Synthesis and anti-influenza evaluation of orally active bicyclic ether derivatives related to zanamivir

Article abstract of DOI:10.1016/S0960-894X(02)01039-9

We synthesized bicyclic ether sialidase inhibitors such as tetrahydro-furan-2-yl, tetrahydro-pyran-2-yl, and oxepan-2-yl derivatives related to zanamivir. These compounds substituted by diol at the C-3′ and C-4′ positions resulted in the retention of low nanomolar inhibitory activities against not only influenza A virus sialidase but also influenza A virus in cell culture. Compound 11a in particular showed comparable efficacy in vivo relative to that of oseltamivir phosphate.

Full text of DOI:10.1016/S0960-894X(02)01039-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 669–673
Synthesis and Anti-Influenza Evaluation of Orally Active Bicyclic
Ether Derivatives Related to Zanamivir
Takeshi Masuda,a Satoshi Shibuya,a Masami Arai,a Shuku Yoshida,b
Takanori Tomozawa,b Akiko Ohno,b Makoto Yamashitab and Takeshi Hondaa,*
aMedicinal Chemistry Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
bBiological Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Received 30 September 2002; accepted 18 November 2002
Abstract—We synthesized bicyclic ether sialidase inhibitors such as tetrahydro-furan-2-yl, tetrahydro-pyran-2-yl, and oxepan-2-yl
derivatives related to zanamivir. These compounds substituted by diol at the C-30 and C-40 positions resulted in the retention of low
nanomolar inhibitory activities against not only influenza A virus sialidase but also influenza A virus in cell culture. Compound 11a
in particular showed comparable efficacy in vivo relative to that of oseltamivir phosphate.
# 2003 Elsevier Science Ltd. All rights reserved.
Sialidase is one of two glycoproteins expressed on the
influenza virus surface and catalyzes the cleavage
of sialic acid residues from glycoproteins, glycolipids
and oligosaccharides. It has been shown that sialidase is
required for release of newly produced virions from
infected cells and that it facilitates the movement of the
virus through the mucus of the respiratory tract.1 The
catalytic activity of sialidase is essential for influenza
virus replication and infectivity. Accordingly, inhibitors
of this enzyme are of interest as potential anti-influenza
agents. Indeed, the effectiveness of sialidase inhibitors as
anti-influenza agents has been demonstrated both in
animal models and in human clinical trials by several
research groups2 and highlighted by recent approval for
human use of zanamivir3 and oseltamivir phosphate4
(Fig. 1). Zanamivir is delivered by inhalation because of
its low oral bioavailability whereas oseltamivir phos-
phate is administered orally. As part of our study of the
structure activity relationships of 4-guanidino-7-sub-
stituted Neu5Ac2en derivatives, we reported that the
appropriate alkyl ether derivatives at the C-7 position of
4-guanidino-Neu5Ac2en had more potent activity
against influenza virus replication compared to zanamivir
and that the multivalent sialidase inhibitor 3 (Fig. 1)
carrying 4-guanidino-Neu5Ac2en analogues via a linker
of alkyl ether at the C-7 position was much more effective
than zanamivir in the mouse/influenza virus infection
model by intranasal administration.5 However, compound
2 and the polyvalent sialidase inhibitor 3 were less
effective than oseltamivir phosphate (data not shown)
when delivered systemically to the mouse. There have
been no reports of orally active derivatives related to
zanamivir. Therefore, in order to developan orally
bioavailable sialidase inhibitor, we designed a newly
synthesized sialidase inhibitor possessing cyclic ether
moieties such as tetrahydro-furan-2-yl, tetrahydro-
pyran-2-yl, and oxepan-2-yl groups in place of the
glycerol side chain of zanamivir. We postulated that
these compounds would possess modified physicochemical
properties which could make them more suitable for
systemic delivery.
*Corresponding author. Tel.: +81-3-3492-3131; fax: +81-3-5436-
8563; e-mail: thonda@shina.sankyo.co.jp
Figure 1.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01039-9

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T. Masuda et al. / Bioorg. Med. Chem. Lett. 13 (2003) 669–673
Herein we report the synthesis of tetrahydro-furan-2-yl,
tetrahydro-pyran-2-yl and oxepan-2-yl derivatives rela-
ted to zanamivir and their biological activities.
removal of the ketals with 80% acetic acid after protec-
tion of the 4-OH groupby the acetyl group, followed by
the formation of the thiocarbonates with thiophosgene
and dimethylaminopyridine provided compounds 6a–
6d, which were then reduced to give the corresponding
di-terminal olefines 7a–7d. Subsequently, ring-closing
metathesis (RCM) reactions with Grubbs’ catalyst
could be successively accomplished to afford the corre-
sponding cyclic compounds 8a–8d. This is the first
example of application of the RCM reaction for sialic
acid modifications. Oxidation of compounds 8a–8d with
OsO4 and NMO provided the diols 9a–9d, respectively,
as single diastereomers. The (30R, 40R)-configurations of
the diols of compounds 9a and 9b were determined by
X-ray crystallographic analysis8 of compounds 11a and
11b, respectively.
Chemistry
The synthesis of the tetrahydro-furan-2-yl, tetrahydro-
pyran-2-yl, and oxepan-2-yl derivatives substituted by
diol at the C-30 and C-40 positions is illustrated in
Scheme 1. Compound 45b was alkylated with toluene-4-
sulfonic acid 2-(2,2-diethyl-[1,3]dioxolan-4-yl)-ethyl
ester,6 allyl iodide, trifluoro-methanesulfonic acid 2,2-
difluoro-but-3-enyl ester,7 or 5-iodo-pent-1-ene in the
presence of NaH in DMF to give the corresponding
compound 5a, 5b, 5c, or 5d, in moderate yield. The
The high diasteroselectivity of osmylation could be
explained in terms of the most preferable conformation
(A) of compound 8a based on molecular orbital calcu-
lation (3-21G* basis set) as shown in Figure 2. Thus, the
oxygen of the tetrahydropyran moiety is orientated
away from the oxygen of the dihydropyran moiety to
avoid electrostatic repulsion and one side of the cyclic
olefin is covered by the acetamide group. Therefore,
osmylation occurred from the sterically more accessible
face, the p-face opposite to that of the preexisting acet-
amide group. Compounds 9a–9d were converted to
compounds 11a–11d under the same conditions as that
described previously.5 The synthesis of the tetrahydro-
furan-2-yl, tetrahydro-pyran-2-yl, and oxepan-2-yl deri-
vatives substituted by diol at the C-40 and C-50 positions
is illustrated in Scheme 2. Removal of the acetonide of
compound 12, followed by treatment of compound 13
with sodium periodate gave the aldehyde 14. Subse-
quently, alkylation of compound 14 was performed with
allyltrimethylsilane in the presence of TiCl4 to provide
the diastereomixture of the homoallyl alcohols (15a/
15b=2:1) which could be separated using a silica gel
column. The (S)-configuration of the C-7 alcohol of
compound 15b was determined by X-ray crystal-
lographic analysis of 19c. Alkylation of the alcohols of
compounds 15a and 15b with allyl trichloroacetimidate
and TfOH, followed by ring closing metathesis reaction
Scheme 1. Reagents and conditions: (a) alkylation reagents
(2.0 equiv), NaH (2.0 equiv), DMF (50–70%); (b) (i) TBAF, THF (70–
80%), (ii) Ac2O, pyridine (90–100%), (iii) AcOH–H2O (4:1) (70–80%),
(iv) thiophosgene (1.2–2.4 equiv), dimethylaminopyridine (2.4–
5.0 equiv), CH2Cl2 (80–90%); (c) P(OCH3)3, 120 ꢀC (60–90%); (d)
benzylidene-bis
(tricyclo-hexylphosphine)
dichlororuthenium
(0.01 equiv), CH2Cl2 (50–100%); (e) OsO4 (0.01 equiv), N-methylmor-
phorine N-oxide (1.2 equiv), acetone–H2O (50–90%); (f) (i) Ac2O,
AcOH, cat. H2SO4 (10:10:1, v/v) (80–90%), (ii) NaN3, Dowex 50W
(H+), t-BuOH (70–80%), (iii) Lindlar cat., H2, EtOH (70–80%), (iv)
1H-pyrazol-1-[N,N0-bis(tert-butoxycarbonyl)] carboxamidine, THF
(95%); (g) (i) CF3COOH–CH2Cl2, (ii) aq NaOH, (90–100%).
Figure 2. The preferred conformation (A) of compound 8a calculated
using Macromodel.

T. Masuda et al. / Bioorg. Med. Chem. Lett. 13 (2003) 669–673
671
of compounds 16a and 16b provided compounds 17a
and 17b, respectively, in good yield. Osmium oxidation
of 17a and 17b each furnished mixtures of two cis diols
(40S, 50R:40R, 50S=2:1), which were separated using a
silica gel column regardless of the C-20 stereochemistry.
Compounds 18a, 18b, 18c, and 18d were thus converted to
the target compounds 19a, 19b, 19c, and 19d, respectively.
The absolute configurations of the diols of compounds
19b and 19c were determined by their X-ray crystal-
lographic analysis.8 The olefin of compound 8a was
reduced with Pd/C under H2 atmosphere to afford the
tetrahydrofuran derivatives 20, which yielded com-
pound 21 as shown in Scheme 3.
Biological Activities
The influenza A virus sialidase inhibitory and plaque
reduction activities9 of a range of the bicyclic sialidase
inhibitors are summarized in Table 1. Tetrahydro-furan-
2-yl, tetrahydro-pyran-2-yl, and oxepan-2-yl derivatives
substituted by diol at the C-30 and C-40 positions, 11a,
11b, 11c, and 11d, exhibited comparable inhibitory
Table 1. Sialidase inhibitory and plaque reduction activities of
bicyclic sialidase inhibitors related to zanamivir IC50 (ng/mL)b
Compd
R
Sialidase inhibitory
assay
Plaque reduction
assay
A/PR/8/34
A/Yamagata/32/89
0.9–6.6 (1.0)a
Zanamivir
0.6–12.0 (1.0)a
11a
10.3 (0.94)
6.1 (1.22)
10.7 (0.88)
12.4 (1.12)
0.6 (0.46)
1.4 (0.82)
1.4 (0.53)
2.2 (1.47)
11b
11c
11d
Scheme 2. Reagents and conditions: (a) AcOH–H2O (4:1) (70%); (b)
NaIO4, acetone–H2O (90%); (c) allyltrimethylsilane (2.0 equiv), TiCl4
(1.5 equiv) (77% as a mixture of R and S diastereomers); (d) allyl
2,2,2-trichloroacetimidate (10 equiv), TfOH (1.5 equiv), CH2Cl2–cyclo-
hexane (20–25%); (e) benzylidene-bis(tricyclohexylphosphine)dichloro-
ruthenium (0.01 equiv), CH2Cl2 (80%); (f) OsO4 (0.01 equiv), N-
methylmorphorine N-oxide (1.2 equiv), acetone–H2O (70–80% as a
mixture of 40S, 50R-diol and 40R, 50S-diol); (g) (i) Ac2O, AcOH, cat.
H2SO4 (10:10:1, v/v) (80–90%), (ii) NaN3, Dowex 50W (H+), t-BuOH
(70–80%), (iii) Lindlar cat., H2, EtOH (70–80%), (iv) 1H-pyrazol-1-
[N,N0-bis(tert-butoxycarbonyl)] carboxamidine, THF (95%), (v)
CF3COOH–CH2Cl2, (vi) aq NaOH, (90–100%).
21
240 (4.00)
2.45 (22.2)
>100
>100
19a
19b
19c
19d
998 (90.7)
370 (30.8)
3600 (300)
>100
>100
>100
aSince IC50 values varied depending on the experiments, the relative
potencies of the compounds to zanamivir are shown in the parentheses
based on the IC50 values of zanamivir as a reference. IC50 values of
zanamivir in enzyme inhibition and plaque reduction were 0.6–12.0 ng/
mL and 0.9–6.6 ng/mL, respectively.
Scheme 3. Reagents and conditions: (a) Pd/C, H2, EtOH (84%); (b)
same conditions as those described in Scheme 1f and g.
bND, not determined.

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T. Masuda et al. / Bioorg. Med. Chem. Lett. 13 (2003) 669–673
activities against influenza A virus sialidase to that of
zanamivir. Compound 11a showed slightly increased
activity (2-fold) in a plaque reduction assay relative to
zanamivir. However, removal of the diol at the C-30 and
C-40 positions resulted in reduced inhibitory activity
(21). Movement of the diol to the C-40 and C-50
positions from the C-30 and C-40 positions, respec-
tively, showed a significant loss of enzyme inhibitory
activity (11a vs 19a). Compounds 19a and 19c pos-
sessing the diol of the 40S and 50R configurations
were relatively more potent than compounds 19b and
19d possessing the diol of the 40R and 50S configur-
ations. This result suggests the position and the
stereochemistry of the diol of the tetrahydro-pyran
side chain must play an important role in binding
affinity with a virus sialidase.
Table 2. The median survival time of compound 11aa and oseltamivir
phosphatea and the results of the Log-rank test
Compd
Median survival
timeb (days)
p valuec compared with
Control
Oseltamivir
phosphate
Control
Oseltamivir phosphate
11a
7.5
10
10
—
<0.0001
<0.0001
—
—
0.1089
Mice were infected with influenza A/PR/8/34 (H1N1) virus.
aCompound 11a and oseltamivir phosphate were administered orally
at doses of 10 mmol/kg once daily for 5 days after infection.
bThe median survival times were calculated by Kaplan–Meier curve.
cStatistical significances of the median survival times were analyzed by
the Log-rank test.
As shown in the model of the structure of compound
11a bound with influenza virus sialidase (Fig. 3), the
carboxylate was held strongly by two arginine residues
(Arg 371 and Arg 118). The guanidino groupforms
strong charge-charge type hydrogen bond interaction
with Glu 119 and Asp151. The diol interacts with the
carboxylate of Glu 276 in a bidentate hydrogen bond
donor-acceptor mode. This binding feature is not sig-
nificantly different from that found in the zanamivir-
sialidase complex.
positions showed comparable sialidase inhibitory activ-
ities relative to that of zanamivir. Furthermore, com-
pound 11a exhibited a similar oral efficacy in the mouse/
infection model to that of oseltamivir. This is the first
example of an in vivo result for derivatives related to
zanamivir.
Acknowledgements
Furthermore, the efficacy of orally administered com-
pound 11a was tested in the influenza virus infected
mouse model on the basis of the survival rates for trea-
ted and infected mice relative to that for control mice.
Compound 11a was administered orally once daily for 5
days after infection. It was found that compound 11a
had similar efficacy relative to oseltamivir phosphate as
shown in Table 2.
We wish to thank Dr. Mutsuo Nakajima and Ms. Reiko
Kametani for performing the sialidase assay, Dr. Shui-
chi Miyamoto and Atsushi Kasuya for molecular mod-
eling studies, and Mr. Youji Furukawa for performing
X-ray crystallographic analysis.
References and Notes
In summary, we prepared the tetrahydro-furan-2-yl,
tetrahydro-pyran-2-yl, and oxepan-2-yl derivatives rela-
ted to zanamivir using an RCM reaction. These bicyclic
ether derivatives substituted by diol at the C-30 and C-40
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Figure 3. Model structure11 of the complex of compound 11a with
influenza virus sialidase.

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6. Toluene-4-sulfonic acid 2-(2,2-diethyl-[1,3]dioxolan-4-yl)-
ethyl ester was prepared as follows: 1,2,4-butanetriol was
reacted with acetone dimethyl acetal in the presence of TsOH,
followed by treatment of TsCl and pyridine to give the target
compound.
7. Trifluoro-methanesulfonic acid 2,2-difluoro-but-3-enyl
ester was prepared as follows: 2,2-difluoro-3-butenoic acid ethyl
ester was treated with LiAlH4 to afford the 2,2-difluoro-3-
butene-1-ol, which was reacted with (TfO)2O in the presence
of pyridine to give the target compound.
Crystallographic data for the structures 11a, 11b, 19b, and 19c
reported in this paper have been deposited with the Cambridge
Crystallographic Data Center as supplementary publication
nos. CCDC 188639, 188640, 188641, 188642, respectively.
Copies of the data can be obtained, free of charge, on appli-
cation to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
(Fax: +44–1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
9. The methods for sialidase inhibitory and plaque reduction
assays are detailed in a previous paper.5a
10. The stereochemistry of the diol of 10d was determined by
1H NMR analysis. J2 ,3 =6.0 Hz and J3 ,4 =2.5 Hz indicated
that the stereochemistry between H-20 and H-30 was a trans
configuration and that between H-30 and H-40 was a cis con-
figuration. Therefore, the configurations of the diol of 10d
were 30R and 40R.
8. ORTEP drawings of compounds 11a, 11b, 19b, and 19c:
0
0
0
0
11. A model of compound 11a complexed with influenza virus
sialidase was created based on the crystal structure of subtype
N9 sialidase complexed with zanamivir (Varghese, J. N.; Epa,
V. C.; Colman, P. M. Protein Sci. 1995, 4, 1081). The glycerol
side chain of zanamivir was replaced by the 3,4-dihydroxy-
tetrahydropyran-2-yl group of 11a, and ligand conformation
was optimized using the molecular mechanics program
CHARMm (Accelrys Inc., San Diego, CA, USA).