Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidases

Article abstract of DOI:10.1016/j.bmc.2012.02.001

A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialylated glycoconjugates, is a promising drug candidate for various infective diseases. The current study demonstrates that the use of an aglycone-focused

Full text of DOI:10.1016/j.bmc.2012.02.001

Bioorganic & Medicinal Chemistry 20 (2012) 2739–2746
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Aglycone-focused randomization of 2-difluoromethylphenyl-type
sialoside suicide substrates for neuraminidases
⇑
Hirokazu Kai, Hiroshi Hinou , Shin-Ichiro Nishimura
Graduate School of Life Science and the Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, N21, W11, Kita-ku, Sapporo 001-0021, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialy-
lated glycoconjugates, is a promising drug candidate for various infective diseases. The current study
Received 10 January 2012
Revised 31 January 2012
Accepted 1 February 2012
Available online 15 February 2012
demonstrates that the use of an aglycone-focused library of 2-difluoromethylphenyl
a-sialosides is an
effective technique to find potent and selective mechanism-based labeling reagents for neuraminidases.
The focused library was constructed from a 4-azide-2-difluoromethylphenyl sialoside (2) and an alkyne-
terminated compound library by a click reaction. The focused library showed different inhibition patterns
for two neuraminidases, Vibrio cholerae neuraminidase (VCNA) and human neuraminidase 2 (hNeu2), and
the most potent inhibitors for each neuraminidase were selected. A kinetic analysis of the selected inhib-
itors demonstrated that the modification of the aglycone moiety improved the KI value with little change
in the t1/2 value of the enzyme activity relative to the basic skeleton (2).
Keywords:
Neuraminidase
Suicide substrate
Mechanism-based inhibitor
Focused library
Aglycone
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
available from crystal structures. Previously, we reported a strategy
to develop a potential inhibitor (Fig. 1) for Vibrio cholerae neuramin-
Neuraminidases (NAs; EC 3.2.1.18), an important family of
glycoside hydrolases, cleave the glycosidic linkages of sialic acid
(Neu5Ac; N-acetylneuraminic acid) and regulate the interactions
between a cell and the extracellular world during infection, develop-
ment, inflammation, antigenicity, and cell–cell adhesion.1 Thus,
inhibition of neuraminidases is a potential target for therapeutic
reagents. Since the first report in 1969, 2,3-dehydroneuraminic acid
(DANA; Fig. 1)2 has been utilized as a transition state mimetic inhib-
itor of neuraminidases, and DANA-based inhibitors have occupied
the main stream of neuraminidase inhibitor design. During the
1990s, highly potent and selective inhibitors for influenza virus
neuraminidases, Zanamivir (Relenza) and GS4071 (Tamiflu), were
successfully developed and approved as anti-influenza drugs
(Fig. 1)3,4; however, similar inhibitors have not been developed for
the pathogenesis of other diseases, such as cholera, in which
neuraminidases are involved. The difficulty in developing a selective
and potent inhibitor for neuraminidases might be due to the flexibil-
ity of the cavities around the active sites of neuraminidases.5,6 The
cavities of neuraminidases are primarily composed of a loop moiety
on a six-bladed b-propeller fold, and despite their low sequence
identities, this structural feature is preserved among every species
among eukaryote, prokaryote, and virus.7,8 The flexibility of the loop
structure makes it difficult to design an inhibitor by a structure-
based drug-design strategy using only the static information
idase (VCNA) using labeling information that was obtained from a
dansyl-modified 2-difluoromethylphenyl-type sialoside, 1a (Fig.
2a).9,10 A focused library was prepared from 9-N3-substituted DANA,
and an alkyne-terminated compound library was designed based on
the labeling information. A tailored inhibitor for VCNA that is selec-
tive and potent (KI = 73 nM) was successfully identified. The use of a
focused library to probe a suitable structure for the inhibition of the
hydrolytic function of neuraminidases is a potential method of find-
ing a potent and selective inhibitor for each neuraminidase.9
2-Difluoromethylphenyl-type sialosides, which are mechanism-
based inactivators for neuraminidases such as the probe 1a, also
provide an attractive alternative to the DANA-based competitive
OH
HO
O
COOH
AcHN
HO
O
COOR
AcHN
HN
H2N
H2N
NH
R = H GS4071
OH
HO
Zanamivir (Relenza)
R = Et Oseltamivir (Tamiflu)
HO
O
COOH
Influenza virus neuraminidase specific inhibitors
AcHN
HO
O
N
OH
DANA
H
N
N
N
O
H
COOH
F3C
N
HO
OH
O
VCNA specific inhibitor
⇑
Corresponding author. Tel.: +81 11 706 9040; fax: +81 11 706 9042.
E-mail address: hinou@sci.hokudai.ac.jp (H. Hinou).
Figure 1. DANA and DANA-based inhibitors.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.001

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H. Kai et al. / Bioorg. Med. Chem. 20 (2012) 2739–2746
aglycone-focused library, 3, prepared from 2 might also improve
(a)
HO
O
S
O
OH
N
this problem. Neuraminidases must have sufficient space in the
active site to recognize large glycoconjugates, such as glycopro-
teins and glycolipids, and the structurally randomized aglycone
moiety of the mechanism-based inhibitor 2. We now report the
preparation of both compound 2 and the focused library 3 and
the evaluation of 3 regarding the inhibition potency and selectivity
for VCNA and human neuraminidase 2 (hNeu2).
CO2H
O
1a
: R =
N
H
CF2H
R
O
AcHN
HO
1b: R = NO2
2: R = N3
HO
N
3: R =
randomized
structures
N
N
(b)
F
Nu
F
F
H
R
F
-
-
neuraminidase
+
H
R
H
O
O
Nu
O
2. Results and discussion
1
~
3
F-
sialic acid
2.1. Preparation and biochemical evaluation of 4-azide-2-
difluoromethylphenyl sialoside 2
R
E + S or E S
E I
E I*
E I
(If Nu = neuraminidase)
The azide-equipped aglycone moiety (6) was prepared from 5-
nitrosalicylaldehyde (4) as shown in Scheme 1. The direct reduc-
tion of the nitro group of 4 yielded a complex mixture due to the
instability of the amine and intermolecular imine formation. To
avoid this instability, the carbonyl group of 4 was converted to a
dithioacetal to yield 5. The nitro group of 5 was then converted
to an azide group by treatment with zinc, sodium nitrite, and so-
dium azide to yield 6.24 The deprotection of the dithioacetal
group25 at the 2-position and the formation of a diazonium salt
at the 4-position were simultaneously accomplished by treatment
with 4 equivalents of sodium nitrite.
in active site
(c)
KI
k1
k2
k3
E + S
E S
E I
E I*
k5
E I
k4
k6
k7
Iinact
I
I*
Figure 2. Structure and mechanism of suicide substrates; (a) Structures of the
reported suicide substrate 1, the p-azide derivative 2, and the aglycone-focused
library 3; (b) plausible reaction mechanism of the selective activation and covalent-
bond formation of the aglycone moiety in the presence of neuraminidases; (c)
plausible kinetics of the inactivation by the 2-difluoromethylphenyl sialosides 1–3.
The azide-equipped skeleton of the suicide substrate 2 was pre-
pared by an established procedure10,11,17 as shown in Scheme 2.
Themethylester of sialicacid 8 was coupledwith 6 in a one-pot reac-
tion26 via a glycosyl chloride intermediate to yield the
a-sialoside 9.
inhibitors. The 2-difluoromethyl-type sialoside is activated by the
hydrolytic cleavage of a phenolic glycoside bond. Following fluo-
ride ion desorption, the activated aglycone moiety forms a Michael
reaction acceptor, which forms a covalent bond with nucleophiles,
and the activated acceptor moiety binds to nucleophilic amino acid
side chains to inhibit the enzymatic activity (Fig. 2b). The function-
specific activation by a neuraminidase (sialidase) and the irrevers-
ible labeling ability of the 2-difluoromethylphenyl-type sialoside
have potential as novel drug and diagnostic tools. This 2-difluo-
romethylphenyl-type glycoside was first reported in 1990 by
Danzin et al. as a glucosidase inhibitor,11 and it has been applied
to various glycosidases, such as galactosidases,12 N-acetyl glucosa-
minidase13; other hydrolases, such as phosphatases,14 sulfatase,15
and proteases16; and neuraminidases.10,17–19 Recently, we reported
that another 2-difluoromethylphenyl-type sialoside (1b) (Fig. 2a)
inhibits the sialidase activity of a Trypanosoma cruzi trans-sialidase
(TcTS) and that a host cell infection by T. cruzi is also inhibited by
treatment with compound 1b.20 A point mutation of an amino acid
residue that is detected by the labeling study with 1a almost com-
pletely abolishes the activity of TcTS. Although there are advanta-
ges to the 2-difluoromethylphenyl-type sialoside, a relatively high
concentration (mM range) of this glycoside is required to inhibit
the target enzymes. This problem may be due to the basic architec-
ture of this type of compound; only a glycan moiety can assume
the potency and specificity of this type of glycoside to be recog-
nized by target enzymes. To overcome this problem, we sought
to design a focused library consisting of the 2-difluoromethylphe-
nyl sialoside skeleton (2) to which we could add the aglycone moi-
ety with potent and specific affinity to each target neuraminidase
by the so-called [2+3] cyclization click reaction21 as demonstrated
by the most potent inhibitor for VCNA.9 This modification of 2 at
the aglycone moiety is expected to strengthen its interaction with
the target neuraminidase not only as the sialoside for hydrolysis
[EÁS] but also as the hydrolyzed aglycone [EÁI] and the activated
aglycone [EÁI⁄] to form a covalent bond with the target enzyme
[EÀI] (Fig. 2b and c).22,23 Previous research has indicated that the
activation rate of the aglycone moiety (k2) is slower than its disso-
ciation rate from an active site on the target enzyme (k4).15,19 The
The aldehyde group of 9 was converted to a difluoromethyl group by
treatment with diethylamino sulfer trifluoride (DAST) to yield 10.
The two-step deprotection of 10, that is, the sodium methoxide-
catalyzed O-acyl replacement in methanol followed by the saponifi-
cation of the methyl neuraminate, yielded the basic skeleton 2.
The kinetic constants of the time-dependent inhibition (KI, ki-
nact, and t1/2) of VCNA and hNeu2 by 2 were explored according
to a method of an irreversible inhibitor reported by Kitz and Wil-
son.11,22,23 Each enzyme was preincubated with 2, and an aliquot
of the solution was added to a solution of 2-a-(4-methylumbellife-
ryl)-N-acetylneuraminic acid (4-MU-NANA) to determine the
residual activity in 5-min intervals. As shown in Table 1, the mM
range of the obtained KI values suggests that a high concentration
of 2 is required to inactivate the neuraminidases because of the
poor interaction between the enzymes and the aglycone moiety.
Compared to VCNA, hNeu2 requires a higher concentration of 2
to be inactivated. The higher KI value for hNeu2 is not surprising,
because the Km value of hNeu2 is generally higher than that of
VCNA with respect to a common substrate.9
2.2. First screening of the focused library 3 prepared by the click
reaction
For the first screening, we adapted a direct analysis of the crude
solution of the click reaction as previously reported.9 The
OH
O
OH
O
OH
S
(i)
(ii)
H
H
S
N3
NO2
NO2
4
5
6
Scheme 1. Preparation of 5-azidosalicylaldehyde (6). Reagents and conditions: (i)
1,3-propanedithiol (1.2 equiv), BF3ÁEt2O (1.2 equiv), CH2Cl2, rt, 30 min, 95%, (ii) (a)
Zn (5.0 equiv), AcOH, rt, 1 h, (b) NaNO2 (4.0 equiv), AcOH, 0 °C, 2 h, (c) NaN3, AcOH,
0 °C?rt, 30 min, 65% (in 3 steps).

H. Kai et al. / Bioorg. Med. Chem. 20 (2012) 2739–2746
2741
OAc
O
CO2Me
OH
AcO
H
CO2Me
OH
HO
(i), (ii)
O
O
AcHN
AcO
O
AcHN
HO
OAc
HO
9
N3
8
OAc
OH
CO2Me
CO2H
O
AcO
HO
CF2H
N3
CF2H
N3
O
O
(iii)
O
(iv)
AcHN
AcO
AcHN
HO
OAc
HO
10
2
Scheme 2. Preparation of 2. Reagents and conditions: (i) AcCl, rt, 24 h, (ii) 6 (1.0 equiv), DIEA (3.0 equiv), MeCN, rt, 24 h, 61%, (iii) DAST (2.5 equiv), CH2Cl2, rt, 3 h, 72%, (iv) (a)
sodium methoxide, MeOH, rt, 30 min, (b) 1 N NaOH (1.2 equiv), H2O, rt, 30 min, 79%.
Table 1
whereas for 45 of the compounds, the yields of the product were
less than 50%. Among these compounds, those possessing a thio-
KI, kinact, and t1/2 values of compound 2 for VCNA and hNeu2
Neuraminidase
KI (mM)
kinact (minÀ1
)
t1/2 (min)
urea group and an imine group tended to produce low yields,
and this tendency differs from the results obtained using an ali-
phatic azide.9 A yield of greater than 70% was obtained for 78 reac-
tion solutions in the ESI-MS analysis, and these compounds were
used for the first screening with VCNA and hNeu2 (Fig. 4a, SI–11).
The levels of neuraminidase activity that remained after treat-
ment with each click reaction solution of 3 with VCNA and hNeu2
are shown in Fig. 4b (and Fig. SI–12) as the result of the first
VCNA
hNeu2
2.7
20
0.11
0.12
5.9
5.7
azide-equipped skeleton 2 was coupled with a commercially avail-
able alkyne compound library (230 compounds, Figs. SI–10) in the
presence of tris-(benzyltriazolylmethyl)amine (TBTA),27 copper
sulfate, and ascorbic acid in 10% dimethyl sulfoxide (DMSO) to af-
ford the aglycone randomized suicide substrate library 3. Before
the first screening, the effect of the reagents of the click reaction
on the hNeu2 activity was evaluated. In the case of VCNA,9 its NA
activity was only slightly affected by the reagents. Itzstein et al. re-
ported that hNeu2 require calcium ion for their NA activity, and the
NA activity decreases by one-half by the addition of 7 mM copper
salt.28 As shown in Figure 3a, the NA activity of hNeu2 disappeared
in the presence of a copper salt even at a very low copper concen-
screening. The triazole derivatives of 3 (30 lM for VCNA, 100 lM
for hNeu2; calculated as though the click reactions produced a
100% yield) were preincubated with VCNA or hNeu2 for 15 min
and diluted with 2 mM of 4-MU-NANA, and the remaining NA
activity was analyzed using a fluorometric assay method.30
A
125-
l
M solution of EDTA was also added to the preincubation
M CuSO4.
solution of hNeu2, which also contained 50
l
As shown in Figure 4b, the different inhibition patterns of
focused library 3 for VCNA and hNeu2 clearly demonstrated the po-
tency of our strategy to give the mechanism-based labeling reagent
selectivity and inhibition potency for each of the neuraminidase.
tration (50 lM CuSO4) in the presence of 4 mM calcium salt. To ne-
gate the effect of the copper salt, the effect of a chelating agent for
the copper ion, 2-({2-[bis(carboxymethyl)amino]ethyl}(carboxy-
methyl)amino) acetic acid (EDTA) was evaluated. Although Itzstein
et al. reported that 1 mM EDTA also decreased the NA activity of
hNeu2 by half,28 addition of 1 equiv of EDTA recovered the NA
activity of hNeu2 completely, and a small excess of EDTA did not
affect the activity (Fig. 3). The reported inhibition effect in the pres-
ence of 1 mM EDTA might be due to the absence of external addi-
tion of calcium ion.28 Based on these results, 2.5 equiv of EDTA
were added to the assay solution to bind the copper ion for the first
screening of hNeu2.
2.3. Determination and evaluation of the best structure of 3 as
an inhibitor of VCNA and hNeu2
To determine the best structure from the focused library 3 to in-
hibit VCNA, the results in Figure 4b were classified into two groups
using 55% of the remaining NA activity as the borderline of the first
screening. The compounds that exhibited less than 55% remaining
NA activity were prepared as purified compounds. Among the
compounds that passed the first screening, the 11 candidates from
the focused library 3 shown in Figure 5a could be isolated and used
for the second screening. For the second screening, a 50 lM solu-
tion of each compound was preincubated with VCNA for 15 min,
and the remaining NA activity was analyzed in 2 mM 4-MU-NANA.
The yield of the click reaction in each crude solution was
roughly estimated from the ratio of 2 and 3 obtained from the
product ion signal of the ESI-MS analysis.9,29 For 90 of the com-
pounds, no product was determined from the ESI-MS analysis,
Figure 3. Optimization of the assay conditions for the first screening of hNeu2; (a) effect of the reagents in the click reaction solution on the NA activity of hNeu2. I: control
(no additive), II: CuSO4, III: CuSO4 and TBTA, IV: CuSO4, TBTA, and ascorbic acid, V: TBTA and ascorbic acid; (b) effect of EDTA in the presence of 50 M CuSO4. I: control
(without CuSO4 or EDTA), II: without EDTA, III: with 50 M EDTA, IV: with 125 M EDTA, V: with 250 M EDTA.
l
l
l
l

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H. Kai et al. / Bioorg. Med. Chem. 20 (2012) 2739–2746
R
Compound 3a (Fig. 5b, click ID: 140) was determined to be the
(230 compounds)
OH
OH
most potent inhibitor for VCNA in the focused library 3. The kinetic
constants of the time-dependent inhibition (KI, kinact, and t1/2) of
CO2H
O
HO
CO2H
O
HO
TBTA, CuSO4
ascorbic acid
CF2H
CF2H
N3
O
O
AcHN
HO
AcHN
HO
VCNA by compound 3a were extrapolated22,23 to be 61
l
M,
HO
HO
H2O-DMSO
(9:1)
0.091 minÀ1 and 7.6 min, respectively.
N
N
R
2
3
For hNeu2, the results in Figure 4b were classified into two
groups using 60% of the remaining activity as the borderline of
the first screening. The 7 candidates shown in Figure 6a were iso-
lated and used for the second screening. For the second screening,
N
3
peak area of
x 100
Yield (%) =
2 +
3
peak area of
peak area of
a 100 lM solution of each compound was preincubated with
(a)
hNeu2 for 15 min, and the remaining NA activity was analyzed in
1 mM 4-MU-NANA. Compound 3b (Fig. 6b, click ID: 60) was deter-
mined to be the most potent inhibitor for hNeu2 among the
focused library 3. The extrapolated kinetic constants of the time-
dependent inhibition (KI, kinact, and t1/2) of hNeu2 by compound
3b were determined to be 215 l
M, 0.054 minÀ1 and 12.6 min,
respectively.
A comparison of the kinetic constants of compounds 2 and 3a
for VCNA and compounds 2 and 3b for hNeu2 indicated that the
KI value changed by approximately one-hundredth owing to the
modifications in both cases. These differences in binding properties
between the basic skeleton and the hit compounds are larger than
that of the DANA-based inhibitor, which was reported previously
to be approximately one-tenth.9 This result indicates that the
modification of the aglycone moiety via the triazole linker greatly
contributed to make sialoside 3 a good substrate to bind to the
active site of the target enzyme, as we expected. In contrast to
the KI values, the change in t1/2 for both neuraminidases was
slightly increased. This result indicates that the modification of
the aglycone moiety did not contribute to the change of the rate
of hydrolysis (k1, Fig. 2c), the activation rate of the released agly-
cone (k2), or the covalent bond formation that inactivated the en-
zyme (k3). Rather, the substituent on the difluoromethylphenyl
moiety is thought to decrease the k1 value by inhibiting the ability
of the enzyme to hydrolyze the substrate, as we observed for our
DANA-based inhibitors.9 Replacement of the azide functional
group on the difluoromethylphenyl group with 1,2,3-triazol might
also affect the kinetics (k1–3) based on the electrostatic change at
the phenolate moiety.11
(b)
Figure 4. Construction and evaluation of the focused library 3; (a) estimated yield
of 3; (b) relative NA activity of VCNA and hNeu2 remaining after preincubation with
each click solution.
Figure 5. Determination and evaluation of the best inhibitor for VCNA from the focused library 3. (a) Relative remaining activity of VCNA after incubation with the 11 isolated
compounds selected from the first screening. (b) Structure and the kinetic constants of the most potent inhibitor, 3a, for VCNA among the click library 3, and the reciprocal
plot to determine the kinetic constants.

H. Kai et al. / Bioorg. Med. Chem. 20 (2012) 2739–2746
2743
Figure 6. Determination and evaluation of the best inhibitor for hNeu2 from the focused library 3. (a) Relative remaining activity of hNeu2 after incubated with 7 isolated
compounds selected by the first screening. (b) Structure and the kinetic constants of the most potent inhibitor, 3b, for hNeu2 among click library 3, and the reciprocal plot to
determine the kinetic constants.
noted. Thin-layer chromatography (TLC) was performed on
0.25 mm pre-coated silica gel plates (Merck 60 F-254) and visual-
ized by UV light charring with a methanol solution of 5% H2SO4.
Column chromatography was performed using silica gel (Kanto
3. Conclusions
An aglycone-focused library of 2-difluoromethylphenyl sialo-
side (3) was efficiently constructed from the azide-equipped
skeleton 2 and an alkyne-terminated compound library using the
click strategy. Two new lead structures, 3a and 3b, were deter-
mined to be potent and selective inhibitors for VCNA and hNeu2,
respectively, by a two-step screening, that is, the crude click reac-
tion mixture was used for the first screening, and the isolated com-
pounds were used for the second screening. Although hNeu2 was
60 N,
u = 40–50 lm; Kanto Chemical Co., Inc.) with air flashing.
The 1H NMR (500 MHz or 600 MHz) and 13C NMR (125 MHz or
150 MHz) spectra were recorded on a BRUKER ADVANCE 500 or
a
BRUKER ADVANCE 600 spectrometer (Bruker BioSpin Co.,
Germany) with CDCl3, D2O, and CD3OD as solvents. The chemical
shifts are reported in d (ppm), and the coupling constants (J) are
in hertz (Hz). The following abbreviations were used for signal
multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet.
The FAB-MS and HR-MS spectra were obtained on a JEOL JMS-
HX110 spectrometer (JEOL, Japan) at the Center for Instrumental
Analysis, Hokkaido University. The reverse-phase HPLC was per-
formed using a HITACHI L-6200 HPLC system equipped with an
Inertsil-ODS3 column (20 Â 250 mm, GL Sciences Inc.) and a HIT-
ACHI L-7405 UV–vis detector with a shallow linear gradient at
room temperature. The activity of neuraminidase was measured
via fluorescence spectrometry in the presence of 4-MU-NANA as
a substrate and using a microplate reader (SpectraMax M5, Molec-
ularevices Co., Sunnyvale, CA).
found to be deactivated almost completely by copper ions (lM
concentration range) during the click reaction of the first screen-
ing, nullification of this effect was achieved by the addition of a
small excess of EDTA. Although it tended to decrease the rate of
inactivation, the substituent on the aglycone moiety greatly con-
tributed to the binding properties of the sialoside 3 in terms of
both potency and selectivity.
Further studies to elucidate the customized lead structure for
other targets, such as the influenza virus neuraminidase andT. cruzi
trans-sialidase, are now in progress.
4. Experimental
4.1. General
4.2. Synthesis of compound 5, 6, 9, 10, and 2
4.2.1. 2-(1,3-Dithian-2-yl)-4-nitrophenol (5)
2-a-(4-Methylumbelliferyl)-N-acetylneuraminic acid (4-MU-
NANA) was synthesized according to published method.31 The
alkyne compounds were purchased from Thermo Fisher Scientific
Inc. (Cornwall, UK) (230 compounds). The ID number of each
alkyne compound assigned to the click reaction solution is shown
in Figures SI–10. V. cholerae neuraminidase (VCNA) was purchased
from Sigma–Aldrich Co. (N6514, St. Louis, MO, USA). Human neur-
aminidase 2 (hNeu2), which was prepared as reported proce-
dure,5,9 was a gift from Dr. X.-D. Gao and Mr. R. Miyoshi. The
solvents and other reagents for the chemical syntheses were
purchased from Sigma–Aldrich Co., Tokyo Chemical Industry Co.,
Ltd (Tokyo, Japan), and Wako Pure Chemical Industries, Ltd (Osaka,
Japan) and used without further purification unless otherwise
BF3ÁEt2O (12.1 mL, 48 mmol) was gradually added to a solution
of 2-hydroxy-5-nitrobenzaldehyde (4) (6.7 g, 40 mmol) and pro-
pane-1,3-dithiol (4.88 mL, 48 mmol) in dry CH2Cl2 (200 mL), and
the mixture was stirred under a N2 gas atmosphere at room tem-
perature. After 2 h, the reaction was quenched by MeOH, and the
solvent was removed under reduced pressure followed by recrys-
tallization from ethyl acetate-hexane to yield a light yellow pow-
der (9.78 g, 95%). The product decomposed at 202 °C. 1H NMR
(500 MHz, CDCl3, 300 K): d 8.26 (d, 1H, J = 2.5 Hz, H-3), 8.13 (dd,
1H, J = 2.6, 9.0 Hz, H-5), 7.43 (s, 1H, OH), 6.98 (d, 1H, J = 9.0 Hz,
H-6), 5.43 (s, 1H, PhCH), 3.09 (dd, 2H, J = 12.4 Hz, S-C(Heq)Hax
CH2), 2.97 (ddd, 2H, J = 3.2, 3.6, 14.0 Hz, S-C(Hax)Heq-CH2), 2.23
-

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H. Kai et al. / Bioorg. Med. Chem. 20 (2012) 2739–2746
(dtt, 1H, J = 2.1, 2.2, 14.3 Hz, CH2-C(Hax)Heq-CH2), 1.97 (dtt, 1H,
J = 2.0, 13.2 Hz, CH2-C(Heq)Hax-CH2); 13C NMR (125 MHz, CDCl3,
300 K): d 160.5, 141.2, 126.1, 125.8, 124.1, 118.0, 46.7, 31.2, 24.5.
reaction mixture at 0 °C, and the mixture was concentrated in va-
cuo. The residue was purified by flash column chromatography on
a silica gel (hexane:acetone = 7:4) to yield compound 10 as a light
yellow powder (738 mg, 72%). 1H NMR (500 MHz, CDCl3, 300 K) d
7.34 (d, 1H, J = 8.4 Hz, H-arom.), 7.21 (d, 1H, J = 2.4 Hz, H-arom.),
7.07 (dd, 1H, J = 2.4, 8.4 Hz, H-arom.), 6.88 (t, 1H, J = 55.2 Hz,
CHF2), 5.43 (d, 1H, J = 10.2 Hz, NH), 5.34-5.38 (m, 2H, H-7, H-8),
4.94 (m, 1H, H-4), 4.44 (d, 1H, J = 10.8 Hz, H-6), 4.28 (dd, 1H,
J = 2.4, 12.8 Hz, H-9a), 4.13 (dd, 1H, J = 4.8, 12.6 Hz, H-9b), 4.09
(q, 1H, J = 10.2 Hz, H-5), 3.66 (s, 3H, COOMe), 2.74 (dd, 1H, J = 4.8,
12.6 Hz, H-3a), 2.24 (t, 1H, J = 6.6 Hz, H-3b), 2.15, 2.14, 2.06, 2.05
(s, 3H each, OAc), 1.92 (s, 3H, NHAc); 13C NMR (125 MHz, CDCl3,
300 K) d 179.2, 170.9, 170.7, 170.4, 167.8, 148.9, 136.7, 127.5,
127.4, 127.2, 122.7, 121.7, 117.1, 112.6, 111.1, 109.5, 101.0, 73.8,
69.0, 68.8, 67.5, 62.5, 53.5, 49.8, 38.5, 23.5, 21.3, 21.1. HRMS (ESI)
Calcd for C27H32N4O13F2Na [M+Na]+ 681.1826, found 681.1827.
HRMS (ESI) Calcd for
256.0111.
C
10H10NO3S2 [MÀH]À 256.0108, found
4.2.2. 5-Azidosalicylaldehyde (6)
Zn powder (5.6 g, 86 mmol) was added to a solution of 2-(1,3-
dithian-2-yl)-4-nitrophenol (5) (4.4 g, 17 mmol) in AcOH
(100 mL), and the mixture was stirred at room temperature for
1 h. The residue was filtered through a celite pad, and the pad
was washed with EtOAc. The combined solutions were removed
under reduced pressure. The residue was dissolved in glacial acetic
acid (100 mL) and cooled on an ice bath to <5 °C. While stirring, a
solution of sodium nitrate (1.72 g, 69 mmol) dissolved in a mini-
mum amount of water was added dropwise, and mixing was con-
tinued for 2 h. Sodium azide (1.70 g, 26 mmol) was dissolved in a
minimal amount of water and added dropwise to the mixture.
After 30 min, the glacial acetic acid was roughly removed under re-
duced pressure. The residue was extracted with EtOAc, and the or-
ganic phases were washed with sat. NaHCO3, brined, dried over
MgSO4, filtered, and concentrated. Purification by flash column
chromatography on a silica gel (hexane:EtOAc = 6:1) yielded 6 as
an orange solid (1.83 g, 65%). 1H NMR (600 MHz, CDCl3, 300 K): d
10.83 (s, 1H, OH), 9.86 (s, 1H, CHO), 7.20 (m, 2H, H-arom.), 7.00
(t, 1H, H-arom.); 13C NMR (150 MHz, CDCl3, 300 K): d 195.7,
158.8, 132.1, 127.9, 122.6, 120.8, 119.4. HRMS (ESI) Calcd for
C7H5N3O2Na [M+Na]+ 186.0274, found 186.0274.
4.2.5. 2-Difluoromethyl-4-azidophenyl 5-acetoamido-3,5-
dideoxy-
D
-glycero-
a-
D-galacto-non-2-ulopyranosidonic acid (2)
A solution of 5 M sodium methoxide in MeOH (450
lL) was
added dropwise to a solution of 10 (620 mg, 0.94 mmol) in MeOH
(10 mL), and the reaction mixture was stirred under a N2 gas
atmosphere at room temperature for 2 h. To neutralize the excess
sodium methoxide, 1% acetic acid was added, and the mixture was
concentrated in vacuo. The residue was dissolved in H2O (8.5 mL),
and 1 N NaOH (1.5 mL) was added dropwise to the mixture, which
was then stirred at room temperature for 1 h. The reaction mixture
was neutralized with 1% acetic acid, and the residue was purified
by reverse-phase chromatography (Wakogel 50C18, H2O:acetoni-
trile = 3:1) and lyophilized to yield compound 2 as a beige solid
(354 mg, 79%). 1H NMR (500 MHz, D2O, 300 K) d 7.25 (d, 1H,
J = 8.9 Hz, H-arom.), 7.21 (d, 1H, J = 1.8 Hz, H-arom.), 7.05 (d, 1H,
J = 8.8 Hz, H-arom.), 6.96 (t, 1H, J = 55.2 Hz, CHF2), 3.61–3.81 (m,
5H, H-4, 5, 6, 9a), 3.46–3.51 (m, 2H, H-7, 9b), 2.78 (dd, 1H,
J = 1.8, 12.6 Hz, H-3a), 1.91 (s, 3H, NHAc), 1.84 (t, 1H, J = 12.5 Hz,
H-3b); 13C NMR (125 MHz, CDCl3, 300 K) d 175.1, 171.9, 136.3,
122.9, 122.1, 116.4, 113.4, 111.6, 109.7, 103.0, 73.5, 71.6, 68.1,
68.0, 62.6, 51.6, 40.3, 22.0. HRMS (ESI) Calcd for C18H22N4O9F2Na
[M+Na]+ 499.1247, found 499.1249.
4.2.3. Methyl [4-azido-2-formylphenyl 5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-
ulopyranoside]onate (9)
A suspension of methyl 5-acetamido-3,5-dideoxy-
-galacto-non-2-ulopyranosonate (8) (969 mg, 3.0 mmol) in acetyl
D-glycero-a-D-galacto-non-2-
D-glycero-a-
D
chloride (10 mL) was vigorously stirred under a N2 gas atmosphere
at room temperature for 36 h. The resulting solution was concen-
trated and co-evaporated with toluene three times. Diisopropyl-
ethylamine (1.57 mL, 9.0 mmol) was added dropwise to
a
suspension of the residue and 5-azido-2-hydroxybenzaldehyde
(6) (490 mg, 3.0 mmol) in acetonitrile (30 mL), and the mixture
was stirred under a N2 gas atmosphere at room temperature for
24 h. After the solvent was evaporated in vacuo, the residue was
purified by flash column chromatography on a silica gel (hex-
ane:acetone = 7:4) to yield compound 9 as a yellow powder
(1.16 g, 61%). 1H NMR (600 MHz, CDCl3, 300 K) d 10.35 (s, 1H,
CHO), 7.49 (d, 1H, J = 3.0 Hz, H-arom.), 7.31 (d, 1H, J = 4.2 Hz, H-
arom.), 7.19 (dd, 1H, H-arom.), 5.35 (m, 3H, NH, H-7, H-8), 4.99
(ddd, 1H, J = 4.8, 10.8, 12.0 Hz, H-4), 4.46 (d, 1H, J = 10.8 Hz, H-6),
4.26 (dd, 1H, J = 2.4, 11.4 Hz, H-9a), 4.06-4.12 (m, 2H, H-5, H-9b),
3.65 (s, 3H, COOMe), 2.79 (dd, 1H, J = 4.8, 12.6 Hz, H-3a), 2.28 (t,
1H, J = 12.0 Hz, H-3b), 2.15, 2.14, 2.06, 2.05 (s, 3H each, OAc),
1.93 (s, 3H, NHAc); 13C NMR (150 MHz, CDCl3, 300 K) d 188.5,
170.9, 170.8, 170.3, 170.1, 170.0, 167.4, 152.9, 136.9, 129.1,
128.5, 128.2, 126.1, 122.5, 117.7, 100.6, 73.5, 68.5, 68.3, 67.1,
62.1, 53.2, 49.5, 38.2, 23.2, 21.0, 20.82, 20.75. HRMS (ESI) Calcd
for C27H32N4O14Na [M+Na]+ 659.1807, found 659.1806.
4.3. Construction of the libraries 3 for first screening
Aqueous solutions of compound 2 (100 mM), sodium ascorbate
(66.7 mM), and CuSO4 (100 mM) and dimethylsulfoxide solutions
of TBTA (100 mM) and the alkyne compounds (100 mM) were pre-
pared. Each alkyne compound (5
l
L) was added to CuSO4 (2.5
l
l
L),
L),
TBTA (2.5 L), compound 2 (5 L), and sodium ascorbate (35
l
l
and the mixture was incubated for 8 h. The yields of the triazole
compounds were roughly estimated by comparison between the
ESI-MS signal intensity of compound 2 and that of each triazole
product 3. The solutions with a greater than 70% yield of the tria-
zole product 3 were used for the following screening assay.
4.4. General procedure for preparation of click compound 3
Solutions of compound
2 (100 mM), sodium ascorbate
(250 mM), and CuSO4 (100 mM) in H2O and solutions of TBTA
(100 mM) and the alkyne compounds (100 mM) in dimethylsulfox-
4.2.4. Methyl [2-difluoromethyl-4-azidophenyl 4,7,8,9-tetra-O-
acetyl-5-acetamido-3,5-dideoxy-
ulopyranoside]onate (10)
D
-glycero-
a
-
D
-galacto-non-2-
ide were prepared. Each alkyne compound (150
CuSO4 (75 L), TBTA (75 L), compound 2 (150
ascorbate (150 L), and the mixture was incubated for 18 h. The
l
L) was added to
l
l
lL), and sodium
Diethylamino sulfur trifluoride (530
lL, 3.88 mmol) was added
l
dropwise to a solution of 9 (1.01 g, 1.55 mmol) in dichloromethane
(15 mL), and the reaction mixture was stirred under a N2 gas atmo-
sphere at room temperature for 3 h. MeOH was added to the
resulting mixture was purified by reverse-phase HPLC using an
H2O-acetonitrile linear gradient system and then lyophilized to
yield 3.

H. Kai et al. / Bioorg. Med. Chem. 20 (2012) 2739–2746
2745
4.4.1. 4-{[2-Methoxy-5-(trifluoromethyl)pyridin-3-ylcarbamoyl
of 60 lL. The VCNA activity was estimated by monitoring the in-
oxy]methyl}-1H-1,2,3-triazol-1-yl-2-(difluoromethyl)phenyl 5-ace
creaseinthefluorescencedue to thehydrolysisof4-MU-NANAusing
an excitation wavelength of 365 nm and an emission wavelength of
450 nm. The residual activity was estimated by comparison with the
control, which was obtained in the absence of the inhibitor.
toamido-3,5-dideoxy-D-glycero-a-D-galacto-non-2-
ulopyranosidonic acid (3a)
White solid (64%). 1H NMR (500 MHz, CD3OD, 300 K) d 8.66 (s,
1H, H-triazole), 8.52 (s, 1H, H-arom.), 8.14 (s, 1H, H-arom.), 8.03
(s, 1H, H-arom.), 7.91 (d, J = 8.6 Hz, H-arom.), 7.78 (d, J = 8.8 Hz,
H-arom.), 7.18 (t, 1H, J = 55.3 Hz, CHF2), 5.42 (s, 2H, CH2), 4.07 (s,
3H, OMe), 3.95 (d, 1H, J = 6.2 Hz, H-6), 3.82–3.90 (m, 4H, H-4, 5,
8, 9a), 3.65 (dd, 1H, J = 5.8, 11.6 Hz, H-9b), 3.57 (d, 1H, J = 9.1 Hz,
H-7), 3.02 (d, 1H, J = 10.6 Hz, H-3a), 2.05 (s, 3H, NHAc), 1.99 (t,
1H, J = 11.4 Hz, H-3b); 13C NMR (125 MHz, CD3OD, 300 K) d
174.1, 153.8, 136.8, 132.4, 123.4, 123.2, 122.8, 122.0, 120.3,
120.0, 119.7, 117.7, 111.2, 74.4, 71.6, 68.8, 67.5, 63.3, 57.7, 53.5,
52.5, 41.2, 21.2. HRMS (ESI) Calcd for C29H30N6O12F5 [MÀH]
À749.1847, found 749.1851.
4.5.3. First and second screening (human neuraminidase 2;
hNeu2)
For the hNeu2 pre-incubation experiment, hNeu2 (10.6 mU),
inhibitor 3 (final concentration 100
EDTA (final concentration 0.25 mM), and BSA (13.2
incubated in 33 mM MES buffer (pH 6.5), 4 mM CaCl2 at 25 °C in fi-
nal volume of 32 L)
lM for 1st and 2nd screening),
lg) were pre-
l
L. At 5-min intervals (0ꢀ15 min), aliquots (4
l
of the pre-incubation enzyme solution were transferred into assay
medium and assayed for the residual activity.
For the hNeu2 residual activity assay, hNeu2 activity was mea-
sured in 33 mM MES buffer (pH 6.5), 4 mM CaCl2and 1 mM 4-MU-
NANA at 37 °C. The enzyme reaction was initiated by addition of
4.4.2. 4-[3-(2,2-Dichloroacetamido)phenyl]-1H-1,2,3-triazol-1-
yl-2-(difluoromethyl)phenyl 5-acetoamido-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosidonic acid (3b)
D
-
pre-incubation enzyme solution in a final volume of 60 lL. The
a-
D
hNeu2 activity was estimated by monitoring the increase of fluo-
rescence due to hydrolysis of 4-MU-NANA using an excitation
wavelength of 365 nm and an emission wavelength of 450 nm.
The residual activity was estimated by comparison with control,
which was obtained without inhibitor.
White solid (46%). 1H NMR (500 MHz, CD3OD, 300 K) d 8.95 (s,
1H, H-triazole), 8.13 (s, 1H, H-arom.), 8.08 (s, 1H, H-arom.), 7.96
(d, 1H, J = 8.5 Hz, H-arom.), 7.79 (d, 1H, J = 8.9 Hz, H-arom.), 7.73
(d, 1H, J = 7.7 Hz, H-arom.), 7.68 (d, 1H, J = 8.1 Hz, H-arom.), 7.48
(dd, J = 7.9 Hz, H-arom.), 7.17 (t, 1H, J = 55.1 Hz, CHF2), 6.43 (s,
1H, CHCl2), 3.94 (d, 1H, J = 9.7 Hz, H-6), 3.78–3.89 (m, 4H, H-4, 5,
8, 9a), 3.64 (dd, 1H, J = 5.4, 11.2 Hz, H-9b), 3.56 (d, 1H, J = 4.5 Hz,
H-7), 3.00 (dd, 1H, J = 4.1, 12.7 Hz, H-3a), 2.03 (s, 3H, NHAc), 1.96
(dd, 1H, J = 11.9 Hz, H-3b); 13C NMR (125 MHz, CD3OD, 300 K) d
174.1, 173.3, 163.1, 147.7, 138.1, 132.5, 131.0, 129.4, 123.0,
122.6, 122.3, 120.2, 119.3, 117.3, 74.4, 71.5, 68.8, 67.5, 66.8, 63.3,
52.5, 41.2, 21.2. HRMS (ESI) Calcd for C28H28N5O10F2Cl2 [MÀH]À
702.1187, found 702.1199.
4.5.4. Determination of KI and kinact and t1/2values (hNeu2)
For the hNeu2 pre-incubation experiment, hNeu2 (10.6 mU),
several concentrations of inhibitor
(20 g) were pre-incubated in 33 mM MES buffer (pH 6.5) with
4 mM CaCl2 at 25 °C in a final volume of 50 L. At 5-min intervals
(0–15 min), aliquots (4 L) of the pre-incubated enzyme solution
3 (50–500 lM), and BSA
l
l
l
were transferred into the assay medium and assayed for residual
activity.
For the hNeu2 residual activity assay, the hNeu2 activity was
measured in 33 mM MES buffer (pH 6.5) containing 4 mM CaCl2
and 1 mM 4-MU-NANA at 25 °C. The enzyme reaction was initiated
by the addition of the pre-incubated enzyme solution in a final vol-
4.5. Biological assay
4.5.1. First and second screening (Vibrio cholerae
neuraminidase; VCNA)
ume of 60 lL. The hNeu2 activity was estimated by monitoring the
For the VCNA pre-incubation experiment, VCNA (8.3 mU) and
increase in the fluorescence due to the hydrolysis of 4-MU-NANA
using an excitation wavelength of 365 nm and an emission wave-
length of 450 nm. The residual activity was estimated by compar-
ison with the control, which was obtained in the absence of the
inhibitor.
inhibitor 3 (30
concentration) were pre-incubated in 100 mM MES buffer (pH 6.5)
with 4 mM CaCl2 at 25 °C in a final volume of 50 L. At 5-min inter-
vals (0–15 min), aliquots (3 L) of the pre-incubated enzyme solu-
lM for 1st screening, 50 lM for 2nd screening; final
l
l
tion were transferred into the assay medium and assayed for
residual activity.
Acknowledgments
For the VCNA residual activity assay, the VCNA activity was mea-
sured in 100 mM MES buffer (pH 6.5) containing 4 mM CaCl2 and
2 mM 4-MU-NANA at 37 °C. The enzyme reaction was initiated by
the addition of the pre-incubated enzyme solution in a final volume
This work was supported by funding from the Ministry of
Education, Culture, Science, Sports and Technology of Japan for
‘Grant-in-Aid for Young Scientists (A) and for Scientific Research
(B)’ and the ‘Innovation COE Program for Future Drug Discovery
and Medical Care’. This work was also supported in part by funding
from Hokkaido University for ‘Special Incentive for Young Scien-
tists’. We thank Ms. M. Kikuchi, Ms. S. Oka, and Mr. T. Hirose at
the Center for Instrumental Analysis, Hokkaido University, for their
help with the ESI-MS measurements. We also thank Dr. X.-D. Gao
and Mr. R. Miyoshi for the gift of hNeu2.
of 60 lL. The VCNA activity was estimated by monitoring the in-
creaseinthefluorescenceduetothehydrolysisof4-MU-NANAusing
an excitation wavelength of 365 nm and an emission wavelength of
450 nm. The residual activity was estimated by comparison with the
control, which was obtained in the absence of the inhibitor.
4.5.2. Determination of KI and kinact and t1/2 values (VCNA)
For the VCNA pre-incubation experiment, VCNA (8.3 mU) and
several concentrations of inhibitor 3 (10–200
bated in 100 mM MES buffer (pH 6.5) with 4 mM CaCl2 at 25 °C
in a final volume of 50 L. At 5-min intervals (0–15 min), aliquots
(3 L) of the pre-incubated enzyme solution were transferred into
lM) were pre-incu-
Supplementary data
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.02.001.
l
the assay medium and assayed for residual activity.
For the VCNA residual activity assay, the VCNA activity was mea-
sured in 100 mM MES buffer (pH 6.5) containing 4 mM CaCl2 and
2 mM 4-MU-NANA at 25 °C. The enzyme reaction was initiated by
the addition of the pre-incubated enzyme solution in a final volume
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