(-)-Carbodine: Enantiomeric synthesis and in vitro antiviral activity against various strains of influenza virus including H5N1 (avian influenza) and novel 2009 H1N1 (swine flu)

Article abstract of DOI:10.1016/j.bmcl.2010.02.074

Enantiomerically pure cyclopentyl cytosine [(-)-carbodine 1] was synthesized from d-ribose and evaluated for its anti-influenza activity in vitro in comparison to the (+)-carbodine, (±)-carbodine and ribavirin. (-)-Carbodine 1 exhibited potent antiviral activity against various strains of influenza A and B viruses.

Full text of DOI:10.1016/j.bmcl.2010.02.074

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2601–2604
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
(ꢀ)-Carbodine: Enantiomeric synthesis and in vitro antiviral activity against
various strains of influenza virus including H5N1 (avian influenza) and
novel 2009 H1N1 (swine flu)
Jagadeeshwar R. Rao a, Ashok K. Jha a, Ravindra K. Rawal a, Ashoke Sharon a, Craig W. Day b, Dale L. Barnard b,
Donald F. Smee b, Chung K. Chu a,
*
a The University of Georgia College of Pharmacy, Athens, GA 30602, USA
b Utah State University Institute for Antiviral Research, Logan, UT, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Enantiomerically pure cyclopentyl cytosine [(ꢀ)-carbodine 1] was synthesized from
D-ribose and
Received 21 January 2010
Revised 16 February 2010
Accepted 18 February 2010
Available online 21 February 2010
evaluated for its anti-influenza activity in vitro in comparison to the (+)-carbodine, ( )-carbodine and
ribavirin. (ꢀ)-Carbodine 1 exhibited potent antiviral activity against various strains of influenza A and
B viruses.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Carbodine
Carbocyclic nucleosides
Anti-influenza activity
In recent years the emergence of the avian influenza A virus
subtype H5N1 has raised concerns for public health worldwide.
Since January 2004, the World Health Organization (WHO) has
been tracking the human cases of avian influenza A (H5N1), and
up to December 30, 2009, 282 deaths (467 cases; 60.4% mortality)
have been reported.1 Recently a new pandemic influenza A (Novel
H1N1 2009) virus originally called ‘swine flu’ has also emerged
which has never been circulated before among humans. WHO offi-
cially raised the phase of pandemic alert to level 6 with regard to
the novel H1N1 2009 influenza on June 11, 2009. As of January
10, 2010, more than 208 countries have reported the laboratory
confirmed cases of the novel influenza H1N1 2009 strains, includ-
ing at least 13,554 deaths.2 The pandemic is unfolding in the south-
ern hemisphere and WHO is nervously monitoring the coming
winter wave in the northern hemisphere.
its clinical use in the future.2b Furthermore, oseltamivir is the only
orally active anti-influenza agent. Therefore, there is an urgent
need for additional orally effective novel antiviral agents and
potentially effective against drug resistant influenza viruses, as
well as agents with different mode of action.
Natural as well as synthetic carbocyclic nucleosides are well
known for their interesting biological properties, including antitu-
mor as well as antiviral activity against a wide variety of RNA and
DNA viruses.4 The carbocyclic analogue of cytosine (carbodine 1;5
Fig. 1) was previously synthesized as a racemic mixture and has
been shown to possess significant antitumor (lymphoid leukemia
L1210 in mice) and antiviral activities against human influenza
type A virus, measles, respiratory syncytial virus, rotavirus, Vene-
zuelan Equine Encephalitis (VEE) virus, Herpes Simplex Viruses
(HSV-1 and HSV-2).6 Carbodine is metabolized to its triphosphate
The use of neuraminidase inhibitors (oseltamivir and zanama-
vir) for the therapy as well as the prophylaxis of influenza A and
B virus infections has been considered a new ‘millennium conun-
drum’ and the stockpiling of oseltamivir has been implemented
worldwide in preparation of an influenza pandemic due to the re-
cent emergence of H5N1 virus. However, the emergence of drug
resistant of H5N1 virus strains with reduced susceptibility to osel-
tamivir3 as well as sporadic cases of oseltamivir-resistant 2009
pandemic influenza A (H1N1) virus infection may potentially limit
* Corresponding author. Tel.: +1 (706) 542 5379; fax: +1 (706) 542 5381.
E-mail address: DCHU@mail.rx.uga.edu (C.K. Chu).
Figure 1. The chemical structure of carbodine and ribavirin.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.074

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J. R. Rao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2601–2604
Scheme 1. Reagents and conditions: (a) MeSO2Cl, TEA, DCM, rt, quantitative; (b) NaN3, DMF, 140 °C, 4 h, 89%; (c) 10% Pd/C, EtOH, rt, 30 psi, 2 h, quantitative; (d) b-
methoxyacryloyl isocyanate, DMF, ꢀ20 °C to rt, 10 h, 72%; (e) 30% NH4OH, EtOH/dioxane (1:1), 100 °C, 18 h, 85%; (f) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et3N,
30% NH4OH, rt, 17 h, 78%; (g) TFA/H2O (2:1), 60 °C, 3 h, 82%.
in mammalian cells and interferes with the viral RNA-dependent
RNA polymerase reaction, which may be the principal mode of ac-
tion for its biological activiy.6c,7 These interesting biological prop-
erties of carbodine prompted us to synthesize enantiomerically
pure (ꢀ)-carbodine (1, Fig. 1) for antiviral evaluations in compari-
son to its racemic and (+)-enantiomer 2.
The
from the cylopentanol key intermediate 4 (Scheme 1), which was
obtained from -ribose in good yield using previously reported
method by our group for
-(+)-carbodine.8 We initially attempted
D
-(ꢀ)-carbodine 1 was synthesized by the linear method
D
L
a convergent approach to couple N6-protected cytosine to com-
pound 4 under the Mitsunobu reaction condition which led to mix-
ture of N1 (minor)- and O2 (major)-regioisomers.9 Therefore, we
used a linear approach8 to synthesize functionalized cyclopentyl-
amine 7, then constructed the heterocyclic base in a step-wise
manner. Compound 4 was reacted with mesyl chloride followed
by SN2 reaction with sodium azide. Reduction of the azide 6 using
Pd/C hydrogenation gave cyclopentyl amine 710 in quantitative
yield, which was reacted with b-methoxyacryloyl isocyanate fol-
lowed by heating with NH4OH to obtain the target uracil com-
Ohno5d synthesized (ꢀ)-carbodine using enzymatic hydrolysis
as the key step for the synthesis of the cyclopentyl intermediate,
whereas Russ et al.5e explored by the double bond reduction of
cyclopentenyl cytosine, which gave a varying ratio of (ꢀ)-carbo-
dine and isocarbodine. Both of these methods resulted in poor
yield and selectivity. Herein, we report the practical synthesis
and antiviral activity of carbodine against various strains of influ-
enza viruses of the current interest.
Table 1
Anti-influenza in vitro activity of (ꢀ)-carbodine, (+)-carbodine, ( )-carbodine and ribavirin against various H5N1 strains
Virus type
Inoculumb Compound
Anti-influenza activity (H5N1)a
SIf
c
d
e
EC50
(lM)
EC90
(l
M)
IC50
(l
M)
Visual CPE Neutral Red Virus Yield Reduction
Visual CPE Neutral Red Virus Yield Reduction
Duck H5N1
60 46
53 40
(ꢀ)-Carbodine
1.8 0.5
>414
2.0 1.4
>414
5.0 1.9
10.5 10.1
>414
17.6 13.8
43.6 15.0
>414
>414
>414
>230
1
>173
>37
>207
1
>83
>40
>39
1
>24
>30
(+)-Carbodine
(
)-Carbodine 2.4 0.9
Ribavirin
35.4 11.0 32.5 8.6
>1310
Gull H5N1
(ꢀ)-Carbodine
0.8 0.3
>41
1.0 0.3
>41
1.4 0.5
27.4 6.8
1.6 1.0
>41
1.6 0.5
20.3 4.7
>414
>414
>414
>1310
>518
<10
>376
>55
>414
<10
>296
>48
>259
<10
>259
>65
(+)-Carbodine
(
)-Carbodine 1.1 0.6
Ribavirin
23.6 1.7
Hong Kong H5N1
Vietnam H5N1
84 91
(ꢀ)-Carbodine
0.7 0.1
>41
0.8 0.04
>41
1.8 1.0
9.4 0.6
2.1 0.4
>41
2.0 0.06
16.8 14.3
>414
>414
>414
>1310
>591
<10
>591
>305
>518
<10
>230
>139
>197
<10
>207
>78
(+)-Carbodine
(
)-Carbodine 0.7 0.2
Ribavirin
4.3 0.9
139 156
(ꢀ)-Carbodine
0.8 0.2
>41
1.5 0.9
>41
2.9 2.3
13.9 10.0
5.7 5.4
>41
12.1 9.3
11.6 9.8
>414
>414
>414
>1310
>518
<10
>414
>156
>276
<10
>143
>94
>73
<10
>34
>113
(+)-Carbodine
(
)-Carbodine 1.0 0.4
Ribavirin
8.4 5.9
a
b
c
d
e
f
Average of triplicate experimental data (duplicate for Hong Kong).
Approximate CCID50 inoculated per well, average SD from triplicate assays.
Effective concentration required to reduce influenza virus-induced cytopathic effect by 50%.
Concentration required to reduce infectious virus titer by 90%.
Concentration of drug for 50% cell inhibition without virus.
In vitro selectivity index (IC50/EC50), and for virus yield reduction (IC50/EC90).

J. R. Rao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2601–2604
2603
Table 2
Anti-influenza in vitro activity of (ꢀ)-carbodine, (+)-carbodine, ( )-carbodine and ribavirin against various H1N1, H3N2 and influenza type B strains
Virus type
Inoculumb
Compounds
Anti-influenza activity (H1N1, H3N2 & FluB)a
SIf
c
d
e
EC50
(
lM)
EC90
(lM)
IC50
(lM)
Visual CPE
Neutral Red
Virus Yield
Reduction
Visual CPE
Neutral Red
Virus Yield
Reduction
A/California/07/2009/H1N1
(swine flu strain)
100
21
(ꢀ)-Carbodine
(+)-Carbodine
( )-Carbodine
Ribavirin
1.3 0.20
>414
1.5 1.0
11.2 6.3
1.6 0.3
>414
1.9 1.0
19.4 1.8
3.8 0.3
>414
4.4 2.3
13.0 4.2
>414
>414
>414
>318
1
>276
>117
>259
1
>218
>68
>109
1
>94
>101
>1310
A/Solomon Island/03/2006/H1N1
A/Wisconsin/67/ 2005/H3N2
B/Malaysia/ 2506/2004
(ꢀ)-Carbodine
(+)-Carbodine
( )-Carbodine
Ribavirin
0.7 0.4
>41
1.9 0.5
23.3 0.7
0.6 0.3
>41
2.0 0.4
25.1 3.6
1.3 0.9
>41
2.3 0.4
27.0 6.4
>414
>414
>414
>1310
>591
<10
>218
>56
>690
<10
>207
>52
>318
<10
>180
>49
10
(ꢀ)-Carbodine
(+)-Carbodine
( )-Carbodine
Ribavirin
1.6 0.7
>41
2.8 0.7
16.4 7.2
2.0 0.9
>41
4.6 3.9
20.2 0.6
2.5 0.4
>41
3.5 1.1
21.7 11.9
>414
>414
>414
>1310
>259
<10
>148
>80
>207
<10
>90
>65
>166
<10
>118
>60
27
(ꢀ)-Carbodine
(+)-Carbodine
( )-Carbodine
Ribavirin
0.6 0.2
>41
0.8 0.0
10.2 5.6
0.8 0.1
>41
1.3 0.3
19.5 5.6
1.5 1.0
>41
3.8 0.9
22.2 1.9
>414
>414
>414
>1310
>690
<10
>518
>128
>518
<10
>318
>67
>276
<10
>109
>59
a
b
c
d
e
f
Average of triplicate experimental data.
Approximate CCID50 inoculated per well; same inoculum used for all replicate tests.
Effective concentration required to reduce influenza virus-induced cytopathic effect by 50%.
Concentration required to reduce infectious virus titer by 90%.
Concentration of drug for 50% cell inhibition without virus.
In vitro selectivity index (IC50/EC50) and for virus yield reduction (IC50/EC90).
pound 9 in good yield. Finally, the compound 9 was converted to a
cytosine using a standard reaction condition followed by removal
of protecting groups with TFA/H2O afford (ꢀ)-carbodine 111 in
good yield (overall 35% yield from intermediate 4).
strains, while (+)-carbodine was devoid of antiviral activity. In
virus yield reduction assays, (ꢀ)-carbodine was about twofold
more active than ( )-carbodine with the Duck and Vietnam strains,
and equally potent as ( )-carbodine with Gull and Hong Kong
strains.
The anti-influenza activity of (ꢀ)-carbodine was evaluated
in vitro in comparison to (+)-carbodine, ( )-carbodine and ribavi-
rin, and it showed significant anti-influenza activity (Table 1).
Virus strains tested were Influenza A/Duck/MN/1525/81/H5N1
and A/Gull/PA/4175/83/H5N1 obtained from R. Webster of St. Jude
Children’s Research Hospital (Memphis, TN) and A/Vietnam/1203/
2004H/H5N1 and A/Hong Kong/213/2003H/H5N1, hybrids of Ann
Arbor/6/60 containing the Asian H5 and N1 segments and provided
by MedImmune. A/California/07/2009/H1N1 (swine flu strain), A/
Solomon Islands/03/2006/H1N1, A/Wisconsin/67/2005/H3N2, and
B/Malaysia/2506/2004 were provided by the Centers for Disease
Control and Prevention (CDC, Atlanta, Ga.). Triplicate wells of Ma-
din-Darby Canine Kidney (MDCK) cells (ATCC CCL-34) were treated
with serial dilutions of the test compounds in 96-well plates then
inoculated with 6300 CCID50 of virus, sufficient to induce ꢁ100%
cytopathic effect (CPE) in control wells within three days. The anti-
viral activity of test compounds was measured by inhibition of
virus-induced CPE,12 neutral red (NR) dye uptake,13 and virus yield
reduction assays,14 essentially as described previously. For com-
parison, ( )-carbodine (SRI 5670 lot 11-30-2000, provided by
Southern Research Institute through the NIAID Antiviral Sub-
stances Program), and ribavirin (obtained from ICN Pharmaceuti-
cals in Costa Mesa, CA), were tested in parallel for each of the
triplicate assays (duplicate for Hong Kong strain). Carbodine sam-
ples were diluted in minimal essential medium, purified water, or
DMSO and held at ꢀ20 °C until tested, and ribavirin was diluted
from powder for each replicate assay.
Table 2 shows the in vitro anti-influenza activity of (ꢀ)-carbo-
dine, (+)-carbodine, ( )-carbodine and ribavirin against novel
2009 H1N1, H1N1, H3N2 and influenza type B strains. In A/Califor-
nia/07/2009/H1N1 strain (swine flu strain), (ꢀ)-carbodine showed
an EC50 value of 1.3
lM in the visual CPE assay, an EC50 of 1.6
lM in
neutral red assay and an EC90 of 3.8
lM in virus yield reduction as-
say with selectivity indexes of >318, >259 and >109, respectively.
The (ꢀ)-carbodine was consistently more active than the other
compounds against all four virus strains in Table 2. In A/Solomon
Island/03/2006/H1N1 strain, (ꢀ)-carbodine was 2 to 3 times more
potent than ( )-carbodine and >20-fold more potent than ribavirin
with high selectivity indices in visual CPE as well as in the neutral
red assay. The (ꢀ)-carbodine was slightly more active than ( )-car-
bodine and ribavirin in all assays against the A/Wisconsin/67/
2005/H3N2 strain. In influenza type B (Malaysia/2506/2004 strain),
(ꢀ)-carbodine was relatively more active than ( )-carbodine, 15–
24-fold more potent than ribavirin in visual CPE, neutral red and
virus yield reduction assays.
In summary, (ꢀ)-carbodine was shown to be the active form of
( )-carbodine as an anti-influenza agent. The ( )-carbodine race-
mic mixture showed less activity than (ꢀ)-carbodine, as expected,
but the differences were not significant by Mann–Whitney analysis
(P >0.05), and further assays with fresh ( )-carbodine may be mer-
ited for this comparison. The (ꢀ)-carbodine was identified as a po-
tent anti-influenza agent against in vitro A/Duck/MN/1525/81/
H5N1, A/Gull/PA/4175/83/H5N1, A/Hong Kong/213/2003H/H5N1
and A/Vietnam/1203/2004H/H5N1 strains, which was further con-
firmed by virus yield reduction assay. Moreover, (ꢀ)-carbodine
showed potent activity against novel H1N1 (A/California/07/
2009/H1N1) (swine), A/Solomon Island/03/2006/H1N1, A/Wiscon-
sin/67/2005/H3N2 and B/Malaysia/2506/2004.
The anti-influenza in vitro activity of (ꢀ)-carbodine, (+)-carbo-
dine, ( )-carbodine and ribavirin against various H5N1 strains is
shown in Table 1. In visual CPE and neutral red assays, the anti-
influenza in vitro activity of (ꢀ)-carbodine (0.7–2.0
lM) was more
potent than that of ( )-carbodine (0.7–5.0 M) against the H5N1
l

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J. R. Rao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2601–2604
R. W. Antimicrob. Agents Chemother. 1981, 20, 769; (d) Hosoya, M.; Shigeta, S.;
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AI-30048.
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