Synthesis and biological evaluation of nucleoside analogues having 6-chloropurine as anti-SARS-CoV agents

Article abstract of DOI:10.1016/j.bmcl.2007.02.026

Nucleoside analogues that have 6-chloropurine as the nucleobase were synthesized and evaluated for anti-SARS-CoV activity by plaque reduction and yield reduction assays in order to develop novel anti-SARS-CoV agents. Among these analogues, two compounds,

Full text of DOI:10.1016/j.bmcl.2007.02.026

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2470–2473
Synthesis and biological evaluation of nucleoside analogues
having 6-chloropurine as anti-SARS-CoV agents
Masahiro Ikejiri,a Masayuki Saijo,b Shigeru Morikawa,b
Shuetsu Fukushi,b Tetsuya Mizutani,b Ichiro Kuraneb and Tokumi Maruyamaa,*
aFaculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan
bSpecial Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases,
4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan
Received 12 December 2006; revised 22 January 2007; accepted 9 February 2007
Available online 13 February 2007
Abstract—Nucleoside analogues that have 6-chloropurine as the nucleobase were synthesized and evaluated for anti-SARS-CoV
activity by plaque reduction and yield reduction assays in order to develop novel anti-SARS-CoV agents. Among these analogues,
two compounds, namely, 1 and 11, exhibited promising anti-SARS-CoV activity that was comparable to those of mizoribine and
ribavirin, which are known anti-SARS-CoV agents. Moreover, we observed several SAR trends such as the antiviral effects of the
6-chloropurine moiety, unprotected 50-hydroxyl group and benzoylated 50-hydroxyl group.
Ó 2007 Elsevier Ltd. All rights reserved.
Severe acute respiratory syndrome (SARS) is an emerg-
ing infectious disease of the 21st century and poses a
global threat to public health, mainly leading to fatal
infection with influenza-like symptoms such as high
fever, dry cough, pneumonia and dyspnoea.1 This dis-
ease appeared in the Guandong province of southern
China in late 2002, and subsequently spread to 29 coun-
tries in early 2003, affecting approximately 8000 persons.
The overall mortality rate of SARS has amounted to
approximately 10%. Following the identification of the
causative pathogen—a new coronavirus (CoV) named
SARS-CoV—in 2003, several compounds have been
reported to exhibit antiviral activity against SARS-
CoV.2 However, thus far, no effective treatment has
been developed.
cious against SARS-CoV. Thus, several nucleoside
analogues, namely, 1–11, which could be readily pre-
pared or are commercially available, were designed as
anti-SARS-CoV agents (Fig. 1). In this report, we
X1
N
N
R1
N
N
X2
O
R2 R3
ribose derivatives
1: R1 = OH, R2 = OH, R3 = OH, X1 = Cl, X2 = H
2: R1 = OBz, R2 = OH, R3 = OH, X1 = Cl, X2 = H
3: R1 = OH, R2 = OH, R3 = OH, X1 = OMe, X2 = H
4: R1 = OH, R2 = OH, R3 = OH, X1 = SMe, X2 = H
5: R1 = OH, R2 = OH, R3 = OH, X1 = Cl, X2 = NH2
In our previous studies, several nucleoside derivatives
having 6-chloropurine as the nucleobase showed potent
antiviral activity against some types of viruses.3 We pre-
sume that the 6-chloropurine moiety could play an
important role in the antiviral activity; in fact, several
6-chloropurine analogues are known to inhibit bacterial
RNA polymerases.4 Therefore, we expected that nucleo-
side analogues that have 6-chloropurine would be effica-
2' or 3'-deoxyribose derivatives
6: R1 = OH, R2 = OH, R3 = H, X1 = Cl, X2 = H
7: R1 = OH, R2 = H, R3 = OH, X1 = Cl, X2 = H
Cl
Cl
N
N
N
N
N
R5
R4
N
N
N
OH
OH
10: R5 = OH
8: R4 = OH
9: R4 = OBz
11: R5 = OBz
Keywords: SARS-CoV; Nucleoside; 6-Chloropurine; Antiviral.
*
Figure 1. Structures of the nucleoside analogues.
Corresponding author. E-mail: maruyama@kph.bunri-u.ac.jp
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.026

M. Ikejiri et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2470–2473
2471
describe the syntheses and anti-SARS-CoV activities of
the nucleoside analogues 1–11.
Cl
N
N
c
N
b
a
O
9
8
N
OH
O
O
Compounds 1, 4 and 5 were purchased from chemical
companies, and 3, 7 and 11 were prepared according
to previous literatures.5 Syntheses of the (deoxy)ribonu-
cleoside derivatives 2 and 6 are illustrated in Scheme 1.
Regioselective protection of the 20,30-diol moiety of 1
yielded acetonide 12,6 which was subsequently benzoy-
lated to afford 13 with a 91% yield over two steps. Cleav-
age of the acetonide group produced the desired product
2 with a 61% yield. Acetylation of both hydroxyl groups
of 20-deoxyadenosine afforded 14,7 which was trans-
formed to 6-chloropurine 15 with a 57% yield over
two steps. The deprotection of the acetyl groups was
accomplished at ice-water temperature to avoid decom-
position of the 6-chloropurine moiety, thereby produc-
ing the desired product 6 with a 76% yield. Syntheses
of the acyclic analogues 8 and 9 and the carbocyclic
oxetanocin analogue 10 are outlined in Scheme 2. The
Mitsunobu reaction of known alcohol 168 with 6-chlo-
ropurine, followed by the removal of the acetonide
group in 17, resulted in the desired diol 8 with a 66%
yield over two steps. Subsequently, monobenzoylation
of 8 was carried out via hydrolysis of the corresponding
orthobenzoate9 to afford the desired benzoate 9 with a
67% yield. Triol 18, which was prepared as described
in a previous literature,10 was converted to bis-silyl ether
19; this compound was then subjected to a Mitsunobu
reaction to yield the 6-chloropurine derivative 20. Final-
ly, cleavage of the TBS groups produced the desired
product10 with a 96% yield. We could not directly con-
vert benzoate 115c to 10 because of the instability of the
6-chloropurine moiety under basic conditions.
16
17
O
Cl
N
N
N
e
f
R1O
N
10
OH
OR2
18: R1 = R2 = H
19: R1 = R2 = TBS
TBSO
OTBS
20
d
Scheme 2. Reagents and conditions: (a) 6-Chloropurine, PPh3, 1,
10-azobis(N,N-dimethylformamide), THF, rt, 2 days; (b) p-TsOH,
MeOH, rt, 24 h, 66% from 16; (c) PhC(OMe)3, p-TsOH, MeCN, rt,
4 h, followed by water, rt, 3 h, 67%; (d) TBSCl, Pyr, ꢀ20 °C to rt, 18 h,
37%; (e) 6-chloropurine, PPh3, 1,10-azobis(N,N-dimethylformamide),
THF, rt to 45 °C, 18 h, 44% (recovery of 19, 40%); (f) p-TsOH, MeOH,
rt, 18 h, 96%.
The antiviral effect of the prepared nucleoside ana-
logues was evaluated by a plaque reduction assay
and a yield reduction assay with SARS-CoV Frank-
furt-1 strain in Vero E6 cells as described in a previ-
ous
report.11
The
anti-SARS-CoV
activities
determined by the plaque reduction assay are shown
in Figure 2 and Table 1. Compounds 1 and 11
showed potent activity (IC50: 48.7 lM and 14.5 lM,
respectively), and compound 2 showed weak activity
(IC50: 108 lM), while the other analogues 3–10 did
not show any significant activity (IC50 > 300 lM).
Notably, the inhibitory activities of compounds 1
and 11 against SARS-CoV were comparable to those
of mizoribine and ribavirin, which were reported as
potential anti-SARS-CoV agents by our group,11
although the antiviral indices of 1 and 11 were smaller
than those of mizoribine and ribavirin. Figure 3 illus-
trates the anti-SARS-CoV activities of 1, 2, and 11
determined by the yield reduction assay. The inhibito-
ry effect of 1 was the greatest among the three com-
pounds; the virus yield at
a concentration of
approximately 20 lM decreased to one-hundredth or
less of the control (Fig. 3, left). It is important to note
that this inhibitory effect was superior to those of
120
100
80
60
1
40
2
20
11
0
0.1
1
10
100
1000
Scheme 1. Reagents and conditions: (a) Me2C(OMe)2, p-TsOH,
acetone, rt, 4 h, 91%; (b) BzCl, Et3N, DMAP, CH2Cl2, 0 °C to rt,
2 h, quant; (c) AcOH/water (4:1), 60 °C, 5 h, 61%; (d) Ac2O, DMAP,
Pyr, 4 h; (e) t-BuONO, Et4NCl, CCl4, CH2Cl2, 0 °C to 50 °C, 4.5 h,
57% from 20-deoxyadenosine; (f) 2 M NH3–MeOH, 0 °C, 9 h, 76%.
Concentration (µM)
Figure 2. Inhibitory effect of compounds 1, 2 and 11 on plaque
formation of SARS-CoV Frankfurt-1 strain in the plaque reduction
assay.

2472
M. Ikejiri et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2470–2473
Table 1. Inhibitory and cytotoxic concentrations of nucleoside ana-
logues 1, 2, 3–10 and 11; ribavirin; and mizoribine in the plaque
reduction assaya
tion of the antiviral activity also supports this possi-
bility. In the case of ribofuranosyl structure,
unprotected 50-hydroxyl group was important in the
antiviral effect (compound 1 vs 2); this would indicate
that following the intracellular phosphorylation of the
50-hydroxyl group to the corresponding triphosphates,
compound 1 exhibited antiviral activity in the same
manner as common nucleoside antivirals.14 The type
of sugar moiety also influenced the anti-SARS-CoV
activity. For example, the 20-deoxy- and 30-deoxyribo-
nucleoside derivatives 6 and 7 showed weak antiviral
activity as compared to the ribonucleoside derivative
1. The conversion to an acyclic backbone that imitates
the antiviral agent penciclovir (i.e., 9-[4-Hydroxy-3-
(hydroxymethyl)butyl]guanine) also decreased the
activity (compound 1 vs 8 and 9). Interestingly, the
carbocyclic oxetanocin analogue 11 whose hydroxyl
group was protected by benzoylation exhibited poten-
tial activity as compared to unprotected 10 that exhib-
ited almost no activity.15 This trend seems to be
different from those of the common nucleoside antivi-
rals (including compound 1), and could indicate that
compound 11 acts on SARS-CoV through another
pathway that does not involve the intracellular
phosphorylation.
Compound
IC50 (lM)b
CC50 (lM)c
Antiviral indexd
1
48.7
108
279
174
—
5.7
1.6
—
2
3–10
>300
14.5
82
11
Ribavirine
Mizoribinee
78
>850
>700
5.4
>10
>52
13.5
a The same experiment was performed at least three times indepen-
dently with SARS-CoV Frankfurt-1 strain and Vero E6 cells.
b Average of 50% inhibitory concentrations or concentration required
to reduce virus plaque formation by 50%.
c Average of 50% cytotoxic concentrations (CC) or concentration
required to reduce cell growth by 50%. The CC was measured using
the WST-1 cytotoxicity assay kit.
d Antiviral index was defined as the 50% toxic dose divided by the 50%
inhibitory dose.
e Extracts obtained from a previous study.11
mizoribine and ribavirin since approximately 35 lM of
mizoribine or 200 lM of ribavirin was required to
decrease the virus yield to one-tenth of the control.11
In the case of compounds 2 and 11, approximately
50–70 lM of each compound was required to reduce
the virus yield to one-hundredth of the control
(Fig. 3, middle and right).
In conclusion, we have synthesized several nucleoside
analogues having 6-chloropurine as the nucleobase.
Among these analogues, two compounds, namely, 1
and 11, were found to be efficacious against SARS-
CoV and showed antiviral activities comparable to
those of mizoribine and ribavirin. This study revealed
several curious SAR trends such as the antiviral effects
of the 6-chloropurine moiety (compounds 1 and 11),
unprotected 50-hydroxyl group (compound 1), and
protected (benzoylated) 50-hydroxyl group (compound
11). Although some issues such as reduction of cyto-
toxicity remain to be resolved, we hope that the re-
sults of the present study will contribute to further
development of antiviral agents against SARS-CoV.
Studies that focus on further optimisation of the syn-
thesized compounds and their biological evaluation
will be reported in due course.
These results revealed several structure-activity rela-
tionship (SAR) trends. A chlorine atom at the 6-posi-
tion of the purine base was considered to be
important for the anti-SARS-CoV activity (compound
1 vs 3 and 4); however, an introduction of the amino
group at the 2-position of 6-chloropurine, which cor-
responds to a guanine derivative, was unfavourable
for the antiviral activity (compound 5). Although the
reason for this trend is unclear, there is a possibility
that due to its electrophilic nature,12 the 6-chloropu-
rine moiety can form a covalent bond with the target
enzyme and can induce an effective irreversible inhibi-
tion.13 The substitution of weak leaving groups (e.g., –
OMe (compound 3) or –SMe (compound 4)) for the
chlorine atom at the 6-position that led to the diminu-
Compound 1
Compound 2
Compound 11
7
7
6
5
4
3
2
1
7
6
5
6
4
3
5
2
1
0.1
4
0.1
1
10
100
1000
0.1
1
10
100
1000
1
10
100
1000
Concentration (µM)
Concentration (µM)
Concentration (µM)
Figure 3. Inhibitory effect of nucleoside analogues 1, 2 and 11 in the yield reduction assay. The same experiment was performed at least three times
independently with SARS-CoV Frankfurt-1 strain and Vero E6 cells.

M. Ikejiri et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2470–2473
2473
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This research was partially supported by a Grant-in-Aid
for Young Scientists (B), No. 18790093, from the Japan
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