Synthesis of enantiomerically pure d- and l-bicyclo[3.1.0]hexenyl carbanucleosides and their antiviral evaluation

Article abstract of DOI:10.1016/j.bmc.2011.05.026

Based upon the fact that l-nucleosides have been generally known to be less cytotoxic than d-counterparts, l-bicyclo[3.1.0]hexenyl carbanucleoside derivatives with a fixed north conformation were designed and synthesized by employing a novel synthetic str

Full text of DOI:10.1016/j.bmc.2011.05.026

Bioorganic & Medicinal Chemistry 19 (2011) 3945–3955
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of enantiomerically pure D- and L-bicyclo[3.1.0]hexenyl
carbanucleosides and their antiviral evaluation
Ah-Young Park a, Won Hee Kim a, Jin-Ah Kang a, Hye Jin Lee a, Chong-Kyo Lee b, Hyung Ryong Moon a,
⇑
a Laboratory of Medicinal Chemistry, College of Pharmacy and Research Institute for Drug Development, Pusan National University, Busan 609-735, Republic of Korea
b Pharmacology Research Center, Korea Research Institute of Chemical Technology, Daejon 305-600, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Based upon the fact that
L-nucleosides have been generally known to be less cytotoxic than
D-counter-
Received 19 April 2011
Revised 16 May 2011
Accepted 17 May 2011
Available online 23 May 2011
parts,
and synthesized by employing a novel synthetic strategy starting from (R)-epichlorohydrin in order to
search for new anti-HIV agents with high potency and less cytotoxicity. A tandem alkylation, -lactoniza-
tion, a chemoselective reduction of ester in the presence of -lactone functional group, a RCM reaction,
and a Mitsunobu coupling reaction were used as key reactions. -Counterpart nucleosides were also pre-
pared according to the same synthetic method. Among the synthesized carbanucleosides, -thymine
nucleoside, -2 and -thymine nucleoside, -2 exhibited excellent anti-HIV-1 and -2 activities, in MT-4
cells, which were higher than those of ddI, an anti-AIDS drug. Whereas -2 exhibited high cytotoxicity
in MT-4 cell lines, -2 did not show any discernible cytotoxicity in all cell lines tested, reflecting that
-2 may be a good candidate for an anti-AIDS drug. -2 also showed weak anti-HSV-2 activity without
L-bicyclo[3.1.0]hexenyl carbanucleoside derivatives with a fixed north conformation were designed
c
c
D
Keywords:
HIV
D
D
L
L
L
-Carbanucleoside
D
DNA virus
D4Ns
Epichlorohydrin
L
L
L
cytotoxicity. However, none of the synthesized nucleosides exhibited antiviral activities against RNA
viruses including coxsakie, influenza, corona and polio viruses, maybe due to their 20,30-dideoxy struc-
ture. Potent antiviral effects of
be an excellent candidate for anti-DNA virus agents. This research strongly supports
D
-2 and
L
-2 indicate that nucleosides belonging to a class of D4Ns can
-nucleosides of a
L
class of D4Ns to be a very promising candidate for antiviral agents due to its low cytotoxicity and a good
antiviral activity.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
ring to be nearly planar and highly rigid. Other potent NRTIs, such
as abacavir (ABC),11 and 20,30-didehydro-20,30-dideoxy-20-fluor-
In the 25 years since the initial discovery that acquired immuno-
deficiency syndrome (AIDS) is caused by the human immunodefi-
ciency virus (HIV), the disease has grown into a global infectious
disease.1 HIV is a pathogenic retrovirus and requires reverse trans-
criptase (RT) to copy its single-stranded RNA genome into a double-
stranded DNA copy for integration in the host cell.2 Therefore, RT of
AIDS virus has been the most important candidate for the treatment
of AIDS. In the search for an effective chemotherapeutic agent of
HIV infections for targeting RT, initial attempts were focused on
the development of 20,30-dideoxynucleosides (ddNs) and 20,30-dide-
hydro-20,30-dideoxynucleosides (d4Ns).3,4 Among them, the nucleo-
side RT inhibitors (NRTIs)5 such as 30-azido-30-deoxythymidine
(AZT),6 20,30-dideoxyinosine (ddI),7 20,30-dideoxycytidine (ddC),8
ocytidine (d4FC),12 have also a double bond on their sugar ring.
In this respect, the 20,30-double bond is considered to be crucial
for plays in enhancing anti-HIV activity (Fig. 2).
However, oxanucleosides belonging to ddNs and d4Ns, have
intrinsic weakness that their glycosidic bond is unstable to acidic
conditions involving a gastric environment and to enzymes such
as pyrimidine and purine phosphorylases.13 In spite of the defect,
ddNs and d4Ns derivatives have been the most promising candi-
dates for AIDS treatment. To overcome easy cleavage of the glyco-
sidic bond of oxanucleosides, carbocyclic nucleosides,14 which
have a methylene group instead of the ring oxygen atom of tetra-
hydrofuran in oxanucleosides, have been synthesized with an
anticipation of searching for better antiviral profile and lower cyto-
toxicity with improved stability.
Conformationally rigid methanocarba (MC) nucleosides built on
a bicyclo[3.1.0]hexane template have a south (S, 3E)15 or north
(N, 2E)16 conformation in the pseudorotational cycle,17 which is a
conformation normal nucleosides take for the most time (Fig. 3).
When nucleosides bind to the active site of enzymes, each con-
former exhibits different affinity. Therefore, rigid MC nucleosides
have been used to investigate the conformational preferences of
L
-20,30-dideoxy-30-thiacytidine (3TC)9 and 20,30-didehydro-20,30-
dideoxythymidine (d4T)10 have been widely used to treat AIDS
patients (Fig. 1).
Particularly, an important structural characteristic of d4T is the
presence of a 20,30-double bond, which renders the pseudosugar
⇑
Corresponding author. Tel.: +82 51 510 2815; fax: +82 51 513 6754.
E-mail address: mhr108@pusan.ac.kr (H.R. Moon).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.026

3946
A.-Y. Park et al. / Bioorg. Med. Chem. 19 (2011) 3945–3955
O
N
NH2
N
NH2
O
NH
H
N
N
N
N
HO
O
HO
N
O
HO
O
N
O
OH
O
O
N
O
S
3TC
N3
ddC
ddI
AZT
Figure 1. Anti-HIV NRTIs.
O
NH2
NH2
HN
F
F
N
NH
N
N
N
NH2
HO
N
O
HO
O
N
O
OH
HO
N
O
N
N
O
O
d4T
Abacavir
L-d4FC
D-d4FC
O
NH2
NH2
F
F
NH
N
N
HO
N
O
O
N
O
OH
O
N
O
OH
S
D
-N-MCd4T
L
-5-FddC
FTC
Figure 2. Several potent anti-HIV NRTIs with 20,30-double bond or
L-type.
highly potent anti-HIV activity (Figs. 1 and 2).
been generally known to be less cytotoxic than
-N-MCd4T,22 bearing a locked north conformation and a 20,30-
L
-Nucleosides have
HO
Base
D-counterparts.
D
double bond in a single structure has been synthesized and its
anti-HIV activity was compared with that of d4T (Fig. 2). Although
north conformation
D
-N-MCd4T was less potent than d4T, it showed significant antivi-
3T2
ral activity against HIV-1 and HIV-2 with less cytotoxicity in varied
cell types, and was stable under conditions that would cleave d4T.
On the basis of these findings, as a part of our ongoing efforts to
search for antiviral agents with better potency, less cytotoxicity and
improved stability, it was of interest to synthesize conformationally
north-locked and enantiopure
nucleoside derivatives involving
antiviral activity of -carbanucleosides with that of
sides. - and -Bicyclo[3.1.0]hexenyl carbanucleoside derivatives
with a natural nucleobase were prepared using (S)- and (R)-epichlo-
rohydrin, respectively, as a starting material and their antiviral
activities against HIV-1 and -2, coxsackie B1 and B3 and HSV-1
and -2 were evaluated.
A new strategy for the synthesis of
banucleoside analogs ( -1– -5) and its
fully accomplished by using cyclopropanation via
alkylation, -lactonization, a chemoselective reduction of ester in
3E
2E
0o
1T2
3T4
north
1E
4E
0T4
1T0
0E
D
- and
L
-bicyclo[3.1.0]hexenyl carba-
-2),23 and compare
-carbanucleo-
L-N-MCd4T (
L
90o
270o
east
0E
0T1
west
D
L
D
L
4T0
4E
1E
south
180o
4T3
2T1
L
-bicyclo[3.1.0]hexenyl car-
-counterpart were success-
tandem
2E
3E
2T3
L
L
D
a
HO
Base
c
the presence of lactone functional group, a Grignard reaction, a
ring-closing metathesis (RCM) reaction and a Mitsunobu reaction
as the key steps, starting from (R)- and (S)-epichlorohydrin,
respectively.
south conformation
Figure 3. Conformationally locked bicyclo[3.1.0]hexenyl nucleosides in the
pseudorotational cycle.
2. Results and discussion
2.1. Chemistry
various enzymes associated with nucleoside metabolism and
nucleotide polymerization.18,19
On the other hand, a number of
L
-nucleosides such as 3TC,
Synthesis of a glycosyl donor with a locked conformation, bicy-
clo[3.1.0]hexenol (2S)-14 is outlined in Scheme 1. Compound 6
FTC,20 -5-FddC21 have also been reported to show
L
-d4FC and
L

A.-Y. Park et al. / Bioorg. Med. Chem. 19 (2011) 3945–3955
3947
was synthesized in two steps according to the procedure reported
by Tsuji and co-workers.24,25 Reaction of (R)-epichlorohydrin with
diethyl malonate and sodium metal gave cyclopropane-fused
sides. (2R)-14 can be also potential starting material for a palla-
dium-mediated coupling reaction with nucleobases to produce
the desired b-anomer.
c-lactone 6 via tandem alkylation followed by lactonization. The
Synthesis of uracil and thymine
L-nucleoside derivatives, L-1
lactone moiety of 6 might be more susceptible to hydrolysis than
the ester, maybe due to the structural constraint caused by a fused
cyclopropane ring. Treatment of 6 with 1 equiv of sodium hydrox-
ide in EtOH afforded monocarboxylate sodium salt 6a, the ester of
which was selectively reduced by sodium borohydride under
and -2 is shown in Scheme 2. Condensation of the glycosyl donor,
L
bicyclo[3.1.0]hexenol (2S)-14 with N3-benzoyluracil and N3-ben-
zoylthymine under Mitsunobu conditions28 smoothly proceeded
to give protected N3-benzoyluracil nucleoside 15 and protected
N3-benzoylthymine nucleoside 16, respectively, in 21% and 40%
yield. Finally, deprotection of N3-benzoyl group of compounds 15
and 16 with ammonium hydroxide aqueous solution in methanol
followed by desilylation with TBAF (n-tetrabutylammonium fluo-
reflux and then recyclized back to
c-lactone 7 under acidic
conditions. Protection of hydroxyl group of 7 with tert-butyldi-
phenylsilyl chloride produced silyl ether 8, which was reduced
with Dibal-H at ꢀ78 °C to afford the corresponding lactol 9 in
97% yield. Wittig reaction of lactol 9 with methyltriphenylphos-
phonium bromide in the presence of potassium tert-butoxide affor-
ded hydroxy olefin 10 in 88% yield after quenching with saturated
aqueous NH4Cl solution. However, when the reaction mixture was
partitioned between EtOAc and water without quenching with sat-
urated NH4Cl aqueous solution after the Wittig reaction, a new by-
product appeared at a higher Rf value than that of the desired
compound 10 in 10–20% yield. It turned out to be an acetylated
compound 10a by 1H, 13C, COSY (Correlation spectroscopy) and
HMBC (Heteronuclear multiple bond correlation) NMR experi-
ments. The acetyl group might be derived from ethyl acetate used
for extraction due to basic reaction conditions. Swern oxidation of
10 with oxalyl chloride and dimethyl sulfoxide at ꢀ78 °C smoothly
proceeded to produce aldehyde 11, which was subjected to a Grig-
nard reaction with vinylmagnesium bromide to afford allylic alco-
hols 12 (48%) and 13 (39%), easily separated by normal silica gel
column chromatography. The stereochemistry of 12 and 13 was
not determined until the corresponding bicyclo[3.1.0]hexenol scaf-
fold was formed via a RCM (ring-closure metathesis) reaction in
the next step. RCM reaction26 of dienes 12 and 13 with a second
generation Grubbs catalyst produced the bicyclo[3.1.0]hex-3-en-
2-ol analogs, (2R)-14 and (2S)-14, respectively. The stereochemis-
try of them was confirmed by comparing their 1H NMR spectra
with that of the authentic enantio-counterpart, an enantiomer of
(2S)-14, prepared from a chiral bicyclo[3.1.0]hexane template,27
indicating that the RCM product (2S)-14 derived from compound
13 has the desired (2S)-stereochemistry for a Mitsunobu reaction
with nucleobases to yield various conformationally locked nucleo-
ride) gave the desired uracil and thymine
L-nucleosides,
L-1 and
L-2, respectively, in 85% and 91% yield. On the other hand, the cor-
responding cytosine analog
L-3 was synthesized starting from the
uracil nucleoside analog
L-1, via acetylation of the free hydroxyl
group followed by successive three conventional reactions ((i)
1,2,4-triazole, phosphorus oxychloride, pyridine; (ii) ammonium
hydroxide, 1,4-dioxane; (iii) methanolic ammonia),29 in overall
42% yield (Scheme 3).
Synthesis of adenine L-nucleoside L-4 is depicted in Scheme 4.
Under Mitsunobu conditions, coupling of (2S)-14 with 6-chloropu-
rine generated protected 6-chloropurine nucleoside 18 in 21%
yield. Amination with methanolic ammonia at 80 °C to adenine
nucleoside followed by desilylation of TBDPS group with TBAF gave
the final adenine
L-nucleoside
L-4 in 44% yield from 18. For the syn-
thesis of guanine nucleoside
L-5, condensation of (2S)-14 with
2-amino-6-chloropurine under Mitsunobu conditions afforded
protected 2-amino-6-chloropurine nucleoside 19 (Scheme 4),
which was subjected to desilylation with 1 M TBAF and conversion
of 2-amino-6-chloropurine nucleobase into guanine with 2-
mercaptoethanol and 1 N NaOMe under reflux to produce guanine
L-nucleoside L-5. The yields of each Mitsunobu condensation are
generally low, probably because the fixed conformation induced
by the fused cyclopropyl group and the double bond causes the
backside attack of nucleobases to be disfavorable. In case of the
locked conformation, increased steric hindrance between a methi-
nyl hydrogen or a 40-methylene group and nucleobase is expected.
D-Bicyclo[3.1.0]hexenyl carbanucleoside derivatives D-1–D-4
were also synthesized starting from (S)-epichlorohydrin
(Scheme 5) instead of (R)-epichlorohydrin used for the synthesis of
OH
O
O
H
CO2Na
O
O
b
a
Cl
62%
67%
O
OEt
OEt
6a
OH
O
O
6
7
(R)-epichlorohydrin
c
84%
O
RO
O
O
OH
d
O
e
f
OTBDPS
97%
90%
88%
OTBDPS
10 (R = H)
OTBDPS
OTBDPS
9
8
11
10a (R = Ac)
g
HO
X
H
OTBDPS
Y
X
h
+
HO
H
Y
OTBDPS
OTBDPS
13 (39%)
OTBDPS
14
(2S)- (X = OH, Y = H)
14
(2R)- (X = H, Y = OH)
12 (48%)
Scheme 1. Reagents and conditions: (a) (EtO2C)2CH2, Na, EtOH, 80 °C, 20 h; (b) (i) 1 equiv NaOH, EtOH, rt, 16 h; (ii) NaBH4, reflux, 3 h, then 2 N HCl, rt, 18 h; (c) TBDPSCl,
imidazole, CH2Cl2, rt, overnight; (d) Dibal-H, CH2Cl2, ꢀ78 °C, 30 min; (e) CH3PPh3Br, t-BuOK, THF, rt, 3 h; (f) oxalyl chloride, DMSO, CH2Cl2, ꢀ78 °C, 1 h, then Et3N, rt, 1 h; (g)
vinylmagnesium bromide, THF, ꢀ78 °C, 1 h; (h) 2nd generation Grubbs catalyst, CH2Cl2, rt, 1.5 h, 85% for (2S)-14 from 13, 84% for (2R)-14 from 12.

3948
A.-Y. Park et al. / Bioorg. Med. Chem. 19 (2011) 3945–3955
O
N
O
X
X
HN
BzN
OTBDPS
a
b, c
OH
OTBDPS
O
O
N
HO
(2S)-14
15
1
(X = H)
16 (X = CH3)
L- (X = H)
L-2 (X = CH3)
Scheme 2. Reagents and conditions: (a) PPh3, DEAD, N-benzoyluracil or N-benzoylthymine, THF, 0 °C, 1 h, 21% for 15, 28% for 16; (b) 28% NH4OH/MeOH, rt, 6 h and then TBAF,
THF, rt, 1 h, 82% for 1, 76% for 2.
O
N
O
N
NH2
HN
HN
N
a
b
OH
O
OAc
OH
O
O
N
17
3
L-
1
L-
Scheme 3. Reagents and conditions: (a) (i) Ac2O, pyridine, rt, overnight, 100% from
L-1; (b) (i) 1,2,4-triazole, POCl3, pyridine, rt, overnight; (ii) 28% NH4OH, 1,4-dioxane,
MeOH, rt, overnight; (iii) NH3/MeOH, rt, overnight, 42%.
X
X
N
N
N
OTBDPS
N
a
b
OTBDPS
OH
N
N
N
N
HO
Y
Y
(2S)-14
18 (X = Cl, Y = H)
L-4 (X = NH2, Y = H)
L-5 (X = OH, Y = NH2)
19
(X = Cl, Y = NH2)
Scheme 4. Reagents and conditions: (a) PPh3, DEAD, 6-chloropurine or 2-amino-6-chloropurine, THF, 0 °C, 1 h, 21% for 17, 17% for 18; (b) NH3/MeOH, 80 °C, 4 h and then
TBAF, THF, rt, 1 h, 44% for
L-4; TBAF, THF, rt, 1.5 h and then 2-mercaptoethanol, 1 N NaOMe, MeOH, 80 °C, 7 h, 97% for L-5.
Y
NH2
X
N
N
N
N
N
H
Cl
HO
HO
N
O
O
1
D- : X = H, Y = OH
2
D- : X = CH3, Y = OH
(S)-epichlorohydrin
3
4
D-
D- : X = H, Y = NH2
Scheme 5.
L
-nucleosides by employing the same methodology used for the
that both thymine nucleosides
D
-2 and L-2 exhibit more potent
synthesis of L-bicyclo[3.1.0]hexenyl carbanucleosides.
anti-HIV-1 and -2 activities than ddI used as a positive control.
Although anti-HIV activity of -2 exceeds that of -2, -2 (CC50
>100 -2 (CC50 = 8.558
g mLꢀ1) is less cytotoxic than g mLꢀ1),
which corresponds to the fact that -nucleosides have been gener-
ally known to be less toxic than -counterpart. Selective indexes
(CC50/EC50) toward HIV-1 and -2 showed 263 and 76, respectively,
for -2 and more than 85.09 and more than 44.05, respectively, for
-2. -Adenine nucleoside, -4 exhibited weak anti-HIV-1 activity
(IC50 = 40.47
g mLꢀ1) but does not show anti-HIV-2 activity.
-Cytosine nucleoside, -3 showed cytotoxicity-derived anti-HIV-
The structures of the synthesized compounds were determined
by 1H, 13C, HMBC and COSY NMR, mass and UV spectrometric
analyses.
D
L
L
l
D
l
L
Antiviral activities with the final conformation-locked D- and L-
D
carbanucleosides were evaluated against HIV-1 (IIIB) and -2 (ROD),
coxsackie B1 and B3 viruses, and HSV-1 and -2. First, when tested
in MT-4 (HTLV-1 and -2-infected human T lymphocyte) cells,
D
L
D
D
among the final nucleosides thymine nucleoside analogs
D
-2 and
l
L-2 showed the most potent anti-HIV-1 and -2 activity and their
D
D
IC50 values against HIV-1 and -2 were 0.033 and 0.112 lg/mL for
1 and -2 activities. The other nucleoside derivatives showed nei-
ther anti-HIV-1 and -2 activities nor cytotoxicity. According to
the literature,16a bicyclo[3.1.0]hexyl nucleosides with no double
bond showed no or weak anti-HIV activity, indicating that the
internal double bond is essential for high anti-HIV activity. Other
antiviral effects for the final nucleoside derivatives were evaluated
against coxsackie B1 and B3 viruses, and poliovirus 3. However, all
nucleosides neither exhibited antiviral activities nor cytotoxicity
D
-2, and 1.175 and 2.270
The bigger difference in the two strains (IIIB and ROD) in the activ-
ity of -2 than in that of -2 reflects that -nucleosides might fit
almost equally at both the active sites of the two strains, whereas
-nucleosides might fit better at the active site for IIIB strain than
that for ROD strain. Comparison of IC50 values of -2 and -2
against HIV-1 and -2 with those (2.31 and 2.76 g/mL) of ddI,
which is clinically used for the treatment of AIDS patients indicates
lg/mL for L-2, respectively (Table 1).
D
L
L
D
D
L
l
up to 100 l
g mLꢀ1. The final nucleosides belong to 20,30-didehy-

A.-Y. Park et al. / Bioorg. Med. Chem. 19 (2011) 3945–3955
3949
Table 1
Anti-HIV-1 and -2 activities of D- and L-bicyclo[3.1.0]hexenyl carbanucleoside derivatives
a
b
Compound
Toxicity (CC50
,
l
g/mL)
HIV (IC50
,
lg/mL)
Selective indexe
IIIBc
RODd
IIIB
ROD
ND
>100.00
>100.00
>100.00
>100.00
>100.00
>100.00
8.558
>100.00
1.175
>100.00
2.270
ND
L
L
L
L
L
-1
-2
-3
-4
-5
>85.09
ND
>44.05
ND
ND
ND
ND
76
>100.00
>100.00
>100.00
>100.00
0.033
>100.00
>100.00
>100.00
>100.00
0.112
ND
ND
ND
D
D
D
D
-1
-2
-3
-4
263
<1
61.80
>61.80
40.47
>61.80
>100.00
<1
>100.00
>2.47
ND
ddC
ddI
10.66
>100.00
0.043
2.31
0.040
5.76
248.73
>43.29
266.70
>17.38
a
b
c
50% cytotoxic concentration.
50% inhibitory concentration.
HIV-1 strain.
d
e
HIV-2 strain.
CC50/EC50
.
dro-20,30-dideoxynucleosides whereas coxsakie viruses and polio-
virus belong to RNA viruses. This point might result in lack of
antiviral activities against coxsakie viruses and poliovirus. Antivi-
ral evaluation against HSV-1 and -2 was conducted and only
tested, but all compounds did not show any significant antiviral
activities, maybe due to a 20,30-dideoxy structure of the com-
pounds.
influenza and corona viruses, but showed neither antiviral activity
nor cytotoxicity. It is interesting to note that all -nucleosides did
not show cytotoxicity up to 100
g mLꢀ1 in all cells tested. Proba-
bly, the low cytotoxicity of -nucleosides may be closely associated
with a -type, an unnatural type. Potent antiviral activities of
- and -thymine carbanucleosides, -2 and -2 indicate that a
L-2 was also evaluated against other RNA viruses such as
L
-thymine nucleoside,
L
-2 showed weak anti-HSV-2 activity
g mLꢀ1) without discernible cytotoxicity. Antiviral
-thymine nucleoside, -2 was also performed against
L
(IC50 = 59.23
inhibition of
l
L
l
L
L
FluA (influenza virus type A) (H1N1) strain Taiwan, FluA (H3N2)
strain Johannesburg, FluB (influenza virus type B) strain Panama,
FCV (feline coronavirus) strain WSU, and FIP (feline infectious peri-
tonitis virus) strain WSU, but it neither showed antiviral activity
L
D
L
D
L
nucleoside belonging to a class of D4Ns can be an excellent candi-
date for anti-AIDS drug and anti-DNA virus agents. This research
strongly supports L-nucleosides of a class of D4Ns to be a very
nor cytotoxicity up to 100
both influenza and corona viruses are viruses with a RNA genome.
All -nucleosides did not show any cytotoxicity up to 100
g mLꢀ1
in all cell lines tested.
According to the obtained antiviral and cytotoxic results,
-bicyclo[3.1.0]hexenyl carbanucleosides synthesized exerts their
l
g mLꢀ1 (not shown), maybe because
promising candidate for antiviral agents due to its low cytotoxicity.
L
l
4. Experimental section
4.1. General methods
D
- and
L
antiviral activity for DNA virus such as HSV-2 and AIDS virus
requiring a RNA-dependent DNA polymerization process during
All chemical reagents were commercially available. Melting
points are uncorrected. Ultraviolet (UV) spectra were recorded in
methylene chloride or MeOH on a JASCO V-530 UV–vis spectro-
photometer. Optical rotations were measured in chloroform,
MeOH or DMF on a JASCO DIP-370 digital polarimeter. Nuclear
Magnetic Resonance (NMR) data were recorded on a Bruker AC
200, Varian Unity INOVA 400 spectrometer and Varian Unity AS
500 spectrometer, using CDCl3 or CD3OD and chemical shifts were
reported in parts per million (ppm) with reference to the respec-
tive residual solvent or deuterated peaks (dH 3.30 and dC 49.0 for
CD3OD, dH 7.26 and dC 77.0 for CDCl3). Coupling constants are re-
ported in hertz. The abbreviations used are as follows: s (singlet),
d (doublet), q (quartet), qd (quartet of doublets), m (multiplet),
dd (doublet of doublets), br s (broad singlet), or br d (broad dou-
blet). All the reactions described below were performed under
nitrogen or argon atmosphere and monitored by thin-layer chro-
matography (TLC). TLC was performed on Merck precoated 60
F254 plates. Column chromatography was performed using Silica
Gel 60 (230–400 mesh, Merck). All anhydrous solvents were dis-
tilled over CaH2 or Na/benzophenone prior to use.
its life-cycle and
L
-nucleoside is less cytotoxic than
-4 vs -4).
D-nucleoside
(e.g., -2 vs -2 and
L
D
L
D
3. Conclusions
In order to search for new anti-HIV agents with high potency
and less cytotoxicity, - and -bicyclo[3.1.0]hexenyl carbanucleo-
side derivatives were designed and synthesized by a novel syn-
thetic strategy starting from (S)- and (R)-epichlorohydrin,
respectively. A tandem alkylation,
tive reduction of ester in the presence of
group, a Grignard reaction, a RCM reaction, and a Mitsunobu cou-
pling reaction were used as key reactions to achieve the synthesis
of north conformation-locked
D
L
c-lactonization, a chemoselec-
c
-lactone functional
D- and L-carbanucleoside analogs.
The synthesized bicyclic carbanucleosides were characterized by
1H, 13C, HMBC, HSQC and COSY NMR, mass and UV spectra, melting
point and optical rotation.
D
-Thymine nucleoside,
found to show more potent anti-HIV-1 and -2 activities than ddI
in MT-4 cells. Whereas -2 exhibited cytotoxicity (CC50 = 8.558
g mLꢀ1),
-2 did not show any discernible cytotoxicity, suggesting
that -2 may be a better candidate for the development of novel
anti-AIDS drug than -2. -2 was also found to exhibit weak anti-
D-2 and L-thymine nucleoside, L-2 were
D
l
L
4.1.1. (ꢀ)-(1S,5R)-Ethyl 2-oxo-3-oxabicyclo[3.1.0]hexane-1-
carboxylate (6)
Sodium (2.42 g, 105 mmol) was carefully dissolved in EtOH
(195 mL) at 0 °C, and diethyl malonate (16.7 mL, 110 mmol) was
added at 0 °C over 5 min to the solution. (R)-(ꢀ)-Epichlorohydrin
L
D
L
HSV-2 activity without cytotoxicity. In addition, antiviral activities
against RNA viruses such as coxsakie viruses, and poliovirus were

3950
A.-Y. Park et al. / Bioorg. Med. Chem. 19 (2011) 3945–3955
(7.8 mL, 100 mmol) in EtOH (5 mL) was added dropwise to the
solution at room temperature over 1 h, and the reaction mixture
was refluxed for 20 h. The mixture was filtered, and the filtrate
was evaporated under reduced pressure. The residue was dissolved
in methylene chloride and washed with H2O. The organic layer was
dried over anhydrous MgSO4 and evaporated under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy using hexanes and ethyl acetate (3:1) as the eluent to give
cyclopropane-fused lactone 6 (11.40 g, 67%) as a colorless oil:
same temperature. MeOH (42.06 mL), hexanes (84.12 mL) and
ethyl acetate (84.12 mL) were added successively and the resulting
mixture was stirred overnight, allowing it to reach room tempera-
ture. The generated gel was filtered off through a pad of Celite, and
the filtrate collected was concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
using hexanes and ethyl acetate (4:1) as the eluent to give lactol
9 (13.67 g, 97%) as a colorless oil: 1H NMR (200 MHz, CDCl3) d
7.72–7.37 (m, 10H, 2 ꢂ Ph), 5.32 (s, 1H, 2-H), 4.18 (d, 1H,
J = 11.2 Hz, TBDPSOCHH), 4.12 (dd, 1H, J = 3.2, 8.4 Hz, 4-HH), 3.77
(d, 1H, J = 8.4 Hz, 4-HH), 3.67 (d, 1H, J = 11.2 Hz, TBDPSOCHH),
1.47 (m, 1H, 5-H), 1.09 (s, 9H, tert-butyl), 0.60–0.50 (m, 2H, 6-
H2); 13C NMR (50 MHz, CDCl3) d 135.95, 133.25, 133.18, 130.32,
130.27, 128.18, 100.09, 67.79, 64.59, 34.21, 27.18, 20.51, 19.50,
13.04; m/z (ESI) 391 (M+Na)+.
½
a 2D5
ꢁ
ꢀ121.44 (c 1.32, CHCl3), (lit.24
½
a 2D5
ꢁ
ꢀ146.58 (c 1.22, EtOH));
1H NMR (400 MHz, CDCl3) d 4.32 (dd, 1H, J = 4.8, 9.2 Hz, 4-HH),
4.22 (qd, 2H, J = 2.4, 6.8 Hz, CH2), 4.15 (d, 1H, J = 9.2 Hz, 4-HH),
2.70 (m, 1H, 5-H), 2.04 (dd, 1H, J = 4.8, 8.4 Hz, 6-HH), 1.34 (t, 1H,
J = 4.8 Hz, 6-HH), 1.27 (t, 3H, J = 6.8 Hz, CH3); 13C NMR (100 MHz,
CDCl3) d 170.76, 166.90, 67.21, 62.22, 29.56, 28.12, 20.93, 14.28;
m/z (FAB+) 170 (M+).
4.1.5. (1R,2S)-(ꢀ)-2-[(tert-Butyl-diphenyl-silanyloxymethyl)-2-
vinylcyclopropyl]methanol (10)
4.1.2. (ꢀ)-(1S,5R)-1-(Hydroxymethyl)-3-oxa-bicyclo[3.1.0]
hexan-2-one (7)
To a stirred suspension of methyltriphenylphosphonium bro-
mide (8.92 g, 24.96 mmol) in anhydrous THF (100 mL) was added
potassium tert-butoxide (2.80 g, 24.96 mmol) at 0 °C, and the mix-
ture was stirred at room temperature for 1 h to give a yellow sus-
pension. The lactol 9 (4.60 g, 12.48 mmol) in anhydrous THF
(30 mL) was added dropwise at 0 °C, and the reaction mixture
was allowed to reach room temperature. After being stirred for
an additional 3 h, it was treated with saturated aqueous NH4Cl
solution (30 mL). The aqueous layer was extracted with methylene
chloride and the organic layer was dried over anhydrous MgSO4,
filtered, and evaporated in vacuo. The residue was purified by silica
gel column chromatography using hexanes and ethyl acetate (3:1)
as the eluent to give hydroxyl olefin 10 (4.03 g, 88%) as a colorless
A solution of 6 (10.56 g, 62.04 mmol) in EtOH (204 mL) was
treated with sodium hydroxide (2.48 g, 62.04 mmol) in EtOH
(204 mL). After stirring for 16 h at room temperature, the reaction
mixture was treated with sodium borohydride (11.74 g,
310.21 mmol) and refluxed for 3 h. After cooling to room temper-
ature, 2 N HCl (186 mL) was added slowly at 0 °C. EtOH in the reac-
tion mixture was evaporated off under reduced pressure and to the
resulting solution was added 2 N HCI (408 mL). After being stirred
for 18 h at room temperature, the aqueous layer was extracted
with methylene chloride several times. The organic layer was dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography using
methylene chloride and MeOH (20:1) as the eluent to give lactone
oil: ½a 2D5
ꢁ
ꢀ37.24 (c 1.25, CHCl3); 1H NMR (500 MHz, CDCl3) d 7.68–
7 (4.97 g, 62%) as a colorless oil: ½a D25
ꢁ
ꢀ71.81 (c 2.07, CHCl3); 1H
7.39 (m, 10H, 2 ꢂ Ph), 5.95 (dd, 1H, J = 11.0, 17.5 Hz, –CH@CH2),
5.20 (d, 1H, J = 11.0 Hz, –CH@CHH), 5.17 (d, 1H, J = 17.5 Hz, –
CH@CHH), 3.75 (d, 1H, J = 10.0 Hz, TBDPSOCHH), 3.72 (dd, 1H,
J = 6.0, 11.0 Hz, CHHOH), 3.57 (d, 1H, J = 10.5 Hz, TBDPSOCHH),
3.44 (dd, 1H, J = 9.0, 12.0 Hz, CHHOH), 1.33 (m, 1H, 1-H), 1.07 (s,
9H, tert-butyl), 0.84 (dd, 1H, J = 5.0, 8.5 Hz, 3-HH), 0.68 (t, 1H,
J = 5.5 Hz, 3-HH); 13C NMR (50 MHz, CDCl3) d 136.85, 135.73,
133.74, 133.71, 129.72, 129.70, 127.71, 115.99, 68.62, 62.67,
29.44, 26.97, 25.53, 19.40, 13.70; m/z (FAB+) 349 (MꢀH2O)+.
NMR (400 MHz, CDCl3) d 4.30 (dd, 1H, J = 4.8, 9.2 Hz, 4-HH), 4.13
(d, 1H, J = 9.6 Hz, 4-HH), 4.06 (d, 1H, J = 12.0 Hz, CHHOH), 3.57 (d,
1H, J = 12.4 Hz, CHHOH), 3.41 (br s, 1H, OH), 2.28 (m, 1H, 5-H),
1.28 (dd, 1H, J = 4.8, 7.6 Hz, 6-HH), 0.94 (t, 1H, J = 5.2 Hz, 6-HH);
13C NMR (100 MHz, CDCl3) d 177.72, 69.13, 60.65, 30.78, 22.01,
16.45.
4.1.3. (1S,5R)-(ꢀ)-1-(tert-Butyl-diphenyl-silanyloxymethyl)-3-
oxa-bicyclo[3.1.0]hexan-2-one (8)
To a solution of 7 (4.70 g, 36.66 mmol) and imidazole (4.99 g,
73.31 mmol) in anhydrous methylene chloride (70 mL) was added
tert-butyldiphenylsilyl chloride (9.38 mL, 36.66 mmol) dropwise at
0 °C. After being stirred at room temperature for 2 h, the reaction
mixture was extracted with methylene chloride. The organic layer
was dried over anhydrous MgSO4, filtered, and evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography using hexanes and ethyl acetate (7:1) as the elu-
ent to give silyl ether 8 (11.35 g, 84%) as a white solid: mp 61.5–
4.1.6. (1R,2S)-2-[(tert-Buthyl-diphenyl-silanyloxymethyl)-2-
vinylcyclopropyl]methylacetate (10a)
1H NMR (500 MHz, CDCl3) d 7.68–7.39 (m, 10H, 2 ꢂ Ph), 5.96
(dd, 1H, J = 10.5, 17.5 Hz, –CH@CH2), 5.19 (dd, 1H, J = 1.5, 10.5 Hz,
–CH@CHH), 5.13 (dd, 1H, J = 1.5, 17.5 Hz, –CH@CHH), 4.17 (dd,
1H, J = 7.0, 12.0 Hz, AcOCHH), 3.85 (dd, 1H, J = 9.0, 11.0 Hz,
AcOCHH), 3.73 (d, 1H, J = 10.0 Hz, TBDPSOCHH), 3.55 (d, 1H,
J = 10.0 Hz, TBDPSOCHH), 2.06 (s, 3H, COCH3), 1.35 (m, 1H, 1-H),
1.07 (s, 9H, tert-butyl), 0.83 (dd, 1H, J = 5.5, 8.0 Hz, 3-HH), 0.70 (t,
1H, J = 5.0 Hz, 3-HH); 13C NMR (50 MHz, CDCl3) d 171.10, 136.25,
135.64, 133.67, 129.62, 127.62, 116.37, 68.56, 64.67, 29.88, 26.84,
21.09, 21.00, 19.35, 12.83.
63.3 °C; ½a 2D5
ꢁ
ꢀ29.09 (c 1.39, CHCl3); 1H NMR (200 MHz, CDCl3) d
7.68–7.38 (m, 10H, 2 ꢂ Ph), 4.34 (d, 1H, J = 10.8 Hz, TBDPSOCHH),
4.25 (dd, 1H, J = 4.4, 9.2 Hz, TBDPSOCHH), 4.13 (d, 1H, J = 9.2 Hz,
4-HH), 3.68 (d, 1H, J = 11.0 Hz, 4-HH), 2.10 (m, 1H, 5-H), 1.32 (dd,
1H, J = 4.8, 7.6 Hz, 6-HH), 1.04 (s, 9H, tert-butyl), 0.87 (t, 1H,
J = 4.8 Hz, 6-HH); 13C NMR (50 MHz, CDCl3) d 176.28, 135.70,
135.62, 133.36, 133.06, 129.97, 129.95, 127.88, 68.60, 60.57,
30.37, 26.90, 21.22, 19.33, 15.28; m/z (ESI) 389 (M+Na)+.
4.1.7. (1R,2S)-(ꢀ)-2-(tert-Butyl-diphenyl-silanyloxymethyl)-2-
vinylcyclopropanecarbaldehyde (11)
To a stirred solution of oxalyl chloride (1.63 mL, 18.68 mmol) in
anhydrous methylene chloride (100 mL) was added dimethyl sulf-
oxide (2.81 mL, 39.60 mmol) in anhydrous methylene chloride
(10 mL) at ꢀ78 °C, and the mixture was stirred at the same temper-
ature for 20 min. To this mixture was added a solution of 10 (4.03 g,
11.00 mmol) in anhydrous methylene chloride (50 mL), and the
reaction mixture was stirred at ꢀ78 °C for 1 h. After the addition
of triethylamine (10.58 mL, 75.91 mmol) at ꢀ78 °C, the mixture
was gradually warmed to room temperature and stirred for 1 h.
4.1.4. (1S,5R)-1-(tert-Butyl-diphenyl-silanyloxymethyl)-3-oxa-
bicyclo[3.1.0]hexan-2-ol (9)
To a stirred solution of 8 (14.02 g, 38.24 mmol) in anhydrous
methylene chloride (200 mL) was added diisobutylaluminum hy-
dride (Dibal-H, 42.06 mL, 42.06 mmol, 1.0 M solution in hexanes)
at ꢀ78 °C, and the reaction mixture was stirred for 30 min at the

A.-Y. Park et al. / Bioorg. Med. Chem. 19 (2011) 3945–3955
3951
The reaction mixture was quenched with saturated aqueous NH4Cl
solution (50 mL) and then extracted with methylene chloride. The
combined organic layers were dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The residue was purified
by silica gel column chromatography using hexanes and ethyl ace-
tate (15:1) as the eluent to give aldehyde 11 (3.60 g, 90%) as a
TBDPSOCHH), 1.58 (m, 1H, 1-H), 1.06 (s, 9H, tert-butyl), 0.73 (dd,
1H, J = 3.5, 7.5 Hz, 6-HH), 0.65 (t, 1H, J = 4.0 Hz, 6-HH); 13C NMR
(50 MHz, CDCl3) d 133.62, 131.24, 131.22, 129.56, 129.51, 126.78,
125.62, 123.74, 77.06, 64.82, 40.30, 29.26, 25.45, 22.99, 22.19;
m/z (EI) 346 (7, MꢀH2O)+. Compound (2R)-14 : (2R)-14 was synthe-
sized in 84% yield using similar procedure to the synthesis of
colorless oil: ½a D25
ꢁ
ꢀ36.81 (c 1.06, CHCl3); 1H NMR (200 MHz, CDCl3)
(2S)-14: ½a 2D5
ꢁ
ꢀ30.90 (c 1.05, CHCl3); 1H NMR (500 MHz, CDCl3) d
d 9.12 (d, 1H, J = 6.0 Hz, CHO), 7.67–7.35 (m, 10H, 2 ꢂ Ph), 5.91 (dd,
1H, J = 10.4, 17.5 Hz, –CH@CH2), 5.26 (d, 1H, J = 2.8 Hz, –CH@CHH),
5.19 (dd, 1H, J = 1.2, 3.2 Hz, –CH@CHH), 3.81 (d, 1H, J = 10.4 Hz,
TBDPSOCHH), 3.65 (d, 1H, J = 10.4 Hz, TBDPSOCHH), 2.13 (m, 1H,
1-H), 1.65 (t, 1H, J = 5.2 Hz, 3-HH), 1.46 (dd, 1H, J = 5.2, 8.4 Hz, 3-
HH), 1.05 (s, 9H, tert-butyl); 13C NMR (50 MHz, CDCl3) d 135.97,
134.95, 133.47, 133.45, 130.22, 128.16, 118.07, 66.27, 36.40,
33.44, 27.21, 19.69, 16.53; m/z (ESI) 365 (M+H)+.
7.70–7.39 (m, 10H, 2 ꢂ Ph), 6.21 (d, 1H, J = 5.5 Hz, 3-H), 5.57 (br
d, 1H, J = 5.5 Hz, 4-H), 4.39 (d, 1H, J = 1.5 Hz, 2-H), 3.97 (d, 1H,
J = 11.0 Hz, TBDPSOCHH), 3.74 (d, 1H, J = 11.5 Hz, TBDPSOCHH),
1.64 (dd, 1H, J = 4.0, 8.5 Hz, 1-H), 1.08 (s, 9H, tert-butyl), 1.00 (dd,
1H, J = 4.0, 8.0 Hz, 6-HH), 0.20 (t, 1H, J = 4.0 Hz, 6-HH); 13C NMR
(50 MHz, CDCl3) d 140.37, 135.81, 135.76, 133.98, 133.87, 130.11,
129.83, 127.82, 127.81, 77.58, 65.11, 37.16, 30.05, 27.04, 25.53,
19.46; m/z (EI) 346 (12, MꢀH2O)+.
4.1.8. (1R)-(ꢀ)-1-[(1R,2S)-2-(tert-Butyl-diphenyl-silanyloxy
methyl)-2-vinylcyclopropyl]prop-2-en-1-ol (12) and (1S)-(ꢀ)-1-
[(1R,2S)-2-(tert-butyl-diphenyl-silanyloxymethyl)-2-vinylcyclo
propyl]prop-2-en-1-ol (13)
4.1.10. (ꢀ)-3-Benzoyl-1-[(1R,2R,5S)-5-(tert-butyl-diphenyl-
silanyloxymethyl)bicyclo[3.1.0]hex-3-en-2-yl]pyrimidine-
2,4(1H,3H)-dione (15)
To a stirred solution of (2S)-14 (163.7 mg, 0.44 mmol), triphenyl
phosphine (294.5 mg, 1.12 mmol), and N3-benzoyluracil (145.6
mg, 0.67 mmol) in anhydrous THF (5 mL) was added DEAD (diethyl
azodicarboxylate, 0.18 mL, 1.12 mmol) dropwise at 0 °C. The reac-
tion mixture was stirred at 0 °C for 1 h. After the volatiles were re-
moved in vacuo, the resulting residue was purified by silica gel
column chromatography using hexanes and ethyl acetate (6:1) as
the eluent to give N3-benzoyluracil nucleoside 15 (52.5 mg, 21%)
To a stirred solution of 11 (3.60 g, 9.88 mmol) in anhydrous THF
(40 mL) was added dropwise vinylmagnesium bromide (14.81 mL,
14.81 mmol, 1.0 M solution in THF) at ꢀ78 °C, and the reaction
mixture was stirred at the same temperature for 1 h. After the mix-
ture was quenched with saturated aqueous NH4Cl solution
(20 mL), the mixture was allowed to warm to room temperature
and then extracted with ether. The organic layers were dried over
anhydrous MgSO4, filtered, and evaporated under reduced pres-
sure. The resulting oil was purified by silica gel column chromatog-
raphy using hexanes and ethyl acetate (18:1?5:1) as the eluent to
give allylic alcohols 12 (1.86 g, 48%) and 13 (1.51 g, 39%) as a col-
as a colorless sticky oil: UV (CH2Cl2) kmax 254 nm; ½a D25
ꢀ55.22 (c
ꢁ
1.13, CHCl3); 1H NMR (500 MHz, CDCl3) d 8.00–7.42 (m, 15H,
3 ꢂ Ph), 7.55 (d, 1H, J = 8.0 Hz, H-6), 6.43 (d, 1H, J = 5.5 Hz, 4-H),
5.59 (d, 1H, J = 8.5 Hz, H-5), 5.53 (br s, 1H, 2-H), 5.45 (m, 1H, 3-
H), 4.38 (d, 1H, J = 11.5 Hz, TBDPSOCHH), 3.43 (d, 1H, J = 11.0 Hz,
TBDPSOCHH), 1.68 (m, 1H, 1-H), 1.12 (s, 9H, tert-butyl), 1.07 (dd,
1H, J = 4.5, 8.0 Hz, 6-HH), 0.46 (t, 1H, J = 4.5 Hz, 6-HH); 13C NMR
orless oil, respectively. Compound 12 (more polar material): ½a D25
ꢁ
ꢀ22.80 (c 1.06, CHCl3); 1H NMR (200 MHz, CDCl3) d 7.69–7.35
(m, 10H, 2 ꢂ Ph), 6.06–5.87 (m, 2H, 2 ꢂ –CH@CH2), 5.37–5.09
(m, 4H, 2 ꢂ –CH@CH2), 3.75 (d, 1H, J = 10.4 Hz, TBDPSOCHH),
3.75–3.66 (m, 1H, CHOH), 3.50 (d, 1H, J = 10.4 Hz, TBDPSOCHH),
1.25–1.13 (m, 1H, 1-H), 1.07 (s, 9H, tert-butyl), 0.89 (s, 1H, 3-
HH), 0.85 (s, 1H, 3-HH); 13C NMR (50 MHz, CDCl3) d 140.53,
137.33, 136.03, 133.95, 130.02, 130.01, 128.01, 116.32, 114.54,
73.48, 68.93, 30.11, 29.23, 27.23, 19.71, 13.62; m/z (ESI) 415 (83,
(125 MHz, CDCl3)
d 169.37, 162.53, 150.02, 143.62, 141.93,
135.88, 135.71, 135.28, 133.38, 133.16, 131.88, 130.77, 130.34,
130.27, 129.39, 128.17, 128.14, 126.25, 102.80, 65.72, 60.76,
38.74, 28.15, 27.19, 24.18, 19.58; LRMS (FAB+) m/z 563 (20,
M+H)+, 585 (10, M+Na)+; HRMS (FAB+) calcd for C34H35N2O4Si
(M+H)+: 563.2366, found: 563.2386.
M+Na)+. Compound 13 (less polar material): ½a 2D5
ꢀ55.94 (c 1.84,
ꢁ
CHCl3); 1H NMR (200 MHz, CDCl3) d 7.71–7.36 (m, 10H, 2 ꢂ Ph),
6.13–5.90 (m, 2H, 2 ꢂ –CH@CH2), 5.33–5.09 (m, 4H,
2 ꢂ –CH@CH2), 3.76 (d, 1H, J = 10.4 Hz, TBDPSOCHH), 3.67–3.65
(m, 1H, CHOH), 3.58 (d, 1H, J = 10.4 Hz, TBDPSOCHH), 1.23–1.11
(m, 1H, 1-H), 1.09 (s, 9H, tert-butyl), 0.86 (dd, 1H, J = 5.2, 8.8 Hz,
3-HH), 0.72 (t, 1H, J = 5.2 Hz, 3-HH); 13C NMR (50 MHz, CDCl3) d
140.06, 137.28, 136.07, 136.05, 134.02, 133.96, 130.05, 130.02,
128.03, 128.02, 116.63, 114.67, 73.45, 69.00, 30.35, 30.20, 27.27,
19.70, 14.19; m/z (ESI) 415 (60, M+Na)+.
4.1.11. (ꢀ)-1-[(1R,2R,5S)-5-(Hydroxymethyl)bicyclo[3.1.0]hex-
3-en-2-yl]pyrimidine-2,4(1H,3H)-dione (L-1)
A solution of 15 (68.7 mg, 0.12 mmol) in MeOH (4 mL) was trea-
ted with concentrated aqueous ammonium hydroxide solution
(28%, 0.5 mL) and stirred at room temperature for 6 h. After evap-
oration under reduced pressure, the residue was purified by silica
gel chromatography using hexanes and ethyl acetate (2:1) as the
eluent to give TBDPS-protected uracil nucleoside (54.4 mg, 97%)
as a sticky oil: UV (CH2Cl2) kmax 267 nm; ½a D25
ꢀ69.51 (c 1.45,
ꢁ
CHCl3); 1H NMR (400 MHz, CDCl3) d 9.78 (br s, 1H, NH), 7.64–
7.35 (m, 10H, 2 ꢂ Ph), 7.47 (d, 1H, J = 8.4 Hz, H-6), 6.34 (d, 1H,
J = 5.2 Hz, 4-H), 5.52 (br s, 1H, 2-H), 5.47 (dd, 1H, J = 1.6, 8.0 Hz,
H-5), 5.36 (m, 1H, 3-H), 4.30 (d, 1H, J = 11.2 Hz, TBDPSOCHH),
3.36 (d, 1H, J = 11.2 Hz, TBDPSOCHH), 1.57 (m, 1H, 1-H), 1.05 (s,
9H, tert-butyl), 1.01 (dd, 1H, J = 4.8, 8.8 Hz, 6-HH), 0.40 (t, 1H,
J = 4.4 Hz, 6-HH); 13C NMR (100 MHz, CDCl3) d 163.95, 151.25,
143.16, 142.14, 135.82, 135.65, 133.35, 133.14, 130.24, 130.17,
128.08, 128.04, 126.42, 102.88, 65.64, 60.39, 38.60, 28.14, 27.10,
24.06, 19.50; LRMS (FAB+) m/z 459 (7, M+H)+, 481 (3, M+Na)+;
HRMS (FAB+) calcd for C27H31N2O3Si (M+H)+: 459.2104, found:
459.2098. To a stirred solution of TBDPS-protected uracil nucleo-
side (45 mg, 0.10 mmol) in THF (2 mL) was added TBAF (n-tetrabu-
tylammonium fluoride, 0.12 mL, 0.12 mmol, 1 M solution in THF).
And the reaction mixture was stirred at room temperature for
1 h. After the reaction mixture was concentrated in vacuo, the
4.1.9. (1R,2S,5S)-(+)-5-(tert-Butyl-diphenyl-silanyloxymethyl)-
bicyclo[3.1.0]hex-3-en-2-ol ((2S)-14) and (1R,2R,5S)-(ꢀ)-5-(tert-
butyl-diphenyl-silanyloxymethyl)-bicyclo[3.1.0]-hex-3-en-2-ol
((2R)-14)
Compound (2S)-14
: To a stirred solution of 13 (1.51 g,
3.85 mmol) in anhydrous methylene chloride (40 mL) was added
Grubbs catalyst 2nd generation (229 mg, 0.07 mmol) at 0 °C, and
the reaction mixture was stirred for 1.5 h at room temperature.
After the volatiles were removed, the resulting residue was purified
by column chromatography using hexanes and ethyl acetate (4:1)
as the eluent to give bicyclo[3.1.0]hexenol (2S)-14 (1.19 g, 85%) as
a colorless oil: ½a D25
ꢁ
+10.30 (c 1.13, CHCl3); 1H NMR (500 MHz,
CDCl3) d 7.68–7.38 (m, 10H, 2 ꢂ Ph), 6.03 (d, 1H, J = 5.5 Hz, 3-H),
5.30 (d, 1H, J = 5.5 Hz, 4-H), 5.22 (d, 1H, J = 7.0 Hz, 2-H), 3.85
(d, 1H, J = 10.5 Hz, TBDPSOCHH), 3.76 (d, 1H, J = 10.5 Hz,

3952
A.-Y. Park et al. / Bioorg. Med. Chem. 19 (2011) 3945–3955
resulting residue was purified by silica gel column chromatogra-
phy using methylene chloride and MeOH (15:1) as the eluent to
oside 16 (151.0 mg, 40%) as a sticky oil: UV (CH2Cl2) kmax 254 nm;
½
a 2D5
ꢁ
ꢀ42.37 (c 0.77, CHCl3); 1H NMR (500 MHz, CDCl3) d 7.97–7.39
give the final uracil nucleoside
L
-1 (18.2 mg, 85%) as a white solid:
(m, 15H, 3 ꢂ Ph), 7.19 (d, 1H, J = 1.5 Hz, H-6), 6.46 (d, 1H, J = 5.0 Hz,
4-H), 5.49 (br s, 1H, 2-H), 5.41 (m, 1H, 3-H), 4.28 (d, 1H, J = 11.0 Hz,
TBDPSOCHH), 3.55 (d, 1H, J = 11.5 Hz, TBDPSOCHH), 1.71 (m, 1H,
1-H), 1.68 (d, 3H, J = 1.5 Hz, CH3), 1.12 (s, 9H, tert-butyl), 1.07
(dd, 1H, J = 4.5, 8.5 Hz, 6-HH), 0.42 (t, 1H, J = 4.5 Hz, 6-HH); 13C
NMR (100 MHz, CDCl3) d 169.56, 163.19, 150.06, 143.52, 137.14,
135.75, 135.64, 135.11, 133.65, 133.46, 132.01, 130.69, 130.23,
130.14, 129.32, 128.09, 128.06, 125.86, 111.18, 65.44, 61.04,
38.75, 28.70, 27.27, 24.00, 19.68, 12.41; LRMS (FAB+) m/z 577
(17, M+H)+, 599 (12, M+Na)+; HRMS (FAB+) calcd for C35H37N2O4Si
(M+H)+: 577.2523, found: 577.2525.
mp 151.2–152.2 °C; UV (MeOH) kmax 266 nm; ½a D25
ꢀ125.08 (c 1.17,
ꢁ
MeOH); 1H NMR (400 MHz, CD3OD) d 7.48 (d, 1H, J = 8.0 Hz, H-6),
6.46 (d, 1H, J = 4.4 Hz, 4-H), 5.64 (d, 1H, J = 7.6 Hz, H-5), 5.39–
5.37 (m, 2H, 2-H, 3-H), 4.04 (d, 1H, J = 12.0 Hz, CHHOH), 3.43 (d,
1H, J = 11.6 Hz, CHHOH), 1.70 (m, 1H, 1-H), 1.15 (dd, 1H, J = 4.4,
8.8 Hz, 6-HH), 0.44 (t, 1H, J = 4.4 Hz, 6-HH); 13C NMR (400 MHz,
CD3OD) d 165.17, 151.40, 143.24, 142.66, 125.68, 101.43, 62.95,
60.90, 38.82, 27.62, 23.81; LRMS (FAB+) m/z 221 (5, M+H)+; HRMS
(FAB+) calcd for C11H13N2O3 (M+H)+: 221.0926, found: 221.0916.
4.1.12. (ꢀ)-4-Amino-1-[(1R,2R,5S)-5-(hydroxymethyl)bicyclo
[3.1.0]hex-3-en-2-yl]pyrimidin-2(1H)-one (
L-3)
4.1.14. (ꢀ)-1-[(1R,2R,5S)-5-(Hydroxymethyl)bicyclo[3.1.0]hex-
To a stirred solution of uracil nucleoside 1 (18.7 mg, 0.08 mmol)
in anhydrous pyridine (2 mL) was added acetic anhydride
(0.016 mL, 0.17 mmol) at room temperature, and the reaction mix-
ture was stirred at the same temperature overnight. The reaction
mixture was evaporated in vacuo and partitioned between ethyl
acetate and dilute aqueous HCl solution. The organic layer was
washed with saturated aqueous NaHCO3 solution, dried over anhy-
drous MgSO4, filtered, and evaporated under reduced pressure. The
resulting residue was purified by silica gel column chromatogra-
phy using methylene chloride and MeOH (30:1) as the eluent to
give the acetylated nucleoside 17 (22.3 mg, 100%) as a sticky oil.
To a stirred suspension of the acetylated nucleoside 17 (22.3 mg,
0.09 mmol) and 1,2,4-triazole (93.6 mg, 1.28 mmol) in anhydrous
pyridine (2 mL) was added phosphorus oxychloride (0.084 mL,
8.50 mmol) at 0 °C, and the reaction mixture was stirred at room
temperature overnight. After water (0.02 mL) was added slowly
to the reaction mixture to destroy the excess phosphorus oxychlo-
ride the volatiles were evaporated in vacuo. 1,4-Dioxane (3 mL)
and concentrated aqueous ammonium hydroxide solution (28%,
1.5 mL) were added to the reaction mixture at 0 °C and the reaction
mixture was stirred at room temperature overnight. After the vol-
atiles were evaporated under reduced pressure, methanol (1.5 mL)
and methanolic ammonia (1.5 mL) was added to the resulting res-
idue, and the reaction mixture was stirred at room temperature
overnight. After the volatiles were removed in vacuo, the resulting
residue was purified by silica gel column chromatography using
methylene chloride and MeOH (7:1) as the eluent to give the final
3-en-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dione (L-2)
A solution of 16 (151.0 mg, 0.26 mmol) in MeOH (12.5 mL) was
treated with concentrated aqueous ammonium hydroxide solution
(28%, 0.9 mL) and stirred at room temperature for 4 h. After evap-
orating under reduced pressure, the residue was purified by silica
gel column chromatography using hexanes and ethyl acetate
(2:1) as the eluent to give TBDPS-protected thymine nucleoside
(109.4 mg, 88%) as a white foam: UV (CH2Cl2) kmax 270 nm; ½a D25
ꢁ
ꢀ64.15 (c 1.30, CHCl3); 1H NMR (500 MHz, CDCl3) d 7.67–7.36
(m, 10H, 2 ꢂ Ph), 7.08 (d, 1H, J = 1.0 Hz, H-6), 6.42 (dd, 1H, J = 1.0,
5.0 Hz, 4-H), 5.51 (d, 1H, J = 1.5 Hz, 2-H), 5.37 (m, 1H, 3-H), 4.23
(d, 1H, J = 11.5 Hz, TBDPSOCHH), 3.52 (d, 1H, J = 11.0 Hz, TBDPS-
OCHH), 1.66 (s, 3H, CH3), 1.65 (m, 1H, 1-H), 1.09 (s, 9H, tert-butyl),
1.05 (dd, 1H, J = 4.5, 8.5 Hz, 6-HH), 0.42 (t, 1H, J = 4.5 Hz, 6-HH); 13
C
NMR (100 MHz, CDCl3) d 164.22, 151.21, 143.12, 137.35, 135.75,
135.62, 133.64, 133.46, 130.17, 130.08, 128.04, 128.01, 126.05,
111.14, 65.41, 60.79, 38.66, 28.70, 27.22, 23.94, 19.63, 12.39; LRMS
(FAB+) m/z 473 (36, M+H)+, 495 (20, M+Na)+; HRMS (FAB+) calcd
for C28H33N2O3Si (M+H)+: 473.2260, found: 473.2278. To a stirred
solution of TBDPS-protected thymine nucleoside (105.6 mg,
0.22 mmol) in THF (7 mL) was added TBAF (0.26 mL, 0.26 mmol,
1 M solution in THF) and the reaction mixture was stirred at room
temperature for 1 h. After the reaction mixture was concentrated
in vacuo, the resulting residue was purified by silica gel column
chromatography using methylene chloride and MeOH (15:1) as
the eluent to give the final thymine nucleoside L-2 (47.8 mg, 91%)
as a white solid: mp 183.3–184.2 °C; UV (MeOH) kmax 270 nm;
cytosine nucleoside
L
-3 (7.9 mg, 42%) as a light brownish solid: mp
½
a 2D5
ꢁ
ꢀ165.60 (c 1.05, MeOH); 1H NMR (CD3OD) d 7.38 (q, 1H,
194.3–195.2 °C dec.; UV (MeOH) kmax 274 nm; ½a D25
ꢁ
ꢀ105.27 (c
J = 1.5 Hz, H-6), 6.46 (m, 1H, 4-H), 5.41–5.39 (m, 2H, 2-H, 3-H),
4.14 (d, 1H, J = 12.5 Hz, CHHOH), 3.41 (d, 1H, J = 12.5 Hz, CHHOH),
1.83 (d, 3H, J = 1.5 Hz, CH3), 1.70 (m, 1H, 1-H), 1.16 (dd, 1H, J = 4.5,
8.5 Hz, 6-HH), 0.45 (t, 1H, J = 4.0 Hz, 6-HH); 13C NMR (100 MHz,
CD3OD) d 165.31, 151.52, 143.00, 138.39, 126.02, 110.23, 63.10,
60.58, 38.83, 27.56, 23.80, 11.22; LRMS (FAB+) m/z 235 (3,
M+H)+; HRMS (FAB+) calcd for C12H15N2O3 (M+H)+: 235.1083,
found: 235.1092.
0.46, MeOH); 1H NMR (400 MHz, CD3OD) d 7.50 (d, 1H, J = 7.2 Hz,
H-6), 6.43 (d, 1H, J = 5.6 Hz, 4-H), 5.85 (d, 1H, J = 7.2 Hz, H-5),
5.48 (br s, 1H, 2-H), 5.39–5.37 (m, 1H, 3-H), 4.04 (d, 1H,
J = 12.0 Hz, CHHOH), 3.42 (d, 1H, J = 12.4 Hz, CHHOH), 1.66 (m,
1H, 1-H), 1.14 (dd, 1H, J = 4.4, 8.8 Hz, 6-HH), 0.44 (t, 1H,
J = 4.0 Hz, 6-HH); 13C NMR (100 MHz, CD3OD) d 166.15, 157.47,
142.98, 142.66, 126.34, 95.00, 63.08, 61.77, 38.72, 28.23, 23.83;
LRMS (FAB+) m/z 220 (5, M+H)+, 242 (4, M+Na)+; HRMS (FAB+)
calcd for C11H14N3O2 (M+H)+: 220.1086, found: 220.1095.
4.1.15. (ꢀ)-9-[(1R,2R,5S)-5-(tert-Butyl-diphenyl-
silanyloxymethyl)bicyclo[3.1.0]hex-3-en-2-yl]-6-chloro-9H-
purine (18)
4.1.13. (ꢀ)-3-Benzoyl-1-[(1R,2R,5S)-5-(tert-butyl-diphenyl-
silanyloxymethyl) bicyclo[3.1.0]hex-3-en-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione (16)
To a stirred solution of (2S)-14 (102.7 mg, 0.28 mmol), triphenyl
phosphine (184.7 mg, 0.70 mmol), and 6-chloropurine (65.3 mg,
0.42 mmol) in anhydrous THF (5 mL) was added DEAD (0.11 mL,
0.70 mmol) dropwise at 0 °C and the reaction mixture was stirred
at 0 °C for 1 h. After the volatiles were removed in vacuo, the
resulting residue was purified by silica gel column chromatogra-
phy using hexanes and ethyl acetate (7:1) as the eluent to give
6-chloropurine nucleoside 18 (29.4 mg, 21%) as a colorless sticky
A solution of triphenylphosphine (427.7 mg, 1.63 mmol) in
anhydrous THF (5 mL) was treated with DEAD (0.28 mL,
1.61 mmol) dropwise at 0 °C. After the mixture was stirred for
30 min at 0 °C, a suspension of (2S)-14 (237.8 mg, 0.65 mmol)
and N3-benzoylthymine (225.3 mg, 0.98 mmol) in THF (15 mL)
was added. The reaction mixture was stirred at 0 °C for 1 h. After
the volatiles were removed in vacuo, the resulting residue was
purified by silica gel column chromatography using hexanes and
ethyl acetate (6:1) as the eluent to give N3-benzoylthymine nucle-
oil: UV (CH2Cl2) kmax 266 nm; ½a D25
ꢁ
ꢀ57.34 (c 0.97, CHCl3); 1H
NMR (500 MHz, CDCl3) d 8.79 (s, 1H, H-8), 8.27 (s, 1H, H-2),
7.67–7.32 (m, 10H, 2 ꢂ Ph), 6.50 (d, 1H, J = 5.0 Hz, 4-H), 5.60–

A.-Y. Park et al. / Bioorg. Med. Chem. 19 (2011) 3945–3955
3953
5.57 (m, 2H, 2-H, 3-H), 4.20 (d, 1H, J = 11.0 Hz, TBDPSOCHH), 3.62
(d, 1H, J = 11.5 Hz, TBDPSOCHH), 1.73 (m, 1H, 1-H), 1.19 (dd, 1H,
J = 4.5, 8.5 Hz, 6-HH), 1.09 (s, 9H, tert-butyl), 0.56 (t, 1H,
J = 4.5 Hz, 6-HH); 13C NMR (125 MHz, CDCl3) d 152.05, 151.38,
151.15, 144.36, 143.71, 135.87, 135.76, 133.41, 133.40, 132.22,
130.15, 130.08, 128.04, 128.00, 125.19, 64.83, 60.40, 38.81, 28.66,
27.16, 24.55, 19.43; LRMS (FAB+) m/z 501 (30, M+H)+; HRMS
(400 MHz, CDCl3) d 7.81 (s, 1H, H-8), 7.60–7.22 (m, 10H, 2 ꢂ Ph),
6.92 (br s, 2H, NH2), 6.36 (d, 1H, J = 5.6 Hz, 4-H), 5.45 (br s, 1H,
2-H), 5.25 (m, 1H, 3-H), 4.04 (d, 1H, J = 10.8 Hz, TBDPSOCHH),
3.56 (d, 1H, J = 11.2 Hz, TBDPSOCHH), 1.62 (m, 1H, 1-H), 1.07 (dd,
1H, J = 4.4, 8.4 Hz, 6-HH), 1.00 (s, 9H, tert-butyl), 0.42 (t, 1H,
J = 4.4 Hz, 6-HH). To a stirred solution of impure 19 (86.5 mg,
0.17 mmol) in THF (5 mL) was added TBAF (0.18 mL, 0.18 mmol,
1 M solution in THF) and the reaction mixture was stirred at room
temperature for 1.5 h. After the reaction mixture was concentrated
in vacuo, the resulting residue was purified by silica gel column
chromatography using methylene chloride and MeOH (20:1) as
the eluent to give 2-amino-6-chloropurine nucleoside (14.1 mg,
(FAB+) calcd for
501.1857.
C
28H30ClN4OSi (M+H)+: 501.1877, found:
4.1.16. (ꢀ)-[(1S,4R,5R)-4-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]
hex-2-en-1-yl]methanol ( -4)
L
A solution of 18 (41.8 mg, 0.08 mmol) in methanolic ammonia
(5 mL) was heated to 80 °C in a glass bomb for 4 h. After cooling
to room temperature, the volatiles were removed in vacuo. The
mixture was filtered and washed with methylene chloride and
MeOH (15:1), and the resulting filtrate was evaporated under re-
duced pressure. The resulting residue was purified by silica gel col-
umn chromatography using hexanes and ethyl acetate (1:2) as the
eluent to give TBDPS-protected adenine nucleoside (37.6 mg),
which was contaminated by triphenylphosphine oxide, as a color-
less sticky oil: 1H NMR (400 MHz, CDCl3) d 8.36 (s, 1H, H-8), 7.93 (s,
1H, H-2), 7.63–7.27 (m, 10H, 2 ꢂ Ph), 6.40 (d, 1H, J = 7.0 Hz, 4-H),
5.54 (m, 1H, 3-H), 5.47 (br s, 1H, 2-H), 4.14 (d, 1H, J = 11.2 Hz,
TBDPSOCHH), 3.55 (d, 1H, J = 11.2 Hz, TBDPSOCHH), 1.68 (m, 1H,
1-H), 1.10 (dd, 1H, J = 4.8, 8.0 Hz, 6-HH), 1.05 (s, 9H, tert-butyl),
17% from (2S)-14) as a white solid: UV (MeOH) kmax 310,
248 nm; 1H NMR (500 MHz, CD3OD) d 8.06 (s, 1H, H-8), 6.53 (dd,
1H, J = 1.0, 5.5 Hz, 2-H), 5.59 (m, 1H, 3-H), 5.38 (br s, 1H, 4-H),
4.10 (d, 1H, J = 12.5 Hz, CHHOH), 3.52 (d, 1H, J = 11.5 Hz, CHHOH),
1.90 (dd, 1H, J = 3.5, 7.5 Hz, 5-H), 1.26 (dd, 1H, J = 4.5, 8.5 Hz, 6-
HH), 0.57 (t, 1H, J = 4.5 Hz, 6-HH); 13C NMR (100 MHz, CD3OD) d
160.30, 153.21, 150.32, 142.76, 141.81, 125.51, 123.98, 63.08,
60.30, 38.86, 27.82, 24.19. To a solution of 2-amino-6-chloropurine
nucleoside (15.2 mg, 0.05 mmol) in MeOH (4 mL) were added 2-
mercaptoethanol (0.03 mL, 0.44 mmol) and 1 N sodium methoxide
(0.5 mL, 0.49 mmol) and the mixture was stirred under reflux for
7 h. After cooling to room temperature, the reaction mixture was
neutralized with glacial acetic acid and evaporated under reduced
pressure. The resulting residue was purified by silica gel column
chromatography using methylene chloride and MeOH (7:1) as
0.48 (t, 1H, J = 4.4 Hz, 6-HH); 13C NMR (100 MHz, CDCl3)
d
155.83, 152.94, 149.45, 142.78, 139.49, 135.82, 135.72, 133.45,
130.05, 130.00, 127.97, 127.93, 125.81, 119.81, 65.05, 59.71,
38.58, 29.90, 27.10, 24.51, 19.39; LRMS (FAB+) m/z 482 (55,
M+H)+, 504 (5, M+Na)+; HRMS (FAB+) calcd for C28H32N5OSi
(M+H)+: 482.2376, found: 482.2375. To a stirred solution of impure
TBDPS-protected adenine nucleoside (37.6 mg, 0.08 mmol) in THF
(2 mL) was added TBAF (0.09 mL, 0.09 mmol, 1 M solution in
THF) and the reaction mixture was stirred at room temperature
for 1 h. After the reaction mixture was concentrated in vacuo, the
resulting residue was purified by silica gel column chromatogra-
phy using methylene chloride and MeOH (15:1) as the eluent to
the eluent to give the final guanine nucleoside L-5 (14.1 mg, 99%)
as a white solid: mp 202.4–204.0 °C dec.; UV (MEOH) kmax
254 nm; ½a 2D5
ꢁ
ꢀ100.08 (c 0.29, DMF); 1H NMR (500 MHz, CD3OD)
d 7.72 (s, 1H, H-8), 6.49 (d, 1H, J = 5.0 Hz, 4-H), 5.57 (m, 1H, 3-H),
5.28 (br s, 1H, 2-H), 4.06 (d, 1H, J = 12.0 Hz, CHHOH), 3.53 (d, 1H,
J = 12.0 Hz, CHHOH), 1.86 (dd, 1H, J = 4.5, 8.5 Hz, 1-H), 1.25 (dd,
1H, J = 4.5, 8.5 Hz, 6-HH), 0.54 (t, 1H, J = 4.0 Hz, 6-HH); 13C NMR
(125 MHz, CD3OD) d 158.59, 154.29, 150.97, 142.36, 137.06,
125.87, 116.47, 63.17, 60.08, 38.78, 28.22, 24.29; LRMS (FAB+)
m/z 260 (12, M+H)+, 282 (43, M+Na)+.
give the final adenine nucleoside
L
-4 (8.9 mg, 44% from 18) as a
4.1.18. Synthesis of
Synthesis of -1,
procedures used for the synthesis of
data were identical to those of -nucleosides, except the values of
optical rotation, which revealed same values as those of the corre-
sponding -nucleosides with an opposite sign.
D
-1,
D
D
-2,
D
-3 and
-4 was accomplished by the same
-1, -2, -3 and -4. All spectral
D-4
white solid: mp 199.8–201.5 °C (dec.); UV (MeOH) kmax 262 nm;
D
D-2,
-3 and
D
½
a 2D5
ꢁ
ꢀ152.02 (c 0.34, MeOH); 1H NMR (400 MHz, CD3OD) d 8.16
L
L
L
L
(s, 1H, H-8), 8.08 (s, 1H, H-2), 6.49 (dd, 1H, J = 1.2, 5.2 Hz, 2-H),
5.56 (m, 1H, 3-H), 5.41 (br s, 1H, 4-H), 4.11 (d, 1H, J = 11.6 Hz,
CHHOH), 3.48 (d, 1H, J = 11.6 Hz, CHHOH), 1.90 (m, 1H, 5-H), 1.25
(dd, 1H, J = 4.4, 8.4 Hz, 6-HH), 0.56 (t, 1H, J = 4.4 Hz, 6-HH); 13C
NMR (100 MHz, CD3OD) d 156.17, 152.23, 148.66, 142.53, 140.18,
125.77, 119.21, 63.37, 60.99, 38.85, 28.11, 24.25; LRMS (FAB+) m/
z 244 (12, M+H)+; HRMS (FAB+) calcd for C12H14N5O (M+H)+:
244.1198, found: 244.1210.
L
L
4.1.19. Cells and medium
HIV-infected and uninfected MT-4 cells, a human T4-positive
cell line carrying human T-lymphotropic virus type 1 (HTLV-1),
were obtained through the Pharmaceutical Screening Center of
the Korea Research Institute of Chemical Technology in Korea.
The cell line, having an RPMI-1640 medium supplemented with
4.1.17. 9-[(1R,2R,5S)-5-(tert-Butyl-diphenyl-silanyloxymethyl)
bicyclo[3.1.0] hex-3-en-2-yl]-6-chloro-9H-purin-2-amine (19)
and (ꢀ)-2-amino-9-[(1R,2R,5S)-5-(hydroxymethyl)bicyclo
10% (w/v) fetal bovine serum (FBS), gentamycin (40
lg/mL), and
2 mM -glutamine (growth medium), was incubated at 37 °C in
L
[3.1.0]hex-3-en-2-yl]-1H-purin-6(9H)-one (
L
-5)
a humidified atmosphere containing 5% CO2. The viability of the
cells was investigated by the trypan blue dye exclusion method.
To a stirred suspension of (2S)-14 (147.5 mg, 0.40 mmol),
triphenyl phosphine (265.3 mg, 1.01 mmol), and 2-amino-6-
chloropurine (102.9 mg, 0.61 mmol) in anhydrous THF (7 mL)
was added DEAD (0.16 mL, 1.01 mmol) dropwise at 0 °C and the
reaction mixture was stirred at 0 °C for 1 h. After the volatiles were
removed in vacuo, the mixture was filtered and washed with
methylene chloride and MeOH (15:1) and the resulting filtrate
was evaporated under reduced pressure. The resulting residue
was purified by silica gel column chromatography using hexanes
and ethyl acetate (3:1) as the eluent to give 2-amino-6-chloropu-
rine nucleoside 19 (116.9 mg), which was contaminated by tri-
For an assay, the cells were seeded at
a
density of 1.0 ꢂ
105 cells/well.
4.1.20. Virus
HIV-1HTLV-IIIB and HIV-2ROD were used in the anti-HIV assay.
HIV-1 was obtained from the culture supernatant of H9 cells per-
sistently infected with HTLV-IIIB (derived from a pool of American
patients with AIDS). To harvest HIV-1, the cells were pelleted by
centrifugation, and the supernatant containing infectious HIV-1
was aliquoted and stored at ꢀ70 °C in a refrigerator (deep freezer;
ULT-1685, REVOC).
phenylphosphine oxide, as
a
colorless sticky oil: 1H NMR

3954
A.-Y. Park et al. / Bioorg. Med. Chem. 19 (2011) 3945–3955
Newmark, P. Nature 1987, 327, 458; (e) Malin, A. A.; Ostrovskii, V. A. Russ. J. Org.
4.1.21. Antiviral assay on an MT-4 cell line
Chem. 2001, 37, 759.
The MT-4 cells30 (1.0 ꢂ 105 cells/mL) were exposed to cell-free
HIV-1HTLV-IIIB or HIV-2ROD at a dose of 100 TCID50/mL (50% tissue
culture infectious dose) and cultured at 37 °C for 1.5 h. The com-
pounds were tested and compared to ddI and ddC obtained from
Sigma for cytotoxicity and for their ability to inhibit HIV replica-
tion. The compounds were first dissolved in 100% DMSO and then
diluted with RPMI-1640/10% FBS just before use. The maximum fi-
nal concentration of DMSO added to the cell cultures was 0.5% at
the highest concentration of the compound. We have determined
that at that concentration DMSO does not interfere with cell
growth. The HIV-1-induced cytopathic effects were monitored by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
3. (a) Mitsuya, H.; Weinhold, K. J.; Furman, P. A.; St Clair, M. H.; Lehrman, S. N.;
Gallo, R. C.; Bolognesi, D.; Barry, D. W.; Broder, S. Proc. Natl. Acad. Sci. U.S.A.
1985, 82, 7096; (b) Mitsuya, H.; Broder, S. Proc. Natl. Acad. Sci. U.S.A. 1986, 83,
1911.
4. (a) Balzarini, J.; Kang, G.-J.; Dalal, M.; Herdewijn, P.; De Clercq, E.; Broder, S.;
Johns, D. G. Mol. Pharmacol. 1987, 32, 162; (b) Herdewijn, P.; Balzarini, J.; De
Clercq, E.; Pauwels, R.; Baba, M.; Broder, S.; Vanderhaeghe, H. J. Med. Chem.
1987, 30, 1270; (c) Balzarini, J.; Baba, M.; Pauwels, R.; Herdewijn, P.; De Clercq,
E. Biochem. Pharmacol. 1988, 37, 2847; (d) Mitsuya, H.; Yarchoan, R.; Broder, S.
Science 1990, 249, 1533; (e) De Clercq, E. AIDS Res. Hum. Retroviruses 1992, 8,
119; (f) De Clercq, E. Clin. Microbiol. Rev. 1997, 10, 674; (g) Roey, P. V.; Taylor, E.
W.; Chu, C. K.; Schinazi, R. F. J. Am. Chem. Soc. 1993, 115, 5365; (h) De Clercq, E.
J. Med. Chem. 2005, 48, 1297; (i) Wang, J.; Jin, Y.; Rapp, K. L.; Schinazi, R. F.; Chu,
C. K. J. Med. Chem. 2007, 50, 1828.
5. Lee, H.; Hanes, J.; Johnson, K. A. Biochemistry 2003, 42, 14711.
6. Furman, P. A.; Fyfe, J. A.; Chair, M. H., St.; Weinhold, K.; Rideout, J. L.; Freeman,
G. A.; Lehrman, S. N.; Bolognesi, D. P.; Broder, S.; Mitsuya, H.; Barry, D. W. Proc.
Natl. Acad. Sci. U.S.A. 1986, 83, 8333.
7. Collier, A. C.; Bozzette, S.; Coombs, R. W.; Causey, D. M.; Schoenfeld, D. A.;
Spector, S. A.; Pettinelli, C. B.; Davies, G.; Richman, D. D.; Leedom, J. M.; Kidd, P.;
Corey, L. N. Eng. J. Med. 1990, 323, 1015.
(MTT) viability assay. In the microplate tests (96 wells), 50 lL of
each compound diluted or phosphate-buffered 0.85% (w/v) sodium
chloride (PBS) alone was distributed in triplicate. The cells were
adjusted to 1.0 ꢂ 105 cells/mL and then were plated in each well
at the rate of 200 lL per well. A virus suspension (200 lL) was
8. Yarchoan, R.; Thomas, R. V.; Allain, J.-P.; Mcatee, N.; Dubinsky, R.; Mitsuya, H.;
Lawley, T. J.; Safai, B.; Myers, C. E.; Perno, C. F.; Klecker, R. W.; Wills, R. J.; Fischl,
M. A.; Mcneely, M. C.; Pluda, J. M.; Leuther, M.; Collins, J. M.; Broder, S. Lancet
1988, 331, 76.
added to the cells with or without drugs and cultured for 6 days.
4.1.22. Cytotoxicity by MTT assay31,32
9. (a) Soudeyns, H.; Yao, X. J.; Gao, Q.; Belleau, B.; Kraus, J.-L.; Nguyen-Ba, N.;
Spira, B.; Wainberg, M. A. Antimicrob. Agents Chemother. 1991, 35, 1386; (b)
Coates, J. A. V.; Cammack, N.; Jenkinson, H. J.; Jowett, A. J.; Jowett, M. I.;
Pearson, B. A.; Penn, C. R.; Rouse, P. L.; Viner, K. C.; Cameron, J. M. Antimicrob.
Agents Chemother. 1992, 36, 733; (c) Schinazi, R. F.; Chu, C. K.; Peck, A.;
McMillan, A.; Mathis, R.; Cannon, D.; Jeong, L. S.; Beach, J. W.; Choi, W.-B.;
Yeola, S.; Liotta, D. C. Antimicrob. Agents Chemother. 1992, 36, 672; (d) Beach, J.
W.; Jeong, L. S.; Alves, A. J.; Pohl, D.; Kim, H. O.; Chang, C.-N.; Doong, S.-L.;
Schinazi, R. F.; Cheng, Y.-C.; Chu, C. K. J. Org. Chem. 1992, 57, 2217.
10. (a) Baba, M.; Pauwels, R.; Herdewijn, P.; De Clercq, E.; Desmyter, J.; Vandeputte,
M. Biochem. Biophys. Res. Commun. 1987, 142, 128; (b) Lin, T.-S.; Schinazi, R. F.;
Prusoff, W. H. Biochem. Pharmacol. 1987, 36, 2713; (c) Balzarini, J.; Kang, G. J.;
Dalal, M.; Herdewijn, P.; De Clercq, E.; Broder, S.; Johns, D. G. Mol. Pharmacol.
1987, 32, 162; (d) Hamamoto, Y.; Nakashima, H.; Matsui, T.; Matsuda, A.; Ueda,
T.; Yamamoto, N. Antimicrob. Agents Chemother. 1987, 31, 907; (e) Cheer, S. M.;
Goa, K. L. Drugs 2002, 62, 2667.
Infected cultures were carried out in parallel to determine the
cytotoxicity of the compound. Briefly, 100
lL of a cell suspension
was collected and mixed with 10 L of a solution of MTT at
l
7.5 mg/mL in PBS. After 1.5 h of incubation at 37 °C, most of the
supernatant was removed, and the formazan precipitate was dis-
solved in 100 lL of 0.04 N HCl in 2-propanol. The absorbance at
540 and 690 nm was measured on a multiwall scanning spectro-
photometer (enzyme-linked immunosorbent assay plate reader;
ERA-400, SLT). The percentage of toxicity was defined with unin-
fected and untreated control cells. The 50% cytotoxic concentration
(CC50) was defined as the concentration required to reduce the via-
bility of uninfected cells at 5 days of incubation in the presence of
the compounds. The concentration achieving 50% protection or the
concentration required to inhibit HIV-induced destruction of MT-4
cells by 50% was defined as the 50% inhibitory concentration (IC50).
HIV-infected or uninfected MT-4 cells (1.0 ꢂ 105 cells/mL) as target
cells were suspended with various concentrations of the samples
and cultured for 5 days in a CO2 incubator at 37 °C. The anti-AIDS
activities of the samples were evaluated after 5 days of HIV
infection.
11. Daluge, S. M.; Good, S. S.; Faletto, M. B.; Miller, W. H.; Clair, M. H., St.; Boone, L.
R.; Tisdale, M.; Parry, N. R.; Krenitsky, T. A. Antimicrob. Agents Chemother. 1997,
41, 1082.
12. Shi, J.; McAtee, J. J.; Wirtz, S. S.; Tharnish, P.; Juodawlkis, A.; Liotta, D. C.;
Schinazi, R. F. J. Med. Chem. 1999, 42, 859.
13. Saunders, J.; Cameron, J. M. Med. Res. Rev. 1995, 15, 497.
14. (a) Marquez, V. E.; Lim, M.-I. Med. Res. Rev. 1986, 6, 1; (b) Borthwick, A. D.;
Biggadike, K. Tetrahedron 1992, 48, 571; (c) Agrofoglio, L.; Suhas, E.; Farese, A.;
Condom, R.; Challand, S. R.; Earl, R. A.; Guedj, R. Tetrahedron 1994, 50, 10611;
(d) Crimmins, M. T. Tetrahedron 1998, 54, 9229; (e) Zhu, X.-F. Nucleosides,
Nucleotides Nucleic Acids 2000, 19, 651.
15. (a) Ezzitouni, A.; Barchi, J. J., Jr.; Marquez, V. E. J. Chem. Soc., Chem. Commun.
1995, 1345; (b) Shin, K. J.; Moon, H. R.; George, C.; Marquez, V. E. J. Org. Chem.
2000, 65, 2172.
Acknowledgment
16. (a) Rodriguez, J. B.; Marquez, V. E.; Nicklaus, M. C.; Mitsuya, H.; Barchi, J. J., Jr. J.
Med. Chem. 1994, 37, 3389; (b) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.;
Russ, P.; Wang, J.; Wagner, R. W.; Matteucci, M. D. J. Med. Chem. 1996, 39, 3739;
(c) Marquez, V. E.; Ezzitouni, A.; Russ, P.; Siddiqui, M. A.; Ford, H., Jr.; Feldman,
R. J.; Mitsuya, H.; George, C.; Barchi, J. J., Jr. J. Am. Chem. Soc. 1998, 120, 2780; (d)
Moon, H. R.; Kim, H. O.; Chun, M. W.; Jeong, L. S.; Marquez, V. E. J. Org. Chem.
1999, 64, 4733; (e) Moon, H. R.; Ford, H., Jr.; Marquez, V. E. Org. Lett. 2000, 2,
3793; (f) Joshi, B. V.; Moon, H. R.; Fettinger, J. C.; Marquez, V. E.; Jacobson, K. A.
J. Org. Chem. 2005, 70, 439.
This work was supported by the Korea Research Foundation
Grant funded by the Korean Government (MOEHRD, Basic Research
Promotion Fund) (KRF-2007-521-E00188).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.05.026.
17. (a) Saenger, W. Principles in Nucleic Acid Structure; Springer: New York, NY,
1984; (b) Thibaudeay, C.; Chattopadhyaya, J. Stereoelectronic Effects in
Nucleosides and Nucleotides and Their Structural Implications; Uppsala
University Press: Uppsala, Sweden, 1999; (c) Altona, C.; Sundaralingam, M. J.
Am. Chem. Soc. 1972, 94, 8205.
References and notes
18. Marquez, V. E.; Ezzitouni, A.; Siddiqui, M. A.; Russ, P. L.; Ikeda, H.; George, C.
Nucleosides Nucleotides 1997, 16, 1431.
1. (a) Gottlieb, M. S.; Schroff, H. M.; Weisman, J. D.; Fan, P. T.; Wolf, R. A.; Saxon, A.
N. Eng. J. Med. 1981, 305, 1425; (b) Barre-Sinoussi, F.; Chermann, J. C.; Rey, F.;
Nugeyre, M. T.; Chamaret, S.; Gruest, J.; Dauguet, C.; Axler-Blin, C.; Vezinet-
Brun, F.; Rouzioux, C.; Rozenbaum, W.; Montagnier, L. Science 1983, 220, 868;
(c) Gallo, R. C.; Salahuddin, S. Z.; Popovic, M.; Shearer, G. M.; Kaplan, M.;
Haynes, B. F.; Palker, T. J.; Redfield, R.; Oleske, J.; Safai, B.; White, G.; Foster, P.;
Markham, P. D. Science 1984, 224, 500; (d) Levy, J. A.; Hoffman, A. D.; Kramer, S.
M.; Landis, J. A.; Shimabukuro, J. M.; Oshiro, L. S. Science 1984, 225, 840; (e)
Nair, V.; Guochen, C. Rev. Med. Virol. 2007, 17, 277.
2. (a) Barre-Sinoussi, F.; Chermann, J. C.; Rey, F.; Nugeyre, M. T.; Chamatet, S.;
Gruest, J.; Dauguet, C.; Axler-Bin, C.; Vezinet-Brun, F.; Rouzioux, C.;
Rozenbaum, W.; Montagnier, L. Science 1983, 220, 868; (b) De Clercq, E. J.
Med. Chem. 1986, 29, 1561; (c) Traut, T. W. Mol. Cell. Biochem. 1994, 140, 1; (d)
19. (a) Jacobson, K. A.; Ji, X.-d.; Li, A.-H.; Melman, N.; Siddiqui, M. A.; Shin, K.-J.;
Marquez, V. E.; Ravi, R. G. J. Med. Chem. 2000, 43, 2196; (b) Wang, P.; Brank, A.
S.; Banavali, N. K.; Nicklaus, M. C.; Marquez, V. E.; Christman, J. K.; MacKerell, A.
D., Jr. J. Am. Chem. Soc. 2000, 122, 12422; (c) Kim, H. S.; Ravi, R. G.; Marquez, V.
E.; Maddileti, S.; Wihlborg, A.-K.; Erlinge, D.; Malsmjo, M.; Boyer, J. L.; Harden,
T. K.; Jacobson, K. A. J. Med. Chem. 2002, 45, 208; (d) Zalah, L.; Huleihel, M.;
Manor, E.; Konson, A.; Ford, H., Jr.; Marquez, V. E.; Johns, D. G.; Agbaria, R.
Antiviral Res. 2002, 55, 63.
20. Schinazi, R. F.; McMillan, A.; Cannon, D.; Mathis, R.; Lloyd, R. M.; Peck, A.;
Sommadossi, J.-P.; Clair, M., St.; Wilson, J.; Furman, P. A.; Painter, G.; Choi, W.-
B.; Liotta, D. C. Antimicrob. Agents Chemother. 1992, 36, 2423.
21. (a) Gosselin, G.; Schinazi, R. F.; Sommadossi, J. P.; Mathe, C.; Bergogne, M. C.;
Aubertin, A. M.; Kim, A.; Imbach, J. L. Antimicrob. Agents Chemother. 1994, 38,

A.-Y. Park et al. / Bioorg. Med. Chem. 19 (2011) 3945–3955
3955
1292; (b) Lin, T. S.; Luo, M. Z.; Liu, M. C.; Pai, S. B.; Dutschman, G. E.; Cheng, Y. C.
Biochem. Pharmacol. 1994, 47, 171.
26. (a) Grubbs, R. H.; Chang, S. Tetrahedron 1998, 54, 4413; (b) Grubbs, R. H.
Tetrahedron 2004, 60, 7117.
22. Choi, Y.; George, C.; Comin, M. J.; Barchi, J. J., Jr.; Kim, H. S.; Jacobson, K. A.;
Balzarini, J.; Mitsuya, H.; Boyer, P. L.; Hughes, S. H.; Marquez, V. E. J. Med. Chem.
2003, 46, 3292.
27. Marquez, V. E.; Russ, P.; Alons, R.; Siddiqui, M. A.; Hernandez, S.; George, C.;
Nicklaus, M. C.; Dai, F.; Ford, H., Jr. Helv. Chim. Acta 1999, 82, 2119.
28. Yamada, K.; Sakata, S.; Yoshimura, Y. J. Org. Chem. 1998, 63, 6891.
29. Shi, J.; Du, J.; Ma, T.; Pankiewicz, K. W.; Patterson, S. E.; Tharnish, P. M.;
McBrayer, T. R.; Stuyver, L. J.; Otto, M. J.; Chu, C. K.; Schinazi, R. F.; Watanabe, K.
A. Bioorg. Med. Chem. 2005, 13, 1641.
23. We have reported a preliminary result related to the synthesis and anti-HIV
effect of an L-thymine nucleoside, L-N-MCd4T as a communication form: Park,
A.-Y.; Moon, H. R.; Kim, K. R.; Chun, M. W.; Jeong, L. S. Org. Biomol. Chem. 2006,
4, 4065.
30. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.;
Desmyter, J.; De Clercq, E. J. Biol. Methods 1988, 20, 309.
24. Sekiyama, T.; Hatsuya, S.; Tanaka, Y.; Uchiyama, M.; Ono, N.; Iwayama, S.;
Oikawa, M.; Suzuki, K.; Okunishi, M.; Tsuji, T. J. Med. Chem. 1998, 41,
1284.
31. Balzarini, J.; Kang, G. J.; Dalal, M.; Herdewijn, P.; De Clerq, E.; Broder, S.; Johns,
D. G. Mol. Pharmacol. 1987, 32, 162.
25. Onishi, T.; Matsuzawa, T.; Nishi, S.; Tsuji, T. Tetrahedron Lett. 1999, 40,
32. Balzarini, J.; Baba, M.; Pauwels, R.; Herdewijn, P.; Clercq, E. D. Biochem.
Pharmacol. 1988, 37, 2847.
8845.