H), 7.55 (t, J 7.7 Hz, 2H, Ar-
H), 7.66 (t, J 7.4 Hz, 1H, Ar-H), 7.68–7.74 (m, 2H, Ar-H), 8.04–8.08 (m, 2H, Ar-H); 13C NMR (125 MHz, CD3OD): 
62.6 (C-6), 68.2 (C-4), 71.7 (C-2), 72.3 (C-3), 76.1 (C-5), 101.0 (C-1), 116.6 (d, JC,F 20 Hz), 119.0, 125.2 (d, JC,F 4
Hz), 129.1, 130.0, 134.9, 136.5, 148.5 (d, JC,F 11 Hz), 153.9 (d, JC,F 246 Hz; 12C, Ar-C), 168.7 (CONH), 196.3
(C=O); IR (KBr):  3412 (vs, OH, NH), 1646 (s, C=O) cm-1; HRMS: m/z: Calcd for C21H23FNNaO8 [M+Na]+:
458.1227, found: 458.1227.
Supplementary Material
For Surface Plasmon Resonance Experiments and Fluorescence Polarization Assay please refer to the
Supporting Information for a detailed description.
References
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