Synthesis and anti-influenza virus activity of 7-O-alkylated derivatives related to zanamivir

Article abstract of DOI:10.1016/S0960-894X(02)00329-3

A series of 7-alkyl ether derivatives related to zanamivir were synthesized using direct alkylation of the C-7 alcohol of sialic acid. Alkyl ether moiety of less than 12 carbons in length showed low nanomolar inhibitory activity against influenza A virus sialidase. Furthermore, their moiety improved influenza A virus plaque reduction activity compared to zanamivir. However, removal of the 8,9-diol of the 7-O-alkyl derivatives resulted in loss of antiviral potency. This result suggests that 8,9-diol must play an important role in binding with both influenza A and B virus sialidases.

Full text of DOI:10.1016/S0960-894X(02)00329-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1925–1928
Synthesis and Anti-Influenza Virus Activity of 7-O-Alkylated
Derivatives Related to Zanamivir
Takeshi Honda,a,* Takeshi Masuda,a Shuku Yoshida,b Masami Arai,a Satoru Kanekoa
and Makoto Yamashitab
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 27 March 2002; accepted 6May 2002
Abstract—A series of 7-alkyl ether derivatives related to zanamivir were synthesized using direct alkylation of the C-7 alcohol of
sialic acid. Alkyl ether moiety of less than 12 carbons in length showed low nanomolar inhibitory activity against influenza A virus
sialidase. Furthermore, their moiety improved influenza A virus plaque reduction activity compared to zanamivir. However,
removal of the 8,9-diol of the 7-O-alkyl derivatives resulted in loss of antiviral potency. This result suggests that 8,9-diol must play
an important role in binding with both influenza A and B virus sialidases. # 2002 Elsevier Science Ltd. All rights reserved.
Influenza virus sialidase promotes virus release from
infected cells and facilitates virus spread within the
respiratory tract.1 Several potential specific inhibitors of
this enzyme have been developed and two (zanamivir 12
and oseltamivir phosphate 23) have been approved for
human use. Zanamivir 1 is delivered by inhalation
because of its low oral bioavailability, small volume of
distribution, and rapid renal elimination, whereas osel-
tamivir phosphate 2, an ethyl ester prodrug of GS4071,
is administered orally. In the search for further clinical
candidates, a number of reports have also documented
other potent sialidase inhibitors.4
deoxy-ManNAc, which possesses alkyl ether of more
than three carbons in length, with sodium pyruvate in
the presence of sialic acid aldolase does not proceed.
Therefore, in our attempts to find a synthetic method to
7-modified sialic acids substituted by alkyl ether, we
focused our attention on a chemical approach towards
them using direct alkylation of the C-7 alcohol of sialic
acid analogues.
Herein we report the synthesis of a series of alkyl ether 3
at the C-7 position of zanamivir which could not be
prepared using a chemoenzymatic reaction, and fur-
thermore, their 8,9-dideoxy derivatives 4 and biological
activities (Fig. 1).
In an accompanying paper,5 we report that the synthesis
of the 7-modified analogues related to zanamivir using a
chemoenzymatic reaction and the replacement of the
7-OH moiety by small lipophilic groups (F, N, OMe,
OEt) resulted in the retaining of excellent inhibitory
activity against influenza A virus sialidase. As part of
our study of the structure–activity relationship (SAR) of
this series, we are interested in the corresponding ana-
logues possessing alkyl ether further homologated at the
C-7 position in order to investigate structural features
important for binding within this region of the sialidase
active site. These compounds could possess modified
physicochemical properties which could make them
more suitable for systemic delivery. However, we have
found that the chemoenzymatic reaction of 4-alkoxy-4-
*Corresponding author. Tel.: +81-3-3492-3131; fax: +81-3-5436-
8563; e-mail: thonda@shina.sankyo.co.jp
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00329-3

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T. Honda et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1925–1928
Chemistry
16a–16e under the same conditions as those described
above.
The syntheses of 4-guanidino-Neu5Ac2en derivatives 3
are shown in Schemes 1 and 2. The alcohol at the C-7
position of 56 could be successfully alkylated with a
variety of (RO)2SO27 in the presence of NaH (2.0 equiv)
in DMF to afford the corresponding alkyl ethers 6a–6i
in moderate yield. The treatment of 6a–6i with Ac2O,
AcOH, and H2SO4 (10:10:1, v/v) followed by introduc-
tion of N3 at the C-4 position via oxazoline gave 7a–7i,
respectively.8 Guanidine formation with N, N0-bis (tert-
butoxycarbonyl)-1H-pyrazole-1-carboxyamidine pro-
vided 8a–8i. Subsequent basic hydrolysis and treatment
of CF3COOH–CH2Cl2 afforded the targets 3a–3i. As an
extension of the series of 3, we modified the terminal of
the alkyl groups at the C-7 position. Treatment of 5
with (BnOCH2CH2O)2SO2 in the presence of NaH and
successive removal of the benzyl group with Pd/C under
H2 atmosphere provided 9. Compound 9 was converted
to 10 under the same conditions as those described
above. The terminal alcohol of 10 was tosylated, fol-
lowed by introduction of azide gave 11. Reduction of
the azide of 11 gave the amine 12 and its subsequent
acetylation provided 13. Compounds 10, 11, 12, and 13
were converted to 3j, 3k, 3l, and 3m, respectively, under
the same conditions as those described above.
Biological Activities
The influenza A virus sialidase inhibitory10 and plaque
reduction activities11 of compounds 3 are summarized
in Table 1. In an accompanying paper, we demonstrated
that 7-modified derivatives related to zanamivir posses-
sing relatively small lipophilic substituents such as F,
The synthesis of 8,9-dideoxy derivatives 4 is shown in
Scheme 3. Compounds 6a–6e were converted to thio-
carbonate 14a–14e after replacement of TBDMS ether
by the acetyloxy group at the C-4 position. Subsequent
9
treatment of 14a–14e with P(OMe)3 as a solvent,
followed by hydrogenation of compounds 15a–15e
afforded the corresponding alkyl ethers 16a–16e,
respectively. Compounds 4a–4e were prepared from
Scheme 2. Reagents and conditions: (a) (i) NaH (2.0 equiv),
(BnOCH2CH2O)2SO2 (1.2 equiv), DMF (50%), (ii) Pd/C, H2, EtOH
(80%); (b) (i) Ac2O, AcOH, concd H2SO4 (10:10:1, v/v) (40%), (ii)
NaN3, Dowex 50W (H+), DMF (80%), (iii) Lindlar cat., H2, EtOH
(70%), (iv) N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carbox-
yamidine, THF (90%); (v) NaOMe, MeOH (85%), (vi) acetone dime-
thyl acetal, cat. TsOH (70%); (c) (i) NaOH, H2O (80–90%), (ii)
CF3COOH–CH2Cl2 (1:3, v/v) (60–70%); (d) (i) TsCl, Pyridine,
CH2Cl2 (90%), (ii) NaN3, DMF (70%); (e) Lindlar cat., H2, EtOH
(70%); (f) Ac2O, pyridine (90%).
Scheme 1. Reagents and conditions: (a) NaH (2.0 equiv), (RO)2SO2
(1.2 equiv), DMF (30–80%); (b) (i) Ac2O, AcOH, concd H2SO4
(10:10:1, v/v), (ii) NaN3, Dowex 50W (H+), DMF (30–80%); (c) (i)
Lindlar cat., H2, EtOH (40–80%), (ii) N,N0-bis(tert-butoxycarbonyl)-
1H-pyrazole-1-carboxyamidine, THF (70–90%); (d) (i) NaOH, H2O
(80–90%), (ii) CF3COOH–CH2Cl2 (1:3, v/v) (60–80%).

T. Honda et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1925–1928
1927
N3, OMe, and OEt groups showed similar sialidase
inhibitory activity to zanamivir. In this paper, we
investigated the extensive SAR of a variety of alkyl
ether analogues at the C-7 position related to zanamivir.
Linear alkyl ether chains of less than 12 carbons in
length exhibited similar inhibitory activity to zanamivir
against influenza A virus sialidase, regardless of the
carbon chain length of the alkyl ether. These com-
pounds, 3a, 3b, 3c, 3d, 3e, and 3f, showed a pronounced
improvement in activity against influenza A virus pla-
que reduction assay compared to zanamivir. However,
the moiety of linear chains of more than 12 carbons
in length (3g) resulted in a slight decrease in sialidase
inhibitory and plaque reduction activities, probably
because of unfavorable steric and electrostatic inter-
action within the enzyme binding site. Addition of a
terminal OH, N3, NH2, or NHAc did not significantly
affect the binding as these compounds, 3j, 3k, 3l, and
3m, had a similar potency to 3b. Removal of the 8,9-diol
of the C-7 alkyl ether derivatives resulted in similar
activity against influenza A virus sialidase to zanamivir
Table 1. Sialidase inhibitory and plaque reduction activities of com-
pounds 3 [IC50 (ng/mL)]
Scheme 3. Reagents and conditions: (a) (i) TBAF, THF (70–80%), (ii)
Ac2O, pyridine (90%), (iii) SCCl2, DMAP, CH2Cl2 (60–70%); (b)
P(OMe)3 (60–70%); (c) Pd/C, H2 MeOH (80%); (d) (i) Ac2O, AcOH,
concd H2SO4 (10:10:1, v/v) (50–60%), (ii) NaN3, Dowex 50W (H+),
DMF (40–80%), (iii) Lindlar cat., H2, EtOH (40–80%), (iv) N,N0-
bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxyamidine, THF (70–
90%), (v) NaOH, H2O (80–90%), (vi) CF3COOH–CH2Cl2 (1:3, v/v)
(60–80%).
R
Sialidase Plaque
inhibitory assay reduction assay
A/PR/8/34
A/Yamagata/32/89
Zanamivir
3a
OH
5.1–10.2 (1.0)a
6.1 (1.2)
1.9–20 (1.0)a
3.0 (0.15)
Table 2. Sialidase inhibitory and plaque reduction activities of com-
pounds 4 [IC50 (ng/mL)]
3b
3c
3d
3e
3f
26.4 (2.6)
30.6(3.0)
14.3 (2.6)
20.2 (2.0)
49.6(4.9)
262 (26)
0.7 (0.14)
1.5 (0.35)
2.0 (0.23)
1.8 (0.42)
2.2 (0.51)
11.0 (5.8)
3.0 (1.6)
R
Sialidase Plaque
inhibitory assay reduction assay
3g
3h
3i
A/PR/8/34
A/Yamagata/32/89
1.9–12 (1.0)a
36.6 (3.6)
26.3 (2.6)
10.3 (1.4)
14.8 (2.0)
47.7 (6.5)
9.8 (1.4)
Zanamivir
5.4–10.2 (1.0)a
1.9 (1.0)
4a
35.1 (1.9)
14.1 (1.9)
9.1 (1.7)
16.0 (8.4)
9.0 (0.75)
3.8 (1.3)
3j
1.8 (0.21)
1.5 (0.13)
7.5 (0.63)
2.4 (0.2)
4b
4c
4d
4e
3k
3l
16.6 (2.3)
28.4 (5.3)
40.0 (3.3)
>50 (>5.9)
3m
aSince IC50 values varied depending on the experiments, the relative
potencies of the compounds to zanamivir are shown in the par-
entheses, based on the IC50 values of zanamivir as a reference. IC50
values of zanamivir in enzyme inhibition and plaque reduction were
5.1–10.2 and 1.9–20 ng/mL, respectively.
aSince IC50 values varied depending on experiments, the relative
potencies of the compounds to zanamivir are shown in the par-
entheses, based on the IC50 value of zanamivir as a reference. IC50
values of zanamivir in enzyme inhibition and plaque reduction were
5.4–10.2 and 1.9–12 ng/mL, respectively.

1928
T. Honda et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1925–1928
as shown in Table 2, but relatively little effect on activity
against the influenza B virus sialidase (data not shown).
Evaluation of a plaque reduction assay demonstrated
that 7-alkyl ether derivatives 3 related to zanamivir were
more active than the 8,9-dideoxy derivatives 4. These
results suggest that 8,9-diol must play an important role
in the binding affinity with sialidase of both A and B
viruses. A computational study to rationalize the potent
activity and high specificity of 7-alkyl ether derivatives
is currently in progress.
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In summary, a series of 7-alkyl ether derivatives related
to zanamivir were synthesized using direct alkylation of
the C-7 alcohol of sialic acid analogue. Chains of less
than 12 carbon in length showed similar activity against
influenza A virus sialidase. Furthermore, their moieties
improved influenza A virus plaque reduction activity
compared to zanamivir. However, removal of the 8,9-
diol of the 7-O-alkyl derivatives showed loss of antiviral
activity. This result suggests that 8,9-diol must play an
important role in binding with both influenza A and B
viruses enzymes.
Acknowledgements
We wish to thank Dr. Mutsuo Nakajima and Ms. Reiko
Kametani for performing the sialidase assays.
5. Honda, T.; Masuda, T.; Yoshida, S.; Arai, M.; Kobayashi,
Y.; Yamashita, M. Bioorg. Med. Chem. Lett. 2002, 12, 1921.
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References and Notes
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in CH2Cl2 to afford (RO)2SO, which was oxidized to
(RO)2SO2 by treatment of NaIO4 (1.2 equiv) and cat. RuCl3.
8. Kok, G. B.; Campbell, M.; Mackey, B.; von Itzstein, M. J.
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10. The method for sialidase inhibitory assay is detailed in an
accompanying paper; Bioorg. Med. Chem. Lett. 2002, 12, 1921.
11. The method for plaque reduction assay is detailed in an
accompanying paper; Bioorg. Med. Chem. Lett. 2002, 12, 1921.
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