Synthesis and in vitro activity of 4′ and 5′-modified analogues of apiosyl nucleosides as potent anti-HCV agents

Article abstract of DOI:10.1080/15257770903396725

Novel doubly branched apio dideoxynucleosides were synthesized starting from 1,3-dihydroxyacetone using an ozonolysis and Grignard addition as key steps, and evaluated for anti-hepatitis C virus (HCV) activity. The adenine derivative 24 showed significant

Full text of DOI:10.1080/15257770903396725

Nucleosides, Nucleotides and Nucleic Acids, 28:1104–1116, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903396725
SYNTHESIS AND IN VITRO ACTIVITY OF 4ꢀ AND 5ꢀ-MODIFIED
ANALOGUES OF APIOSYL NUCLEOSIDES AS POTENT ANTI-HCV
AGENTS
Hua Li, Wonjae Lee, and Joon Hee Hong
BK21-Project Team, College of Pharmacy, Chosun University, Kwangju, Republic of Korea
Novel doubly branched apio dideoxynucleosides were synthesized starting from 1,3-
2
dihydroxyacetone using an ozonolysis and Grignard addition as key steps, and evaluated for
anti-hepatitis C virus (HCV) activity. The adenine derivative 24 showed significant anti-HCV
activity, indicating that the branches at the 4ꢁ,5ꢁ-position of the apiosyl ring led to favorable
interaction with HCV polymerase.
Keywords anti-HCV agent; ozonolysis; Grignard addition
INTRODUCTION
Apiosyl nucleosides[1,2] such as 3TC that belong to a novel class of
nucleosides, which have the oxygen of the furanose and C2-methylene
transposed, show anti-HIV activity and resistance to enzymatic deamination.
Similarly, adenine analogues such as apio-ddA, 1[3] and aminoapio-ddA, 2[4]
(Figure 1) show comparable anti-HIV and anti-HBV activity to the parent
2ꢁ,3ꢁ-dideoxy adenosine. Apio-ddA is more resistant to adenosine deaminase
(ADA) and shows enhanced stability of the glycosidic bond under acidic
and enzymatic conditions compared to natural 2ꢁ,3ꢁ-dideoxynucleosides
(ddNs) nucleosides.[5] Apio nucleoside phosphonates can be assembled
from natural precursor molecules and form duplexes with DNA and RNA
with thermal stability, similar to that of the natural nucleic acid
association.[6] Diphosphoryl phosphonate of apio nucleoside 3, a substrate
of several polymerases, can be enzymatically incorporated into DNA.[7,8]
These nucleosides can substitute for ribonucleosides in the catalytic site of
Received 28 August 2009; accepted 8 October 2009.
Address correspondence to Joon Hee Hong, College of Pharmacy, Chosun University, Kwangju
501-759, Republic of Korea. E-mail: hongjh@chosun.ac.kr
1104

Synthesis of 4ꢁ and 5ꢁ-Modified Apiosyl Nucleosides
1105
NH2
N
NH2
N
N
N
N
N
N
N
HO
HO
H2N
O
O
1
2
NH2
N
N
O
N
N
HO P
HO
O
O
3
FIGURE 1 Examples of apiosyl nucleoside analogues as potent antiviral agents.
a hammerhead ribozyme, although the catalytic efficiency of the ribozyme
is significantly reduced.[9]
Because branched nucleoside derivatives are a drug of choice in
curing viral infections, including hepatitis C virus (HCV), a number of
nucleoside derivatives have been synthesized and evaluated for anti-HCV
activity. For example, 2ꢁ-C-methylcytidine,[10] 2ꢁ-C-methyladenosine,[11] and
2ꢁ-C-hydroxymethyladenosine[12] are potent and selective anti-HCV agents.
These nucleosides are converted into their triphosphates and incorporated
into proviral RNA, resulting in viral RNA chain termination, because subse-
quent incorporation of the substrate, nucleoside triphosphate, is sterically
hindered by the 2ꢁ-methyl group. On the basis of potent anti-HCV activity
of furanosyl nucleosides, it is known that the hydroxyl functional group of
3ꢁ-position and 2ꢁ (β)-methyl group were essential for nucleosides to show
anti-HCV activity. Therefore, we applied similar structural environments to
the design of novel doubly branched apiosyl nucleosides. We introduced not
only hydroxylmethy funtional group at 4ꢁ-position but also methyl group
at 5ꢁ-position for the purpose of causing the favorable interaction of 4ꢁ-C-
hydroxymethyl with NS5b RNA dependent RNA polymerase and the steric
repulsion like the 5ꢁ-methyl group.
RESULTS AND DISCUSSION
To synthesize the target branched apiosyl nucleosides, we utilized the
unsaturated ester intermediate 5 as a starting material, which was readily
prepared by previously reported procedure from 1,3-dihydroxyacetone

1106
H. Li et al.
4.[13] Ester 5 was subjected to diisobutylaluminum hydride (DIBALH)
reduction to give the alcohol 6 in high yield. The primary hydroxyl
group of 6 was protected as a temporary p-methoxybenzyl ether (PMB) by
reaction[14] with PMBCl and NaH in DMF to afford the protected olefin 7 in
a yield of 95%. The olefin of 7 was treated with ozone in methylene chloride
at –78◦C, followed by the decomposition of the ozonide by dimethylsulfide
(DMS) to give the aldehyde 8. Compound 8 was subjected to carbonyl
addition with methylmagnesium bromide to provide the secondary alcohol
derivative 9, which was acetylated with acetic anhydride in pyridine to give
the ester 10. Oxidative deprotection of the PMB ether of 10 was effected
with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)[15] in CH2Cl2 with
a small amount of water to give the alcohol 11, which was subjected to
Swern oxidation [DMSO,(COCl)2, TEA][16] to provide aldehyde 12 in a
quantitative yield. Deacetylation of 12 using NaOMe/MeOH provided the
lactol derivative 13, and subsequent acetylation with acetic anhydride in
pyridine yielded a glycosyl donor 14, which is ready for the condensation
with bases Scheme 1.
O
HO
HO
PO
PO
ref.13
PO
PO
i
O
OH
OEt
P = TBDMS
4
6
7
5
ii
PO
PO
PO
PO
PO
PO
iii
7
iv
OPMB
OPMB
OPMB
OH
CH3
O
8
9
v
PO
PO
PO
PO
PO
PO
O
OAc
OH
OAc
vii
vi
OPMB
OAc
CH3
CH3
CH3
10
12
11
SCHEME 1 Synthesis of aldehyde intermediate 12. Reagents: i) BIBAL-H, CH2Cl2; ii) PMBCl, naH,
THF; iii) O3/DMS, CH2Cl2; iv) CH3MgBr, THF; v) Ac2O, pyridine; vi) DDQ, CH2Cl2-H2O; vii) (COCl)2,
DMSO, CH2Cl2, −78◦C.

Synthesis of 4ꢁ and 5ꢁ-Modified Apiosyl Nucleosides
1107
OH
PO
PO
PO
PO
PO
PO
i
O
OH
O
OAc
O
CH3
13'
CH3
H3C
13
12
ii
OAc
PO
PO
B
B
PO
PO
PO
PO
iii
and
O
O
O
H3C
14
H3C
H3C
15: B = 6-chloropurine
17: B = N4-Bz-cytosine
16: B = 6-chloropurine
18: B = N4-Bz-cytosine
iv
iv
B
B
HO
HO
HO
and
HO
O
O
H3C
H3C
20: B = 6-chloropurine
22: B = N4-Bz-cytosine
19: B = 6-chloropurine
21: B = N4-Bz-cytosine
v
v
vi
24: B = adenine
26: B = cytosine
23: B = adenine
25: B = cytosine
vi
SCHEME 2 Synthesis of target nucleosides. Reagents: i) NaOMe, MeOH; ii) Ac2O, DMAP, pyridine; iii)
persilylated bases, TMSOTf, ClCH2CH2Cl; iv) TBAF, THF/CH3CN; v) NH3/MeOH, steel bomb, 90◦C;
vi) NH3/MeOH, room temperature.
Synthesis of the desired nucleosides is depicted in Scheme 2. The
glycosyl donor 14 was condensed with silylated 6-chloropurine in the pres-
ence of trimethylsilyl trifluoromethansulfonate (TMSOTf) and separated
by silica gel column chromatography to provide the protected nucleosides
15 and 16. As shown in Figure 2, the stereochemistries of 15 and 16
were unambiguously determined on the basis of the NOE correlations. On
irradiation of C2-H, a relatively weak nuclear Overhauser effect (NOE) was
observed at C5-H of 15 (0.3%), but not at C5-H of 16 (0.8%).
Desilylation of 15 and 16 with n-tetrabutylammonium fluoride (TBAF)
afforded the nucleoside analogues 19 and 20, respectively. Chloropurine
derivatives 19 and 20 were individually transformed to 23 and 24 by treating
with methanolic ammonia in a steel bomb at 90◦C.

1108
H. Li et al.
B
0.3%
PO
H3C
B
2
3
PO
PO
O
H
0.8%
PO
H
4
H
O
H
5
1
16
H3C
15
FIGURE 2 NOE comparisons of compounds 15 and 16.
For the synthesis of cytosine analogues, the glycosyl donor 14 was
condensed with silylated N 4-benzoyl cytosine in the presence of TMSOTf
and separated by silica gel column chromatography to give the protected
nucleosides 17 and 18. The stereochemistries of 17 and 18 were as described
for 15 and 16 on the basis of the NOE correlations. Desilylation of each
anomer was performed by TBAF in the THF-CH3CN co-solvent system
followed by debenzolyation with methanolic ammonia to afford the final
cytosine nucleosides 25 and 26, respectively.
All the synthesized compounds were tested for anti-HCV activity using
an in vitro assay that is suitable for monitoring anti-HCV activities of
compounds. This system is composed of a human hepatocarcinoma cell line
(Huh-7) supporting multiplication of an HCV replicon named NK-R2AN.
Compound 24 significantly inhibited the replication of the replicon NK-
R2AN in Huh-7 cells by 50% at 19 µM. Therefore, two branches at the 4ꢁ,5ꢁ-
position make the apiosyl ring conformation favorable for phosphorylation
at the 4ꢁ-hydroxyl group and subsequent incorporation into the growing
RNA chain by the polymerase.
EXPERIMENTAL
Melting points were determined on a Mel-temp II laboratory device
and are uncorrected. NMR spectra were recorded on a JEOL 300 Fourier
transform spectrometer (JEOL, Tokyo, Japan); chemical shifts are reported
in parts per million (δ) and signals are reported as s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet) and dd (doublet of doublets). UV
spectra were obtained on a Beckman DU-7 spectrophotometer (Beckman,
South Pasadena, CA, USA). The elemental analyses were performed using
a Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT, USA). Thin
layer chromatography (TLC) was performed on Uniplates (silica gel)
purchased from Analtech Co. (7558, Newark, DE, USA). All reactions were
performed under an atmosphere of nitrogen unless otherwise specified. Dry
dichloromethane, benzene, and pyridine were obtained by distillation from
CaH2. Dry THF was obtained by distillation from Na and benzophenone
immediately prior to use.

Synthesis of 4ꢁ and 5ꢁ-Modified Apiosyl Nucleosides
1109
3,3-Bis-(tert-butyl-dimethyl-silanyloxymethyl)-pent-4-en-1-ol (6): To a so-
lution of 5 (5.5 g, 13.19 mmol) in CH2Cl2 (100 mL), DIBAL-H (27.71 mL,
1.0 M solution in hexane) was added slowly at −20◦C, and the mixture was
stirred for 2 hours at the same temperature. To the mixture, methanol
(28 mL) was added. The mixture was stirred at room temperature for
2 hours, and the resulting solid was filtered through a Celite pad. The filtrate
was concentrated under vacuum and the residue was purified by silica gel
column chromatography (EtOAc/hexane, 1:7) to give alcohol 6 (4.55 g,
1
92%) as a colorless oil: H NMR (CDCl3, 300 MHz) δ 5.70–5.63 (m, 1H),
4.91–4.98 (m, 2H), 3.71–3.64 (m, 4H), 3.52 (m, 2H), 1.45–1.39 (m, 2H),
0.81 (m, 18H), 0.01 (m, 12H); 13C NMR (CDCl3, 75 MHz) δ 149.6, 107.9,
70.5, 59.1, 38.7, 34.1, 25.3, 18.7, −5.4; Anal. Calc. for C19H42O3Si2: C, 60.90;
H, 11.30. Found: C, 60.94; H, 11.26.
1-[3,3-Bis-(tert-butyl-dimethyl-silanyloxymethyl)-pent-4-enyloxymethyl]-
4-methoxy-benzene (7): Compound 6 (5.2 g, 13.87 mmol) was dissolved
in dry DMF (50 mL). After cooling the solution to 0◦C, NaH (0.4 g, 60%
◦
in mineral oil, 16.6 mmol) was added. The solution was stirred at 0 C
for 30 minutes and then 4-methoxybenzyl chloride (2.6 g, 16.6 mmol)
was slowly added. After warming the solution to room temperature, it was
stirred for 3 hours. The solvent was removed under reduced pressure and
the residue was quenched with H2O followed by extraction with EtOAc
(2 × 60 mL). The organic layers were combined, dried over anhydrous
magnesium sulfate, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (EtOAc/hexane, 1:15)
to give 7 (5.83 g, 85%) as a colorless oil. 1H NMR (CDCl3, 300 MHz) δ 7.25
(d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz, 2H), 5.82–5.05 (m, 2H), 4.65 (s,
2H), 3.73–3.63 (m, 7H), 3.38 (t, J = 6.8 Hz, 2H), 1.43–1.36 (m, 2H), 0.82
(m, 18H), 0.02 (m, 12H); 13C NMR (CDCl3, 75 MHz) δ 159.7, 148.5, 130.4,
129.7, 113.6, 109.3, 76.2, 70.6, 65.6, 57.4, 39.8, 31.8, 25.5, 18.4, −5.5; Anal.
Calc. for C27H50O4Si2: C, 65.53; H, 10.18. Found: C, 65.59; H, 10.21.
2,2-Bis-(tert-butyl-dimethyl-silanyloxymethyl)-4-(4-methoxy-benzyloxy)-
butyraldehyde (8): A solution of compound 7 (4.5 g, 9.1 mmol) in
anhydrous CH2Cl2 (60 mL) was cooled down to −78◦C, and ozone gas was
then bubbled into the reaction mixture until a blue color persisted for an
additional 5 minutes. The reaction mixture was degassed with nitrogen,
and dimethyl sulfide (2.79 mL, 38 mmol) was slowly added at –78◦C. The
mixture was stirred for 1 hour at room temperature under argon gas and
concentrated under reduced pressure. The residue was purified by silica gel
column chromatography (EtOAc/hexane, 1:30) to give compound 8 (1.79
1
g, 80%) as a colorless oil: H NMR (CDCl3, 300 MHz) δ 9.78 (s, 1H), 7.26
(d, J = 8.2 Hz, 2H), 6.97 (d, J = 8.2 Hz, 2H), 4.62 (s, 2H), 4.02–3.94 (m,
4H), 3.75 (s, 3H), 3.36 (dd, J = 7.0, 1.2 Hz, 2H), 1.75–1.68 (m, 2H), 0.81

1110
H. Li et al.
(m, 18H), 0.02 (m, 12H); 13C NMR (CDCl3, 75 MHz) δ 203.2, 160.2, 130.1,
129.2, 114.1, 75.7, 65.3, 62.3, 56.8, 55.8, 25.6, 24.5, 18.3, −5.6.
3,3-Bis-(tert-butyl-dimethyl-silanyloxymethyl)-5-(4-methoxy-benzyloxy)-
pentan-2-ol (9): To a solution of 8 (2.1 g, 4.22 mmol) in dry THF (30 mL)
was slowly added CH3MgBr (5.0 mL, 1.0 M solution in THF) at −78◦C. After
5 hours, saturated NH4Cl solution (5 mL) and water (50 mL) were sequen-
tially added, and the reaction mixture was slowly warmed to room temper-
ature. The mixture was extracted with EtOAc (60 mL) two times. The com-
bined organic layer was dried over MgSO4, filtered, and evaporated. The
residue was purified by silica gel column chromatography (EtOAc/hexane,
1:15) to give 9 (1.86 g, 86%) as colorless oil: 1H NMR (CDCl3, 300 MHz) δ
7.22 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 8.1 Hz, 2H), 4.65 (s, 2H), 3.72–3.63
(m, 7H), 3.36–3.30 (m, 3H), 1.37–1.29 (m, 2H), 1.20 (d, J = 6.8 Hz, 3H),
0.82 (m, 18H), 0.02 (m, 12H); 13C NMR (CDCl3, 75 MHz) δ 160.1, 131.2,
128.4, 112.9, 75.8, 68.5, 65.6, 62.8, 56.5, 45.5, 25.7, 24.8, 18.4, 17.2, −5.4;
Anal. Calc. for C27H52O5Si2: C, 63.23; H, 10.22. Found: C, 63.26; H, 10.19.
2,2-Bis-(tert-butyl-dimethyl-silanyloxymethyl)-4-(4-methoxy-benzyloxy)-
1-methyl-butyl acetate (10): To a solution of compound 9 (2.5 g, 4.87 mmol)
in anhydrous pyridine (30 mL), Ac2O (497 mg, 1.53 mmol) was slowly
added, and the mixture was stirred overnight under nitrogen. The pyridine
was evaporated under reduced pressure and co-evaporated with toluene.
The residue was poured into water (50 mL) and extracted with EtOAc
(50 mL) two times. The combined organic layer was dried over MgSO4
and filtered. The filtrate was concentrated under reduced pressure and the
residue was purified by silica gel column chromatography (EtOAc/hexane,
1:25) to give compound 10 (2.46 g, 91%) as a colorless oil: 1H NMR (CDCl3,
300 MHz) δ 7.26 (d, J = 8.1 Hz, 2H), 6.89 (d, J = 8.1 Hz, 2H), 4.65 (s, 2H),
4.13 (q, J = 6.8 Hz, 1H), 3.75–3.67 (m, 7H), 3.34 (dd, J = 6.2, 1.2 Hz, 2H),
2.03 (s, 3H), 1.39–1.31 (m, 5H), 0.83 (s, 18H), 0.02 (m, 12H); 13C NMR
(CDCl3, 75 MHz) δ 171.1, 159.9, 130.7, 129.1, 113.6, 76.5, 72.5, 66.3, 64.2,
56.8, 43.1, 25.7, 24.9, 18.4, 17.4, 14.3, −5.6; Anal. Calc. for C29H54O6Si2: C,
62.77; H, 9.81. Found: C, 62.81; H, 9.78.
2,2-Bis-(tert-butyl-dimethyl-silanyloxymethyl)-4-hydroxy-1-methyl-butyl
acetate (11): To a solution of compound 10 (2.2 g, 3.96 mmol) in
CH2Cl2/H2O mixture (119 mol CH2Cl2, 5.94 mol H2O) was added
DDQ (1.08 g, 4.75 mmol) and the mixture was stirred for 2 hour at
room temperature. Saturated NaHCO3 (20 mL) was added to quench
the reaction. The organic layer was separated, washed with brine, dried
over MgSO4, and filtered. The filtrate was concentrated under reduced
pressure and the residue was purified by silica gel column chromatography
(EtOAc/hexane, 1:15) to give compound 11 (2.46 g, 83%) as a colorless

Synthesis of 4ꢁ and 5ꢁ-Modified Apiosyl Nucleosides
1111
1
oil: H NMR (CDCl3, 300 MHz) δ 4.13 (m, 1H), 3.73–3.65 (m, 4H), 3.51
(t, J = 6.6 Hz, 2H), 2.02 (s, 3H), 1.42–1.36 (m, 5H), 0.81 (m, 18H), 0.01
(s, 12H); 13C NMR (CDCl3, 75 MHz) δ 171.2, 71.6, 64.2, 58.7, 41.8, 27.9,
25.4, 18.4, 17.5, 15.6, −5.5; Anal. Calc. for C21H46O5Si2: C, 58.01; H, 10.66.
Found: C, 58.09; H, 10.70.
2,2-Bis-(tert-butyl-dimethyl-silanyloxymethyl)-1-methyl-4-oxo-butyl
acetate (12): To a stirred solution of oxalyl chloride (0.06 mL, 0.68 mmol)
in CH2Cl2 (6 mL) was added a solution of DMSO (0.06 mL, 0.9 mmol) in
CH2Cl2 (0.34 mL) dropwise at –78◦C. The resulting solution was stirred at
–78◦C for 5 minutes, and a solution of alcohol 11 (197 mg, 0.454 mmol) in
CH2Cl2 (3 mL) was added dropwise. The mixture was stirred at –78◦C for
20 minutes and triethylamine (0.32 mL, 2.26 mmol) was added. The
◦
resulting mixture was warmed to 0 C and stirred for 30 minutes. H2O (6
mL) was added, and the solution was stirred at room temperature for 20
minutes. The mixture was poured into water (40 mL), extracted with EtOAc
(40 mL) two times. The combined organic layer was dried over MgSO4
and filtered. The filtrate was concentrated under reduced pressure and the
residue was purified by silica gel column chromatography (EtOAc/hexane,
1
1:30) to give compound 12 (193 mg, 99%) as a colorless oil: H NMR
(CDCl3, 300 MHz) δ 9.75 (s, 1H), 4.12 (m, 1H), 3.74–3.67 (m, 4H), 2.34
(dd, J = 8.4, 6.2 Hz, 1H), 2.03 (s, 3H), 0.81 (s, 18H), 0.01 (s, 12H); 13C
NMR (CDCl3, 75 MHz) δ 201.5, 171.5, 71.8, 65.0, 58.7, 38.4, 37.3, 25.5, 18.6,
15.7, −5.4.
(rel)-(2ꢁS,5ꢁR and 5ꢁS)-4,4-Bis-(tert-butyl-dimethyl-silanyloxymethyl)-5-
methyl-tetrahydro-furan-2-ol (13): To a solution of 12 (2.3 g, 5.31 mmol)
in methanol (10 mL) was added NaOMe (1.0 mmol, 1.0 M in MeOH). The
mixture was stirred for 4 hours at room temperture and neutralized with
acetic acid (0.1 mL). The mixture was concentrated under reduced pres-
sure. The residue was poured into water (60 mL) and extracted with EtOAc
(60 mL) two times. The combined organic layer was dried over MgSO4
and filtered. The filtrate was concentrated under reduced pressure and the
residue was purified by silica gel column chromatography (EtOAc/hexane,
1:25) to give compound 13 (1.74 g, 84%) as a colorless oil: 1H NMR (CDCl3,
300 MHz) δ 5.67 (m, 1H), 3.91 (m, 1H), 3.72–3.63 (m, 4H), 1.94–1.89 (m,
1H), 1.82 (m, 1H), 1.22 (d, J = 6.2 Hz, 3H), 0.81 (m, 18H), 0.01 (m, 12H);
13C NMR (CDCl3, 75 MHz) δ 93.1, 92.9, 68.0, 67.8, 63.8, 63.7, 47.3, 38.4,
34.3, 34.2, 25.5, 25.4, 18.6, −5.7, −5.6.
(rel)-(2ꢁS,5ꢁR and 5ꢁS)-Acetic acid 4,4-bis-(tert-butyl-dimethyl-silanyloxy-
methyl)-5-methyl-tetrahydro-furan-2-yl ester (14): To a solution of com-
pound 13 (3.1 g, 7.93 mmol) in anhydrous pyridine (30 mL), Ac2O (1.21
g, 11.9 mmol) and DMAP (36 mg, 0.3 mmol) were slowly added, and the

1112
H. Li et al.
mixture was stirred overnight under nitrogen. The pyridine was evaporated
under reduced pressure and co-evaporated with toluene. The residue was
purified by silica gel column chromatography (EtOAc/hexane, 1:25) to give
diastereomeric compound 14 (2.95 g, 86%) as a syrup: 1H NMR (CDCl3, 300
MHz) δ 6.13 (dd, J = 5.4, 1.2 Hz, 1H), 3.93 (m, 1H), 3.74–3.65 (m, 4H),
2.09–2.01 (m, 4H), 1.83–1.76 (m, 1H), 1.22 (dd, J = 6.6, 4.2 Hz, 3H), 0.81
(m, 18H), 0.01 (m, 12H); 13C NMR (CDCl3, 75 MHz) δ 170.9, 170.8, 97.3,
68.1, 64.2, 64.1, 47.0, 46.9, 31.3, 25.5, 18.4, 17.5, 15.2, −5.7.
(rel)-(2ꢁS,5ꢁR)-6-Chloro-9-[4,4-Bis-(tert-butyl-dimethyl-silanyloxymethyl)-
5-methyl-tetrahydro-furan-2-yl] purine (15) and (rel)-(2ꢁS,5ꢁS)-6-chloro-9-[4,
4-bis-(tert-butyl-dimethyl-silanyloxymethyl)-5-methyl-tetrahydro-furan-2-yl]
purine (16): 6-Chloropurine (541 mg, 3.5 mmol), anhydrous HMDS
(20 mL), and a catalytic amount of ammonium sulfate (20 mg) were
refluxed to a clear solution (overnight), and the solvent was distilled
under anhydrous conditions. The residue was dissolved in anhydrous
1,2-dichloroethane (DCE, 10 mL). To this mixture, a solution of 14 (757
mg, 1.75 mmol) in dry DCE (10 mL) and TMSOTf (778 mg, 3.5 mmol)
was added, and the resulting mixture was stirred at room temperature
for 2 hours. The reaction mixture was quenched with 20 mL of saturated
NaHCO3 and stirred for 20 minutes. The resulting solid was filtered
through a Celite pad, and the filtrate was extracted twice with CH2Cl2. The
combined organic layers were dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by silica gel
column chromatography (EtOAc/hexane, 2:1) to give compound 15(286
mg, 31%) and 16 (304 mg, 33%) as white solids, respectively: compound
1
for 15: H NMR (CDCl3, 300 MHz) δ 8.43 (s, 1H), 8.11 (s, 1H), 6.14 (br
s, 2H), 6.01 (dd, J = 5.4, 1.4 Hz, 1H), 3.92 (m, 1H), 3.72–3.64 (m, 4H),
2.25 (dd, J = 12.6, 6.6 Hz, 1H), 1.87 (dd, J = 12.6, 8.6 Hz, 1H), 1.19 (d, J
= 6.8 Hz, 3H), 0.82 (m, 18H), 0.01 (s, 12H); 13C NMR (CDCl3, 75 MHz) δ
155.7, 152.6, 148.4, 147.8, 129.5, 82.5, 68.4, 64.2, 48.1, 31.9, 25.6, 18.7, 14.7,
−5.5; Anal. Calc. for C24H43ClN4O3Si2: C, 54.67; H, 8.22; N, 10.63. Found:
C, 54.71; H, 8.19; N, 10.69; compound for 16: 1H NMR (CDCl3, 300 MHz) δ
8.51 (s, 1H), 8.29 (s, 1H), 6.11 (br s, 2H), 6.00 (d, J = 5.8 Hz, 1H), 3.87 (q, J
= 6.2 Hz, 1H), 3.78–3.69 (m, 4H), 2.31 (dd, J = 12.8, 7.2 Hz, 1H), 1.90 (dd,
J = 12.7, 6.8 Hz, 1H), 1.16 (d, J = 6.6 Hz, 3H), 0.81 (m, 18H), 0.01 (s, 12H);
13C NMR (CDCl3, 75 MHz) δ 155.2, 151.7, 147.4, 145.8, 128.1, 83.3, 67.9,
63.6, 49.7, 32.2, 25.4, 18.3, 14.9, −5.6; Anal. Calc. for C24H43ClN4O3Si2: C,
54.67; H, 8.22; N, 10.63. Found: C, 54.65; H, 8.25; N, 10.59.
(rel)-(2ꢁS,5ꢁR)-1-[4,4-Bis-(tert-butyl-dimethyl-silanyloxymethyl)-5-methyl-
tetrahydro-furan-2-yl] N4-benzoylcytosine (17) and (rel)-(2ꢁS,5ꢁS)-1-[4,4-
bis-(tert-butyl-dimethyl-silanyloxymethyl)-5-methyl-tetrahydro-furan-2-yl]
N4-benzoylcytosine (18): The glycosyl donor 14 (389 mg, 0.9 mmol) was
condensed with N 4-benzoylcytosine (387 mg, 1.8 mmol) by the same

Synthesis of 4ꢁ and 5ꢁ-Modified Apiosyl Nucleosides
1113
procedure as described for the preparation of 15 and 16 to give 17 (148mg,
28%) and 18 (142 mg, 27%) as white solids, respectively: compound for
1
17: H NMR (CDCl3, 300 MHz) δ 7.96–7.91 (m, 2H), 7.51–7.42 (m, 4H),
5.89 (d, J = 5.6, 1.8 Hz, 1H), 5.77 (d, J = 6.8 Hz, 1H), 3.89 (q, J = 6.8 Hz,
1H), 3.70–3.62 (m, 4H), 2.21 (dd, J = 12.8, 6.8 Hz, 1H), 1.88 (dd, J = 12.7,
8.4 Hz, 1H), 1.18 (d, J = 6.8 Hz, 3H), 0.81 (m, 18H), 0.01 (s, 12H); 13C
NMR (CDCl3, 75 MHz) δ 170.3, 164.9, 159.7, 134.7, 133.4, 131.2, 129.5,
127.4, 99.3, 79.2, 68.9, 63.6, 63.1, 49.3, 30.5, 25.5, 18.7, 15.1, −5.6; Anal.
Calc. for C30H49N3O5Si2: C, 61.29; H, 8.40; N, 7.15. Found: C, 61.34; H,
8.37; N, 7.19; compound for 18: 1H NMR (CDCl3, 300 MHz) δ 8.12 (d, J =
6.2 Hz, 7.90 (m, 1H), 7.49–7.40 (m, 4H), 5.93 (t, J = 5.4, Hz, 1H), 5.71 (d,
J = 7.0 Hz, 1H), 3.91 (q, J = 6.7 Hz, 1H), 3.73–3.61 (m, 4H), 2.19 (dd, J =
13.0, 8.2 Hz, 1H), 1.91 (dd, J = 12.9, 6.2 Hz, 1H), 1.16 (d, J = 6.4 Hz, 3H),
0.83 (m, 18H), 0.02 (m, 12H); 13C NMR (CDCl3, 75 MHz) δ 170.2, 164.5,
158.2, 133.8, 132.6, 131.4, 128.1, 127.4, 124.5, 98.6,80.1, 67.4, 63.7, 63.3,
48.8, 30.7, 25.6, 18.4, 14.9, −5.7; Anal. Calc. for C30H49N3O5Si2: C, 61.29;
H, 8.40; N, 7.15. Found: C, 61.26; H, 8.44; N, 7.11.
(rel)-(2ꢁS,5ꢁR)-6-Chloro-9-[4,4-Bis-(hydroxymethyl)-5-methyl-tetrahydro-
furan-2-yl] purine (19): To a solution of 15 (322 mg, 0.61 mmol) in tetrahy-
drofurane/acetonitrile (1/1 co-mixture) (12 mL), tetrabutylammonium
fluoride (1.83 mL, 1.0 M solution in THF) was added at 0◦C. The mixture
was stirred overnight at room temperature, and concentrated under
reduced pressure. The residue was purified by silica gel column chromatog-
raphy (MeOH/CH2Cl2, 1:7) to give compound 19 (144 mg, 79%) as a white
solid: UV (MeOH) λmax 264.5 nm; 1H NMR (DMSO-d6, 300 MHz) δ 8.96 (s,
1H), 8.67 (s, 1H), 5.96 (dd, J = 5.6, 1.8 Hz, 1H), 3.92 (m, 1H), 3.46–3.35 (m
4H), 2.21 (dd, J = 12.8, 6.8 Hz, 1H), 1.89 (dd, J = 12.8, 9.2 Hz, 1H), 1.20
(d, J = 6.7 Hz, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 154.9, 152.1, 147.2,
127.6, 81.4, 69.1, 61.7, 61.3, 47.1, 31.5, 14.5; Anal. Calc. for C12H15ClN4O3:
C, 48.25; H, 5.06; N, 18.76. Found: C, 48.32; H, 5.12; N, 18.69.
(rel)-(2ꢁS,5ꢁS)-6-Chloro-9-[4,4-Bis-(hydroxymethyl)-5-methyl-tetrahydro-
furan-2-yl] purine (20): Purine derivative 20 was synthesized from 16 by the
same procedure described for 19: yield 75%; UV (MeOH) λmax 264.0 nm;
1H NMR (DMSO-d6, 300 MHz) δ 8.85 (s, 1H), 8.36 (s, 1H), 5.99 (d, J =
5.4 Hz, 1H), 3.92 (m, 1H), 3.45–3.33 (m 4H), 2.24 (dd, J = 12.6, 8.2 Hz,
1H), 1.91 (dd, J = 12.6, 6.2 Hz, 1H), 1.19 (d, J = 6.6 Hz, 3H); 13C NMR
(DMSO-d6, 75 MHz) δ 155.3, 153.2, 146.4, 128.1, 81.8, 68.9, 61.2, 60.8,
47.5, 30.8, 14.6; Anal. Calc. for C12H15ClN4O3: C, 48.25; H, 5.06; N, 18.76.
Found: C, 48.18; H, 4.96; N, 18.82.
(rel)-(2ꢁS,5ꢁR)-1-[4,4-Bis-(hydroxymethyl)-5-methyl-tetrahydro-furan-2-
yl]N4-benzoylcytosine (21): Compound 21 was synthesized from 17 by the
same procedure described for 19: yield 79%; UV (MeOH) λmax 259.0 nm;

1114
H. Li et al.
1H NMR (CDCl3, 300 MHz) δ 8.24 (d, J = 6.7 Hz, 1H), 7.87 (m, 2H),
7.49–7.41 (m, 3H), 5.85 (m, 1H), 5.62 (d, J = 7.0 Hz, 1H), 3.86 (q, J = 6.7
Hz, 1H), 3.51–3.43 (m, 4H), 2.19 (dd, J = 13.2, 8.8 Hz, 1H), 1.84 (dd, J =
13.1, 7.4 Hz, 1H), 1.16 (d, J = 7.4 Hz, 3H); 13C NMR (CDCl3, 75 MHz) δ
170.7, 164.3, 158.5, 135.2, 132.4, 130.3, 127.5, 124.4, 100.4, 78.7, 67.3, 61.7,
61.3, 46.1, 30.1, 14.3; Anal. Calc. for C18H21N3O5: C, 60.16; H, 5.89; N,
11.69. Found: C, 60.09; H, 5.78; N, 11.77.
(rel)-(2ꢁS,5ꢁS)-1-[4,4-Bis-(hydroxymethyl)-5-methyl-tetrahydro-furan-2-
yl]N4-benzoylcytosine (22): Compound 22 was synthesized from 18 by the
same procedure described for 19: yield 79%; UV (MeOH) λmax 259.5 nm;
1H NMR (CDCl3, 300 MHz) δ 8.19 (d, J = 6.6 Hz, 1H), 7.89 (d, J = 5.2
Hz, 1H), 7.47–7.42 (m, 4H), 5.93 (dd, J = 5.2, 1.2 Hz, 1H), 5.48 (d, J =
6.8 Hz, 1H), 3.86 (q, J = 6.8 Hz, 1H), 3.46–3.40 (m, 4H), 2.24 (dd, J =
12.8, 8.2 Hz, 1H), 1.89 (m, 1H), 1.14 (d, J = 7.0 Hz, 3H); 13C NMR (CDCl3,
75 MHz) δ 170.1, 165.1, 157.7, 134.5, 131.4, 129.1, 126.9, 123.4, 98.9, 79.1,
68.2, 63.3, 63.0, 47.2, 30.6, 15.7. Anal. Calc. for C18H21N3O5: C, 60.16; H,
5.89; N, 11.69. Found: C, 60.22; H, 5.91; N, 11.65.
(rel)-(2ꢁS,5ꢁR)-9-[4,4-Bis-(hydroxymethyl)-5-methyl-tetrahydro-furan-
2-yl] adenine (23): A solution of 19 (180 mg, 0.6 mmol) in saturated
methanolic ammonia (15 mL) was stirred in a steel bomb at 90◦C overnight
and the mixture was concentrated. The residue was purified by silica gel
column chromatography (MeOH/CH2Cl2, 1:4) to give compound 23
(144 mg, 86%) as a white solid: m.p. 199∼201◦C; UV (MeOH) λmax 261.0
1
nm; H NMR (DMSO-d6, 300 MHz) δ 8.29 (s, 1H), 8.15 (s, 1H), 5.99 (d, J
= 6.2 Hz, 1H), 4.93 (t, J = 5.2 Hz, 1H, D2O exchangeable), 4.87 (t, J = 5.4
Hz, 1H, D2O exchangeable), 3.93 (q, J = 6.8 Hz, 1H), 3.48–3.39 (m 4H),
2.18 (dd, J = 12.6, 6.6 Hz, 1H), 1.86 (dd, J = 12.7, 8.8 Hz, 1H), 1.19 (d, J
= 6.8 Hz, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 155.7, 152.5, 149.8, 141.3,
119.4, 80.8, 68.3, 61.2, 60.9, 46.3, 30.1, 15.0; Anal. Calc. for C11H17N3O4
(+0.5 MeOH): C, 50.83; H, 6.48; N, 23.71. Found: C, 50.91; H, 6.36; N,
23.77.
(rel)-(2ꢁS,5ꢁS)-9-[4,4-Bis-(hydroxymethyl)-5-methyl-tetrahydro-furan-2-yl]
adenine (24): Compound 24 was synthesized from 20 by the same procedure
described for 23: yield 82%: m.p. 199 ∼ 201◦C; UV (MeOH) λmax 261.5
1
nm; H NMR (DMSO-d6, 300 MHz) δ 8.31 (s, 1H), 8.22 (s, 1H), 5.96 (t,
J = 5.8 Hz, 1H), 4.91 (t, J = 5.3 Hz, 1H, D2O exchangeable), 4.85 (t, J =
5.4 Hz, 1H, D2O exchangeable), 3.89 (m, 1H), 3.46–3.33 (m 4H), 2.15 (m,
1H), 1.85 (dd, J = 12.8, 8.2 Hz, 1H), 1.21 (d, J = 6.9 Hz, 3H); 13C NMR
(DMSO-d6, 75 MHz) δ 155.6, 152.8, 148.4, 142.0, 118.5, 79.2, 68.6, 60.9,
60.4, 46.5, 30.6, 14.6; Anal. Calc. for C12H17N5O3 (+1.0 H2O): C, 48.47; H,
6.44; N, 23.55. Found: C, 48.53; H, 6.50; N, 23.51.

Synthesis of 4ꢁ and 5ꢁ-Modified Apiosyl Nucleosides
1115
(rel)-(2ꢁS,5ꢁR)-1-[4,4-Bis-(hydroxymethyl)-5-methyl-tetrahydro-furan-
2-yl] cytosine (25): A solution of 21 (123 mg, 0.34 mmol) in saturated
methanolic ammonia (10 mL) was stirred overnight at room temperature
and the mixture was concentrated. The residue was purified by silica gel
column chromatography (MeOH/CH2Cl2, 1:4) to give compound 25
(73 mg, 84%) as a white solid: m.p. 202∼204◦C; UV (MeOH) λmax 271.0
1
nm; H NMR (DMSO-d6, 300 MHz) δ 7.86 (d, J = 7.4 Hz, 1H), 5.87 (dd,
J = 5.8, 1.8 Hz, 1H), 5.68 (d, J = 7.5 Hz, 1H), 4.89 (t, J = 5.4 Hz, 1H,
D2O exchangeable), 4.79 (t, J = 5.2 Hz, 1H, D2O exchangeable), 3.89 (m,
1H), 3.47–3.38 (m, 4H), 2.20 (dd, J = 13.0, 6.2 Hz, 1H), 1.89 (dd, J =
12.9, 8.7 Hz, 1H), 1.24 (d, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6, 75 MHz)
δ 165.2, 156.3, 142.3, 95.5, 79.0, 68.2, 60.7, 60.3, 47.2, 30.1; Anal. Calc. for
C11H17N3O4 (+1.0 MeOH): C, 50.16; H, 7.37; N, 14.62. Found: C, 50.09; H,
7.33; N, 14.71.
(rel)-(2ꢁS,5ꢁS)-1-[4,4-Bis-(hydroxymethyl)-5-methyl-tetrahydro-furan-2-yl]
cytosine (26): Compound 26 was synthesized from 22 by the same procedure
described for 25: yield 87%: m.p. 198∼201◦C; UV (MeOH) λmax 271.5 nm;
1H NMR (DMSO-d6, 300 MHz) δ 7.84 (d, J = 7.6 Hz, 1H), 5.92 (d, J =
6.0 8 Hz, 1H), 5.62 (d, J = 7.7 Hz, 1H), 4.87 (t, J = 5.2 Hz, 1H, D2O
exchangeable), 4.77 (t, J = 5.2 Hz, 1H, D2O exchangeable), 3.92 (q, J =
7.0 Hz, 1H), 3.49–3.39 (m, 4H), 2.18 (dd, J = 12.6, 7.2 Hz, 1H), 1.86 (dd,
J = 12.7, 8.8 Hz, 1H), 1.22 (d, J = 6.9 Hz, 3H); 13C NMR (DMSO-d6, 75
MHz) δ 165.4, 156.5, 143.0, 96.1, 79.6, 69.6, 61.8, 61.5, 47.8, 29.9; Anal.
Calc. for C11H17N3O4 (+1.0 H2O): C, 48.34; H, 7.00; N, 15.37. Found: C,
48.26; H, 7.03; N, 15.42.
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