Efficient synthesis of nucleoside aryloxy phosphoramidate prodrugs utilizing benzyloxycarbonyl protection

Article abstract of DOI:10.1016/j.tet.2011.05.046

An efficient method for the synthesis of nucleoside phosphoramidates prodrugs (6a-f) has been developed that employs a simple protection/deprotection sequence of the nucleoside with benzyloxycarbonyl (Cbz). The coupling reaction of Cbz-protected derivativ

Full text of DOI:10.1016/j.tet.2011.05.046

Tetrahedron 67 (2011) 5487e5493
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Efficient synthesis of nucleoside aryloxy phosphoramidate prodrugs utilizing
benzyloxycarbonyl protection
,
Jong Hyun Cho a, Franck Amblard a, Steven J. Coats b, Raymond F. Schinazi a
*
a Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta,
GA 30033, USA
b RFS Pharma, LLC, 1860 Montreal Road, Tucker, GA 30084, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient method for the synthesis of nucleoside phosphoramidates prodrugs (6aef) has been
developed that employs a simple protection/deprotection sequence of the nucleoside with benzylox-
ycarbonyl (Cbz). The coupling reaction of Cbz-protected derivatives (5aef) with phenyl-(ethoxy-L-ala-
ninyl)-phosphorochloridate (7), followed by Cbz group removal by hydrogenolysis provided the phenyl
phosphoramidate ProTides (6aef) in excellent overall yields.
Received 29 March 2011
Received in revised form 9 May 2011
Accepted 10 May 2011
Available online 17 May 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
ProTide
Nucleoside
Monophosphate prodrug
Benzyloxycarbonyl
Antiviral
1. Introduction
For instance, 20,30-didehydro-30-deoxythymidine mono-
phosphate prodrug (d4T-MP-PD, 1a) demonstrated 100-fold more
Over the past 40 years, nucleoside analogs have established
themselves as important agents for first-line antiviral therapy and
cancer therapy. In most cases, the observed therapeutic activity
does not result directly from the nucleoside itself, but from the
corresponding nucleoside 50-triphosphate (NuTP). However, this
three step intracellular conversion by kinases is often limited by an
inefficient initial phosphorylation step. Nucleotides cannot be
considered as therapeutic agents due to their polar nature, their
inability to cross the cell membrane and their dephosphorylation in
extracellular fluids. In order to overcome these problems, consid-
erable efforts have focused on monophosphate prodrugs1 that
mask the monophosphate moiety with various chemically or en-
zymatically cleaved groups once the compound is transported into
the cell. This strategy has the potential to reveal potent compounds
that were inactive in their nucleoside form because of a lack of
phosphorylation. Among all reported prodrugs approach, the class
of aryl phosphoramidate nucleoside prodrug (ProTide) developed
by McGuigan2 has been proven to enhance, in vitro, the activity of
parent nucleosides by increasing the formation rate of NuTP by
improved intracellular transport and/or bypassing the rate limiting
monophosphorylation step.
potency against HIV than d4T.3 The phosphoramidate (d4A-MP-PD,
1b) of 20,30-didehydro-20,30-dideoxyadenosine (d4A) has markedly
enhanced anti-HIV activity (ca. 2000-fold) compared to d4A.4 Re-
cently, application of the ProTide approach significantly improved
antiviral potency of the carbocyclic adenosine analog L-Cd4A-MP-
PD, 1c (9000-fold) against HIV.5 The antiviral potency of both
abacavir and carbovir ProTides (1f and 1g) was boosted by 10- and
20-fold, respectively, against hepatitis B virus (HBV).6 In the same
manner, ProTide derivative (1h) of BVDU (brivudin) showed en-
hanced potency compared to parent compound against colon
cancer.7 40-Azidoadenosine phosphoramidate derivative (1e) also
exhibited a more potent antiviral activity against HCV replication
than the parent compound.8 More interestingly, phosphoramidates
(1d),9 (1iek),10 and (1l)11 have been shown to exhibit significant
antiviral activity against hepatitis C virus (HCV) replication, while
their parent nucleoside was devoid of activity (Fig. 1).
In general, aryl phosphoramidate ProTides are synthesized by
direct coupling of an unprotected nucleoside with a phosphoro-
chloridate derivative using either N-methyl imidazole (NMI) or
t-butyl magnesium halide (Br or Cl) in tetrahydrofuran (THF).
However, the synthesis of most ProTides is plagued with low to
moderate yields due to poor chemoselectivity in the ProTide
forming reaction and/or poor solubility of nucleoside in THF.
Acid labile protective groups, such as isopropylidene12 or
* Corresponding author. E-mail address: rschina@emory.edu (R.F. Schinazi).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.05.046

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J.H. Cho et al. / Tetrahedron 67 (2011) 5487e5493
cyclopentylidene,8,13 have been used in the past to prepare phos-
phoramidate prodrugs of ribonucleosides. However, the phos-
phorochloridate/nucleoside coupling yields remain moderate and
the relative instability of the phosphoramidate function to acidic
conditions lead to low yields for the deprotection step. Now that
pharmaceutical companies, such as Inhibitex,12a Gilead,14 Phar-
masset,10 and Idenix15 are developing their own HCV nucleoside
ProTides, there is need for an efficient high yielding synthetic route
to prepare these compounds involving simple purifications steps.
Herein, we are reporting an efficient method to prepare phenyl
phosphoramidate ProTides by protection of polar functional groups
(OH and NH2) with benzyloxycarbonyl chloride (CbzCl) followed by
coupling with phosphoramidate 7 and final deprotection under
neutral conditions. The benzyloxycarbonyl (Cbz) group was a par-
ticularly attractive protective group for its facile introduction on the
sugar moiety (and the base moiety in case of a cytidine analog) and
also for its clean removal by simple hydrogenation under mild and
neutral conditions without affecting the phosphoramidate func-
tional group.
2. Results and discussions
Cytidine (2a) was used as a model system and we synthesized
the Cbz-protected cytidine analog (3a) following previously
reported synthetic methods.16 The selective silylation of the
50-hydroxyl group of 2a was conducted with TBSCl and imidazole in
pyridine. Additionally, protection of the two remaining hydroxyl
groups along with the amino group was preformed with CbzCl to
provide a 93% yield after purification of the fully protected cytidine
derivative (3a) (Scheme 1). The silyl group of 3a was removed with
Et3Ne3HF to afford Cbz-derivative 4a in quantitative yield. With
the liphophilic cytidine derivative (4a) in hand, we investigated its
Fig. 1. Antiviral phosphoramidate ProTides (1ael).
to the direct coupling of cytidine 2a with phosphorochloridate (7)
(10% yield).
coupling with phenyl-(ethoxy-
which was synthesized by the reaction of phenyl-
phosphodichloridate with
-alanine ethyl ester hydrochloric salt.7
The reaction of Cbz-cytidine 4a with phosphorochloridate (7), us-
ing the previously reported procedure,7 provided Cbz-cytidine
phosphoramidate 5a in 97% yield. Finally, the hydrogenolysis of
Cbz-protected cytidine ProTide (5a) in the presence of Pd/C in EtOH
provided the cytidine ProTide (6a) in 98% yield. With an overall
yield of 86% from cytidine (2a), it is evident that our new CBz
protection/deprotection approach represents a valuable alternative
L
-alaninyl)-phosphorochloridate (7),
In order to generalize our strategy, this efficient synthetic route
was applied to other natural nucleosides including adenosine (A),
guanosine (G), and uridine (U) as summarized in Table 1. The re-
action of A and U with TBSCl in pyridine gave 50-O-silylated A and
U in high yield. Due to its low solubility in pyridine, the silylation of
G was conducted in DMSO. The reaction of silylated nucleosides
(U, A, G) with CbzCl in the presence of DMAP in CH2Cl2 afforded
their corresponding Cbz-protected compounds in good yields. In
L
NH
NHCbz
N
NHCbz
N
2
N
i.TBSCl,imidazole,
pyridine,rt,24h
HO
TBSO
Et N-3HF
3
THF,rt,12h
HO
N
O
N
O
N
O
O
O
O
ii.CbzCl,DMAP
97 %
HO
OH
CH Cl ,rt,48h
Cbz O
OCbz
CbzO
OCbz
2
2
2a
3a
4a
93%
NHCbz
NH
2
O
P
HN
Cl
N
N
O
P
O
P
OPh
H
(1.0atm)
P d/C
2
HN
O
7
N
O
HN
O
CO E t
N
O
2
O
OP h
O
O Ph
CO Et
CO Et
2
NM M, THF
EtOH,rt,12h
98%
2
o
CbzO
OCbz
HO
OH
-78 C, 2 h,
rt,12h
97%
5a
6a
Scheme 1. Synthesis of the cytidine ProTide 6a.

J.H. Cho et al. / Tetrahedron 67 (2011) 5487e5493
5489
Table 1
Synthesis of nucleoside aryloxy phosphoramidate prodrugs 6ae6f
Entry Yields
Protection (three steps)
Coupling reaction
NHCbz
Hydrogenolysis (Method A/B)a
NH
Overall yield (%)
NHCbz
N
2
N
N
O
P
O
P
HN
O
HO
N
O
HN
O
N
N
O
O
1
86(10)b
O
O
O
OPh
OPh
92%
96%
98%
92%
90%
96%
93%
97% (A)
98% (B)
94% (A)
CO Et
2
CO Et
2
CbzO
OCbz
O
OH OH
Cb z O
OCbz
O
5a
6a
4a
O
NH
NH
NH
O
P
O
P
HN
HN
O
HO
N
O
N
O
O
N
O
O
2
3
4
5
92
O
O
OPh
CO Et
2
OPh
CO Et
96%
2
OH OH
6b
CbzO
OCbz
CbzO
OCbz
5b
4b
NH2
N
NH2
NH
2
N
N
N
N
N
N
N
O
P
O
P
HO
HN
O
HN
O
N
O
N
N
N
O
79
O
OPh
OPh
CO Et
90%
CO2Et
2
CbzO
OCbz
OH OH
CbzO
OCbz
4c
6c
5c
O
N
O
N
O
N
N
N
N
NH
NH
NH
NH
NH
O
O
HO
HN P O
HN
P O
OPh
CO Et
2
N
N
N
NH
2
2
2
73
O
O
O
OPh
85%
95% (A)
96% (B)
95% (A)
CO Et
2
OH OH
6d
CbzO
OCbz
CbzO
OCbz
5d
4d
O
N
O
N
O
NH
O
NH
NH
O
P
O
P
HN
O
O
N
HO
O
HN
O
O
87(19.6)c
O
OPh
O
OPh
96%
CO2Et
CO Et
Me
Me
2
Me
F
OH
6e
CbzO
F
CbzO
F
4e
5e
NHCbz
N
NH2
NHCbz
N
N
N
O
P
O
P
HO
HN
O
N
O
N
O
HN
O
OPh
O
O
O
O
OPh
CO2Et
95%
6
86(17)b
Me
CO2Et
M e
Me
CbzO
F
CbzO
F
OH
6f
F
4f
5f
a
Method A: Pd/C, H2 (1 atm), EtOH; Method B: Pd/C, 1,4-cyclohexadiene, EtOH.
Yield for the coupling of unprotected nucleoside with 7.
Yield for the coupling of unprotected nucleoside with 7, see Ref. 18.
b
c
,
accordance with previously reported results,16a 16b formation of N6-
Cbz-protected A and O6-/N2-Cbz-protected G derivatives was never
observed, even though an excess of CbzCl was used. The treatment of
50-O-silylated Cbz-nucleosides (A, G, and U) with Et3Ne3HF pro-
vided Cbz-protected nucleosides (4bed) in excellent yields (Table
1). Finally, the coupling reaction of 4bed with phenoxy
phosphorochloridate (7), followed by hydrogenolysis using either
Pd/C, H2 (A and G) or catalytic hydrogen transfer (U),16a afforded
their corresponding phosphoramidate ProTides (6bed) in excellent
yield.
agents17 previously prepared by direct coupling of the nucleosides
with phosphorochloridate 7 (17% and 20% yields, respectively).18
The key Cbz protected intermediates (4eef), prepared from their
parent nucleosides using the procedure described above (Scheme
1), were reacted with the phenyl-(ethoxy-L-alaninyl)-phosphoro-
chloridate (7) to give Cbz-protected ProTides (5eef) in excellent
yield. Subsequently, they were converted to their corresponding
ProTides (6eef) by hydrogenolysis in quantitative yield (Table 1).
3. Conclusion
The Cbz approach was also applied to the preparation of both
20-deoxy-20-
oro-20-
a
-fluoro-20-
b
-C-methyl uridine and 20-deoxy-20-
a
-flu-
In summary, an efficient synthetic procedure for the preparation
of aryl phosphoramidate ProTides was successfully developed by
b
-C-methyl-cytidine ProTide 6e and 6f; two anti HCV-

5490
J.H. Cho et al. / Tetrahedron 67 (2011) 5487e5493
utilizing a Cbz protection/deprotection sequence. This method
appears to be general and can be applied to ribo or 20-deoxyribo
nucleosides bearing various bases (A, G, C, U). The coupling reaction
of a variety of Cbz-protected nucleosides (4aef) with phenyl-
0 ꢀC under N2 atmosphere. The solution was stirred for 12 h at rt
and all volatiles were removed using a rotary evaporator. The res-
idue was dissolved in EtOAc (100 mL) and washed with cold satu-
rated NaHCO3 solution (30 mLꢂ2), and brine (30 mL). The resulting
solution was dried over Na2SO4 for 3 h and filtered. The filtrate was
adsorbed on silica gel and purified by silica gel column chroma-
tography (CH2Cl2/MeOH¼40:1 to 20:1 v/v) to give 4a (3.71 g,
(ethoxy- -alaninyl)-phosphorochloridate (7) afforded the Cbz-
L
protected ProTides (5aef) in excellent yields (w90%). The
removal of the Cbz group from 5aef was successfully conducted
under hydrogenolysis with Pd/C, hydrogen (1 atm) or catalytic
hydrogen transfer to provide the aryl phosphoramidate ProTides
(6aef) in near quantative overall yields. To our knowledge, this
approach is one of the most efficient and potentially most general
sequences to prepare phosphoramidate nucleoside prodrugs. Its
development could significantly impact the multikilo production of
clinically relevant compounds, such as 6e and 6f.
5.74 mmol) in 97% yield. 1H NMR (400 MHz, CDCl3)
d 7.87 (d,
J¼7.6 Hz, 1H), 7.85 (br, 1H), 7.40e7.30 (m, 15H), 7.27 (d, J¼7.6 Hz,
1H), 5.80e5.75 (m, 2H), 5.51 (t, J¼4.8 Hz,1H), 5.21 (s, 2H), 5.14e5.07
(m, 4H), 4.33 (m, 1H), 3.99 (d, J¼12.0 Hz, 1H), 3.83e3.79 (m, 1H),
3.60 (br, 1H); 13C NMR (100 MHz, CDCl3)
d 174.3, 163.1, 155.2, 154.3,
153.9, 152.3, 146.8, 135.0, 134.8, 128.9, 128.9, 128.8, 128.8, 128.7,
128.6, 95.9, 92.3, 83.8, 75.9, 73.9, 70.6, 70.6, 68.3, 61.5; MS-ESIþ m/z
646 (MþHþ).
4. Experimental section
4.1. General
4.1.3. 50-O-[Phenyl-(ethoxy-
benzyloxycarbonylcytidine (5a). To
L
-alaninyl)]phosphoryl-N4-20,30-O- tris-
solution of 4a (0.50 g,
a
0.78 mmol) and 7 (0.46 g, 1.56 mmol) in 10 mL of anhydrous THF
was added 1-methylimidazole (0.15 mL, 2.0 mmol) over 10 min at
ꢁ78 ꢀC under argon atmosphere. After stirring for 2 h at ꢁ78 ꢀC, the
reaction was maintained for 12 h at rt. The solvent was removed
under reduced pressure and the residue was dissolved in CH2Cl2
(20 mL), washed with cold 0.5 M HCl solution (5 mLꢂ2), cold water
(10 mL), and brine (5 mL). The organic layer was dried over MgSO4,
filtered, and concentrated under reduced pressure. The residue was
purified by silica gel chromatography (CH2Cl2/MeOH¼20:1 to
10:1 v/v) to give 5a (0.68 g, 0.76 mmol), as 1:1 diastereomeric
Nuclear magnetic resonance (NMR) spectra (1H, 13C, 19F, and 31P)
were recorded on a Varian Unity Plus 400 MHz fourier transform
spectrometer at rt, with tetramethylsilane (TMS) as an internal
standard. Chemical shifts (d) are reported in parts per million
(ppm), and signals are quoted as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), br (broad), dd (doublet of doublets), or ddd
(doublet of doublets of doublets). The phosphoramidates are an
approximate 50:50 mixture of diastereomers and the 13C NMR data
is reported as observed, that is, some carbon signals overlap. High-
resolution mass spectra (HRMS) were recorded on a Micromass
Autospec high-resolution mass spectrometer with electrospray
ionization (ESI). Thin-layer chromatography (TLC) was performed
(RP/SP) mixture, in 97% yield. 1H NMR (400 MHz, CDCl3)
d 8.05 (br,
1H), 7.92e7.34 (m, 1H), 7.38e7.09 (m, 21H), 6.12e6.08 (m, 1H),
5.38e5.30 (m, 1H), 5.21 (br, 2H), 5.15e5.06 (m, 4H), 5.06e5.01 (m,
1H), 4.49e4.30 (m, 1H), 4.20e4.08 (m, 3H), 4.07e3.96 (m, 2H),
3.66e3.51 (m, 1H), 1.43e1.33 (m, 3H), 1.28 (m, 3H); 13C NMR
on 0.25 mm silica gel. Purifications were carried out on silica gel
ꢀ
column chromatography (60 A, 63e200
mm, or 40e75
mm).
(100 MHz, CDCl3)
d 173.4, 173.5, 162.9, 154.8, 154.0, 153.9, 152.3,
4.1.1. 50-O-tert-Butyldimethylsilyl-N4-20,30-O-tris-benzylox-
ycarbonylcytidine (3a). To a solution of cytidine (3.60 g, 14.3 mmol)
in 30 mL of anhydrous pyridine was added imidazole (1.25 g,
18.40 mmol) and tert-butyldimethylsilyl chloride (TBDMSCl, 2.40 g,
15.7 mmol) at 0 ꢀC under a N2 atmosphere. The reaction mixture
was stirred at rt for 12 h and then treated with methanol (8.0 mL).
After stirring for 60 min, the solvent was removed under reduced
pressure. The residue was purified by silica gel column chroma-
tography (hexane/EtOAc¼10:1 to 1:4 v/v) to give (5.12 g,
12.5 mmol) in 97% yield. Subsequently, to a solution of protected
cytidine (2.68 g, 7.50 mmol) and DMAP (5.50 g, 45.0 mmol) in
50 mL of anhydrous CH2Cl2 was added benzyl chloroformate
(CbzCl, 4.76 mL, 33.74 mmol) at 0 ꢀC under N2 atmosphere. After
stirring for 72 h at rt, the reaction mixture was diluted with CH2Cl2
(200 mL) and then washed with cold 1.0 M HCl aqueous solution
(50 mL) then water (100 mL). The solution was dried over anhy-
drous MgSO4, filtered, and concentrated under reduced pressure.
The residue was purified on silica gel column chromatography
(hexane/EtOAc¼10:1 to 1:1 v/v) to give 3a (5.47 g, 7.20 mmol) in
150.6, 144.4, 135.2, 134.7, 130.1, 130.0, 129.8, 128.8, 128.8, 128.7,
128.6, 128.5, 128.5, 125.3, 125.0, 120.3, 120.2, 95.7 89.1, 80.1, 76.7,
72.6, 70.9, 68.1, 64.9, 61.8, 50.5, 21.1,14.2; 31P NMR (162 MHz, CDCl3)
d
3.14, 3.00; MS-ESIþ m/z 901 (MþHþ); HRMS-ESIþ: m/z calcd for
C44H45N4O15NaP (MþNaþ) 923.2530, found 923.2525.
4.1.4. 50-O-[Phenyl-(ethoxy-
-alaninyl)]phosphorylcytidine (6a). To
L
a solution of 5a (0.37 g, 0.41 mmol) in 10 mL of EtOH was added
Pd/C (0.02 g, 10% Pd on activated carbon) at rt. The mixture was
stirred for 2 h under an atmosphere of H2 (1 atm) and then treated
with Celite (1.0 g) and stirred for 30 min. The suspension was fil-
tered and washed with MeOH (10 mLꢂ3). The collected solution
was concentrated under reduced pressure and the residue was
purified on silica gel column chromatography (MeOH/
EtOAc¼1:10 v/v) to give 6a (0.20 g, 0.40 mmol), as 1:1. di-
astereomeric (RP/SP) mixture, in 98% yield. 1H NMR (400 MHz,
CD3OD)
d 7.75e7.67 (m, 1H), 7.36e7.31 (m, 2H), 7.24e7.13 (m, 3H),
5.88e5.79 (m, 3H), 4.46e4.26 (m, 2H), 4.18e4.06 (m, 3H),
4.06e4.02 (m, 1H), 3.94e3.85 (m, 1H), 1.33e1.28 (m, 3H), 1.24e1.14
93% yield. 1H NMR (400 MHz, CDCl3)
d
8.32 (d, J¼8.0 Hz, 1H), 7.58
(m, 3H); 13C NMR (100 MHz, CD3OD)
d
175.1, 167.7, 158.6, 142.5,
(br, 1H), 7.42e7.30 (m, 15H), 7.21 (d, J¼8.0 Hz, 1H), 6.27 (d, J¼3.2 Hz,
1H), 5.35 (t, J¼4.0 Hz, 1H), 5.28 (t, J¼5.6 Hz, 1H), 5.23 (s, 2H), 5.13 (d,
J¼2.4 Hz, 2H), 5.10 (d, J¼7.6 Hz, 2H), 4.33 (d, J¼5.6 Hz, 1H), 4.06 (d,
J¼10.8 Hz, 1H), 3.79 (d, J¼10.8 Hz, 1H), 0.93 (s, 9H), 0.12 (s, 3H), 0.11
131.0,130.2,126.4,124.0,121.6,121.5, 96.5, 92.1, 83.6, 76.1, 70.8, 67.2,
62.5, 51.8, 20.6, 14.6; 31P (162 MHz, CD3OD) 4.89, 4.74; MS-ESIþ m/
d
z 499 (MþHþ); HRMS-ESIþ: m/z calcd for C20H27N4O9NaP (MþNaþ)
521.1403, found 521.1408.
(s, 3H); 13C NMR (100 MHz, CDCl3)
d 162.8, 154.7, 153.9, 153.8, 152.5,
144.0,137.2,135.1,134.7, 134.7,130.7,129.2,128.9,128.8,128.7,128.7,
128.6, 128.5, 128.4, 128.3, 117.2, 95.2, 87.8, 81.9, 77.5, 72.7, 70.4, 70.4,
67.9, 61.5, 25.9, 18.4, ꢁ5.5, ꢁ5.6; MS-ESIþ m/z 760 (MþHþ).
4.1.5. 50-O-tert-Butyldimethylsilyl-20,30-O-bis-benzyloxycarbonylur-
idine (3b). Compound 3b was prepared using the same procedure
as 3a: yield 96%; 1H NMR (400 MHz, CDCl3)
d 9.25 (s, 1H), 7.83 (d,
J¼8.0 Hz, 1H), 7.34e7.31 (m, 10H), 6.28 (d, J¼4.8 Hz, 1H), 5.72 (d,
J¼8.0 Hz, 1H), 5.30e5.26 (m, 3H), 5.16e5.09 (m, 4H), 4.29 (q,
J¼1.6 Hz, 1H), 3.95 (dd, J¼2.0, 11.6 Hz, 1H), 3.82 (dd, J¼2.0, 11.6 Hz,
1H), 0.94 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H); 13C NMR (100 MHz,
4.1.2. N4-20,30-O-Tris-benzyloxycarbonylcytidine (4a). To a solution
of 3a (4.50 g, 5.92 mmol) in 20 mL of anhydrous THF was added
triethylamine trihydrofluoride (Et3Ne3HF, 6.0 mL, 36.8 mmol) at

J.H. Cho et al. / Tetrahedron 67 (2011) 5487e5493
5491
CDCl3)
d
163.2, 154.2, 154.0, 150.4, 139.6, 134.7, 134.7, 128.9, 128.8,
(d, J¼10.8 Hz, 2H), 6.85 (br, 2H), 6.10 (dd, J¼4.8, 7.6 Hz, 1H), 6.03 (d,
J¼7.6 Hz, 1H), 5.70 (d, J¼4.8 Hz, 1H), 5.16e5.01 (m, 4H), 4.43 (s, 1H),
3.97 (d, J¼12.4 Hz, 1H), 3.83 (t, J¼12.4 Hz, 1H); 13C NMR (100 MHz,
128.7, 128.5, 103.2, 85.7, 83.0, 74.3, 70.6, 70.5, 62.8, 60.6, 26.0, 18.5,
14.35, ꢁ5.4; MS-ESIþ m/z 627 (MþHþ).
CDCl3)
d 156.4, 154.2, 153.6, 152.7, 148.3, 140.2, 134.7, 134.5, 128.8,
4.1.6. 20,30-O-Bis-benzyloxycarbonyluridine (4b). Compound 4b was
prepared using the same procedure as 4a: yield 99%; 1H NMR
(400 MHz, CDCl3)
128.7, 128.7, 128.6, 128.5, 87.7, 85.8, 76.1, 75.6, 70.6, 70.3, 62.6, 60.4,
21.1, 14.2; MS-ESIþ m/z 536 (MþHþ).
d
9.69 (s, 1H), 7.64 (d, J¼8.0 Hz,1H), 7.35e7.30 (m,
10H), 5.96 (d, J¼6.0 Hz, 1H), 5.74 (d, J¼8.0 Hz, 1H), 5.54 (dd, J¼5.2,
6.0 Hz, 1H), 5.45 (dd, J¼3.6, 5.2 Hz, 1H), 5.12e5.05 (m, 4H), 4.25 (d,
J¼2.4 Hz, 1H), 3.89 (d, J¼12.0 Hz, 1H), 3.80 (m, 1H), 3.69 (s, 1H); 13C
4.1.11. 50-O-[Phenyl-(ethoxy- -alaninyl)]phosphoryl-20,30-O-bis-benzyl-
L
oxycarbonyladenosine (5c). Compound 5c was prepared using the
same procedure as 5a: yield 92% (1:1 diastereomeric (RP/SP) mixture);
NMR (100 MHz, CDCl3)
d
163.8, 154.3, 154.0, 150.6, 141.7, 134.7,
1H NMR (400 MHz, CDCl3)
d 8.29 (s, 0.5H), 8.26 (s, 0.5H), 7.99 (s, 0.5H),
134.6, 128.8, 128.8, 128.7, 128.5, 103.3, 88.6, 83.3, 75.8, 74.4, 70.7,
7.93 (s, 0.5H), 7.34e7.19 (m, 15H), 6.25 (br, 2H), 6.19e6.17 (m, 1H),
6.02e5.93 (m, 1H), 5.72 (m, 1H), 5.13e5.03 (m, 7H), 4.54e4.37 (m,
70.5, 61.7, 39.2; MS-ESIþ m/z 513 (MþHþ).
2H), 4.14e3.96 (m, 2H),1.35e1.25 (m, 3H), 1.18 (m, 3H);
d
ꢁ158.03; 13
C
4.1.7. 50-O-[Phenyl-(ethoxy-
L
-alaninyl)]phosphoryl-20,30-O-bis-benzyl-
NMR (100 MHz, CDCl3) d 173.7, 173.6, 155.8, 154.1, 153.8, 153.3, 150.7,
oxycarbonyluridine (5b). Compound 5b was prepared using the same
149.8, 139.6, 134.6, 129.9, 128.9, 128.8, 128.7, 128.6, 125.1, 120.4, 120.3,
120.2, 85.9, 80.7, 75.8, 73.6, 70.6, 65.3, 50.4, 47.9, 21.0, 14.2; 31P
procedure as 5a: yield 98% (1:1 diastereomeric (RP/SP) mixture). 1H
NMR (400 MHz, CDCl3)
d
10.11e10.00 (m, 1H), 7.89 (d, J¼8.0 Hz,
(162 MHz, CDCl3)
d
3.23, 3.16; MS-ESIþ m/z 791 (MþHþ); HRMS-ESIþ:
0.5H), 7.46 (d, J¼8.8 Hz, 0.5H), 7.31e7.22 (m, 15H), 6.13e6.10 (m, 1H),
5.75e5.69 (m, 0.5H), 5.56e5.48 (m, 0.5H), 5.46e5.41 (m, 1H), 5.30 (t,
J¼5.6 Hz, 0.5H), 5.20 (t, J¼5.6 Hz, 0.5H), 5.14e5.04 (m, 4H), 4.48e4.32
(m, 2H), 4.23e4.08 (m, 3H), 4.08e3.96 (m, 1H), 3.85e3.76 (m, 1H),
1.34e1.33 (m, 3H), 1.26e1.18 (m, 3H); 13C NMR (100 MHz, CDCl3)
m/z calcd for C37H40N6O12P (MþHþ) 791.2435, found 791.2436.
4.1.12. 50-O-[Phenyl-(ethoxy-
L-alaninyl)]phosphoryladenosine
(6c). Compound 6c was prepared using the same procedure as 6a:
yield 94% (1:1 diastereomeric (RP/SP) mixture); 1H NMR
d
173.6, 173.4, 163.4, 153.9, 150.4, 139.8, 134.5, 129.9, 129.8, 129.6,
(400 MHz, CD3OD) d 8.25e8.16 (m, 2H), 7.31e7.28 (m, 2H),
129.6, 128.7, 128.6, 128.6, 128.4, 128.4, 128.3, 103.4, 86.9, 86.3, 83.3,
80.2, 76.0, 75.5, 74.6, 73.1, 70.4, 70.2, 65.3, 61.6, 61.4, 60.4, 53.6, 50.2,
7.20e7.11 (m, 3H), 6.02 (t, J¼4.8 Hz, 1H), 4.66e4.61 (m, 1H),
4.45e4.30 (m, 3H), 4.29e4.23 (m, 1H), 4.09e4.01 (m, 2H),
3.91e3.08 (m, 1H), 1.27e1.20 (m, 3H), 1.18e1.13 (m, 3H); 13C NMR
20.8, 14.0; 31P (162 MHz, CDCl3)
d
4.13, 4.07; MS-ESIþ m/z 768
(MþHþ); HRMS-ESIþ: m/z calcd for C36H38N3O14NaP (MþNaþ)
(100 MHz, CD3OD)
d 175.1, 157.4, 154.1, 152.2, 150.8, 141.1, 130.9,
790.2001, found 790.1997.
130.1, 126.3, 121.5, 121.5, 120.6, 90.0, 84.5, 75.5, 71.7, 67.4, 62.5,
51.7, 20.5, 14.6; 31P (162 MHz, CD3OD)
d
4.93, 4.78; MS-ESIþ m/z
4.1.8. 50-O-[Phenyl-(ethoxy-
L
-alaninyl)]phosphoryluridine (6b). To
523 (MþHþ); HRMS-ESIþ: m/z calcd for C21H27N6O8NaP (MþNaþ)
a solution of 5b (0.50 g, 0.65 mmol) in 10 mL of EtOH was added
1,4-cyclohexadiene (1.30 mL) and Pd/C (0.03 g, 10% Pd on activated
carbon) at rt. After stirring for 3 h at rt, the reaction mixture was
treated with Celite (2.0 g) and stirred for 30 min. The suspension
was filtered and washed with MeOH (15 mLꢂ3). The collected so-
lution was concentrated under reduced pressure and the residue
was purified by silica gel column chromatography (MeOH/
EtOAc¼1:10 v/v) to give 6b (0.32 g, 0.64 mmol), as a 1:1 di-
astereomeric (RP/SP) mixture, in 98% yield. 1H NMR (400 MHz,
545.1517, found 545.1515.
4.1.13. 50-O-tert-Butyldimethylsilyl-20,30-O-bis-benzyloxycarbonyl-
guanosine (3d). To a solution of guanosine (2.0 g, 7.06 mmol) in
20 mL of anhydrous DMSO was added DMAP (0.09 g, 0.71 mmol),
Et3N (1.07 g, 10.6 mmol), and TBSCl (1.17 g, 7.76 mmol) at 0 ꢀC
under N2 atmosphere. After stirring for 24 h at rt, 20 mL of an-
hydrous CH2Cl2, CbzCl (4.82 g, 28.24 mmol), and DMAP (4.32 g,
35.30 mmol) was added to the solution at 0 ꢀC under N2 atmo-
sphere. After stirring for 48 h at rt, the reaction mixture was
concentrated under reduced pressure and the residue was dis-
solved in CH2Cl2 (100 mL) and washed with cold 0.5 M HCl solu-
tion (30 mLꢂ2), cold water (30 mL), and brine (30 mL). The
organic layer was dried over Na2SO4, and filtered. The filtrate was
concentrated and purified by silica gel column chromatography
(CH2Cl2/MeOH¼30:1 to 10:1 v/v) to give 3d (4.32 g, 6.49 mmol) in
CD3OD)
d
7.70 (d, J¼8.0 Hz, 0.5H), 7.63 (d, J¼8.0 Hz, 0.5H), 7.39e7.33
(m, 2H), 7.27e7.18 (m, 3H), 5.91 (d, J¼8.8 Hz, 0.5H), 5.89 (d,
J¼8.8 Hz, 0.5H), 5.69 (d, J¼8.0 Hz, 0.5H), 5.61 (d, J¼8.0 Hz, 0.5H),
4.44e4.29 (m, 2H), 4.19e4.09 (m, 5H), 3.98e3.91 (m, 1H),
3.87e3.76 (m, 1H), 1.36e1.30 (m, 3H), 1.25e1.21 (m, 3H); 13C NMR
(100 MHz, CD3OD)
d 175.2, 166.2, 152.2, 142.4, 131.0, 130.8, 126.4,
126.2, 121.5, 103.2, 90.8, 84.0, 75.3, 71.1, 67.4, 62.5, 62.4, 51.7, 20.6,
14.6; 1P (162 MHz, CD3OD)
d
4.94, 4.76; MS-ESIþ m/z 500 (MþHþ);
92% yield. 1H NMR (400 MHz, DMSO-d6)
d 10.74 (br, 1H), 7.85 (s,
HRMS-ESIþ: m/z calcd for C20H26N3O10NaP (MþNaþ) 522.1253,
1H), 7.40e7.29 (m, 10H), 6.57 (br, 2H), 5.99 (d, J¼7.2 Hz, 1H), 5.67
(dd, J¼5.2, 7.2 Hz, 1H), 5.40 (dd, J¼2.8, 5.2 Hz, 1H), 5.18e5.10 (m,
4H), 4.30 (q, J¼3.6 Hz, 1H), 3.87 (d, J¼3.6 Hz, 2H), 0.89 (s, 9H), 0.09
found 522.1256.
4.1.9. 50-O-tert-Butyldimethylsilyl-20,30-O-bis-benzylox-
ycarbonyladenosine (3c). Compound 3c was prepared using the
same procedure as 3a: yield 92% (two steps); 1H NMR (400 MHz,
(3H), 0.08 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
d 173.7, 156.6,
154.1, 153.5, 153.1, 151.3, 135.0, 134.8, 134.3, 128.7, 128.6, 128.5,
128.4, 128.2, 116.4, 82.9, 82.4, 76.1, 74.6, 69.9, 69.7, 62.8, 47.3, 33.9,
25.8, 25.5, 18.0, ꢁ5.5, ꢁ5.6; MS-ESIþ m/z 666 (MþHþ); HRMS-
ESIþ: m/z calcd for C32H40N5O9Si (MþHþ) 666.2601, found
666.2603.
CDCl3)
d 8.33 (s, 1H), 8.15 (s, 1H), 7.37e7.29 (m, 10H), 6.33 (d,
J¼6.4 Hz, 1H), 5.81 (dd, J¼4.8, 6.0 Hz, 1H), 5.66 (br, 2H), 5.52 (dd,
J¼4.8, 6.0 Hz, 1H), 5.17e5.05 (m, 4H), 4.37 (q, J¼2.8 Hz, 1H), 3.95
(dd, J¼2.8, 11.6 Hz, 1H), 3.80 (dd, J¼2.8, 11.6 Hz, 1H), 0.94 (s, 9H),
0.13 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
155.6, 154.4, 153.9, 153.6,
4.1.14. 20,30-O-Bis-benzyloxycarbonylguanosine (4d). Compound 4d
150.3, 142.8, 138.9, 134.8, 134.7, 128.9, 128.9, 128.9, 128.8, 128.6,
120.0, 85.0, 83.3, 77.0, 74.7, 70.6, 70.5, 62.9, 26.1, ꢁ5.2, ꢁ5.3; MS-
ESIþ m/z 650 (MþHþ).
was prepared using the same procedure as 4a: yield 92%; 1H NMR
(400 MHz, DMSO-d6)
d 10.72 (br, 1H), 7.99 (s, 1H), 7.38e7.29 (m,
10H), 6.55 (br, 2H), 5.98 (d, J¼7.6 Hz, 1H), 5.72 (dd, J¼5.2, 7.6 Hz,
1H), 5.47 (t, J¼5.2 Hz,1H), 5.42 (dd, J¼2.0, 5.2 Hz,1H), 5.17e5.06 (m,
5H), 4.26 (q, J¼2.0 Hz,1H), 3.68 (m, 2H); 13C NMR (100 MHz, DMSO-
4.1.10. 20,30-Bis-benzyloxycarbonyladenosine (4c). Compound 4c
was prepared using the same procedure as 4a: yield 98%; 1H NMR
d6) d 186.5, 175.4, 173.7, 156.6, 154.0, 153.6, 153.1, 151.2, 135.2, 135.0,
(400 MHz, CDCl3)
d
8.25 (s,1H), 7.71 (s,1H), 7.36e7.27 (m,10H), 7.00
134.9, 128.6, 128.6, 128. 5, 128.5, 128.4, 128.2, 116.5, 83.1, 83.1, 76.1,

5492
J.H. Cho et al. / Tetrahedron 67 (2011) 5487e5493
75.1, 69.8, 69.6, 60.9, 47.3; MS-ESIþ m/z 552 (MþHþ); HRMS-ESIþ:
m/z calcd for C26H26N5O9 (MþHþ) 552.2601, found 552.2603.
(m, 6H), 1.28e1.20 (m, 3H); 13C NMR (100 MHz, CDCl3)
d 174.2,
162.9, 154.4, 150.3, 139.3, 134.7, 130.1, 129.8, 129.1, 129.1, 128.9,
128.9, 128.7, 128.6, 125.4, 125.0, 120.3, 120.2, 103.4, 100.1, 98.3, 77.7,
4.1.15. 50-O-[Phenyl-(ethoxy-
L
-alaninyl)]phosphoryl-20,30-O-bis-benzyl-
74.7, 71.1, 61.9, 50.5, 21.2, 17.6, 14.3; 19F (376 MHz, CDCl3)
d
ꢁ159.56;
oxycarbonylguanosine (5d). Compound 5d was prepared using the
31P (162 MHz, CDCl3)
d
5.87, 5.90; MS-ESIþ m/z 650 (MþHþ);
same procedure as 5a: yield 90% (1:1 diastereomeric (RP/SP) mixture);
HRMS-ESIþ: m/z calcd for C29H34FN3O11P (MþHþ) 650.1918, found
1H NMR (400 MHz, CD3OD)
d
7.73 (s, 1H), 7.31e7.13 (m, 15H), 7.10 (s,
650.1923.
1H), 7.00 (s, 1H), 6.07 (t, J¼6.0 Hz, 1H), 5.89 (t, J¼6.0 Hz, 0.5H), 5.81 (t,
J¼6.0 Hz, 0.5H), 5.69e5.64 (m, 1H), 5.16e5.01 (m, 5H), 4.45e4.32 (m,
3H), 4.09e4.04 (m, 2H), 3.94e3.85 (m, 1H), 1.29e1.23 (m, 3H), 1.16 (t,
4.1.20. 50-O-[Phenyl-(ethoxy- -alaninyl)]phosphoryl-20-fluoro-20-
L
methyluridine (6e). Compound 6e was prepared using the same
J¼5.8 Hz, 3H); 13C NMR (100 MHz, CD3OD)
d 175.2, 175.0, 159.4, 155.6,
procedure as 6b: yield 96% (1:1 diastereomeric (RP/SP) mixture); 1H
155.3, 153.0, 152.2, 138.9, 138.4, 136.6, 131.0, 130.9, 130.0, 129.8, 129.8,
129.6, 129.6, 129.5, 128.1, 126.4, 122.3, 121.6, 87.2, 82.0, 77.2, 75.2, 71.6,
NMR (400 MHz, CDCl3) d 10.05 (br, 1H), 7.50e7.14 (m, 6H), 6.17 (d,
J¼18.4 Hz, 1H), 5.70e5.54 (m, 1H), 4.59e4.31 (m, 3H), 4.30e4.22
(m, 1H), 4.20e4.11 (m, 2H), 4.07e3.96 (m, 1H), 3.94e3.78 (m, 1H),
3.46 (m, 1H), 1.41e1.28 (m, 6H), 1.27e1.22 (m, 3H); 19F (376 MHz,
66.8, 62.5, 51.7, 34.1, 20.5,14.6; 31P (162 MHz, CD3OD)
d 4.98, 4.84; MS-
ESIþ m/z 807 (MþHþ); HRMS-ESIþ: m/z calcd for C37H40N6O13
P
(MþHþ) 807.2399, found 807.2399.
CDCl3)
d
ꢁ162.41; 31P (162 MHz, CDCl3)
d
4.01, 3.58; MS-ESIþ m/z
516 (MþHþ).
4.1.16. 50-O-[Phenyl-(ethoxy-
L-alaninyl)]phosphorylguanosine
(6d). Compound 6d was prepared using the same procedure as 6a:
4.1.21. 50-O-tert-Butyldimethylsilyl-N4-30-O-bis-benzyloxycarbonyl-
20-fluoro-20-methylcytidine (3f). Compound 3f was prepared using
the same procedure as 3a: yield 93% (two steps); 1H NMR
yield 95% (1:1 diastereomeric (RP/SP) mixture); 1H NMR (400 MHz,
DMSO-d6)
d 10.73 (br, 1H), 7.84 (s, 1H), 7.38e7.14 (m, 5H), 6.57 (br,
2H), 6.11e6.02 (m, 1H), 5.76e5.71 (m, 1H), 5.56 (m, 1H), 5.37e5.33
(m, 1H), 4.43e4.39 (m, 1H), 4.24e3.98 (m, 6H), 3.80 (m, 1H),
1.23e1.18 (m, 3H), 1.15e1.10 (m, 3H); 13C NMR (100 MHz, DMSO-d6)
(400 MHz, CDCl3)
d
8.44 (d, J¼7.2 Hz, 1H), 7.40e7.26(m, 11H) 7.20
(br, 1H), 6.39 (d, J¼17.2 Hz, 1H), 5.24e5.12 (m, 5H), 4.28 (dd, J¼1.2,
9.2 Hz, 1H), 4.12 (dd, J¼1.6, 12.0 Hz, 1H), 3.72 (dd, J¼1.6, 12.0 Hz,
1H), 1.37 (d, J¼24.4 Hz, 3H), 0.93 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H);
d
173.3, 173.2, 156.8, 153.8, 151.5, 150.7, 135.3, 130.0, 129.6, 124.6,
120.2, 116.7, 86.3, 82.5, 73.2, 70.2, 66.0, 60.6, 49.8, 19.7, 14.0; 31P
13C NMR (100 MHz, CDCl3)
d 174.3, 162.7, 154.4, 144.1, 137.3, 135.1,
(162 MHz, DMSO-d6)
d
4.66, 4.65; MS-ESIþ m/z 539 (MþHþ);
134.6, 130.5, 129.4, 129.0, 128.9, 128.8, 128.7, 128.6, 128.4, 127.6,
127.1, 117.3, 101.0, 99.1, 89.8 (d, J¼3.9 Hz), 79.5, 73.6 (d, J¼1.6 Hz),
70.8, 68.1, 65.3, 60.0, 26.0, 18.6, 17.0 (d, J¼1.6 Hz), ꢁ5.5, ꢁ5.6; MS-
ESIþ m/z 642 (MþHþ); HRMS-ESIþ: m/z calcd for C32H41FN3O8Si
(MþHþ) 642.2665, found 642.2642.
HRMS-ESIþ: m/z calcd for C21H27N6O9NaP (MþNaþ) 561.1468,
found 561.1469.
4.1.17. 50-O-tert-Butyldimethylsilyl-30-O-benzyloxycarbonyl-20-flu-
oro-20-methyluridine (3e). Compound 3e was prepared using the
same procedure as 3a: yield 98% (two steps); 1H NMR (400 MHz,
4.1.22. N4-30-O-Bis-benzyloxycarbonyl-20-fluoro-20-methylcytidine
(4f). Compound 4f was prepared using the same procedure as 4a:
CDCl3)
d
9.90 (br, 1H), 8.05 (d, J¼8.0 Hz, 1H), 7.40e7.35 (m, 5H), 6.27
(d, J¼17.6 Hz, 1H), 5.72 (dd, J¼1.6, 8.0 Hz, 1H), 5.20 (dd, J¼12.0,
25.6 Hz, 2H), 5.16 (dd, J¼9.6, 22.0 Hz, 1H), 4.26 (dd, J¼1.2, 9.6 Hz,
1H), 4.10 (dd, J¼1.2, 12.0 Hz, 1H), 3.71 (dd, J¼1.2, 12.0 Hz, 1H), 1.42
(d, J¼22.4 Hz, 3H), 0.91 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H); 13C NMR
yield 99%; 1H NMR (400 MHz, CDCl3)
d 8.45 (br, 1H), 8.16 (s, 1H),
7.45e7.32(m, 10H) 7.05 (s, 1H), 6.33 (d, J¼7.6 Hz, 1H), 5.41 (s, 1H),
5.20e5.15 (m, 4H), 4.26 (d, J¼9.2 Hz, 1H), 4.12 (d, J¼12.8 Hz, 1H),
3.79 (d, J¼12.0 Hz, 1H), 1.38 (d, J¼22.4 Hz, 1H), 1.381 (d, J¼22.4 Hz,
(100 MHz, CDCl3)
d
163.5, 154.4, 150.7, 139.5, 134.6, 129.0, 128.8,
3H); 13C NMR (100 MHz, CDCl3)
d 162.9, 154.9, 148.7, 144.8, 137.3,
128.7, 103.1, 100.7, 98.9, 89.0 (d, J¼39.3 Hz), 79.6, 77.4, 73.7 (d,
135.1, 134.4, 130.6, 129.3, 129.0, 128.9, 128.8, 128.8, 128.6, 128.5,
J¼15.4 Hz), 70.9, 60.0, 25.9, 18.5, 17.1 (d, J¼24.7 Hz), ꢁ5.5, ꢁ5.7; 19F
117.3, 101.0, 95.9, 80.0, 74.2 (d, J¼1.6 Hz), 70.8, 70.1, 68.1, 65.3, 59.2,
(376 MHz, CDCl3)
d
ꢁ159.70, ꢁ159.74; MS-ESIþ m/z 509 (MþHþ);
17.0 (d, J¼2.6 Hz); 19F (376 MHz, CDCl3)
d
ꢁ159.89; MS-ESIþ m/z 528
HRMS-ESIþ: m/z calcd for C24H34FN2O7Si (MþHþ) 509.2126, found
(MþHþ); HRMS-ESIþ: m/z calcd for C26H27FN3O8 (MþHþ) 528.1776,
509.2127.
found 528.1777.
4.1.18. 30-Benzyloxycarbonyl-20-fluoro-20-methyluridine
(4e). Compound 4e was prepared using the same procedure as 4a:
4.1.23. 50-O-[Phenyl-(ethoxy- -alaninyl)]phosphoryl-N4-30-O-bis-
L
benzyloxycarbonyl-20-fluoro-20-methylcytidine (5f). Compound 5f
was prepared using the same procedure as 5a: yield 93% (1:1 di-
yield 98%; 1H NMR (400 MHz, CD3OD)
d
8.06 (d, J¼8.0 Hz, 1H),
7.40e7.31 (m, 5H), 6.13 (d, J¼18.4 Hz, 1H), 5.73 (d, J¼8.0 Hz, 1H),
5.20 (s, 2H), 5.18 (dd, J¼8.8, 20.8 Hz, 1H), 4.17 (dd, J¼1.2, 8.8 Hz, 1H),
3.95 (dd, J¼2.0, 13.2 Hz, 1H), 3.71 (dd, J¼2.8, 13.2 Hz, 1H), 1.36 (d,
astereomeric (RP/SP) mixture); 1H NMR (400 MHz, CDCl3)
d 8.14 (s,
1H), 7.86 (m, 1H), 7.46e7.06(m, 16H) 6.35 (br, 1H), 5.24e5.15 (m,
4H), 5.14e5.05 (m, 1H), 4.62e4.55 (m, 1H), 4.42e4.33 (m, 2H),
4.29e4.22 (m, 1H), 4.20e4.12 (m, 2H), 4.09e3.93 (m, 1H), 1.41e1.35
(m, 3H), 1.34e1.31 (m, 3H), 1.26e1.20 (m, 3H); 13C NMR (100 MHz,
J¼22.8 Hz, 3H); 13C NMR (100 MHz, CD3OD)
d 165.8, 156.0, 152.3,
142.0, 136.7, 129.8, 129.8, 129.5, 103.4, 102.0, 100.2, 91.1 (d,
J¼38.2 Hz), 81.4, 75.9 (d, J¼15.5 Hz), 71.6, 60.1, 17.8 (d, J¼25.5 Hz);
CDCl3) d 173.7, 162.8, 154.9, 154.3, 152.3, 150.7, 143.8, 137.3, 135.1,
19F (376 MHz, CD3OD)
d
ꢁ159.56; MS-ESIþ m/z 395 (MþHþ);
134.4, 130.8, 130.1, 130.0, 129.4, 129.1, 128.9, 128.6, 128.6, 128.5,
125.4, 120.2, 120.1, 117.3, 100.4, 95.8, 77.5, 74.7, 71.0, 70.0, 68.2, 65.5,
HRMS-ESIþ: m/z calcd for C18H19FN2O7Na(MþNaþ) 417.1069, found
417.1069.
64.9, 61.8, 50.5, 21.1, 17.4, 14.3; 19F (376 MHz, CDCl3)
d
ꢁ158.94; MS-
ESIþ m/z 783 (MþHþ); HRMS-ESIþ: m/z calcd for C37H41FN4O12
(MþHþ) 783.2447, found 783.2451.
P
4.1.19. 50-O-[Phenyl-(ethoxy- -alaninyl)]phosphoryl-30-O-benzyloxy-
L
carbonyl-20-fluoro-20-methyluridine (5e). Compound 5e was pre-
pared using the same procedure as 5a: yield 96% (1:1
diastereomeric (RP/SP) mixture); 1H NMR (400 MHz, CDCl3)
4.1.24. 50-O-[Phenyl-(ethoxy- -alaninyl)]phosphoryl-20-fluoro-20-
L
methylcytidine (6f). Compound 6f was prepared using the same
d
9.42e9.28 (m, 1H), 7.49e7.42 (m, 1H), 7.39e7.28 (m, 6H),
procedure as 6a: yield 95% (1:1 diastereomeric (RP/SP) mixture); 1H
7.24e7.13 (m, 4H), 6.20 (br, 1H), 5.71e5.50 (m, 1H), 5.23e5.14 (m,
2H), 5.12e5.03 (m, 1H), 4.61e4.55 (m, 1H), 4.38e4.33 (m, 2H),
4.27e4.21 (m, 1H), 4.19e4.11 (m, 2H), 4.08e3.96 (m, 1H), 1.43e1.30
NMR (400 MHz, CD3OD) d 7.66e7.50 (m, 1.0 H), 7.40e7.29 (m, 2H),
7.26e7.16(m,3H), 6.22(br,1H), 5.86e5.78(m,1H), 4.60e4.47(m,1H),
4.45e4.35 (m, 1H), 4.33e4.22 (m, 1H), 4.17e4.06 (m, 3H), 3.97e3.77

J.H. Cho et al. / Tetrahedron 67 (2011) 5487e5493
5493
(m, 2H), 1.38e1.18 (m, 9H); 19F (376 MHz, CD3OD)
d
ꢁ162.81; 31P
7. (a) McGuigan, C.; Thiery, J.-C.; Daverio, F.; Jiang, W. G.; Davies, G.; Mason, M.
Bioorg. Med. Chem. 2005, 13, 3219e3227; (b) Congiatu, C.; Brancale, A.; Mason,
M. D.; Jiang, W. G.; McGuigan, C. J. Med. Chem. 2006, 49, 452e455.
8. Perrone, P.; Daverio, F.; Valente, R.; Rajyaguru, S.; Martin, J. A.; Leveque, V.;
Pogam, S. L.; Najera, I.; Klumpp, K.; Smith, D. B.; McGuigan, C. J. Med. Chem.
2007, 50, 5463e5470.
9. Perrone, P.; Luoni, G. M.; Kelleher, M. R.; Daverio, F.; Angell, A.; Mulready, S.;
Congiatu, C.; Rajyaguru, S.; Martin, J. A.; Leveque, V.; Pogam, S. L.; Najera, I.;
Klumpp, K.; Smith, D. B.; McGuigan, C. J. Med. Chem. 2007, 50, 1840e1849.
10. Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.; Rachakondfa, S.; Reddy,
P. G.; Ross, B. S.; Wang, P.; Zhang, H.-R.; Bansal, S.; Espiritu, C.; Keilman, M.;
Lam, A. M.; Steuer, H. M. M.; Niu, C.; Otto, M. J.; Furman, P. A. J. Med. Chem. 2010,
53, 7202e7218.
(162 MHz, CD3OD)
d
4.94, 4.80; MS-ESIþ m/z 515 (MþHþ).
Acknowledgements
This work was supported in part by NIH grant 1RO1-AI071846,
5R37-AI-025899, 5R37-AI-041980, 5P30-AI-50409 (CFAR), and by
the Department of Veterans Affairs. Dr. Schinazi is the founder and
a major shareholder of RFS Pharma, LLC. Emory received no funding
from RFS Pharma, LLC to perform this work and vice versa.
11. Rondla, R.; Coats, S. J.; McBrayer, T. R.; Grier, J.; Johns, M.; Tharnish, P. M.;
Whitaker, T.; Zhou, L.; Schinazi, R. F. Antiviral Chem. Chemother. 2009, 20,
99e106.
12. (a) McGuigan, C.; Gilles, A.; Madela, K.; Aljarah, M.; Holl, S.; Jones, S.; Vernachio,
J.; Hutchins, J.; Ames, B.; Bryant, K. D.; Gorovits, E.; Ganguly, B.; Hunley, D.; Hall,
A.; Kolykhalov, A.; Liu, Y.; Muhammad, J.; Raja, N.; Walters, R.; Wang, J.;
Chamberlain, S.; Henson, G. J. Med. Chem. 2010, 53, 4949e4957; (b) McGuigan,
C.; Perrone, P.; Madela, K.; Neyts, J. Bioorg. Med. Chem. Lett. 2009, 19,
4316e4320.
13. Derudas, M.; Brancale, A.; Naesens, L.; Neyts, J.; Balzarini, J.; McGuigan, C. Bi-
oorg. Med. Chem. 2010, 18, 2748e2755.
14. Birkus, G.; Kutty, N.; He, G.-X.; Mulato, A.; Lee, W.; McDermott, M.; Cihlar, T.
References and notes
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18. Our attempts to apply this method to ProTides with a 20-
b-C-methyl sugar have
been unsuccessful due to formation of a 20,30-carbonate during the CbzCl
reaction.