Anti-influenza virus activities of 2-alkoxyimino-N-(2-isoxazolin-3-ylmethyl)acetamides

Article abstract of DOI:10.1016/S0960-894X(01)00362-6

A series of 2-alkoxyimino-N-(2-isoxazolin-3-ylmethyl)acetamides and related compounds were synthesized and their antiviral activities against human influenza A virus were assessed. Studies of the structure-activity relationships revealed the strongest antiviral activity when position-5 of the isoxazoline ring was substituted with a tert-butyl group. When the alkoxyimino moiety was substituted with a methyl, ethyl, isopropyl or allyl group, good antiviral activity was obtained. Among the geometrical isomers at the oxime moiety, the E-isomers were more active than the Z-isomers. Among the compounds examined, (E)-2-allyloxyimino-2-cyano-N-(5-tert-butyl-2-isoxazolin-3-ylmethyl)acetamide (1j) was the most active inhibitor with an EC₅₀ of 3 μg/mL in vitro.

Full text of DOI:10.1016/S0960-894X(01)00362-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1997–2000
Anti-influenza Virus Activities of 2-Alkoxyimino-
N-(2-isoxazolin-3-ylmethyl)acetamides
Hiroyuki Kai,a,* Hiroshi Matsumoto,b Naohiko Hattori,c Akira Takase,a
Tamio Fujiwarab and Hirohiko Sugimotob
aAburahi Laboratories, Shionogi & Co., Ltd., Koka-cho, Koka-gun, Shiga 520-3423, Japan
bShionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553-0002, Japan
cKikukawa Laboratories, Jokoh & Co., Ltd. Kikukawa-cho, Ogasa-gun, Shizuoka 439-0037, Japan
Received 30 March 2001; accepted 16 May 2001
Abstract—A series of 2-alkoxyimino-N-(2-isoxazolin-3-ylmethyl)acetamides and related compounds were synthesized and their
antiviral activities against human influenza A virus were assessed. Studies of the structure–activity relationships revealed the
strongest antiviral activity when position-5 of the isoxazoline ring was substituted with a tert-butyl group. When the alkoxyimino
moiety was substituted with a methyl, ethyl, isopropyl or allyl group, good antiviral activity was obtained. Among the geometrical
isomers at the oxime moiety, the E-isomers were more active than the Z-isomers. Among the compounds examined, (E)-2-
allyloxyimino-2-cyano-N-(5-tert-butyl-2-isoxazolin-3-ylmethyl)acetamide (1j) was the most active inhibitor with an EC50 of 3 mg/
mL in vitro. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
were randomly screened. Certain 2-alkoxyimino-N-(2-
isoxazolin-3-ylmethyl)acetamides (1, Fig. 1) were found
to show antiviral activities against human influenza
virus. Since potent antiviral activities of isoxazolines
were observed, a series of isoxazoline derivatives were
synthesized with various substituents to find the optimal
structure exhibiting the most potent antiviral activity
against human influenza virus.
Influenza is caused by a group of enveloped RNA vir-
uses that belong to the Orthomyxoviridae family. There
are three types of influenza viruses: A, B, and C. The
first two types are responsible for annual epidemics or
pandemic outbreaks, while influenza C is endemic. The
most severe pandemic human influenza outbreak of
recent times occurred in 1918–1919, when ‘Spanish’
influenza killed more than 20 million people world-
wide.1 Vaccination provides limited protection based on
the ability to predict the exact strains of influenza virus
that will predominate a year in advance of the influenza
season. The antiviral agents, Amantadine and Riman-
tadine, are the only two drugs approved for the pro-
Chemistry
The 3-aminomethyl-2-isoxazolines 6a–e were prepared
as outlined in Scheme 1. The 3-hydroxymethyl-2-iso-
xazolines 4 were prepared by 1,3-dipolar cycloaddition
of the chloro oxime 2 and the alkenes followed by
reduction with sodium borohydride. The alcohols 4
were converted via the modified Gabriel method to their
amines 6a–e by successive treatment with thionyl chloride,
potassium phthalimide and hydrazine.4
phylactic treatment of human influenza
A virus
infection.2 Recently, a novel series of neuraminidase
inhibitors, exemplified by Zanamivir and Oseltamivir,
have emerged.3 These agents are effective against both
type A and B human influenza infections and their suc-
cess has spurred a new wave of research in fighting the
infection.
To discover novel antiviral compounds with different
pharmacophores from currently used antiviral agents,
many compounds from our institute’s chemical library
*Corresponding author. Tel.:+81-748-88-3281; fax: +81-748-88-
6536; e-mail: hiroyuki.kai@shionogi.co.jp
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00362-6

1998
H. Kai et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1997–2000
The 2-cyano-N-(2-isoxazolin-3-ylmethyl)acetamides 1a–n
were prepared as outlined in Scheme 2. The E-isomer
esters 8 were prepared by alkylation of the oxime 7.4
The Z-isomer ester 11 was prepared by amidation of the
dicarboxylate 10 followed by dehydration.5 The iso-
xazoline derivatives 1a–m were prepared by reaction of
the esters (8 or 11) and the amines 6a–e.
The regio isomer 15 was prepared as outlined in Scheme
4. The amide 14 was prepared by reaction of the ester 8a
and allylamine. Compound 15 was prepared by 1,3-
dipolar cycloaddition of tert-butyl oxime chloride9 and
the amide 14. The isoxazole analogue 16 was prepared
as described for the synthesis of 1. Compounds 17–21
were synthesized as described in the literature.4,10,11
The
2-allyloxyimono-N-(5-tert-butyl-2-isoxazolin-3-
ylmethyl)acetamides 1n–p were prepared as outlined in
Scheme 3. The 2-hydroxyimonoacetates 12 were syn-
thesized as described in the literature.6ꢀ8 The esters 13
were prepared by alkylation of the oximes 12. The iso-
xazoline derivatives 1n–p were prepared by reaction of
the esters 13 and the amines 6c.
Results and Discussion
Antiviral activity and cytotoxicity of the 2-alkoxyimino-
N-(5-substituted 2-isoxazolin-3-ylmethyl)acetamides and
related compounds are shown in Tables 1 and 2. Anti-
viral activity (or cytotoxicity) is expressed as an index of
Scheme 1. Synthesis of 3-aminomethyl-2-isoxazolines (6). Reagents and conditions: (i) R1(R2)C¼CH2, NaHCO3, i-PrOH, 40 ꢁC 24 h; (ii) NaBH4,
MeOH, reflux 1 h; (iii) SOCl2, PhH, rt 1 h, reflux 1 h; (iv) PhtNK, DMF, rt overnight; (v) NH2NH2, MeOH, 60 ꢁC 2 h (PhtNK=potassium
phthalimide).
Scheme 2. Synthesis of 2-cyano-N-(2-isoxazolin-3-ylmethyl)acetamides (1a–m). Reagents and conditions: (i) alkyl halides (Me2SO4), K2CO3, DMF,
rt 5 h; (ii) amines (6), Et3N, MeOH, rt overnight; (iii) NaNO2, AcOH, H2O, rt overnight; (iv) Me2SO4, K2CO3, acetone, reflux 4 h; (v) 28% NH3 aq,
MeOH, rt 3h; (vi) (CF 3CO)2O, pyridine, rt 0.5 h.
Scheme 3. Synthesis of 2-allyloxyimino-N-(5-tert-butyl-2-isoxazolin-3-ylmethyl)acetamides (1n–q). Reagents and conditions: (i) allyl chloride,
K2CO3, DMF, rt 5 h; (ii) 3-aminomethyl-5-tert-butyl-2-isoxazoline (6c), 150 ꢁC 3 h.

H. Kai et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1997–2000
1999
Scheme 4. Synthesis of the regio isomer 15. Reagents and conditions: (i) allylamine, Et3N, MeOH, rt 24 h; (ii) t-butyl oxime chloride,
NaHCO3, i-PrOH, rt 65 h.
+++, ++, +, or ꢀ, corresponding to 1–10, 10–100,
100, and >100 ppm EC50 (or CC50) values, respectively.
The EC50 of A/WSN/33 strain of influenza virus
(H1N1 subtype) in cell protection assay in MDBK
cells is measured in mg/mL. The CC50 of the com-
pound in MDBK cells is given.12,13
groups (cyanomethyl, 1k), reduced the activity. The
benzyl (1l) derivative showed weak cytotoxicity. Among
the geometrical isomers at the oxime moiety of dimethyl
and tert-butyl derivatives, E-isomers (1b and 1c) were
more active than Z-isomers (1f and 1g).
The effects of substituent (R4) at position-2 on the
acetamide were examined. Among the compounds (1j
and 1n–q), cyano (1j) and methyl (1o) derivatives were
most active. In the case of smaller (H, 1n) or larger (Et,
1p; Ph, 1q) substituents, the activity was decreased.
Table 1 shows some 2-alkoxyimino-N-(2-isoxazolin-3-
ylmethyl)acetamides and their anti-influenza virus
activities. When position-5 on the isoxazoline ring was
substituted with a tert-butyl (1c) group, good antiviral
activity was obtained. Methyl (1a), dimethyl (1b) and
phenyl (1d) derivatives showed weak activity, whereas
phenyl (1d) and diphenyl (1e) derivatives showed cyto-
toxicity. These results suggested that the physicochem-
ical characteristics or steric effects of the tert-butyl
group might lead to enhancement of the activity,
although no definitive explanations for these observations
have yet been obtained.
Since
2-alkoxyimino-N-(5-tert-butyl-2-isoxazolin-3-
ylmethyl)acetamides were found to be favorable for the
antiviral activity, the 5-tert-butyl-2-isoxazolin-3-ylme-
thyl moiety of compound 1c was modified structurally.
Table 2 shows the effects of group Z on the antiviral
activity examined with various kinds of derivatives.
Isomeric isoxazoline ring (15) or isoxazole analogues
(16) had lower activity than the corresponding
compound 1c. Compound 17, produced by deletion of
–CH2– between the isoxazoline ring and amide moiety
of compound 1c, was inactive. Thienylmethyl (18), pyr-
idylmethyl (19), ethylcarbamoyl (20) and tert-butox-
ycarbonyl (21) derivatives were inactive. These results
The effects of substituent (R3) on the oxime moiety were
examined. Among the compounds 1c and 1h–m, methyl
(1c), ethyl (1h), isopropyl (1i), and allyl (1j) derivatives
were the most active, whereas larger substituent groups
(benzyl; 1l, pyridylmethyl; 1m) or nitrogen-containing
Table 1. 2-Alkoxyimino-N-(2-isoxazolin-3-ylmethyl)acetamides and their antiviral activities
Compound no.
R1
R2
R3
R4
E/Z
Antiviral activitya
Cytotoxicitya
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
Me
Me
t-Bu
Ph
Ph
Me
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
H
Me
H
Me
Me
Me
Allyl
Allyl
Me
Me
Et
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
H
E
E
E
E
E
Z
Z
E
E
E
E
E
E
E
E
E
E
++
++
+++
++
NDb
ꢀ
ꢀ
ꢀ
ꢀ
H
++
++
ꢀ
Ph
Me
H
H
H
H
H
H
H
H
H
H
H
++
+++
+++
+++
++
++
ꢀ
ꢀ
ꢀ
i-Pr
Allyl
ꢀ
ꢀ
CH2CN
Benzyl
3-Pyridylmethyl
Allyl
ꢀ
+
ꢀ
+
ꢀ
Allyl
Allyl
Allyl
Me
Et
Ph
+++
+
+
ꢀ
ꢀ
ꢀ
aAntiviral activity (or Cytotoxicity) is expressed as an index of +++, ++, or ꢀ, corresponding to 1–10, 10–100, 100, and >100 ppm EC50 (or
CC50) values, respectively. The EC50 of a A/WSN/33 strain of influenza virus (H1N1 subtype) in cell protection assay in MDBK cells in m/mL. The
CC50 of the compound in MDBK cells.
bND, >CC50
.

2000
H. Kai et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1997–2000
Table 2. 2-Alkoxyiminoacetamides and their antiviral activities
dose of 3 mg/mL, with stronger activity than Amanta-
dine. However, these compounds showed less or no
antiviral activities against influenza B and respiratory
syncytial virus (unpublished data).
In conclusion, our studies indicated that these 2-
alkoxyimino-N-(5-tert-butyl-2-isoxazolin-3-ylmethyl)-
acetamides, a new class of antiviral agents, are highly
effective against human influenza A. However, the
mechanisms of action of the isoxazoline derivatives are
unknown at present.
Compound
no.
Z
R3
Antiviral Cytotoxicitya
activitya
1c
Me
+++
ꢀ
15
16
17
18
19
Me
Allyl
Me
++
++
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
Acknowledgements
The authors wish to express their thanks to Dr. Masa-
fumi Fujimoto, Director of Aburahi Laboratories,
Shionogi & Co., Ltd., for encouragement and permis-
sion to publish this work. We also wish to thank Drs.
Masaru Ogata and Toshikazu Ohtsuka for invaluable
discussions and encouragement.
Me
ꢀ
References and Notes
Me
ꢀ
1. Taubenberger, J. K.; Reid, A. H.; Karafft, A. E.; Bijwaard,
K. E.; Fanning, T. G. Science 1997, 275, 1793.
2. Scholtissek, C.; Webter, R. G. Antiviral Res. 1998, 38, 213.
3. Gubareva, L. V.; Kaiser, L.; Hayden, F. G. Lancet 2000,
355, 827.
4. Kai, H.; Ichiba, T.; Nishida, K.; Masuko, M.; Takase, A. J.
Pesticide Sci. 1998, 23, 262.
20
21
EtNHCO
t-BuOCO
Me
Me
ꢀ
ꢀ
ꢀ
ꢀ
aAntiviral activity or cytotoxicity are expressed as in Table 1.
5. Goto, J.; Sakane, K.; Teraji, T. J. Antibiot. 1984, 37, 557.
6. Rave, T. W.; Breslow, D. S. J. Org. Chem. 1971, 36, 3813.
7. Armand, J.; Guette, J.-P. Bull. Soc. Chim. Fr. 1969, 2894.
8. Kornblum, N.; Eicher, J. H. J. Am. Chem. Soc. 1956, 1494.
9. Liu, K. C.; Shelton, B. R.; Howe, R. K. J. Org. Chem.
1980, 45, 3916.
10. Kai, H.; Nakayama, K.; Tsuji, H.; Masuko, M.; Takase,
A. J. Pesticide Sci. 1998, 23, 44.
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Table 3. Antiviral activities of selected compounds
Compound no.
EC50 (mg/mL)a
CC50 (mg/mL)b
1j
1o
3
6
>100
>100
>100
Amantadine
10–100
aThe EC50 of A/WSN/33 strain of influenza virus (H1N1 subtype) in
cell protection assay in MDBK cells in mg/mL.
bThe CC50 of the compound in MDBK cells.
12. Human influenza virus A/WSN/33 (H1N1) strain was
grown in 10-day-old chick embryonated eggs and its infectious
titer was assayed on Mardy–Darby kidney (MDBK) cells
using the 50% tissue culture infectious dose (TCID50)
method.13 Anti-influenza virus activity of each compound was
determined as described previously.13 The 50% effective dose
(EC50) and 50% cytotoxic dose (CC50) were determined as
described previously.13
suggested that the 2-isoxazolin-3-ylmethyl group on the
amide nitrogen atom of 2-alkoxyiminoacetamides
played a major role in the potent antiviral activity.
Based on the results described above, compounds 1j and
1o were selected for further evaluation (Table 3). Com-
pound 1j showed the most potent activity among this
series of derivatives. It showed excellent control at a
13. Yoshimoto, J.; Kakui, M.; Iwasaki, H.; Fujiwara, T.;
Sugimoto, H.; Hattori, N. Arch. Virol. 1999, 144, 865.