Synthesis of 5-isoxazol-5-yl-2′-deoxyuridines exhibiting antiviral activity against HSV and several RNA viruses

Article abstract of DOI:10.1016/j.bmcl.2008.12.103

This paper describes a simple method for synthesizing a small library of 5-isoxazol-5-yl-2′-deoxyuridines from 5-iodo-2′-deoxyuridine. Nitrile oxides were generated in situ from oximes using a commercial bleaching agent; their cycloaddition with 5-ethynyl-2′-deoxyuridine yielded isoxazoles possessing activity against herpes simplex viruses 1 and 2, Encephalomyocarditis virus, Coxsackie B3, and vesicular stomatitis virus; these isoxazoles were, however, inactive against corona virus, influenza virus, and HIV.

Full text of DOI:10.1016/j.bmcl.2008.12.103

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1126–1128
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 5-isoxazol-5-yl-20-deoxyuridines exhibiting antiviral activity
against HSV and several RNA viruses
*
Yoon-Suk Lee, Sun Min Park, Byeang Hyean Kim
Department of Chemistry, BK School of Molecular Science, Pohang University of Science and Technology, Pohang 790-784, South Korea
a r t i c l e i n f o
a b s t r a c t
This paper describes a simple method for synthesizing a small library of 5-isoxazol-5-yl-20-deoxyuridines
from 5-iodo-20-deoxyuridine. Nitrile oxides were generated in situ from oximes using a commercial
bleaching agent; their cycloaddition with 5-ethynyl-20-deoxyuridine yielded isoxazoles possessing activ-
ity against herpes simplex viruses 1 and 2, Encephalomyocarditis virus, Coxsackie B3, and vesicular sto-
matitis virus; these isoxazoles were, however, inactive against corona virus, influenza virus, and HIV.
Ó 2009 Published by Elsevier Ltd.
Article history:
Received 8 October 2008
Revised 5 December 2008
Accepted 26 December 2008
Available online 31 December 2008
Keywords:
HSV
Antiviral
EMCV
Cox. B3
VSV
Isoxazole
Isoxazole nucleosides
Antiviral nucleoside
[3+2] Cycloaddition
Herpes simplex viruses (HSVs) are among the most widespread
viruses on Earth. HSV-1 and HSV-2 are two common strains of the
herpes virus family, Herpesviridae. About 50–70% of young adults
have HSV-1 antibodies in their blood. The persistent nature of her-
pes infection is the reason for the high probability of infection.1
HSVs cause oral and facial lesions in humans, beginning on the skin
or mucosal epithelium. Because the resistance of HSV toward anti-
viral drugs is increasing, the development of new drugs possessing
potent antiviral activity is very important. Acyclic nucleosides,
including Acyclovir (ACV), Penciclovir, and Ganciclovir, are the
largest class of compounds used as anti-herpes drugs.2 Another
nucleoside drug class is the C5-modified pyrimidine nucleosides,
including C5-iodo-20-deoxyuridine (IdU) and Brivudine (BVDU),3
which inhibits the action of DNA polymerases after its incorpora-
tion into the viral DNA, thereby preventing viral replication. Herd-
ewijn’s group synthesized, from 5-iodo-20-deoxyuridine, a large
number of C5-heteroaromatic-substituted 20-deoxyuridines
posses- sing anti-HSV activity. In 1993, they synthesized 5-iso-
Previously, we reported the use of [3+2] cycloadditions to effi-
ciently synthesize sugar-modified isoxazole nucleosides and back-
bone-modified DNA.6 In this paper, we describe the synthesis of
novel C5-modified nucleosides 2 through [3+2] cycloadditions.
We performed [3+2] cycloadditions using a commercial bleach-
ing agent (containing ca. 4% NaOCl) as a practical reagent for gen-
erating the nitrile oxide. As the key intermediate and dipolarophile,
we synthesized the acetyl-protected 5-ethynyl-20-deoxyuridine 5
through Sonogashira coupling (87% yield) of 5-iodo-20-deoxyuri-
dine and TMS-acetylene (See Scheme 1).7,8 The oximes 6 were ob-
tained from the reactions of several aldehydes with hydroxylamine
(see Scheme 2). For the [3+2] cycloadditions, we employed previ-
ously established reaction conditions.6 Dropwise addition of the
commercial bleaching agent provided higher yields—ranging from
59% to 77%—than did the use of reagent-grade 30% NaOCl (see
Fig. 2 and Scheme 3).
O
R'
xazol-5-yl-20-deoxyuridine
1
from 5-(3-oxo-propyn-l-yl)-20-
O
N
O
N
O
N
H
deoxy-uridine (Fig. 1) and hydroxylamine-O-sulfonic acid.4 The
[3+2] cycloaddition is a very powerful reaction for synthesizing a
diverse range of heteroaromatic five-membered rings.5
N
N
NH
NH
O
NH
O
RO
RO
O
O
RO
O
O
O
O
NH2OH
OR
OR
1
2
OR
* Corresponding author. Tel.: +82 54 279 2115; fax: +82 54 279 3399.
E-mail address: bhkim@postech.ac.kr (B.H. Kim).
Figure 1. C-5 isoxazole nucleosides.
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.12.103

Y.-S. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1126–1128
1127
Table 1
R'
O
O
N
In vitro EC50 data for HSV-1 and HSV-2 assays
I
NH
NH
Toxicity
CC50
Antiviral activity (EC50
)
Selectivity index
AcO
N
O
RO
b
O
HSV-1
HSV-2
HSV-1
HSV-2
O
O
7c
7d
8c
ACV>
Ara-C>
0.15
0.06
0.26
>10.00
>10.00
0.08
>0.06
0.09
0.18
0.20
0.07
0.03
0.09
0.36
0.52
0.07
<1.0
2.88
55.76
49.13
2.03
1.81
2.83
>27.78
>19.38
OAc
OR
4
: R' = TMS
: R' = H
R = H
a
c
3
: R = Ac
5
EC50
(
l
g/mL): Concentration of test compound required to inhibit virus-induced
Scheme 1. Synthesis of 3–5. Reagents and conditions: (a) Ac2O, pyridine, rt, 8 h;
yield 92%. (b) TMS-acetylene, Pd(PPh3)4, Et3N, DMF, 40 °C, 8 h; yield 87%. (c) KF, 10%
MeOH in CH2Cl2; yield 80%.
cytopaticity by 50%; CC50
cytotoxicity to uninfected cells; selectivity index: CC50/EC50
(
lg/mL): concentration of test compound that causes 50%
.
OH
Table 2
O
N
a
In vitro EC data for EMCV, Cox. B3, and VSV assays
R''
H
R''
H
Toxicity
CC50
Antiviral activity (EC50
)
Selectivity index
Cox. B3
6
EMCV
Cox. B3
VSV
0.03
BMCV
VSV
R'' = methyl
= propyl
7c
7d
8c
0.05
0.04
0.07
0.30
0.01
0.01
0.02
0.02
0.01
0.01
0.03
0.04
4.91
5.94
5.38
5.30
1.65
1.50
<1
0.03
>0.07
0.08
3.30
2.76
= 4-isopropylphenyl
= 4-tert-butylphenyl
Rib.
>13.91
>7.69
>35.22
EC50
(
l
g/mL): Concentration of test compound required to inhibit virus-induced
g/mL): concentration of test compound that causes 50%
cytotoxicity to uninfected cells; selectivity index: CC50/EC50
Scheme 2. Synthesis of oximes 6. Reagents and conditions: (a) HONH2ꢀHCl, 1 N
cytopaticity by 50%; CC50 (l
NaOH, THF (1:1), rt, 6 h; yield 90–99%.
.
R''
We investigated the antiviral activities for the various com-
pounds against 12 different viruses, namely HSV-1, HSV-2, HIV-1,
HIV-2, EMCV, Cox. B3, VSV, three different influenza viruses (Tai-
wan, Seoul, and Panama), and two corona viruses. The isoxazole
nucleosides all exhibited anti-HSV activity. The nucleoside ana-
logues presenting bulky R00 groups possessed anti-herpes activity
(Table 1). On the other hand, compound 7d9 exhibits only HSV-2
activity. The antiviral activity for HSV-1 of 7d couldn’t be deter-
O
N
O
N
N
NH
O
NH
6
RO
AcO
O
O
a
O
O
OAc
5
OR
7: R = Ac
b
mined due to the high toxicity (CC50 = 0.06 lg/mL). The anti-herpes
8: R = H
activities of the isoxazole nucleoside analogues are better than
those of the reference drugs ACV and Cytarabine (Ara-C); for exam-
ple, 8c9 and the acetylated prodrug 7c9 are ca. twice as active
against HSV-1, and ca. 4-fold more active than ACV and 6- to 7-fold
more active than Ara-C against HSV-2. Normally the acetyl groups
increase the liphophilicity and support the cell penetration due to
the cleavage of the acetyl groups by carboxyesterase in the cell.
The 7d, one of isoxazole nucleoside analogues exhibited the best
anti-HSV-2 activity: 12- and 17-fold more active than ACV and
Ara-C, respectively.
Scheme 3. Synthesis of 7 and 8. Reagents and conditions: (a) 4% NaOCl, THF, slow
dropwise addition, rt, 10 h; yield 59–77%. (b) LiOH, MeOH/water (3:1), rt 10 h; yield
60–89%.
The isoxazole nucleosides displayed anti-RNA virus activities
(Table 2) against Coxsackie B3 (Cox. B3), vesicular stomatitis virus
(VSV), and Encephalomyocarditis virus (EMCV). In the RNA virus
assay, compound 8c exhibited almost the same anti-EMCV and
anti-Cox. B3 potencies as that of the reference, ribavirin (Rib);
compounds 7c and 7d, however, possessed 2- to 4-fold better anti-
viral activities against the three RNA viruses. Although some of the
isoxazole nucleosides are more active than the reference drug, the
bioassay results were disappointing in terms of their cytotoxicity.
The selectivity indexes for the isoxazole nucleosides were lower
than those of the reference drugs. None of the compounds exhib-
ited antiviral activity against HIV, influenza, or corona viruses. In
future studies, we will use the [3+2] cycloaddition strategy to syn-
thesize different C5-triazole-substituted nucleosides from azides,
rather than oximes.
O
N
O
N
O
N
O
N
N
N
N
N
O
NH
NH
O
O
NH
O
NH
AcO
AcO
AcO
AcO
O
O
O
O
O
O
O
O
OAc
OAc
OAc
OAc
7d
7b
7c
7a
O
N
O
N
O
N
O
N
N
N
N
NH
O
NH
O
NH
O
NH
AcO
HO
HO
HO
O
O
O
O
O
O
O
O
Acknowledgments
OAc
OH
OH
OH
8b
8a
8c
5
We are grateful to KOSEF (Gene Therapy R&D, KNRRC, and NCRC
programs) for financial support. We thank Dr. J.K. Lee for perform-
ing the antiviral bioassay at KRICT.
Figure 2. Compounds tested for their antiviral properties against 12 different
viruses.

1128
Y.-S. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1126–1128
2.54 (m, 2H), 2.28 (s, 3H), 2.19 (s, 3H), 2.10 (s, 3H); 13C NMR (75 MHz,
References and notes
CDCl3) d 172.0, 171.8, 163.6, 162.0, 161.3, 151.0, 139.5, 126.3, 105.1, 104.4,
87.5, 84.4, 76.1, 65.4, 39.2, 21.4, 21.3, 11.8; MS (EI+) m/z 393.12, calcd
393.096.
1. Bloom, D. C.; Devi-Rao, G. B.; Hill, J. M.; Stevens, J. G.; Wagner, E. K. J. Virol. 1994,
68, 1283.
2. De Clercq, E. J. Med. Chem. 2005, 48, 1297.
Compound 7b: 1H NMR (300 MHz, CDCl3) d 8.30 (br, 1H), 6.79 (s, 1H), 6.39
(t, J = 5.71 Hz, 1H), 5.26 (d, J = 6.32 Hz, lH), 4.46–4.41 (m, 1H), 4.33–4.28 (m,
2H), 2.66–2.61 (m, 2H), 2.53–2.51 (m, 1H), 2.32–2.29 (m, 1H), 2.27 (s, 3H),
2.11 (s, 3H); 13C NMR (75 MHz, CDCl3) d 170.8, 170.5, 165.2, 161.1, 159.1,
149.1, 137.4, 126.3, 105.1, 101.3, 85.9, 83.3, 74.6, 64.1, 38.8, 28.3, 21.9, 21.2,
21.1, 14.0; MS (EI+) m/z 421.15, calcd 421.149.
3. (a) Bergstrom, D. E.; Ruth, J. L.; Reddy, P. A.; De Clercq, E. J. Med. Chem. 1984, 27,
279; (b) De Clercq, E.; Descamps, J.; Verheht, G.; Walker, R. T.; Jones, A. S.;
Torrence, P. F.; Shugar, D. J. Infect. Dis. 1980, 141, 563; (c) Cheng, Y. C.; Grill, S.;
Ruth, J. L.; Bergstrom, D. E. Antimicrob. Agents Chemother. 1980, 18, 957.
4. (a) Creuven, I.; Evrard, C.; Olivier, A.; Evrard, G.; Van Aerschot, A.; Wigerinck, P.;
Herdewijn, P.; Durant, F. Antiviral Res. 1996, 30, 63; (b) Wigerinck, P.;
Pannecouque, C.; Snoeck, R.; Claes, R.; De Clercq, E.; Herdewijn, P. J. Med.
Chem. 1991, 34, 2383; (c) Wigerinck, P.; Kerremans, L.; Claes, P.; Snoeck, R.;
Maudgal, J. P.; De Clercq, E.; Herdewijn, P. J. Med. Chem. 1993, 36, 538.
5. (a) Cecchi, L.; De Sarlo, F.; Machetti, F. Eur. J. Org. Chem. 2006, 4852; (b) Himo, F.;
Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.; Sharpless, K. B.; Fokin, V.
V. J. Am. Chem. 2005, 127, 210.
Compound 7c: 1H NMR (300 MHz, CDCl3)
d 8.85 (br, 1H), 8.39 (s, 1H),
7.68 (d, J = 8.21 Hz, 2H), 7.23 (d, J = 8.08 Hz, 2H), 7.25 (s, 1H), 7.24 (s, 1H),
6.41 (t, J = 5.62 Hz, 1H), 5.27 (s, lH), 4.42–4.37 (m, 1H), 4.36–4.32 (m, 2H),
2.90–2.85 (m, 1H), 2.54–2.48 (m, 1H), 2.26–2.23 (m, 1H), 2.21 (s, 3H), 2.05
(s, 3H), 1.20 (s, 3H), 1.18 (s, 3H); 13C NMR (75 MHz, CDCl3) d 170.7, 170.4,
163.4, 161.7, 159.1, 151.5, 149.1, 137.4, 127.2, 126.6, 104.9, 101.5, 86.0,
83.2, 74.5, 64.0, 38.7, 34.3, 21.1, 21.0; MS (EI+) m/z 497.18, calcd
497.180.
6. (a) Lee, Y.-S.; Kim, B. H. Bioorg. Med. Chem. Lett. 2002, 12, 1395; (b) Kim, S. J.; Lee,
J. Y.; Kim, B. H. Bioorg. Med. Chem. Lett. 1998, 8, 1313; (c) Kong, J. R.; Kim, S. K.;
Moon, B. J.; Kim, S. J.; Kim, B. H. Nucleosides, Nucleotides & Nucleic Acids 2001, 20,
1751; (d) Park, S. M.; Lee, Y. S.; Kim, B. H. Chem. Commun. 2003, 2912.
7. (a) McGuigan, C.; Yarnold, C. J.; Jones, G.; Velazquez, S.; Barucki, H.; Brancale, A.;
Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J. J. Med. Chem. 1991, 34, 2275.
8. (a) Cristofoli, W. A.; Wiebe, L. I.; De Clercq, E.; Andrei, G.; Snoeck, R.; Balzarini, J.;
Knaus, E. E. J. Med. Chem. 2007, 50, 2851; (b) Terrence, P. F.; Spencer, J. W.; Bobst,
A. M.; Descamps, J.; De Clercq, E. J. Med. Chem. 1978, 21, 228; (c) Robins, M. J.;
Manfredini, S.; Wood, S. G.; Wanklin, R. J.; Rennie, B. A.; Sacks, S. L. J. Med. Chem.
1991, 34, 2275.
Compound 7d: 1H NMR (300 MHz, CDCl3)
d 8.95 (br, 1H), 8.39 (s, 1H),
7.77 (d, J = 8.19 Hz, 2H), 7.46 (d, J = 8.09 Hz, 2H), 7.26 (s, 1H), 7.24 (s, 1H),
6.41 (t, J = 5.76 Hz, 1H), 5.27 (s, 1H), 4.47–4.44 (m, 1H), 4.36–4.32 (m, 2H),
2.57–2.53 (m, 1H), 2.29–2.33 (m, 1H), 2.26 (s, 3H), 2.12 (s, 3H), 1.33 (s,
9H); 13C NMR (75 MHz, CDCl3)
d 170.7, 170.4, 163.4, 161.7, 159.1, 151.5,
149.1, 137.4, 127.2, 126.6, 104.9, 101.5, 86.0, 83.2, 74.5, 64.0, 38.7, 34.3,
21.1, 21.0; MS (EI+) m/z 511.20, calcd 511.195.
Compound 8c: 1H NMR (300 MHz, acetone-d6) d 8.90 (br, 1H), 8.39 (s, 1H),
7.82 (d, J = 8.27 Hz, 2H), 7.37 (d, J = 8.20 Hz, 2H), 7.21 (s, 1H), 6.37 (t,
J = 6.59 Hz, 1H), 5.27 (s, 1H), 4.57–4.54 (m, 21H), 4.51–4.49 (m, 1H), 4.05–
4.03 (m, 1H), 3.90–3.87 (m, 2H), 2.98–2.91 (m, 1H), 2.41–2.37 (m, 2H),
1.26 (s, 3H), 1.24 (s, 3H); 13C NMR (75 MHz, CDCl3) d 170.7, 170.4, 163.4,
161.7, 159.1, 151.5, 149.1, 137.4, 127.2, 126.6, 104.9, 101.5, 86.0, 83.2,
74.5, 64.0, 38.7, 34.3, 21.1, 21.0; MS (EI+) m/z 413.16, calcd 413.159.
9. Selected spectroscopic data. 5: 1H NMR (300 MHz, acetone-d6) d 10.34 (br, 1H),
8.01 (s, 1H), 6.24 (t, J = 7.20 Hz, 1H), 5.31–5.27 (m, lH), 4.35–4.29 (m, 3H), 2.51–
2.47 (m, 2H), 2.10 (s, 3H), 2.05 (s, 3H), 2.03–2.01 (m, 1H).
Compound 7a: 1H NMR (300 MHz, CDCl3) d 8.37 (br, 1H), 6.74 (s, 1H), 6.35
(t, J = 6.77 Hz, 1H), 5.35 (br, 1H), 4.46–4.39 (m, 2H), 4.38–4.35 (m, 1H), 2.58–