Analogs of zanamivir with modified C4-substituents as the inhibitors against the group-1 neuraminidases of influenza viruses

Article abstract of DOI:10.1016/j.bmc.2010.04.010

Unlike the group-2 neuraminidase, the group-1 neuraminidase of influenza virus possesses a flexible loop (the 150-loop) and a cavity (the 150-cavity) adjacent to the active site, and renders a conformational change from the 'open' form to the 'closed' form on binding with substrate (sialo-glycoprotein) or inhibitor (e.g., zanamivir). Zanamivir derivative 8a having an extended (piperazinocarbonyl)propyl substituent at the internal N-position of the guanidino group is designed as a possible inhibitor on the basis of computer docking to the open form of N1 subtype neuraminidase. Indeed, compound 8a exhibits strong neuraminidase inhibition and good anti-influenza activity against H1N1 virus with IC₅₀ = 2.15 μM and EC₅₀ = 0.77 μM, respectively. This study may provide a clue to future design of better group-1 neuraminidase inhibitors.

Full text of DOI:10.1016/j.bmc.2010.04.010

Bioorganic & Medicinal Chemistry 18 (2010) 4074–4084
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Analogs of zanamivir with modified C4-substituents as the inhibitors against
the group-1 neuraminidases of influenza viruses
Wen-Hsien Wen a, Shi-Yun Wang b, Keng-Chang Tsai b, Yih-Shyun E. Cheng b, An-Suei Yang b,
b
Jim-Min Fang a,b, , Chi-Huey Wong
*
a Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
b The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Unlike the group-2 neuraminidase, the group-1 neuraminidase of influenza virus possesses a flexible loop
(the 150-loop) and a cavity (the 150-cavity) adjacent to the active site, and renders a conformational
change from the ‘open’ form to the ‘closed’ form on binding with substrate (sialo-glycoprotein) or inhib-
itor (e.g., zanamivir). Zanamivir derivative 8a having an extended (piperazinocarbonyl)propyl substituent
at the internal N-position of the guanidino group is designed as a possible inhibitor on the basis of com-
puter docking to the open form of N1 subtype neuraminidase. Indeed, compound 8a exhibits strong neur-
Received 17 February 2010
Accepted 2 April 2010
Available online 8 April 2010
Keywords:
Zanamivir derivative
Neuraminidase inhibitor
Influenza
aminidase inhibition and good anti-influenza activity against H1N1 virus with IC50 = 2.15
EC50 = 0.77 M, respectively. This study may provide a clue to future design of better group-1 neuramin-
idase inhibitors.
lM and
l
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
4-deoxy-4-guanidino-Neu5Ac2en (zanamivir, 3, Ki = 2 ꢀ 10ꢁ10 M),
by replacing the C4-hydroxy group in DANA with a basic group.
The enhanced affinity is attributable to the strong electrostatic
interactions of the aminium or guanidinium substituents with
the two glutamate residues in physiological conditions. Zanamivir,
in the trade name Relenza, is the first NA inhibitor approved to be
used as anti-influenza drug.1c,6
Influenza is a long-standing health problem causing significant
mortality in annual epidemics by infection of the human respira-
tory system. The worldwide spread of H5N1 avian flu and the re-
cent outbreak of the new type H1N1 human flu have raised
public concerns of the global influenza pandemics. Neuraminidase
(NA), a glycoprotein expressed on the influenza virus surface, is
essential for virus replication and infectivity by breaking the link-
age between the progeny virus from the surface sialo-receptor of
host cells. Thus, inhibition of NA by the structure-based strategy
has been applied in discovery of anti-influenza drugs.1 Though lit-
tle success has been achieved in development of the substrate
mimetics, for example, the thioglycoside,2a 3-deoxy-3-fluoro,2b
and phosphonate2c of sialic acid (Neu5Ac), as the influenza virus
NA inhibitors, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid
(Neu5Ac2en, DANA, 1)3 is identified as the first effective NA inhib-
itor (Ki = 4 ꢀ 10ꢁ6 M) to mimic the transition state in the enzy-
matic cleavage of the sialo-moiety. The X-ray crystal structures4
of the influenza virus NA (N2 subtype) and its complex with the
inhibitor DANA reveal that the C4-hydroxy group is positioned in
the negatively charged binding pocket S2 containing two gluta-
mate residues (Glu119 and Glu227). This structural analysis has
thus led to a finding of more potent NA inhibitors,5 such as 4-ami-
no-4-deoxy-Neu5Ac2en (amino-DANA, 2, Ki = 4 ꢀ 10ꢁ8 M) and
Generally speaking, the flu genes encoding NA are more con-
served than hemagglutinin (HA), another essential glycoprotein on
the surface of influenza virus for recognition and attachment to
the host cell. There are two genetically distinct groups in neurami-
nidases of influenza A viruses: group-1 includes N1, N4, N5, and
N8 subtypes, and group-2 includes N2, N3, N6, N7, and N9 subtypes.
The N1 and N2 are prominent subtypes in human influenza viruses,
in particular, the current pandemic H1N1 and H5N1 viruses belong
to the N1 subtype. A recent X-ray crystallographic study7 reveals
that group-1 and group-2 NAs are not only genetically distinct but
also structurally distinct. Group-1 NA possesses a flexible loop
(known as the 150-loop) and a cavity (the 150-cavity) adjacent to
the active site. Group-1 NA would render the conformational change
from the ‘open’ form to the ‘closed’ form on binding with substrate
(or inhibitor), whereas group-2 NA always exists in the closed form.
The NA inhibitors such as zanamivir and oseltamivir carboxylic acid8
are originally designed based on the crystal structures of group-2
NAs (N2 and N9) in the closed form. Because group-1 NA would
change the conformation from the ‘open’ form to the ‘closed’ form
on binding, an insight into such conformational change can provide
a novel approach to design effective inhibitors specifically against
* Corresponding author. Tel.: +886 2 33661663; fax: +886 2 27325090.
E-mail address: jmfang@ntu.edu.tw (J.-M. Fang).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.010

W.-H. Wen et al. / Bioorg. Med. Chem. 18 (2010) 4074–4084
4075
the group-1 NAs, such as those exist in the avian H5N1 and the 2009
human H1N1 viruses, by exploiting additional interactions with the
150-cavity of the open form.
AcO
OAc
AcO
OAc
AcO
AcHN
H
O
a
AcO
AcHN
H
O
b
CO2Me
CO2Me
H2N
NsHN
2. Molecular modeling
10
9
Accordingly, a substituent extended from the amino or guani-
dino groups at the C-4 position of zanamivir may exert an addi-
tional binding to the 150-cavity. Docking zanamivir to the crystal
structure of the N1 neuraminidase in the open form (Fig. 1A, PDB
code 2HU0) reveals room for possible side chain extension from
the C-4 position of zanamivir to fit in with the 150-cavity. Further
examination of the docking structure suggests that the prominent
guanidinium side chain of Arg156 and the carbonyl group of the
backbone of Gly147 around the 150-cavity are prospective binding
partners by a salt-bridge or hydrogen bond for drug design. We
thus propose compound 8a bearing an amide group and an amino
group on its side chain as a target molecule. The docking simula-
tion of compound 8a with the open form N1 neuraminidase shows
that the carbonyl group of the 8a side chain has a hydrogen bond
(3.64 Å) with residue Arg156 in the cavity, and the terminal amino
group at the tip of the side chain also displays a strong hydrogen
bond (3.29 Å) with residue Gly147 in the same cavity (Fig. 1B).
As a consequence, the designed side chain of 8a may provide addi-
tional interactions with the 150-cavity in the open form of N1
neuraminidase.
AcO
AcO
OAc
6
HO
OH
H
O
HO
AcHN
H
O
c (for 11a, b)
or d (for 11c, d)
AcHN
CO2Me
1
CO2H
2
4
Y(CH2)nHN
Y(CH2)nHN
4a Y = NH2 n = 3
4b Y = NH2 n = 4
4c Y = CO2H n = 3
4d Y = CO2H n = 4
11a Y = NHBoc n = 3
11b Y = NHBoc n = 4
11c Y = CO2Me n = 3
11d Y = CO2Me n = 4
Scheme 1. Synthesis of compound 4a–d bearing modified amino substituents at
C-4 position. Reagents and conditions: (a) o-O2NC6H4SO2Cl, CH2Cl2, rt, 83%; (b) (i)
Y(CH2)nOH, DIAD, PPh3, THF, rt; (ii) PhSH, Cs2CO3, DMF, rt, 80ꢁ86%; (c) (i) NaOH,
MeOH, H2O, rt; (ii) CF3CO2H, CH2Cl2, rt, 92ꢁ95%; (d) NaOH, MeOH, H2O, rt, 67ꢁ85%.
sulfonamide 10 as a key intermediate (Scheme 1). After Mitsunobu
reaction of 10 with appropriate alcohols, the Ns group was subse-
quently removed by thiophenol in the presence of cesium carbonate
to afford 11a–d in high yields (80–86%). Saponification of 11a–d, fol-
lowed by removal of the Boc group, produced the desired com-
pounds 4a–d.
To prepare guanidine derivatives, amine 9 was treated with
N-Boc-2-methylthio-2-imidazoline, followed by saponification
and removal of the Boc group, to afford a cyclic guanidine com-
pound 5 (Scheme 2A). Alternatively, amine 9 was reacted with
9-fluorenylmethoxycarbonyl isocyanate (FmocNCS) to give a thio-
urea 13 (Scheme 2B). After the Fmoc group was switched to a Boc
group, the thiourea 14 was reacted with appropriate alkylidenedi-
amine mono-carbamates in the presence of HgCl2 to give guani-
dines 15a and 15b. By saponification and a subsequent treatment
with TFA, the derivatives 6a and 6b bearing substituents at a ter-
minal N-position of the guanidine moiety were obtained in high
yields.
In this paper, we describe the synthesis and bioactivities of a
zanamivir derivative 8a having an extended substituent at the inter-
nal N-position of the guanidino group. We also prepared the analo-
gous compounds 4a–d, 5, 6a–b, 7a–c, and 8b–d (Schemes 1–3) for
comparison of their NA inhibitory and anti-influenza activities.
3. Chemistry
Amine9 wasprepared fromsialicacidaccordingtothepreviously
reported method.9 Although alkylamination of 9 has been realized,10
attempts in direct alkylation of 9 with various halides in basic condi-
tions resulted in complicated mixture. Therefore, amine 9 was first
treated with o-nitrobenzenesulfonyl chloride (NsCl)11 to give
Figure 1. Zanamivir (3) and compound 8a binding to the active sites of group-1 neuraminidase (N1) in the ‘open’ form. (A) Molecular surface of N1 neuraminidase with
bound zanamivir; (B) The docking result of compound 8a with neuraminidase in the open form. Zanamivir and compound 8a are highlighted in atom-colored stick model.
Individual atoms are displayed in gray (carbon), blue (nitrogen), and red (oxygen). The neuraminidases are shown in cyan, whereas R118, E119, G147, V149, D151, R152,
R156, W178, E227, E276, R292, and R371 are also highlighted in atom-colored stick model. Hydrogen bonds are represented by dotted lines.

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W.-H. Wen et al. / Bioorg. Med. Chem. 18 (2010) 4074–4084
activity. Among them, the zanamivir analog 8a bearing a (piperaz-
inocarbonyl)propyl substituent at the internal position of guani-
dino group was the most potent NA inhibitor with IC50 = 2.15
and EC50 = 0.77 M. In comparison, compound 8a showed about
AcO
OAc
HO
OH
AcO
AcHN
H
O
HO
AcHN
H
O
l
M
a
b
CO2Me
CO2H
9
l
88-fold potency than DANA (1) in NA inhibition and twofold po-
tency than compound 2 in anti-influenza activity. However, com-
pound 8a was still inferior to zanamivir with regard to the
enzyme inhibition and anti-influenza activity.
HN
HN
HN
N
N
BocN
12
5
5. Discussion
AcO
OAc
AcO
OAc
From the binding model (Fig. 1), we can find that there is a spa-
tially restricted region in the S2 pocket defined by Glu119, Trp178
and Glu227 of the active site in open form. Thus the N-substituted
guanidino group of 5, 6a, and 6b may be too large to be accommo-
dated into the S2 pocket. In the case of 7c, the carboxylic acid of the
side chain could make a salt-bridge interaction with Arg156,
whereas the amino group of 7a,b could result in repulsive interac-
tion. Compound 8a is the most potent NA inhibitor among the test
samples, presumably because the piperazino group of the side
chain in 8a can exert a strong hydrogen bond interaction with
Gly147. The analogs 8b–d having N-methylpiperazino, morpho-
lino, and piperidino groups lack such hydrogen bonding with
Gly147, and their inhibitory activities were reduced.
AcO
AcHN
H
O
AcO
AcHN
H
O
d
c
CO2Me
CO2Me
9
HN
S
HN
S
NHFmoc
13
NHBoc
14
AcO
OAc
HO
OH
AcO
H
O
HO
AcHN
H
O
e
f
AcHN
CO2Me
CO2H
HN
HN
NHBoc
NH
BocHN(CH2)nN
H2N(CH2)nHN
In order to understand the activity discrepancy between com-
pound 8a and zanamivir, we also examined the 3D model of
zanamivir binding to neuraminidase (N1) in closed form6 (Fig. 2)
to compare with that of compound 8a binding to neuraminidase
in open form (Fig. 1B). In the closed form of neuraminidase, the
guanidinium group of zanamivir makes tight salt-bridge interac-
tions with negatively charged S2 pocket created by Glu119,
Asp151 and Glu227 in the interaction distances of 3.33, 2.92 and
3.04 Å, respectively. On the other hand, when compound 8a binds
to the open form of neuraminidase, the interaction between the
guanidinium group and the S2 pocket will weaken to 4.21, 4.24
and 3.24 Å, respectively. This change may explain why compound
8a shows less potency than zanamivir in NA inhibition, even
though compound 8a possesses additional interaction with the
150-cavity adjacent to the active site of NA.
15a n = 3
15b n = 4
6a n = 3
6b n = 4
Scheme 2. Synthesis of zanamivir derivatives 5, 6a, and 6b with modification at
terminal positions of the guanidino group. Reagents and conditions: (a) N-Boc-2-
methylthio-2-imidazoline, AcOH, EtOH, 50 °C, 62%; (b) (i) CF3CO2H, CH2Cl2, rt; (ii)
NaOH, MeOH, H2O, rt, 94%; (c) FmocNCS, CH2Cl2, rt, 90%; (d) (i) piperidine, CH2Cl2,
rt; (ii) Boc2O, CH2Cl2, rt, 87%; (e) BocHN(CH2)nNH2, HgCl2, Et3N, DMF, rt, 66ꢁ74%; (f)
(i) NaOH, MeOH, H2O, rt; (ii) CF3CO2H, CH2Cl2, rt, 90ꢁ94%.
In another approach, the alkylamino groups in 11a–c were
effectively transformed into di-Boc-alkylguanidino groups by
treatment with N,N0-di-Boc-S-methylisothiourea in the presence
of HgCl2 (Scheme 3A). After saponification, the Boc groups in
16a–c were removed by TFA to afford the desired products 7a–c
bearing substituents at the internal N-position of the guanidine
group. By a similar procedure, 16d was prepared and its allyl group
was subsequently removed by the catalysis of Pd(PPh3)4 to give
acid 18 (Scheme 3B). The acid was converted to amides 20a–d by
coupling with N-Boc-piperazine, N-methylpiperazine, morpholine,
and piperidine, respectively, using benzotriazol-1-yl-oxytripyrroli-
dinophosphonium hexafluorophosphate (PyBOP) as the promoter.
Saponification, followed by removal of Boc groups, culminated in
the synthesis of the target zanamivir derivatives 8a–d.
From a different perspective, Stanislav and co-workers12 have
recently carried out the molecular dynamics simulation for the
complexes of group-1 NA with zanamivir and its phosphonate con-
gener that contains a phosphonyl group to replace the carboxylate
group in zanamivir. Their study indicates that the neuraminidase
N1 quickly interchanges between the closed and open conforma-
tions on binding with inhibitors. Zanamivir prefers the closed NA
form, whereas its phosphonate congener having a negative total
charge can repel the Asp151 residue to induce opening of
the150-loop of NA. In combination of Stanislav’s and our studies,
one may consider design of better group-1 NA inhibitors such as
the phosphonate congener of 8a, in which the strong electrostatic
interactions of phosphonyl group with arginine residues13 can
compensate the reduced salt-bridge interaction due to the ex-
tended side chain.
4. Bioassay
All the target compounds were evaluated for their abilities to
inhibit the hydrolysis of a fluorogenic substrate 20-(4-methyl-
umbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA) by the N1
neuraminidase of influenza A/WSN/1933 (H1N1) virus. The active
inhibitors were further evaluated by their anti-influenza activities
using cytopathic prevention assays. The results are listed in Table 1
including the values of DANA (1), compound 2 and zanamivir (3)
for comparison. Compounds 4a–b, 6a–b, and 7a–b with the amino
or guanidino group at C-4 bearing a substituent annexed with ami-
no group showed no significant activities (IC50 >200 lM), whereas
compounds 4c–d and 7c having the terminal amino group replaced
by a carboxylic group improved the NA inhibitory activities consid-
erably (e.g., the IC50 of 7c was 7.2 lM). The zanamivir analog 5 hav-
6. Conclusion
On the basis of computer docking to the open form of N1 sub-
type neuraminidase, we have designed and prepared compound
8a with an extended substituent at the internal N-position of the
guanidino group, in addition to other analogous compounds 4a–
d, 5, 6a–b, 7a–c, and 8b–d, as a possible inhibitor specifically
against the group-1 neuraminidases. Though direct alkylation of
amine 9 did not give clean product, introduction of an extended
substituent at the N-position is achieved by Mitsunobu reaction
of the o-nitrobenzenesulfonamide 10 in good yields. Compound
ing the guanidino group confined by an ethylene unit exhibited no
NA inhibitory activity. Elaboration of the terminal carboxylic group
in 7c to amides 8a and 8b, further improved the NA inhibitory

W.-H. Wen et al. / Bioorg. Med. Chem. 18 (2010) 4074–4084
4077
AcO
OAc
HO
OH
AcO
AcHN
H
O
HO
AcHN
H
O
a
b
11a−c
CO2Me
CO2H
Y(CH2)nN
BocHN
Y(CH2)nN
H2N
NBoc
NH
16a Y = NHBoc n = 3
16b Y = NHBoc n = 4
16c Y = CO2Me n = 3
7a Y = NH2 n = 3
7b Y = NH2 n = 4
7c Y = CO2H n = 3
AcO
OAc
AcO
OAc
AcO
AcHN
H
O
c
AcO
AcHN
H
O
d
10
CO2Me
CO2Me
N
NH
NHBoc
O
BocN
O
O
O
16d
17
AcO
OAc
f
AcO
AcHN
H
O
e
CO2Me
NH
N
X
NHBoc
19a X = NBoc
19b X = NMe
19c X = O
HO
BocN
O
18
19d X = CH2
HO
OH
AcO
OAc
HO
AcHN
H
O
AcO
AcHN
H
O
g
CO2H
CO2Me
N
N
NH
NHBoc
X
N
H2N
X
N
BocN
O
O
20a X = NBoc
20b X = NMe
20c X = O
8a X = NH
8b X = NMe
8c X = O
20d X = CH2
8d X = CH2
Scheme 3. Synthesis of zanamivir derivatives 7a–c and 8a–c with modification at the internal N-position of guanidino group. Reagents and conditions: (a) N,N0-di-Boc-S-
methylisothiourea, HgCl2, Et3N, DMF, rt, 86ꢁ91%; (b) (i) NaOH, MeOH, H2O, rt; (ii) CF3CO2H, CH2Cl2, rt, 92ꢁ95%; (c) (i) allyl 4-hydroxybutanoate, DIAD, PPh3, THF, rt; (ii) PhSH,
Cs2CO3, DMF, rt, 81%; (d) N,N0-di-Boc-S-methylisothiourea, HgCl2, Et3N, DMF, rt, 89%; (e) Pd(PPh3)4, morpholine, THF, rt, 85%; (f) 19a–d, PyBOP, Et3N, DMF, rt, 73ꢁ89%; (g) (i)
NaOH, MeOH, H2O, rt, (ii) CF3CO2H, CH2Cl2, rt, 88ꢁ96%.
8a exhibited strong NA inhibition and good anti-influenza activity
with IC50 = 2.15 M and EC50 = 0.77 M, respectively. According to
argon unless otherwise noted. Reactions were magnetically stirred
and monitored by thin-layer chromatography on silica gel using
aqueous p-anisaldehyde as visualizing agent. Silica gel (0.040–
0.063 mm particle sizes) was used for column chromatography.
Flash chromatography was performed on silica gel of 60–200 lm
particle size. Molecular sieves were activated under high vacuum
l
l
the molecular modeling, the extended side chain of 8a can provide
additional interactions with the 150-cavity in the open form of N1
neuraminidase. However, this disposition also weakens the salt-
bridge or hydrogen bond interaction between the guanidino group
of 8a and the S2 pocket in the active site. This study may provide a
clue to future design of better group-1 neuraminidase inhibitors
against influenza virus.
at 220 °C over 6 h.
Melting points were recorded on a Yanaco or Electrothermal
MEL-TEMP 1101D apparatus in open capillaries and are not cor-
rected. Optical rotations were measured on digital polarimeter of
Japan JASCO Co. DIP-1000. ½a D
ꢂ
values are given in units of
7. Experimental
7.1. Chemistry
10ꢁ1 deg cm2 gꢁ1. Infrared (IR) spectra were recorded on Nicolet
Magna 550-II or Thermo Nicolet 380 FT-IR spectrometers. Nuclear
magnetic resonance (NMR) spectra were obtained on Varian Unity
Plus-400 (400 MHz) or Bruker Avance-400 (400 MHz) spectrome-
ter. Chemical shifts (d) are given in parts per million (ppm) relative
to dH 7.24/dC 77.0 (central line of t) for CHCl3/CDCl3, dH 4.80
for H2O/D2O, dH 3.31/dC 48.2 for CD3OD-d4, or dH 2.49/dC 39.5
for DMSO-d6. The splitting patterns are reported as s (singlet),
All the reagents and solvents were reagent grade and were used
without further purification unless otherwise specified. All sol-
vents were anhydrous grade unless indicated otherwise. CH2Cl2
was distilled from CaH2. All non-aqueous reactions were carried
out in oven-dried glassware under a slight positive pressure of

4078
W.-H. Wen et al. / Bioorg. Med. Chem. 18 (2010) 4074–4084
Table 1
under reduced pressure, the residue was purified by silica gel col-
umn chromatography (CH2Cl2/MeOH = 25:1) to give the sulfon-
amide product 10 (3.0 g, 92%). C24H29N3O14S; colorless solid, mp
Neuraminidase inhibitory and anti-influenza activities of compounds 1–8
a
b
Inhibitor
IC50
(l
M)
EC50 (lM)
105–107 °C; TLC (CH2Cl2/MeOH = 9:1) Rf = 0.23; ½a D26
ꢂ
+28.9 (c
1 (DANA)
2 (amino-DANA)
3 (zanamivir)
190c
—
—
0.89, EtOAc); IR mmax (neat) 1739, 1660, 1542, 1370, 1222 cmꢁ1
;
1.5d
1H NMR (400 MHz, CDCl3) d 8.07–8.05 (1H, m), 7.83–7.80 (1H,
m), 7.74–7.69 (2H, m), 6.20 (1H, d, J = 9.2 Hz), 6.05 (1H, br s),
5.71 (1H, d, J = 2.0 Hz), 5.44 (1H, dd, J = 4.8, 1.2 Hz), 5.32–5.29
(1H, m), 4.63 (1H, dd, J = 12.0, 2.0 Hz), 4.54–4.49 (2H, m), 4.15
(1H, dd, J = 12.0, 6.8 Hz), 4.05–3.98 (1H, m), 3.74 (3H, s), 2.08
(3H, s), 2.05 (3H, s), 2.04 (3H, s), 1.82 (3H, s); 13C NMR (100 MHz,
CDCl3) d 171.4, 169.9 (2ꢀ), 169.2, 161.1, 146.8, 144.0, 134.2,
133.1, 132.6, 129.8, 124.6, 110.2, 77.1, 71.8, 68.6, 62.3, 53.6, 52.5,
47.1, 23.1, 21.1 (2ꢀ), 21.0; ESI-HRMS calcd for C24H29N3O14NaS:
638.1268, found: m/z 638.1250 [M+Na]+.
0.004
>200
>200
ꢃ70
0.013
NDe
NDe
NDe
NDe
NDe
NDe
NDe
NDe
NDe
52.0
0.77
10.7
37.6
45.0
4a
4b
4c
4d
5
6a
6b
7a
7b
7c
8a
8b
8c
8d
ꢃ80
>200
>200
>200
>200
>200
7.12
2.15
6.50
33.3
23.1
7.1.2. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-(3-tert-but
oxycarbonylamino)propylamino-3,4,5-trideoxy-D-glycero-D-gal
a
Neuraminidase inhibition against influenza virus A/WSN/1933 (H1N1).
Concentration of NA inhibitors for 50% protection of the cytopathic effects due
cto-non-2-enonic acid methyl ester (11a)
b
To a solution of sulfonamide 10 (245 mg, 0.4 mmol) in anhy-
drous THF (5 mL) were added PPh3 (157 mg, 0.6 mmol), 4-(tert-
butoxycarbonylamino)propanol (84 mg, 0.6 mmol) and diisopropyl
azodicarboxylate (DIAD, 0.169 mL, 0.6 mmol). The mixture was
stirred at room temperature for 21 h, and evaporated under re-
duced pressure. The residue was purified by silica gel column chro-
matography (CH2Cl2/MeOH = 40:1) to afford N-alkyl product with
contamination of triphenylphosphine oxide.
to influenza (A/WSN/1933) infection.
c
Neuraminidase inhibition against influenza virus A/Vienna/83/58 (H2N2) which
3
adapted from Ref.
d
Concentration required to reduce 50% plaque formation in MDCK cells due to flu
5a
(A/Singapore/1/57, H2N2) which adapted from Ref.
e
Not determined.
To the above-prepared N-alkyl compound in DMF (4 mL)
were added Cs2CO3 (362 mg, 1.11 mmol) and PhSH (0.23 mL,
2.22 mmol). The reaction mixture was stirred at room temperature
for 2.5 h, concentrated, and then dissolved in CH2Cl2. The mixture
was filtered; the filtrate was concentrated and purified by silica
gel column chromatography (gradients CH2Cl2/MeOH = 30:1 to
CH2Cl2/MeOH/Et3N = 15:1:0.5) to give amine 11a (202 mg, 86% for
two steps). C26H41N3O12; pale yellow solid, mp 139–140 °C; ½a D22
ꢂ
+49.5 (c 2.0 EtOAc); IR mmax (neat) 1743, 1677, 1530, 1368, 1250,
1223 cmꢁ1 1H NMR (400 MHz, CDCl3) d 6.22 (1H, d, J = 8.8 Hz),
;
6.02 (1H, d, J = 1.2 Hz), 5.47 (1H, dd, J = 3.6, 3.2 Hz), 5.31–5.27 (1H,
m), 5.01 (1H, br), 4.63 (1H, d, J = 12.0 Hz), 4.22 (1H, d, J = 7.2 Hz),
4.13 (1H, dd, J = 12.0, 7.2 Hz), 4.04–4.00 (1H, m), 3.74 (3H, s), 3.40
(1H, d, J = 6.8 Hz), 3.27–3.22 (1H, m), 3.05–3.00 (1H, m), 2.76–2.72
(1H, m), 2.60–2.55 (1H, m), 2.07 (3H, s), 2.01 (6H, s), 1.95 (1H, br
s), 1.93 (3H, s), 1.56–1.53 (2H, m), 1.39 (9H, s); 13C NMR
(100 MHz, CDCl3) 170.6, 170.4, 170.0, 169.9, 162.0, 156.0, 143.4,
111.8, 79.0, 76.9, 71.0, 68.1, 62.2, 55.6, 52.3, 46.8, 42.6, 38.0, 30.0,
28.5 (3ꢀ), 23.3, 21.0, 20.9 (2ꢀ); ESI-HRMS calcd for C26H42N3O12
:
588.2763, found: m/z 588.2768 [M+H]+.
Figure 2. A crystal complex of zanamivir with N1 neuraminidase in ‘closed’ form.
Hydrogen bonds are represented by dotted lines. The neuraminidase and zanamivir
are displayed in the colors as that described in Figure 1.
7.1.3. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-(4-tert-but
oxycarbonylamino)butylamino-3,4,5-trideoxy-D-glycero-D-gala
cto-non-2-enonic acid methyl ester (11b)
By a procedure similar to that for 11a, Mitsunobu reaction of
sulfonamide 10 (430 mg, 0.7 mmol) with 4-(tert-butoxycarbonyla-
mino)butanol (159 mg, 0.84 mmol) gave amine 11b (345 mg, 82%)
after removal of the sulfonamide group by PhSH/Cs2CO3.
d (doublet), t (triplet), q (quartet), m (multiplet), dd (double of
doublets) and br (broad). Coupling constants (J) are given in Hz.
Distortionless enhancement polarization transfer (DEPT) spectra
were taken to determine the types of carbon signals. The ESI-MS
experiments were conducted on a Bruker Daltonics BioTOF III
high-resolution mass spectrometer.
C27H43N3O12; pale yellow solid, mp 135–136 °C; ½a D24
ꢂ
+12.2 (c 2.0,
EtOAc); IR mmax (neat) 1746, 1684, 1532, 1368, 1251, 1220 cmꢁ1
;
1H NMR (400 MHz, CDCl3) d 6.70 (1H, d, J = 9.2 Hz), 5.98 (1H, s),
5.43 (1H, dd, J = 4.4, 3.2 Hz), 5.25–5.22 (1H, m), 5.01 (1H, br s),
4.59 (1H, d, J = 11.6 Hz), 4.22 (1H, d, J = 8.0 Hz), 4.08 (1H, dd,
J = 11.6, 7.2 Hz), 4.04–4.00 (1H, m), 3.70 (3H, s), 3.47 (1H, d,
J = 6.4 Hz), 3.35 (1H, br s), 3.00 (2H, br s), 2.72–2.68 (1H, m),
2.52–2.47 (1H, m), 2.01 (3H, s), 1.97 (6H, s), 1.86 (3H, s), 1.41
(4H, br), 1.35 (9H, s); 13C NMR (100 MHz, CDCl3) 171.0, 170.6,
170.2, 170.1, 162.1, 156.1, 143.8, 111.2, 78.9, 76.7, 71.1, 68.0,
62.0, 55.4, 52.2, 46.1, 44.6, 40.0, 28.3 (3ꢀ), 27.1, 26.6, 22.9, 20.7,
7.1.1. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-(2-nitrobe
nzenesulfonamido)-3,4,5-trideoxy-D-glycero-D-galacto-non-2-e
nonic acid methyl ester (10)
A
solution of amine 9 (2.38 g, 5.34 mmol) and 2-nitro-
benzenesulfonyl chloride (NsCl, 1.18 g, 5.34 mmol) in CH2Cl2
(50 mL) was stirred at room temperature for 6 h. The mixture
was washed with saturated NaHCO3 and brine. After concentration

W.-H. Wen et al. / Bioorg. Med. Chem. 18 (2010) 4074–4084
4079
20.6 (2ꢀ); ESI-HRMS calcd for C27H43N3O12: 602.2920, found: m/z
7.1.7. 5-Acetamido-2,6-anhydro-4-(4-aminobutyl)amino-3,4,5-
trideoxy- -glycero- -galacto-non-2-enonic acid (4b)
602.2919 [M+H]+.
D
D
By a procedure similar to that for 4a, saponification of ester
11b (153 mg, 0.25 mmol), followed by treatment with TFA, gave
4b (114 mg, 67%). C15H27N3O7ꢄ2(CF3CO2H); pale yellow solid,
mp 140–142 °C; 1H NMR (400 MHz, D2O) d 5.93 (1H, s), 4.49
(1H, dd, J = 10.0, 9.2 Hz), 4.40 (1H, d, J = 10.0 Hz), 4.32 (1H, d,
J = 9.2 Hz), 3.95–3.91 (1H, m), 3.87 (1H, dd, J = 12.0, 2.4 Hz),
3.69 (1H, d, J = 9.2 Hz), 3.64 (1H, dd, J = 12.0, 6.0 Hz), 3.27 (1H,
m), 3.11 (1H, m), 3.02 (2H, m), 2.06 (3H, s), 1.75 (4H, m); 13C
NMR (100 MHz, D2O) d 174.9, 165.7, 162.9 (CO2 of TFA, q,
J = 34.9 Hz), 149.2, 117.9 (CF3 of TFA, q, J = 289.6 Hz), 100.9,
75.9, 69.9, 67.9, 63.2, 56.2, 44.0, 43.2, 39.0, 24.2, 23.3, 22.4; ESI-
HRMS calcd for C15H28N3O7: 362.1927, found: m/z 362.1924
[M+H]+.
7.1.4. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-(3-methxy
carbonyl)propylamino-3,4,5-trideoxy-
2-enonic acid methyl ester (11c)
D-glycero-D-galacto-non-
By a procedure similar to that for 11a, Mitsunobu reaction of
sulfonamide 10 (332 mg, 0.54 mmol) with methyl 4-hydroxybut-
anoate (77 mg, 0.65 mmol) gave amine 11c (170 mg, 80%) after re-
moval of the sulfonamide group by PhSH/Cs2CO3. C23H34N2O12
;
pale yellow foam; ½a D23
ꢂ
+60.0 (c 1.0, CH2Cl2); IR mmax (neat) 1738,
1660, 1544, 1439, 1371, 1251, 1222 cmꢁ1
;
1H NMR (400 MHz,
CDCl3) d 5.98 (1H, br), 5.98 (1H, d, J = 2.8 Hz), 5.46 (1H, dd J = 4.0,
3.6 Hz), 5.26–5.23 (1H, m), 4.62 (1H, dd, J = 12.0, 2.8 Hz), 4.20
(1H, dd, J = 8.8, 3.6 Hz), 4.10 (1H, dd, J = 12.0, 7.6 Hz), 4.00 (1H,
dd, J = 8.8, 8.0 Hz), 3.71 (3H, s), 3.59 (3H, s), 3.33 (1H, J = 8.0,
2.8 Hz), 2.72–2.69 (1H, m), 2.52–2.49 (1H, m), 2.32–2.28 (2H, m),
2.04 (3H, s), 1.98 (6H, s), 1.89 (3H, s), 1.69–1.65 (2H, m)1.60 (1H,
br); 13C NMR (100 MHz, CDCl3) d 173.8, 170.3, 170.1, 169.9 (2ꢀ),
161.9, 143.2, 111.9, 76.7, 71.0, 68.0, 62.0, 55.7, 52.3, 51.5, 46.7,
44.9, 31.6, 25.3, 23.3, 20.9, 20.8 (2ꢀ); ESI-HRMS calcd for
C23H35N2O12: 531.2185, found: m/z 531.2189 [M+H]+.
7.1.8. 5-Acetamido-2,6-anhydro-4-(3-carboxypropyl)amino-3,4,
5-trideoxy-D-glycero-D-galacto-non-2-enonic acid (4c)
A solution of 11c (80 mg, 0.15 mmol) in MeOH (1 mL) and
aqueous NaOH (1 M, 1 mL) was stirred at room temperature for
1 h. The mixture was chromatographed on a Dowex 50 W ꢀ 8
(H+) ion exchange column. The column was first eluted with
H2O to discard impurities, and then with ammonia water to
afford the desired product 4c (45 mg, 79%). C15H24N2O9;
colorless solid, mp 90–91 °C; 1H NMR (400 MHz, D2O) d 5.77
(1H, d, J = 2.4 Hz), 4.47 (1H, dd, J = 10.0, 9.6 Hz), 4.37 (1H, d,
J = 10.0 Hz), 4.30 (1H, dd, J = 8.8, 1.6 Hz), 3.99–3.94 (1H, m), 3.90
(1H, dd, J = 11.6, 2.4 Hz), 3.72–3.64 (2H, m), 3.33–3.26 (1H, m),
3.18–3.13 (1H, m), 2.53 (2H, t, J = 7.2 Hz), 2.09 (3H, s), 2.06–
1.96 (2H, m); 13C NMR (100 MHz, D2O) d 177.0, 174.5, 167.5,
162.6 (CO2 of TFA, q, J = 34.9 Hz), 151.3, 116.4 (CF3 of TFA, q,
J = 288.3 Hz), 98.6, 75.8, 70.1, 68.2, 63.5, 56.5, 44.7, 43.8, 31.9,
23.0, 21.9; ESI-HRMS calcd for C15H25N2O9: 377.1555, found: m/
z 377.1567 [M+H]+.
7.1.5. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-(4-methox
ycarbonyl)butylamino-3,4,5-trideoxy-D-glycero-D-galacto-non-
2-enonic acid methyl ester (11d)
By a procedure similar to that for 11a, Mitsunobu reaction of
sulfonamide 10 (430 mg, 0.7 mmol) with methyl methyl 5-hydro-
xypentanoate (110 mg, 0.84 mmol) gave amine 11d (310 mg, 82%)
after removal of the sulfonamide group by PhSH/Cs2CO3. C24H36
-
N2O12; colorless solid, mp 70–72 °C; ½a D23
ꢂ
+26.6 (c 1.0, CH2Cl2); IR
m
max (neat) 1746, 1621, 1371, 1217 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d 6.02 (1H, d, J = 2.8 Hz), 5.96 (1H, d, J = 9.2 Hz), 5.49 (1H, dd J = 4.4,
3.6 Hz), 5.30–5.27 (1H, m), 4.62 (1H, dd, J = 12.4, 2.8 Hz), 4.21 (1H,
dd, J = 8.8, 3.6 Hz), 4.12 (1H, dd, J = 12.4, 7.2 Hz), 4.06–4.00 (1H,
m), 3.73 (3H, s), 3.61 (3H, s), 3.35 (1H, J = 8.0, 2.8 Hz), 2.72–2.67
(1H, m), 2.56–2.51 (1H, m), 2.30–2.26 (2H, m), 2.06 (3H, s), 2.00
(6H, s), 1.97 (1H, br s), 1.91 (3H, s), 1.65–1.57 (2H, m), 1.44–1.38
(2H, m); 13C NMR (100 MHz, CDCl3) d 173.9, 170.3, 170.2, 170.0,
169.9, 162.0, 143.1, 111.7, 76.7, 71.0, 68.0, 62.1, 55.5, 52.3, 51.5,
46.8, 45.3, 33.7, 29.3, 23.3, 22.3, 20.9, 20.8 (2ꢀ); ESI-HRMS calcd
for C24H37N2O12: 545.2341, found: m/z 545.2333 [M+H]+.
7.1.9. 5-Acetamido-2,6-anhydro-4-(4-carboxybutyl)amino-3,4,5
-trideoxy-D-glycero-D-galacto-non-2-enonic acid (4d)
By a procedure similar to that for 4c, saponification of ester
11d (90 mg, 0.16 mmol) gave 4d (68 mg, 84%). C16H26N2O9;
colorless solid, mp 100–101 °C; 1H NMR (400 MHz, D2O) d 5.72
(1H, d, J = 2.0 Hz), 4.15–4.06 (2H, m), 3.93–3.85 (2H, m),
3.66–3.56 (3H, m), 2.70–2.64 (1H, m), 2.60–2.55 (1H, m), 2.16
(2H, t, J = 7.2 Hz), 2.03 (3H, s), 1.57–1.50 (2H, m), 1.49–1.42
(2H, m); ESI-HRMS calcd for C16H27N2O9: 391.1711, found: m/z
391.1704 [M+H]+.
7.1.6. 5-Acetamido-2,6-anhydro-4-(3-aminopropyl)amino-3,4,5
-trideoxy-D-glycero-D-galacto-non-2-enonic acid (4a)
A solution of ester 11a (60 mg, 0.10 mmol) in MeOH (1 mL) and
aqueous NaOH (1 M, 1 mL) was stirred at room temperature for
30 min. The mixture was neutralized by Dowex 50 W ꢀ 8 (H+), fil-
tered, and rinsed with ammonia water. The combined aqueous
solution was concentrated under reduced pressure, and the residue
of acid product was dissolved in CH2Cl2 (1 mL) and TFA (1 mL). The
mixture was stirred at room temperature for 5 h, and concentrated
under reduced pressure. The residue was triturated with Et2O, and
centrifuged to give the desired product 4a (42 mg, 74%).
C14H25N3O7ꢄ2(CF3CO2H); pale yellow foam; 1H NMR (400 MHz,
D2O) d 5.99 (1H, d, J = 1.2 Hz), 4.50 (1H, dd, J = 10.0, 9.2 Hz), 4.41
(1H, d, J = 10.0 Hz), 4.36 (1H, dd, J = 9.2, 1.2 Hz), 3.95–3.91 (1H,
m), 3.86 (1H, dd, J = 12.0, 2.4 Hz), 3.70 (1H, d, J = 9.6 Hz), 3.65
(1H, dd, J = 12.0, 6.0 Hz), 3.39–3.32 (1H, m), 3.20–3.13 (1H, m),
3.08 (2H, t, J = 8.0 Hz), 2.15–2.02 (5H, m); 13C NMR (100 MHz,
D2O) d 174.9, 164.9, 162.9 (CO2 of TFA, q, J = 35.7 Hz), 148.6,
116.4 (CF3 of TFA, q, J = 288.8 Hz), 101.4, 76.0, 69.9, 67.8, 63.1,
56.3, 43.9, 40.9, 36.8, 24.2, 22.4; ESI-HRMS calcd for C14H26N3O7:
348.1765, found: m/z 348.1763 [M+H]+.
7.1.10. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-(1-tert-bu
toxycarbonyl-4,5-dihydroimidazol-2-yl)amino-3,4,5-trideoxy-
-glycero- -galacto-non-2-enonic acid methyl ester (12)
D
D
A solution of amine 9 (210 mg, 0.47 mmol) and N-Boc-2-
methylthio-2-imidazoline (66 mg, 0.38 mmol) in EtOH/AcOH (v/
v = 9:1, 3 mL) was stirred at 50 °C for 24 h. The mixture was con-
centrated, and then partitioned between CH2Cl2 and saturated
NaHCO3 aqueous solution. The organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (CH2Cl2/MeOH = 15:1) to give 12 (175 mg, 62%).
C26H38N4O12; colorless solid, mp 145–147 °C; 1H NMR (400 MHz,
D2O) d 7.09 (1H, br s), 6.27 (1H, br s), 5.95 (1H, d, J = 2.0 Hz),
5.39 (1H, dd, J = 4.8, 1.6 Hz), 5.30–5.26 (1H, m), 4.66 (1H, br s),
4.64 (1H, dd, J = 12.4, 2.8 Hz), 4.27–4.17 (2H, m), 4.13 (1H, dd,
J = 12.4, 7.2 Hz), 3.75 (3H, s), 3.77–3.66 (2H, m), 3.59–3.54 (2H,
m), 2.10 (3H, s), 2.03 (3H, s), 2.02 (3H, s), 1.87 (3H, s), 1.46 (9H,
s); ESI-HRMS calcd for C26H39N4O12
: 599.2559, found: m/z
599.2626 [M+H]+.

4080
W.-H. Wen et al. / Bioorg. Med. Chem. 18 (2010) 4074–4084
7.1.11. 5-Acetamido-2,6-anhydro-4-(4,5-dihydroimidazol-2-yl)
(3ꢀ), 23.6, 21.3, 21.1 (2ꢀ); ESI-HRMS calcd for C24H34N3O12S:
588.1863, found: m/z 588.1856 [MꢁH]ꢁ.
amino-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic acid
(5)
A solution of 12 (107 mg, 0.18 mmol) was treated with trifluo-
roacetic acid (1 mL) in CH2Cl2 (1 mL) at room temperature for 5 h
to remove the t-Boc group. The mixture was concentrated under
reduced pressure. The residue was triturated with Et2O, and the
precipitated product was collected by centrifuge. The precipitate
was subjected to saponification in aqueous NaOH (1 M, 1 mL)
and MeOH (1 mL). After stirring at room temperature for 5 min,
the mixture was neutralized by Dowex 50 W ꢀ 8 (H+), filtered,
and rinsed with ammonia water. The combined aqueous solution
7.1.14. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-[N0-(tert-
butoxycarbonyl)-N00-(3-tert-butoxycarbonylaminopropyl)gua
nidino]-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic acid
methyl ester (15a)
To a solution of thiourea 14 (150 mg, 0.254 mmol) in DMF
(3 mL) were added tert-butyl N-(3-aminopropyl) carbamate
(66 mg, 0.38 mmol), HgCl2 (83 mg, 0.305 mmol) and Et3N
(0.054 mL, 0.38 mmol). The mixture was stirred at room tempera-
ture for 2 h, and then concentrated under reduced pressure. The
residue was dissolved in EtOAc, and filtered through a pad of Celite.
The filtrate was concentrated, and the crude product was purified
by silica gel column chromatography (EtOAc/hexane = 2:1) to give
15a (122 mg, 66%). C32H51N5O14; colorless solid, mp 108–110 °C;
was concentrated to give compound 5 (79 mg, 94%). C14H22
-
N4O7ꢄ2(CF3CO2H); colorless solid, mp 167–168 °C; 1H NMR
(400 MHz, D2O) d 5.72 (1H, s), 4.41–4.36 (2H, m), 4.25 (1H, dd,
J = 10.0, 9.6 Hz), 3.97–3.88 (2H, m), 3.72–3.64 (6H, m), 2.05 (3H,
s); 13C NMR (100 MHz, D2O) d 173.6, 167.2, 162.2 (CO2 of TFA, q,
J = 34.9 Hz), 158.9, 147.5, 116.0 (CF3 of TFA, q, J = 287.3 Hz),
104.8, 75.5, 69.8, 68.0, 63.1, 52.5, 49.0, 47.5, 42.9, 22.3; ESI-HRMS
calcd for C14H23N4O7: 359.1561, found: m/z 359.1642 [M+H]+.
TLC (EtOAc/hexane = 4:1) Rf = 0.39; ½a D25
ꢂ
+56.6 (c 1.43, EtOAc); IR
mmax (neat) 2976, 1746, 1638, 1603, 1368, 1250, 1218,
1147 cmꢁ1 1H NMR (400 MHz, CDCl3) d 9.05 (1H, br), 6.53 (1H,
;
d, J = 9.2 Hz), 5.91 (1H, s), 5.44 (1H, dd, J = 5.2, 2.0 Hz), 5.40 (2H,
br), 5.28 (1H, m), 4.65 (1H, d, J = 12.4 Hz), 4.27–4.17 (2H, m),
4.12 (1 H, dd, J = 12.4, 7.2 Hz), 3.75 (3H, s), 3.21–3.02 (4 Y, m),
2.06 (3H, s), 2.03 (3H, s), 2.02 (3H, s), 1.85 (3H, s), 1.77 (1H, m),
1.75 (1H, m), 1.43 (9H, s), 1.39 (9H, s); 13C NMR (100 MHz, CDCl3)
d 171.1, 169.8, 169.3, 169.0, 162.6, 161.0, 159.4, 155.8, 143.4,
110.9, 79.1, 78.2, 77.2, 70.9, 67.8, 62.1, 52.4, 49.7, 47.8, 38.6,
37.6, 30.0, 28.6 (3ꢀ), 28.5 (3ꢀ), 23.2, 21.1, 21.0, 20.9; ESI-HRMS
calcd for C32H52N5O14: 730.3511, found: m/z 730.3512 [M+H]+.
7.1.12. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-[N0-(9-flu
orenylmethoxycarbonyl)thiourea]-3,4,5-trideoxy-
D-glycero-D-
galacto-non-2-enonic acid methyl ester (13)
To a solution of 9 (366 mg, 0.82 mmol) in anhydrous CH2Cl2
(5 mL) was added a solution of 9-fluorenylmethxoxycarbonyl iso-
cyanate (FmocNCS, 253 mg, 0.9 mmol) in anhydrous CH2Cl2
(5 mL) dropwise. The mixture was stirred at room temperature
for 15 min, concentrated, and purified by silica gel column chroma-
tography (EtOAc/hexane = 1:1) to give thiourea 13 (524 mg, 90%).
C34H37N3O12S; yellow solid, mp 103–105 °C; 1H NMR (400 MHz,
CDCl3) d 9.76 (1H, d, J = 8.8 Hz), 8.53, (1H, s), 7.72 (2H, d,
J = 7.2 Hz), 7.51 (2H, dd, J = 7.6, 0.8 Hz), 7.37 (2H, dd, J = 7.6,
7.2 Hz), 7.28 (2H, ddd, J = 7.6, 7.2, 0.8 Hz), 6.14 (1H, d,
J = 10.0 Hz), 5.97 (1H, d, J = 2.4 Hz), 5.49 (1H, dd, J = 4.8, 2.0 Hz),
5.45 (1H, ddd, J = 10.8, 8.8, 2.4 Hz), 5.31 (1H, m), 4.69 (1H, dd,
J = 12.4, 2.8 Hz), 4.50–4.33 (4H, m), 4.18–4.14 (2H, m), 3.73 (3H,
s), 2.09 (3H, s), 2.05 (3H, s), 2.03 (3H, s), 1.87 (3H, s); 13C NMR
(100 MHz, CDCl3) d 180.0, 170.5, 170.4, 170.1, 169.8, 161.3,
151.9, 144.7, 142.6, 142.5, 141.0 (2ꢀ), 127.8 (2ꢀ), 127.8 (2ꢀ),
127.0 (2ꢀ), 124.6 (2ꢀ), 120.0, 108.8, 71.3, 68.2, 67.6, 62.1, 54.3,
52.5, 46.6, 46.4, 23.2, 21.0, 20.8 (2ꢀ); ESI-HRMS calcd for
C34H38N3O12S: 712.2176, found: m/z 712.2180 [M+H]+.
7.1.15. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-[N0-(tert-
butoxycarbonyl)-N00-(4-tert-butoxycarbonylaminobutyl)guani
dino]-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic acid me
thyl ester (15b)
The reaction of thiourea 14 (190 mg, 0.32 mmol) with N-(4-
aminobutyl) carbamic tert-butyl ester (73 mg, 0.39 mmol) was car-
ried out by a procedure similar to that for 15a to give 15b (176 mg,
74%). C33H53N5O14; colorless solid, mp 87–89 °C; TLC (EtOAc/hex-
ane = 4:1) Rf = 0.36; ½a D25
ꢂ
+50.4 (c 1.0, EtOAc); IR mmax (neat) 2976,
1745, 1639, 1604, 1368, 1250, 1219, 1147 cmꢁ1
;
1H NMR
(400 MHz, CDCl3) d 9.02 (1H, br), 6.71 (1H, d, J = 9.2 Hz), 5.91
(1H, d, J = 2.0 Hz), 5.42 (1H, dd, J = 5.2, 2.0 Hz), 5.25 (1H, m), 5.11
(1H, br), 4.99 (2H, m), 4.60 (1H, d, J = 11.6 Hz), 4.25–4.12 (2H, m),
4.07 (1H, dd, J = 11.6, 7.2 Hz), 3.74 (3H, s), 3.06 (4 Y, m), 2.03
(3H, s), 2.01 (3H, s), 2.00 (3H, s), 1.84 (3H, s), 1.54 (2H, m), 1.48
(2H, m), 1.41 (9H, s), 1.37 (9H, s); 13C NMR (100 MHz, CDCl3) d
171.6, 170.4, 169.9, 169.7, 163.4, 161.6, 159.8, 156.0, 144.0,
111.1, 79.1, 78.2, 77.2, 71.2, 67.9, 62.2, 52.4, 49.7, 40.8, 39.7,
29.7, 28.5 (3ꢀ), 28.4 (3ꢀ), 27.4, 26.2, 23.0, 20.9, 20.8, 20.7; ESI-
HRMS calcd for C33H52N5O14: 742.3511, found: m/z 742.3507
[MꢁH]ꢁ.
7.1.13. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-[N0-(tert-
butoxycarbonyl)thiourea]-3,4,5-trideoxy-
non-2-enonic acid methyl ester (14)
D-glycero-D-galacto-
To a solution of 13 (4.5 g, 6.3 mmol) in CH2Cl2 (50 mL) was
added piperidine (12 mL). The mixture was stirred at room tem-
perature for 15 min, concentrated, and washed with hexane to re-
move 9-methylidenefluorene. The crude product was dissolved in
CH2Cl2, and di-tert-butyl dicarbonate (Boc2O, 2.06 g, 9.45 mmol)
and 4-dimethylaminopyridine (DMAP, 1.15 g, 9.45 mmol) were
added. The mixture was stirred at room temperature for 7 h, con-
centrated, and purified by silica gel column chromatography
(EtOAc/hexane = 3:2) to give thiourea 14 (3.23 g, 87%).
C24H35N3O12S; colorless solid, mp 100–102 °C; TLC (EtOAc/hex-
7.1.16. 5-Acetamido-2,6-anhydro-4-[N0-(3-aminopropyl)guani
dino]-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic acid
(6a)
By a procedure similar to that for 4a, saponification of 15a
(120 mg, 0.16 mmol), followed by removal of the Boc group with
TFA, gave 6a (92 mg, 94%). C15H27N5O7ꢄ2(CF3CO2H); pale yellow
ane = 4:1) Rf = 0.55; ½a D20
ꢂ
+58.2 (c 2.0, EtOAc); IR mmax (neat) 2980,
foam; IR mmax (neat) 3411, 1682, 1638, 1400, 1205, 1138 cmꢁ1
;
1743, 1525, 1370, 1249, 1223, 1143 cmꢁ1
;
1H NMR (400 MHz,
1H NMR (400 MHz, D2O) d 5.83 (1H, s), 4.50 (1H, d, J = 8.4 Hz),
4.41 (1H, d, J = 10.4 Hz), 4.29 (1H, dd, J = 10.4, 8.4 Hz), 3.94–3.86
(2H, m), 3.71–3.62 (2 Y, m), 3.32 (2 Y, m), 3.05 (2 Y, t, J = 8.0 Hz),
2.03 (3H, s), 1.98 (2H, m); 13C NMR (100 MHz, D2O) d 174.2,
166.7, 163.0 (CO2 of TFA, q, J = 35 Hz), 156.0, 146.8, 116.4 (CF3 of
TFA, q, J = 289.6 Hz), 106.9, 75.9, 70.0, 68.1, 63.2, 51.5, 47.7, 38.7,
37.0, 26.4, 22.2; ESI-HRMS calcd for C15H28N5O7: 390.1989, found:
m/z 390.2004 [M+H]+.
CDCl3)
d 9.78 (1H, d, J = 8.4 Hz), 7.90 (1H, s), 5.94 (1H, d,
J = 2.4 Hz), 5.86 (1H, d, J = 5.2 Hz), 5.56 (1H, m), 5.45 (1H, dd,
J = 4.8, 2.0 Hz), 5.29 (1H, m), 4.65 (1H, dd, J = 12.4, 2.4 Hz), 4.42–
4.31 (2H, m), 4.13 (1H, dd, J = 12.4, 7.2 Hz), 3.77 (3H, s), 2.10 (3H,
s), 2.06 (3H, s), 2.04 (3H, s), 1.90 (3H, s), 1.46 (9H, s); 13C NMR
(100 MHz, CDCl3) 180.3, 170.0, 169.9, 169.6, 169.4, 160.9, 150.6,
144.6, 108.7, 84.3, 77.6, 71.3, 67.6, 62.2, 54.1, 52.6, 47.5, 28.2

W.-H. Wen et al. / Bioorg. Med. Chem. 18 (2010) 4074–4084
4081
7.1.17. 5-Acetamido-2,6-anhydro-4-[N0-(4-aminobutyl)guani
7.1.20. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-[N0,N00-
di(tert-butoxycarbonyl)-N-(3-methoxycarbonylpropyl)guani
dino]-3,4,5-trideoxy-
(6b)
D-glycero-D-galacto-non-2-enonic acid
dino]-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic acid
By a procedure similar to that for 4a, saponification of 15b
(176 mg, 0.24 mmol), followed by removal of the Boc group with
TFA, gave 6b (136 mg, 90%). C16H29N5O7ꢄ2(CF3CO2H); pale yellow
methyl ester (16c)
By a procedure similar to that for 16a, the reaction of 11c
(90 mg, 0.17 mmol) with 1,3-bis(tert-butoxycarbonyl)-2-methyl-
2-thiopseudourea in the presence of HgCl2 and Et3N gave 16c
(120 mg, 91%). C34H52N4O16; colorless solid, mp 75–77 °C; TLC
foam; ½a 2D0
ꢂ
+21.6 (c 3.39, H2O); IR mmax (neat) 3457, 1694, 1400,
; d 5.89 (1H, d,
1203, 1136 cmꢁ1 1H NMR (400 MHz, D2O)
J = 2.0 Hz), 4.47 (1H, dd, J = 9.2, 2.0 Hz), 4.31 (1H, d, J = 10.8 Hz),
4.19 (1H, dd, J = 10.8, 9.2 Hz), 3.85–3.81 (1H, m), 3.79 (1H, dd,
J = 12.0, 2.4 Hz), 3.64 (1H, d, J = 9.6 Hz), 3.58 (1H, dd, J = 12.0,
6.0 Hz), 3.15 (2 Y, t, J = 6.4 Hz), 2.93 (2 Y, t, J = 6.8 Hz), 1.94 (3H,
s), 1.62–1.56 (4H, m); 13C NMR (100 MHz, D2O) d 174.4, 164.8,
162.5 (CO2 of TFA, q, J = 35.6 Hz), 155.9, 144.6, 116.2 (CF3 of TFA,
q, J = 289.7 Hz), 109.0, 76.1, 69.9, 67.9, 63.0, 50.7, 47.5, 40.7, 39.0,
25.0, 23.9, 21.9; ESI-HRMS calcd for C16H30N5O7: 404.2145, found:
m/z 404.2238 [M+H]+.
(EtOAc/hexane) Rf = 0.29; ½a D23
ꢂ
+11.4 (c 1.0, CH2Cl2); 1H NMR
(400 MHz, CDCl3) d 9.17 (1H, br s), 6.3 (1H, br s), 5.9 (1H, s), 5.45
(1H, br s), 5.41 (1H, 5.2 Hz), 5.30–5.26 (1H, m), 4.63 (1H, d,
J = 12.0 Hz), 4.33 (1H, d, J = 9.6 Hz), 4.24 (1H, br s), 4.13 (1H, dd,
J = 12.4, 7.2 Hz), 3.78 (3H, s), 3.65 (3H, s), 3.20 (2H, br), 2.34–2.28
(2H, m), 2.10 (3H, s), 2.07 (6H, s), 1.85 (3H, s), 1.85–1.82 (2H, m),
1.46 (18H, s); 13C NMR (100 MHz, CDCl3) d 172.4, 170.4, 169.6,
169.3, 160.7, 159.8, 153.9, 149.5, 145.3, 109.9, 84.7, 81.8, 79.4,
77.7, 77.2, 71.0, 67.8, 62.2, 56.4, 52.4, 51.7, 44.7, 44.2, 30.9, 28.2
(6ꢀ), 23.2, 21.1, 21.0, 20.9; ESI-HRMS calcd for C34H53N4O16
:
7.1.18. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-[N0,N00-
di(tert-butoxycarbonyl)-N-(3-tert-butoxycarbonylaminopro
773.3451, found: m/z 773.3450 [M+H]+.
pyl)guanidino]-3,4,5-trideoxy-
D
-glycero-
D
-galacto-non-2-enon
7.1.21. 5-Acetamido-2,6-anhydro-4-[N-(3-aminopropyl)guani
ic acid methyl ester (16a)
dino]-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic acid
To a solution of 11a (104 mg, 0.177 mmol) in DMF (2 mL) were
(7a)
added
1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea
(61.5 mg, 0.21 mmol), HgCl2 (57.5 mg, 0.21 mmol) and Et3N
(0.05 mL, 0.35 mmol). The mixture was stirred at room tempera-
ture for 3 h, and then concentrated in vacuo. The residue was dis-
solved in EtOAc and filtered through a pad of Celite. The organic
solution was washed with 1 M HCl, saturated NaHCO3 and brine,
dried over MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy (EtOAc/hexane = 2:1) to give 16a (134 mg, 91%).
C37H59N5O16; colorless solid, mp 85–86 °C; TLC (EtOAc) Rf = 0.48;
By a procedure similar to that for 4a, saponification of 16a
(110 mg, 0.13 mmol), followed by removal of the Boc group with
TFA, gave 7a (72 mg, 92%). C15H27N5O7ꢄ2(CF3CO2H); colorless solid,
mp >200 °C (decomposed); 1H NMR (400 MHz, D2O) d 5.70 (1H, s),
4.89 (1H, m), 4.42–4.35 (2H, m), 3.98–3.85 (2H, m), 3.69–3.63 (2H,
m), 3.44–3.36 (1H, m), 3.30 (1H, br), 3.04 (2H, t, J = 7.2 Hz), 2.15–
2.09 (1H, m), 2.07–1.97 (1H, m), 2.02 (3H, s); 13C NMR (100 MHz,
D2O) d 174.1, 168.0, 163.0 (CO2 of TFA, q, J = 35.0 Hz), 157.7,
151.5, 117.9 (CF3 of TFA, q, J = 288.1 Hz), 103.4, 75.8, 70.0, 68.2,
63.3, 58.4, 45.4, 41.2, 37.1, 25.2, 22.2; ESI-HRMS calcd for
C15H28N5O7: 390.1983, found: m/z 390.1977 [M+H]+.
½
a 2D4 +43.5 (c 1.0, CH2Cl2); 1H NMR (400 MHz, CDCl3, 50 °C) d 9.51
ꢂ
(1H, br s), 6.20 (1H, br s), 5.94 (1H, s), 5.39 (1H, d, J = 6.8 Hz),
5.30–5.26 (1H, m), 5.21 (1H, br s), 5.06 (1H, br s), 4.63 (1H, dd,
J = 12.4, 2.8 Hz), 4.35 (1H, J = 8.4 Hz), 4.17–4.12 (2H, m), 3.76 (3H,
s), 3.30–3.24 (2H, m), 3.15–3.10 (1H, m), 2.99–2.91 (1H, m), 2.10
(3H, s), 2.02 (3H, s), 2.00 (3H, s), 1.84 (3H, s), 1.79–1.73 (1H, m),
1.69–1.62 (1H, m), 1.45 (18H, s), 1.40 (9H, s); 13C NMR
(100 MHz, CDCl3) d 171.0, 170.3, 170.1, 170.0, 161.3, 161.2,
156.1, 146.0, 109.9, 82.8, 80.1, 79.1, 78.0, 77.2, 71.4, 67.9, 62.2,
57.3, 52.5, 45.7, 37.7, 29.1, 28.5 (6ꢀ), 28.2 (3ꢀ), 23.2, 21.1, 21.0,
20.9; ESI-HRMS calcd for C37H60N5O16: 830.4030, found: m/z
830.4033 [M+H]+.
7.1.22. 5-Acetamido-2,6-anhydro-4-[N-(4-aminobutyl)guani
dino]-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic acid
(7b)
By a procedure similar to that for 4a, saponification of 16b
(114 mg, 0.13 mmol), followed by removal of the Boc group with
TFA, gave 7b (77 mg, 94%). C16H29N3O7ꢄ2(CF3CO2H); colorless solid,
mp 167–168 °C; 1H NMR (400 MHz, D2O) d 5.73 (1H, s), 4.89 (1H,
m), 4.43–4.34 (2H, m), 4.00–3.95 (1H, m), 3.93–3.86 (1H, m),
3.70–3.64 (2H, m), 3.36–3.33 (1H, m), 3.20 (1H, br), 3.02 (2H, t,
J = 7.2 Hz), 2.03 (3H, s), 1.84 (1H, br), 1.71–1.70 (3H, m); 13C
NMR (100 MHz, D2O) d 174.0, 168.1, 163.0 (CO2 of TFA, q,
J = 35.0 Hz), 157.6, 150.9, 116.5 (CF3 of TFA, q, J = 289.6 Hz),
103.8, 75.8, 70.0, 68.2, 63.3, 58.2, 45.5, 43.5, 39.3, 24.4, 23.9,
22.2; ESI-HRMS calcd for C16H30N5O7: 404.2140, found: m/z
404.2135 [M+H]+.
7.1.19. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-[N0,N00-
di(tert-butoxycarbonyl)-N-(4-tert-butoxycarbonylaminobu
tyl)guanidino]-3,4,5-trideoxy-
onic acid methyl ester (16b)
D-glycero-D-galacto-non-2-en
By a procedure similar to that for 16a, the reaction of 11b
(120 mg, 0.2 mmol) with 1,3-bis(tert-butoxycarbonyl)-2-methyl-
2-thiopseudourea in the presence of HgCl2 and Et3N gave 16b
(148 mg, 88%). C38H61N5O16; colorless solid, mp 69–71 °C; TLC
(EtOAc) Rf = 0.49; 1H NMR (400 MHz, CDCl3, 50 °C) d 6.33 (1H, br
s), 5.92 (1H, s), 5.38 (1H, dd, J = 4.8, 2.0 Hz), 5.26–5.23 (1H, m),
5.25 (1H, br s), 5.05 (1H, br s), 4.63 (1H, dd, J = 12.4, 2.8 Hz), 4.30
(1H, J = 10.0 Hz), 4.18 (1H, br s), 4.11 (1H, dd, J = 12.4, 7.2 Hz),
3.75 (3H, s), 3.15 (2H, m), 3.05–3.04 (2H, m), 2.08 (3H, s), 2.02
(3H, s), 2.00 (3H, s), 1.82 (3H, s), 1.58–1.48 (2H, m), 1.43 (18H, s),
1.38 (9H, s), 1.38–1.30 (2H, m); 13C NMR (100 MHz, CDCl3) d
171.8, 170.2, 169.9, 169.8, 161.1, 155.7, 155.4, 150.2, 145.7,
110.1, 82.1, 80.1, 78.7, 77.9, 77.2, 71.3, 67.8, 62.1, 57.0, 52.4,
45.4, 44.8, 39.5, 29.6, 28.4 (6ꢀ), 28.0 (3ꢀ), 27.0, 23.0, 20.9 (2ꢀ),
20.7; ESI-HRMS calcd for C38H62N5O16: 844.4186, found: m/z
844.4184 [M+H]+.
7.1.23. 5-Acetamido-2,6-anhydro-4-[N-(3-carboxypropyl)gua
nidino]-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic acid
(7c)
By a procedure similar to that for 4a, saponification of 16c
(50 mg, 0.06 mmol), followed by removal of the Boc group with
TFA, gave 7c (30 mg, 95%). C16H26N4O9ꢄCF3CO2H; colorless solid,
mp 152–153 °C; 1H NMR (400 MHz, D2O) d 5.74 (1H, s), 4.90 (1H,
m), 4.42 (1H, d, J = 10.4 Hz), 4.35–4.34 (1H, br), 3.99–3.95 (1H,
m), 3.92–3.89 (1H, m), 3.71–3.64 (2H, m), 3.32–3.28 (2H, m),
2.45 (2H, t, J = 6.8 Hz), 2.03 (3H, s), 2.00–1.93 (2H, m); 13C NMR
(100 MHz, D2O) d 177.6, 173.8, 167.7, 162.6 (CO2 of TFA, q,
J = 35.0 Hz), 157.4, 151.6, 116.5 (CF3 of TFA, q, J = 288.3 Hz),
104.0, 75.9, 70.2, 68.5, 63.6, 58.0, 45.9, 43.6, 31.2, 23.1, 22.7; ESI-
HRMS calcd for C16H25N4O9: 417.1622, found: m/z 417.1624
[MꢁH]ꢁ.

4082
W.-H. Wen et al. / Bioorg. Med. Chem. 18 (2010) 4074–4084
7.1.24. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-[N-(3-
To a solution of 18 (200 mg, 0.26 mmol) in DMF (3 mL)
were added the above-prepared N-Boc-piperazine (19a, 58 mg,
0.31 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-
fluorophosphate (PyBOP, 160 mg, 0.31 mmol) and Et3N (0.054 mL,
0.39 mmol). The mixture was stirred for 3 h, and then concentra-
tion under reduced pressure. The residue was dissolved in EtOAc,
and washed sequentially with 1 M HCl, saturated NaHCO3 and
brine. The organic phase was dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (CH2Cl2/MeOH = 20:1) to give
the amide product 20a (198 mg, 82%). C42H66N6O17; colorless solid,
allyloxycarbonylpropyl)guanidino]-3,4,5-trideoxy-
D-glycero-D-
galacto-non-2-enonic acid methyl ester (17)
By a procedure similar to that for 11a, Mitsunobu reaction of
sulfonamide 10 (1.2 g, 1.95 mmol) with allyl 3-hydroxypropanoate
(420 mg, 2.9 mmol) gave amine 17 (878 mg, 81%) after removal of
the sulfonamide group by PhSH/Cs2CO3. C25H36N2O12; colorless
foam; TLC (CH2Cl2/MeOH = 3:1) Rf = 0.12;
CH2Cl2); IR mmax (neat) 1740, 1659, 1544, 1440, 1371, 1248,
1221 cmꢁ1 1H NMR (400 MHz, CDCl3) d 6.02 (1H, d, J = 2.8 Hz),
½
a 2D3
ꢂ
+58.2 (c 1.0,
;
5.96–5.81 (1H, m), 5.78 (1H, br s), 5.49 (1H, dd, J = 4.0, 3.6 Hz),
5.30–5.17 (3H, m), 4.62 (1H, dd, J = 12.0, 2.8 Hz), 4.53–4,51 (2H,
m), 4.24 (1H, dd, J = 8.8, 3.6 Hz), 4.14 (1H, dd, J = 12.0, 7.2 Hz), 4.0
(1H, m), 3.74 (3H, s), 3.36 (1H, dd, J = 8.0, 2.8 Hz), 2.77–2.71 (1H,
m), 2.59–2.52 (1H, m), 2.35 (2H, t, J = 8.4 Hz), 2.07 (3H, s), 2.01
(6H, s), 1.92 (3H, s), 1.76–1.68 (2H, m); 13C NMR (100 MHz, CDCl3)
172.8, 170.2, 170.1, 169.7, 161.7, 143.0, 131.7, 117.7, 112.0, 76.6,
71.0, 67.8, 64.7 (2ꢀ), 61.9, 55.8, 52.0, 46.3, 44.5, 31.5, 25.2, 23.0,
20.6 (3ꢀ); ESI-HRMS calcd for C25H37N2O12: 557.2341, found: m/z
557.2340 [M+H]+.
mp 115–117 °C; TLC (EtOAc) Rf = 0.32; ½a D23
ꢂ
ꢁ3.9 (c 0.5, CH2Cl2); 1H
NMR (400 MHz, CDCl3) d 9.45 (1H, br s), 6.53 (1H, br s), 5.87 (1H, s),
5.64 (1H, br s), 5.40 (1H, d, J = 5.2 Hz), 5.29–5.26 (1H, m), 4.59 (1H,
dd, J = 12.4, 2.0 Hz), 4.33–4.26 (2H, br s), 4.10 (1H, dd, J = 12.4,
7.2 Hz), 3.75 (3H, s), 3.55–3.53 (2H, m), 3.41–3.37 (6H, m), 3.09
(2H, br s), 2.33–2.20 (2H, m), 2.07 (3H, s), 2.04 (3H, s), 2.02 (3H,
s), 1.86 (3H, s), 1.44 (27H, s); 13C NMR (100 MHz, CDCl3) 171.6,
170.4, 169.9, 169.8, 161.4, 160.0, 154.2, 153.6, 150.6, 145.7,
110.3, 81.5, 80.3, 79.1, 78.0, 77.2, 76.7, 71.0, 67.9, 62.2, 54.5,
52.5, 45.3, 44.9, 43.6, 41.5, 29.7, 29.4, 28.4 (6ꢀ), 28.2 (3ꢀ), 24.9,
23.2, 21.0, 20.9 (2ꢀ); ESI-HRMS calcd for C42H67N6O17: 927.4557,
found: m/z 927.4562 [M+H]+.
7.1.25. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-[N0,N00-
di(tert-butoxycarbonyl)-N-(3-allyloxycarbonylpropyl)guani
dino]-3,4,5-trideoxy-
methyl ester (16d)
D-glycero-D-galacto-non-2-enonic acid
7.1.27. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-{N0,N00-
di(tert-butoxycarbonyl)-N-[3-(4-methylpiperazino)carbonyl
By a procedure similar to that for 16a, the reaction of 17
(683 mg, 1.21 mmol) with 1,3-bis(tert-butoxycarbonyl)-2-methyl-
2-thiopseudourea in the presence of HgCl2 and Et3N gave 16d
(800 mg, 83%). C36H54N4O16; colorless solid, mp 59–60 °C; TLC
propyl]guanidino}-3,4,5-trideoxy-D-glycero-D-galacto-non-2-
enonic acid methyl ester (20b)
By a procedure similar to that for 20a, the coupling reaction of
acid 18 (237 mg, 0.31 mmol) with 1-methylpiperazine (19b,
37 mg, 0.37 mmol) in the presence of PyBOP and Et3N gave amide
20b (190 mg, 73%). C38H60N6O15; colorless solid, mp 121–122 °C;
1H NMR (400 MHz, CDCl3) d 9.48 (1H, br s), 6.82 (1H, br s), 5.81
(1H, s), 5.61 (1H, br), 5.35 (1H, d, J = 5.2 Hz), 5.21–5.15 (1H, m),
4.53 (1H, d, J = 12.4 Hz), 4.28 (2H, br s), 4.04 (1H, dd, J = 12.4,
7.2 Hz), 3.69 (3H, s), 3.57–3.48 (2H, m), 3.38–3.29 (2H, m), 3.06–
2.95 (2H, m), 2.82 (2H, br s), 2.32–2.28 (4H, m), 2.26–2.21 (5H,
m), 2.01 (3H, s), 1.96 (6H, s), 1.79 (3H, s), 1.37 (18H, s); ESI-HRMS
calcd for C38H61N6O15: 841.4195, found: m/z 841.4196 [M+H]+.
(EtOAc/hexane = 3:1) Rf = 0.39; ½a D23
ꢂ
+8.9 (c 1.0, CH2Cl2); 1H NMR
(400 MHz, CDCl3) d 9.00 (1H, br s), 6.34 (1H, br s), 5.88–5.77 (2H,
m), 5.36 (1H, d, J = 5.2 Hz), 5.35 (1H, br s), 5.28–5.14 (3H, m),
4.58 (1H, d, J = 12.0 Hz), 4.49 (2H, dd, J = 5.6, 0.8 Hz), 4.30 (1H, d,
J = 9.6 Hz), 4.21 (1H, br s), 4.08 (1H , dd, J = 12.0, 6.8 Hz), 3.72
(3H, s), 3.16 (2H, br s), 2.27–2.23 (2H, m), 2.05 (3H, s), 1.99 (3H,
s), 1.98 (3H, s), 1.80 (5H, s), 1.4 (18H, s); 13C NMR (100 MHz, CDCl3)
171.7, 170.4, 169.6, 169.3, 160.7, 160.0, 153.9, 149.7, 145.3, 131.4,
117.9, 109.9, 81.9, 79.3, 77.8, 77.2, 71.1, 67.8, 65.1, 62.2, 56.2, 52.4,
45.3, 44.2, 31.0, 28.2 (6ꢀ), 24.2, 23.2, 21.1 (2ꢀ), 20.9; ESI-HRMS
calcd for C36H55N4O16: 799.3608, found: m/z 799.3603 [M+H]+.
7.1.28. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-{N0,N00-
di(tert-butoxycarbonyl)-N-[3-(morpholinocarbonyl)propyl]gua
7.1.26. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-{N0,N00-
di(tert-butoxycarbonyl)-N-[3-(4-tert-butoxycarbonylpiperaz
nidino}-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic acid
ino)carbonylpropyl]guanidino}-3,4,5-trideoxy-
acto-non-2-enonic acid methyl ester (20a)
D-glycero-D-gal
methyl ester (20c)
By a procedure similar to that for 20a, the coupling reaction of
acid 18 (185 mg, 0.24 mmol) with morpholine (19c, 25 mg,
0.29 mmol) in the presence of PyBOP and Et3N gave amide 20c
(170 mg, 85%). C37H57N5O16; colorless solid, mp 133–134 °C; 1H
NMR (400 MHz, CDCl3) d 9.45 (1H, br s), 6.51 (1H, br s), 5.87 (1H,
s), 5.64 (1H, br s), 5.40 (1H, d, J = 5.2 Hz), 5.29–5.25 (1H, m), 4.60
(1H, dd, J = 12.4, 2.8 Hz), 4.33 (2H, br s), 4.10 (1H, dd, J = 12.4,
7.2 Hz), 3.75 (3H, s), 3.67–3.61 (4H, m), 3.58–3.56 (2H, m), 3.39–
3.38 (2H, m), 3.31–3.07 (2H, m), 2.37–2.17 (4H, m), 2.07 (3H, s),
2.04 (3H, s), 2.02 (3H, s), 1.86 (3H, s), 1.44 (18H, s); ESI-HRMS calcd
for C37H58N5O16: 828.3873, found: m/z 828.3870 [M+H]+.
To a solution of 16d (800 mg, 1.0 mmol) in THF (10 mL) were
added Pd(PPh3)4 (230 mg, 0.2 mmol) and morpholine (1.74 mL,
20 mmol). The mixture was stirred at room temperature for
3.5 h, and then filtered through a pad of Celite. The filtrate was
concentrated, and partitioned between 1 M HCl and CH2Cl2. The or-
ganic layer was washed with brine, dried over MgSO4, and concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography (CH2Cl2/MeOH = 20:1) to give 18
(645 mg, 85%). ESI-HRMS calcd for C13H49N4O16: 757.3149, found:
m/z 757.3143 [MꢁH]ꢁ.
To a solution of piperazine (4.3 g, 50 mmol) in CH2Cl2 (125 ml)
was added a solution of Boc2O (5.45 g, 25 mmol) in CH2Cl2 (50 mL)
dropwise at ice-bath temperature. The mixture was warmed to
room temperature, stirred for 1 h, and filtered. The filtrate was
concentrated in vacuo. The residue was added H2O, and filtered.
The filtrate was saturated with K2CO3 and extracted with Et2O
(3ꢀ). The combined organic phase was dried over MgSO4, filtered
and concentrated to afford N-Boc-piperazine 19a (2.95 g, 63%).
C9H18N2O2; colorless oil; 1H NMR (400 MHz, CDCl3) d 3.34 (4H, t,
J = 4.8 Hz), 2.76 (4H, t, J = 4.8 Hz), 1.42 (9H, s); ESI-HRMS calcd
for C9H19N2O2: 187.1447, found: m/z 187.1446 [M+H]+.
7.1.29. 5-Acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-{N0,N00-
di(tert-butoxycarbonyl)-N-[3-(piperidinocarbonyl)propyl]gua
nidino}-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonic acid
methyl ester (20d)
By a procedure similar to that for 20a, the coupling reaction of
acid 18 (163 mg, 0.21 mmol) with piperidine (19d, 22 mg,
0.25 mmol) in the presence of PyBOP and Et3N gave amide 20d
(154 mg, 89%). C38H59N5O15; TLC (EtOAc) Rf = 0.42; colorless solid,
mp 116–117 °C; 1H NMR (400 MHz, CDCl3) d 9.65 (1H, br s), 6.57
(1H, br s), 5.87 (1H, s), 5.68 (1H, br s), 5.41 (1H, d, J = 5.2 Hz),

W.-H. Wen et al. / Bioorg. Med. Chem. 18 (2010) 4074–4084
4083
5.30–5.26 (1H, m), 4.59 (1H, dd, J = 12.4, 2.8 Hz), 4.33 (2H, br s),
4.11 (1H, dd, J = 12.4, 7.2 Hz), 3.75 (3H, s), 3.57–3.46 (2H, m),
3.32–3.30 (2H, m), 3.12–3.01 (2H, m), 2.25–2.16 (2H, m), 2.08
(3H, s), 2.04 (3H, s), 2.02 (3H, s), 1.87 (3H, s), 1.87–1.78 (2H, m),
1.64–1.60 (2H, m), 1.54–1.48 (4H, m), 1.44 (18H, s); 13C NMR
(100 MHz, CDCl3) d 171.8, 170.4, 169.8, 169.7, 161.4, 159.5,
153.4, 150.7, 145.5, 110.5, 81.2, 78.9, 78.2, 77.2, 71.0, 67.9, 62.2,
54.3, 52.5, 46.1, 45.3, 43.6, 42.8, 29.1, 28.3 (3ꢀ), 28.2 (3ꢀ), 26.3,
25.4, 24.5, 23.2 (2ꢀ), 21.0 (2ꢀ), 20.9; ESI-HRMS calcd for
C38H60N5O15: 826.4080, found: m/z 826.4083 [M+H]+.
(6H, m); ESI-HRMS calcd for C21H36N5O8: 486.2558, found: m/z
486.2557 [M+H]+.
7.2. Biology
7.2.1. Materials
Influenza A/WSN/1933 (H1N1) (from Dr. Shin-Ru Shih, Chang
Gung University, Taiwan) was cultured in the allantoic cavities of
10-day-old embryonated chicken eggs for 72 h, and purified by su-
crose gradient centrifugation. Madin-Darby canine kidney (MDCK)
cells were obtained from American Type Culture Collection
(Manassas, Va), and were grown in DMEM (Dulbecco’s modified
Eagle medium, GibcoBRL) containing 10% fetal bovine serum (Gib-
coBRL) and penicillin–streptomycin (GibcoBRL) at 37 °C under 5%
CO2.
7.1.30. 5-Acetamido-2,6-anhydro-4-[N-(3-piperazinocarbon
yl)propyl]guanidino-3,4,5-trideoxy-D-glycero-D-galacto-non-2-
enonic acid (8a)
By a procedure similar to that for 4a, saponification of ester 20a
(76 mg, 0.082 mmol), followed by removal of the Boc groups with
TFA, gave 8a (51 mg, 88%). C20H34N6O8ꢄ2(CF3CO2H), colorless solid,
7.2.2. Determination of NA activity by a fluorescent assay
The neuraminidase activity was measured using diluted allan-
toic fluid harvest from influenza A/WSN/1933 (H1N1) infected
embryonated eggs. A fluorometric assay was used to determine
the NA activity with the fluorogenic substrate 20-(4-methylumbel-
mp 160–161 °C; ½a D22
ꢂ
+26.6 (c 1.0 H2O); 1H NMR(400 MHz, D2O) d
5.74 (1H, s), 4.86 (1H, m), 4.41 (1H, d, J = 10.8 Hz), 4.36–4.32 (1H,
m), 3.99–3.95 (1H, m), 3.92–3.79 (5H, m), 3.70–3.63 (2H, m),
3.35–3.24 (5H, m), 3.20–3.18 (1H, m), 2.54 (2H, m), 2.02 (4H, m),
1.92–1.89 (1H, m), 13C NMR (100 MHz, CDCl3) d 174.1, 173.4,
167.5, 162.9 (CO2 of TFA, q, J = 35.0 Hz), 157.6, 150.3, 116.5 (CF3
of TFA, q, J = 289.6 Hz), 104.4, 75.9, 70.0, 68.2, 63.3, 57.5, 45.5,
43.5, 43.2 (2ꢀ), 42.4, 38.8, 29.2, 22.6, 22.3; ESI-HRMS calcd for
C20H35N6O8: 487.2511, found: m/z 487.2510 [M+H]+.
liferyl)-a-D-N-acetylneuraminic acid (MUNANA; Sigma). The fluo-
rescence of the released 4-methylumbelliferone was measured in
Envision plate reader (Perkin–Elmer, Wellesley, MA) using excita-
tion and emission wavelengths of 365 and 460 nm, respectively.
Neuraminidase activity was determined at 200 lM of MUNANA.
Enzyme activity was expressed as the fluorescence increase during
15 min incubation at room temperature.
7.1.31. 5-Acetamido-2,6-anhydro-4-{N-[3-(4-methylpiperaz
inocarbonyl)propyl]guanidino}-3,4,5-trideoxy-
galacto-non-2-enonic acid (8b)
D-glycero-D-
7.2.3. Determination of IC50 of NA inhibitor
NA inhibition was determined by mixing inhibitor and neur-
aminidase for 10 min at room temperature followed by the addi-
By a procedure similar to that for 4a, saponification of ester 20b
(63 mg, 0.075 mmol), followed by removal of the Boc groups with
TFA, gave 8b (49 mg, 91%). C21H36N6O8ꢄ2(CF3CO2H); colorless solid,
tion of 200 lM of substrate. Inhibitor IC50 value were determined
mp 155–156 °C; ½a D22
ꢂ
+43.3. (c 1.0 H2O); 1H NMR (400 MHz, D2O) d
from the dose–response curves by plotting the percent inhibition
of NA activity versus inhibitor concentrations using Graph Pad
Prism 4.
5.96 (1H, s), 4.59 (1H, d, J = 11.6 Hz), 4.47 (1H, d, J = 10.8 Hz), 4.37
(1H, br s), 4.18 (1H, d, J = 14.4 Hz), 3.98–3.86 (2H, m), 3.73–3.51
(6H, m), 3.29–3.05 (5H, m), 2.96 (3H, s), 2.56–2.54 (2H, m), 2.06
(3H, s), 2.03 (1H, br s), 1.91 (1H, br s); ESI-HRMS calcd for
C21H37N6O8: 501.2673, found: m/z 501.2671 [M+H]+.
7.2.4. Determination of influenza virus TCID50
The TCID50 (50% tissue culture infectious dose) was determined
by serial dilution of the influenza virus stock onto 100 lL MDCK
cells at 1 ꢀ 105 cells/mL in 96-well microplates. The infected cells
were incubated at 37 °C under 5.0% CO2 for 48 h and added to each
7.1.32. 5-Acetamido-2,6-anhydro-4-{N-[3-(morpholinocar
bonyl)propyl]guanidino}-3,4,5-trideoxy-
non-2-enonic acid (8c)
D-glycero-D-galacto-
wells with 100 l
L per well of CellTiter 96Ò AQueous Non-Radioac-
tive Cell Proliferation Assay reagent (Promega). After incubation at
37 °C for 15 min, absorbance at 490 nm was read on a plate reader.
Influenza virus TCID50 was determined using Reed–Muench
method.14
By a procedure similar to that for 4a, saponification of ester 20c
(47 mg, 0.057 mmol), followed by removal of the Boc groups with
TFA, gave 8c (33 mg, 96%). C20H33N5O9ꢄCF3CO2H; colorless solid;
mp 170–171 °C; ½a D22
ꢂ
+11.9 (c 0.5 H2O); 1H NMR (400 MHz, D2O)
d 5.85 (1H, s), 4.75–4.70 (1H, m), 4.32 (1H, d, J = 10.8 Hz), 4,23
(1H, br s), 3.84–3.80 (1H, m), 3.76 (1H, d, J = 12.4 Hz), 3.61–3.49
(6H, m), 3.45–3.41 (4H, m), 3.18–3.13 (1H, m), 3.05 (1H, br s),
2.41–2.36 (2H, m), 1.92–1.00 (1H, m), 1.89 (3H, s), 1.80–1.68 (1H,
m); ESI-HRMS calcd for C20H34N5O9: 488.2351, found: m/z
488.2342 [M+H]+.
7.2.5. Determination of EC50 of NA inhibitor
The anti-flu activities of neuraminidase inhibitors were mea-
sured by the EC50 values that were the concentrations of NA inhib-
itor for 50% protection of the H1N1 CPE activities. Fifty microliter
diluted H1N1 at 100 TCID50 were mixed with equal volumes of
NA inhibitors at varied concentrations. The mixtures were used
to infect 100
l
L of previously seeded MDCK cells at 1 ꢀ 105 cells/
7.1.33. 5-Acetamido-2,6-anhydro-4-{N-[3-(peperidinocarbonyl)
mL in 96-wells. After 48 h incubation at 37 °C under 5% CO2, the
cytopathic effects (CPE) were determined with CellTiter 96Ò AQue-
ous Non-Radioactive Cell Proliferation Assay reagent as described
above. Inhibitor EC50 values were determined by fitting the curves
of percent CPE versus the concentrations of NA inhibitor using
Graph Pad Prism 4.
propyl]guanidino}-3,4,5-trideoxy-D-glycero-D-galacto-non-2-en
onic acid (8d)
By a procedure similar to that for 4a, saponification of ester 20d
(73 mg, 0.088 mmol), followed by removal of the Boc groups with
TFA, gave 8d (49 mg, 93%). C21H35N5O8ꢄCF3CO2H; colorless solid;
mp 152–153 °C; ½a D22
ꢂ
+17.0 (c 1.0 H2O); 1H NMR (400 MHz, D2O)
d 5.96 (1H, s), 4.78–4.75 (1H, m), 4.48 (1H, d, J = 10.8 Hz), 4.39–
4.37 (1H, m), 4.00–3.95 (1H, m), 3.91 (1H, dd, J = 12.0, 2.4 Hz),
3.73–3.66 (2H, m), 3.56–3.49 (4H, m), 3.30–3.27 (1H, m), 3.20
(1H, br s), 2.51 (2H, br s), 2.04 (4H, s), 1.88 (1H, br s), 1.67–1.55
7.3. Computer modeling
The model of compound 8a in complex with the NA was con-
structed through docking compound 8a to the crystallographic

4084
W.-H. Wen et al. / Bioorg. Med. Chem. 18 (2010) 4074–4084
8. (a) Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.; Swaminathan, S.;
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C. J. Am. Chem. Soc. 1997, 119, 681; (b) McClellan, K.; Perry, C. M. Drugs 2001, 61,
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Steeples, I. P.; Storer, R.; Weir, N. G.; Wright, M.; Willamson, C. J. J. Chem. Soc.,
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structure of N1 neuraminidase (PDB code 2HU0). The 3D structure
of compound 8a was build by modifying the 3D structure of
zanamivir (PDB code 1NNC) with SYBYL 8.0 program (Tripos Associ-
ates). GOLD 4.0.115,16 was used to dock compound 8a onto the pro-
tein with flexible docking option turned on. Kollmann-all atom
charges17 were assigned to the protein atoms, and Gasteiger–Hüc-
kel charges18–20 were assigned to ligand atoms using the SYBYL 8.0
program. During the following docking procedure, the side chain
structure of the Asp151 and Arg156 amino acid residues remained
flexible, modeled with the built-in rotamer libraries of the GOLD
4.0.1 package. Initial 1000 independent genetic algorithm cycles
of computation were carried out with ligand torsion angles varying
between ꢁ180° and 180°. The search efficiency was set at 200% to
ensure the most exhaustive search for the docking conformational
space. All other parameters were kept the same as the default set-
tings. The docking processes were distributed to a 40-processor Li-
nux cluster with Intel(R) Xeon(TM) CPU 3.00 GHz CPUs. The
resultant ligand–protein complex structures were ranked with
the GOLDSCORE scoring function to determine the top 1000 hits.
Acknowledgments
HO
OH
HO
OH
HO
OH
We thank the National Science Council for financial support. We
are grateful to the National Center for High-performance Comput-
ing for computer time and facilities. The SYBYL computation was
conducted at the National Center for High Performance Computing,
Taiwan. The GOLD 4.0.1 computation was conducted at the Genom-
ics Research Center of Academia Sinica.
HO
AcHN
H
O
CO2H
HO
AcHN
H
O
HO
AcHN
H
O
CO2H
CO2H
HN
NH
HO
H2N
H2N
1 (DANA)
2 (Amino-DANA)
3 (Zanamivir)
Supplementary data
HO
OH
HO
OH
HO
OH
Supplementary data (1H and 13C NMR spectra of new com-
pounds) associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2010.04.010.
HO
AcHN
H
O
HO
H
O
HO
AcHN
H
O
CO2H AcHN
CO2H
CO2H
Y(CH2)nHN
HN
HN
HN
References and notes
NH
N
4a Y = NH2 n = 3
4b Y = NH2 n = 4
4c Y = CO2H n = 3
4d Y = CO2H n = 4
H2N(CH2)nHN
1. (a) Moscona, A. N. Eng. J. Med. 2005, 353, 1363; (b) De Clercq, E. Nat. Rev. Drug
Disc. 2006, 5, 1015; (c) Schmidt, A. C. Drugs 2004, 64, 2031.
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Hagiwara, T.; Kijima-Suda, I.; Ido, T.; Ohrui, H.; Tomita, K. Carbohydr. Res. 1994,
263, 167; (c) White, C. L.; Janakiraman, M. N.; Laver, W. G.; Philippon, C.;
Vasella, A.; Air, G. M.; Luo, M. J. Mol. Biol. 1995, 245, 623.
6a n = 3
6b n = 4
5
HO
OH
HO
OH
HO
H
O
3. Meindl, P.; Bodo, G.; Palese, P.; Schulman, J.; Tuppy, H. Virology 1974, 58, 457.
4. Colman, P.; Varghese, J. N.; Laver, W. G. Nature 1983, 303, 41.
5. (a) von Itzstein, M.; Wu, W.-Y.; Kok, G. B.; Pegg, M. S.; Dyason, J. C.; Jin, B.; Phan,
T. V.; Smythe, M. L.; White, H. F.; Oliver, S. W.; Colman, P. M.; Varghese, J. N.;
Ryan, D. M.; Woods, J. M.; Bethell, R. C.; Hotham, V. J.; Cameron, J. M.; Penn, C.
R. Nature 1993, 363, 418; (b) Taylor, N. R.; von Itzstein, M. J. Med. Chem. 1994,
37, 616; (c) von Itzstein, M.; Dyason, J. C.; Oliver, S. W.; White, H. F.; Wu, W.-Y.;
Kok, G. B.; Pegg, M. S. J. Med. Chem. 1996, 39, 388.
AcHN
CO2H
HO
AcHN
H
O
CO2H
N
NH
Y(CH2)nN
H2N
X
N
H2N
NH
O
8a X = NH
8b X = NMe
8c X = O
7a Y = NH2 n = 3
7b Y = NH2 n = 4
7c Y = CO2H n = 3
6. Dunn, C. J.; Goa, K. L. Drugs 1999, 58, 761.
7. Russell, R. J.; Haire, L. F.; Stevens, D. J.; Collins, P. J.; Lin, Y. P.; Blackurn, G.-M.;
Hay, A. J.; Gamblin, S. J.; Skehel, J. J. Nature 2006, 443, 45.
8d X = CH2