Design, Synthesis, and Evaluation of Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir: Exomethylene Acycloguanine Nucleosides and Their Monophosphate Derivatives

Article abstract of DOI:10.1080/15257770.2015.1037456

Exomethylene acycloguanine nucleosides 4, 6 and its monophosphate derivatives 5, 7, and 8 have been synthesized. Mitsunobu-type coupling of 2-N-acetyl-6-O-diphenylcarbamoylguanine (11) with primary alcohols proceeded regioselectively to furnish the desire

Full text of DOI:10.1080/15257770.2015.1037456

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Design, Synthesis, and Evaluation of
Anti-HBV Activity of Hybrid Molecules of
Entecavir and Adefovir: Exomethylene
Acycloguanine Nucleosides and Their
Monophosphate Derivatives
a
bc
a
d
Shuhei Imoto , Satoru Kohgo , Ryoh Tokuda , Hiroki Kumamoto ,
e
f
f
Manabu Aoki , Masayuki Amano , Nobuyo Kuwata-Higashi , Hiroaki
cfg
b
Mitsuya & Kazuhiro Haraguchi
a
Click for updates
Faculty of Pharmaceutical Sciences, Sojo University, Nishi-ku,
Kumamoto, Japan
b
Department of Pharmaceutical Sciences, Nihon Pharmaceutical
University, Inamachi, Kita-adachi-gun, Saitama, Japan
c
Center for Clinical Sciences, National Center for Global Health and
Medicine, Shinju-ku, Tokyo, Japan
d
School of Pharmacy, Showa University, Shinagawa-ku, Tokyo, Japan
e
Department of Medical Technology, Kumamoto Health Science
University, Kumamoto, Japan
f
Departments of Infectious Diseases and Hematology, Kumamoto
University School of Medicine, Kumamoto, Japan
g
Experimental Retrovirology Section, HIV and AIDS Malignancy
Branch, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland, USA
Published online: 13 Jul 2015.
To cite this article: Shuhei Imoto, Satoru Kohgo, Ryoh Tokuda, Hiroki Kumamoto, Manabu Aoki,
Masayuki Amano, Nobuyo Kuwata-Higashi, Hiroaki Mitsuya & Kazuhiro Haraguchi (2015) Design,
Synthesis, and Evaluation of Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir:
Exomethylene Acycloguanine Nucleosides and Their Monophosphate Derivatives, Nucleosides,
Nucleotides and Nucleic Acids, 34:8, 590-602, DOI: 10.1080/15257770.2015.1037456
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Nucleosides, Nucleotides and Nucleic Acids, 34:590–602, 2015
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2015.1037456
DESIGN, SYNTHESIS, AND EVALUATION
OF ANTI-HBV ACTIVITY OF HYBRID MOLECULES OF ENTECAVIR
AND ADEFOVIR: EXOMETHYLENE ACYCLOGUANINE NUCLEOSIDES
AND THEIR MONOPHOSPHATE DERIVATIVES
1
2,3
1
4
Shuhei Imoto, Satoru Kohgo, Ryoh Tokuda, Hiroki Kumamoto, Manabu
5
6
6
3,6,7
Aoki, Masayuki Amano, Nobuyo Kuwata-Higashi, Hiroaki Mitsuya,
and Kazuhiro Haraguchi
Faculty of Pharmaceutical Sciences, Sojo University, Nishi-ku, Kumamoto, Japan
Department of Pharmaceutical Sciences, Nihon Pharmaceutical University, Inamachi,
Kita-adachi-gun, Saitama, Japan
2
1
2
3
Center for Clinical Sciences, National Center for Global Health and Medicine, Shinju-ku,
Tokyo, Japan
4
School of Pharmacy, Showa University, Shinagawa-ku, Tokyo, Japan
Department of Medical Technology, Kumamoto Health Science University, Kumamoto,
5
Japan
6
Departments of Infectious Diseases and Hematology, Kumamoto University School of
Medicine, Kumamoto, Japan
7
Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer
Institute, National Institutes of Health, Bethesda, Maryland, USA
2
Received 14 January 2015; accepted 31 March 2015.
Address correspondence to Kazuhiro Haraguchi, Department of Pharmaceutical Sciences, Nihon
Pharmaceutical University, 10281 Komuro, Inamachi, Kita-adachi-gun, Saitama 362-0806, Japan. E-mail:
harakazu@nichiyaku.ac.jp
5
90

Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir
591
Exomethylene acycloguanine nucleosides 4, 6 and its monophosphate derivatives 5, 7, and 8
have been synthesized. Mitsunobu-type coupling of 2-N-acetyl-6-O-diphenylcarbamoylguanine (11)
9
with primary alcohols proceeded regioselectively to furnish the desired N -substituted products in
moderate yield. Evaluation of 4-8 for anti-HBV activity in HepG2 cells revealed that the phospho-
nate derivative 8 was found to exhibit moderated activity (EC50 value of 0.29 μM), but cytotoxicity
(
CC50 value of 39 μM) against the host cells was also observed.
Keywords Nucleoside; acyclonucleoside; nucleoside phosphonate; pro-drug; hybrid;
entecavir; adefovir; anti-HBV activity
INTRODUCTION
Hepatitis B is one of the most prevalent viral diseases in the world
and is known to be a major cause of chronic disease, leading to cirrho-
[
1]
sis/hepatocellular carcinoma. Among the most frequently used drugs for
treatment of the disease[ are the nucleoside analogue entecavir (1)
2]
[3a,b]
and
the nucleotide analogue adefovir (2)[ (Figure 1). Entecavir 1 is especially
4]
considered as one of the best choices for chronic patients due to its lack of
[5]
significant adverse effects. Entecavir is structurally a carbocyclic analogue
ꢁ
ꢁ
of 2 -deoxyguanosine. The exomethylene functionality at the 5 -position of 1
would appear to be an important pharmacophore for significant antiviral ac-
tivity because the potency of carbocyclic dG that truncates the double bond
is 10 times less than that of 1.[ Meanwhile, adefovir 2 is the phosphonate
analogue of monophosphate of acycloadenine nucleoside. The feature of
this class of nucleotide analogues is that the requisite first phosphorylation,
which is a crucial step for the activation of biologically active nucleoside
derivatives, has been bypassed.
3a]
Further studies on the structure–activity relationship of these classes
of nucleosides should increase our knowledge of structural requirements
for developing novel antiviral agents for HBV, and will aid in the search
for better anti-HBV agents. In this context, we have envisioned combin-
ing the above two structural features in one molecule and designed the
exomethylene acyclic guanine nucleosides and its monophosphate deriva-
tives as shown in Figure 2. The initial target molecules are exomethylene
FIGURE 1 Structure of entecavir (1) and adefovir (2).

5
92
S. Imoto et al.
FIGURE 2 Structure of A-MEP (3) and the target molecules (4–8).
propyl-(4, MEP-G) and butyl-(6, MEB-G) guanine nucleosides. The number
ꢁ
ꢁ
of constituted atoms (1 to 4 -position) in the acyclic side chain of MEB-G 6
ꢁ
ꢁ
ꢁ
ꢁ
corresponds to the structure comprising C1 , C5 , C4 , and C7 in entecavir
, whereas MEP-G truncates one-carbon atom in acyclic moiety. -ala-P-MEP-
1
L
G 5 and -ala-P-MEB-G 7 are the respective phosphoalaninate pro-drugs of
L
the monophophates of 4 and 6. Moreover, the phosphonate analogue Piv-
P-MEP-G 8 of 5 was also designed. The phosphonate 8 has a one-carbon
ꢁ
ꢁ
elongated side chain (C1 to C5 ) compared with that of adefovir 2. Herein,
we describe the results of the synthesis of 4-8 and evaluation of their anti-HBV
activity.
RESULTS AND DISCUSSION
Chemistry
Initially, synthesis of G-MEP (4) was carried out (Scheme 1). Synthe-
sis of the adenine counterpart 3 (A-MEP) of the target molecule 4 has
been reported.[ Therefore, according to the procedure given in literature,
6]

Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir
593
SCHEME 1 Synthesis of G-MEP (4) and its monophosphate pro-drug L-ala-P-MEP-G (5).
2
-methylenepropane-1,3-diol (9) was utilized as a starting material. Com-
pound 9 was converted into 2-O-(tert-butyldimethylsilyloxymethyl)prop-2-en-
-ol (10). The literature procedure for the coupling of adenine with acyclic
1
moiety involved the mesylation of 10 followed by nucleophilic substitution
of respective mesylate with nucleobase under basic reaction conditions. To
reduce synthetic steps to the target G-MEP 4, Mitsunobu-type reaction of 10
[7]
with 2-N -acetyl-6-O-diphenylcarbamoylguanine (11) was examined. Thus,
when 10 was reacted with 11 in the presence of DIAD/Ph P in THF at
3
◦
7
0 C, the desired protected acyclopurine nucleoside 12 could be obtained
in 53% yield. Removal of protecting groups in the base moiety was car-
ried out by the treatment of 12 with ammonium hydroxide in methanol
to give guanine derivative 13 in 88% isolated yield. In the Heteronuclear
Multiple-Bond Correlation (HMBC) spectra of 13, the correlation between
ꢁ
ꢁ
9
CH -1 /C-4 and CH -1 /C-8 was observed, by which 13 was assigned as N -
2
2
isomer. Compound 13 was converted to MEP-G 4 in 33% yield by treating
with Bu NF. Finally, 4 was transformed into the phosphoalaninate pro-drug
4
5
(16%) by reaction with methyl chlorophenylphosphoryl P→N - -alaninate
L
[8]
and N -mehtylimidazole in pyridine.
Next, synthesis of G-MEB (6) was performed (Scheme 2). Initially, 4-(tert-
butyldiphenylsilyloxy)-2-methylenebutan-1-ol (16) was prepared from 14 in
the following three steps: (1) silylation of 14, (2) epoxidation of the resulting
silylated alkene, (3) β-elimination of the obtained epoxide 15 with diethy-
[9]
laluminium 2,2,6,6-tetramethylpiperidide. When 16 was reacted with 11

5
94
S. Imoto et al.
SCHEME 2 Synthesis of G-MEB (6) and L-ala-P-MEB-G (7).
9
under the above-mentioned reaction conditions, the desired N -substituted
17 was obtained in 69% isolated yield as a single regio-isomer. Compound
1
7 was converted into 18 in 81% yield by ammonolysis in methanol, and
ꢁ
its HMBC spectra revealed the correlation between CH -1 /C-4 and CH -
2
2
ꢁ
1
/C-8. Desilylation of 18 gave G-MEB (6) in 60% yield. As described above
for 4, MEB-G 6 was transformed into phosphoralaninate pro-drug 7 in 57%
yield.
Finally, synthesis of the phosphonate analogue 8 of -ala-P-MEB-G 7
L
was accomplished as illustrated in Scheme 3. Phosphonate alcohol 19 was
[
6]
prepared from 9 according to the published procedure. Reaction of the
alcohol 19 with 11 under the identical conditions for the synthesis of 17
gave the desired 20 in 62% isolated yield. Treatment of 20 with aqueous
ammonia in methanol provided acycloguanine phosphonate derivative 21
in 54% yield, and at this stage, the regiochemistry was confirmed on the
basis of HMBC spectrum, in which the same correlations as for 4 and 6
were observed. The phosphonate 21 was transformed into phosphonic acid
(
22) in 74% yield by treatment with TMSBr in CH Cl , and the resulting
2 2
phosphonic acid 22 was converted into its pivaloyloxymethyl (POM) ester
8; Piv-P-MEP-G) in 10% yield.[
10]
(
Biological Evaluation
Evaluation of anti-HBV activity of the novel acyclonucleosides synthe-
sized in this study was conducted with HepG2 2.2.15 cells transfected with
the HBV genome.[ As shown in Table 1, G-MEP 4 and G-MEB 6 neither
11]

Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir
595
SCHEME 3 Synthesis of Piv-P-MEP-G (8).
exhibit anti-HBV activity nor display any cytotoxicity toward HepG2 cells
entries 1 and 2). These results revealed that the conformationally rigid cy-
(
clopentane ring of entecavir is an essential structure for antiviral activity.
Moreover, inactivity of phosphoalaninates 5 and 7 suggested that the initial
phosphorylation step of 4 and 6 is not responsible for the lack of anti-HBV
activity.
In contrast with the above results, the POM-protected phosphonate
derivative 8 was found to possess antiviral activity with an EC50 of 0.29 μM,
which is 1500 times less potent than that of entecavir. However, cytotoxic-
ity toward HepG2 cells was also observed. With POM-protected prodrugs,
it has been pointed out that the toxicity could be attributed to pivalic acid
[10b]
generated during the release of parent compounds.
Therefore, we have
evaluated the toxicity of pivalic acid in this assay system and found that the
TABLE 1 Anti-HBV activity and cytotoxicity of 4-8
Compound
EC50 (μM)
CC50 (μM) (MT-4)
4
5
6
7
>1
>1
>1
>1
0.29
0.00077
>100
>100
>100
>100
39
8
a
Entecavir
ND
aND: not determined.

5
96
S. Imoto et al.
acid did not show any cytotoxicity up to 100 μM, which most likely ruled out
this issue.
CONCLUSIONS
We have synthesized the novel exomethylene acycloguanine nucleosides
MEP-G (4) and MEB-G (6). The respective monophosphate derivatives, that
is, phosphoalaninate pro-drugs -ala-P-MEP-5 and -ala-P-MEB-G 7 of the
L
L
monophosphate of 4 and 6 were also synthesized. Furthermore, the phos-
phonate analog Piv-P-MEP-G (8) of 7 was also evaluated for anti-HBV activity.
Mitsunobu-type alkylation of 2-N -acetyl-6-O-diphenylcarbamoylguanine (11)
9
with respective primary alcohols proceeded regioselectively at its N -position
and this synthetic strategy enabled us to shorten the synthetic route for the
target molecules compared with the previously reported method. Evaluation
of anti-HBV activity and its cytotoxicity toward HepG2 cells revealed that the
rigidity of aglycon moiety is an important requirement for the inhibition of
HBV replication. The phosphonate derivative 8 showed moderate activity
with an EC50 of 0.29 μM, and the selectivity index (SI) was 137, although
some cytotoxicity (CC50 of 39 μM) was observed.
EXPERIMENTAL
General
Melting points are uncorrected. Reagents and solvents were used without
any further purification unless otherwise noted. Thin layer chromatography
(
0
TLC) was performed using precoated TLC plates (Merck, Silica gel 60 F254,
.25 mm). H and C NMR spectra were recorded on a JEOL ECA 500
1
13
spectrometer operating at room temperature. Chemical shifts are reported
in parts per million (δ) relative to the residual solvent peak. DSS served as
13
internal standard for C NMR measurements in D O. Mass spectra analysis
2
was performed on a JEOL JMS-T100LP.
2-Acetyl -9-[2-(tert-butyldimethylsilyloxymethyl)allyl]-6-O-
diphenylcarbamoyl (12)
To a solution of 10 (989 mg, 5.68 mmol), triphenylphosphine (1.49 g,
5
(
.68 mmol) and 11 (2.10 g, 5.41 mmol) in THF (50 mL) was added DIAD
◦
1.15 g, 5.68 mmol). The reaction mixture was heated to 70 C and stirred for
2
h. After cooling, the mixture was filtered through a celite pad. The filtrate
was evaporated, and the residue was purified by silica gel column chromatog-
raphy (Hexane-EtOAc 3:2 to 2:3, v/v) to provide 12 (1.71 g, 2.99 mmol, 53%
1
yield) as a white foam. H NMR (500 MHz, CDCl ) δ 8.05 (brs, 1H), 7.95
3
(s, 1H), 7.43-7.25 (m, 10H), 5.23 (s, 1H), 4.96 (s, 1H), 4.80 (s, 2H), 4.10 (s,

Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir
597
1
3
2
1
1
H), 2.56 (s, 3H), 0.89 (s, 9H), 0.05 (s, 6H); C NMR (126 MHz, CDCl ) δ
3
71.08, 156.09, 155.13, 152.13, 150.36, 144.18, 142.43, 141.69, 129.15, 127.08,
25.86, 120.52, 114.27, 64.08, 45.50, 25.77, 25.13, 18.22, −5.46. MS (ESI) m/z
+
(
M+Na) calcd. 595.2465, found 595.2485.
9-[2-(tert-Butyldimethylsilyloxymethyl)allyl]guanine (13)
To a solution of 12 (868 mg, 1.51 mmol) in methanol (6 mL) was added
8% ammonia solution (3 mL) and heated to 60 C in a sealed tube. After
◦
2
2
h, the reaction mixture was concentrated and purified by silica gel column
chromatography (CH Cl -MeOH 92:8 to 84:16, v/v) to provide 13 (446 mg,
2
2
1
1
1
4
.33 mmol, 88% yield) as a white solid. H NMR (500 MHz, DMSO-d ) δ
6
0.58 (brs, 1H), 7.60 (s, 1H), 6.42 (brs, 2H), 5.12 (s, 1H), 4.65 (s, 1H),
.54 (s, 2H), 4.11 (s, 2H), 0.86 (s, 9H), 0.03 (s, 6H); 13C NMR (126 MHz,
DMSO-d ) δ 157.05, 153.80, 151.52, 144.56, 137.67, 116.57, 110.84, 63.69,
6
+
4
3
4.20, 25.96, 18.15, –5.31; MS (ESI) m/z (M+Na) calcd. 358.1675, found
58.1714.
9-[(Hydroxymethyl)allyl]guanine (4)
To a suspension of 13 (421 mg, 1.26 mmol) in THF (15 mL) was added
M TBAF in THF solution (3.14 mL, 3.14 mmol). After 2 h, the reaction
1
mixture was concentrated. The residue was recrystalized from MeOH to
1
provide 3 (91 mg, 0.41 mmol, 33% yield) as a white solid. H NMR (500 MHz,
DMSO-d ) δ 10.57 (brs, 1H), 7.63 (s, 1H), 6.45 (brs, 2H), 5.07 (s, 1H), 5.05
6
13
(
t, J = 5.4 Hz, 1H), 4.56 (s, 3H), 3.91(d, J = 5.7 Hz, 2H); C NMR (126 MHz,
DMSO-d ) δ 157.01, 153.78, 151.51, 145.86, 137.82, 116.53, 110.25, 62.19,
6
+
4
4.44; MS (ESI) m/z (M+Na) calcd. 244.0810, found 244.0827.
Synthesis of -ala-P-MEP-G (5)
L
To a solution of 4 (100 mg, 0.452 mmol) in pyridine (15 mL) was
added a solution of methyl chlorophenylphosphoryl P→N - -alaninate in
L
THF (0.2 M, 11.3 mL, 2.26 mmol). After addition of N -methylimidazole
(
0.36 mL, 4.52 mmol), the mixture was stirred for 16 h. The solvents were
evaporated and the residue was re-dissolved into AcOH (6 mL) and stirred at
room temperature overnight. The solution was evaporated, and the residue
was purified by silica gel column chromatography (EtOAc-MeOH 9:1 to 7:3,
1
v/v) to provide 5 (33 mg, 0.072 mmol, 16%). H NMR (DMSO-d , 500 MHz)
6
δ 10.62 (brs, 1H), 7.63 and 7.61 (2s, 1H), 7.41-7.35 (m, 2H), 7.22-7.16 (m,
3
4
2
H), 6.48 (brs, 2H), 6.16-6.05 (m, 1H), 5.22 and 5.18 (2s, 1H), 4.70 (s, 1H),
.61 and 4.58 (2s, 2H), 4.54 (d, J = 6.3 Hz, 2H) and 4.49 (d, J = 5.7 Hz,
13
H), 3.91-3.81 (m, 1H), 3.61 and 3.59 (2s, 3H), 1.26-1.19 (m, 3H); C NMR
(
DMSO-d , 126 MHz) δ 173.98 and 173.90 (2d, J = 4.8 Hz), 157.06, 153.91,
6
1
51.56, 150.86 and 150.83, 140.63 and 140.53, 137.76, 129.91, 124.90, 120.50

5
98
S. Imoto et al.
and 120.47, 116.61, 114.00 and 113.74, 66.56 and 66.40 (2d, J = 4.8 Hz),
+
5
4
2.16, 50.03 and 49.90, 44.12 and 44.07, 19.87; MS (ESI) m/z (M+H) calcd.
63.1495; found 463.1540.
2
-Acetylamino-9-[4-(tert-butyldiphenylsilyloxy)-2-
methylenebutyl]-9H-purin-6-yl diphenylcarbamate
17)
(
To a solution of 16 (1.50 g, 4.40 mmol), triphenylphosphine (1.38 g,
.28 mmol) and 11 (2.05 g, 5.28 mmol) in THF (35 mL) was added DIAD
5
(
7
1.07 g, 5.28 mmol) in THF (7 mL). The reaction mixture was heated to
◦
0 C and stirred for 2 h. After cooling, the mixture was filtered through a
celite pad. The filtrate was evaporated, and the residue was purified by silica
gel column chromatography (Hexane-EtOAc 6:4 to 3:7, v/v) to provide
1
compound 17 (2.17 g, 3.05 mmol, 69% yield) as a white foam. H NMR
(
CDCl , 500 MHz) δ 7.92 (brs, 1H), 7.82 (s, 1H), 7.65-7.62 (m, 4H), 7.47-
3
7
2
1
1
3
7
.34 (m, 16H), 5.04 (s, 1H), 4.84 (s, 1H), 4.70 (s, 2H), 3.80 (t, J = 6.3 Hz,
1
3
H), 2.50 (s, 3H), 2.24 (t, J = 6.3 Hz, 2H), 1.05 (s, 9H); C NMR (CDCl ,
3
26 MHz) δ 171.11, 156.12, 155.10, 152.16, 150.38, 143.98, 141.72, 140.85,
35.49, 133.31, 129.82, 129.17, 127.73, 125.92, 120.48, 115.42, 62.58, 48.48,
+
6.48, 26.82, 25.12, 19.14; MS (ESI) m/z (M+Na) calcd. 733.2935; found
33.2924.
2-Amino-9-[4-((tert-butyldiphenylsilyloxy)-2-methylenebutyl)-1H-
purin-6(9H)-one (18)
A solution of compound 17 (1.00 g, 1.41 mmol) in 2 M NH (MeOH
3
◦
sol) (8 mL) was stirred at 70 C for 2 h. The solution was evaporated, and the
residue was purified by silica gel column chromatography (CH Cl -MeOH
2
2
9
7:3 to 94:6, v/v) to provide 18 (540 mg, 1.14 mmol, 81% yield) as a white
1
foam. H NMR (DMSO-d , 500 MHz) δ 10.56 (brs, 1H), 7.64-7.58 (m, 4H),
6
7
.54 (s, 1H), 7.50-7.41 (m, 6H), 6.39 (brs, 2H), 4.90 (s, 1H), 4.60 (s, 1H), 4.50
13
(
s, 2H), 3.74 (t, J = 6.7 Hz, 2H), 2.26 (t, J = 6.7 Hz, 2H), 0.98 (s, 9H); C
NMR (DMSO-d , 126 MHz) δ 157.01, 153.78, 151.46, 142.15, 137.58, 135.22,
6
1
33.25, 130.08, 128.12, 116.56, 113.03, 63.23, 47.08, 36.51, 26.84, 18.94; MS
+
(ESI) m/z (M+Na) calcd. 496.2145; found 496.2187.
2-Amino-9-(4-hydroxy-2-methylenebutyl)-1H-purin-6(9H)-one (6)
To a solution of 18 (437 mg, 0.923 mmol) in THF (35 mL) was added
M TBAF in THF (1.11 mL, 1.11 mmol). The mixture was stirred at room
1
temperature for 1 h. The solvent was removed, and the residue was purified
by silica gel column chromatography (CH Cl -MeOH 9:1 to 2:8, v/v) to
2
2
1
provide 6 (130 mg, 0.55 mmol, 60% yield) as a white foam. H NMR (DMSO-
d6, 500 MHz) δ 10.58 (brs, 1H), 7.61 (s, 1H), 6.45 (brs, 2H), 4.88 (s, 1H),

Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir
599
4
2
1
.60 (t, J = 5.2 Hz, 1H), 4.53 (s, 2H), 4.50 (s, 1H), 3.52 (dt, J = 6.8, 5.2 Hz,
13
H), 2.14 (t, J = 6.8 Hz, 2H); C NMR (DMSO-d , 125 MHz) δ 157.08,
6
53.82, 151.54, 143.07, 137.85, 116.51, 111.97, 59.64, 47.15, 36.99; MS (ESI)
+
m/z (M+Na) calcd. 258.0967; found 258.0966.
Synthesis of L-ala-P-MEB-G (7)
To a solution of 6 (50 mg, 0.213 mmol) in pyridine (7.5 mL) was added
a solution of methyl chlorophenylphosphoryl P→N -L-alaninate in THF
(
2
0.2 M, 5.3 mL, 1.06 mmol). After addition of N -methylimidazole (0.17 mL,
.13 mmol), the mixture was stirred for 16 h. The solvents were evaporated
and the residue was re-dissolved into AcOH (6 mL) and stirred at room
temperature overnight. The solution was evaporated, and the residue was
purified by silica gel column chromatography (CH Cl -MeOH 97:3 to 91:9,
2
2
1
v/v) to provide 7 (58 mg, 0.12 mmol, 57%). H NMR (DMSO-d , 500 MHz)
6
δ 10.59 (brs, 1H), 7.61 and 7.59 (2s, 1H), 7.44-7.28 (m, 2H), 7.25-7.08 (m,
3
H), 6.45 (s, 2H), 6.09-5.91 (m, 1H), 4.95 and 4.93 (2s, 1H), 4.58 and 4.57
(
2s, 1H), 4.55 and 4.53 (2s, 2H), 4.21-4.07 (m, 2H), 3.89-3.79 (m, 1H), 3.59
and 3.58 (2s, 3H), 2.39-2.30 (m, 2H), 1.26-1.18 (m, 3H); C NMR (DMSO-
13
d , 126 MHz) δ 174.02 and 173.90 (2d, J = 4.8 Hz), 157.06, 153.85, 151.55,
6
1
50.94, 141.38 and 141.30, 137.77, 129.82, 124.74, 120.41, 116.54, 113.07
and 113.01, 64.29 and 64.23 (2d, J = 4.8 Hz), 52.10, 50.04 and 49.87, 46.82
+
and 46.80, 34.02 and 33.98, 19.88; MS (ESI) m/z (M+H) calcd. 477.1651;
found 477.1700.
2
-Acetylamino-9-[(diisopropoxyphosphorylmethoxy)allyl]-6-
diphenylcarbamoylpurine (20)
To a solution of (2-hydroxymethylallyloxymethyl)phosphonic acid diiso-
propyl ester 19 (2.13 g, 8.00 mmol), triphenylphosphine (2.73 g, 10.4 mmol)
and 11 (3.73 g, 9.60 mmol) in THF (40 mL) was added DIAD (1.94 g,
◦
9
.60 mmol). The reaction mixture was heated to 70 C and stirred for 2 h.
After cooling, the mixture was filtered through a celite pad. The filtrate was
evaporated, and the residue was purified by silica gel column chromatog-
raphy (EtOAc-MeOH 100:0 to 92:8, v/v) to provide 20 (3.14 g, 4.93 mmol,
1
6
1
2% yield) as a pale yellow foam. H NMR (500 MHz, CD OD) δ 8.31 (s,
3
H), 7.49-7.27 (m, 10H), 5.31 (s, 1H), 5.10 (s, 1H), 4.97 (s, 2H), 4.79-4.65
(
8
m, 2H), 4.10 (s, 2H), 3.74 (d, J = 8.6 Hz, 2H), 2.31 (s, 3H), 1.36 (dd, J =
13
.0, 6.3 Hz, 12H); C NMR (126 MHz, CD OD) δ 172.63, 157.21, 156.76,
3
1
54.14, 152.58, 147.49, 143.55, 141.76, 130.61, 128.61, 121.78, 117.98, 72.24
(
d, J = 12.0 Hz), 73.46 (d, J = 6.0 Hz), 65.53 (d, J = 167.9 Hz), 47.06, 25.06,
+
2
6
4.62 (d, J = 3.6 Hz), 24.58 (d, J = 4.8 Hz); MS (ESI) m/z (M+H) calcd.
37.2540, found 637.2569.

6
00
S. Imoto et al.
9-[(Diisopropoxyphosphorylmethoxy)allyl]guanine (21)
Compound 20 (361 mg, 0.567 mmol) was dissolved in ca. 9 M ammonia in
◦
methanol (5 mL) and heated to 70 C in a sealed tube. After 2 h, the reaction
mixture was concentrated and purified by silica gel column chromatography
(
CH Cl -MeOH 95:5 to 88:12, v/v) to provide 21 (123 mg, 54% yield) as a
2 2
1
white solid. H NMR (500 MHz, CDCl ) δ 11.81 (brs, 1H), 7.60 (s, 1H), 7.00
3
(
brs, 2H), 5.22 (s, 1H), 4.86-4.79 (m, 3H), 4.62 (s, 2H), 4.12 (s, 2H), 3.78 (d,
13
J = 9.7 Hz, 2H), 1.36 (dd, J = 8.0 and 6.3 Hz, 12H); C NMR (126 MHz,
CDCl ) δ 158.40, 154.49, 151.16, 139.67, 137.52, 117.12, 116.53, 74.41 (d, J
3
=
14.4 Hz), 71.85 (d, J = 6.0 Hz), 64.78 (d, J = 171.5 Hz), 44.69, 24.07 (d,
+
J = 3.6 Hz), 24.03(d. J = 4.8 Hz); MS (ESI) m/z (M+Na) calcd. 422.1569,
found 422.1581.
9-[2-(Hydroxymethyl)allyloxymethylphosphonic acid]guanine (22)
To a solution of 21 (475 mg, 1.19 mmol) in CH Cl (10 mL) was added
2
2
TMSBr (910 mg, 5.95 mmol). The reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with CH Cl and
2
2
extracted with water. The aqueous layer was lyophilized and the residue was
purified by ODS column chromatography (H O-MeOH 95:5 to 7:3, v/v) to
2
1
provide 22 (278 mg, 74% yield) as a white solid. H NMR (500 MHz, D O) δ
2
8
.72 (s, 1H), 5.38 (s, 1H), 5.11 (s, 1H), 4.88 (s, 2H), 4.10 (s, 2H), 3.58 (d, J
13
=
9.2 Hz, 2H); C NMR (126 MHz, D O) δ 158.52, 157.93, 152.92, 141.08,
2
1
40.82, 121.33, 111.85, 76.00 (d, J = 13.2 Hz), 68.34 (d, J = 158.4 Hz), 49.37;
−
MS (ESI) m/z (M−H) calcd. 314.0654 found 314.0690.
9-[Bis-O-(pivaloyoxymethyl)phosphorylmethoxy]allyl]guanine (8)
To a solution of 22 (101 mg, 0.320 mmol), triethylamine (179 μL,
.28 mmol) and tetrabutylammonium bromide (103 mg, 0.320 mmol) in
1
N -methylpyrrolidone (3 mL) was added chloromethyl pivalate (241 mg,
◦
1
.60 mmol). The reaction mixture was heated to 50 C and stirred for 20 h.
The reaction was quenched with MeOH and directly applied to silica gel
column chromatography (CH Cl -MeOH 98:2 to 9:1, v/v). Appropriate frac-
2
2
tions were evaporated, triturated from acetonitrile/water = 1/1 to provide
1
8
(17.0 mg, 10% yield) as a white solid. H NMR (500 MHz, CD OD) δ 7.72
3
(
2
s, 1H), 5.74-5.69 (m, 4H), 5.26 (s, 1H), 4.94 (s, 1H), 4.71 (s, 2H), 4.11 (s,
13
H), 3.93 (d, J = 8.6 Hz, 2H), 1.22 (s, 18H); C NMR (126 MHz, CD OD)
3
δ 178.42, 159.69, 155.64, 153.53, 142.19, 140.25, 117.75, 117.37, 83.52 (d, J
=
6.0 Hz), 75.22 (d, J = 14.4 Hz), 64.81 (d, J = 166.74 Hz), 46.04, 40.03,
+
2
7.51; MS (ESI) m/z (M+Na) calcd. 566.1992 found 566.1963.

Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir
601
Cell Culture, Anti-HBV Assay, and Cytotoxicity Assay
HepG2 2.2.15 human hepatoblastoma cell line, which can stably produce
HBV particles,[ was kindly gifted from B. Korba of Georgetown Univer-
sity. The HepG2 2.2.15 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM; Gibco, USA) and MT-4 cells were grown in RPMI 1640-
based culture medium, which supplemented with 10% (v/v) fetal calf serum
11]
(
FCS; Gibco, USA) and 50 U/mL penicillin and 50 μg/mL streptomycin.
5
The HepG2 2.2.15 cells were plated at 1 × 10 /mL in the presence or
absence of various concentrations of a test compound in 96-well microtiter
culture plates, then followed by incubation at 37 C for six days. After incu-
◦
bation, the concentration of HBV DNA in the supernatant was determined.
Cytotoxicity of a compound in MT-4 cells was also determined. Cells
5
were plated in a 96-well plate at a density of 1 × 10 /mL and cultured in
the absence or the presence of various concentrations of a compound at
◦
3
7 C for seven days. After 100 mL of the medium was removed from each
well, 10 mL of 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(
MTT) solution (Nacalai Tesque, Kyoto, Japan) was added to each well in
◦
the plate, followed by incubation at 37 C for 2 h. After incubation to dissolve
formazan crystals, 100 mL of acidified isopropanol containing 4% (v/v)
Triton X-100 was added to each well, and the optical density was measured
in a kinetic microplate reader (Vmax; Molecular Devices, Sunnyvale, CA).
Detection of HBV DNA by Real-Time Quantitative Polymerase
Chain Reaction
Viral DNA in the supernatants was extracted using QIAamp MinElute
Virus Spin Kit (Qiagen, Valencia, CA) according to the manufacture’s in-
struction. HBV DNA was quantified by real-time PCR relative to an external
ꢀ
R
plasmid DNA standard on a Light Cycler instrument using LightCycler
PLUS
FastStart DNA Master
SYBR Green I (Roche, Mannheim, Germany) and
primers HBV-RT-F (5 -GAGTCTAGACTCGTGGTGGA-3 ) and HBV-RT-R
5 -TGAGGCATAGCAGCAGGATG- 3 ), which amplified a 184-bp fragment
ꢁ
ꢁ
ꢁ
ꢁ
(
in the RT region of the HBV genome. The PCR conditions used were an
◦
◦
◦
initial 3 min at 95 C, followed by 40 cycles of 95 C for 10 s, 55 C for 10 s,
and 72 C for 10 s.
◦
FUNDING
Financial support from a Health and Labor Sciences Research Grant
Practical Research on Hepatitis (Research on the innovative development
[
and the practical application of new drugs for hepatitis B)] is gratefully
acknowledged.

6
02
S. Imoto et al.
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