Synthesis and in vitro anti-hepatitis B virus activity of six-membered azanucleoside analogues

Article abstract of DOI:10.1016/j.bmc.2010.11.063

Fifteen novel six-membered azanucleoside derivatives were prepared and evaluated for their anti-hepatitis B virus (HBV) activity and cytotoxicity in human hepatoblastoma-derived liver Hep-G2 cells. The most potent compound 16b with an IC₅₀ valu

Full text of DOI:10.1016/j.bmc.2010.11.063

Bioorganic & Medicinal Chemistry 19 (2011) 41–51
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro anti-hepatitis B virus activity of six-membered
azanucleoside analogues
Dan Wang a, Yu-Huan Li b, Yu-Ping Wang b, Rong-Mei Gao b, Li-He Zhang a, Xin-Shan Ye a,
⇑
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xue Yuan Rd. No. 38, Beijing 100191, China
b Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Fifteen novel six-membered azanucleoside derivatives were prepared and evaluated for their anti-hepatitis
B virus (HBV) activity and cytotoxicity in human hepatoblastoma-derived liver Hep-G2 cells. The most
potent compound 16b with an IC50 value of 2.74 lg/mL (lower than 3TC) and a SI value of 13.5 was
disclosed. The key synthetic steps involved the rearrangement of lactones (which were readily obtained
from monosaccharides) and the Lewis acid-catalyzed condensation of nucleobases with azasugar donors.
Using the versatile acetylated azasugar donors, azanucleosides covering three types of azasugars and four
types of natural nucleobases were successfully obtained. The experimental results showed that some
six-membered azanucleosides may find applications in the discovery of new anti-viral agents.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 1 October 2010
Revised 25 November 2010
Accepted 30 November 2010
Available online 4 December 2010
Keywords:
Azanucleoside
Azasugar
Hepatitis B virus
Anti-viral agent
Synthesis
1. Introduction
with one more carbon atom in azasugar ring than pyrrolidine
nucleosides. Few attempts have been reported so far to synthesize
Considerable interests have been focused on the synthesis of new
types of nucleosides since the appearance of many compounds such
as acyclovir, ribavirine, AZT, showdomycin as antitumor, antibacte-
rial and antiviral drugs.1 Traditional modifications of nucleosides
include changes on nucleobases and changes on sugar moiety.2
Modifications of nucleosides have given a plethora of biologically
active compounds,3 and in this way many modified nucleosides
exhibiting remarkable antiviral and anticancer properties have been
discovered.4 Recently, many sugar modified nucleosides,2,5 includ-
ing thionucleosides,6 carbocyclic nucleosides7 and azanucleosides,8
have received a great interest for their biological and medical
applications.
this type of nucleosides. We reasoned that piperidine nucleosides
are likely to mimic the naturally occurring nucleosides because of
the increasing flexibility in sugar rings.
For this purpose, we report herein the synthesis of fifteen novel
six-membered azanucleosides covering three types of azasugars
and four types of nucleobases. In previous studies, very limited
types of nucleobases were introduced to any types of azasugars
due to difficult synthetic access. In our work, more types of nucle-
obases (both pyrimidine type and purine type) were introduced to
azasugar rings to further increase the structural diversity. Further-
more, the anti-hepatitis B virus (HBV) activity of these synthetic
azanucleoside analogues was evaluated.
Azanucleosides, in which the oxygen atom of furanose or
pyranose ring is replaced by nitrogen atom, represent an important
class of modified nucleosides. D- and L-nucleoside analogues with a
2. Results and discussion
2.1. Chemistry
pyrrolidine,9 pyridine,10 piperidine,11,12 or five-, six-, seven-mem-
bered lactam13–17 ring have been synthesized and biologically
evaluated. Among all types of azanucleosides, five-membered
pyrrolidine-type nucleosides have been most investigated because
of their high comparability with naturally occurring nucleosides.
However, no pyrrolidine nucleosides with outstanding bioactivity
have been reported to date. Six-membered azanucleosides, which
are also called piperidine nucleosides, are the type of nucleosides
The synthesis of thymine-containing azanucleosides was shown
in Scheme 1. The glucose alkene 1a was easily obtained from
D
-glucose through three steps18 or five steps in high overall yield
(77%),19 and the mannose and galactose alkenes, 1b and 1c, respec-
tively, were prepared in the similar way. Following the one-pot
tandem procedure with lactone as intermediate, which was re-
ported by our group,20 the alkenes 1a–c were smoothly converted
to N-p-methoxybenzyl (PMB) substituted d-lactams 2a–c. To facil-
itate the subsequent reduction, the substituent group in nitrogen
⇑
Corresponding author. Tel.: +86 10 62014949; fax: +86 10 82802724.
E-mail address: xinshan@bjmu.edu.cn (X.-S. Ye).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.063

42
D. Wang et al. / Bioorg. Med. Chem. 19 (2011) 41–51
R4
R4
R4
O
OH
R3
O
R4
R3 PMB
N
R3
NBoc
R3
O
O
c, d
a, b
OBn
R2
HO
R2
BnO
R2
BnO
R1
R2
R1
R1
R1
OH
OMe
1a-c
2a-c
3a-c
Glucose: R1=R2=OH, R3=R4=H
Manose: R2=R3=OH, R1=R4=H
Galactose: R1=R4=OH, R2=R3=H
O
N
T
T
4
R4
NH
R
NBoc
R3
T =
NBoc
R3
OBn
OH
O
OAc
R4
R2
R2
R1
R1
NBoc
R3
e, f
5a-c
7a-c
g, h
i
Compounds 1-6, a: R1=R2=OBn, R3=R4=H
b: R2=R3=OBn, R1=R4=H
OBn
T
T
R2
R2
R2
c: R1=R4=OBn, R2=R3=H
BocN
R1
BocN
R1
R1
1
2
3
4
7,8
,
a
: R =R =OH, R =R =H
Compounds
4a-c
2
3
1
4
R4
OBn
R4
b
: R =R =OH, R =R =H
c: R1=R4=OH, R2=R3=H
R3
6a,c
OH
R3
8a,8c
Scheme 1. Reagents and conditions: (a) O3, Me2S, MeOH; (b) ZnCl2, NaBH3(CN), PMBNH2, MeOH, 87%–99% for two steps; (c) CAN, H2O/CH3CN = 1:2, 68%–95%; (d) (Boc)2O,
pyridine, DMAP, 91%–92%; (e) NaBH4, MeOH, 68%–91%; (f) Ac2O, CH2Cl2, pyridine, 73%–99%; (g) Thymine (T), BSA, CH3CN, reflux; (h) TMSOTf, CH3CN, 81%–88% for two steps;
(i) Pd/C, H2, HCl in MeOH, 74%–94%.
atom was manipulated from PMB group, an electron donating
group to t-butyl carbamate (Boc), an electron withdrawing group.
After the introduction of Boc group, we calculated each dihedral
angle from Karplus equation based on their 3J-coupling constants
in the 1H NMR and we got enough proof that the conformation
of the resulting six-membered azasugar ring was neither chair
form nor other usual conformations of six-membered rings (such
as boat or twisted boat). Instead, the ring seemed to be quite flat
and unusual (that’s why we used Haworth presentation to show
the structures). For example, in compound 3a, the J value between
Using NaBH4 as reducing agent, the lactam group in compounds
3a–c were selectively reduced to the corresponding unstable
N,O-hemiacetal intermediates, which were subjected to acetylation
to afford compounds 4a–c, the azasugar donors of the following
coupling reaction. It should be noted that compounds 4a–c are
not so stable either, all of which cannot be stored more than a week
even at very low temperature. With the glucose type (4a), mannose
type (4b), and galactose type (4c) azasugar donors in hands, the
coupling reactions were carried out. Coupling of 4a–c and silylated
thymine under Vorbrueggen’s conditions (glycosylation reaction)21
afforded the protected azanucleosides 5a–c, 6a, and 6c. It is
noteworthy that the coupling reactions of both the glucose and
galactose type sugar donors resulted in two anomeric isomers,
but the ratios of two anomers were different, which was verified
by analyses of their H–H COSY and NOESY NMR spectra. In con-
trast, the glycosylation of mannose type sugar donor 4b provided
only one coupling product 5b. Finally, global debenzylation by
hydrogenolysis under acidic conditions led to target molecules
7a, 7b, 7c, 8a, and 8c.
H4 and H3 (Fig. 1) was 4.2 Hz, which means the dihedral angle be-
tween these two C–H bonds was about 55° or 110° (according to
0
Karplus equation); one of the two J values between H6/H6 and
H5 was nearly 0, which indicated a dihedral angle of about 80°,
while the other J value was 5.4, which corresponded to a dihedral
angle of 50° or 115°. Since benzyl groups are very bulky compared
with hydrogens, we assumed that the possibility that all three ben-
zyl groups were in axial bonds was very low, which means the
dihedral angles of H5–H6 and H3–H4 shouldn’t be less than 60°.
0
Considering the ring restriction, UH6 –C6–C5–H5 should be smaller
Encouraged by the success of preparation of five thymidine
compounds, we tried the coupling of the benzylated azasugar
donors with other nucleobases such as uracil, cytosine, and ade-
nine. The coupling reaction proceeded smoothly. However, the
following deprotection (cleavage of the benzyl groups) turned
out to be extremely difficult. The base moieties of some nucleo-
sides such as uracil and cytosine would be reduced in the hydrog-
enolysis conditions that cleaved the benzyl groups. Meanwhile, the
glycosidic bonds in the synthesized adenosine compounds were
very vulnerable under the acidic conditions we used in hydrogen-
olysis reactions. Realizing the restrictions benzyl groups brought to
us, we decided to alter the benzyl groups before coupling reactions.
After several attempts, an effective and convenient route that
avoided the use of benzyl groups in azanucleosides was achieved.
The debenzylation of compounds 3a–c over Pd–C under neutral
hydrogenolysis conditions proceeded very efficiently, affording
products 9a–c in high yields (92%–99%). Compounds 9a–c were
reduced to N,O-hemiacetals followed by acetylation to produce
10a–c in the similar manner as described in the preparation of
4a–c (Scheme 2). Comparing with the former donors 4a–c, the
than 60°. Based on all the information and reasonable assumptions
listed above, the only possible conformation of compound 3a was
0
the flat conformation we mentioned, with UH6 –C6–C5–H5 = 50°,
UH6–C6–C5–H5 = 80° and UH3–C3–C4–H4 = 110° (as shown in Fig. 1).
Similar phenomena were also observed in compounds 3b and 3c.
Boc
H3
O
H6'
N
H5
2
BnO
BnO
H6
H4
OBn
Figure 1. The assumed conformation of compound 3a.

D. Wang et al. / Bioorg. Med. Chem. 19 (2011) 41–51
43
OAc
OAc
O
O
R4
R2
R4
R4
R2
NBoc
R3
NBoc
R3
NBoc
R3
b, c
a
OBn
OH
R2
R1
R1
R1
3a: R1=R2=OBn, R3=R4=H
9a: R1=R2=OH, R3=R4=H
9b: R2=R3=OH, R1=R4=H
9c
: R =R =H, R =R =OH
10a: R1=R2=OAc, R3=R4=H
10b: R2=R3=OAc, R1=R4=H
10c
: R =R =H, R =R =OAc
2
3
1
4
3b
: R =R =OBn, R =R =H
2
3
1
4
2
3
1
4
2
3
1
4
3c
: R =R =H, R =R =OBn
Scheme 2. Reagents and conditions: (a) Pd/C, H2, EtOAc, 92%–99%; (b) NaBH4, MeOH; (c) Ac2O, pyridine, DMAP, 75%–87% for two steps.
new glycosyl donors 10a–c had many advantages. They were more
stable (could be stored under 0 °C for more than one month) and
the cleavage of protective groups affected neither the glycosidic
bond nor the base groups, which meant they were universal donors
that could be coupled with almost all kinds of nucleobases. As
shown in Scheme 3, coupling reactions of the new donors 10a–c
with nucleobases such as N6-benzoyladenine, uracil, 4-acetam-
ido-cytosine, and 5-fluoro-uracil proceeded uneventfully. In most
cases, only one anomeric isomer was obtained as the coupling
product via the neighboring group participation of the acetyl
group.22 But in some cases, the reaction did not provide a prepon-
derant anomer. Instead, the desired nucleosides were obtained as a
mixture of anomeric isomers (11b and 11b0; 11c and 11c’). Finally,
the acyl groups were removed by using saturated ammonia in
methanol23 to yield the target molecules 15–18. All structures of
synthetic azanucleosides were identified by analyses of their 1D
and/or 2D NMR spectra.
traditional chair- or boat-shaped conformation, the six atoms in
the rings of these azasugars are almost in a plane. This factor
may lead to the dramatic increase of their activity. In addition,
the antiviral activities of nucleosides with pyrimidine nucleobases
are slightly better than those with purine nucleobases.
3. Conclusion
In summary, to increase the structural diversity and study the
structure–activity relationships, a versatile method for synthesiz-
ing azanucleoside analogues was developed and a series of novel
six-membered azanucleosides were prepared. The main difficulties
of the synthetic work lied in the glycosyl coupling reactions and
the deprotection procedure. In our approach, suitable donors (com-
pounds 10a–c) with high reactivity were adopted to overcome the
problems. The anti-HBV activities of azanucleosides were tested
in vitro. Among all the compounds being tested, the most potent
one was compound 16b (IC50 2.74
lg/mL), which was even more
potent than Lamivudine (IC50 4.51
lg/mL). These findings provide
2.2. Anti-HBV activity
a starting point for the discovery of new anti-HBV therapeutic
agents and may warrant further investigation on the mechanism
of action as well as other biological evaluation of these analogues.
Stably HBV-transfected Hep-G2 2.2.15 cell line was derived
from hepatoblastoma Hep-G2 cells and was used as in vitro model
for evaluation of anti-HBV agents.24,25 Azanucleosides 7a–c, 8a, 8c,
15a–c, 15b0, 15c0, 16a–c, 17a, and 18a, along with the reference
antiviral drug Lamivudine (3TC) as the positive control, were eval-
uated in vitro against the Hep-G2 2.2.15 cells. The levels of HBV
DNA and the Hepatitis B surface antigen (HBsAg and HBeAg) in
the culture supernatants were detected to determine the inhibitory
effect of each compound. Toxic effects of the tested compounds
were quantified using TC50 values. And the experimental results
were shown in Table 1.
The present studies investigated the anti-HBV activity of azanu-
cleoside analogues with different sugar types and different nucle-
obases. None of the compounds including 3TC showed any
HBsAg or HBeAg inhibitory effects. However, some synthetic aza-
nucleosides such as compounds 7c, 8c, 15c, 15c0, 15b and 16b
inhibited the replication of viral DNA to different extent. Among
all the compounds we synthesized, the 5-fluoro-uridine-containing
mannose type azasugar (compound 16b), with an IC50 value of
4. Experimental
4.1. General
All chemicals were purchased and used without further purifi-
cation. Tetrahydrofuran (THF), toluene, and diethyl ether (Et2O)
were distilled over sodium/benzophenone, methylene chloride
(CH2Cl2) and acetonitrile (CH3CN) were distilled over calcium hy-
dride. All reactions were carried out under an argon atmosphere
with dry, freshly distilled solvents under anhydrous conditions,
unless otherwise noted. Reactions were monitored with analytical
TLC on Silica Gel 60-F254 precoated on aluminum plates and
visualized under UV (254 nm) and/or by staining with acidic ceric
ammonium molybdate. Column chromatography was performed
on silica gel (35–75 l
m). 1H NMR spectra were recorded on a
Varian VXR-300 M, Bruker 400 M or Varian INOVA-500 M spec-
trometer at 20 °C. Chemical shifts (in ppm) were referenced to
tetramethylsilane (d = 0 ppm) in deuterated chloroform or residual
proton solvent as internal standard. 13C NMR spectra were ob-
tained by using the same NMR spectrometers and were calibrated
with CDCl3 (d = 77.00 ppm). Mass spectra were recorded using a PE
SCLEX QSTAR spectrometer. Elemental analysis data were recorded
on PE-2400C elemental analyzer.
2.74 lg/mL and selective index value of 13.5, was found to be
the most potent one (Table 1). The IC50 value of compound 16b
was even lower than the widely used anti-HBV drug 3TC, and the
moderate selectivity of 16b could potentially be increased by fur-
ther structural modifications. Furthermore, based on the experi-
mental results, some pilot conclusions of the structure–activity
relationship of synthetic azanucleosides could be drawn. It ap-
peared that the antiviral activities of mannose type and galactose
type azanucleosides are better than those of glucose type, which
might result from their different conformations of sugar moieties.
Based on the NMR data (see Supplementary data for details), it is
easy to conclude that the conformations of most mannose type
and galactose type azanucleosides are quite flat. Instead of the
4.2. (3S,4R,5R)-3,4,5-Tris(benzyloxy)-1-(4-methoxybenzyl)piperi-
din-2-one (2a)
A solution of glucose alkene 1a20 (40 mg, 0.089 mmol) in MeOH
(10 mL) at ꢀ78 °C was bubbled with O3 until the solution became

44
D. Wang et al. / Bioorg. Med. Chem. 19 (2011) 41–51
AcO
B
B
NBoc
OAc
NBoc
OAc
NBoc
OH
a,b
c
OAc
OAc
+
B
OAc
OAc
OH
OH
11a: B=BzA
12a: B=5F-U
13a: B=U
15a: B=A
16a
17a: B=U
18a: B=C
10a
: B=5F-U
14a: B=Ac
C
OAc
OAc
B
OAc
OAc
BocN
BocN
BocN
a,b
+
B
+
B
OAc
OAc
OAc
OAc
OAc
10b
OAc
11b: B=BzA
11b': B=Bz
A
12b: B=5F-U
c
BzA =
c
A =
NH2
NHBz
B
N
N
N
N
OH
OH
N
BocN
BocN
N
N
N
B
OH
OH
OH
OH
15b: B=A
15b': B=A
16b
: B=5F-U
AcO
AcO
B
B
AcO
NBoc
OAc
NBoc
BocN
OAc
a,b
OAc
+
B
OAc
+
OAc
OAc
: B=BzA
10c
OAc
: B=BzA
11c
12c
11c'
AcC =
C =
: B=5F-U
NH2
NHAc
N
c
c
N
B
B
N
O
N
O
OH
BocN
OH
NBoc
OH
U =
5FU =
O
O
N
OH
OH
: B=A
16c: B=5F-U
F
NH
NH
OH
: B=A
15c
15c'
N
O
O
Scheme 3. Reagents and conditions: (a) BSA, CH3CN, reflux, 30 min; (b) TMSOTf, 0 °C to rt, 1–3.5 h, 57%–87%; (c) NH3 in MeOH, rt, 5 h, 41%–93%.
pale blue. The solution was stirred at ꢀ78 °C for 3 min and then bub-
bled with N2 until the solution became colorless. PMBNH2 (24 L,
ica gel (petroleum ether/ethyl acetate 6:1) to provide 2a (51 mg,
99%) as a colorless oil. Rf = 0.45 (petroleum ether/ethyl acetate,
2:1). 1H NMR (300 MHz, CDCl3) d: 3.20 (dd, J = 7.2 Hz, J = 12.9 Hz,
1H), 3.29 (dd, J = 4.5 Hz, J = 12.9 Hz, 1H), 3.67 (dt, J = 4.8 Hz,
J = 6.0 Hz, 1H), 3.77 (s, 3H), 3.83 (t, J = 6.6 Hz, 1H), 4.04
(d, J = 7.2 Hz, 1H), 4.39 (d, J = 11.7 Hz, 1H), 4.44–4.56 (m, 3H), 4.65
(d, J = 11.4 Hz, 1H), 4.72–4.79 (m, 2H), 5.15 (d, J = 11.4 Hz, 1H),
6.80–6.84 (m, 2H), 7.16–7.46 (m, 17H). 13C NMR (75 MHz, CDCl3)
d: 46.53, 49.36, 55.22, 71.90, 73.63, 74.22, 75.95, 79.34, 82.05,
113.99, 127.56, 127.73, 127.90, 128.35, 129.66, 137.66, 137.95,
l
0.178 mmol), NaCNBH3 (10 mg, 0.134 mmol) and ZnCl2 (2.5 mg,
0.0178 mmol) were added, and the mixture was heated under reflux
for 1 h, followed by quenching with saturated NaHCO3 aqueous
solution. After removal of the solvent, the product mixture was dis-
solved in ethyl acetate (80 mL) and washed by saturated brine
(20 mL ꢁ 2). The organic phase was dried over anhydrous sodium
sulfate. The dried solution was filtered and the filtrate was concen-
trated. The residue was purified by column chromatography on sil-

D. Wang et al. / Bioorg. Med. Chem. 19 (2011) 41–51
45
Table 1
Anti-HBV activity and toxicity of the target compounds
a
Compound
Type of sugar
Type of base
TC50
(
lg/mL)
DNA replication
g/mL)
b
IC50
(l
SIc
d
7a
8a
glu
glu
glu
glu
glu
glu
gal
gal
gal
gal
gal
T
T
A
U
FU
C
T
T
A
A
FU
T
A
1000
1000
>1000
>1000
192.45
>1000
429.52
577.35
693.36
1000
29.01
>1000
1000
—
—
—
—
—
—
—
4.6
17.5
3.6
3.2
—
—
4.0
—
—
—
—
—
—
15a
17a
16a
18a
7c
93.00
33.03
280.99
315.90
—
8c
15c
15c0
16c
7b
15b
15b0
16b
3TC
man
man
man
man
—
247.42
—
2.74
4.51
A
FU
1000
37.04
1000
13.5
221.9
a
b
c
TC50 is 50% cytotoxic concentration in HepG2.2.15 cells.
IC50 is 50% inhibitory concentration.
Selectivity index (SI: TC50/IC50).
d
That means no antiviral activity at the concentration lower than its TC50
.
137.97, 159.07, 168.80. Anal. Calcd for C34H35NO5: C, 75.95; H, 6.56;
N, 2.61. Found: C, 75.83; H, 6.26; N, 2.56; ESI-MS: 474 [M+H]+.
by saturated brine (20 mL ꢁ 2). The organic phase was dried over
anhydrous sodium sulfate. The dried solution was filtered and
the filtrate was concentrated. The residue was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate 6:1)
to provide 2c (183 mg, 87%) as a colorless oil. Rf = 0.40 (petroleum
ether/ethyl acetate, 2:1). 1H NMR (300 MHz, CDCl3) d: 3.11 (dd,
J = 2.4 Hz, J = 12.9 Hz, 1H), 3.51 (dd, J = 4.2 Hz, J = 13.2 Hz, 1H),
3.77–3.80 (m, 4H), 3.97–3.99 (m, 1H), 4.28–4.37 (m, 4H), 4.59 (d,
J = 12.0 Hz, 1H), 4.71–4.78 (m, 2H), 4.85 (d, J = 12.0 Hz, 1H), 5.16
(d, J = 12.3 Hz, 1H), 6.76–6.79 (m, 2H), 7.09–7.45 (m, 17H). 13C
NMR (75 MHz, CDCl3) d: 47.78, 48.95, 55.21, 71.42, 72.95, 73.30,
73.92, 75.24, 77.22, 113.88, 127.51, 127.59, 127.72, 127.85,
128.03, 128.30, 128.41, 128.57, 129.21, 137.40, 138.23, 138.33,
158.83, 169.24. HRMS (ESI) Anal. Calcd for C34H36NO5 [M+H]+:
538.2588; found 538.2578.
4.3. (3S,4R,5S)-3,4,5-Tris(benzyloxy)-1-(4-
methoxybenzyl)piperidin-2-one (2b)
A solution of mannose alkene 1b20 (502 mg, 1.12 mmol) in
MeOH (20 mL) at ꢀ78 °C was bubbled with O3 until the solution
became pale blue. The solution was stirred at ꢀ78 °C for 3 min
and then bubbled with N2 until the solution became colorless.
PMBNH2 (0.436 mL, 3.36 mmol), NaCNBH3 (150 mg, 2.24 mmol)
and ZnCl2 (32 mg, 0.224 mmol) were added, and the mixture was
heated under reflux for 1.5 h, followed by quenching with satu-
rated NaHCO3 aqueous solution. After removal of the solvent, the
product mixture was dissolved in ethyl acetate (80 mL) and washed
by saturated brine (20 mL ꢁ 2). The organic phase was dried over
anhydrous sodium sulfate. The dried solution was filtered and the
filtrate was concentrated. The residue was purified by column chro-
matography on silica gel (petroleum ether/ethyl acetate 6:1) to pro-
vide 2b (536 mg, 89%) as a colorless oil. Rf = 0.51 (petroleum ether/
ethyl acetate, 2:1). 1H NMR (300 MHz, CDCl3) d: 3.14 (dd, J = 3.9 Hz,
J = 12.0 Hz, 1H), 3.36 (dd, J = 6.3 Hz, J = 12.6 Hz, 1H), 3.77 (s, 3H),
3.85 (dd, J = 2.1 Hz, J = 6.6 Hz, 1H), 4.01 (m, 1H), 4.26 (d, J = 6.6 Hz,
1H), 4.35 (d, J = 14.4 Hz, 1H), 4.43–4.54 (m, 2H), 4.60–4.69 (m,
3H), 4.79 (d, J = 11.1 Hz, 1H), 5.14 (d, J = 11.1 Hz, 1H), 6.81 (m, 2H),
7.15–7.42 (m, 17H). 13C NMR (75 MHz, CDCl3) d: 46.68, 49.09,
55.21, 71.53, 71.73, 72.38, 74.64, 77.12, 77.24, 113.94, 127.56,
127.69, 128.18, 128.30, 128.35, 129.30, 137.78, 138.06, 138.11,
158.92, 168.14. HRMS (ESI) Anal. Calcd for C34H36NO5 [M+H]+:
538.2588; found 538.2587.
4.5. (3S,4R,5R)-tert-Butyl 3,4,5-tris(benzyloxy)-2-oxopiperidine-
1-carboxylate (3a)
To a solution of 2a (101 mg, 0.19 mmol) in CH3CN/H2O (2:1,
9 mL) was added cerium (IV) ammonium nitrate (312 mg,
0.57 mmol). The mixture was stirred for 3 h at rt and then diluted
with water (5 mL). The aqueous layer was extracted with ethyl
acetate (20 mL ꢁ 2). The combined organic layers were washed suc-
cessively with saturated sodium bicarbonate and brine, dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo. Flash
column chromatography (EtOAc/petroleum ether = 1:1) of the resi-
due afforded the N-deprotected product (74 mg, 0.18 mmol, 95%) as
a colorless oil. A mixture of the N-deprotected product, di-tert-butyl
dicarbonate (136 mg, 0.72 mmol) and DMAP (cat.) in pyridine was
stirred for 3 h under N2. The residue obtained after removal of the
solvent was purified by column chromatography (EtOAc/petroleum
ether = 1:10) to afford 3a (84 mg, 92%) as a colorless oil. Rf = 0.37
(petroleum ether/ethyl acetate, 3:1). 1H NMR (300 MHz, CDCl3) d:
1.54 (s, 9H), 3.54 (d, J = 13.8 Hz, 1H), 3.75–3.81 (m, 2H), 4.05 (d,
J = 6.9 Hz, 1H), 4.34 (dd, J = 4.2 Hz, J = 14.1 Hz, 1H), 4.47 (d,
J = 12.0 Hz, 1H), 4.55–4.69 (m, 4H), 5.04 (d, J = 11.7 Hz, 1H), 7.29–
7.44 (m, 15H). 13C NMR (75 MHz, CDCl3) d: 27.99, 42.81, 70.53,
72.85, 73.37, 75.30, 80.98, 82.09, 83.62, 127.62, 127.77, 127.84,
127.91, 128.14, 128.32, 128.40, 137.35, 137.59, 137.63, 151.69,
4.4. (3R,4R,5R)-3,4,5-Tris(benzyloxy)-1-(4-
methoxybenzyl)piperidin-2-one (2c)
A solution of galactose alkene 1c20 (175 mg, 0.39 mmol) in
MeOH (20 mL) at ꢀ78 °C was bubbled with O3 until the solution
became pale blue. The solution was stirred at ꢀ78 °C for 3 min
and then bubbled with N2 until the solution became colorless.
PMBNH2 (59 lL, 0.78 mmol), NaCNBH3 (31 mg, 0.47 mmol) and
ZnCl2 (7 mg, 0.047 mmol) were added, and the mixture was heated
under reflux for 1.5 h, followed by quenching with saturated
NaHCO3 aqueous solution. After removal of the solvent, the prod-
uct mixture was dissolved in ethyl acetate (80 mL) and washed
168.37; HRMS (ESI) Anal. Calcd for
540.2357; found 540.2373.
C
31H35NO6Na [M+Na]+:

46
D. Wang et al. / Bioorg. Med. Chem. 19 (2011) 41–51
4.6. (3S,4R,5S)-tert-Butyl 3,4,5-tris(benzyloxy)-2-oxopiperidine-
1-carboxylate (3b)
anhydrous sodium sulfate and filtered. The filtrate was concen-
trated and purified by column chromatography on silica gel
(petroleum ether/ethyl acetate 9:1) to provide the unstable N,O-
hemiacetal product (68 mg, 0.13 mmol, 91%) as a colorless oil. To
the solution of N,O-hemiacetal product in dry dichloromethane
(3 mL) were added anhydrous pyridine (0.22 mL, 2.6 mmol) and
Ac2O (0.14 mL, 1.3 mmol). The mixture was then stirred at rt over-
night. The residue obtained after removal of the solvent was puri-
fied by column chromatography (EtOAc/petroleum ether = 1:9) to
afford 4a (73 mg, 99%) as a colorless oil. Rf = 0.35 (petroleum
ether/ethyl acetate, 3:1). Compound 4a was actually a pair of ano-
mers (3:1) and the above name and the following analytical data
were from the major product. 1H NMR (500 MHz, DMSO-d6) d:
1.37 (s, 9H), 2.07(s, 3H), 2.78 (t, J = 11.5 Hz, 1H), 3.48–3.53 (m,
1H), 3.57 (dd, J = 3.5 Hz, J = 9.5 Hz, 1H), 3.64 (t, J = 9.5 Hz, 1H),
4.00–4.04 (m, 1H), 4.57 (d, J = 11.5 Hz, 1H), 4.67–4.69 (m, 3H),
4.72–4.77 (m, 2H), 6.88 (br s, 1H), 7.24–7.33 (m, 15H). 13C NMR
(125 MHz, CDCl3) d: 20.92, 28.09, 41.33, 73.00, 73.13, 75.79,
77.50, 78.83, 81.45, 81.71, 127.54, 127.81, 127.91, 128.17, 128.29,
128.36, 128.45, 137.69, 138.05, 138.78, 153.55, 169.22. HRMS
(ESI) Anal. Calcd for C33H39NO7Na [M+Na]+: 584.2619; found
584.2608.
To a solution of 2b (0.99 g, 1.86 mmol) in CH3CN/H2O (2:1,
30 mL) was added cerium (IV) ammonium nitrate (3.06 g,
5.59 mmol). The mixture was stirred for 3 h at rt and then diluted
with water (10 mL). The aqueous layer was extracted with ethyl
acetate (60 mL ꢁ 2). The combined organic layers were washed
successively with saturated sodium bicarbonate and brine, dried
over anhydrous sodium sulfate, filtered, and concentrated in vacuo.
Flash column chromatography (EtOAc/petroleum ether = 1:1) of
the residue afforded the N-deprotected product (606 mg,
1.45 mmol, 77%) as a colorless oil. A mixture of the N-deprotected
product, di-tert-butyl dicarbonate (1.57 g, 7.2 mmol) and DMAP
(cat.) in pyridine was stirred for 5 h under N2. The residue ob-
tained after removal of the solvent was purified by column chro-
matography (EtOAc/petroleum ether = 1:10) to afford 3b (690 mg,
92%) as a colorless oil. Rf = 0.42 (petroleum ether/ethyl acetate,
3:1). 1H NMR (300 MHz, CDCl3) d: 1.52 (s, 9H), 3.47 (dd,
J = 3.6 Hz, J = 13.2 Hz, 1H), 3.80 (dd, J = 2.1 Hz, J = 7.2 Hz, 1H),
3.88 (dd, J = 6.3 Hz, J = 12.9 Hz, 1H), 4.02–4.06 (m, 1H), 4.26 (d,
J = 6.9 Hz, 1H), 4.61–4.71 (m, 5H), 5.06 (d, J = 11.1 Hz, 1H), 7.21–
7.45 (m, 15H). 13C NMR (75 MHz, CDCl3) d: 27.97, 45.53, 71.31,
71.80, 72.57, 74.48, 79.14, 83.38, 127.70, 127.88, 128.23, 128.33,
128.37, 128.47, 137.72, 137.66, 137.86, 152.32, 169.48; HRMS
(ESI) Anal. Calcd for C31H35NO6Na [M+Na]+: 540.2357; found
540.2375.
4.9. (2R,3S,4R,5S)-tert-Butyl 2-acetoxy-3,4,5-tris(benzyloxy)-
piperidine-1-carboxylate (4b)
To a solution of 3b (610 mg, 1.18 mmol) in MeOH (10 mL) was
added NaBH4 (190 mg, 5 mmol) at 0 °C. After 15 min, another por-
tion of NaBH4 (76 mg, 2 mmol) was added. The mixture was stirred
at 0 °C for another 30 min. After removal of the solvent, the residue
was dissolved in ethyl acetate (60 mL) and washed by saturated
brine (15 mL ꢁ 2). The organic phase was dried over anhydrous
sodium sulfate and filtered. The filtrate was concentrated and
purified by column chromatography on silica gel (petroleum
ether/ethyl acetate 9:1) to provide the unstable N,O-hemiacetal
product (545 mg, 1.05 mmol, 89%) as a colorless oil. To the solution
of N,O-hemiacetal product in dry dichloromethane (10 mL) were
added anhydrous pyridine (1.3 mL, 15.4 mmol) and Ac2O (1 mL,
9.63 mmol). The mixture was then stirred at rt overnight. The res-
idue obtained after removal of the solvent was purified by column
chromatography (EtOAc/petroleum ether = 1:9) to afford 4b
(589 mg, 99%) as a colorless oil. Rf = 0.35 (petroleum ether/ethyl
acetate, 3:1). Compound 4b was actually a pair of anomers (3:1)
and the above name and the following analytical data were from
the major product. 1H NMR (500 MHz, CDCl3) d: 1.45 (s, 9H), 1.96
(s, 3H), 3.37 (br s, 1H), 3.80 (s, 3H), 3.92 (br s, 1H), 4.16 (br s,
1H), 4.49–4.54 (m, 3H), 4.63–4.71 (m, 3H), 6.61 (br s, 1H),
7.21–7.35 (m, 15H). 13C NMR (75 MHz, CDCl3) d: 20.99, 28.21,
37.58, 71.12, 72.18, 72.34, 72.76, 73.80, 74.21, 81.22, 127.32,
127.46, 127.60, 127.90, 128.22, 128.36, 128.42, 137.55, 138.33,
138.45, 154.46, 170.12. Anal. Calcd for C33H39NO7: C, 70.57; H,
7.00; N, 2.49. Found: C, 70.30; H, 6.86; N, 2.41; ESI-MS: 584
[M+H]+.
4.7. (3R,4R,5R)-tert-Butyl 3,4,5-tris(benzyloxy)-2-oxopiperidine-
1-carboxylate (3c)
To a solution of 2c (464 mg, 0.864 mmol) in CH3CN/H2O (2:1,
30 mL) was added cerium (IV) ammonium nitrate (1.42 g,
2.59 mmol). The mixture was stirred for 3 h at rt and then diluted
with water (10 mL). The aqueous layer was extracted with ethyl
acetate (50 mL ꢁ 2). The combined organic layers were washed
successively with saturated sodium bicarbonate and brine, dried
over anhydrous sodium sulfate, filtered, and concentrated in vacuo.
Flash column chromatography (EtOAc/petroleum ether = 1:1) of
the residue afforded the N-deprotected product (245 mg,
0.59 mmol, 68%) as a colorless oil. A solution of the N-deprotected
product, di-tert-butyl dicarbonate (654 mg, 2.95 mmol) and DMAP
(cat.) in pyridine was stirred for 3.5 h under N2. The residue ob-
tained after removal of the solvent was purified by column chro-
matography (EtOAc/petroleum ether = 1:9) to afford 3c (278 mg,
91%) as a colorless oil. Rf = 0.32 (petroleum ether/ethyl acetate,
3:1). 1H NMR (300 MHz, CDCl3) d: 1.52 (s, 9H), 3.62 (dd,
J = 3.9 Hz, J = 13.5 Hz, 1H), 3.78 (dd, J = 3.9 Hz, J = 7.8 Hz, 1H),
3.99–4.05 (m, 2H), 4.33 (d, J = 3.0 Hz, 1H), 4.44 (ABq, J = 11.7 Hz,
2H), 4.60 (d, J = 12.0 Hz, 1H), 4.61 (d, J = 12.0 Hz, 1H), 4.83 (d,
J = 12.0 Hz, 1H), 5.10 (d, J = 12.0 Hz, 1H), 7.15–7.41 (m, 15H). 13C
NMR (75 MHz, CDCl3) d: 28.00, 46.02, 71.35, 73.04, 73.50, 73.76,
77.58, 83.21, 127.60, 127.75, 127.89, 127.93, 127.99, 128.33,
128.51, 137.22, 137.91, 137.94, 152.35, 170.01; HRMS (ESI) Anal.
Calcd for C31H35NO6Na [M+Na]+: 540.2357; found 540.2354.
4.10. (2R,3R,4R,5R)-tert-Butyl 2-acetoxy-3,4,5-tris(benzyloxy)-
piperidine-1-carboxylate (4c)
4.8. (2R,3S,4R,5R)-tert-Butyl 2-acetoxy-3,4,5-
tris(benzyloxy)piperidine-1-carboxylate (4a)
To a solution of 3c (218 mg, 0.422 mmol) in MeOH (10 mL) was
added NaBH4 (76 mg, 2 mmol) at 0 °C. After 15 min, another por-
tion of NaBH4 (76 mg, 2 mmol) was added. The mixture was stirred
at 0 °C for another 30 min. After removal of the solvent, the residue
was dissolved in ethyl acetate (40 mL) and washed by saturated
brine (10 mL ꢁ 2). The organic phase was dried over anhydrous
sodium sulfate and filtered. The filtrate was concentrated and
purified by column chromatography on silica gel (petroleum
To a solution of 3a (75 mg, 0.15 mmol) in MeOH (3 mL) was
added NaBH4 (6 mg, 0.15 mmol) at 0 °C. After 15 min, another por-
tion of NaBH4 (3 mg, 0.075 mmol) was added. The mixture was
stirred at 0 °C for another 30 min. After removal of the solvent,
the residue was dissolved in ethyl acetate (20 mL) and washed
by saturated brine (10 mL ꢁ 2). The organic phase was dried over

D. Wang et al. / Bioorg. Med. Chem. 19 (2011) 41–51
47
ether/ethyl acetate 9:1) to provide the unstable N,O-hemiacetal
product (200 mg, 0.385 mmol, 91%) as a colorless oil. To the solu-
tion of N,O-hemiacetal product in dry dichloromethane (10 mL)
were added anhydrous pyridine (0.65 mL, 7.7 mmol) and Ac2O
(0.50 mL, 3.85 mmol). The mixture was then stirred at rt overnight.
The residue obtained after removal of the solvent was purified by
column chromatography (EtOAc/petroleum ether = 1:9) to afford
4c (157 mg, 73%) as a colorless oil. Rf = 0.30 (petroleum ether/ethyl
acetate, 3:1). Compound 4c was actually a pair of anomers (4:1)
and the above name and the following analytical data were from
the major product. 1H NMR (500 MHz, CDCl3) d: 1.46 (s, 9H), 2.03
(s, 3H), 3.38–3.42 (m, 1H), 3.71 (br s, 1H), 3.84 (t, J = 3.0 Hz, 1H),
3.90 (dd, J = 3.0 Hz, J = 4.0 Hz, 1H), 4.14 (br s, 1H), 4.38 (d,
J = 11.5 Hz, 1H), 4.53 (d, J = 12.0 Hz, 1H), 4.58–4.62 (m, 2H), 4.78
(br s, 1H), 4.86 (d, J = 12.0 Hz, 1H), 7.03 (br s, 1H), 7.22–7.34 (m,
15H). 13C NMR (75 MHz, CDCl3) d: 21.11, 28.19, 37.28, 70.57,
72.02, 72.94, 74.17, 74.68, 75.34, 77.19, 81.14, 127.20, 127.31,
127.58, 127.70, 128.16, 128.30, 128.36, 137.77, 138.15, 138.97,
154.37, 169.82. Anal. Calcd for C32H39NO7: C, 70.57; H, 7.00; N,
2.49. Found: C, 70.29; H, 7.17; N, 2.45; ESI-MS: 402 [M+H]+.
137.98, 149.75, 154.30, 163.46. HRMS (ESI) Anal. Calcd for
36H42NO7 [M+H]+: 628.3017; found 628.3023.
C
4.13. (2S,3R,4R,5S)-tert-Butyl 3,4,5-tris(benzyloxy)-2-(thymine-
1-yl)piperidine-1-carboxylate (5b)
The coupling reaction of 4b with thymine afforded 5b (85%
yield, colorless foam). Rf = 0.35 (petroleum ether/ethyl acetate,
1:1). 1H NMR (500 MHz, CDCl3) d: 1.38 (s, 9H), 1.55 (s, 3H), 3.33
(dd, J = 11.0 Hz, 12.5 Hz, 1H), 3.82 (dd, J = 2.0 Hz, J = 4.0 Hz, 1H),
3.91 (d, J = 3.0 Hz, 1H), 4.01 (ddd, J = 2.0 Hz, J = 6.0 Hz, J = 10.5,
1H), 4.31 (dd, J = 6.5 Hz, J = 13.0 Hz, 1H), 4.35 (d, J = 11.5 Hz, 1H),
4.58–4.70 (m, 4H), 4.85 (d, J = 12.0 Hz, 1H), 6.19 (d, J = 1.0 Hz,
1H), 6.91 (d, J = 1.0 Hz, 1H), 7.09–7.11 (m, 2H), 7.24–7.39 (m,
12H), 8.25 (br s, 1H). 13C NMR (75 MHz, CDCl3) d: 12.28, 28.08,
41.38, 65.50, 71.48, 71.55, 72.30, 73.23, 74.22, 74.86, 82.15,
109.46, 127.76, 127.83, 127.88, 127.97, 128.43, 128.49, 137.12,
137.60, 137.88, 150.31, 154.34, 163.79. HRMS (ESI) Anal. Calcd
for C36H42NO7 [M+H]+: 628.3017; found 628.3028.
4.14. (2S,3S,4R,5R)-tert-Butyl 3,4,5-tris(benzyloxy)-2-(thymine-
1-yl)piperidine-1-carboxylate (5c) and (2R,3S,4R,5R)-tert-butyl
3,4,5-tris(benzyloxy)-2-(thymine-1-yl)piperidine-1-carboxylate
(6c)
4.11. General procedure for the synthesis of aza-thymidines
To a stirred solution of thymine (50 mg, 0.39 mmol) in anhy-
drous acetonitrile (2 mL) was added N,O-bis(trimethylsilyl)acet-
amide (0.24 mL, 0.975 mmol) under argon. The reaction mixture
was stirred under reflux for 15 min. After cooled to 0 °C, donors
(compounds 4a–c) (20 mg, 0.036 mmol) in anhydrous acetonitrile
These two compounds were products of the coupling reaction of
4c with thymine. The less polar one was confirmed by NMR to be
compound 6c (53% yield, colorless foam). Rf = 0.28 (petroleum
ether/ethyl acetate, 1:1). 1H NMR (300 MHz, CDCl3) d: 1.38 (s,
9H), 1.86 (s, 3H), 3.54 (dd, J = 9.3 Hz, 13.2 Hz, 1H), 3.78–3.83 (m,
2H), 4.35 (d, J = 12.0 Hz, 2H), 4.41–4.54 (m, 3H), 4.57–4.60 (m,
3H), 5.42 (d, J = 8.4 Hz, 1H), 7.04 (br s, 1H), 7.17–7.36 (m, 14H),
9.10 (br s, 1H). 13C NMR (75 MHz, CDCl3) d: 12.10, 28.16, 43.01,
71.02, 71.70, 72.28, 72.93, 73.91, 75.00, 75.78, 81.47, 108.73,
127.58, 128.14, 128.25, 128.37, 137.08, 137.65, 137.84, 142.06,
150.13, 154.39, 164.47. HRMS (ESI) Anal. Calcd for C36H42NO7
[M+H]+: 628.3017; found 628.3012. The more polar compound
was confirmed by NMR to be compound 5c (35% yield, colorless
foam). Rf = 0.26 (petroleum ether/ethyl acetate, 1:1). 1H NMR
(300 MHz, CDCl3) d: 1.36 (s, 9H), 1.80 (s, 3H), 3.62 (dd, J = 3.3 Hz,
14.7 Hz, 1H), 3.67 (dd, J = 2.1 Hz, J = 3.9 Hz, 1H), 3.93 (t, J = 3.6 Hz,
1H), 4.28–4.29 (m, 1H), 4.41–4.51 (m, 3H), 4.67–4.80 (m, 4H),
6.19 (d, J = 4.2 Hz, 1H), 6.92 (s, 1H), 7.11–7.14 (m, 2H), 7.26–7.34
(m, 12H), 9.12 (s, 1H). 13C NMR (75 MHz, CDCl3) d: 12.34, 28.00,
42.98, 65.29, 70.54, 72.09, 73.66, 75.51, 78.71, 80.83, 81.74,
109.38, 127.45, 127.73, 127.83, 128.06, 128.20, 128.32, 128.39,
136.99, 137.21, 137.60, 137.73, 150.16, 154.13, 163.86. HRMS
(ESI) Anal. Calcd for C36H42NO7 [M+H]+: 628.3017; found 628.3006.
(5 mL) was added and TMSOTf (13 lL, 0.072 mmol) was added
dropwise. The mixture was stirred at 0 °C for further 3 h. The reac-
tion was quenched with cold saturated sodium bicarbonate aque-
ous solution (6 mL) and the resulting mixture was extracted with
CH2Cl2 (40 mL ꢁ 3). The combined organic phases were washed
with brine and dried over anhydrous sodium sulfate. After removal
of the solvent, the resulting residue was purified by flash column
chromatography (petroleum ether/ethyl acetate, 3:1?petroleum
ether/acetone, 4:1) to give the products 5a–c, 6a, 6c.
4.12. (2R,3R,4R,5R)-tert-Butyl 3,4,5-tris(benzyloxy)-2-(thymidine-
1-yl)piperidine-1-carboxylate (5a) and (2S,3R,4R,5R)-tert-butyl
3,4,5-tris(benzyloxy)-2-(thymidine-1-yl)piperidine-1-carboxy-
late (6a)
These two compounds were products of the coupling reaction of
4a with thymine. The less polar one was confirmed by NMR to be
compound 5a (70% yield, colorless foam). Rf = 0.30 (petroleum
ether/ethyl acetate, 1:1). 1H NMR (500 MHz, CDCl3) d: 1.38 (s, 9H),
1.83 (s, 3H), 3.63–3.67 (m, 2H), 3.79 (d, J = 1.0 Hz, 1H), 4.28–4.32
(m, 1H), 4.38–4.42 (m, 2H), 4.52 (d, J = 12.0 Hz, 1H), 4.68–4.71 (m,
3H), 4.80 (d, J = 12.0 Hz, 1H), 5.02 (br s, 1H), 6.90 (br s, 1H), 7.12–
7.14 (m, 2H), 7.23–7.37 (m, 12H), 7.93 (br s, 1H). 13C NMR
(75 MHz, CDCl3) d: 12.13, 28.20, 41.26, 70.17, 72.87, 74.51, 74.91,
80.10, 81.59, 84.77, 108.76, 127.78, 127.85, 127.95, 128.14,
128.39, 128.42, 128.80, 137.17, 137.61, 137.95, 141.97, 149.69,
154.60, 163.68. HRMS (ESI) Anal. Calcd for C36H42NO7 [M+H]+:
628.3017; found 628.3017. The more polar compound was con-
firmed by NMR to be compound 6a (11% yield, colorless foam).
Rf = 0.29 (petroleum ether/ethyl acetate, 1:1). 1H NMR (500 MHz,
CDCl3) d: 1.35 (s, 9H), 1.82 (s, 3H), 3.42 (dd, J = 10.0 Hz, J = 12.5 Hz,
1H), 3.73 (br s, 1H), 3.83 (br s, 1H), 4.05 (br s, 1H), 4.31 (d,
J = 11.5 Hz, 1H), 4.44 (br s, 1H), 4.49–4.60 (m, 5H), 6.24 (d,
J = 4.5 Hz, 1H), 7.05 (s, 1H), 7.12–7.14 (m, 2H), 7.27–7.37 (m, 12H),
7.93 (s, 1H). 13C NMR (75 MHz, CDCl3) d: 12.33, 28.06, 41.98,
63.99, 71.42, 71.94, 73.32, 75.15, 81.97, 109.16, 127.7 4, 127.94,
128.04, 128.30, 128.46, 128.50, 128.53, 136.50, 137.35, 137.71,
4.15. (2R,3R,4R,5R)-tert-Butyl 3,4,5-trihydroxy-2-(thymidine-1-
yl)piperidine-1-carboxylate (7a)
To the solution of 5a (21 mg, 0.033 mmol) in MeOH (4 mL) were
added hydrogen chloride solution (1.25 M in methanol, 0.2 mL)
and Pd/C catalyst (10% wt, 8 mg). The reaction mixture was stirred
under the atmosphere of hydrogen gas for 60 h. The reaction
mixture was filtered through celite pad and the filtrate was con-
centrated. The residue was subjected to a C-18 reverse-phase col-
umn chromatography (H2O/CH3OH = 2:1) to give 7a (12 mg, 94%)
as a solid after lyophilization. 1H NMR (300 MHz, CD3OD) d: 1.40
(s, 9H), 1.87 (s, 3H), 3.40 (dd, J = 3.0 Hz, J = 9.9 Hz, 1H), 3.77–3.96
(m, 3H), 4.15 (t, J = 11.1 Hz, 1H), 5.20 (d, J = 8.4 Hz, 1H), 7.33 (s,
1H). 13C NMR (75 MHz, CD3OD) d: 12.55, 28.56, 47.62, 70.42,
74.88, 76.28, 77.89, 82.53, 109.98, 143.24, 154.28, 156.45. HRMS
(ESI) Anal. Calcd for C15H23N3O7Na [M+Na]+: 380.1428; found
380.1421.

48
D. Wang et al. / Bioorg. Med. Chem. 19 (2011) 41–51
4.16. (2S,3R,4R,5S)-tert-Butyl 3,4,5-trihydroxy-2-(thymine-1-yl)
piperidine-1-carboxylate (7b)
76.32, 86.31, 152.54, 173.63. HRMS (ESI) Anal. Calcd for
C
10H17NO6Na [M+Na]+: 270.0948; found 270.0950.
Compound 7b was prepared from 5b as described in the prepa-
ration of 7a from 5a, yielding 7b (89% yield) as colorless solid after
lyophilization. 1H NMR (500 MHz, C5D5N) d: 1.39 (s, 9H), 1.95 (s,
3H), 4.15 (dd, J = 10.5 Hz, J = 12.5 Hz, 1H), 4.60 (t, J = 3.5 Hz, 1H),
4.80 (dd, J = 6.0 Hz, J = 12.5 Hz, 1H), 4.92–4.96 (m, 2H), 5.06 (m,
1H), 6.74 (d, J = 3.0 Hz, 1H), 6.89 (br s, 1H), 7.27 (br s, 1H), 7.84
(s, 1H), 8.08 (br s, 1H), 13.17 (br s, 1H). 13C NMR (75 MHz, D2O)
d: 12.21, 28.01, 45.21, 64.77, 69.85, 70.18, 71.17, 84.43, 111.49,
139.88, 152.96, 156.71, 167.98. HRMS (ESI) Anal. Calcd for
4.21. (3S,4R,5S)-tert-Butyl 3,4,5-trihydroxy-2-oxopiperidine-1-
carboxylate (9b)
Compound 9b was prepared from 3b as described in the prepa-
ration of 9a, yielding 9b (92% yield) as a colorless oil. 1H NMR
(300 MHz, D2O) d: 1.35 (s, 9H), 3.54 (d, J = 14.1 Hz, 1H), 3.76 (d,
J = 11.7 Hz, 2H), 4.10 (s, 1H), 4.19 (d, J = 9.9 Hz, 1H). 13C NMR
(75 MHz, D2O) d: 27.63, 49.84, 66.73, 71.70, 71.82, 86.21, 153.40,
174.37. HRMS (ESI) Anal. Calcd for
286.0682; found 286.0693.
C
10H17NO6K [M+K]+:
C
15H24N3O7 [M+H]+: 358.1609; found 358.1604.
4.17. (2S,3S,4R,5R)-tert-Butyl 3,4,5-trihydroxy-2-(thymine-1-
4.22. (3R,4R,5R)-tert-Butyl 3,4,5-trihydroxy-2-oxopiperidine-1-
yl)piperidine-1-carboxylate (7c)
carboxylate (9c)
Compound 7c was prepared from 5c as described in the prepara-
tion of 7a from 5a, yielding 7c (92% yield) as a solid after lyophiliza-
tion. 1H NMR (500 MHz, D2O) d: 1.36 (s, 9H), 1.89 (s, 3H), 3.68 (dd,
J = 2.0 Hz, J = 5.0 Hz, 1H), 3.81 (dd, J = 4.5 Hz, J = 15.0 Hz, 1H), 4.01
(t, J = 4.5 Hz, 1H), 4.09 (d, J = 14.5 Hz, 1H), 4.27 (dd, J = 2.5 Hz,
J = 4.5 Hz, 1H), 6.09 (d, J = 4.5 Hz, 1H), 7.31 (d, J = 1.5 Hz, 1H). 13C
NMR (75 MHz, D2O) d: 12.18, 27.85, 47.78, 66.92, 68.68, 72.01,
73.45, 83.94, 111.34, 138.89, 153.00, 156.30, 168.28; HRMS (ESI)
Calcd for C15H23N3O7Na [M+Na]+: 380.1428; found 380.1434.
Compound 9c was prepared from 3c as described in the prepa-
ration of 9a, yielding 9c (98% yield) as a colorless oil. 1H NMR
(300 MHz, D2O) d: 1.38 (s, 9H), 3.47 (dd, J = 6.6 Hz, J = 15.3 Hz,
1H), 3.98–4.06 (m, 3H), 4.43 (d, J = 3.0 Hz, 1H). 13C NMR (75 MHz,
D2O) d: 27.66, 48.45, 67.80, 70.35, 72.78, 86.17, 152.91, 174.99.
HRMS (ESI) Anal. Calcd for C10H17NO6Na [M+Na]+: 270.0948; found
270.0946.
4.23. (2R,3S,4R,5R)-1-(tert-Butoxycarbonyl)piperidine-2,3,4,5-
tetrayl tetraacetate (10a)
4.18. (2S,3R,4R,5R)-tert-Butyl 3,4,5-trihydroxy-2-(thymidine-1-
yl) piperidine-1-carboxylate (8a)
To the cooled solution of 9a (33 mg, 0.13 mmol) in MeOH
(2 mL) was added NaBH4 (4 mg, 0.4 mmol). The reaction mixture
was stirred at 0 °C for 10 min. After removal of the solvent, the res-
idue was subjected to a C-18 reverse-phase column chromatogra-
phy (H2O/CH3OH = 2:1) to give the intermediate product (32 mg,
96%) as a colorless oil. To the solution of the intermediate product
in dry pyridine (5 mL) was added Ac2O (1 mL). The mixture was
then stirred at rt overnight. The residue obtained after removal
of the solvent was purified by column chromatography (EtOAc/
petroleum ether = 1:5) to afford 10a (48 mg, 91%) as a colorless
oil. Rf = 0.45 (petroleum ether/ethyl acetate, 2:1). 1H NMR
(300 MHz, CDCl3) d: 1.47 (s, 9H), 2.01–2.09 (m, 12H), 3.52 (dd,
J = 2.7 Hz, J = 14.1 Hz, 1H), 3.96 (d, J = 14.7 Hz, 1H), 5.00–5.06 (m,
3H), 6.62 (br s, 1H). 13C NMR (75 MHz, CDCl3) d: 20.61, 20.72,
20.79, 20.83, 28.13, 39.76, 68.36, 68.80, 69.09, 77.17, 81.71,
153.67, 168.66, 169.03, 169.11, 169.77. HRMS (ESI) Anal. Calcd
for C18H27NO6K [M+K]+: 456.1261; found 456.1258.
Compound 8a was prepared from 6a as described in the prepa-
ration of 7a from 5a, yielding 8a (74% yield) as a solid after lyoph-
ilization. 1H NMR (500 MHz, CD3OD) d: 1.39 (s, 9H), 1.87 (s, 3H),
3.40 (dd, J = 10.5 Hz, J = 12.5 Hz, 1H), 3.63–3.67 (m, 1H), 3.85 (dd,
J = 4.0 Hz, 5.5 Hz, 1H), 4.02 (t, J = 5.0 Hz, 1H), 4.20 (dd, J = 6.5 Hz,
J = 13.0 Hz, 1H), 6.16 (d, J = 5.0 Hz, 1H), 7.34 (d, J = 1.0 Hz, 1H).
13C NMR (75 MHz, CD3OD) d: 12.44, 28.42, 45.84, 67.06, 71.59,
72.35, 76.01, 82.73, 110.09, 141.02, 152.61, 156.08, 166.79. HRMS
(ESI) Anal. Calcd for C15H23N3O7Na [M+Na]+: 380.1428; found
380.1419.
4.19. (2R,3S,4R,5R)-tert-Butyl 3,4,5-trihydroxy-2-(thymine-1-
yl)piperidine-1-carboxylate (8c)
Compound 8c was prepared from 6c as described in the prepara-
tion of 7a from 5a, yielding 8c (89% yield) as a solid after lyophiliza-
tion. 1H NMR (300 MHz, D2O) d: 1.22 (s, 9H), 1.73 (s, 3H), 3.03–3.13
(m, 1H), 3.77–3.81 (m, 2H), 4.13 (dd, J = 7.2 Hz, J = 13.5 Hz, 1H), 4.28
4.24. (2R,3S,4R,5S)-1-(tert-Butoxycarbonyl)piperidine-2,3,4,5-
tetrayl tetraacetate (10b)
(dd, J = 2.7 Hz, J = 8.1 Hz, 1H), 5.50 (d, J = 7.8 Hz, 1H), 7.25 (s, 1H); 13
C
NMR (75 MHz, D2O) d: 12.08, 27.94, 44.88, 68.12, 69.71, 71.03,
73.79, 84.34, 111.42, 141.09, 153.37, 156.29, 168.65; HRMS (ESI)
Calcd for C15H23N3O7Na [M+Na]+: 380.1428; found 380.1421.
To the cooled solution of 9b (31 mg, 0.13 mmol) in MeOH
(2 mL) was added NaBH4 (4 mg, 0.4 mmol). Then, at the 10th,
15th, and 20th min, NaBH4 (4 mg, 0.4 mmol) was added respec-
tively. After removal of the solvent at the 25th min, the residue
was subjected to a C-18 reverse-phase column chromatography
(H2O/CH3OH = 2:1) to give the intermediate product (30 mg, 95%)
as a colorless oil. To the solution of the intermediate product in
dry pyridine (5 mL) was added Ac2O (1 mL). The mixture was then
stirred at rt overnight. The residue obtained after removal of the
solvent was purified by column chromatography (EtOAc/petro-
leum ether = 1:5) to afford 10b (46 mg, 91%) as a colorless oil.
Rf = 0.45 (petroleum ether/ethyl acetate, 2:1). 1H NMR (300 MHz,
CDCl3) d: 1.47 (s, 9H), 2.04 (s, 3H), 2.08 (s, 3H), 2.12 (s, 3H), 2.13
(s, 3H), 3.28 (t, J = 11.7 Hz, 1H), 4.08 (br s, 1H), 5.05–5.15 (m,
2H), 5.25 (s, 1H), 6.57 (s, 1H). 13C NMR (75 MHz, CDCl3) d: 19.15,
20.71, 28.10, 64.97, 66.63, 67.88, 75.66, 77.21, 81.95, 153.59,
4.20. (3S,4R,5R)-tert-Butyl 3,4,5-trihydroxy-2-oxopiperidine-1-
carboxylate (9a)
To the solution of 3a (205 mg, 0.397 mmol) in anhydrous EtOAc
(5 mL) was added Pd/C catalyst (10% wt, 20 mg). The mixture was
stirred under the atmosphere of hydrogen gas for 20 h. The reac-
tion mixture was filtered through celite pad and the filtrate was
concentrated to afford 9a (97 mg, 99%) as a colorless oil. No further
purification by chromatography was needed. 1H NMR (300 MHz,
D2O) d: 1.38 (s, 9H), 3.50 (dd, J = 4.5 Hz, J = 9.3 Hz, 1H), 3.67 (dd,
J = 3.0 Hz, J = 13.5 Hz, 1H), 3.78–3.89 (m, 2H), 4.04 (d, J = 9.3 Hz,
1H). 13C NMR (75 MHz, CDCl3) d: 27.69, 47.71, 69.75, 73.89,

D. Wang et al. / Bioorg. Med. Chem. 19 (2011) 41–51
49
168.44, 168.63, 169.38, 169.60. HRMS (ESI) Anal. Calcd for
18H27NO6K [M+K]+: 456.1261; found 456.1256.
1H), 5.30 (dd, J = 3.2 Hz, J = 4.4 Hz, 1H), 5.78 (dd, J = 2.8 Hz,
J = 5.6 Hz, 1H), 6.96 (d, J = 5.6 Hz, 1H), 7.52–7.55 (m, 2H),
7.60–7.62 (m, 1H), 8.03 (d, J = 7.2 Hz, 2H), 8.17 (s, 1H), 8.80 (s, 1H),
9.01 (s, 1H). 13C NMR (100 MHz, CDCl3) d: 20.47, 20.67, 20.92,
27.94, 42.80, 62.18, 65.78, 68.09, 68.93, 82.86, 121.82, 127.81,
128.88, 132.82, 133.55, 141.31, 149.42, 152.32, 152.82, 153.59,
164.52, 168.98, 169.05, 169.66. HRMS (ESI) Anal. Calcd for
C
4.25. (2R,3R,4R,5R)-1-(tert-Butoxycarbonyl)piperidine-2,3,4,5-
tetrayl tetraacetate (10c)
Compound 10c was prepared from 9c as described in the prepa-
ration of 10b, yielding 10c (75% yield for two steps) as a pale yellow
oil. 1H NMR (300 MHz, CDCl3) d: 1.46 (s, 9H), 2.02 (s, 3H), 2.09 (s, 3H),
2.10 (s, 3H), 2.13 (s, 3H), 3.42 (m, 1H), 4.13 (d, J = 14.7 Hz, 1H), 4.99
(br s, 1H), 5.26–5.28 (m, 2H), 6.92 (br s, 1H). 13C NMR (75 MHz,
CDCl3) d: 20.50, 20.69, 20.83, 28.02, 40.40, 61.79, 65.48, 66.29,
69.43, 81.93, 153.61, 169.00, 169.26, 169.35, 169.45. HRMS (ESI)
Anal. Calcd for C18H27NO6Na [M+Na]+: 440.1527; found 440.1527.
C
28H33N6O9 [M+H]+: 597.2304; found 597.2306.
4.29. (2S,3R,4R,5R)-1-(tert-Butoxycarbonyl)-2-(5-fluoro-uracil-
1-yl) piperidine-3,4,5-triyl triacetate (12a)
To a stirred solution of 5-fluoro-uracil (50 mg, 0.36 mmol) in
anhydrous acetonitrile (2 mL) under argon was added N,O-bis(tri-
methylsilyl)acetamide (0.14 mL, 0.58 mmol). The reaction mixture
was stirred under reflux for 20 min. After cooled to 0 °C, compound
10a (30 mg, 0.072 mmol) in anhydrous acetonitrile (5 mL) was
4.26. (2S,3R,4R,5R)-2-(N6-Benzoyl-adenine-9-yl)-1-(tert-
butoxycarbonyl)piperidine-3,4,5-triyl triacetate (11a)
added and TMSOTf (26 lL, 0.144 mmol) was added dropwise. The
To a stirred solution of N6-benzoyladenine (75 mg, 0.317 mmol)
in anhydrous acetonitrile (2 mL) under argon was added
N,O-bis(trimethylsilyl)acetamide (0.12 mL, 0.48 mmol). The reac-
tion mixture was stirred under reflux for 20 min. After cooled to
0 °C, compound 10a (33 mg, 0.079 mmol) in anhydrous acetonitrile
solution was stirred at 0 °C for 1 h. The reaction was then quenched
with cold saturated sodium bicarbonate aqueous solution (6 mL)
and the resulting mixture was extracted with CH2Cl2 (40 mL ꢁ 3).
The combined organic phases were washed with brine and dried
over anhydrous Na2SO4. After removal of the solvent, the resulting
residue was purified by flash column chromatography (petroleum
ether/ethyl acetate, 5:1?CH2Cl2/MeOH, 80:1) to give 12a (31 mg,
87%) as a colorless foam. Rf = 0.30 (CH2Cl2/MeOH, 20:1). Only crude
product of 12a was obtained because some inseparable impurity
accompanied 12a all the time, which was directly used for the next
reaction.
(5 mL) was added and TMSOTf (29 lL, 0.160 mmol) was added
dropwise. The solution was stirred at 0 °C for 1 h and at rt for fur-
ther 1.5 h. The reaction was then quenched with cold saturated so-
dium bicarbonate aqueous solution (6 mL) and the resulting
mixture was extracted with CH2Cl2 (40 mL ꢁ 3). The combined or-
ganic phases were washed with brine and dried over anhydrous
Na2SO4. After removal of the solvent, the resulting residue was
purified by flash column chromatography (petroleum ether/ethyl
acetate, 5:1?CH2Cl2/MeOH, 70:1) to give 11a (29 mg, 61%) as a
colorless foam. Rf = 0.25 (CH2Cl2/MeOH, 25:1). Only crude product
of 11a was obtained because about 10% of its anomer and other
impurity accompanied 11a all the time, which was directly used
for the next reaction.
4.30. (2S,3R,4R,5S)-1-(tert-Butoxycarbonyl)-2-(5-fluoro-uracil-
1-yl)piperidine-3,4,5-triyl triacetate (12b)
Compound 12b was prepared from 10b as described in the
preparation of 12a from 10a, yielding 12b (83% yield) as a colorless
foam. Rf = 0.32 (CH2Cl2/MeOH, 30:1). 1H NMR (400 MHz, CDCl3) d:
1.47 (s, 9H), 2.02 (s, 3H), 2.04 (s, 3H), 2.11 (s, 3H), 3.86
(d, J = 14.0 Hz, 1H), 4.24 (dd, J = 3.2 Hz, J = 14.4 Hz, 1H), 5.33 (dd,
J = 8.4 Hz, J = 10.0 Hz, 1H), 5.45 (d, J = 1.6 Hz, 1H), 5.83
(dd, J = 3.2 Hz, J = 10.0 Hz, 1H), 6.13 (d, J = 8.4 Hz, 1H), 7.38 (d,
J = 5.2 Hz, 1H), 9.26 (s, 1H). 13C NMR (100 MHz, CDCl3) d: 20.39,
20.67, 20.82, 28.14, 45.14, 67.18, 67.24, 69.05, 70.19, 83.01,
130.60, 130.94, 138.00, 140.37, 149.65, 154.68, 156.88, 157.15,
169.63, 169.80, 170.69. HRMS (ESI) Anal. Calcd for C20H26FN3O10Na
[M+Na]+: 510.1494; found 510.1501.
4.27. (2S,3R,4R,5R)-2-(N6-Benzoyl-adenine-9-yl)-1-(tert-
butoxycarbonyl)piperidine-3,4,5-triyl triacetate (11b) and
(2R-3R,4R,5R)-2-(N6-benzoyl-adenine-9-yl)-1-(tert-
butoxycarbonyl)piperidine-3,4,5-triyl triacetate (11b0)
Compounds 11b and 11b0 were prepared from 10b as described
in the preparation of 11a, yielding the inseparable mixture of 11b
and 11b0 (11:7, 68% yield in total) as a colorless oil, which was di-
rectly used for the next reaction. Rf = 0.28 (CH2Cl2/MeOH, 25:1).
4.31. (2R,3S,4R,5R)-1-(tert-Butoxycarbonyl)-2-(5-fluoro-uracil-
1-yl)piperidine-3,4,5-triyl triacetate (12c)
4.28. (2R,3S,4R,5R)-2-(N6-Benzoyl-adenine-9-yl)-1-(tert-
butoxycarbonyl)piperidine-3,4,5-triyl triacetate (11c) and
(2S,3S,4R,5R)-2-(N6-benzoyl-adenine-9-yl)-1-(tert-
butoxycarbonyl)piperidine-3,4,5-triyl triacetate (11c0)
Compound 12c was prepared from 10c as described in the prep-
aration of 12a from 10a, yielding 12c (57% yield) as a colorless
foam. Rf = 0.30 (CH2Cl2/MeOH, 30:1). 1H NMR (400 MHz, CDCl3)
d: 1.49 (s, 9H), 2.06 (s, 3H), 2.12 (s, 6H), 3.59 (dd, J = 9.6 Hz,
J = 14.0 Hz, 1H), 4.44 (dd, J = 7.6 Hz, J = 14.0 Hz,1H), 4.89–4.94 (m,
1H), 5.37–5.39 (m, 2H), 5.98 (dd, J = 2.8 Hz, J = 8.4 Hz, 1H), 7.37
(d, J = 5.6 Hz, 1H), 9.06 (d, J = 3.2 Hz, 1H). 13C NMR (100 MHz,
CDCl3) d: 20.58, 20.64, 20.75, 28.13, 42.42, 67.02, 69.50, 70.65,
72.13, 82.91, 128.93, 129.26, 138.62, 140.99, 148.92, 154.12,
156.59, 156.86, 169.20, 169.48, 169.60. HRMS (ESI) Anal. Calcd
for C20H26FN3O10Na [M+Na]+: 510.1494; found 510.1482.
Compounds 11c and 11c0 were prepared from 10c as described
in the preparation of 11a, yielding 11c (51%, the less polar product)
as a colorless oil. Rf = 0.25 (CH2Cl2/MeOH, 25:1). 1H NMR (300 MHz,
CDCl3) d: 1.46 (s, 9H), 2.01 (s, 3H), 2.13 (s, 3H), 2.17 (s, 3H), 3.80 (dd,
J = 10.5 Hz, J = 13.2 Hz, 1H), 4.51 (m, 1H), 5.09–5.16 (m, 1H), 5.61 (s,
1H), 6.31–6.32 (m, 2H), 7.51–7.65 (m, 3H), 8.02–8.12 (m, 3H), 8.83
(s, 1H), 9.03 (s, 1H). 13C NMR (75 MHz, CDCl3) d: 20.56, 20.68,
20.89, 28.09, 65.99, 68.04, 68.76, 70.11, 77.20, 82.76, 123.64,
127.83, 128.89, 132.85, 133.45, 149.57, 151.54, 164.49, 169.31,
169.42, 169.83. HRMS (ESI) Anal. Calcd for C28H33N6O9 [M+H]+:
597.2304; found 597.2306. The more polar product was 11c0 (30%,
colorless oil). Rf = 0.23 (CH2Cl2/MeOH, 25:1). 1H NMR (400 MHz,
CDCl3) d: 1.28 (s, 9H), 1.85 (s, 3H), 2.03 (s, 3H), 2.16 (s, 3H), 3.90
(dd, J = 2.4 Hz, J = 15.2 Hz, 1H), 4.47 (d, J = 15.2 Hz,1H), 5.20 (br s,
4.32. (2S,3R,4R,5R)-tert-Butyl 2-(uracil-1-yl)-3,4,5-
trihydroxypiperidine-1-carboxylate (13a)
To a stirred solution of uracil (40 mg, 0.36 mmol) in anhydrous
acetonitrile (2 mL) under argon was added N,O-bis(trimethylsilyl)

50
D. Wang et al. / Bioorg. Med. Chem. 19 (2011) 41–51
acetamide (0.14 mL, 0.58 mmol). The reaction mixture was stirred
under reflux for 15 min. After cooled to 0 °C, compound 10a
(30 mg, 0.072 mmol) in anhydrous acetonitrile (5 mL) was added
(400 MHz, D2O) d: 1.10 (s, 9H), 3.48 (dd, J = 9.6 Hz, J = 13.2 Hz, 1H),
3.91 (dd, J = 3.6 Hz, J = 6.0 Hz, 1H), 4.05 (dd, J = 6.0 Hz, J = 13.2 Hz,
1H), 4.14–4.18 (m, 1H), 4.31 (dd, J = 4.0 Hz, J = 6.0 Hz, 1H), 6.08 (d,
J = 4.0 Hz, 1H), 8.15 (s, 1H), 8.20 (s, 1H). 13C NMR (75 MHz, D2O) d:
27.66, 43.56, 49.38, 64.50, 67.50, 70.00, 70.46, 84.23, 118.41,
141.31, 149.27, 153.10, 156.08, 156.60. HRMS (ESI) Anal. Calcd for
and TMSOTf (26 lL, 0.144 mmol) was added dropwise. The solution
was stirred at 0 °C for 1 h. The reaction was then quenched with cold
saturated sodium bicarbonate aqueous solution (6 mL) and the
resulting mixture was extracted with CH2Cl2 (40 mL ꢁ 3). The
combined organic phases were washed with brine and dried over
anhydrous Na2SO4. After removal of the solvent, the resulting
residue was purified by flash column chromatography (petroleum
ether/ethyl acetate, 5:1?CH2Cl2/MeOH, 80:1) to give 13a (35 mg
P 100%) as a colorless foam. Rf = 0.35 (CH2Cl2/MeOH, 25:1). Only
crude product of 13a was obtained because some inseparable impu-
rity accompanied 13a all the time, which was directly used for the
next reaction.
C
15H22N6O5Na [M+Na]+: 389.1544; found 389.1550. The more polar
product was 15b0 (6 mg, 37%, white solids after lyophilization).
1H NMR (400 MHz, D2O) d: 1.13 (s, 9H), 3.56 (dd, J = 1.6 Hz,
J = 14.8 Hz, 1H), 4.02 (dd, J = 3.2 Hz, J = 10.8 Hz, 1H), 4.09
(dd, J = 2.4 Hz, J = 14.8 Hz, 1H), 4.17–4.18 (m, 1H), 4.26 (dd,
J = 6.4 Hz, J = 10.4 Hz, 1H), 6.71 (d, J = 6.4 Hz, 1H), 8.13 (s, 1H), 8.19
(s, 1H). 13C NMR (75 MHz, D2O) d: 27.76, 46.99, 65.96, 66.90,
67.81, 69.73, 84.19, 118.15, 141.33, 150.35, 153.09, 156.14,
156.52. HRMS (ESI) Anal. Calcd for
389.1544; found 389.1555.
C
15H22N6O5Na [M+Na]+:
4.33. (2S,3R,4R,5R)-2-(4-Acetamido-cytosine-1-yl)-1-(tert-
butoxycarbonyl)piperidine-3,4,5-triyl triacetate (14a)
4.36. (2R,3S,4R,5R)-tert-Butyl 2-(adenine-9-yl)-3,4,5-
trihydroxypiperidine-1-carboxylate (15c)
Toastirredsolutionof4-acetamido-cytosine(101 mg, 0.66 mmol)
in anhydrous acetonitrile (2 mL) under argon was added N,
O-bis(trimethylsilyl)acetamide (0.19 mL, 0.79 mmol). The reaction
mixture was stirred under reflux for 15 min. After cooled to 0 °C,
compound 10a (55 mg, 0.132 mmol) in anhydrous acetonitrile
Compound 11c (14 mg, 0.023 mmol) was dissolved in MeOH sat-
urated with ammonia (5 mL) and stirred at rt overnight. The reaction
mixture was concentrated, and the residue was purified by C-18 re-
verse-phase column chromatography (H2O/CH3OH = 2:1) to afford
15c (8 mg, 93%) as colorless solids after lyophilization. 1H NMR
(400 MHz, D2O) d: 1.17 (s, 9H), 3.17 (dd, J = 10.0 Hz, J = 13.6 Hz,
1H), 3.81 (dd, J = 3.2 Hz, J = 6.0 Hz, 1H), 3.94 (td, J = 6.0 Hz,
J = 10.0 Hz, 1H), 4.28 (dd, J = 6.4, J = 13.6 Hz, 1H), 4.57 (dd,
J = 2.8 Hz, J = 5.2 Hz, 1H), 6.19 (d, J = 5.6 Hz, 1H), 8.14 (s, 2H). 13C
NMR (100 MHz, D2O) d: 27.19, 44.64, 67.42, 67.44, 69.10, 71.92,
83.76, 118.46, 140.44, 149.00, 152.76, 155.60, 155.72. HRMS (ESI)
Anal. Calcd for C15H23N6O5 [M+H]+: 367.1724; found 367.1725.
(5 mL) was added and TMSOTf (48 lL, 0.264 mmol) was added
dropwise. The mixture was stirred at 0 °C for 1.5 h. The reaction
was then quenched with cold saturated sodium bicarbonate aque-
ous solution (6 mL) and the resulting mixture was extracted with
CH2Cl2 (40 mL ꢁ 3). The combined organic phases were washed
with brine and dried over anhydrous Na2SO4. After removal of
the solvent, the resulting residue was purified by flash column
chromatography (petroleum ether/ethyl acetate, 3:1?CH2Cl2/
MeOH, 80:1) to give 14a (51 mg, 76%) as a colorless foam.
Rf = 0.32 (CH2Cl2/MeOH, 25:1). Only crude product of 14a was ob-
tained because some inseparable impurity accompanied 14a all the
time, which was directly used for the next reaction.
4.37. (2S,3S,4R,5R)-tert-Butyl 2-(adenine-9-yl)-3,4,5-
trihydroxypiperidine- 1-carboxylate (15c0)
Compound 15c0 was prepared from 11c0 as described in the
preparation of 15c from 11c, yielding 15c0 (90% yield) as colorless
solids after lyophilization. 1H NMR (300 MHz, CD3OD) d: 1.22 (s,
9H), 3.75–3.84 (m, 2H), 4.02–4.11 (m, 2H), 4.31 (dd, J = 2.7 Hz,
J = 5.7 Hz, 1H), 6.61 (d, J = 5.7 Hz, 1H), 8.19 (s, 1H), 8.34 (s, 1H);
13C NMR (75 MHz, CD3OD)d: 28.31, 45.43, 65.76, 67.66, 71.07,
72.64, 82.61, 118.89, 142.64, 151.35, 153.41, 156.35, 157.11; HRMS
(ESI) Calcd for C15H23N6O5 [M+H]+: 367.1724; found 367.1715.
4.34. (2S,3R,4R,5R)-tert-Butyl 2-(adenine-9-yl)-3,4,5-
trihydroxypiperidine-1-carboxylate (15a)
The crude product obtained in the previous reaction (compound
11a, 29 mg, 0.049 mmol) was dissolved in MeOH saturated with
ammonia (5 mL) and stirred at rt overnight. The reaction mixture
was concentrated, and the residue was purified by C-18 reverse-
phase column chromatography (H2O/CH3OH = 2:1). Further purifi-
cation was carried out using preparative HPLC (C-18, 40% MeOH in
water) to yield compound 15a (15 mg, 82%, white solids). 1H NMR
(300 MHz, D2O) d: 1.00 (s, 9H), 3.51 (dd, J = 3.9 Hz, J = 10.5 Hz, 1H),
3.85–3.90 (m, 3H), 4.13 (dd, J = 9.0 Hz, J = 10.2 Hz, 1H), 5.71 (d,
J = 9.0 Hz, 1H), 8.08 (s, 1H), 8.15 (s, 1H). 13C NMR (100 MHz, D2O)
d: 27.15, 45.70, 68.75, 70.77, 72.98, 75.76, 83.36, 118.33, 141.21,
149.03, 152.67, 155.52, 155.68. HRMS (ESI) Anal. Calcd for
4.38. (2S,3R,4R,5R)-tert-Butyl 2-(5-fluoro-uracil-1-yl)-3,4,5-
trihydroxypiperidine-1-carboxylate (16a)
The crude product obtained in the previous reaction (compound
12a, 31 mg, 0.064 mmol) was dissolved in MeOH saturated with
ammonia (5 mL) and stirred at rt overnight. The reaction mixture
was concentrated, and the residue was purified by C-18 reverse-
phase column chromatography (H2O/CH3OH = 3:1). Further purifi-
cation was carried out using preparative HPLC (C-18, 40% MeOH in
water) to yield compound 16a (20 mg, 85%, white solids). 1H NMR
(300 MHz, D2O) d: 1.20 (s, 9H), 3.34 (dd, J = 3.3 Hz, J = 10.2 Hz, 1H),
3.49 (dd, J = 2.7 Hz, J = 14.7 Hz, 1H), 3.76–3.92 (m, 3H), 5.32 (d,
J = 9.3 Hz, 1H), 7.67 (d, J = 6.0 Hz, 1H). 13C NMR (75 MHz, D2O) d:
28.41, 46.76, 70.36, 73.39, 74.22, 77.00, 84.71, 129.10, 129.53,
140.07, 143.16, 151.90, 156.87, 161.21, 161.54. HRMS (ESI) Anal.
Calcd for C14H20FN3O7Na [M+Na]+: 384.1178; found 384.1170.
C
15H22N6O5Na [M+Na]+: 389.1544; found 389.1549.
4.35. (2S,3R,4R,5S)-tert-Butyl 2-(adening-9-yl)-3,4,5-trihydroxy-
piperidine-1-carboxylate (15b) and (2R,3R,4R,5S)-tert-butyl 2-
(adening-9-yl)-3,4,5-trihydroxypiperidine-1-carboxylate (15b0)
The product obtained in the previous reaction (compounds 11b
and 11b0, 29 mg, 0.049 mmol) was dissolved in MeOH saturated
with ammonia (5 mL) and stirred at rt overnight. The reaction
mixture was concentrated, and the residue was purified by C-18 re-
verse-phase column chromatography (H2O/CH3OH = 2:1). Further
purification was carried out using column chromatography
(CHCl3/CH3OH/NH3ꢂH2O = 100:10:1). The less polar product was
15b (9 mg, 51%, white solids after lyophilization). 1H NMR
4.39. (2S,3R,4R,5S)-tert-Butyl 2-(5-fluoro-uracil-1-yl)-3,4,5-
trihydroxy piperidine-1-carboxylate (16b)
Compound 16b was prepared from 12b as described in the prep-
aration of 15c from 11c, yielding 16b (41% yield) as white solids after

D. Wang et al. / Bioorg. Med. Chem. 19 (2011) 41–51
51
lyophilization. 1H NMR (300 MHz, C5D5N) d: 1.35 (s, 9H), 4.18–4.21
(m, 1H), 4.57 (br s, 1H), 4.65–4.74 (m, 2H), 5.05 (t, J = 6.0 Hz, 1H),
6.98 (d, J = 5.4 Hz, 1H), 8.23 (d, J = 6.9 Hz, 1H); 13C NMR (75 MHz,
C5D5N) d: 16.12, 36.03, 54.76, 56.86, 57.09, 58.05, 68.75, 117.04,
117.48, 127.08, 130.14, 138.93, 143.78, 146.29, 146.63; HRMS
(ESI) Calcd for C14H20FN3O7Na [M+Na]+: 384.1178; found 384.1178.
well flat-bottomed tissue culture and treated with eight consecu-
tive daily doses of tested compounds and lamivudine (purchased
from Glaxo & Welcome Co.) in RPMI1640 medium with 2% fetal bo-
vine serum. HBV nucleic acids and proteins were analyzed at the
end of the treatment period (day 8). HBsAg and HBeAg were ana-
lyzed in culture medium by semi-quantitative EIA. Intracellular
HBV DNA forms were extracted from culture media and analyzed
by a slot blot hybridization technique.
4.40. (2R,3S,4R,5R)-tert-Butyl 2-(5-fluoro-uracil-yl)-3,4,5-
trihydroxy piperidine-1-carboxylate (16c)
Acknowledgments
Compound 16c was prepared from 12c as described in the prep-
aration of 15c from 11c, yielding 16c (81% yield) as white solids after
lyophilization. 1H NMR (400 MHz, D2O) d: 1.30 (s, 9H), 3.07 (dd,
J = 9.6 Hz, J = 14.0 Hz, 1H), 3.81–3.86 (m, 2H), 4.19 (dd, J = 7.6 Hz,
J = 14.4 Hz, 1H), 4.30 (dd, J = 2.4 Hz, J = 7.6 Hz, 1H), 5.60 (d,
J = 7.6 Hz, 1H), 7.72 (d, J = 6.4 Hz, 1H). 13C NMR (100 MHz, D2O) d:
27.36, 44.32, 67.64, 68.93, 70.61, 73.08, 83.96, 128.15, 128.49,
139.34, 141.67, 150.43, 155.58, 159.64, 159.90. HRMS (ESI) Anal.
Calcd for C14H20FN3O7Na [M+Na]+: 384.1178; found 384.1182.
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 90713010) and the Grant
(2009ZX09501-011) from the Ministry of Science and Technology
of China.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.11.063.
4.41. (2S,3R,4R,5R)-tert-Butyl 2-(uracil-1-yl)-3,4,5-
trihydroxypiperidine-1-carboxylate (17a)
References and notes
The crude product obtained in the previous reaction (compound
13a, 35 mg, 0.075 mmol) was dissolved in MeOH saturated with
ammonia (5 mL) and stirred at rt overnight. The reaction mixture
was concentrated, and the residue was purified by C-18 reverse-
phase column chromatography (H2O/CH3OH = 3:1). Further purifi-
cation was carried out using preparative HPLC (C-18, 40% MeOH in
water) to yield compound 17a (16 mg, 62%, white solids). 1H NMR
(300 MHz, D2O) d: 1.25 (s, 9H), 3.39 (d, J = 10.5 Hz, 1H), 3.56 (d,
J = 13.8 Hz, 1H), 3.82–3.87 (m, 2H), 3.97 (t, J = 9.6 Hz, 1H), 5.36
(d, J = 9.3 Hz, 1H), 5.75 (d, J = 7.8 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H).
13C NMR (75 MHz, D2O) d: 28.35, 46.78, 70.18, 73.66, 74.21,
77.00, 84.62, 102.83, 145.94, 152.72, 156.83, 167.56. HRMS (ESI)
Anal. Calcd for C14H21N3O7Na [M+Na]+: 366.1272; found 366.1265.
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The crude product obtained in the previous reaction (compound
14a, 51 mg, 0.10 mmol) was dissolved in MeOH saturated with
ammonia (5 mL) and stirred at rt overnight. The reaction mixture
was concentrated, and the residue was purified by C-18 reverse-
phase column chromatography (H2O/CH3OH = 3:1). Further
purification was carried out using preparative HPLC (C-18, 40%
MeOH in water) to yield compound 18a (24 mg, 71%, white solids).
1H NMR (400 MHz, D2O) d: 1.24 (s, 9H), 3.41 (dd, J = 3.6 Hz,
J = 10.0 Hz, 1H), 3.66 (dd, J = 3.6 Hz, J = 15.2 Hz, 1H), 3.82–3.85
(m, 2H), 4.05 (t, J = 9.6, 1H), 5.25 (d, J = 9.2 Hz, 1H), 5.91 (d,
J = 7.6 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H). 13C NMR (125 MHz, D2O)
d: 28.21, 46.66, 69.92, 74.10, 74.99, 76.96, 84.16, 96.41, 145.96,
146.48, 156.78, 158.25, 166.95. HRMS (ESI) Anal. Calcd for
C
14H22N4O6Na [M+Na]+: 365.1432; found 365.1431.
4.43. HBV antiviral assay
Anti-HBV assays were conducted as described previously.24,25
Briefly, confluent cultures of 2.2.15 cells were maintained on 96-