Synthesis and in vivo influenza virus-inhibitory effect of ester prodrug of 4-guanidino-7-O-methyl-Neu5Ac2en

Article abstract of DOI:10.1016/j.bmcl.2009.04.067

A series of ester prodrugs of 7-O-methyl derivative of Zanamivir (compound 3) was synthesized and their efficacy was evaluated in an influenza infected mice model by intranasal administration. Compound 7c (CS-8958), octanoyl ester prodrug of the C-9 alcoh

Full text of DOI:10.1016/j.bmcl.2009.04.067

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2938–2940
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vivo influenza virus-inhibitory effect of ester prodrug
of 4-guanidino-7-O-methyl-Neu5Ac2en
c
b
a
d
Takeshi Honda a, , Shuku Kubo , Takeshi Masuda , Masami Arai , Yoshiyuki Kobayashi ,
*
Makoto Yamashita b
a Medicinal Chemistry Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Medicinal Chemistry Research Laboratories II, Daiichi Sankyo Co., Ltd, 16-13 Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan
c Biological Research Laboratories IV, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Global Project Management Department, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of ester prodrugs of 7-O-methyl derivative of Zanamivir (compound 3) was synthesized and their
efficacy was evaluated in an influenza infected mice model by intranasal administration. Compound 7c
(CS-8958), octanoyl ester prodrug of the C-9 alcohol of compound 3, was found to be much longer-acting
than Zanamivir. Furthermore, the in vivo efficacies of compounds 12a, 12b, and 12c, the linear alkyl ester
prodrug of the carboxylic acid, were comparable to that exerted by compound 7c.
Ó 2009 Published by Elsevier Ltd.
Received 2 March 2009
Revised 14 April 2009
Accepted 16 April 2009
Available online 20 April 2009
Keywords:
Ester prodrug
CS-8958
Zanamivir
Influenza
Long-acting NA inhibitor
Influenza is a respiratory infection associated with significant
morbidity in the general population and mortality in elderly and
high-risk patients.1 Influenza A and B viruses have two major
membrane-associated surface glycoproteins, hemagglutinin and
sialidase, which are both essential for infectivity.2 Sialidase cleaves
terminal sialic acid residues from glycoconjugates and promotes
the release of newly formed virus particles from infected cells. Re-
cently, several potent and specific inhibitors of this enzyme have
been developed,3 and two (Zanamivir 14 and Oseltamivir phos-
phate 25) have been approved for human use (Fig. 1). Unlike aman-
tadine6 and rimantadine, which target the M2 protein of influenza
A viruses, these drugs inhibit replication of both influenza A and B
viruses. Zanamivir (RelenzaTM) is delivered by inhalation because
of its low oral bioavailability, whereas Oseltamivir phosphate
(TamifluTM) is administered orally. Early treatment with either
drug reduces the severity and duration of influenza symptoms
and associated complications. Both agents are effective for chem-
prophylaxis. Because of a broader antiviral spectrum, better toler-
ance, and less potential for the emergence of resistance than is
seen with the M2 inhibitors, the sialidase inhibitors represent an
important advance in the treatment of influenza.
In the course of our study on the synthesis of sialidase inhibi-
tors, we focused our attention on a novel sialidase inhibitor admin-
istered by inhalation because an inhaled compound has the
advantage of a rapid onset of action, very few side effects and
the ability to use a lower dose than that administered orally. How-
ever, Zanamivir is administered twice daily in spite of the inhaled
administration because of its low maintenance of activity. There-
fore, our main goal is to develop a new inhaled sialidase inhibitor
which has longer lasting activity than that of Zanamivir. Thus, it
would have slower clearance of the drug from the target tissue
and the advantage of a single inhalation for treatment.
OH OR
H
H
O
COOEt
O
COOH
OH
AcHN
AcHN
NH2H3PO4
HN
NH
Oseltamivir phosphate 2
H2N
1
3
R : H (Zanamivir)
R : Me
We recently reported that the replacement of the 7-OH moiety
of Zanamivir by small lipophilic groups (F, N3, OMe, OEt) resulted
in an enhancement of anti-viral activity in an influenza A virus pla-
que reduction assay.7 In particular, compound 38 effectively inhib-
Figure 1.
* Corresponding author.
E-mail address: honda.takeshi.sx@daiichisankyo.co.jp (T. Honda).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.04.067

T. Honda et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2938–2940
2939
HO MeO
ited the enzymatic activities of several influenza A (H1N1, H2N2,
H3N2) and B viruses including an avian influenza H5N1 virus
and Tamiflu resistant virus. The IC50s against their influenza virus
replication were better than that of Zanamivir. However, the effi-
cacy of intranasally administered compound 3 showed the only
slight increase (2–3-fold) compared to that of Zanamivir in an
influenza infected mice model.9 Therefore, in our attempt to syn-
thesize a more potent sialidase inhibitor than compound 3 in vivo,
we designed the ester prodrugs of compound 3 on the basis of our
hypothesis that a lipophilic ester prodrug could suppress its clear-
ance from lung tissue to plasma and the active form is hydrolyzed
and retain in the lung for a long time. Herein, we report that the
synthesis of the ester prodrug of compound 3 and their in vivo
evaluations in an infected mice model.
RCOO MeO
H
H
O
COOCHPh2
NBoc
O
COOCHPh2
HO
RCOO
AcHN
AcHN
a
HN
HN
NBoc
BocHN
BocHN
5
9
RCOO MeO
H
O
COOH
NH
10a: R = (CH2)2CH3
10b: R = (CH2)4CH3
10c: R = (CH2)6CH3
b
RCOO
AcHN
HN
H2N
The synthetic pathway to the ester prodrug at the C-9 position
of compound 3 is shown in Scheme 1. Compound 410 was treated
with diphenyl diazomethane in THF to provide the diphenyl ester
5 in a 85% yield. Subsequently, the C-9 alcohol of compound 5
was selectively acylated with the appropriate linear alkyl carbox-
ylic acid chlorides or 2-ethylhexanoyl chloride in the presence of
TEA, followed by deprotection with CF3COOH–CH2Cl2 gave the tar-
get compounds 7a–7g, respectively, accompanied by the unsepara-
ble esters 8a–8g at the C-8 position (7:8 = 10:1–20:1) after
purification by reverse phase chromatography.
The diester formation at the C-8, 9 positions and the deprotec-
tion with CF3COOH–CH2Cl2 gave compounds 10a–10c as shown in
Scheme 2. The synthetic route for the ester prodrug of the carbox-
ylic acid of compound 3 is shown in Scheme 3. Compound 4 was
treated with 1.1 equiv of KOH to afford the potassium salt of the
carboxylic acid of compound 4. The potassium salt was reacted
with the appropriate linear alkyl bromides or carbonic acid alkyl
ester 1-iodo-ethyl esters11 to provide the corresponding ester 11,
respectively. Compounds 12a–12c and 12d–12f were obtained as
trifluoroacetic acid salts by deprotection with CF3COOH–CH2Cl2.
Scheme 2. Reagents: (a) RCOCl (3.0 equiv), TEA (3.0 equiv), 4-N,N-
dimethylaminopyridine (3.0 equiv), CH2Cl2 (80–90%); (b) CF3COOH–Cl2Cl2 (60–
70%).
HO MeO
HO MeO
H
H
O
COOR
NBoc
O
COOH
NBoc
a
HO
HO
AcHN
AcHN
HN
HN
BocHN
BocHN
11
4
HO MeO
H
b
12a: R = (CH2)11CH3
12b: R = (CH2)13CH3
12c: R = (CH2)15CH3
O
COOR
NH
HO
AcHN
12d: R = CH(CH3)OCOOCH2CH3
12e: R = CH(CH3)OCOOc-hexyl
12f: R = CH(CH3)OCOOCH10CH21
HN
H2N
CF3COOH
Scheme 3. Reagents and conditions: (a) (i) KOH (1.1 equiv), MeOH, (ii) 12a–12c:
RBr (1.1 equiv), DMF 50 °C, 4 h (50–80%); 12d–12f: ICH(CH3)OCOOR (1.5 equiv),
DMF, 20 °C, 1 h (50–60%); (b) CF3COOH–Cl2Cl2 (60–70%).
HO OMe
HO MeO
H
H
O
COOH
O
COOCHPh2
NBoc
a
The efficacy of the intranasally administered ester prodrugs of
compound 3 was evaluated on the basis of the survival rate mea-
sured for 20 days post infection in treated infected mice relative to
untreated infected (control) mice. These prodrugs showed very
weak or no inhibition of sialidase activity. They were administered
intranasally 4 h before and 4 h, 17 h after the infection. All of the lin-
ear alkyl esters prepared at the C-9 alcohol position had longer last-
ing efficacy than Zanamivir and compound 3 (data not shown), as
shown in Table 1. Their prolonged in vivo activities were supposed
to be due to slower clearance of the active form from the lung. Thus,
a high concentration of compound 3, the active form of the esters,
could be presumably maintained for a long time at the site of virus
HO
HO
AcHN
AcHN
HN
HN
NBoc
BocHN
BocHN
5
4
RCOO OMe
H
O
COOCHPh2
c
b
HO
AcHN
HN
NBoc
BocHN
6
RCOO OMe
HO OMe
Table 1
H
H
Survival rates of infected micea intranasally administered compounds 7a–7gb and
O
COOH
NH
O
COOH
NH
Zanamivirb
HO
RCOO
AcHN
AcHN
No. of survivors/total no. of mice
+
HN
HN
At 8 days after infection
At 8 days after infection
H2N
H2N
Zenamivir
2/12
2/12
3/11
9/12
10/11
6/11
8/12
8/11
0/12
0/12
1/11
9/12
6/11
2/11
6/12
4/11
7a: R = (CH2)2CH3
7b: R = (CH2)4CH3
7c: R = (CH2)6CH3
7d: R = (CH2)8CH3
7e: R = (CH2)10CH3
7f: R = (CH2)12CH3
7g: R = (CH2)14CH3
7h: R = CH(Et)(CH2)3CH3
7a
7b
7c
7d
7e
7f
8a: R = (CH2)2CH3
8b: R = (CH2)4CH3
8c: R = (CH2)6CH3
8d: R = (CH2)8CH3
8e: R = (CH2)10CH3
8f: R = (CH2)12CH3
8g: R = (CH2)14CH3
8h: R = CH(Et)(CH2)3CH3
7g
a
Twelve mice were infected with influenza A/PR/8/34 (H1N1) virus.
Compounds 7a–7g and Zanamivir were intranasally administered at doses of
b
Scheme 1. Reagents: (a) Ph2CN2, THF (85%); (b) RCOCl (1.1 equiv), TEA (1.2 equiv),
CH2Cl2, (50–70%); (c) CF3COOH–CH2Cl2 (60–70%).
0.3 lmol/kg 4 h before and 4 h, 17 h after the infection.

2940
T. Honda et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2938–2940
2004, 14, 1589; (h) MacDonald, S. J. F.; Cameron, R.; Demaine, D. A.; Fenton, R.
Table 2
Survival rates of infected micea intranasally administered compounds 7cb, 12a,b 12b,b
12c,b and Zanamivirb
J.; Foster, G.; Gower, D.; Hamblin, J. N.; Hamilton, S.; Hart, G. J.; Hill, A. P.; Inglis,
G. G. A.; Jin, B.; Jones, H. T.; McConnell, D. B.; McKimm-Breschkin, J.; Mills, G.;
Nguyen, V.; Owens, I. J.; Parry, N.; Shanahan, S. E.; Smith, D.; Watson, K. G.; Wu,
W.-Y.; Tucker, S. P. J. Med. Chem. 2005, 48, 2964.
No. of survivors/total no. of mice
4. (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) Ryan, D. M.; Ticehurst, J.; Dempsey, M. H.; Penn, C.
R. Antimicrob. Agents Chemother. 1994, 38, 2270; (c) Woods, J. M.; Bethell, R. C.;
Coates, J. A.; Healy, N.; Hiscox, S. A.; Pearson, B. A.; Ryan, D. M.; Ticehurst, J.;
Tilling, J.; Walcott, S. M.; Penn, C. R. Antimicrob. Agents Chemother. 1993, 37,
1473; (d) Hayden, F. G.; Treanor, J. J.; Betts, R. F.; Lobo, M.; Esinhart, J. D.;
Hussey, E. E. JAMA 1996, 275, 295.
5. (a) Williams, M. A.; Lew, W.; Liu, H.; Mendel, D. B.; Tai, C. Y.; Escarpe, P. A.;
Laver, W. G.; Stevens, R. C.; Kim, C. U. Bioorg. Med. Chem. Lett. 1997, 14, 1837;
(b) Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.; Swaminathan, S.;
Bischofberger, N.; Chen, M. S.; Mendel, D. B.; Tai, C. Y.; Laver, W. G.; Stevens, R.
C. J. Am. Chem. Soc. 1997, 119, 681; (c) Kim, C. U.; Lew, W.; Williams, M. A.; Wu,
H.; Zhang, L.; Chen, X.; Escarpe, P. A.; Mendel, D. B.; Laver, W. G.; Stevens, R. C. J.
Med. Chem. 1998, 41, 2451; (d) Mendel, D. B.; Tai, C. Y.; Escarpe, P. A.; Li, W.;
Sidwell, R. W.; Huffmann, J. H.; Sweet, C.; Jakeman, K. J.; Merson, J.; Lacy, S. A.;
Lew, W.; Williams, M. A.; Zhang, L.; Chen, X.; Bischofberger, N.; Kim, C. U.
Antimicrob. Agents Chemother. 1998, 640.
At 8 days after infection
At 8 days after infection
Zenamivir
7c
12a
12b
12c
3/12
0/12
12/12
12/12
12/12
12/12
12/12
11/12
10/12
10/12
a
Twelve mice were infected with influenza A/PR/8/34 (H1N1) virus.
Compounds 7c, 12a, 12b, 12c, and Zanamivir were intranasally administered at
doses of 0.3 lmol/kg 4 h before and 4 h, 17 h after the infection.
b
replication compared to Zanamivir and compound 3. Their pharma-
cokinetic study is currently in progress. However, the alkyl branched
ester 7h resulted in a decreased activity12 compared to the linear al-
kyl esters. The reason for this remains unclear: it could be due to the
resistance of enzymatic hydrolysis of the prodrugs. Among the pro-
drugsof theC-9positioncompound7c(CS-8958), octanoylesterpro-
drug of compound 3, was found to be most effective. On the other
hand, the diacyl esters13 at the C-8, 9 positions 10a, 10b, and 10c
showed a loss of activity compared to the former prodrugs. The eval-
uation of the linear alkyl esters of the carboxylic acid demonstrated
that compounds 12a, 12b, and 12c were much more potent than
Zanamivir and were comparable to compound 7c (Table 2). The
shorter carbon chain in length than that of compound 12a resulted
in decreased activity. A series of 1-alkoxy carbonyloxy ethylester
prodrugs14 12d, 12e, and 12f were less effective than the linear alkyl
ester prodrugs at the carboxylic acid.
In summary, a series of the ester prodrugs of compound 3 was
synthesized and evaluated in a mouse model by intranasal admin-
istration. The linear alkyl esters at the C-9 position showed long
lasting activity relative to Zanamivir and compound 3. Among
them compound 7c (CS-8958), octanoyl ester prodrug at the C-9
position of 3, was the most potent. Furthermore, the long efficacies
of compounds 12a, 12b, and 12c, the linear alkyl ester of carboxylic
acid, were comparable to that of compound 7c (CS-8958). In con-
trast to the currently available drugs, it is expected that a single
inhalation of the prodrugs such as compound 7c (CS-8958) would
provide effective treatment for influenza virus infections. A phase
III clinical trial by a single inhalation of CS-8958 is currently under-
way to determine its potential as a LANI (long-acting NA inhibitor)
for the treatment of seasonal influenza. Further in vivo evaluation
of the ester prodrugs of compound 3,15 including a pharmacoki-
netic investigation, is currently in progress and the obtained re-
sults will be reported in due course.
6. Hay, A. J. Semin. Virol. 1992, 3, 21.
7. Honda, T.; Masuda, T.; Yoshida, S.; Arai, M.; Kobayashi, Y.; Yamashita, M. Bioorg.
Med. Chem. Lett. 2002, 12, 1921.
8. Sialidase inhibitory and plaque reduction activities of compound 3 against
various influenza virus strains. IC50 (nM) sialidase inhibitory and plaque
reduction activities of compounds were determined by previously reported
methods.
Virus
Subtrain
Sialidase
Zanamivir
Virus replication
3
3
Zanamivir
A/PR/8/34
H1N1
H1N1
H2N2
H3N2
H3N2
B
5.97 3.62
3.42 2.55
11.4 3.66
16.8 7.33
23.5 9.30
31.3 12.0
4.54 2.99
2.2
2.4
0.43 0.79
1.3
1.7
4.2
11
10
A/Yamagata/32/89
A/Singapore/1/57
A/Aichi/2/68
A/Kitakyushu/159/93
B/Mie/1/93
A/R(duck/mongolia/54/01-
duck/mongolia/47/01)
A/Yokohama/67/2006(Tamiflu
resistant virus)
1.6
3.3
9.2
H5N1
0.34 0.79
H1N1
5.62 3.05
NT NT
9. Compound
3 and Zanamivir were administered intranasally at doses of.
0.2 mol/kg 4 h before and 4 h, 17 h after the infection in mice. Compound3
l
was more effective than Zanamivir (2/10 survived in the 3 treated infected
group while there was no survivor in the case of Zanamivir at 20 days after
infection).
10. Honda, T.; Masuda, T.; Yoshida, S.; Arai, M.; Kaneko, S.; Yamashita, M. Bioorg.
Med. Chem. Lett. 2002, 12, 1925.
11. Okazaki, T.; Suga, A.; Watanabe, T.; Kikuchi, K.; Kurihara, H.; Shibasaki, M.;
Fujimori, A.; Inagaki, O.; Yanagisawa, I. Chem. Pharm. Bull 1998, 46, 287.
12. Survival rates of infected micea intranasally administered compounds 7hb and
Zanamivirb
No.of survivors/total No.of mice
References and notes
At 8 days after infection
At 20 days after infection
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7h
3/10
4/10
0/10
0/10
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a
Ten mice were infected with influenza A/PR/8/34 (H1N1) virus.
Compounds 7h and Zanamivir were intranasally administered at doses of
b
0.3 lmol/kg 4 h before and 4 h, 17 h after the infection.
13. Compounds 10a–10c were administered intranasally at doses of 0.3 lmol/kg
4 h before and 4 h, 17 h after the infection in mice. There was no survivor in the
10a, 10b, and 10c-treated infected groups at 20 days after infection.
14. Compounds 12d, 12e,12f, and Zanamivir were administered intranasally at
doses of 0.3 lmol/kg 4 h before and 4 h, 17 h after the infection in mice. Their
efficacies were found to be almost same as that of Zanamivir (there were no
survivors in the 12d, 12e, 12f, and Zanamivir-treated infected groups at 9 days
after infection).
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