Synthesis of nucleotide analogues, EFdA, EdA and EdAP, and the effect of EdAP on hepatitis B virus replication

Article abstract of DOI:10.1080/09168451.2019.1673696

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) and 4′-ethynyl-2′-deoxyadenosine (EdA) are nucleoside analogues which inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. EdAP, a cyclosaligenyl (cycloSal) phosphate derivative of EdA, inhibits the replication of the influenza A virus. The common structural feature of these compounds is the ethynyl group at the 4′-position. In this study, these nucleoside analogues were prepared by a common synthetic strategy starting from the known 1,2-di-O-acetyl-D-ribofuranose. Biological evaluation of EdAP revealed that this compound reduced hepatitis B virus (HBV) replication dose-dependently without cytotoxicity against host cells tested in this study.

Full text of DOI:10.1080/09168451.2019.1673696

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20
Synthesis of nucleotide analogues, EFdA, EdA and
EdAP, and the effect of EdAP on hepatitis B virus
replication
Mai Kamata, Toshifumi Takeuchi, Ei Hayashi, Kazane Nishioka, Mizuki
Oshima, Masashi Iwamoto, Kota Nishiuchi, Shogo Kamo, Shusuke
Tomoshige, Koichi Watashi, Shinji Kamisuki, Hiroshi Ohrui, Fumio Sugawara
& Kouji Kuramochi
To cite this article: Mai Kamata, Toshifumi Takeuchi, Ei Hayashi, Kazane Nishioka, Mizuki
Oshima, Masashi Iwamoto, Kota Nishiuchi, Shogo Kamo, Shusuke Tomoshige, Koichi Watashi,
Shinji Kamisuki, Hiroshi Ohrui, Fumio Sugawara & Kouji Kuramochi (2019): Synthesis of nucleotide
analogues, EFdA, EdA and EdAP, and the effect of EdAP on hepatitis B virus replication,
Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2019.1673696
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2019.1673696
Synthesis of nucleotide analogues, EFdA, EdA and EdAP, and the effect of EdAP
on hepatitis B virus replication
Mai Kamataa, Toshifumi Takeuchi a, Ei Hayashia, Kazane Nishiokaa,b, Mizuki Oshimaa,b, Masashi Iwamotoa,b,
c
Kota Nishiuchia, Shogo Kamo a, Shusuke Tomoshige a, Koichi Watashia,b, Shinji Kamisuki
,
a
Hiroshi Ohruid, Fumio Sugawara a and Kouji Kuramochi
aDepartment of Applied Biological Science, Tokyo University of Science, Chiba, Japan; bDepartment of Virology II, National Institute of
Infectious Diseases, Tokyo, Japan; cSchool of Veterinary Medicine, Azabu University, Sagamihara, Japan; dFaculty of Pharmacy, Yokohama
University of Pharmacy, Yokohama, Japan
ABSTRACT
ARTICLE HISTORY
Received 29 August 2019
Accepted 23 September 2019
4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) and 4′-ethynyl-2′-deoxyadenosine (EdA) are
nucleoside analogues which inhibit human immunodeficiency virus type 1 (HIV-1) reverse
transcriptase. EdAP, a cyclosaligenyl (cycloSal) phosphate derivative of EdA, inhibits the repli-
cation of the influenza A virus. The common structural feature of these compounds is the
ethynyl group at the 4′-position. In this study, these nucleoside analogues were prepared
by a common synthetic strategy starting from the known 1,2-di-O-acetyl-D-ribofuranose.
Biological evaluation of EdAP revealed that this compound reduced hepatitis B virus
(HBV) replication dose-dependently without cytotoxicity against host cells tested in this study.
KEYWORDS
Nucleotide analogue; EdAP;
synthesis; hepatitis B virus
4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA, 1)
inhibits human immunodeficiency virus type 1
(HIV-1) reverse transcriptase (Figure 1) [1–9]. This
compound inhibits the replication of a wide spectrum
of HIV-1 strains resistant to nucleoside reverse tran-
scriptase inhibitors (NRTIs) [10–14]. Furthermore,
EFdA has a long half-life in vivo and is currently in
human clinical trials [15–20]. 4′-Ethynyl-2′-
deoxyadenosine (EdA, 2) also inhibits HIV-1 reverse
transcriptase [9,21]. However, this compound is sus-
ceptible to degradation by adenosine deaminase
(ADA) [22–24]. To overcome the sensitivity against
ADA, the fluorine atom was introduced at the 2-posi-
ton in 2 to generate 1 [3,4,7,20,22]. The susceptibility
of 1 to ADA was drastically decreased compared to
that of 2. EdAP (3) is a cyclosaligenyl (cycloSal) phos-
phate derivative of EdA [25,26]. EdAP inhibits the
replication of the influenza virus A both in vivo and
in vitro. The cycloSal moiety generally serves as
a lipophilic masking unit to increase membrane-
permeability of the nucleoside or nucleoside 5′-
monophosphate [27–29]. The cycloSal moiety is
hydrolyzed in cells to release the nucleoside 5′-
monophosphate.
nucleophilic base (Scheme 1). Kuwahara and Ohrui
synthesized 1 through a direct N-glycosylation
between 4 and 2-fluoroadenine (5) (Scheme 1(a))
[30]. Ohrui also used 4 as the glycosyl donor for the
synthesis of 2 (Scheme 1(b)) [21]. The silyl-Hilbert-
Johnson reaction [31,32] between 4 and adenine (8)
afforded 9. The N-glycosylation reactions between 4
and the bases proceeded selectively. The high β-
selectivity is explained by the neighboring group effect
of the acetyl group at the C-2 position [31]. However,
the syntheses of 1 and 2 included the deprotection of
the benzyl groups using hazardous ammonia and
highly reactive lithium metal. In the synthesis of 2,
the deprotection of the benzyl groups in 10 under the
Birch reduction conditions gave 1, together with the
deaminated product 11 as a byproduct [21]. The use of
2-deoxyribose derivatives as glycosyl donors decreased
the diastereoselectivity of the N-glycosylations due to
the lack of neighboring group participation at the C-2
position (Scheme 1(c,d)). Kuwahara and Ohrui
reported that the silyl-Hilbert-Johnson reaction
between 2-deoxyribose derivative 12 and 5 gave the
desired β-anomer 13 and undesired α-anomer in 46%
and 25% yields, respectively (Scheme 1(c)) [33,34].
After separation of 12 by silica gel chromatography,
this compound was converted into 1. MacLaughlin
used 3,5-di-O-(p-toluoyl)-2-deoxyribose derivative
14 as the glycosyl donor [35]. The coupling between
14 and 5 gave a 1.8:1 mixture of β- and α-anomers
EFdA (1) was designed and first synthesized begin-
ning with 2-amino-2′-deoxyadenosine via 4′-ethynyl
-2-amino-2′-deoxyadenosine by Ohrui [1,2]. Other
previous syntheses of
1 and 2 included the
N-glycosylation between an electrophilic sugar and
CONTACT Kouji Kuramochi
kuramoch@rs.tus.ac.jp
Supplemental data for this article can be accessed here.
© 2019 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
M. KAMATA ET AL.
Figure 1. Structures of EFdA (1), EdA (2), and EdAP (3).
Scheme 1. Previous syntheses of EFdA (1) and EdA (2). (a) synthesis of 1 reported by Kuwahara and Ohrui [30]. (b) synthesis of 2
reported by Ohrui [21]. (c) alternative synthesis of 1 reported by Kuwahara and Ohrui [33,34]. (d) synthesis of 1 reported by
MacLaughlin [35].
(Scheme 1(d)). After separation of the desired β-
anomer 15 by crystallization, deprotection of the pro-
tective groups in 15 afforded 1.
not reproducible, and the reactions were often accom-
panied by the formations of unidentified byproducts.
Chronic infection of hepatitis B virus (HBV) affects
approximately 257 million people worldwide and is
a risk factor for developing liver cirrhosis and hepato-
cellular carcinoma [36]. To solve this public health
problem, antiviral agents that eliminate the infection
are needed. Because EFdA (2) shows anti-HBV activ-
ity with IC50 value of 0.16 μM [37], we have been
interested in the anti-HBV activity of EdA and EdAP.
In this paper, we report the syntheses of EFdA (1),
EdA (2), and EdAP (3) starting from the 1,2-di-O-acetyl-
D-ribofuranose 16 and the anti-HBV activity of EdAP.
We optimized the conditions of the silyl-Hilbert-Johnson
Takeuchi, Sugawara and Ohrui designed 16 as
a glycosyl donor and used this compound for the
synthesis of EdAP (3) (Scheme 2) [25,26]. The silyl-
Hilbert-Johnson reaction between 16 and 8 afforded
17 in 78% yield. After conversion of 17 into 18, the
introduction of the cycloSal group into the hydroxy
group in 18, followed by deprotection of the p-meth-
oxybenzyl (PMB) group gave 3. However, we suffered
from the lack of the reproducibility of the silyl-Hilbert
-Johnson reaction between 16 and 8, and the intro-
duction of the cycloSal group into 18. The yields were

SYNTHESIS OF NUCLEOTIDE ANALOGUES, EFDA, EDA AND EDAP
3
Scheme 2. Synthesis of EdAP (3) reported by Takeuchi, Sugawara and Ohrui.
reaction and the cycloSal-introduction. The utility of 16
as a glycosyl donor has been demonstrated by the present
synthesis of these nucleoside analogues.
5-[(triethylsilyl)ethynyl]tetrahydrofuran-3-yl acetate
(17) and (2R,3R,4S,5R)-2-(6-amino-3H-purin-3-yl)-
5-(tert-butyldiphenylsilyloxymethyl)-4-(4-methoxyben-
zyloxy)-5-[(triethylsilyl)ethynyl]tetrahydrofuran-3-yl
acetate (21)
Materials and methods
N,O-Bis(trimethylsilyl)acetamide (BSA) (198 μL,
0.810 mmol) was added to a solution of adenine (8,
54.7 mg, 0.405 mmol) in 1,2-dichloroethane (810 μL).
The mixture was stirred under an argon atmosphere at
70°C for 5.5 h. A solution of 16 [25,26] (98.8 mg, 0.135
mmol) in 1,2-dichloroethane (1.9 mL) was added to
the mixture at room temperature. Trimethylsilyl tri-
flate (TMSOTf) (73.3 μL, 0.405 mmol) was added to
the resultant mixture at 0°C. The mixture was stirred
under an argon atmosphere at 90°C for 19.5 h. The
reaction was quenched by the addition of saturated
aqueous NaHCO3 solution. The mixture was diluted
with CHCl3 at 0°C to give a biphasic solution. The
aqueous layer was extracted with CHCl3. The com-
bined organic layer was dried over Na2SO4, and con-
centrated to give a residue. The residue was purified by
silica gel column chromatography (hexane/EtOAc =
1/1, then MeOH) to afford 17 (49.9 mg, 46%) as a pale
yellow oil and 21 (38.2 mg, 35%) as a pale yellow oil.
The NMR spectroscopic data for 17 were identical
General information
Melting point (Mp) data were determined using
a Micro Melting Point Determination Apparatus
Type MM-2 instrument (Shimadzu Seisakusyo, Ltd)
and were uncorrected. IR spectra were recorded on
a Horiba FT-720 spectrometer using KBr pellets.
1H and 13C NMR spectra were recorded on a Bruker
Avance 400 (400 and 100 MHz, respectively) spectro-
meter, using chloroform-d (CDCl3), methanol-d4
(CD3OD) or dimethyl sulfoxide-d6 (DMSO-d6) as
a solvent. Chemical shift values are expressed in δ
(ppm) relative to tetramethylsilane (TMS, δ 0.00
ppm) or the solvent resonance (CDCl3, δ 7.26 ppm
for 1H NMR and δ 77.0 ppm for 13C NMR; CD3OD, δ
3.30 ppm for 1H NMR and δ 49.0 ppm for 13C NMR;
DMSO-d6, δ 2.49 ppm for 1H NMR). Data are
reported as follows: chemical shift, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, br =
broad, m = multiplet), coupling constants (J; Hz),
and integration. Mass spectra were obtained by on
a JEOL high-resolution double-focusing mass spectro-
meter (JMS-700, “MStation”) using fast atom bom-
bardment (FAB). Specific rotations were recorded on
a JASCO polarimeter (P-1010), and recorded as [α]D
values (concentration in g/100 mL).
23
with the reported data [25,26]. Compound 21: [α]D
− 17.1 (c 0.34, CHCl3). IR (KBr) νmax = 3438, 3363,
3072, 3049, 2956, 2933, 2873, 2858, 2171, 1749, 1658,
1
1614, 1589, 1556, 1514, 1471 cm−1. H NMR (400
MHz, CDCl3; relative to TMS) δ 8.43 (s, 1H), 7.95 (s,
1H), 7.64 (m, 4H), 7.46–7.40 (m, 2H), 7.38–7.32 (m,
4H), 7.22 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H),
6.50 (d, J= 4.0 Hz, 1H), 5.85 (dd, J= 6.0, 4.0 Hz, 1H),
4.75 (d, J= 6.0 Hz, 1H), 4.60 (d, J = 11.0 Hz, 1H), 4.52
(d, J = 11.0 Hz, 1H), 4.14 (d, J= 11.4 Hz, 1H), 3.85 (d,
J = 11.4 Hz, 1H), 3.80 (s, 3H), 2.05 (s, 3H), 1.07 (s, 9H),
0.94 (t, J= 7.9 Hz, 9H), 0.58 (q, J = 7.9 Hz, 6H).
Synthesis
(2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-5-(tert-
butyldiphenylsilyloxymethyl)-4-(4-methoxybenzyloxy)-

4
M. KAMATA ET AL.
13C NMR (100 MHz, CDCl3; relative to the solvent
resonance) δ 169.9, 159.2, 154.4, 154.2, 149.5, 140.4,
135.7 (2C), 135.5 (2C), 132.4, 132.3, 130.0, 129.9,
129.7, 129.2 (2C), 127.9 (2C), 127.8 (2C), 120.7,
113.5 (2C), 101.2, 92.1, 91.4, 84.2, 76.3, 74.4, 73.3,
66.5, 55.2, 26.9 (3C), 20.7, 19.2, 7.4 (3C), 4.1 (3C).
HRMS (FAB) m/z calcd. for C44H56N5O6Si2 ([M +
H]+) 806.3769, found 806.3768.
spectroscopic data for 24 were identical with the
reported data [25,26].
[(2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-2-ethynyl-
3-(4-methoxybenzyloxy)tetrahydrofuran-2-yl]metha-
nol (18)
A 1.0 M solution of tetra-n-butylammonium fluor-
ide (TBAF) in THF (1.2 mL, 1.20 mmol) was added to
a solution of 24 (0.41 g, 0.548 mmol) in THF (5.5 mL).
The mixture was stirred under an argon atmosphere at
room temperature for 2 h. The reaction was quenched
by the addition of water. The mixture was diluted with
EtOAc to give a biphasic solution. The layers were
separated. The aqueous layer was extracted with
EtOAc. The combined organic layer was dried over
Na2SO4, and concentrated to a residue. The residue
was purified by silica gel column chromatography
(EtOAc/MeOH = 15/1) to afford 18 (0.196 g, 95%) as
a white solid. Mp = 135–136°C. The NMR spectro-
scopic data for 18 were identical with the reported
data [25,26].
Isomerization of 21 to 17.
p-Toluenesulfonic acid monohydrate (p-TsOH·H2
O) (2.75 mg, 14.5 μmol) was added to a solution of 21
(38.9 mg, 48.3 μmol) in chlorobenzene (1.0 mL). The
reaction mixture was refluxed for 50 min. The mixture
was diluted with CHCl3, and concentrated to
a residue. The residue was purified by silica gel column
chromatography (hexane/EtOAc = 2/1) to afford 17
(32.5 mg, 84%).
(2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-5-{[(tert-
butyldiphenylsilyl)oxy]methyl}-4-(4-methoxybenzy-
loxy)-5-[(triethylsilyl)ethynyl]tetrahydrofuran-3-ol (22)
A solution of 17 (1.32 g, 1.64 mmol) in a 2:5 mix-
ture of Et3N and MeOH (6.3 mL) was stirred at 45°C
for 21 h. The mixture was diluted with EtOAc, and
concentrated to a residue. The residue was purified by
silica gel column chromatography (EtOAc only) to
afford 22 (1.23 g, 98%) as a pale yellow oil. The
NMR spectroscopic data for 22 were identical with
the reported data [25,26].
EdA,
(2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-
2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (2)
Cerium ammonium nitrate (CAN) (122.7 mg,
0.224 mmol) was added to a solution of 18 (35.4 mg,
89.5 μmol) in a 5:1 mixture of MeCN and water
(0.9 mL) at 0°C. The mixture was stirred at 0°C for
5.5 h. The mixture was diluted with MeOH, and con-
centrated to a residue. The residue was purified by
silica gel column chromatography (EtOAc/MeOH =
10/1) to afford EdA (2) (20.3 mg, 82%) as a white solid.
O-((2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-
5-(tert-butyldiphenylsilyloxymethyl)-4-(4-methoxyben-
zyloxy)-5-{[(triethylsilyl)ethynyl]tetrahydrofuran-3-yl}
O-phenyl carbonothioate (23)
Phenyl chlorothionoformate (37.3 μL, 0.276
mmol) was added to a solution of 22 (0.211 mg,
23
Mp = 154–155°C. [α]D + 13.7 (c 0.14, MeOH). IR
(KBr) νmax = 3392, 3273, 2927, 2112, 1649, 1604, 1506,
1
1479, 1421 cm−1. H NMR (400 MHz, DMSO-d6;
relative to the solvent resonance) δ 8.31 (s, 1H), 8.12
(s, 1H), 7.32 (br s, 2H), 6.34 (t, J = 6.3 Hz, 1H),
5.58–5.55 (2H, overlapped), 4.57 (q, J = 6.1 Hz, 1H),
3.66 (dd, J = 12.0, 5.3 Hz, 1H), 3.55 (dd, J = 11.8, 7.4
Hz, 1H), 3.51 (s, 1H), 2.78–2.72 (m, 1H), 2.43–2.38 (m,
0.276
mmol)
and
4-dimethylaminopyridine
(DMAP) (67.4 mg, 0.552 mmol) in CH2Cl2 (1.8 mL)
at 0°C. The reaction mixture was stirred under an
argon atmosphere at room temperature for 5 h. The
mixture was diluted with EtOAc, and concentrated to
a residue. The residue was purified by silica gel col-
umn chromatography (hexane/EtOAc = 1/1) to
afford 23 (0.215 g, 87%) as a colorless oil. The NMR
spectroscopic data for 23 were identical with the
reported data [25,26].
1
1H). H NMR (400 MHz, CD3OD; relative to the
solvent resonance) δ 8.51 (s, 1H), 8.34 (s, 1H), 6.49
(dd, J = 7.2, 4.5 Hz, 1H), 4.75 (t, J = 7.1 Hz, 1H), 3.85
(d, J = 12.2 Hz, 1H), 3.77 (d, J = 12.2 Hz, 1H), 3.11 (s,
1H), 2.84–2.78 (m, 1H), 2.70–2.63 (m, 1H). 13C NMR
(100 MHz, CD3OD; relative to the solvent resonance)
δ 153.3, 149.6, 147.6, 143.3, 120.4, 87.0, 85.0, 80.6, 78.9,
71.5, 65.7, 40.1. HRMS (FAB) m/z calcd. for C12H14N5
O3 ([M + H]+) 276.1097, found 276.1095.
9-((2R,4S,5R)-5-(tert-Butyldiphenylsilyloxymethyl)
-4-(4-methoxybenzyloxy)-5-{[(triethylsilyl)ethynyl]tet-
rahydrofuran-2-yl}-9H-purin-6-amine (24)
Azobisisobutyronitrile (AIBN) (9.84 mg, 59.9
μmol) was added to a solution of 23 (0.216 g, 0.236
mmol) and tri-(n-butyl)tin hydride (387 μL, 1.44
mmol) in toluene (6.0 mL). The reaction mixture
was refluxed under an argon atmosphere for 3 h. The
mixture was diluted with EtOAc, and concentrated to
a residue. The residue was purified by column chro-
matography using silica gel containing 10% w/w anhy-
drous potassium carbonate (hexane/EtOAc = 1/2) to
afford 24 (0.163 g, 91%) as a pale yellow oil. The NMR
2-{[(2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-2-ethy-
nyl-3-(4-methoxybenzyloxy)tetrahydrofuran-2-yl]meth-
oxy}-4H-benzo[d] [1,3,2]dioxaphosphinine 2-oxide (20)
N,N-Diisopropylethylamine (61.5 μL, 0.353 mmol)
and 19 (66.5 μL, 0.353 mmol) were added to a solution
of 18 (69.7 mg, 0.176 mmol) in THF (2.7 mL). The
mixture was stirred under an argon atmosphere at –
40°C for 1 h. An aqueous 30% hydrogen peroxide
solution (39.9 μL, 0.353 mmol) was added at – 40°C.
The mixture was stirred at room temperature for 50

SYNTHESIS OF NUCLEOTIDE ANALOGUES, EFDA, EDA AND EDAP
5
min. The mixture was diluted with EtOAc, and con-
centrated to a residue. The residue was purified by
silica gel column chromatography (EtOAc/MeOH =
15/1) to afford a crude product. This purification was
repeated three time to afford pure 20 (40.4 mg, 41%)
as a 1.43:1 diastereomeric mixture as a pale yellow oil.
The NMR spectroscopic data for 20 were identical
with the reported data [25,26].
EdAP, 2-{[(2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-
2-ethynyl-3-hydroxytetrahydrofuran-2-yl]methoxy}-
4H-benzo[d] [1,3,2]dioxaphosphinine 2-oxide (3)
CAN (80.7 mg, 0.108 mmol) was added to
a solution of 20 (30.4 mg, 53.9 μmol) in a 5:1 mixture
of MeCN and water (0.9 mL) at 0°C. The mixture was
stirred at 0°C for 4.5 h. The mixture was diluted with
MeOH, and concentrated to a residue. The residue
was purified by silica gel column chromatography
(CHCl3/MeOH = 10/1) to afford 3 (15.5 mg, 65%) as
a 1.25:1 diasteromeric mixture as a pale yellow solid.
Mp = 128–130°C (dec.). [α]D22 – 2.67 (c 0.54, MeOH).
The NMR spectroscopic data for 3 were identical with
the reported data [25,26].
= 19.9 Hz), 151.1 (d, JC-F = 19.5 Hz), 139.5 (d, JC-F
=
2.3 Hz), 135.6 (2C), 135.5 (2C), 132.6, 132.4, 129.9,
129.8, 129.7, 129.4 (2C), 127.8 (4C), 118.1 (d, JCF = 4.0
Hz), 113.6 (2C), 101.6, 91.8, 86.3, 83.5, 76.3, 73.8, 73.3,
66.7, 55.2, 26.7 (3C), 20.6, 19.2, 7.41 (3C), 4.13 (3C).
HRMS (FAB) m/z calcd. for C44H55FN5O6Si2 ([M +
H]+) 824.3675, found 824.3676.
(2R,3R,4S,5R)-2-(6-Amino-2-fluoro-9H-purin
-9-yl)-5-(tert-butyldiphenylsilyloxymethyl)-4-(4-meth-
oxybenzyloxy)-5-[(triethylsilyl)ethynyl]tetrahydro-
furan-3-ol (26)
A solution of 25 (61.6 mg, 74.8 μmol) in a 2:5
mixture of Et3N and MeOH (1.4 mL) was stirred at
45°C for 19 h. The mixture was diluted with EtOAc,
and concentrated to a residue. The residue was pur-
ified by silica gel column chromatography (EtOAc
only) to afford 26 (53.5 mg, 91%) as a pale yellow oil.
21
[α]D − 2.1 (c 0.27, CHCl3). IR (KBr) νmax = 3333,
3183, 3072, 3049, 2998, 2955, 2933, 2912, 2874, 2858,
2169, 1648, 1611, 1588, 1559, 1515, 1490 cm−1.
1H NMR (400 MHz, CDCl3; relative to TMS) δ 7.81
(s, 1H), 7.62–7.60 (m, 4H), 7.44–7.39 (m, 2H),
7.36–7.32 (m, 6H), 6.88 (d, J = 8.7 Hz, 2H), 6.22 (br
s, 2H), 6.00 (d, J = 5.0 Hz, 1H), 4.89 (d, J = 11.2 Hz,
1H), 4.75 (ddd, J = 8.2, 6.0, 5.0 Hz, 1H), 4.63 (d, J =
11.2 Hz, 1H), 4.55 (d, J = 6.0 Hz, 1H), 3.97 (d, J = 11.0
Hz, 1H), 3.83 (d, J = 11.0 Hz, 1H), 3.80 (s, 3H), 3.45 (br
s, 1H), 1.03 (s, 9H), 0.97 (t, J = 7.8 Hz, 9H), 0.62 (q, J =
7.8 Hz, 6H). 13C NMR (100 MHz, CDCl3; relative to
the solvent resonance) δ159.6, 158.9 (d, JC-F = 209.9
Hz), 156.9 (d, JC-F = 20.0 Hz), 151.1 (d, JC-F = 19.5 Hz),
139.9, 135.6 (2C), 135.4 (2C), 132.6, 132.4, 129.93,
129.90, 129.8 (2C), 128.9, 127.8 (4C), 118.3, 113.9
(2C), 102.2, 91.2, 89.4, 83.0, 77.2, 73.1, 72.9, 66.8,
55.3, 26.7 (3C), 19.2, 7.42 (3C), 4.10 (3C). HRMS
(FAB) m/z calcd. for C42H53FN5O5Si2 ([M + H]+)
782.3569, found 782.3568.
(2R,3R,4S,5R)-2-(6-Amino-2-fluoro-9H-purin
-9-yl)-5-(tert-butyldiphenylsilyloxymethyl)-4-(4-meth-
oxybenzyloxy)-5-[(triethylsilyl)ethynyl]tetrahydro-
furan-3-yl acetate (25)
BSA (128 μL, 0.524 mmol) was added to a solution
of 2-fluoroadenine (5, 40.1 mg, 0.262 mmol) in
1,2-dichloroethane (550 μL). The mixture was stirred
under an argon atmosphere at 70°C for 7 h. A solution
of 16 (63.9 mg, 87.4 μmol) in 1,2-dichloroethane
(1.2 mL) was added to the mixture at room tempera-
ture. TMSOTf (47.5 μL, 0.262 mmol) was added to the
resultant mixture at 0°C, and the resultant mixture
was stirred under an argon atmosphere at 90°C for
13 h. The reaction was quenched by the addition of
saturated aqueous NaHCO3 solution. The mixture was
diluted with CHCl3 to give a biphasic solution. The
layers were separated. The aqueous layer was extracted
with CHCl3. The combined organic layer was dried
over Na2SO4, and concentrated to a residue. The resi-
due was purified by silica gel column chromatography
(hexane/EtOAc = 2/1) to afford 25 (61.6 mg, 86%) as
a white solid. Mp = 144–146°C. [α]D15 − 33.3 (c 0.28,
CHCl3). IR (KBr) νmax = 3324, 3176, 3072, 2955, 2933,
2873, 2859, 1747, 1647, 1612, 1587, 1559, 1515,
O-((2R,3R,4S,5R)-2-(6-Amino-2-fluoro-9H-purin
-9-yl)-5-(tert-butyldiphenylsilyloxymethyl)-4-(4-meth-
oxybenzyloxy)-5-{[(triethylsilyl)ethynyl]tetrahydro-
furan-3-yl} O-phenyl carbonothioate (27)
Phenyl chlorothionoformate (9.3 μL, 68.4 μmol)
was added to a solution of 26 (53.5 mg, 68.4 μmol)
and DMAP (16.7 mg, 0.137 mmol) in dichloro-
methane (1.0 mL) at 0°C. The reaction mixture was
stirred under an argon atmosphere at room tempera-
ture for 2 h. The mixture was diluted with CHCl3, and
concentrated to a residue. The residue was purified by
silica gel column chromatography (hexane/EtOAc =
1/1) to afford 27 (56.9 mg, 91%) as a pale yellow oil.
1
1472 cm−1. H NMR (400 MHz, CDCl3; relative to
TMS) δ 7.86 (s, 1H), 7.64–7.60 (m, 4H), 7.43–7.39
(m, 2H), 7.36–7.31 (m, 4H), 7.27 (d, J = 8.6 Hz, 2H),
6.85 (d, J = 8.6 Hz, 2H), 6.22 (d, J = 5.0 Hz, 1H), 5.97
(br s, 2H), 5.74 (dd, J = 6.1, 5.0 Hz, 1H), 4.74 (d, J = 6.1
Hz, 1H), 4.70 (d, J = 11.2 Hz, 1H), 4.53 (d, J = 11.2 Hz,
1H), 3.97 (d, J = 11.2 Hz, 1H), 3.802 (d, J = 11.2 Hz,
1H), 3.797 (s, 3H), 2.04 (s, 3H), 1.03 (s, 9H), 0.96 (t, J =
7.9 Hz, 9H), 0.59 (q, J = 7.9 Hz, 6H). 13C NMR (100
MHz, CDCl3; relative to the solvent resonance) δ
169.9, 159.2, 159.1 (d, JC-F = 209.9 Hz), 157.1 (d, JC-F
21
[α]D − 37.5 (c 0.30, CHCl3). IR (KBr) νmax = 3327,
3176, 3070, 2999, 2955, 2932, 2873, 2858, 2171, 1722,
1
1649, 1610, 1590, 1514, 1490 cm−1. H NMR (400
MHz, CDCl3; relative to TMS) δ 7.87 (s, 1H),
7.64–7.61 (m, 4H), 7.42–7.27 (m, 11H), 6.93 (d, J =
8.7 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 6.35 (d, J = 4.9 Hz,
1H), 6.22 (dd, J = 6.0, 4.9 Hz, 1H), 6.13 (br s, 2H), 5.01

6
M. KAMATA ET AL.
(d, J = 6.0 Hz, 1H), 4.83 (d, J = 11.1 Hz, 1H), 4.62 (d,
J = 11.1 Hz, 1H), 4.02 (d, J = 11.2 Hz, 1H), 3.86 (d, J =
11.2 Hz, 1H), 3.80 (s, 3H), 1.03 (s, 9H), 0.97 (t, J = 7.9
Hz, 9H), 0.61 (q, J = 7.9 Hz, 6H). 13C NMR (100 MHz,
CDCl3; relative to the solvent resonance) δ 194.4,
159.3, 159.0 (d, JC-F = 210.5 Hz), 157.0 (d, JC-F = 19.8
Hz), 153.4, 151.2 (d, JC-F = 19.5 Hz), 140.0 (d, JC-F = 2.7
Hz), 135.6 (2C), 135.5 (2C), 132.5 (2C), 129.9, 129.8,
129.7 (2C), 129.6, 129.5 (2C), 127.8 (2C), 127.7 (2C),
126.7, 121.7 (2C), 118.3 (d, JC-F = 4.0 Hz), 113.7 (2C),
101.3, 92.4, 86.1, 83.6, 80.8, 76.0, 73.8, 66.5, 55.3, 26.7
(3C), 19.2, 7.44 (3C), 4.10 (3C). HRMS (FAB) m/
z calcd. for C49H57FN5O6SSi2 ([M + H]+) 918.3552,
found 918.3552.
9-((2R,4S,5R)-5-(tert-Butyldiphenylsilyloxymethyl)
-4-(4-methoxybenzyloxy)-5-{[(triethylsilyl)ethynyl]tet-
rahydrofuran-2-yl}-2-fluoro-9H-purin-6-amine (28)
AIBN (4.38 mg, 26.7 μmol) was added to a solution
of 27 (98.1 mg, 0.107 mmol) and tri-(n-butyl)tin
hydride (172 μL, 0.641 mmol) in toluene (2.7 mL).
The reaction mixture was refluxed under an argon
atmosphere for 6 h. The mixture was diluted with
EtOAc, and concentrated to a residue. The residue
was purified by column chromatography using silica
gel containing 10% w/w anhydrous potassium carbo-
combined organic layer was dried over Na2SO4, and
concentrated to a residue. The residue was purified by
silica gel column chromatography (hexane/EtOAc =
1/3) to afford 29 (66.4 mg, 94%) as a pale yellow
23
amorphous. [α]D – 24.8 (c 0.49, CHCl3). IR (KBr)
νmax = 3294, 3180, 3012, 2935, 2836, 2114, 1685, 1614,
1
1514, 1442 cm−1. H NMR (400 MHz, CD3OD; rela-
tive to the solvent resonance) δ 8.20 (s, 1H), 7.33 (d,
J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 6.33 (dd, J =
7.0, 5.6 Hz, 1H), 4.69 (d, J = 11.3 Hz, 1H), 4.59 (d, J =
6.4 Hz, 1H), 4.58 (d, J = 11.3 Hz, 1H), 3.85 (d, J = 13.3
Hz, 1H), 3.76 (s, 3H), 3.74 (d, J = 13.3 Hz, 1H), 3.11 (s,
1H), 2.80–2.74 (m, 1H), 2.63–2.57 (m, 1H). 13C NMR
(100 MHz, CDCl3; relative to the solvent resonance) δ
159.4, 158.5 (d, JC-F = 212.3 Hz), 157.3 (d, JC-F = 20.0
Hz), 149.9, 140.3, 129.53, 129.49 (2C), 119.1, 113.9
(2C), 86.4, 86.1, 79.6, 79.5, 77.2, 72.6, 67.9, 55.3, 38.3.
HRMS (FAB) m/z calcd. for C20H21FN5O4 ([M + H]+)
414.1578, found 414.1576.
EFdA,
(2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin
-9-yl)-2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-
3-ol (1)
CAN (96.3 mg, 0.176 mmol) was added to
a solution of 29 (24.2 mg, 58.5 μmol) in a 5:1 mixture
of MeCN and water (0.9 mL) at 0°C. The mixture was
stirred at 0°C for 3 h. The mixture was diluted with
MeOH, and concentrated to a residue. The residue
was purified by silica gel column chromatography
nate (hexane/EtOAc = 2/1) to afford 28 (65.4 mg,
19
80%) as a white solid. Mp = 163–165°C. [α]D
−
21.4 (c 0.13, CHCl3). IR (KBr) νmax = 3437, 3317,
3166, 3072, 2954, 2932, 2873, 2858, 2165, 1674, 1648,
(CHCl3/MeOH = 8/1) to afford EFdA (1) (10.0 mg,
58%) as a white solid. Mp = 219–220°C (dec.). [α]D +
1
19
1613, 1585, 1514, 1498 cm−1. H NMR (400 MHz,
CDCl3; relative to TMS) δ 7.94 (s, 1H), 7.64–7.60 (m,
4H), 7.43–7.39 (m, 2H), 7.36–7.31 (m, 4H), 7.28 (d, J =
8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.36 (dd, J = 6.7,
5.2 Hz, 1H), 6.00 (br s, 2H), 4.68 (d, J = 11.6 Hz, 1H),
4.61 (d, J = 6.7 Hz, 1H), 4.55 (d, J = 11.6 Hz, 1H), 4.00
(d, J = 11.1 Hz, 1H), 3.87 (d, J = 11.1 Hz, 1H), 3.81 (s,
3H), 2.73–2.61 (m, 2H), 1.04 (s, 9H), 0.97 (t, J = 7.8 Hz,
9H), 0.61 (q, J = 7.8 Hz, 6H). 13C NMR (100 MHz,
CDCl3; relative to the solvent resonance) δ 159.2,
158.9 (d, JC-F = 209.3 Hz), 156.9 (d, JC-F = 20.1 Hz),
150.7 (d, JC-F = 19.6 Hz), 139.5 (d, JC-F = 2.6 Hz), 135.6
(2C), 135.5 (2C), 132.7, 132.5, 129.90, 129.86 (2C),
129.1 (2C), 127.8 (4C), 118.2, 113.7 (2C), 102.3, 91.2,
84.6, 83.4, 77.1, 72.1, 66.3, 55.3, 37.3, 26.8 (3C), 19.2,
7.45 (3C), 4.21 (3C). HRMS (FAB) m/z calcd. for C42
H53FN5O4Si2 ([M + H]+) 766.3620, found 766.3619.
[(2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl)-
2-ethynyl-3-(4-methoxybenzyloxy)tetrahydrofuran-
2-yl]methanol (29)
12.7 (c 0.50, MeOH). IR (KBr) νmax = 3321, 3167, 3001,
2116, 1695, 1618, 1496 cm−1. 1H NMR (400 MHz, CD3
OD; relative to the solvent resonance) δ 8.25 (s, 1H),
6.34 (dd, J = 7.2, 4.9 Hz, 1H), 4.73 (t, J = 6.9 Hz, 1H),
3.85 (d, J = 12.2 Hz, 1H), 3.76 (d, J = 12.2 Hz, 1H), 3.08
(s, 1H), 2.81–2.74 (m, 1H), 2.63–2.56 (m, 1H).
13C NMR (100 MHz, CD3OD; relative to the solvent
resonance) δ 160.4 (d, JC-F = 207.7 Hz), 159.1 (d, JC-F
=
19.9 Hz), 151.5 (d, JC-F = 19.3 Hz), 141.4, 118.7, 86.8,
84.8, 80.7, 78.8, 71.7, 65.9, 39.9. HRMS (FAB) m/
z calcd. for C12H13FN5O3 ([M + H]+) 294.1002,
found 294.1000.
Cell culture
Hep38.7-Tet cells were cultured at 37°C, 5% CO2 as
described previously [38,39]. The Hep38.7-Tet cells
produce HBV replication under depletion of tetracy-
cline from medium.
A 1.0 M solution of TBAF in THF (377 μL, 0.377
mmol) was added to a solution of 28 (0.131 g, 0.171
mmol) in THF (1.7 mL). The mixture was stirred
under an argon atmosphere at room temperature for
2 h. The reaction was quenched by the addition of
water. The mixture was diluted with EtOAc to give
a biphasic solution. The layers were separated. The
aqueous layer was extracted with EtOAc. The
Quantification of cell viability
At 3 days after seeding, Hep38.7-Tet cells were incu-
bated in the absence of tetracycline to induce HBV
replication and treated with or without the compound
for 6 days. 2,3-Bis-(2-methoxy-4-nitro-5-sulfophe-
nyl)-2H-tetrazolium-5-carboxyanilide salt (XTT) cell

SYNTHESIS OF NUCLEOTIDE ANALOGUES, EFDA, EDA AND EDAP
7
viability assays were performed with XTT cell prolif-
eration kit II (Roche), according to the manufacturer’s
instructions [40]. The absorbance values were then
measured at 450 nm with a 96-well plate reader. Cell
growth inhibition was evaluated as the ratio of the
absorbance of the sample to that of the control.
the following two reactions: silylation of a base and gly-
cosylation between the silyated base and an electrophilic
sugar. In the previous protocol [25,26], the silylation of
adenine (8) with BSA was performed in the presence of
1,2-di-O-acetyl-D-ribofuranose 16. Following this proto-
col, we frequently observed decomposition of 16 during
the silylation step. Thus, we considered that the silylation
of 8 with BSA in the absence of 14 will improve the
reproducibility of the results. Actually, we obtained
reproducible results according to the following proce-
dure. First, 8 was reacted with an excess amount of BSA
in 1,2-dicloroethane at 70°C. Then, a mixture of the
silylated adenine and 16 (α:β = 7.7:1) was treated with
TMSOTf and heated at 90°C. This reaction afforded the
desired N-glycoside 17 in 46% yield, together with iso-
adenine derivative 21 in 35% yield [42,43]. The structure
of 21 was confirmed by HMBC correlation from H-2 (δ
8.42) at the adenine moiety to C-1′ (δ 91.5) at the ribose
moiety (Figure 2 and Figure S10 in Supplementary mate-
rial). Although compound 21 was obtained, this com-
pound easily isomerized to 17 by heating with a catalytic
amount of p-TsOH·H2O in refluxing chlorobenzene [44].
Deprotection of the acetyl group in 17 under basic con-
ditions gave alcohol 22. The hydroxy group in 22 was
replaced with a hydrogen atom by a Barton-McCombie
HBV replication assay
At 3 days after seeding, Hep38.7-Tet cells were incu-
bated in the absence of tetracycline to induce HBV
replication and treated with or without the compound
for 6 days. HBV DNA in the culture supernatant were
recovered and quantified by real-time PCR using 5′-
AAGGTAGGAGCTGGAGCATTCG-3′ (Forward) and
5′-AGGCGGATTTGCTGGCAAAG-3′ (Reverse) as
a primer set and 5′-AGCCCTCAGGCTCAGGGC
ATAC-3′ as a probe [41].
Results
Synthesis of EdA and EdAP
The syntheses of EdA (2) and EdAP (3) are depicted in
Scheme 3. The silyl-Hilbert-Johnson reaction consists of
Scheme 3. Synthesis of EdA (2) and EdAP (3).

8
M. KAMATA ET AL.
Synthesis of EFdA
The glycosyl donor 16 was also used for the synthesis
of EFdA (1) (Scheme 4). The silyl-Hilbert-Johnson
reaction between 5 and 16 afforded the desired β-
anomer 25 as a sole product in 86% yield. In the
synthesis of EdA, the N-glycoside 17 and isoadenine
derivative 21 were obtained in the glycosylation step.
Because the nucleophilicity of the nitrogen at the
three-position in 2-fluoroadenine will be suppressed
by the electron withdrawing fluorine at the C-2 posi-
tion, the silylated 2-fluoroadenine will selectively react
with 16 at the nitrogen at the nine-position (Figure 3).
According to the same protocol for the synthesis of 2,
compound 25 was converted into compound 29.
Deprotection of the PMB group in 29 with CAN
gave 1 in 58% yield.
Figure 2. A selected HMBC correlation in compound 21.
deoxygenation reaction [45,46]. The alcohol 22 was con-
verted into phenyl thionocarbonate 23, which was heated
with tri-(n-butyl)tin hydride and AIBN to give 24.
Removal of the tert-butyldiphenylsilyl (TBDPS) and
triethylsilyl (TES) groups in 24 with TBAF gave 18 in
95% yield. Deprotection of the PMB group in 18 with
CAN gave EdA (2) in 82% yield. We found that the lack
of reproducibility in the introduction of the cycloSal into
18 was due to the low solubility of 18 in acetonitrile. The
use of THF as a solvent instead of acetonitrile improved
the reproducibility of the cycloSal-introduction.
Coupling of 18 with chlorophosphite 19 [27] in THF,
followed by oxidation of the resultant phosphite ester
with 30% hydrogen peroxide gave 20 as a 1.43:1 diaster-
eomeric mixture in 41% yield. The difficulty in removal of
trace impurities from the product by silica gel chromato-
graphy caused the low-isolated yield. Deprotection of the
PMB group in 20 with CAN gave 3 as a 1.25:1 diaster-
eomeric mixture in 65% yield.
Effect of EdAP on hepatitis B virus replication
The inhibitory effect of EdAP on the replication of
HBV is depicted in Figure 4. EdAP did not show
significant cytotoxic activity against Hep38.7-Tet
Figure 3. Comparison of the reactivity between adenine and
2-fluoroadenine.
Scheme 4. Synthesis of EFdA (1).

SYNTHESIS OF NUCLEOTIDE ANALOGUES, EFDA, EDA AND EDAP
9
Figure 4. Effect of EdAP on the replication of HBV. (a) Cytotoxicity of EdAP against Hep38.7-Tet cells after 6 days was determined
by XTT assay [40]. (b) Anti-HBV activity of EdAP was evaluated by real-time PCR quantifying HBV DNA in the culture supernatant of
Hep38.7-Tet cells treated with or without the compound for 6 days [41]. The data indicate the means SD of five samples from an
experiment.
cells at a concentration below 100 μM (Figure 4(a)).
EdAP reduced the HBV DNA secreted from the cells
in a dose-dependent manner (Figure 4(b)). The half-
maximal inhibitory concentration (IC50) value of
EdAP was determined to be 14.5 μM. On the other
hand, the effect of EdA on the replication of HBV is
weak (Figure S21 in Supplementary material). EdA at
30 μM reduced HBV DNA to only 78%. These results
suggest that the cycloSal moiety installed in EdAP
improved the anti-HBV activity of EdA.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Core Research for
Evolutional Science and Technology [JPMJCR1413]; Japan
Agency for Medical Research and Development [JP18
fk0210036j0001, JP18fk0310101j1002, JP18fk0310103j0202,
JP18fk0310114j0002, JP18fm0208019j0002]; Japan Society
for the Promotion of Science [KAKENHI/JP17H04085,
KAKENHI/JP66KT0111].
Conclusion
The syntheses of EFdA, EdA, and EdAP were achieved
using a common synthetic strategy starting from
known 1,2-diacetylribose derivative 16. The present
method provides easy and reproducible access to
these nucleoside analogues. The present biological
studies indicate that EdAP reduced HBV replication
with an IC50 value of 14.5 μM, whereas this compound
did not influence the proliferation of the host cells.
The present findings provide valuable information for
the design and development of novel nucleoside/
nucleotide drugs against HBV.
ORCID
Toshifumi Takeuchi
0857
Shogo Kamo
Shusuke Tomoshige
5809
Shinji Kamisuki
Fumio Sugawara
Kouji Kuramochi
http://orcid.org/0000-0002-5096-
http://orcid.org/0000-0002-5799-4937
http://orcid.org/0000-0002-4948-
http://orcid.org/0000-0001-8773-3909
http://orcid.org/0000-0002-5419-2955
http://orcid.org/0000-0003-0571-9703
References
[1] Kohgo S, Ohrui H, Kodama E, et al. 4ʹ-C-substituted-
2-haloadenosine
2005:2502109.
derivative.
Can
Pat
CA.
Author contributions
[2] Kohgo S, Yamada K, Kitano K, et al. Design, efficient
synthesis, and anti-HIV activity of 4ʹ-C-cyano- and 4ʹ-
C-ethynyl-2ʹ-deoxy purine nucleosides. Nucleosides
Nucleotides Nucleic Acids. 2004;23:671–690.
S.Kamisuki., H.O., F.S., and K.K. designed the research; M.
K., T.T., E.H., K.N., M.O., M.I., and K.N. and K.K. performed
the research. S.Kamo., S.T., K.W., S.Kamisuki., and K.
K. analyzed the data; S.T. and K.K. wrote the article.
[3] Ohrui H. 2′-Deoxy-4′-C-ethynyl-2-fluoroadenosine,
a nucleoside reverse transcriptase inhibitor, is highly
potent against all human immunodeficiency viruses
type 1 and has low toxicity. Chem Rec. 2006;6:133–143.
[4] Ohrui H, Hayakawa H, Kohgo S, et al. Creation of low
toxic reverse-transcriptase inhibitory nucleosides that
prevent the emergence of drug-resistant HIV
variants. J Synth Org Chem Jpn. 2006;64:716–723.
[5] Ohrui H, Kohgo S, Hayakawa H, et al. 2′-Deoxy-4′-
C-ethynyl-2-fluoroadenosine: a nucleoside reverse
transcriptase inhibitor with highly potent activity
Acknowledgments
This study was supported by the Japan Society for the
Promotion of Science KAKENHI (JP17H04085, JP66K
T0111); the JST CREST program (JPMJCR1413); the Japan
Agency for Medical Research and Development, AMED
(JP18fk0310114j0002, JP18fk0310101j1002, JP18fk0310103j0
202, JP18fm0208019j0002, JP18fk0210036j0001).

10
M. KAMATA ET AL.
against wide spectrum of HIV-1 strains, favorable
toxic profiles, and stability in plasma. Nucleosides
Nucleotides Nucleic Acids. 2007;26:1543–1546.
[6] Michailidis E, Marchand B, Kodama EN, et al.
Mechanism of inhibition of HIV-1 reverse transcrip-
tase by 4′-Ethynyl-2-fluoro-2′-deoxyadenosine tripho-
sphate, a translocation-defective reverse transcriptase
inhibitor. J Biol Chem. 2009;284:35681–35691.
[7] Ohrui H. Development of modified nucleosides that
have supremely high anti-HIV activity and low toxi-
city and prevent the emergence of resistant HIV
mutants. Proc Jpn Acad Ser B Phys Biol Sci.
2011;87:53–65.
[8] Michailidis E, Huber AD, Ryan EM, et al. 4′-Ethynyl
-2-fluoro-2′-deoxyadenosine (EFdA) inhibits HIV-1
reverse transcriptase with multiple mechanisms.
J Biol Chem. 2014;289:24533–24548.
[9] Kodama EI, Kohgo S, Kitano K, et al. 4′-Ethynyl nucleo-
side analogs: potent inhibitors of multidrug-resistant
human immunodeficiency virus variants in vitro.
Antimicrob Agents Chemother. 2001;45:1539–1546.
mice and favorable pharmacokinetic properties in
mice and the rhesus macaque. Antimicrob Agents
Chemother. 2015;59:4190–4198.
[20] Markowitz M, Sarafianos SG. 4′-Ethynyl-2-fluoro-2′-
deoxyadenosine, MK-8591: a novel HIV-1 reverse
transcriptase translocation inhibitor. Curr Opin HIV
AIDS. 2018;13:294–299.
[21] Ohrui H, Kohgo S, Kitano K, et al. Syntheses of 4′-
C-ethynyl-β-D-arabino- and 4′-C-ethynyl-2′-deoxy
-β-D-ribo-pentofuranosylpyrimidines and -purines
and evaluation of their anti-HIV activity. J Med
Chem. 2000;43:4516–4525.
[22] Ohrui H. A new paradigm for developing antiviral
drugs exemplified by the development of supremely
high anti-HIV active EFdA. J Antivir Antiretrovir.
2014;6:32–39.
[23] Nakata H, Amano M, Koh Y, et al. Activity against
human immunodeficiency virus type 1, intracellular
metabolism, and effects on human DNA polymerases
of 4ʹ-ethynyl-2-fluoro-2ʹ-deoxyadenosine. Antimicrob
Agents Chemother. 2007;51:2701–2708.
[10] Kawamoto A, Kodama E, Sarafianos SG, et al. 2′-
Deoxy-4′-C-ethynyl-2-halo-adenosines active against
drug-resistant human immunodeficiency virus type 1
variants. Int J Biochem Cell Biol. 2008;40:2410–2420.
[11] Michailidis E, Ryan EM, Hachiya A, et al.
[24] Kirby KA, Michailidis E, Fetterly TL, et al. Effects of
substitutions at the 4′ and 2 positions on the bioactivity
of 4′-ethynyl-2-fluoro-2′-deoxyadenosine. Antimicrob
Agents Chemother. 2013;57:6254–6264.
[25] Sugawara F, Takeuchi T, Kamisuki S, et al. Jpn Kokai
Tokkyo Koho. JP. 2015;151346A.
Hypersusceptibility
mechanism
of
Tenofovir-resistant HIV to EFdA. Retrovirology.
2013;10:65.
[26] Takeuchi T, Sriwilaijaroen N, Sakuraba A, et al. Design,
synthesis, and biological evaluation of EdAP, a 4′-
ethynyl-2′-deoxyadenosine 5′-monophosphate analog,
[12] Maeada K, Desai DV, Aoki M, et al. Delayed emer-
gence of HIV-1 variants resistant to 4ʹ-ethynyl
-2-fluoro-2ʹ-deoxyadenosine: comparative sequential
passage study with lamivudine, tenofovir, emtricita-
bine and BMS-986001. Antivir Ther. 2014;19:179–189.
[13] Wu VH, Smith RA, Masoum S, et al. MK-8591 (4′-
ethynyl-2-fluoro-2′-deoxyadenosine) exhibits potent
activity against HIV-2 isolates and drug-resistant
HIV-2 mutants in culture. Antimicrob Agents
Chemother. 2017;61:e00714–17.
[14] Njenda DT, Aralaguppe SG, Singh K, et al.
Antiretroviral potency of 4′-ethnyl-2′-fluoro-2′-
deoxyadenosine, tenofovir alafenamide and second-
generation NNRTIs across diverse HIV-1 subtypes.
J Antimicrob Chemother. 2018;73:2721–2728.
[15] Takamatsu Y, Das D, Kohgo S, et al. The high genetic
barrier of EFdA/MK-8591 stems from strong interac-
tions with the active site of drug-resistant HIV-1 reverse
transcriptase. Cell Chem Biol. 2018;25:1268–1278.
[16] Murphey-Corb M, Rajakumar P, Michael H, et al.
Response of simian immunodeficiency virus to the
novel nucleoside reverse transcriptase inhibitor 4′-
ethynyl-2-fluoro-2′-deoxyadenosine in vitro and
as
a potent influenza A inhibitor. Molecules.
2019;24:2603.
[27] Meier C. 2-Nucleos-5′-O-yl-4H-1,3,2-benzodioxa
phos-phinin-2-oxides—A new concept for lipophilic,
potential prodrugs of biologically active nucleoside
monophosphate. Angew Chem Int Ed Engl.
1996;35:70–72.
[28] Meier C, Knispel T, Clercq ED, et al. cycloSal-
pronucleotides of 2′,3′-dideoxyadenosine and 2′,3′-
dideoxy-2′,3′-didehydroadenosine: synthesis and
antiviral evaluation of a highly efficient nucleotide
delivery system. J Med Chem. 1999;42:1604–1614.
[29] Meier C, Balzarini J. Application of the
cycloSal-prodrug approach for improving the biolo-
gical potential of phosphorylated biomolecules.
Antiviral Res. 2006;71:282–292.
[30] Fukuyama K, Ohrui H, Kuwahara S. Synthesis of EFdA
via a diastereoselective aldol reaction of a protected
3-keto furanose. Org Lett. 2015;17:828–831.
[31] Vorbrüeggen H. Adventures in silicon-organic
chemistry. Acc Chem Res. 1995;28:509–520.
[32] Niedballa U, Vorbrüggen H. A general synthesis of
pyrimidine nucleosides. Angew Chem Int Ed Engl.
1970;9:461–462.
in
vivo.
Antimicrob
Agents
Chemother.
2012;56:4707–4712.
[17] Hachiya A, Reeve AB, Marchand B, et al.
Evaluation of combinations of 4′-ethynyl-2-fluoro-
2′-deoxyadenosine with clinically used antiretro-
viral drugs. Antimicrob Agents Chemother.
2013;57:4554–4558.
[18] Zhang W, Parniak MA, Mitsuya H, et al.
Preformulation studies of EFdA, a novel nucleoside
reverse transcriptase inhibitor for HIV prevention.
Drug Dev Ind Pharm. 2014;40:1101–1111.
[33] Kageyama M, Nagasawa T, Yoshida M, et al.
Enantioselective total synthesis of the potent
anti-HIV
nucleoside
EFdA.
Org
Lett.
2011;13:5264–5266.
[34] Kageyama M, Miyagi T, Yoshida M, et al. Concise
synthesis of the anti-HIV nucleoside EFdA. Biosci
Biotechnol Biochem. 2012;76:1219–1225.
[35] McLaughlin M, Kong J, Belyk KM, et al.
Enantioselective synthesis of 4′-ethynyl-2-fluoro-2′-
deoxyadenosine (EFdA) via enzymatic desymmetri-
zation. Org Lett. 2017;19:926–929.
[19] Stoddart CA, Galkina SA, Joshi P, et al. Oral
administration of the nucleoside EFdA (4′-
ethynyl-2-fluoro-2′-deoxyadenosine)
rapid suppression of HIV viremia in humanized
provides
[36] Geneva: World Health Organization. Global hepatitis
report 2017; 2017. Available from: https://www.who.

SYNTHESIS OF NUCLEOTIDE ANALOGUES, EFDA, EDA AND EDAP
11
int/hepatitis/publications/global-hepatitis-report2017
/en/
[41] Passioura T, Watashi K, Fukano K, et al. De
novo macrocyclic peptide inhibitors of hepatitis
[37] Takamatsu Y, Tanaka Y, Kohgo S, et al. 4ʹ-Modified
nucleoside analogs: potent inhibitors active against
entecavir-resistant hepatitis B virus. Hepatology.
2015;62:1024–1036.
B
virus cellular entry. Cell Chem Biol.
2018;25:906–915.
[42] Leonard NJ, Laursen RA. The synthesis of 3-β-
D-ribofuranosyladenine.
J
Am Chem Soc.
[38] Ogura N, Watashi K, Noguchi T, et al. Formation of
covalently closed circular DNA in Hep38.7-Tet cells,
a tetracycline inducible hepatitis B virus expression
cell line. Biochem Biophys Res Commun.
2014;452:315–321.
1963;85:2026–2028.
[43] Leonard NJ, Laursen RA. Synthesis of 3-β-
D-ribofuranosyladenine and (3-β-
D-ribofuranosyladenine)-5′-phosphate.
Biochemistry. 1965;4:354–365.
[39] Iwamoto M, Watashi K, Tsukuda S, et al. Evaluation
and identification of hepatitis B virus entry inhibitors
using HepG2 cells overexpressing a membrane trans-
porter NTCP. Biochem Biophys Res Commun.
2014;443:808–813.
[40] Roehm NW, Rodgers GH, Hatfield SM, et al. An
improved colorimetric assay for cell proliferation
and viability utilizing the tetrazolium salt XTT.
J Immunol Methods. 1991;142:257–265.
[44] Framski G, Gdaniec Z, Gdaniec M, et al.
A reinvestigated mechanism of ribosylation of ade-
nine under silylating conditions. Tetrahedron.
2006;62:10123–10129.
[45] Barton DHR, McCombie SW. A new method for the
deoxygenation of secondary alcohols. J Chem Soc
Perkin Trans. 1975;1:1574–1585.
[46] Crich D, Quintero L. Radical chemistry associated with
the thiocarbonyl group. Chem Rev. 1989;89:1413–1432.