Antiviral activity of cyclopentenyl nucleosides against orthopox viruses (smallpox, monkeypox and cowpox)

Article abstract of DOI:10.1016/S0960-894X(02)00841-7

An improved method for the synthesis of enantiomerically pure D-cyclopentenyl nucleosides has been accomplished and their antiviral activity against orthopox viruses have been evaluated. The key intermediate, L-cyclopent-2-enone 13 was prepared from D-ribose using a ring closing metathesis reaction in eight steps. Among the synthesized nucleosides, the adenine 2 (Neplanocin A), cytosine 14, and 5-F-cytosine 15 analogues exhibited potent anti-orthopox virus activity, including smallpox virus.

Full text of DOI:10.1016/S0960-894X(02)00841-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 9–12
Antiviral Activity of Cyclopentenyl Nucleosides Against Orthopox
Viruses (Smallpox, Monkeypox and Cowpox)
a,
a
b
b
C. K. Chu, * Y. H. Jin, R. O. Baker and J. Huggins
aDepartment of Pharmaceutical and Biomedical Science, College of Pharmacy,
The University of Georgia, Athens, GA 30602, USA
US Army Medical Research Institute of Infectious Diseases, Ft Detrick, MD 21702, USA
b
Received 29 August 2002; accepted 9 October 2002
Abstract—An improved method for the synthesis of enantiomerically pure d-cyclopentenyl nucleosides has been accomplished and
their antiviral activity against orthopox viruses have been evaluated. The key intermediate, l-cyclopent-2-enone 13 was prepared
from d-ribose using a ring closing metathesis reaction in eight steps. Among the synthesized nucleosides, the adenine 2 (Neplanocin
A), cytosine 14, and 5-F-cytosine 15 analogues exhibited potent anti-orthopox virus activity, including smallpox virus.
#
2002 Elsevier Science Ltd. All rights reserved.
Due to their interesting biological activity, carbocyclic
nucleosides have received much attention in recent
years. Various synthetic methodologies have been
reported not only for naturally occurring carbocyclic
and neplanocin A, using l-cyclopent-2-enone 13 as the
10
key intermediate. Optically pure starting material,
such as d-ribose, d-mannose and d-ribonolactone, have
1
1
been used for the synthesis of l-cyclopent-2-enone 13.
1
nucleosides, such as (À)-aristeromycin 1 and neplano-
However, the synthesis of 13 from these carbohydrates
has been problematic due to low and inconsistent yield.
Some of the reactions are very sensitive to the reaction
conditions, such as moisture and temperature, resulting
in low yield. Therefore, an efficient synthetic method for
the key intermediate, such as d-cyclopent-2-enone 13, is
highly desirable in order to carry out studies for struc-
tural–activity relationships of optically pure carbocyclic
nucleosides. Recently, we have developed an efficient
and practical methodology for the synthesis of d-cyclo-
pent-2-enone using ring closure metathesis (RCM)
2
cin A 2 but also for synthetic analogues, including
3
4
abacavir 3 and carbovir 4, which demonstrated potent
anti-HIV activity (Fig. 1). Neplanocin A 2, an unsatu-
rated analogue of (À)-aristeromycin 1, has potent anti-
5
viral activity as well as cytotoxicity. The antiviral effect
may be due to the inhibition of S-adenosylhomocysteine
6
(
AdoHcy) hydrolase. AdoHcy is a potential feedback
inhibitor of cellular transmethylation using S-adenosyl-
l-methionine as a methyl donor, which is essential for
mRNA maturation. Therefore, inhibition of AdoHcy
7
hydrolase would provide broad-spectrum of antiviral
activity for DNA as well as RNA viruses. Although
neplanocin A 2 itself is not an effective antiviral agent
due to its cytotoxicity to the host cell, it is a lead com-
pound for the discovery of structurally related chemo-
8
therapeutic agents. Therefore, a number of neplanocin
A analogues have been synthesized and evaluated as
9
potential antiviral and antitumor agents.
Previously, we have developed a synthetic method for
enantiomerically pure d- and l-cyclopentenyl and
cyclopentyl nucleosides, including (+)-aristeromycin
*Corresponding author. Tel.: +1-706-542-5379; fax: +1-706-542-5381;
e-mail: dchu@rx.uga.edu
Figure 1. Biologically active natural and synthetic carbocyclic nucleo-
sides cyclopentenyl nucleosides as reported in this article.
0
960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00841-7

10
C. K. Chu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 9–12
reaction from d-ribose in excellent overall yield in pre-
using NaH, DMSO and methyltriphenylphosphonium
bromide in THF to give olefin 6 in 91% yield (Scheme
1). The carbonyl compound 7 was obtained by oxida-
tion of the secondary hydroxyl group. Several oxidation
conditions were investigated to optimize the yield.
Among them, Moffatt oxidation with dicyclohexyl
carbodiimide, DMSO, pyridine and trifluoroacetic acid
in toluene gave the best result to afford ketone 7 in 75%
yield. To introduce another olefin moiety, Grignard
reaction was carried out with vinylmagnesium bromide
to provide inseparable mixture of diene 8 in 80% yield.
This underwent the ring-closing metathesis (RCM)
reaction using 5% Grubbs catalyst at refluxing tem-
perature in anhydrous CH Cl to provide a and b
1
2
parative scale. Therefore, our efforts were directed
toward the synthesis of the l-cyclopent-2-enone 13 as
the key intermediate for the synthesis of d-cyclopente-
nyl nucleosides as reported in this article.
Smallpox (Variola virus),13 which has only human as
the natural host, has been responsible for worldwide
epidemics. Smallpox is highly contagious virus with a
case fatality rate of 30% in unvaccinated individuals.14
Under the massive vaccination program by World
Health Organization (WHO), it has been more than 22
1
5
years since the last case of smallpox was confirmed,
and WHO finally declared in 1980 that the global eradi-
cation had been achieved. Smallpox is a particularly
dangerous biological threat because of its clinical and
2
2
cyclopentanol 9 and 10 (9/10 ratio=10/1) in 88% yield.
1
The a- and b-configurations were assigned by H NMR
1
6
epidemiological properties, and no clinically proven
antiviral treatment exists at this time. The recent
increasing concern of biological terrorism affects all
segments of the population and the growing concern of
smallpox virus as a prime bioterrorism agent prompted
us to search for antiviral agents to prevent and to treat
the smallpox virus.
and NOE experiments. The removal of primary
hydroxyl protecting group using 1 M solution of tetra-
butylammonium fluoride in THF to form a vicinal diol
11 and 12, followed by an oxidative cleavage with
sodium periodate to afford the key intermediate cylo-
1
7
pentenone 13 in 95% yield. The prepared cyclopente-
none 13 was converted to adenine (Neplanocin A) 2,
cytosine 14, 5-Fluoro-cytosine 15, and inosine 16 ana-
1
0a
In this communication, we describe an improved syn-
thesis of the key intermediate l-cyclopenten-2-one 13
for d-cyclopentenyl nucleosides and their potent anti-
orthopox virus (smallpox, monkeypox, cowpox and
vaccinia virus) activity.
logues as we previously reported.
Antiviral Activity
The synthesized nucleosides were evaluated for their
antiviral activity against smallpox, monkeypox, cow-
pox, and vaccinia viruses as well as their cytotoxicity in
Vero and MK2 cells. The results are summarized in
Table 1. It was found that adenine 2 (Neplanocin A),
cytosine 14, and 5-fluoro-cytosine 15 analogues exhibited
Chemistry
1
8
The protected d-ribose 5 was readily available from
d-ribose in two steps, which underwent Wittig reaction
1
1
ꢀ
Scheme 1. (a) NaH, DMSO, methyltriphenylphosphonium bromide, THF, 0 C to reflux, 2 h; (b) dicyclohexyl carbodiimide, DMSO, pyridine, tri-
ꢀ
fluoroacetic acid, toluene, rt, 4 h; (c) vinylmagnesium bromide, anhydrous THF, À78 C to rt, 1 h; (d) Grubbs catalyst, anhydrous CH
2 2
Cl , reflux, 24
4 2
h; (e) TBAF, THF, rt, 1 h; (g) NaIO , H O, rt, 0.5 h.

C. K. Chu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 9–12
11
Table 1. Antiviral activity and cytotoxicity against orthopox viruses
of d-cyclopentenyl nucleosides in MK2 and Vero cells
rated carbocyclic nucleosides, and it was found that
adenine, cytosine and 5-F-cytosine derivatives possess
potent antiviral activity against orthopox viruses,
including smallpox virus.
Compd
Cell Virus Activity EC50 Cytotoxicity
mg/mL)
TIh
(
IC50 (mg/mL)
Adenine 2
MK2a 7124c
0.10
0.10
100
0.26
2.62
0.03
0.14
>100
0.21
>100
23
36
100
42
31
50
10
20
230
360
1.0
161
12
1667
71
<0.2
271
1.0
d
BSH
CPXe
f
Acknowledgements
MPX
VACg
Verob 7124
BSH
This research was supported by grants from National
Institute of Allergy and Infectious Diseases (UO1
AI48495).
CPX
MPX
VAC
57
>100
Cytosine 14
MK2 7124
BSH
CPX
MPX
VAC
Vero 7124
BSH
CPX
0.08
0.03
0.06
0.1
0.12
<0.05
44
3
10
21
550
100
>166
210
300
References and Notes
36
>100
30
1
. Kusaka, T.; Yamamoto, H.; Muroi, M.; Kishi, T.; Mizuno,
K. J. Antibiot. 1968, 21, 255.
. Yaginuma, S.; Muto, N.; Tsujino, M.; Sudate, Y.; Hatashi,
M.; Otani, M. J. Antibiot. 1981, 34, 359.
. (a) Daluge, S. M.; Good, S. S.; Faletto, M. B.; Miller,
>2000
>100
0.3
2
0.05
<0.05
0.04
1
4
40
>20
>80
1000
MPX
VAC
3
W. H.; St. Clair, M. H.; Boone, L. R.; Tisdale, M.; Parry,
N. R.; Reardon, J. E.; Dornsife, R. E.; Averett, D. R.; Kre-
nitsky, T. A. Antimicrob. Agents Chemother. 1997, 41, 1082.
(b) Weller, S.; Radomski, K. M.; Lou, Y.; Stein, D. S. Anti-
microb. Agents Chemother. 2000, 44, 2052.
5
-F-Cytosine 15 MK2 7124
1.73
0.51
0.43
0.61
0.53
2.63
1.17
1.35
2.05
1.46
>100
>100
70
>100
100
39
>58
>196
162
>164
189
15
86
>74
49
BSH
CPX
MPX
VAC
4
5
. Vince, R.; Hua, M. J. Med. Chem. 1990, 33, 17.
. Bennett, L. L., Jr.; Brockman, R. W.; Rose, L. M.; Allan,
Vero 7124
BSH
CPX
100
P. W.; Shaddix, S. F.; Clayton, J. D. Mol. Pharmacol. 1985,
7, 666.
6. Wolfe, M. S.; Borchardt, R. T. J. Med. Chem. 1991, 34,
521.
7. De Clercq, E. Antimicrob. Agents Chemother. 1985, 28, 84.
. Shuto, S.; Obara, T.; Saito, Y.; Andrei, G.; Snoeck, R.; De
Clercq, E.; Matsuda, J. Med. Chem. 1996, 39, 2392.
. (a) Shuto, S.; Minakawa, N.; Niizuma, S.; Kim, H. S.;
>100
100
>100
2
MPX
VAC
>69
1
Inosine 16
MK2 7124
BSH
CPX
MPX
VAC
Vero 7124
BSH
CPX
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
—
—
—
—
—
—
—
—
—
—
>100
>100
>100
>100
>100
>100
>100
>100
>100
8
9
Wataya, Y.; Matsuda, A. J. Med. Chem. 2002, 45, 748. (b)
Ono, M.; Nishimura, K.; Tsubouchi, H.; Nagaoka, Y.;
Tomioka, K. J. Org. Chem. 2001, 66, 8199.
10. (a) Song, G. Y.; Paul, V.; Choo, H.; Morrey, J.; Sidwell,
R. W.; Schinazi, R. F.; Chu, C. K. J. Med. Chem. 2001,
MPX
VAC
4
4, 3958. (b) Song, G. Y.; Naguib, F. N.; El Kouni, M. H.;
Chu, C. K. Nucleosides Nucleotides Nucleic Acid 2001, 20,
915. (c) Wang, P.; Gullen, B.; Newton, M. G.; Cheng, Y. C.;
a
LLC-MK2 (ATTC CRL 7).
Vero 76 (ATTC CRL 1587).
b
1
c,d
Smallpox virus.
Cowpox virus.
Schinazi, R. F.; Chu, C. K. J. Med. Chem. 1999, 42, 3390.
11. (a) Wolfe, M. S.; Borcherding, D. R.; Borchardt, R. T.
Tetrahedron Lett. 1989, 30, 1453. (b) Borcherding, D. R.;
Scholtz, S. A.; Borchardt, R. T. J. Org. Chem. 1987, 52, 5457.
e
f
Monkeypox virus.
g
Vaccinia virus.
h
TI=IC50/EC50
.
(
c) Ali, S. M.; Ramesh, K.; Borchardt, R. T. Tetrahedron Lett.
1
1
4
990, 31, 1509.
2. (a) Jin, Y. H.; Chu, C. K. Tetrahedron Lett. 2002, 43,
141. (b) Similar strategies using RCM reaction have been
potent anti-smallpox, -cowpox, -monkeypox, and -vac-
cinia virus activity.
published in Seepersaud, M.; Al-Abed, Y. Tetrahedron Lett.
000, 41, 7801. (c) Choi, W. J.; Park, J. G.; Yoo, S. J.; Kim,
2
These preliminary studies against orthopoxviruses
showed that d-cyclopentenyl nucleoside analogues, par-
ticularly adenine, cytosine and 5-F-cytosine derivatives,
possess potent anti-smallpox virus activities. Therefore
we plan to further investigate the structure–activity
relationships as well as biochemical studies of these
carbocyclic nucleosides.
H. O.; Moon, H. R.; Chun, M. W.; Jung, Y. H.; Jeong, L. S. J.
Org. Chem. 2001, 61, 6490. (d) Lee, K.; Cass, C.; Jacobson,
K. A. Org. Lett. 2001, 3, 597.
13. Davis, B. D.; Dulbecco, R.; Eisen, H. N.; Ginsberg, H. S.;
Wood, W. G. Jr.; McCarty, M. In Microbiology, 2nd ed.;
Harper & Row: Hagerstown, MD, 1973; p 1258.
1
3
1
4. Klietmann, W. F.; Ruoff, K. L. Clin. Micro. Rev. 2001, 14,
64.
5. De Clercq, E. Clin. Micro. Review 2001, 14, 382.
In summary, we have developed an improved synthesis
of l-cyclopentenone 13 using the ring closure metathesis
reaction which was applied to synthesize several unsatu-
16. (a) Henderson, D. A. Emerg. Infect. Dis. 1999, 5, 537.
(b) Steven, R. R.; Michael, M.; Cynthia, K.; Karen, L. G.
Emerg. Infect. Dis. 2001, 7, 920.

12
C. K. Chu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 9–12
1
(
7. Data for 6: [a]
D
À9.97 (c 0.39, MeOH); 1H NMR
ꢀ
1H), 5.64 (d, J=5.8 Hz, 1H), 5.25 (d, J=5.8 Hz, 1H), 4.49 (d,
J=5.8 Hz, 1H), 3.84 (dd, J=4.2 and 11.4 Hz, 1H), 3.56 (dd,
CDCl ) d 6.07–5.98 (m, 1H), 5.41 (dd, J=1.26 and 17.1 Hz,
3
1
1
H), 5.28 (dd, J=0.97 and 10.4 Hz, 1H), 4.68 (t, J=6.0 Hz,
H), 4.05 (t, J=7.7 Hz, 1H), 3.70–3.64 (m, 3H), 2.53 (d,
J=8.7 and 11.2 Hz, 1H), 2.89 (s, OH, D
2.32 (dd, J=4.7 and 8.6 Hz, OH, D O exchangeable, 1H), 1.38
2
O exchangeable, 1H),
2
1
3
J=4.7 Hz, 1H), 1.46 (s, 3H), 1.36 (s, 3H), 0.90 (s, 9H), 0.08 (s,
6
6
(s, 3H), 1.29 (s, 3H); C NMR (CDCl ) d 135.31, 135.12,
3
H); 13C NMR (CDCl ) d 134.16, 117.55, 108.74, 78.80, 77.44,
112.94, 86.34, 84.42, 65.84, 27.08, 25.41. Anal. calcd for
3
9.55, 64.32, 27.82, 25.42, 18.31, À5.37, À5.45. Anal. calcd for
9 14 4
C H O : C, 58.05; H, 7.58. Found: C, 58.06; H, 7.61.
+88.18 (c 0.27, MeOH); 1H NMR
ꢀ
C
15
H
29
O
4
Si: C, 59.56; H, 10.00. Found: C, 60.17; H, 9.60.
Data for 12: [a]
D
ꢀ
1
Data for 7: [a]D À20.34 (C 0.70, MeOH); H NMR (CDCl )
(CDCl ) d 5.95 (dd, J=1.7 and 5.8 Hz, 1H), 5.72 (d, J=5.8
3
3
d 5.74–5.66 (m, 1H), 5.41 (d, J=16.6 Hz, 1H), 5.24 (d, J=10.5
Hz, 1H), 4.92–4.87 (m, 2H), 4.47 (d, J=18.9 Hz, 1H), 4.22 (d,
J=18.9 Hz, 1H), 1.61 (s, 3H), 1.40 (s, 3H), 0.92 (s, 9H), 0.08 (s,
Hz, 1H), 5.07 (d, J=5.5 Hz, 1H), 4.61 (d, J=5.6 Hz, 1H), 3.73
(d, J=11.5 Hz, 1H), 3.31 (bs, OH, D
3.26 (d, J=11.5 Hz, 1H), 2.13 (bs, OH, D
1H), 1.46 (s, 3H), 1.41 (s, 3H); C NMR (CDCl ) d 136.42,
2
O exchangeable, 1H),
O exchangeable,
2
H); 13C NMR (CDCl ) d 132.70, 118.89, 81.90, 78.22, 68.62,
13
6
3
3
3
1.42, 26.99, 25.83, 24.86, 22.64, 13.71, À5.48. Anal. calcd for
133.60, 112.83, 83.43, 82.58, 79.12, 66.38, 27.67, 26.48. Anal.
C
15
H
28
O
4
Si: C, 59.96; H, 9.39. Found: C, 59.92; H, 9.17.
9 14 4
calcd for C H O : C, 58.05; H, 7.58. Found: C, 58.10; H,
1
Data for 8: H NMR (CDCl ) d 6.14–5.88 (m, 2H), 5.43–
7.63.
3
ꢀ ꢀ
Data for 13: mp68.1–69.4 C; [a]D +69.1 (c 0.77, CHCl );
3
10c
5
0
2
.14 (m, 4H), 4.66 (t, J=7.1 Hz, 0.9H), 4.54 (t, J=6.6 Hz,
.1H), 4.38 (d, J=6.4 Hz, 0.1H), 4.29 (d, J=6.9 Hz, 0.9H),
.77 (s, OH, D
ꢀ
ꢀ
D 3
+69.1 (c 1.98, CHCl )];
[reported:
mp68.7–69.8 C; [a]
H NMR (CDCl ) d 7.57 (dd, J=2.0 and 5.8 Hz, 1H), 6.17 (d,
J=5.9 Hz, 1H), 5.23 (dd, J=2.3 and 5.4 Hz, 1H), 4.42 (d,
1
2
O exchangeable, 0.9H), 2.51 (s, OH, D
2
O
3
exchangeable, 0.1H), 1.53 (s, 0.3H), 1.51 (s, 2.7H), 1.38 (s,
1
3
0
5
1
7
2
.3H), 1.36 (s, 2.7H), 0.89 (s, 8.1H), 0.87 (s, 0.9H), 0.05 (s,
1
J=5.4 Hz, 1H), 1.37 (s, 3H), 1.36 (s, 3H); C NMR (CDCl
d 203.17, 159.65, 134.28, 115.46, 78.56, 76.46, 27.37, 26.10.
3
)
3
3
.4H), 0.03 (s, 0.6H); C NMR (CDCl ) d 138.07, 138.01,
35.70, 135.29, 117.56, 117.05, 115.80, 115.70, 108.16, 107.92,
9.33, 78.57, 78.32, 75.08, 76.69, 75.08, 74.79, 68.15, 67.99,
7.67, 27.29, 25.82, 25.80, 25.43, 24.88, 18.31, 18.20, À5.40,
18. Neutral red uptake assay. Stocks of antiviral compounds
were made by dissolving each compound in DMSO to a con-
centration of 20 mg/mL. Drugs were then diluted to 400 mg/
mL in RPMI-1640, serially diluted 3-fold in RPMI-1640, and
50 mL added to 96-well microtiter plates of confluent Vero 76
and LLC-MK2 cells already containing 100 mL of medium. At
À5.49, À5.54. Anal. calcd for C17
32 4
H O Si: C, 62.15; H, 9.82.
Found: C, 62.05; H, 9.76.
Data for 9: [a]D +55.97 (c 0.37, MeOH); 1H NMR
ꢀ
5
(
CDCl
3
) d 5.98 (d, J=5.7 Hz, 1H), 5.74 (d, J=5.7 Hz, 1H),
each drug concentration, three wells were infected with 10
5
J=9.9 Hz, 1H), 3.62 (d, J=9.9 Hz, 1H), 3.22 (s, OH, D O
.31 (d, J=5.3 Hz, 1H), 4.47 (d, J=5.4 Hz, 1H), 3.92 (d,
pfu/well (MOI=0.1) of orthopoxvirus in 50 mL of medium,
while three were left uninfected for toxicity determination (50
mL of medium added to each well). Plates were examined
daily, and were stained once virus-infected, untreated cells
showed 4+ cytopathic effect (CPE). 50 mL neutral red (1.11
mg/mL) was added to the medium to give a final concentra-
tion of 0.22 mg/mL, and cells returned to the incubator for 90
min. The medium was removed, the wells were rinsed twice
with buffered saline solution, and retained stain was solubi-
lized by adding 100 mL of developing solution (50% ethanol, 5
2
exchangeable, 1H), 1.38 (s, 3H), 1.32 (s, 3H), 0.92 (s, 9H), 0.10
3
1
(
s, 3H), 0.09 (s, 3H); C NMR (CDCl
3
) d 135.20, 135.02,
1
12.06, 84.82, 84.66, 64.97, 27.49, 25.95, 25.88, 18.38, À5.38,
À5.41. Anal. calcd for C H O Si: C, 59.96; H, 9.39. Found:
1
5
29
4
C, 60.05; H, 9.48.
ꢀ
); 1H NMR
Data for 10: [a]
D 3
+72.04 (c 0.35, CHCl
(
5
CDCl
.00 (d, J=5.3 Hz, 1H), 4.47 (d, J=5.2 Hz, 1H), 3.69 (ddd,
J=1.5, 9.7 and 38.7 Hz, 2H), 3.12 (s, OH, D O exchangeable,
H), 1.43 (s, 3H), 1.39 (s, 3H), 0.85 (s, 9H), 0.03 (s, 3H), 0.01
s, 3H); 13C NMR (CDCl ) d 136.90, 133.15, 112.41, 84.13,
3
) d 5.90 (d, J=5.7 Hz, 1H), 5.66 (d, J=5.7 Hz, 1H),
2
4 2 4
mM ammonium phosphate (NH H PO ), pH 3.5). Plates were
1
(
rocked for 30 min at 150 RPM, and the optical density (OD)
of the wells at a wavelength of 450 nm was measured on a
plate reader. The data were graphed and analyzed by using the
four parameter–log it curve fit option of a curve-fitting pro-
gram (Molecular Devices, Menlo Park, CA, USA) to deter-
mine the 50% inhibitory and cytotoxic drug concentration.
3
8
2.47, 80.84, 67.04, 27.85, 26.75, 25.80, À5.49. Anal. calcd for
15 29 4
C H O
Si: C, 59.96; H, 9.39. Found: C, 60.10; H, 9.39.
ꢀ
ꢀ
Data for 11: mp103–104 C; [a]
D
+104.12 (c 0.28,
1
MeOH); H NMR (CDCl ) d 5.96 (dd, J=1.6 and 5.8 Hz,
3