Synthesis of (R)- and (S)-β-hydroxyphosphonate acyclonucleosides: Structural analogues of Adefovir (PMEA)

Article abstract of DOI:10.1016/j.tetasy.2011.08.010

A synthetic pathway to new acyclonucleoside phosphonates, as analogues of Adefovir, is described. The reduction of an acyclonucleoside β-ketophosphonate, readily available from the nucleobase and benzylacrylate, afforded a mixture of (R)- and (S)-β-hydrox

Full text of DOI:10.1016/j.tetasy.2011.08.010

Tetrahedron: Asymmetry 22 (2011) 1505–1511
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis of (R)- and (S)-b-hydroxyphosphonate acyclonucleosides:
structural analogues of Adefovir (PMEA)
Mahesh Kasthuri a, Laurent Chaloin b, Christian Périgaud a, Suzanne Peyrottes a,
⇑
a IBMM, UMR 5247 CNRS-UM1-UM2, Equipe Nucléosides & Effecteurs Phosphorylés, Université Montpellier 2, cc 1705, place E. Bataillon, 34095 Montpellier, France
b CPBS, UMR 5236 CNRS-UM1-UM2, Equipe de Biophysique et Bioinformatique, 1919 route de Mende, 34293 Montpellier cedex 5, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A synthetic pathway to new acyclonucleoside phosphonates, as analogues of Adefovir, is described. The
reduction of an acyclonucleoside b-ketophosphonate, readily available from the nucleobase and benzyl-
acrylate, afforded a mixture of (R)- and (S)-b-hydroxyphosphonate derivatives which was resolved. The
assignment of the absolute configuration was proposed on the basis of NMR studies and was supported
by molecular modelling studies.
Received 20 June 2011
Accepted 11 August 2011
Available online 1 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
NH2
N
NH2
N
Over the past 30 years, antiviral drug discoveries1,2 have at-
tracted the interest of both organic and medicinal chemists to pro-
pose new potential drugs for the treatment of viral infections,
including DNA viruses and retroviruses. Among the various classes
of antiviral drugs, acyclic nucleoside and nucleotide derivatives3–5
have received considerable attention due to their ability to inhibit
viral DNA polymerases and reverse transcriptases, which play key
roles in the viral cycles.
N
N
N
N
N
N
O
P
O
P
OH
OH
OH
OH
O
O
1
1
3
3
2
2
4
4
Adefovir 1
Tenofovir 2
(PMEA)
(PMPA)
Acyclic nucleoside phosphonates (ANPs) belong to a family of
modified nucleotide analogues, in which the sugar moiety has been
replaced by a functionalized acyclic chain linking the nucleobase at
one end and the phosphonic acid group at the other. Therefore,
ANPs are metabolically stable due to the presence of a phospho-
nate linkage (P–C) instead of a phosphoester (P–O), making these
compounds resistant towards phosphatases. Furthermore, they
do not require the first intracellular phosphorylation step which
is often considered as a limiting-step and essential for the antiviral
effect. Indeed, ANPs share their mechanism of action with other
nucleoside analogues. Briefly, after two subsequent phosphoryla-
tion steps the ANP is converted to its corresponding triphosphate
derivative, which can then interfere with nucleic acid biosynthesis
as a DNA chain terminator.5
NH2
N
NH2
N
N
N
N
N
N
OH
N
OH
O
O
P
ONa
ONa
ONa
ONa
P
1
1
3
3
2
4
4
2
(S)-3
(R)-3
Figure 1. Examples of adenine containing ANPs (PMEA and PMPA) and the
structures of targeted analogues.
Amongst the antiviral agents that exhibited a broad spectrum of
activity and bear a phosphonate group, adenine containing ANPs
are well represented (Fig. 1). Prodrugs of Adefovir (PMEA, 1) and
Tenofovir (PMPA, 2) have been approved for the treatment of hep-
atitis B and HIV infections,6 respectively. Herein, we aimed to ob-
tain novel ANPs structurally related to Adefovir (PMEA) which
contained a carbinol moiety that replaced the oxygen atom of
the phosphonomethoxyethyl chain (Fig. 1). The CH(OH) functional-
ity is not an isostere for the C–O–C linkage, however, the resulting
b-hydroxyphosphonate derivative may present some structural
features (distance between the nucleobase and the phosphonate
moiety, flexibility and so on) and the presence of the hydroxyl
group may allow the formation of supplementary hydrogen bonds.
Enantiomerically pure b-hydroxyphosphonate derivatives have
received significant attention due to their potential biological
⇑
Corresponding author. Tel.: +33 467 144964; fax: +33 467 042029.
E-mail address: peyrottes@univ-montp2.fr (S. Peyrottes).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.08.010

1506
M. Kasthuri et al. / Tetrahedron: Asymmetry 22 (2011) 1505–1511
activity.7 Consequently, several synthetic routes using the corre-
sponding b-ketophosphonates as intermediates have been re-
ported and may involve stereoselective reduction,8,9 enzymatic
reduction,10 as well as the use of chiral derivatizing agents.7,11
Herein, we report the synthesis and resolution of enantiomerically
pure (R)- and (S)-b-hydroxyphosphonate bearing adenine as a
nucleobase, and the use of a chiral derivatizing agent such as (S)-
methoxyphenyl acetic acid [(S)-MPA].
Na in methanol.13 Finally, diisopropylethylamine (DIEA) was se-
lected as a suitable base, allowing the exclusive formation of the
N-9 alkylated product in good yield (77%). It is noteworthy that this
step could be easily scaled up to a 25 g batch. After removal of the
solvent, the addition of ethyl acetate to the crude afforded pure 5
as a precipitate without further purification. The exclusive forma-
tion of the N-9 alkylated product was confirmed by NMR and UV
absorption studies.14
The exocyclic amino group of 5 was next protected with a
monomethoxytrityl (MMTr) group to give
6 in 88% yield
2. Results and discussion
2.1. Chemistry
(Scheme 2). The MMTr moiety was chosen as the nucleobase pro-
tecting group to enhance the solubility of intermediate 6 in the less
polar solvents used in the next step of the synthesis (such as THF).
Compound 6 was treated with an excess of the lithium salt of
dimethylmethylphosphonate at ꢀ78 °C in THF and afforded the
desired key intermediate b-ketophosphonate 4 in 55% yield.
Reduction of b-ketophosphonate 4 was performed with NaBH4 in
methanol and led to a racemic mixture of b-hydroxy-phosphonate
( )-7 in 89% yield.
With the racemic mixture of ( )-7 in hand, we next resolved the
(R)- and (S)-isomers using (S)-methoxyphenylacetic acid [(S)-
MPA], a well known chiral derivatizing agent. Thus, coupling of
( )-7 with (S)-MPA in the presence of 1,3-dicyclohexylcarbodiim-
ide (DCC) and a catalytic amount of 4-N,N-(dimethylamino)pyri-
dine (DMAP), in dichloromethane at room temperature, provided
an equimolar (a 1:1 ratio was observed in the 31P NMR spectra,
Figure 2) mixture of the two mandelates [Scheme 2, (R,S)-8 as less
polar and (S,S)-9 as more polar], which were separated by silica gel
The retro-synthesis proposed for the preparation of the target
b-hydroxyphosphonate derivatives (R)-3 and (S)-3 is shown in
Scheme 1. Both isomers may be obtained via reduction of the cor-
responding b-ketophosphonate intermediate 4 and resolution of
the diastereomeric pair with a suitable chiral derivatizing agent.
The assignment of the absolute configuration would then be ad-
dressed using NMR spectroscopy. The key intermediate 4 could
be prepared through treatment of 5 with the lithium salt of dim-
ethylmethylphosphonate. Compound 5 would be available through
the Michael addition of adenine to benzyl acrylate as the Michael
acceptor.
In the literature, such Michael additions suffer from low yields
and a lack of regioselectivity, with the formation of both N-7 and
N-9 substituted products. Initially the reaction was conducted in
the presence of various bases such as K2CO3,12 Cs2CO3, or catalytic
B
OH
O
P
ONa
ONa
O
(R)-3
OBn
B
O
O
P
B
O
OMe
OMe
OBn
B
OH
O
P
Adenine
4
5
ONa
ONa
(S)-3
Scheme 1. Proposed retrosynthesis for derivatives (R)-3 and (S)-3.
MMTr
MMTr
NH
N
NH2
N
NH
N
NH2
N
O
N
N
N
N
N
N
N
N
N
N
DIEA
N
MMTrCl
CH3P(O)(OMe)2
OBn
+
DMF, 80oC
77%
O
O
O
P
Pyridine, 60oC
88%
O
N
H
n-BuLi, THF
-78oC, 55%
O
O
OBn
OBn
5
6
4
MMTr
NH
N
MMTr
NH
MMTr
NH
N
N
N
O
O
O
O
N
N
N
N
N
N
NaBH4
(S)-MPA
N
OH
O
4
+
O
O
O
O
O
O
MeOH, rt
89%
DCC, DMAP
CH2Cl2
N
P
P
P
O
O
7
( )-
O
O
less polar-(R,S)-8
more polar-(S,S)-9
35%
38%
Scheme 2. Synthesis of the b-ketophosphonate intermediate 4 and the diastereomeric pair: (R,S)-8 and (S,S)-9.

M. Kasthuri et al. / Tetrahedron: Asymmetry 22 (2011) 1505–1511
1507
P(OCH3)2
31P-NMR of
the mixture
O
P
OCH3
H
CH3O
CH3O
H
O
H4,H4'
1H-NMR
O
H2
H2'
H1
H1'
H8
N
H1,H1'
H2,H2'
N
N
N
MMTrHN
Less Polar diastereomer
(R,S) -8
(S,S) -9
OCH3,OCH3'
N
N
OCH3
H1'
H
1 H
O
H
MMTrHN
1H-NMR
N
H4,H4'
N
O
H4'
OCH3
H4
O
H8
P
H1,H1'
OCH3
H2,H2'
More Polar diastereomer
Figure 2. Assignment of the absolute configuration of the diastereomeric pair based on the NMR chemical shift differences due to the shielding effect exerted by the
anisotropy cone of the phenyl ring from (S)-MPA.
column chromatography. The less polar and more polar diastereo-
mers were obtained in 35% and 38% yields, respectively.
Once the absolute configuration was determined by NMR spec-
troscopy, the final steps of the synthesis were separately carried
out for both derivatives (Scheme 3). Treatment of the mandelates
(R,S)-8 or (S,S)-9 with LiOHꢁH2O, in a mixture of MeOH/H2O (8:2)
at room temperature, led to enantiomerically pure (R)-10 or (S)-
10 after flash column purification in 74% and 70% yields, respec-
tively. Finally, the (R)- and (S)-10 isomers were treated with an ex-
cess of TMSBr in dry dichloromethane at room temperature to
MMTr
N
MMTr
NH2
NH
N
NH
N
O
O
N
N
N
N
N
N
N
LiOH·H2O
TMSBr, DCM
N
OH
O
P
O
O
OH
O
P
N
H2O, Et3N
DowexNa+, dialysis
quantitative
ONa
ONa
MeOH:H2O, rt
74%
O
O
P
O
(R,S)-8
(R)-3
O
(R)-10
MMTr
N
MMTr
N
NH2
NH
NH
N
O
O
N
N
N
N
N
LiOH·H2O
TMSBr, DCM
N
OH
O
P
N
O
O
N
OH
O
P
N
H2O,Et3N
MeOH:H2O, rt
70%
ONa
ONa
O
O
Dowex Na+, dialysis
quantitative
P
O
(S,S)-9
(S)-3
O
(S)-10
Scheme 3. Final steps of the synthesis of acyclic phosphonate analogues (R)- and (S)-3.

1508
M. Kasthuri et al. / Tetrahedron: Asymmetry 22 (2011) 1505–1511
Table 1
remove both the dimethylphosphonate and MMTr protecting
groups in a single step, and afford the desired derivatives (R)-3
and (S)-3 in quantitative yields after passing through a Dowex
Na+ ion exchange resin and dialysis.
Chemical shift data of the diastereomeric pair of b-hydroxyphosphonates 8 and 9
Entry
Group
Less polar (dRS
)
More polar (dSS
)
Dd
1H NMR (300 MHz, CDCl3)
1
2
CH2P(O)
2.10
2.02
3.57
3.61
2.16
3.59
6.94
1.99
1.87
3.49
3.51
2.28
4.04
7.42
0.11
0.15
0.08
0.1
0.12
0.45
0.48
2.2. Assignment of the absolute configuration by NMR studies
(CH3O)2P
The assignment of the absolute configuration of the diastereo-
meric pair was achieved by the Trost model,15 which is based on
the chemical shift differences in the NMR spectra between the
two diastereomers (Table 1). The origin of the chemical shift differ-
ence is attributed to the shielding cone of the phenyl ring from (S)-
MPA; the signals of the substituents under the shielding cone are
moved upfield.16 When the phenyl group is eclipsed with the ade-
nine–CH2–CH2 moiety in the less polar diastereomer (Fig. 2), the
3
4
5
Ad–CH2CH2
Ad–CH2CH2
H-8 (adenine)
13C NMR (75 MHz, CDCl3)
7
8
9
CH2P(O)
Ad–CH2CH2
Ad–CH2CH2
29.9
34.7
39.6
29.6
34.4
40
0.3
0.3
0.4
Arom(MPA)
H-8
A
N
N
OCH3
(CH3O)2P
H1'
H
1 H
O
H
MMTrHN
N
N
H4'
O
OCH3
H4
O
H8
P
OCH3
Ad-CH2-CH2
More Polar diastereomer
Arom(MPA)
H-8
CH2-P
O
P
OCH3
H
CH3O
CH3O
H
O
B
Ad-CH2-CH2
H2
H2'
O
H1
H1'
H8
CH3-O(MPA)
N
N
N
(CH3O)2P
N
CH3-O(MTrt) + Ad-CH2-CH2
MMTrHN
Less Polar diastereomer
CH-O
Figure 3. Part of the NOESY spectra of the (A) more polar diastereomer (S,S)-9; (B) less polar diastereomer (R,S)-8 in CDCl3.

M. Kasthuri et al. / Tetrahedron: Asymmetry 22 (2011) 1505–1511
1509
two methylene groups are shielded and should appear upfield
compared to the more polar one. Indeed, we observed that the
Ad–CH2–CH2 signal resonates at 2.16 ppm in the less polar diaste-
reomer whereas it resonates at 2.28 ppm in the more polar one (Dd
0.12 ppm). Similarly the Ad–CH2–CH2 resonates at 3.59 ppm in the
partial charges17,18 and Charmm22 force field,19 respectively. Then,
four planar angles were selected corresponding to the bonds of the
methoxy and carbonyl groups of MPA and the proton at the b-po-
sition to the phosphorus atom of each diastereomer, and a sys-
temic conformational search using the AMMP (Another Molecular
Mechanics Program) program20 and Charmm22 as Force field was
carried out on the diastereomeric pair. Molecular modelling stud-
ies supported the NMR chemical shift analysis of each diastereo-
mer due to the geometry predicted by Mosher’s approximation21
in which the methoxy and carbonyl groups of MPA and the proton
at the b-position to the phosphorus atom are in almost the same
plane for both diastereomers (Fig. 4).
less polar diastereomer, whereas it resonates at 4.04 ppm in the
more polar one (Dd 0.45 ppm). On the other hand, when the phenyl
group is eclipsed with the (CH3O)2P and the CH2P(O) groups, as in
the more polar diastereomer, both the methyl and methylene pro-
tons are shielded and should appear upfield with respect to the less
polar one. Indeed, the two methyl protons of the (CH3O)2P group
resonate at 3.51 and 3.49 ppm in the more polar diastereomer
(Fig. 2), whereas in the less polar one, they resonate at 3.61 and
In agreement with the NMR analysis, a
p-stacking effect be-
3.57 ppm (
D
d 0.1 and
D
d 0.08 ppm, respectively). Similarly, the
tween the adenine moiety and phenyl ring from MPA was also ob-
served in our molecular models. In the case of the lowest energy
conformer compound (R,S)-8 (Fig. 4A), the H-8 proton was pro-
jected to be close to the phenyl ring and resonate at a higher field
than for (S,S)-9 (Fig. 4B). These theoretical studies supported the
experimental results and confirmed our hypothesis with regard
to the absolute configurations of both diastereomers.
protons of the CH2P(O) group resonate at 1.87 and 1.99 ppm in
the more polar diastereomer whereas in less polar one, they ap-
peared at 2.02 and 2.1 ppm (Dd 0.15 and Dd 0.11 ppm, respec-
tively). In addition, chemical shift differences were also observed
in the 13C NMR spectra of the two diastereomers and these are
summarized in Table 1.
When the phenyl group was eclipsed with the adenine moiety
as in the less polar diastereomer, the H-8 proton of the nucleobase
was shielded and resonated at 6.94 ppm whereas in the more polar
3. Conclusion
one, it appeared at 7.42 ppm (
Dd 0.48 ppm). This difference can be
In conclusion, we have synthesized, separated and characterized
a newset of enantiomerically pure(R)- and(S)-b-hydroxyphosphon-
ic acids belonging to the adenine acyclonucleoside phosphonate
family. These derivatives were designed as structural analogues of
Adefovir (PMEA), a well-known ANP used for the treatment of HBV
infections. The newly obtained derivatives were assayed in various
cell lines (including HEL, CRFK, Vero, MDCK and HeLa cells) for anti-
viralactivityagainstawidevarietyofDNAandRNAviruses[varicella
zoster virus (VZV), cytomegalovirus (CMV), herpes simplex virus
type 1 (HSV-1, KOS and TK- KOS), HSV-2 (G), vaccinia virus and vesic-
ular stomatitis viruses, feline herpes and corona viruses, parainflu-
enza-3, reovirus-1, Sindbis, Coxsackie B4 and Punta Toro virus,
influenza A (H1N1 and H3N2) and influenza B viruses and respira-
tory syncytial virus]. However, none of them revealed a significant
attributed to the -stacking effect between the phenyl ring and the
p
nucleobase. This observation was supported by molecular model-
ling studies and suggested that the H-8 proton was in close prox-
imity to the phenyl ring. The 2D NMR-NOESY spectra were also
obtained and a selected portion is shown in Figure 3. In the case
of the less polar diastereomer (R,S)-8, we clearly observed cross
peaks between the H-8 proton of the nucleobase and the aromatic
protons of MPA with both the adenine–CH2–CH2 moiety and the
(CH3O)2P groups (Fig. 3B). In comparison with an identical part
of the 2D NOESY spectrum of the more polar diastereomer, due
to the overlapping of the signals of interest, less relevant cross
peaks were observed (Fig. 3A). Consequently, the 1H-, 13C- and
31P-NMR spectra of both isolated diastereoisomers strongly sug-
gested that the absolute configuration of the less polar compound
is (R,S)-8 while the more polar compound is (S,S)-9.
effect up to 100
and MDCKcell culturesand thestudiedcompoundswere foundtobe
non-toxic (up to 100 M). Further studies are currently underway to
lM. Cytotoxicity was evaluated on HEL, Vero, HeLa
l
2.3. Molecular modelling studies
tentatively explain the lack of biological activity.
In addition to the NMR experiments, computational calcula-
tions were carried out in order to determine the minimum energy
conformer for each diastereomeric mandelate. Molecular model-
ling was achieved using the Biopolymer module implemented in
the Insight II suite program (Accelrys). Atomic charges and poten-
tials were assigned using the Gasteiger–Marsili empirical atomic
4. Experimental
Tetrahydrofuran (THF) was distilled from sodium/benzophenone
immediately prior to use, DMF and methanol from CaH2, dichloro-
methane from P2O5, pyridine and Et3N from KOH. Solids were dried
Figure 4. (A) Minimum energy conformer for the less polar diastereomer (R,S)-8. (B) Minimum energy conformer for the more polar diastereomer (S,S)-9.

1510
M. Kasthuri et al. / Tetrahedron: Asymmetry 22 (2011) 1505–1511
over P2O5 under reduced pressure at rt. Moisture sensitive reactions
were performed under argon atmosphere using oven-dried glass-
ware. 1H NMR spectra were recorded at 300 MHz and 13C NMR spec-
tra at 75 MHz with proton decoupling at 25 °C using a Bruker 300
Avance. Chemical shifts are given in d values referenced to the resid-
ual solvent peak (CHCl3 at 7.26 and 77 ppm or DMSO-d5 at 2.49 and
39.5 ppm) relative to TMS. COSY experiments were performed in or-
der to confirm proton assignments. Coupling constants, J, are re-
ported in Hertz (Hz). 2D 1H–3C heteronuclear COSY spectra were
recorded for the attribution of 13C signals. 31P NMR spectra were re-
corded at ambient temperature at 121 MHz with proton decoupling.
Chemical shifts are reported relative to external H3PO4. The ESI-QTof
mass spectra were recorded in the positive-ion or negative-ion
modes using a Micromass Q-TOF spectrometer. Specific rotations
were measured with a Perkin–Elmer Model 341 spectropolarimeter
(path length 1 cm) and are given in units of 10ꢀ1 deg cm 2 gꢀ1. TLC
was performed on pre-coated aluminium sheets of silica gel 60
F254 (Merck, Art. 9385), visualization of products being accom-
plished by UV absorbance followed by charring with 5% ethanolic
sulfuric acid and then heating.
4.3. [N-6-Monomethoxytrityl-N-9-(3-oxo-4-dimethyl-
phosphonobutyl)]adenine 4
A
solution of dimethylmethylphosphonate (0.473 mL, 4.37
mmol) in dry THF (7.5 mL) was cooled to ꢀ78 °C and n-BuLi
(1.74 mL, 4.37 mmol) was added dropwise. The resulting mixture
was stirred at ꢀ78 °C for 1 h and a solution of 6 (1.0 g, 1.75 mmol)
in THF (5 mL) was added dropwise. The reaction mixture was stir-
red at ꢀ78 °C for 2 h, and then the reaction mixture was added to a
cooled saturated NH4Cl solution. The resulting organic layer was
separated and the aqueous layer was extracted with ethyl acetate.
The combined organic layers were washed with water, brine, dried
over MgSO4 and concentrated under reduced pressure. The crude
product was purified by flash column chromatography (4% MeOH
in DCM) to afford 4 (560 mg, 55%) as a colourless solid. Rf (4%
MeOH in DCM) 0.31. 1H NMR (300 MHz, CDCl3) d 3.21 (d,
J = 23.0 Hz, 2H, CH2P), 3.4 (t, J = 6.0 Hz, 2H, Ad–CH2–CH2), 3.77 (d,
J = 11.0 Hz, 6H, (CH3O)2P), 3.92 (s, 3H, OCH3), 4.61 (t, J = 6.0 Hz,
2H, Ad–CH2–CH2), 6.92 (d, J = 8.5 Hz, 2Harom), 7.38 (m, 12Harom),
8.02 (s, 1H, H-2), 8.21 (s, 1H, H-8). 13C NMR (75 MHz, CDCl3) d
39.4 (d, J = 199 Hz, CH2P), 42.7 (d, J = 35 Hz, (CH3O)2P), 70.9 (s, C–
Ph3), 113.1 (s, Carom), 120.7 (s, C-5), 126.8, 127.8, 128.9, 130.2,
137.2, 140.9 (s, Carom) 145.2 (s, C-8), 148.6 (s, C-4), 152.0 (s, C-2),
154.0 (s, C-6), 158.3 (s, Carom), 199.3 (d, J = 6.5 Hz, C@O). 31P
(121 MHz, CDCl3) d 21.5. ESI-QTof > 0: m/z 586 (M+H)+, HRMS
4.1. N-9-(2-Benzyloxycarbonylethyl)adenine 5
To a suspension of adenine (10.0 g, 74 mmol) in dry DMF
(200 mL) diisopropylethylamine (21.48 mL, 148 mmol) was added
and the mixture was heated at 80 °C for 1 h under argon. Then,
the reaction mixture was cooled to room temperature and benzyl-
acrylate (24.0 g, 148 mmol) was added. The mixture was heated at
80 °C for 3–4 days. The solvent was evaporated and the crude was
suspended in ethyl acetate and filtered. The resulting solid was
washed with ethyl acetate to give 5 as a fine powder (17 g, 77%).
Rf (5% MeOH in DCM) 0.4. 1H NMR (300 MHz, DMSO-d6) d 3.21 (t,
J = 6.0 Hz, 2H), 4.59 (t, J = 6.0 Hz, 2H), 5.25 (s, 2H), 7.46 (m, 5H, Har-
om), 8.26 (s, 1H), 8.31 (s, 1H). 13C NMR (75 MHz, DMSO-d6) d 33.5 (s,
CH2), 390 (s, CH2), 65.7 (s, CH2), 118.7 (s, C5), 127.9, 128.0, 128.3,
135.8, 140.8 (s, C8), 149.4 (s, C2), 152.3 (s, C6), 155.9 (s, C4),
170.5 (C@O). ESI-QTof > 0: m/z 298.1 (M+H)+, HRMS calcd for
calcd for C31H32N5O5P: 586.2219, found: 586.2204. UV kmax
275 nm ( 21,100), kmin = 248 nm ( 11,700) (EtOH 95).
=
e
e
4.4. ( )-[N-6-Monomethoxytrityl-N-9-(3-hydroxy-4-dimethyl
phosphonobutyl)]adenine 7
To a solution of 4 (1.2 g, 2.04 mmol) in methanol was added so-
dium borohydride (463 mg, 12.24 mmol) and the resulting mixture
was stirred for 1 h. Then, the reaction mixture was cooled to 0 °C
and quenched with saturated NH4Cl. The solvent was removed un-
der reduced pressure, and the crude was dissolved in water and ex-
tracted with ethyl acetate. The resulting organic layer was washed
with brine, dried over MgSO4 and concentrated under reduced
pressure. The crude product was purified by flash column chroma-
tography (4% MeOH in DCM) to afford ( )-7 (1.05 g, 87%) as a col-
ourless solid. Rf (4% MeOH in DCM) 0.28. 1H NMR (300 MHz, CDCl3)
d 1.86 (d, J = 5.5 Hz, 1H, CHP), 1.93 (d, J = 8.0 Hz, 1H, CHP), 1.96
(dm, J = 41.5 Hz, 2H, Ad–CH2–CH2), 3.62 (s, 3H, (CH3O)2P), 3.66 (s,
3H, (CH3O)2P), 3.73 (s, 3H, OCH3), 3.77 (m, 1H, CH(OH)), 4.3 (dm,
J = 38.0 Hz, Ad–CH2–CH2), 6.73 (d, J = 12.0 Hz, 2Harom), 6.98 (br s,
1H, NH), 7.21 (m, 12Harom), 7.76 (s, 1H, H-2), 7.98 (s, 1H, H-8).
13C NMR (75 MHz, CDCl3) d 32.6 (d, J = 138.0 Hz, CH2P), 38.5 (d,
J = 15.0 Hz, Ad–CH2–CH2), 40.2 (s, Ad–CH2–CH2), 52.4 (d,
J = 6.5 Hz, (CH3O)P), 52.5 (d, J = 6.5 Hz, (CH3O)P), 52.2 (s, Carom),
62.6 (d, J = 4.0 Hz, CH(OH)), 71.0 (s, CPh3), 113.1 (s, Carom), 120.8
(s, C-5), 126.9, 127.9, 128.9, 130.2, 137.6, (s, Carom), 145.1 (s, C-8),
148.9 (s, C-4), 152.1 (s, C-2), 154.2 (s, C-6), 158.3 (s, Carom). 31P
(121 MHz, CDCl3) d 31.8. ESI-QTof > 0: m/z 588 (M+H)+, HRMS
C15H15N5O2: 298.1304, found: 298.1320. UV kmax = 261 nm (
e
21,000), kmin = 230 nm ( 9000) (EtOH 95).
e
4.2. [N-6-Monomethoxytrityl-N-9-(2-benzyloxycarbonyethyl)]
adenine 6
To a solution of 5 (12.0 g, 42 mmol) in dry pyridine (240 mL), 4-
methoxytrityl chloride (25.94 g, 84 mmol) was added portionwise
and the reaction mixture was then heated at 60 °C for 24 h. The sol-
vent was evaporated, and the residue was co-evaporated with a
toluene/methanol (1:1) mixture. The crude was dissolved in ethyl
acetate and washed with saturated NaHCO3, water, brine and dried
over MgSO4, concentrated in vacuo. The crude product was purified
by column chromatography (5% acetone in DCM) and the resulting
solid was passed through neutral alumina to remove unwanted
colouring material to afford 6 (20.4 g, 88%) as a colourless solid.
Rf (5% acetone in DCM) 0.37. 1H NMR (300 MHz, CDCl3) d 2.89 (t,
J = 6.5 Hz, 2H, Ad–CH2–CH2), 3.70 (s, 3H, OCH3), 4.41 (t, J = 6.5 Hz,
Ad–CH2–CH2), 5.03 (s, 2H, CH2Ph), 6.71 (d, J = 15.5 Hz, 2Harom),
7.12 (br s, 1H, NH), 7.18 (m, 17Harom), 7.77 (s, H-2), 7.89 (s, H-8).
13C NMR (75 MHz, CDCl3) d 34.2 (s, Ad–CH2–CH2), 39.3 (s, Ad–
CH2–CH2), 55.2 (s, Carom), 66.9 (s, CH2–Ph), 70.9 (s, C–Ph3), 113.1
(s, Carom), 120.9 (s, C-5), 126.9, 127.1, 127.9, 128.4, 128.5, 128.9,
130.2, 135.2, 137.2, 140.5 (s, Carom), 145.2 (s, C-8), 148.7 (s, C-4),
152.2 (s, C-2), 154.1 (s, C-6), 158.30 (s, Carom), 170.8 (s, C@O).
ESI-QTof > 0: m/z 570 (M+H)+, HRMS calcd for C35H31N5O3:
calcd for C31H34N5O5P: 588.2376, found: 588.2391. UV kmax
276 nm ( 23,900), kmin = 248 nm ( 11,600) (EtOH 95).
=
e
e
4.5. Derivatization of ( )-7 with (S)-MPA
To a solution of ( )-7 (1 g, 1.7 mmol) and (S)-methoxyphenyl
acetic acid (0.40 g, 2.38 mmol) in dichloromethane (15 mL) were
added 1,3-dicyclohexylcarbodiimide (0.49 g) and
a catalytic
amount of dimethylaminopyridine at room temperature. The
resulting mixture was stirred for 1 h, then filtered and the solvent
was evaporated under reduced pressure. The crude product was
purified by column chromatography (5% ethanol in diethyl ether)
to afford the less polar compound 8 (430 mg, 35%) and the more
polar compound 9 (470 mg, 38%).
570.2505, found: 570.2516. UV kmax = 275 nm
kmin = 246 nm ( 7000) (EtOH 95).
(e 18,200),
e

M. Kasthuri et al. / Tetrahedron: Asymmetry 22 (2011) 1505–1511
1511
4.5.1. Less polar diastereomer (R,S)-8
4.8. (R)-9-(3-Hydroxy-4-phosphonobutyl)adenine 3
Rf (5% ethanol in diethyl ether) 0.2. ½a D20
¼ þ53:3 (c 0.89,
ꢂ
MeOH). 1H NMR (300 MHz, CDCl3) d 2.1 (dd, J = 8.0 Hz, 1H, CHP),
2.02 (dd, J = 5.5 Hz, 1H, CHP), 2.16 (dm, J = 67.5 Hz, 2H, Ad–CH2–
CH2), 3.37 (s, 3H, OCH3), 3.57 (d, J = 11.0 Hz, 3H, (CH3O)2P), 3.59
(m, 2H, Ad–CH2–CH2), 3.61 (d, J = 11.0 Hz, 3H, (CH3O)2P), 3.69 (s,
3H, OCH3), 4.74 (s, 1H, CH), 4.94 (m, 1H, CH), 6.7 (d, J = 12.0 Hz,
2Harom), 6.8 (br s, 1H, NH), 6.94 (s, 1H, H8), 7.26 (m, 12Harom),
7.98 (s, 1H, H2). 13C NMR (75 MHz, CDCl3) d 29.9 (d, J = 140.0 Hz,
CH2P), 34.7 (d, J = 7.5 Hz, Ad–CH2–CH2), 39.6 (Ad–CH2–CH2), 52.4
(d, J = 6.5 Hz, (CH3O)2P), 52.7 (d, J = 6.5 Hz, (CH3O)2P), 55.2 (s,
To a cooled solution of (R)-10 (110 mg, 0.187 mmol) in dry
dichloromethane (4 mL) was added trimethylsilylbromide
(0.16 mL, 1.22 mmol), and the reaction mixture was stirred at
room temperature until the completion of the reaction, as indi-
cated by TLC (isopropanol/water/ammonia 30%, 7:2:1, v/v/v). Next,
water was added and the reaction mixture was stirred at room
temperature for 1 h. The aqueous layer was separated, washed
with diethyl ether and concentrated under high vacuum. After
freeze-drying, the crude product was treated with triethylamine
until pH 7 and then passed through a Dowex Na+ ion exchange re-
sin column; the desired fractions were collected and lyophilized to
give (R)-3 quantitatively as a hygroscopic salt. Rf (iPrOH/H2O/
Carom), 57.4 (OCH3), 66.9 (d, J = 1.5 Hz, C-Ph3), 70.9 (CH(OH)), 82.2
(C(OCH3)), 113.1 (s, Carom), 120.9 (C-5), 126.8, 127.1, 127.3, 127.8,
128.9, 128.9, 129.2, 130.2, 136.5, 139.8 (s, Carom), 145.2 (C-8),
148.7 (C-4), 152.1 (C-2), 154.0 (C-6), 158.3 (s, Carom), 170.1
NH4OH 30%, 7:2:1, v/v/v) 0.25. ½a D20
ꢂ
¼ þ10:6 (c 0.47, H2O). 1H
(C@O). 31P (121 MHz, CDCl3)
d
27.6. ESI-QTof > 0: m/z 736
NMR (300 MHz, D2O) d 1.54 (m, 2H, CH2P), 1.9 (dm, J = 42.5 Hz,
2H, Ad–CH2–CH2), 3.84 (m, 1H, CH(OH)), 4.18 (t, J = 8.0 Hz, 2H,
Ad–CH2–CH2), 8.03 (s, 1H, H2), 8.06 (s, 1H, H8). 13C NMR
(75 MHz, D2O) d 35.2 (s, Ad–CH2–CH2), 37.1 (t, J = 13.0 Hz, CH2P),
41.0 (s, Ad–CH2–CH2), 66.2 (d, J = 3.5 Hz, CH(OH)), 118.4 (s, C-5),
142.6 (s, C-8), 148.7 (s, C-2), 152.1 (s, C-6), 155.3 (s, C-4). 31P
(100 MHz, CDCl3) d 18.5. ESI-QTof > 0: m/z 332 (M+H)+, HRMS calcd
for C9H12N5O4Na2P: 332.0501, found: 332.0490. UV kmax = 261 nm
(M+H)+, HRMS calcd for C40H42N5O7P: 736.2900, found:
736.2910. UV kmax = 276 nm (
e 18,700), kmin = 247 nm (e 7500)
(EtOH 95).
4.5.2. More polar diastereomer (S,S)-9
Rf (5% ethanol in diethyl ether) 0.17. ½a D20
¼ þ48:6 (c 0.74,
ꢂ
MeOH). 1H NMR (300 MHz, CDCl3) d 1.87 (ddd, J = 7.5 Hz, 5.0 Hz,
7.5 Hz, 1H, CHP), 1.99 (ddd, J = 5.0 Hz, 10.0 Hz, 5.0 Hz, 1H, CHP),
2.28 (d, J = 65.5 Hz, 2H, Ad–CH2–CH2), 3.32 (s, 3H, OCH3), 3.49 (d,
J = 11.0 Hz, 3H, (CH3O)2P), 3.51 (d, J = 11.0 Hz, 3H, (CH3O)2P), 3.69
(s, 3H, OCH3), 4.04 (m, 2H, Ad–CH2–CH2), 4.59 (s, 1H, CH), 4.97
(m, 1H, CH), 6.7 (d, J = 12.0 Hz, 2Harom), 6.8 (br s, 1H, NH), 7.24
(m, 12Harom), 7.42 (s, 1H, H8), 7.98 (s, 1H, H2). 13C NMR (75 MHz,
CDCl3) d 29.6 (d, J = 138.5 Hz, CH2P), 34.4 (d, J = 5.5 Hz, Ad–CH2–
CH2), 40.0 (s, Ad–CH2–CH2), 52.4 (d, J = 4.0 Hz, (CH3O)2P), 52.5 (d,
J = 4.0 Hz, (CH3O)2P), 55.2 (s, OCH3), 57.5 (s, OCH3), 67.9 (s, C–
Ph3), 70.9 (CH(OH), 82.4 (C(OCH3)), 113.1 (s, Carom), 120.9 (C-5),
126.8, 127.1, 127.8, 128.8, 129.0, 130.2, 135.2, 137.2, 139.8 (s, Car-
om), 145.2 (C-8), 148.9 (C-4), 152.2 (C-2), 154.1 (C-6), 158.3 (s, Car-
om), 170.1 (C@O). 31P (121 MHz, CDCl3), d 27.5. ESI-QTof > 0: m/z
736 (M+H)+, HRMS calcd for C40H42N5O7P: 736.2900, found:
(e 9000), kmin = 228 nm (e 1600) (EtOH 95).
4.9. (S)-N-9-(3-Hydroxy-4-phosphonobutyl)adenine 3
The procedure described above was applied to (S)-10 (120 mg,
0.204 mmol) to afford (S)-3 quantitatively as a hygroscopic salt.
The NMR chemical shifts were identical to (R)-3. ½a D20
¼ ꢀ10:6 (c
ꢂ
0.47, H2O). ESI-QTof > 0: m/z 332 (M+H)+, HRMS calcd for
C9H12N5O4Na2P: 332.0501, found: 332.0494. UV kmax = 261 nm (
8500), kmin = 228 nm (e 1400) (EtOH 95).
e
Acknowledgments
M.K. is grateful to the RTRS-Infectiopôle Sud for a doctoral
fellowship. The authors are indebted to the staff of Professor J.
Balzarini laboratory for the screening of antiviral activity.
736.2928. UV kmax = 276 nm (
e 21,100), kmin = 247 nm (e 8200)
(EtOH 95).
4.6. (R)-[N-6-Monomethoxytrityl-N-9-(3-hydroxy-4-dimethyl
phosphonobutyl)]adenine 10
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(8:2, v/v, 10 mL) was added LiOHꢁH2O (32.47 mg, 0.77 mmol), and
the resulting mixture was stirred at room temperature until com-
pletion of the reaction was indicated by TLC. Then, the solvents
were evaporated under reduced pressure and the crude was dis-
solved in water and extracted with ethyl acetate. The organic layer
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(R)-10 (165 mg, 74%). ½a D20
¼ þ28:3 (c 0.46, MeOH). The NMR
ꢂ
chemical shifts were identical to ( )-7. HRMS calcd for
C31H34N5O5P: 588.2376, found: 588.2380. UV kmax = 276 nm (
e
13. Zhang, Y.-M.; Ding, Y.; Tang, W.; Luo, W.; Gu, M.; Lu, W.; Tang, J.; Zuo, J.-P.;
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e
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4.7. (S)-[N-6-Monomethoxytrityl-N-9-(3-hydroxy-4-dimethyl
phosphonobutyl)]adenine 10
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The above procedure was applied to (S,S)-9 to afford (S)-10
(156 mg, 70%). ½a D20
¼ ꢀ30:4 (c 0.6, MeOH). The NMR chemical
ꢂ
shifts were identical to ( )-7. HRMS calcd for C31H34N5O5P:
588.2376, found: 588.2388. UV kmax = 276 nm
kmin = 246 nm ( 6500) (EtOH 95).
(e 17,000),
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e