Synthesis and anti-HCV activity of β-D-2′-deoxy-2′-α-chloro-2′-β-fluoro and β-D-2′-deoxy-2′-α-bromo-2′-β-fluoro nucleosides and their phosphoramidate prodrugs

Article abstract of DOI:10.1016/j.bmc.2019.01.005

We report herein the synthesis and evaluation of a series of β-D-2′-deoxy-2′-α-chloro-2′-β-fluoro and β-D-2′-deoxy-2′-α-bromo-2′-β-fluoro nucleosides along with their corresponding phosphoramidate prodrugs. Key intermediates, lactols 11 and 12, were obtained by a diastereoselective fluorination of protected 2-deoxy-2-chloro/bromo-ribonolactones 7 and 8. All synthesized nucleosides and prodrugs were evaluated with a hepatitis C virus (HCV) subgenomic replicon system.

Full text of DOI:10.1016/j.bmc.2019.01.005

Accepted Manuscript
Synthesis and anti-HCV activity of β-D-2'-deoxy-2'-α-chloro-2'-β-fluoro and
β-D-2'-deoxy-2'-α-bromo-2'-β-fluoro nucleosides and their phosphoramidate
prodrugs
Reuben Ovadia, Ahmed Khalil, Hao Li, Coralie De Schutter, Seema Mengshetti,
Shaoman Zhou, Leda Bassit, Steven J. Coats, Franck Amblard, Raymond F.
Schinazi
PII:
S0968-0896(18)32026-1
https://doi.org/10.1016/j.bmc.2019.01.005
BMC 14697
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 November 2018
3 January 2019
8 January 2019
Please cite this article as: Ovadia, R., Khalil, A., Li, H., De Schutter, C., Mengshetti, S., Zhou, S., Bassit, L., Coats,
S.J., Amblard, F., Schinazi, R.F., Synthesis and anti-HCV activity of β-D-2'-deoxy-2'-α-chloro-2'-β-fluoro and β-
D-2'-deoxy-2'-α-bromo-2'-β-fluoro nucleosides and their phosphoramidate prodrugs, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.005
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Bioorganic & Medicinal Chemistry
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Synthesis and anti-HCV activity of β-D-2'-deoxy-2'-α-chloro-2'-β-fluoro and β-D-2'-
deoxy-2'-α-bromo-2'-β-fluoro nucleosides and their phosphoramidate prodrugs
Reuben Ovadia¥, Ahmed Khalil¥,≠, Hao Li, Coralie De Schutter, Seema Mengshetti, Shaoman Zhou, Leda
Bassit, Steven J. Coats#, Franck Amblard and Raymond F. Schinazi
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, 30322, USA
#Former employee of Cocrystal Pharma, Inc. 1860 Montreal Road, Tucker, GA,30084, USA
≠Current affiliation: Chemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt
¥ Both authors contributed equally to this manuscript
———
ARTICLE INFO
ABSTRACT
Corresponding author. Tel.: +1-404-727-1414; fax +1-404-727-2012; e-mail: rschina@emory.edu
Article history:
Received
Received in revised form
Accepted
We report herein the synthesis and evaluation of a series of β-D-2'-deoxy-2'-α-chloro-2'-β-fluoro
and β-D-2'-deoxy-2'-α-bromo-2'-β-fluoro nucleosides along with their corresponding
phosphoramidate prodrugs. Key intermediates, lactols 11 and 12, were obtained by a
diastereoselective fluorination of protected 2-deoxy-2-chloro/bromo-ribonolactones 7 and 8. All
synthesized nucleosides and prodrugs were evaluated with a hepatitis C virus (HCV)
subgenomic replicon system.
Available online
Keywords:
Nucleosides
Prodrugs
2009 Elsevier Ltd. All rights reserved.
fluorination
Antivirals
1. Introduction
nucleosides as potential anti-HCV agents. In 2017, Pinho et al.
first reported the discovery of the β-D-2’-deoxy-2’-
According to WHO, an estimated 71 million people are
chronically infected worldwide with Hepatitis C Virus (HCV)
and among them a significant number will develop cirrhosis or
liver cancer.1 However, since 2011 and the first approved direct
acting antivirals (DAAs), boceprevir and telaprevir, a revolution
occurred in the treatment of HCV infection. Today, several oral
regimens combining different pangenotypic DAAs with different
modes of action are available and a sustained virologic response
(SVR) of almost 100% can be reached after 8 to 12 weeks of
treatment.2 To date, sofosbuvir (SOF) is the only nucleoside
analog approved for the treatment of HCV infection and it is the
pillar of the most prescribed treatments (Vozevi and Epcluza).
Because nucleoside analogs targeting the HCV NS5B
polymerase generally display broader activity against HCV
genotypes (GT) along with a favorable safety profile and a high
genetic barrier to resistance, they remain the gold standard for the
treatment of HCV.3 This idea was reinforced by the recent
discovery of treatment naïve patients from Equatorial Guinea,
with a new HCV GT-1 subtype naturally bearing resistance-
associated mutations to NS5A inhibitors, another class of DAA.4
dichlorouridine nucleotide prodrug 3 as a potent inhibitor of
HCV replication.7 Our group also reported the discovery of β-D-
2’-deoxy-2’-β-chloro,2’-α-fluoro-,
β-D-2’-deoxy-2’-dibromo-
and β-D-2’-deoxy-2’-β-bromo,2’-α-fluoro-uridine prodrugs 4a8,
59 and 4b,10 as three potent anti-HCV agents (Figure 1). As part
of our ongoing HCV drug discovery program, we wish to report
herein, the synthesis and antiviral evaluation of new series of 2’-
α-chloro,2’-β-fluoro and 2’-α-bromo,2’-β-fluoro nucleosides and
their corresponding phosphoramidate prodrugs.
Based on the success of 2’-halogeno compounds such as SOF5
but also the anticancer drug gemcitabine 26, our group, and
others, focused on the development of new 2’-dihalogeno

Thus, benzoylated derivatives 13 and 14, formed by reaction
of benzoyl chloride with lactols 11 and 12, were reacted under
classical Vorbrüggen type conditions in presence of persilylated
uracil and trimethylsilyl trifluoromethanesulfonate (TMSOTf)
under microwave irradiation to give  and  anomers 15a-b and
16a-b. At this stage,  and  isomers were separated by column
chromatography and the isolated  isomers 16a-b were
deprotected by treatment with tetra-N-butylammonium fluoride
(TBAF) to give nucleosides 17 and 18 in 70% and 90% yield
respectively. In order to express their therapeutic effect,
nucleoside analogs need to be sequentially phosphorylated onto
their corresponding 5’-triphosphate forms. However, the first
phosphorylation step has often been identified as the limiting step
and that is why nucleoside monophosphate prodrugs, which are
able to deliver intracellularly a nucleoside monophosphate, have
been developed.12 Among all the prodrugs that have emerged,
McGuigan’s Protides remains the most popular and have been
clinically validated with the FDA-approval of drugs such as
sofosbuvir 1 (HCV) or tenofovir disoproxyl fumarate (TDF)
(HIV and HBV). Hence, phosphoramidate prodrugs 19 and 20
(RP:Sp ~ 1:1) were prepared from nucleosides 17 and 18 by
reaction
with
the
phenyl
L-isopropylalaninyl
phosphorochloridate reagent 21 in presence of N-
methylimidazole (Scheme 2).
Fig. 1. Structures of sofosbuvir, gemcitabine, known 2’-deoxy-2’-dihalogeno
nucleoside prodrugs, 3-5 and targeted 2’-β-chloro-2’-α-fluoro and 2’-β-
bromo,2’-α-fluoro nucleotides analogs.
TBDMSO
O
F
TBDMSO
X
O
N
X = Cl
15a
16a
2. Results and Discussion
HN
X = Br
O
TBDMSO
2.1. Chemistry
a
O
b
11
12
OBz
F
O
N
Diastereoselective synthesis of key 2-chloro-2-fluoro and 2-
bromo-2-fluoro lactols 11 and 12 was performed by adapting the
chemistry developed by Cen et al. for the stereoselective
synthesis of the 2-Br, 2-F lactone 1011 (Scheme 1). Thus,
protected lactone 6 was first converted to its corresponding
trimethylsilyl enol ether followed by direct chlorination or
bromination in the presence of either N-chlorosuccimimide
(NCS) or N-bromosuccimimide (NBS) to give the chloro and
bromo lactones 7 and 8, as diastereomeric mixtures. Subsequent
fluorination in presence of lithium hexamethyldisilazide
(LiHMDS) and N-fluorobenzenesulfonimide (NFSI) afforded
selectively the dihalogeno lactones 9 and 10. Reduction with
lithium tri-tert-butoxyaluminium hydride in tetrahydrofuran
(THF) gave the corresponding lactols 11 and 12 as a mixture of
α- and β- anomers (ratio ~2:1). With these two key lactols in
hand, the corresponding pyrimidine and purine nucleoside
analogs along with their phosphoramidate prodrugs were
prepared following the chemistry depicted in Schemes 2-7.
NH
TBDMSO
X
X = Cl
X = Br
13
14
TBDMSO
O
O
F
TBDMSO
X
X = Cl
X = Br
15b
16b
O
O
N
NH
NH
O
P
O
HO
N
O
21
N
O
O
H
c
d
O
O
O
O
F
F
HO
X
HO
X
X = Cl
X = Br
X = Cl
X = Br
19
20
17
18
O
O
N
P
Cl
21=
H
O
O
TBDMSO
TBDMSO
O
O
Scheme 2. Synthesis of uridine analog phosphoramidates 19 and 20. (a)
BzCl, TEA, DCM, 0 ºC to rt, 16 h, 96% (13) and 83% (14); (b) uracil, BSA,
CH3CN, 60 ºC, 15 min, then TMSOTf, MW, 150 ºC, 6 min (15), 10 min (16),
30% (15a), 12% (15b), 22% (16a), 12% (16b); (c) TBAF, THF, rt, 1 h, 70%
(17), 90% (18); (d) 21, NMI, THF, rt, 1 h, 34% (19), 46% (20).
O
O
a
X
TBDMSO
TBDMSO
X = Cl
X = Br
6
7
8
TBDMSO
TBDMSO
O
O
b
O
c
OH
F
F
In
a
similar manner, glycosylation of benzoylated
TBDMSO
X
TBDMSO
X
X = Cl
X = Br
X = Cl
X = Br
derivativatives 13 and 14 with persilylated N-Bz-cytosine in the
presence of TMSOTf at 150 °C for 12 min under microwave
irradiation gave compounds 22 and 23 as a mixture of α/β
anomers, easily separable by flash column chromatography on
silica gel. In this case, in-situ 5'-TBDMS cleavage was observed
probably due to the longer reaction time. Removal of the
remaining TBDMS group with TBAF followed by
debenzoylation in a saturated solution of ammonia in methanol
9
10
11
12
Scheme 1. Synthesis of ribolactols 11 and 12. (a) i) TEA, TMSOTf, DCM, 0
ºC, 30 min; ii) NCS or NBS, DCM, 0 ºC, 1 h, 77% (7), 82% (8) over two
steps; (b) NFSI, LiHMDS, THF, -78 ºC, 1 h, 55% (9), 64% (10); (c)
LiAl(OtBu)3H, THF, 0 ºC to rt, 1 h, 97% (11), 98% (12).

afforded nucleosides 26 and 27 in 71% and 96% yield,
respectively (Scheme 3).
HO
O
F
TBDMSO
X
O
N
X = Cl
22a
23a
N
X = Br
TBDMSO
NHBz
O
a
OBz
NHBz
N
F
TBDMSO
X
X = Cl
X = Br
13
14
HO
N
O
O
F
TBDMSO
22b
X
X = Cl
X = Br
23b
NHBz
NH2
N
N
HO
N
O
O
HO
N
O
b
O
c
F
F
HO
24
25
X
HO
26
27
X
X = Cl
X = Br
X = Cl
X = Br
Scheme 3. Synthesis of cytidine nucleoside analogs 26 and 27. (a) N-Bz-
cytosine, BSA, CH3CN, 60 ºC, 15 min, then TMSOTf, 150 ºC, 12 min, MW,
37% (22a), 12% (22b), 21% (23a), 14% (23b); (b) TBAF, THF, rt, 1 h, 73%
(24), 90% (25); (c) NH3/MeOH, rt,16 h, 71% (26), 96% (27).
Scheme 4. Synthesis of cytidine analog phosphoramidate 32. (a) TBDMSCl,
imidazole, DCM, rt, 16 h, 83%; (b) CbzCl, DMAP, DCM, rt, 1.5 h, 74%; (c)
Et3N.3HF, THF, rt, 36 h 67%; (d) 21, NMI, THF, rt, 4 h, 20%; (e) Pd/C,
EtOH, 1,4-cyclohexadiene, rt, 2 h, 55%.
Due to the low solubility of nucleosides 26 and 27 in solvents
,and combination of solvents, commonly used for the formation
of phosphoramidates (THF, THF/ACN or THF/DMF), alternative
approaches were investigated to synthesize the corresponding
cytidine phosphoramidate prodrugs 32 and 35. We first
introduced the prodrug moiety onto the more soluble N-
benzoylated intermediates 24 and 25 but were finally unable to
find suitable conditions to debenzoylate the protected prodrug
without substantial degradation. The preparation of prodrug 32
was eventually achieved by introduction of a more labile
benzyloxycarbonyl group (Cbz).13 Thus, nucleoside 27 was first
reacted with TBDMSCl in presence of imidazole followed by
treatment with CbzCl and DMAP to give the fully protected
nucleoside 29. TBDMS groups were then removed in the
presence of Et3N.3HF to give the desired 4-N-Cbz protected
nucleoside 30. Finally, reaction with phenyl L-isopropylalaninyl
phosphorochloridate reagent 21 in the presence of N-methyl
imidazole and hydrogenolysis of the Cbz-protected cytidine
For the synthesis of 2’-β-chloro-2’-α-fluoro cytidine
phosphoramidate prodrug 35, we investigated the protection of
the 3’-hydroxyl with a Boc group in order to favor the 5’-
regioselectivity of the phosphorylation and increase the solubility
of the nucleoside.14 Thus, nucleoside 26 was selectively protected
in the presence of di-tert-butyl dicarbonate in dioxane/water (4:1)
to give compound 33 in 30% yield. Compound 33 was then
reacted with phenyl L-isopropylalaninyl phosphorochloridate
reagent 21 in the presence of tert-butyl magnesium chloride to
give the phosphoramidate prodrug 34, in 89% yield. Final
treatment of compound 34 with a 4 M solution of HCl in dioxane
to remove the Boc group gave the desired phosphoramidate
prodrug 35 (Scheme 5).
ProTide
intermediate
31
gave
the
corresponding
phosphoramidate prodrug 32 (Scheme 4).
Scheme 5. Synthesis of cytidine analog phosphoramidate 35. (a) Boc2O,
Na2CO3, dioxane/water 4:1, rt, 48 h, 30%; (b) 21, tBuMgCl, THF, 0 ºC to rt, 1
h, 89%; (c) HCl/dioxane 4M, 2 h, rt, 50%.
Purine nucleosides 36, 37, 44 and 45 were prepared according
to the chemistry described in Scheme 6 and 7. Thus, coupling of
lactols 11 and 12 with bis-N-Boc protected adenine in the
presence of DIAD and PPh3 gave 36 and 37 as a mixture of α/β
anomers (ratio 4:1) separable by flash chromatography on silica

gel. Deprotection of the silyl groups with TBAF in THF lead to
the bis-N-Boc protected adenine derivatives 38 and 39, which
were treated with a 4 M solution of HCl in dioxane to give the
desired adenosine nucleosides 40 and 41. The corresponding
monophosphate prodrugs 42 and 43 were obtained by reaction of
protected intermediates 38 and 40 with phenyl L-
isopropylalaninyl phosphorochloridate 21 in presence of tert-
butyl magnesium chloride followed by treatment with a 4 M
solution of HCl in dioxane (Scheme 6).
low temperature.16 Thus, reaction of compound 47 with
pentafluoro phenyl phosphoramidate 52 in the presence of tert-
butyl magnesium chloride followed by treatment with an aqueous
solution of formic acid gave the desired phosphoramidate
prodrug 51 (Sp isomer) in 22% yield over two steps (Scheme 7).
Cl
N
N
TBDMSO
N
TBDMSO
N
N(Boc)2
O
O
a
11
12
F
F
TBDMSO
X = Cl
X
TBDMSO
44a
X
N
X = Cl
X = Br
44b
45b
N(Boc)2
N
X = Br
45a
N
N
N
(Boc)2N
N
Cl
N
TBDMSO
TBDMSO
N
O
O
N
a
11
12
O
Cl
F
F
N
N
N
NH
NH2
N
TBDMSO
X
TBDMSO
X
N
N
O
HO
HO
N
N
N(Boc)2
X = Cl
X = Br
X = Cl
X = Br
36b
37b
36a
37a
O
b
c
N
F
F
N
HO
X
HO
X
N(Boc)2
N(Boc)2
X = Cl
X = Br
X = Cl
X = Br
48
49
46
47
NH2
N
d
N
N
O
N
N
N
F
F
F
NH
NH2
52=
N
N
O
HO
HO
O
N
N
O
P
O
O
O
O
N
N
H
P
O
b
c
N
O
F
H
O
O
F
F
O
O
F
F
HO
X
HO
X
HO
Cl
X = Cl
X = Br
X = Cl
X = Br
38
39
40
41
50
O
N
N
N
d
NH
NH2
O
P
O
O
NH2
N
N
H
O
O
O
N
F
O
O
HO
Br
N
N
P
O
N
51
H
O
O
O
F
Scheme 7. Synthesis of guanosine analog phosphoramidates 50 and 51. (a) 2-
bis-N-Boc-6-Cl purine, PPh3, DIAD, 70 ºC, 1 h, 54% (44a), 12% (44b), 20%
(45a), 6% (45b); (b) TBAF, THF, rt, 1 h, 89% (46), 60% (48); (c)
HCOOH/H2O (4/1), 60 ºC, 16 h, 54% (48), 50% (49); (d) i) 21 or 52,
tBuMgCl, THF, 0 ºC to rt, 1 h; ii) HCOOH/H2O (4/1), 60 ºC, 4 h, 24% (50),
22% (51) over two steps.
HO
X
X = Cl
X = Br
42
43
Scheme 6. Synthesis of adenosine analog phosphoramidates 42 and 43. (a)
bis-N-Boc A, PPh3, DIAD, 70 ºC, 1 h, 27% (36a), 7% (36b), 44% (37a), 12%
(37b); (b) TBAF, THF, rt, 1 h, 99% (38), 70% (39); (c) 4 M HCl/dioxane, rt,
1 h, 61% (40), 60% (41); (d) i) 21, tBuMgCl, THF, rt, 1 h; ii) 4 M
HCl/dioxane, rt, 1 h, 29% (42), 20% (43) over two steps.
All α and β anomers were identified using 1H 2D-NOESY
experiments. Fig. 2 shows an example of the NOE effect
observed for each isomers. In the case of the β-anomer clear
NOE interaction were observed between H2 of the adenine
nucleobase and H3’ and H5’ of the sugar as well as between H1’
and H4’. On the other hand, NOE interactions between the H2
and H4’, H1’ and H5’ were observed for the α-anomer (Fig. 2).
Similarly, reaction of the 2-bis-N-Boc-amino-6-chloro-9H-
purine with lactols 11 and 12 under Mitsunobu conditions gave
compounds 44 and 45 as a mixture of α/β anomers (ratio 4:1)
separable by flash chromatography. Treatment of 44a and 44b
with TBAF afforded bis-N-Boc protected nucleosides 46 and 47
which were treated with 75% aqueous formic acid to give
targeted nucleosides 48 and 49.15 Compound 46 was reacted with
L-isopropylalaninyl phosphorochloridate 21 in presence of tert-
butyl magnesium chloride and then treated with 75% aqueous
formic acid to give the targeted phophoramidate prodrug 50 as a
Sp/Rp mixture, in 24% yield over two steps.
NH(Boc)2
N
H2
H5’
N
N
N
H5’
TBDMSO
H4’
TBDMSO
H4’
H1’
F
H ’
3O
O
N
F
H1’
TBDMSO
Br
TBDMSO
Br
N
H2
N
N
N(Boc)2
Interestingly, our first attempts to prepare monophosphate
prodrugs 51 from protected intermediates 47 using phenyl L-
isopropylalaninyl phosphorochloridate 21 were unsuccessful and
led to the formation of 3’,5’-di-phosphorylated derivatives. In
order to avoid this problem, we turned our attention to the
pentafluoro phenyl reagent 52. Indeed, this diastereomerically
pure reagent (Sp isomer), used to synthesize sofosbuvir on a large
scale, is known to be selective to the 5’-position when used at
37a
37b
Fig. 2. Anomer assignment for compound 37a and 37b via NOE experiments.
Despite what was reported by Cen et al.,11 the stereochemistry
at the 2’-position could not be clearly established through
common NMR experiments. Therefore, suitable crystals were
grown from compounds 15a and 22a which allowed us to

confirm the R-configuration of C-2’ of our nucleosides through
X-ray diffraction analysis (Fig. 3).
2.2. Antiviral activity and cytotoxicity
Nucleoside analogs 17, 18, 26, 27, 40, 41, 48, 49 and their
corresponding phosphoramidate prodrugs 19, 20, 32, 35, 42, 43,
50, 51 were evaluated for inhibition of HCV genotype 1b RNA
replication in Huh-7 cells using a subgenomic HCV replicon
system.17 Cytotoxicity in Huh-7 cells was determined
simultaneously by extraction and amplification of both HCV
RNA and cellular ribosomal RNA (rRNA).18 In addition,
cytotoxicity was determined in primary human peripheral blood
mononuclear (PBM) cells, human lymphoblastoid cells (CEM),
and African Green monkey Vero cells (Table 1).19,20 Except for
2’-Br,2’-F cytidine derivative 27, which displayed an EC50 of 5.1
M, none of the nucleosides showed anti-HCV activity at
concentration up to 10 M. On the other hand, monophosphate
prodrugs 19, 20, 32, 35 and 42 had EC50’s in the low to sub-
micromolar range (EC50 between 0.4 and 2.7 M). This
difference of potency between the nucleosides and their prodrug
counterparts indicates that, once more, the monophosphorylation
of modified nucleosides can be problematic and that making
prodrug is an unavoidable step in the development of nucleoside
analogs. Unfortunately, the increase in potency also came with an
increase of cytotoxicity, especially towards Huh-7 cells. Indeed,
compounds 19, 20, 27, 32, 35 and 42 displayed CC50 in the low
micromolar range in this specific cell line. Interestingly, neither
the guanosine analogs 48 and 49 nor their corresponding
monophosphate prodrugs (50 and 51) showed anti-HCV activity
at concentration up to 10 M. The unusual anti-HCV activity
profile observed in these two series of 2’-dihalo nucleoside
analogs versus what we have reported previously8,Error!
Bookmark not defined. for related 2’-halo series of nucleoside
analogs is a strong indicator that the observed anti-HCV activity
presented here is largely, if not completely, due to the
cytotoxicity toward the Huh-7 replicon cell line.
Fig. 3. Single crystal X-ray structure of compound 15a (top) and 22a
(bottom).
Table 1
HCV genotype 1b replicon activity and cytotoxicity of nucleosides 17, 18, 26, 27, 40, 41, 48, 49 and their phosphoramidate prodrugs 19, 20, 32, 35, 42, 43, 50,
51.
Anti-HCV activity (μM)
Cytotoxicity, CC50 (μM)
Compound
EC50
> 10
EC90
> 10
Huh-7
> 100
PBM
> 100
CEM
> 100
Vero
17
18
19
20
26
27
32
35
40
41
42
> 100
> 10
0.9 ± 0.3
0.4 ± 0.3
> 10
> 10
3.7 ± 1.1
2.0 ± 0.9
> 10
> 100
1.3
> 100
> 100
> 100
9.3
> 100
90
> 100
> 100
> 100
63
8.0
> 100
11
12
5.1 ± 0.9
2.4 ± 0.1
0.7 ± 0.2
> 10
> 10
2.9
> 100
> 100
> 100
> 100
> 100
26
3.2
2.9
≥ 10
6.8
> 100
> 100
> 100
> 100
15
> 100
> 100
> 100
> 100
> 100
3.4 ± 1.7
> 10
23
> 100
> 100
1.6
> 10
> 10
2.7 ± 1.3
≥ 10

43
48
> 10
> 10
> 10
> 10
6.9
19
29
71
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
84
> 100
> 100
> 100
> 100
> 100
49
> 10
> 10
50
> 10
> 10
51
> 10
> 10
> 100
> 100
SOF
0.05 ± 0.02
0.37 ± 0.25
3. Conclusion
(hexane/ethyl acetate 40:1) to afford compound 7 as a 1:1.3
mixture of diastereoisomers (4.22 g, 77%). H NMR (400 MHz,
1
Sixteen 2’-α-chloro,2’-β-fluoro and 2’-α-bromo,2’-β-fluoro
nucleosides analogs along with their corresponding
phosphoramidate prodrugs were prepared from key dihalogeno
lactols 11 and 12. Among them, compounds 19, 20, 27, 32, 35
and 42 displayed anti-HCV activity in the low to sub-
micromolar range. Unfortunately cytotoxicity was observed for
most of these compounds in Huh-7 cells, precluding them for
further development for HCV. Based on this study as well as
previously reported studies,21 having a 2’-F group in the beta
position seems to significantly increase the probability of
cytotoxicity.
CDCl3): 4.59-4.62 (m, 1.5H), 4.48 (m, 0.5H), 4.38-4.42 (m,
1.5H), 4.16-4.19 (m, 1H), 3.90-3.95 (m, 1.5H), 3.76-3.81 (m,
1.5H), 0.8 and 0.9 (each s, 27H), 0.14-0.08 (m, 18H). 13C NMR
(100 MHz, CDCl3): δ 171.4, 169.7, 86.0, 83.8, 75.1, 70.6, 61.5,
59.9, 59.2, 55.9, 25.9, 25.7, 18.4, 18.0, -4.9, -5.3, -5.4, -5.5. MS
(HR-ESI) for C17H36ClO4Si2 [(M+H)+], Calcd: m/z 395.1841,
Found: m/z 395.1834.
4.2.2
(3R/S,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-bromodihydrofuran-2(3H)-one
(8)
4. Experimental section
4.1. General
To a solution of compound 6 (7.0 g, 19.4 mmol) in DCM (250
mL) at 0 °C was added triethylamine (16.3 mL, 117 mmol)
followed by TMSOTf (10.6 mL, 58.3 mmol). The solution was
stirred at 0 °C for 30 minutes then, at 0 °C, a solution of N-
bromosuccinimide (5.2 g, 29.1 mmol) in DCM (75 mL) was
added. After stirring at 0 °C for another 1 h, the reaction mixture
was poured into a saturated solution of NaHCO3 (300 mL) and
extracted with DCM (2 x 200 mL). The combined organic layers
were washed with brine, dried over anhydrous Na2SO4, filtered
and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel
(hexane/ethyl acetate 40:1) to afford compound 8 as a 1:1.5
Anhydrous solvents were purchased from Aldrich Chemical
Company, Inc. (Milwaukee). Reagents were purchased from
commercial sources. Unless noted otherwise, the materials used
in the examples were obtained from readily available commercial
suppliers or synthesized by standard methods known to one
skilled in the art of chemical synthesis. Microwave reactions
were performed with a CEM discover explorer 12 hybrid. 1H, 13
C
and 31P NMR spectra were taken on a Bruker AscendTM 400
spectrometer at rt, and reported in ppm downfield from internal
tetramethylsilane (for 1H-NMR). NMR processing was
performed with Mnova (Mestrelab Research). Deuterium
exchange and decoupling experiments were utilized to confirm
proton assignments. Signal multiplicities are represented by s
(singlet), d (doublet), dd (doublet of doublets), t (triplet), q
(quadruplet), br (broad), bs (broad singlet), m (multiplet). All J-
values are in Hz and calculated by Mnova programs. Mass
1
mixture of diastereoisomers (7.0 g, 82%). H NMR (400 MHz,
CDCl3): δ 4.71 – 4.67 (m, 1H), 4.49 (d, J = 5.6 Hz, 1H), 4.42 –
4.37 (m, 3H), 4.33 (dt, J = 4.7, 2.2 Hz, 1H), 4.25 – 4.20 (m, 1H),
3.98 – 3.92 (m, 1H), 3.83 – 3.74 (m, 3H), 0.93 – 0.86 (m, 48H),
0.20 – 0.04 (m, 33H).13C NMR (100 MHz, CDCl3): δ 170.6 (d, J
= 80.9 Hz), 85.2 (d, J = 28.2 Hz), 75.6, 69.6, 68.8, 60.3, 46.13 (d,
J = 3.6 Hz), 39.0, 25.8, 25.6, 21.1, 18.2 18.0, 14.21, -4.9, -5.3, -
5.4, -5.5. MS (HR-ESI) for C17H36BrO4Si2 [(M+H)+]. Calcd: m/z
439.1335. Found: m/z 439.1330.
spectra were determined on
a Micromass Platform LC
spectrometer using electrospray ionization. Analytic TLC was
performed on Analtech GHLF silica gel plates, and preparative
TLC on Analtech GF silica gel plates. Column chromatography
was performed on Combiflash Rf 200.
4.2.3
(3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-chloro-3-fluorodihydrofuran-
2(3H)-one (9)
4.2. Synthetic procedures
Compound 7 (4 g, 10.1 mmol) and NFSI (4.78 g, 15.2 mmol)
were dissolved in anhydrous THF (80 mL). The solution was
cooled to -78 °C, and a 1 M solution of LiHMDS in THF (13.2
mL, 13.2 mmol) was added dropwise. The reaction mixture was
stirred at -78 °C for 1 h, and quenched with a saturated solution
of NH4Cl (100 mL). The mixture was allowed to warm to rt, and
the aqueous layer was extracted with ethyl acetate (3 x 100 mL).
The organic layers were combined, washed with a saturated
solution of NaHCO3, water and brine, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel
(hexane/ethyl acetate 40:1) to afford compound 9 (2.25 g, 55%).
1H NMR (400 MHz CDCl3): 4.79 (dd, J = 14.5 Hz, J = 8.4 Hz,
1H), 4.09 (dt, J = 1.9 Hz, 1H), 4.01 (dt, J = 2.3 Hz, 1H), 3.80
(two d, J = 1.9 Hz, 1H), 0.9 (ds, 18H), 0.09 (four s, 12H). 19F
4.2.1
(3R/S,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-chlorodihydrofuran-2(3H)-one
(7)
To a solution of compound 6 (5 g, 13.9 mmol) in DCM (180
mL) at 0 °C was added triethylamine (11.6 mL, 83.1 mmol)
followed by TMSOTf (7.54 mL, 41.6 mmol). The reaction
mixture was stirred at 0 °C for 30 minute and a solution of N-
chlorosuccinimide (2.8 g, 21.0 mmol) in DCM (36 mL) was
added. After stirring at 0 °C for another 1 h, the reaction mixture
was poured into a saturated solution of NaHCO3 (200 mL) and
extracted with DCM (2 x 150 mL). The combined organic layers
were washed with brine, dried over anhydrous Na2SO4, filtered
and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel

NMR (376 MHz, CDCl3): -127.5 (d, J = 14.3 Hz). 13C NMR
(100 MHz, CDCl3): δ 165.5, 104.4 (d, J = 264 Hz), 80.9, 71.9,
58.6, 25.9, 25.6, 18.4, 18.2, -4.6, -5.1, -5.3, -5.4. MS (HR-ESI)
for C17H35ClFO4Si2 [(M+H)+], Calcd: m/z 413.1740. Found: m/z
413.1746.
acetate (100 mL). The organic layer was washed with water,
brine, dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure. The crude residue was purified by flash
column chromatography on silica gel (hexane/ethyl acetate 20:1)
1
to afford compound 12 (3.43 g, 98%). H NMR (400 MHz,
CDCl3): δ 5.32 (dd, J = 9.0, 1.3 Hz, 1H), 5.18 (ddd, J = 12.4,
6.3, 0.9 Hz, 0.5H), 4.49 (dd, J = 13.5, 6.7 Hz, 1H), 4.39 (ddd, J =
12.1, 4.6, 1.0 Hz, 0.5H), 4.06 – 3.99 (m, 0.5H), 3.92 (dt, J = 6.7,
2.0 Hz, 1H), 3.87 (d, J = 9.2 Hz, 1H), 3.79 (t, J = 2.4 Hz, 0.5H),
3.76 (d, J = 2.5 Hz, 1H), 3.67 (m, 1H), 3.63 (d, J = 1.7 Hz, 1H),
3.61 (d, J = 1.7 Hz, 0.5H), 3.49 (d, J = 12.4 Hz, 0.5H), 0.96 –
0.87 (m, 27H), 0.22 – 0.04 (m, 18H).19F NMR (376 MHz,
CDCl3): -120.06 (dd, J = 12.2 Hz, J = 6.4 Hz), -127.23 (d, J =
13.4 Hz). 13C NMR (100 MHz, CDCl3): δ 113.5, 110.8, 99.9 (d,
J = 21.4 Hz), 99.0 (d, J = 31.9 Hz), 83.6, 82.3 (d, J = 8.7 Hz),
74.5 (d, J = 24.9 Hz), 72.1 (d, J = 22.3 Hz), 61.7, 60.9, 28.4 –
24.1 (m), 18.2 (d, J = 29.8 Hz).
4.2.4
(3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-bromo-3-fluorodihydrofuran-
2(3H)-one (10)
Compound 8 (7.0 g, 15.9 mmol) and NFSI (7.5 g, 23.8 mmol)
were dissolved in anhydrous THF (140 mL). The solution was
cooled to -78 °C and a 1 M solution of LiHMDS in THF (20.7
mL, 20.7 mmol) was added dropwise. The reaction mixture was
stirred at -78 °C for another 1 h, and was quenched with a
saturated solution of NH4Cl (200 mL). The mixture was allowed
to warm to rt, and the aqueous layer was extracted with ethyl
acetate (3 x 150 mL). The organic layers were combined, washed
with a saturated solution of NaHCO3, water and brine, dried over
anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by flash column
chromatography on silica gel (hexane/ethyl acetate 40:1) to
afford compound 10 (4.6 g, 64%). 1H NMR (400 MHz CDCl3): δ
4.54 (dd, J = 15.3, 8.0 Hz, 1H), 4.04 – 4.00 (m, 1H), 3.98 (dd, J =
2.6, 1.8 Hz, 1H), 3.81 – 3.75 (m, 1H), 0.94 (s, 9H), 0.87 (s, 9H),
0.18 (s, 3H), 0.14 (s, 3H), 0.07 (d, J = 2.6 Hz, 6H).19F NMR (376
MHz, CDCl3): δ -126.98 (dd, J = 15.5 Hz). 13C NMR (100 MHz,
CDCl3): δ 165.7, 100.7, 97.9, 80.6, 72.1, 58.1, 25.7, 25.6, 18.2,
18.0, -4.6, -5.1, -5.4, -5.5. MS (HR-ESI) for C17H35BrO4Si2
[(M+H)+]. Calcd: m/z 457.1241. Found: m/z 457.1236.
4.2.7 (2R/S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-chloro-3-fluorotetrahydrofuran-
2-yl benzoate (13)
To a solution of compound 11 (4.72 g, 11.4 mmol) in DCM
(100 mL) was added triethylamine (3.96 mL, 28.4 mmol). The
reaction was cooled to 0 °C and benzoyl chloride (1.97 mL, 17.0
mmol) was introduced dropwise. After being stirred for 30 min at
0 °C, the mixture was warmed to rt and stirred for 16 h. The
reaction was quenched with methanol (1 mL) and then was
washed with water and brine. The organic layer was dried over
Na2SO4, filtered and concentrated under reduced pressure. The
crude product was purified by flash column chromatography on
silica gel (hexane/ethyl acetate 20:1) to afford compound 13
4.2.5 (2R/S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-chloro-3-fluorotetrahydrofuran-
2-ol (11)
1
(5.65 g, 96%). H NMR (400 MHz, CDCl3): 8.10 and 8.05 (m,
2.2H), 7.57-7.62 (m, 1.2H), 7.42-7.47 (m, 2.4H), 6.54 and 6.46
(m, 1.25H), 4.80 (dd, J = 12.5 Hz, J = 7.9 Hz, 1H), 4.37 (dd, J =
18.1 Hz, J = 4.7 Hz, 0.25 H), 4.21 (m, 0.25 H), 3.85-3.91 (m,
2H), 3.76-3.84 (m, 0.5H), 3.68-3.72 (m, 1H), 0.95 and 0.78 (each
s, 22H), 0.07, 0.08, 0.16, 0.20 and 0.22 (each s, 15 H). 19F NMR
(376 MHz, CDCl3): -115.15 (dd, J = 17.8 Hz, J = 7.7 Hz) and
128.53 (d, J = 12.1 Hz). 13C NMR (100 MHz, CDCl3): δ 164.9,
133.7, 130.3, 130.2, 129.4, 128.6, 114.0 (d, J = 273 Hz), 97.7,
87.4, 82.5, 72.0, 62.0, 60.8, 25.7, 25.8, 25.9, 26.0, 18.5, 18.2, -
4.5, -4.7, -4.8, -5.2, -5.3, -5.4. MS (HR-ESI) for
C24H40ClFNaO5Si2 [(M+Na)+], Calcd: m/z 541.1985, Found:
m/z 541.1978.
To a solution of compound 9 (5.37 g, 13 mmol) in anhydrous
THF (90 mL) was added dropwise LiAlH[OC(CH3)3]3 (32.5 mL,
32.48 mmol, 1 M in THF) at 0 °C. The reaction mixture was
stirred at rt for 1 h. The reaction was quenched with a saturated
solution of NH4Cl (50 mL) and extracted with ethyl acetate (100
mL). The organic layer was washed with water, brine, dried over
anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The crude residue was purified by flash column
chromatography on silica gel (hexane/ethyl acetate 20:1) to
afford compound 11 (5.23 g, 97%). 1H NMR (400 MHz, CDCl3):
5.25 (ddd, J = 12.5 Hz, J = 6.3 Hz, J = 0.9 Hz, 0.5H), 5.16 (d, J =
9.6 Hz, 1H), 4.66 (dd, J = 12.5 Hz, J = 6.7 Hz, 1H), 4.39 (ddd, J
= 11.7 Hz, J = 4.0 Hz, J = 0.9 Hz, 0.5H), 4.07-4.11 (m, 0.5H),
3.92 (dt, J = 1.9 Hz, 1H), 3.82 (d, J = 9.3 Hz, 1H), 3.78 (dt, J =
2.5 Hz, 1H), 3.72 and 3.69 (each dd, J = 4.0 Hz, J = 1.8 Hz,
0.5H), 3.61-3.66 (m, 1.5H), 3.50 (d, J = 12.2 Hz, 0.5H), 0.93,
0.92, 0.90 (each s, 27 H), 0.19, 0.16, 0.15, 0.1, 0.07, 0.00 (each s,
18 H). 19F NMR (376 MHz, CDCl3): -122.59 (dd, J = 12.2 Hz, J
= 6.4 Hz), -130.55 (d, J = 12.5 Hz). 13C NMR (100 MHz,
CDCl3): δ 116.1 (d, J = 249 Hz), 115.1 (d, J = 263 Hz), 99.5,
99.2, 98.9, 84.5, 82.7, 82.6, 74.7, 74.5, 72.2, 72.0, 62.0, 61.2,
26.0, 25.7, 18.49, 18.47, 18.2, 18.1, -4.4, -4.7, -4.8, -5.3, -5.3, -
5.4. MS (HR-ESI) for C17H36ClFNaO4Si2 [(M+Na)+], Calcd: m/z
437.1722, Found: m/z 437.1717.
4.2.8 (2R/S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-bromo-3-fluorotetrahydrofuran-
2-yl benzoate (14)
To a solution of compound 12 (744 mg, 1.62 mmol) in DCM
(40 mL) was added triethylamine (0.34 mL, 2.43 mmol). The
reaction was cooled to 0 °C and benzoyl chloride (0.23 mL, 1.94
mmol) was introduced dropwise. After being stirred for 30 min at
0 °C, the mixture was warmed to rt and stirred for 16 h. The
reaction was quenched with methanol (0.20 mL) and then washed
with water and brine. The organic layer was dried over Na2SO4,
filtered and concentrated under reduced pressure. The crude
product was purified by flash column chromatography on silica
gel (hexane/ethyl acetate 20:1) to afford compound 14 (760 mg,
4.2.6 (2R/S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-bromo-3-fluorotetrahydrofuran-
2-ol (12)
1
83%). H NMR (400 MHz, CDCl3): 8.13-8.11 (m, 1H), 8.06-
8.03 (m, 2H), 7.62-7.58 (m, 2H), 7.47-7.43 (m, 3H), 6.64 (m,
1H), 6.56 (m, 0.5H), 4.59 (dd, J = 12.5 Hz, J = 7.9 Hz, 1H), 4.37
(dd, J = 18.1 Hz, J = 4.7 Hz, 1H), 4.20 (m, 1H), 3.90-3.86 (m,
2H), 3.81-3.77 (m, 1H), 3.72-3.68 (m, 1H), 0.95 and 0.78 (each s,
22H), 0.25, 0.20, 0.18, 0.09, 0.08, 0.07 (each s, 15 H). 19F NMR
(376 MHz, CDCl3): -115.18 (dd, J = 17.8 Hz, J = 7.7 Hz) and
To a solution of compound 10 (3.5 g, 7.65 mmol) in
anhydrous THF (70 mL) was added dropwise LiAlH[OC(CH3)3]3
(11.5 mL, 11.5 mmol, 1 M in THF) at 0 °C. The reaction mixture
was stirred at rt for 1 h. The reaction was quenched with a
saturated solution of NH4Cl (50 mL) and extracted with ethyl

127.00 (d, J = 12.1 Hz). MS (HR-ESI) for C24H40BrFO5Si2
0.15 (three s, 12H). 19F NMR (376 MHz, CDCl3): -111.15 (t, J =
11.3 Hz). 13C NMR (100 MHz, CDCl3): δ 163.3, 150.3, 140.3,
116.7 (d, J = 252 Hz), 114.1 101.8, 88.4 (d, J = 150 Hz), 85.3,
74.8 (d, J = 103 Hz), 61.1, 25.9, 25.7, 18.5, 18.0, -4.5, -4.7, -4.9,
-5.4, -5.5. 16b: 1H NMR (400 MHz, CDCl3): δ 9.05 (s, 1H),
7.76 (dd, J = 8.3 Hz, J = 1.5 Hz, 1H), 6.56 (d, J = 5.6 Hz, 1H),
5.71 (d, J = 8.0 Hz, 1H), 4.25 (dd, J = 16.3 Hz, J = 7.3 Hz, 1H,
3.97 (dt, J = 2.7 Hz 1H), 3.85 (m, 1H), 3.77 (m, 1H), 0.93 (two s,
18H), 0.12 (three s, 12H). 19F NMR (376 MHz, CDCl3): -120.56
(dd, J = 16.04 Hz, J = 4.7 Hz). 13C NMR (100 MHz, CDCl3): δ
162.8, 150.1, 139.5, 111.8, 109.1, 102.5, 88.7, 81.6, 73.2, 60.0,
25.7, 18.4, 18.0, -4.4, -4.9, -5.4, -5.5 MS (HR-ESI) for
C21H38BrFN2O5Si2 [(M+H)+], Calcd: m/z 553.1565, Found: m/z
553.1567.
[(M+H)+], Calcd: m/z 563.1582, Found: m/z 563.1588.
4.2.9 1-((2S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-chloro-3-fluorotetrahydrofuran-
2-yl)pyrimidine-2,4(1H,3H)-dione (15a) and 1-((2R,3R,4R,5R)-4-
((tert-butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-chloro-3-fluorotetrahydrofuran-
2-yl)pyrimidine-2,4(1H,3H)-dione (15b)
A solution of compound 13 (1.14 g, 2.19 mmol), uracil (0.5 g,
4.46 mmol) and BSA (2.65 mL, 10.8 mmol) in acetonitrile (10
mL) was stirred at 60 °C for 15 minutes before addition of
TMSOTf (2.22 mL, 12.3 mmol). The reaction vessel was then
placed into the cavity of microwave reactor and irradiated for 6.5
min at 150 °C. The reaction was quenched by addition of a 5%
aqueous solution of NaHCO3 at 0 °C. The aqueous layer was
extracted with ethyl acetate (20 mL), and the combined organic
layers were washed with a saturated solution of NaHCO3, water,
and brine. The solution was dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by flash
column chromatography on silica gel (hexanes/ethyl acetate 2:1)
to afford 15a (350 mg, 32%) and 15b (140 mg, 13%). 15a: 1H
NMR (400 MHz, CDCl3): δ 8.58 (s, 1H), 7.49 (d, J = 8.04 Hz,
1H), 6.45 (d, J = 10.5 Hz, 1H), 5.72 (dd, J = 8.3 Hz, J = 2.3 Hz,
1H), 4.61 (dd, J = 12.3 Hz, J = 4.8 Hz, 1H) 4.20 (m, 1H), 3.75
(m, 2H), 0.93 (two s, 18H), 0.08 (four s, 12H). 19F NMR (376
MHz, CDCl3): -114.05 (t, J = 11.3 Hz). 13C NMR (100 MHz,
CDCl3): δ 162.7, 150.2, 140.4, 117.9 (d, J = 252 Hz), 102.0,
88.2, 86.1, 74.8, 61.3, 26.0, 25.7, 18.4, 18.1, -4.7, -4.9, -5.3, -
5.32. MS (HR-ESI) for C21H38ClFN2NaO5Si2 [(M+Na)+], Calcd:
m/z 531.1890, Found: m/z 531.1882. 15b: 1H NMR (400 MHz,
CDCl3): δ 8.64 (s, 1H), 7.73 (dd, J = 8.3 Hz, J = 1.5 Hz, 1H),
6.37 (d, J = 5.6 Hz, 1H), 5.72 (dd, J = 8.3 Hz, J = 2.4 Hz, 1H),
4.42 (dd, J = 16.3 Hz, J = 7.3 Hz, 1H, 3.96 (dt, J = 2.7 Hz 1H),
3.86 (m, 1H), 3.79 (m, 1H), 0.93 (two s, 18H), 0.08 (four s, 12H).
19F NMR (376 MHz, CDCl3): -122.39 (dd, J = 16.04 Hz, J = 4.7
Hz). 13C NMR (100 MHz, CDCl3): δ 162.6, 150.1, 139.6, 114.6
(d, J = 259 Hz), 102.7, 87.8, 82.0, 73.2, 60.3, 26.0, 5.7, 18.5,
18.2, -4.39, -4.91, -5.29, -5.39. MS (HR-ESI) for
C21H38ClFN2NaO5Si2 [(M+Na)+], Calcd: m/z 531.1890. Found:
m/z 531.1884.
4.2.11
1-((2R,3R,4R,5R)-3-Chloro-3-fluoro-4-hydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-
dione (17)
To a solution of compound 15b (21 mg, 0.041 mmol) in
anhydrous THF (1 mL) was added TBAF (91 µL, 0.091 mmol, 1
M in THF). The reaction mixture was stirred for 1 h at rt and then
quenched with a saturated solution of NH4Cl. The aqueous layer
was extracted with ethyl acetate (3 x 5 mL) and the combined
organic layers were dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (CH2Cl2/MeOH 10:1)
to afford compound 17 (8 mg, 70%). 1H NMR (400 MHz,
MeOD): δ 7.95 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 6.33 (d, J = 8.4
Hz, 1H), 5.72 (d, J = 8.8 Hz, 1H), 4.36 (dd, J = 17.0 Hz, J = 6.9
Hz, 1H), 3.9 (m, 2H), 3.78 (m, 1H). 19F NMR (376 MHz,
MeOD): -124.82 (dd, J = 17.4 Hz, J = 8.5 Hz). 13C NMR (100
MHz, MeOD): δ 165.7, 152.1, 142.2, 116.3 (d, J = 258 Hz),
102.8, 88.8, 83.6, 74.1, 60.6. MS (HR-ESI) for C9H11ClFN2O5
[(M+H)+], Calcd: m/z 281.0341, Found: m/z 281.0333.
4.2.12
1-((2R,3R,4R,5R)-3-Bromo-3-fluoro-4-hydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-
dione (18)
To a solution of compound 16b (60 mg, 0.14 mmol) in
anhydrous THF (4 mL) was added TBAF (310 µL, 0.31 mmol, 1
M in THF). The reaction mixture was stirred for 1 h at rt and then
quenched with a saturated solution of NH4Cl. The aqueous layer
was extracted with ethyl acetate (3 x 10 mL) and the combined
organic layers were dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (CH2Cl2/MeOH 10:1) to afford
4.2.10
1-((2S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-
(((tert-butyldimethylsilyl)oxy)methyl)-3-bromo-3-
fluorotetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (16a)
and 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-bromo-3-fluorotetrahydrofuran-
2-yl)pyrimidine-2,4(1H,3H)-dione (16b)
1
compound 18 (41 mg, 90%). H NMR (400 MHz, MeOD): δ
7.97 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 6.49 (d, J = 8.4 Hz, 1H),
5.72 (d, J = 8.8 Hz, 1H), 4.22 (dd, J = 17.0 Hz, J = 6.9 Hz, 1H),
3.90 (m, 2H), 3.78 (m, 1H). 19F NMR (376 MHz, MeOD): -
124.12 (dd, J = 17.4 Hz, J = 8.5 Hz). 13C NMR (100 MHz,
MeOD): δ 164.3, 153.5, 140.8, 128.6, 122.2, 120.1, 112.5,
101.3, 88.3, 81.9, 72.7, 68.1, 59.1, 50.5, 20.4. MS (HR-ESI) for
C9H310BrFN2O5 [(M+H)+], Calcd: m/z 324.9835, Found: m/z
324.9833.
A solution of compound 14 (1.00 g, 1.77 mmol), uracil (0.22
g, 1.95 mmol) and BSA ((1.29 mL, 5.31 mmol) in acetonitrile
(10 mL) was stirred at 60 °C for 15 min before addition of
TMSOTf (0.96 mL, 5.31 mmol). The reaction vessel was then
placed into the cavity of microwave reactor and irradiated for 6.5
min at 150 °C. The reaction was quenched by addition of a 5%
aqueous solution of NaHCO3 at 0 °C. The aqueous layer was
extracted with ethyl acetate (20 mL), and the combined organic
layers were washed with a saturated solution of NaHCO3, water,
and brine. The solution was dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by flash
column chromatography on silica gel (hexanes/ethyl acetate 2:1)
to afford 16a (220 mg, 22%) and 16b (120 mg, 12%). 16a: 1H
NMR (400 MHz, CDCl3): δ 7.50 (d, J = 8.24 Hz, 1H), 6.42 (d, J
= 10.6 Hz, 1H), 5.73 (d, J = 8.3 Hz, 1H), 4.53 (dd, J = 12.3 Hz, J
= 4.8 Hz, 1H) 4.18 (m, 1H), 3.73 (m, 2H), 0.95 (two s, 18H),
4.2.13 Isopropyl ((((2R,3R,4R,5R)-4-chloro-5-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxytetrahydrofuran-
2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate, diastereomeric
mixture (19)
To a solution of compound 17 (30 mg, 0.11 mmol) in THF (2
mL) was added N-methylimidazole (68 µL, 0.85 mmol) then
(2S)-isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate
(0.21 g, 0.69 mmol) in THF (0.69 mL). The mixture was stirred
at rt for 1 h, quenched with water and extracted with ethyl acetate
(5 mL). The organic layer was washed with water twice, dried

over Na2SO4, filtrated and concentrated under reduced pressure.
The crude product was purified by flash chromatography
(CH2Cl2/MeOH 20:1) to afford compound 19 as a RP:SP
diastereomeric mixture (~1:1) (20 mg, 34%).1H NMR (400 MHz,
MeOD): δ 7.66 and 7.58 (each dd, J = 8.3 Hz, J = 2.4 Hz, 1H),
7.35-7.38 (m, 2H), 7.20-7.27 (m, 3H), 6.33-6.37 (m, 1H), 5.69
and 5.64 (each d, J = 8.3 Hz, 1H), 4.93-4.99 (m, 1H), 4.29-4.41
(m, 3H), 4.10 (m, 1H), 3.9 (m, 1H), 1.29-1.36 (m, 3H), 1.22-1.24
(m 6H). 19F NMR (376 MHz, MeOD): -124.88 (m). 31P NMR
(162 MHz, MeOD): 3.65, 3.58. 13C NMR (100 MHz, MeOD): δ
173.2, 162.8,150.5, 150.3, 140.1, 130.1, 125.6, 120.1, 113.7 (d, J
= 257 Hz), 103.0, 87.3, 80.4, 73.4, 69.8, 64.2, 50.6, 21.8, 20.9.
MS (HR-ESI) for C21H27ClFN3O9P [(M+H)+], Calcd: m/z
550.1157, Found: m/z 550.1149.
Hz), 96.6, 88.2, 84.8, 74.5, 60.6, 25.7, 18.3, -4.6, -4.9. MS (HR-
ESI) for C22H30ClFN3O5Si [(M+H)+], Calcd: m/z 498.1627,
Found: m/z 498.1624. 22b: 1H NMR (400 MHz, CDCl3):
δ
8.81 (br s, 1H), 8.17 (d, J = 7.3 Hz, 1H), 7.86 (d, J = 7.3 Hz, 1H),
7.60 (m, 2H), 7.49 (m, 2H), 6.55 (d, J = 6.8 Hz, 1H), 4.51 (dd, J
= 16.6 Hz, J = 7.4 Hz, 1H), 4.09 (m, 1H), 3.95 (m, 1H), 3.84 (m,
1H), 2.7 (br s, 1H), 0.93 (s, 9H), 0.15 (s, 6H). 19F NMR (376
MHz, CDCl3): -122.31 (s). 13C NMR (100 MHz, CDCl3):
δ
170.8, 162.9, 155.1, 145.3, 133.4, 133.0, 129.1, 127.8, 114.5 (d, J
= 260 Hz), 97.4, 89.2, 82.2, 73.4, 59.7, 25.8, 18.1, -4.6, -4.9. MS
(HR-ESI) for C22H30ClFN3O5Si [(M+H)+], Calcd: m/z 498.1627,
Found: m/z 498.1620.
4.2.16
bromo-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)benzamide (23a) and N-(1-
N-(1-((2S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-3-
4.2.14 Isopropyl ((((2R,3R,4R,5R)-4-bromo-5-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxytetrahydrofuran-
2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate, diastereomeric
mixture (20)
((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-bromo-3-
fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide (23b)
To a solution of compound 18 (22 mg, 0.077 mmol) in THF (2
mL) was added N-methylimidazole (31 µL, 0.39 mmol) then
(2S)-isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate
21 (0.071 g, 0.23 mmol) in THF (0.23 mL). The mixture was
stirred at rt for 1 h, quenched with water and extracted with ethyl
acetate (5 mL). The organic layer was washed with water twice,
dried over Na2SO4, filtrated and concentrated under reduced
pressure. The crude product was purified by flash
chromatography (CH2Cl2/MeOH 20:1) to afford compound 20 as
a diastereomeric mixture of RP:SP (~1:1) (18 mg, 46%). 1H NMR
(400 MHz, MeOD): δ 7.66 and 7.60 (each dd, J = 8.3 Hz, J = 2.4
Hz, 1H), 7.38-7.34 (m, 2H), 7.27-7.20 (m, 3H), 6.52-6.47 (m,
1H), 5.69 and 5.64 (each d, J = 8.3 Hz, 1H), 4.99 (m, 1H), 4.40-
4.30 (m, 1H), 4.29-4.20 (m, 1H), 4.15-4.05 (m, 1H), 3.95-3.85
(m, 1H) 1.35-1.31 (m, 3H), 1.25-1.21 (m 6H). 19F NMR (376
MHz, MEOD): -124.53 (m). 31P NMR (162 MHz, MeOD):
3.64, 3.57. 13C NMR (100 MHz, MeOD): δ 173.2, 164.1, 150.5,
140.1, 129.4, 124.9, 120.0, 101.7, 88.0, 80.4, 68.8, 64.8, 50.3,
20.5, 18.9.: MS (HR-ESI) for C21H27BrFN3O9P [(M+H)+], Calcd:
m/z 594.0652, Found: m/z 594.0652
A solution of compound 14 (1.5 g, 2,67 mmol), 4-N-
benzoylcytosine (634 mg, 2.94 mmol) and BSA (1.94 mL, 8.01
mmol) in acetonitrile (10 mL) was stirred at rt for 15 min before
addition of TMSOTf (1.45 mL, 8.01 mmol). The reaction vessel
was then placed into the cavity of microwave reactor and
irradiated for 12 min at 150 °C. The reaction was quenched by
addition of a 5% aqueous solution of NaHCO3 (20 mL) at 0 °C.
The aqueous layer was extracted with ethyl acetate (3 x 20 mL),
and the combined organic layers were washed with a saturated
solution of NaHCO3, water, and brine. The solution was dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel
(hexanes/ethyl acetate 2:1) to afford 23a (300 mg, 21%) and 23b
(200 mg, 14%). 23a: 1H NMR (400 MHz, CDCl3) δ 7.89-7.84
(m, 3H), 7.59 (t, J= 14.8 Hz, 2H), 7.50 (t, J = 15.2 Hz, 2H), 6.68
(d, J = 8.0 Hz, 1H), 4.60 (dd, J = 13.5, 7.2 Hz, 1H), 4.18 – 4.15
(m, 1H), 3.92 – 3.70 (m, 2H), 2.52 (BR S, 1H), 0.91 (s, 9H), 0.17
(s, 3H), 0.15 (s, 3H). 19F NMR (376 MHz, CDCl3) δ -113.25 (s).
13C NMR (100 MHz, CDCl3): δ 170.8, 162.9, 155.1, 144.9,
133.3, 129.1, 127.6, 117.1, 114.4, 96.2, 88.2 (d, J= 137.6 Hz),
83.9, 74.5 (d, J= 94.9 Hz), 60.3, 25.6, 18.0, -4.5, -4.9. . MS (HR-
ESI) for C22H29BrFN3O5Si [(M+H)+]. Calcd: m/z 542.1122.
Found: m/z 542.1117. 23b: 1H NMR (400 MHz, CDCl3) δ 8.28
(d, J = 7.5 Hz, 1H), 7.91 (d, J = 7.5 Hz, 2H), 7.63-7.58 (m, 2H),
7.52 – 7.48 (m, 2H), 6.71 (d, J = 5.6 Hz, 1H), 4.36 (dd, J = 17.1,
7.6 Hz, 1H), 4.14 – 4.08 (m, 1H), 3.95 (dt, J = 4.9, 2.2 Hz, 1H),
3.84 (dd, J = 12.4, 2.8 Hz, 1H), 0.94 (s, 9H), 0.17 (s, 3H), 0.15 (s,
3H). 19F NMR (CDCl3, 376 MHz) δ -121.28 (dd, J = 12.0, 7.8
Hz). 13C NMR (100 MHz, CDCl3): δ 170.9, 162.6, 155.2, 145.4,
133.5, 132.7, 129.0, 127.9, 111.5, 97.2, 81.6, 73.2, 59.4, 29.7,
25.6, 18.0, -4.5, -4.9. MS (HR-ESI) for C22H29BrFN3O5Si
[(M+H)+]. Calcd: m/z 542.1122. Found: m/z 542.1117.
4.2.15
chloro-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)benzamide (22a) and N-(1-
N-(1-((2S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-3-
((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-chloro-3-
fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide (22b)
A solution of compound 13 (1.0 g, 1.92 mmol), 4-N-
benzoylcytosine (660 mg, 3.06 mmol) and BSA (1.9 mL, 7.66
mmol) in acetonitrile (10 mL) was stirred at rt for 15 min before
addition of TMSOTf (3.11 mL, 17.23 mmol). The reaction vessel
was then placed into the cavity of a microwave reactor and
irradiated for 12 min at 150 °C. The reaction was quenched by
addition of 5% aqueous solution of NaHCO3 (20 mL) at 0 °C.
The aqueous layer was extracted with ethyl acetate (3 x 20 mL),
and the combined organic layers were washed with a saturated
solution of NaHCO3, water, and brine. The solution was dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel
(hexanes/ethyl acetate 2:1) to afford 22a (352 mg, 37%) and 22b
(120 mg, 13%). 22a: 1H NMR (400 MHz, CDCl3): δ 8.95 (br s,
1H), 7.91 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.61 (m,
2H), 7.51 (m, 2H), 6.77 (d, J = 8.1 Hz, 1H), 4.73 (dd, J = 12.3
Hz, J = 7.1 Hz, 1H), 4.20 (m, 1H), 3.92 (m, 1H), 3.75 (m, 1H),
2.76 (br s, 1H), 0.92 (s, 9H), 0.17 (s, 6H). 19F NMR (376 MHz,
CDCl3): -116.55 (s). 13C NMR (100 MHz, CDCl3): δ 166.6,
162.9, 155.2, 145.1, 133.5, 133.0, 129.2, 127.8, 117.7 (d, J = 258
4.2.17
N-(1-((2R,3R,4R,5R)-3-Chloro-3-fluoro-4-hydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide (24)
To a solution of compound 22b (70 mg, 0.14 mmol) in
anhydrous THF (2 mL) was added TBAF (180 µL, 0.18 mmol, 1
M in THF). The reaction mixture was stirred for 1 h at rt and then
quenched with a saturated solution of NH4Cl (4 mL). The
aqueous layer was extracted with ethyl acetate (3 x 4 mL) and the
combined organic layers were dried over anhydrous Na2SO4,
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel (CH2Cl2/MeOH 10:1)
1
to afford compound 24 (39 mg, 73%). H NMR (400 MHz,

MeOD): δ 8.51 (d, J = 7.6 Hz, 1H), 7.97 (m, 2H), 7.64 (m, 2H),
7.52 (m, 2H), 6.51 (d, J = 6.1 Hz, 1H), 4.44 (dd, J = 16.1 Hz, J =
7.3 Hz, 1H), 3.93-4.01 (m, 2H), 3.83 (m, 1H). 19F NMR (376
MHz, MeOD): -124.97 (dd, J = 16.6 Hz, J = 5.3 Hz). 13C NMR
(100 MHz, MeOD): δ 169.1, 165.4, 157.7, 146.4, 134.59,
134.17, 129.8, 129.2, 116.2 (d, J = 257 Hz), 98.5, 90.0, 83.4,
73.6, 60.3. MS (HR-ESI) for C16H16ClFN3O5 [(M+H)+], Calcd:
m/z 384.0763, Found: m/z 384.0753.
4.2.21
4-Amino-1-((2R,3R,4R,5R)-3-bromo-4-((tert-
butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-
3-fluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (28)
Imidazole (18 mg, 0.26 mmol) was added to a mixture of
compound 27 (26 mg, 0.086 mmol) and TBDMSCl (26 mg, 0.17
mmol) in DCM (4 mL). The mixture was stirred for 16h at rt,
quenched with water and the crude mixture was extracted with
ethyl acetate (5 mL). The organic layer was washed with a
saturated aqueous solution of NH4Cl, dried over Na2SO4, filtrated
and concentrated under reduced pressure. The crude product was
purified by flash column chromatography on silica gel using
4.2. 18 N-(1-((2R,3R,4R,5R)-3-bromo-3-fluoro-4-hydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide (25)
1
CH2Cl2/MeOH (10:1) to afford compound 28 (27 mg, 83%). H
To a solution of compound 23b (40 mg, 0.074 mmol) in
anhydrous THF (2 mL) was added TBAF (96 µL, 0.096 mmol, 1
M in THF). The reaction mixture was stirred for 1 h at rt and then
quenched with a saturated solution of NH4Cl (4 mL). The
aqueous layer was extracted with ethyl acetate (3 x 4 mL) and the
combined organic layers were dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel
NMR (CDCl3, 400 MHz) δ (ppm) 7.75 (dd, J = 7.5, 1.0 Hz, 1H),
6.69 (d, J = 5.7 Hz, 1H), 5.72 (d, J = 7.5 Hz, 1H), 4.23 (dd, J =
16.8, 7.5 Hz, 1H), 3.97 (dt, J = 11.6, 2.6 Hz, 1H), 3.83 (dd, J =
7.5, 2.3 Hz, 1H), 3.78 (dd, J = 11.7, 2.2 Hz, 1H), 0.93 (s, 9H),
0.92 (s, 9H), 0.16 (s, 3H), 0.11 (s, 9H). 19F NMR (CDCl3, 376
MHz) δ -121.70 (dd, J = 16.9, 4.6 Hz). 13C NMR (CDCl3, 101
MHz) δ 165.98 (s), 155.80 (s), 141.02 (s), 112.36 (s), 109.65 (s),
95.12 (s), 89.34 (d, J = 19.0 Hz), 81.23 (s), 73.50 (d, J = 25.3
Hz), 60.34 (s), 26.14 (s), 25.84 (s), 18.60 (s), 18.25 (s), -4.12 (s),
1
(CH2Cl2/MeOH 10:1) to afford compound 25 (28 mg, 90%). H
NMR (400 MHz, MeOD) δ (ppm) 8.56 (d, J = 7.6 Hz, 1H), 7.98
(d, J = 6.8 Hz, 2H), 7.67 – 7.53 (m, 4H), 6.68 (d, J = 5.6 Hz, 1H),
4.28 (dd, J = 16.8, 7.6 Hz, 1H), 4.01 – 3.93 (m, 2H), 3.83 (dd, J =
12,0, 2.4 Hz, 1H). 19F NMR (376 MHz, MeOD) δ -123.50 (d, J =
18.4 Hz). 13C NMR (100 MHz, MeOD): δ 169.1, 164., 157.7,
144.9, 133.2, 132.7, 128.4, 127.8, 112.3, 97.1, 89.5, 81.7, 72.5,
58.7, 58.1. MS (HR-ESI) for C16H15BrFN3O5 [(M+H)+]. Calcd:
m/z 428.0257. Found: m/z 428.0249.
-4.71 (s), -5.19 (s), -5.26 (s).
.
MS (HR-ESI) for
C21H39BrFN3O4Si2 [(M+H)+]. Calcd: m/z 552.1647. Found: m/z
552.1642.
4.2.22
Benzyl
(1-((2R,3R,4R,5R)-3-bromo-4-((tert-
butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-
3-fluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-
yl)carbamate (29)
4.2.19 4-Amino-1-((2R,3R,4R,5R)-3-chloro-3-fluoro-4-hydroxy-
5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (26)
To a solution of compound 28 (27 mg, 0.049 mmol) and
benzyl chloroformate (21 μL, 0.15 mmol) in DCM (5 mL) was
added DMAP (36 mg, 0.29 mmol). The reaction was stirred at rt
for 1.5 h, quenched with water (5 mL) and extracted with ethyl
acetate (5 mL). The organic layer was washed with HCl 1N,
NaHCO3, brine, dried over Na2SO4, filtrated and concentrated
under reduced pressure. The crude product was purified by flash
chromatography (hexane/ethyl acetate 1:1) to afford compound
29 (25 mg, 74%).1H NMR (CDCl3, 400 MHz) δ (ppm) 8.18 (d, J
= 7.6 Hz, 1H), 7.75 (s, 1H), 7.37 (brs, 5H), 7.22 (d, J = 7.6 Hz,
1H), 6.72 (d, J = 4.5 Hz, 1H), 5.22 (brs, 2H), 4.26 (dd, J = 16.6,
7.8 Hz, 1H), 4.02 (d, J = 11.8 Hz, 1H), 3.90 (d, J = 7.7 Hz, 1H),
3.81 (dd, J = 11.8, 1.9 Hz, 1H), 0.96 (s, 9H), 0.93 (s, 9H), 0.16 (s,
3H), 0.14 (s, 3H), 0.13 (s, 3H), 0.11 (s, 3H). 19F NMR (CDCl3,
376 MHz) δ -122.22 (d, J = 16.6 Hz). MS (HR-ESI) for
C29H46BrFN3O6Si2 [(M+H)+]. Calcd: m/z 686.2014. Found: m/z
686.2009.
A solution of compound 24 (39 mg, 0.1 mmol) in a saturated
solution of ammonia in MeOH (2 mL) was stirred at rt for 16 h
The solution was evaporated to dryness under reduced pressure
and co-evaporated several times with methanol. The residue was
purified by flash column chromatography on silica gel
1
(CH2Cl2/MeOH 4:1) to afford compound 26 (20 mg, 71%). H
NMR (400 MHz, MeOD): δ 7.95 (dd, J = 7.6 Hz, J = 1.9 Hz,
1H), 6.45 (d, J = 8.2 Hz, 1H), 5.93 (d, J = 7.3 Hz, 1H), 4.36 (dd,
J = 17.2 Hz, J = 7.2 Hz, 1H), 3.94 (m, 1H), 3.89 (m, 1H), 3.8 (m,
1H). 19F NMR (376 MHz, MeOD): -124.72 (dd, J = 16.7 Hz, J =
7.6 Hz). 13C NMR (100 MHz, MeOD): δ 167.6, 158.0, 143.7,
116.4 (d, J = 258 Hz), 96.2, 89.3, 83.1, 74.1, 60.7. MS (HR-ESI)
for C9H12ClFN3O4 [(M+H)+], Calcd: m/z 280.0500, Found: m/z
280.0492.
4.2.20 4-Amino-1-((2R,3R,4R,5R)-3-bromo-3-fluoro-4-hydroxy-
5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (27)
4.2.23 Benzyl (1-((2R,3R,4R,5R)-3-bromo-3-fluoro-4-hydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-
dihydropyrimidin-4-yl)carbamate (30)
A solution of compound 25 (32 mg, 0.073 mmol) in a
saturated solution of ammonia in MeOH (2 mL) was stirred at rt
for 16 h. The solution was evaporated to dryness under reduced
pressure and co-evaporated several times with methanol. The
residue was purified by flash column chromatography on silica
gel (CH2Cl2/MeOH 4:1) to afford compound 27 (23 mg, 96%).
1H NMR (400 MHz, MeOD) δ 7.96 (dd, J = 7.6, 1.8 Hz, 1H),
6.60 (d, J = 8.2 Hz, 1H), 5.90 (d, J = 7.6 Hz, 1H), 4.20 (dd, J =
17.6, 7.2 Hz, 1H), 3.96 – 3.89 (m, 1H), 3.89 – 3.84 (m, 1H), 3.78
(dd, J = 12.4, 3.3 Hz, 1H). 19F NMR (376 MHz, MeOD) δ -
123.93 (m). 13C NMR (100 MHz, MeOD): δ 165.6, 155.8, 144.7,
141.6, 132.2, 130.9, 128.5, 112.5, 109.8, 94.7, 93.3, 88.9, 88.8,
81.6, 729, 72.6, 59.1, 29.3 MS (HR-ESI) for C9H12BrFN3O4
[(M+H)+], Calcd: m/z 323.9995, Found: m/z 323.9993.
To a solution of compound 29 (25 mg, 0.036 mmol) in THF (4
mL) was added Et3N.3HF (0.06 mL, 0.36 mmol). The reaction
mixture was stirred for 36 h at rt then Et3N (0.06 mL) was added
to quench the reaction. After removal of the volatiles under
reduced pressure, the crude product was purified using silica gel
chromatography (CH2Cl2/MeOH 10:1) to afford compound 30
(11 mg, 67%).1H NMR (MeOD, 400 MHz) δ (ppm) 8.44 (dd, J =
7.7, 1.1 Hz, 1H), 7.50 – 7.26 (m, 6H), 6.64 (d, J = 6.2 Hz, 1H),
5.23 (s, 2H), 4.25 (dd, J = 17.2, 7.6 Hz, 1H), 3.98 – 3.91 (m, 2H),
3.81 (dd, J = 12.5, 2.9 Hz, 1H). 19F NMR (MeOD, 376 MHz) δ -
124.35 (dd, J = 17.0, 4.8 Hz). 13C NMR (100 MHz, MeOD): δ
163.9, 156.1, 153.1, 144.5, 135.7, 128.2, 127.9, 112.2, 109.5,
95.5, 89.6, 89.4, 81.7 (d, J= 23.56 Hz), 72.4, 72.2, 67.3, 58.8. MS
(HR-ESI) for C17H17BrFN3O6 [(M+H)+]. Calcd: m/z 458.0363.
Found: m/z 458.0355.

4.2.24
Isopropyl
((((2R,3R,4R,5R)-5-(4-
143.0, 113.8 (d, J = 257 Hz), 96.5, 89.1, 85.0, 81.3, 76.9, 60.8,
27.8. MS (HR-ESI) for C14H20ClFN3O6 [(M+H)+], Calcd: m/z
380.1025, Found: m/z 380.1017.
(((benzyloxy)carbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-4-
bromo-4-fluoro-3-hydroxytetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphoryl)-L-alaninate,
mixture (31)
diastereomeric
4.2.27 Isopropyl ((((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-
1(2H)-yl)-3-((tert-butoxycarbonyl)oxy)-4-chloro-4-
fluorotetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-
alaninate diastereomeric mixture (34)
To a solution of compound 30 (35 mg, 0.076 mmol) in THF
(3 mL) was added NMI (30 μL, 0.38 mmol) then (2S)-isopropyl
2-((chloro(phenoxy)phosphoryl)amino)propanoate 21 (70 mg,
0.23 mmol) in THF (0.23 mL). The mixture was stirred at rt for 4
h, quenched with water (1 mL) and extracted with ethyl acetate
(2 mL). The organic layer was washed with water twice, dried
over Na2SO4, filtrated and concentrated under reduced pressure.
The crude product was purified by flash chromatography
(CH2Cl2/MeOH 20:1) to afford compound 31 (11 mg, 20%) as a
diastereomeric mixture of RP/SP (~1:1).1H NMR (MeOD, 400
MHz) δ (ppm) 8.00 (ddd, J = 43.8, 7.7, 1.8 Hz, 1H), 7.47 – 7.15
(m, 11H), 6.65 (dd, J = 9.0 Hz, 1H), 5.24 (s, 2H), 5.04 – 4.94 (m,
1H), 4.59 – 4.35 (m, 2H), 4.26 – 4.19 (m, 1H), 4.16 – 4.12 (m,
1H), 3.97 – 3.86 (m, 1H), 1.38 – 1.31 (m, 3H), 1.22 (dd, J = 6.2,
2.1 Hz, 6H). 19F NMR (MeOD, 376 MHz) δ -124.22 – -124.67
(m). 31P NMR (MeOD, 162 MHz) δ 3.66 (d, J = 13.5 Hz). LR-
MS: calculated for C29H34BrFN4O10P 727.12, found 726.11,
728.20,.
To a solution of compound 33 (44 mg, 0.11 mmol) in THF (3
mL) was added tert-butylmagnesium chloride (180 µL, 0.18
mmol, 1 M in THF) at 0 °C. The reaction mixture was stirred at 0
°C for 20 min and
a solution of L-isopropylalaninyl
phosphorochloridate 21 (105 mg, 0.34 mmol) in THF (0.34 mL)
was added. The reaction mixture was stirred for 1 h at rt. The
solvent was evaporated and the residue was purified by flash
column chromatography on silica gel (CH2Cl2/MeOH 10:1) to
afford compound 34 as a diastereomeric mixture of RP/SP (~1:1)
1
(67 mg, 89%). H NMR (400 MHz, CDCl3): 7.28-7.35 (m, 3H),
7.13-7.23 (m, 3H), 6.51-6.57 (m, 1H), 5.76 and 5.68 (each d, J =
7.6 Hz, 1H), 5.17-5.22 (m, 1H), 4.97-5.00 (m, 1H), 4.29-4.45 (m,
2H), 4.21 (m, 1H), 3.91-3.99 (m, 2H), 1.51 (s, 9H), 1.36-1.41 (m,
3H), 1.16-1.23 (m, 6H). 19F NMR (376 MHz, CDCl3): -125.8 (s),
126.1 (s). 32P NMR (162 MHz, CDCl3): 2.51, 2.38. 13C NMR
(100 MHz, CDCl3): δ 173.0, 165.7, 155.5, 151.7, 150.7, 142.2,
129.93,129.88, 125.3, 120.3, 111.5 (d, J = 254 Hz), 95.5, 87.1,
84.7, 84.7, 69.5, 64.5, 50.5, 27.7, 21.8, 21.0. MS (HR-ESI) for
C26H36ClFN4O10P [(M+H)+], Calcd: m/z 649.1842, Found: m/z
649.1837.
4.2.25 Isopropyl ((((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-
1(2H)-yl)-4-bromo-4-fluoro-3-hydroxytetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphoryl)-L-alaninate,
diastereomeric
mixture (32)
To a round bottom flask charged with compound 31 (23 mg,
0.032 mmol) in ethanol (2 mL) was added 1,4-cyclohexadiene
(0.1 mL, 0.70 mmol) and Pd/C (10 mg, 0.01 mmol). After 2 h at
25 °C, the mixture was filtrated through a pad of celite and the
filtrate was then concentrated under reduced pressure.
Purification by flash column chromatography on silica gel
(CH2Cl2/MeOH 10:1) gave nucleotide 32 in 55% yield (13 mg)
as a diastereomeric mixture of Rp/Sp (~1:1).1H NMR (MeOD, 400
MHz) δ (ppm) 7.61 (ddd, J = 28.8, 7.6, 2.3 Hz, 1H), 7.40 – 7.34
(m, 2H), 7.27 – 7.20 (m, 3H), 6.61 (dd, J = 11.2, 8.4 Hz, 1H),
5.86 (dd, J = 12.0, 7.6 Hz, 1H), 5.04 – 4.95 (m, 1H), 4.54 – 4.31
(m, 2H), 4.19 (ddd, J = 17.5, 6.5, 2.0 Hz, 1H), 4.10 (brs, 1H),
3.93 – 3.88 (m, 1H), 1.37 – 1.31 (m, 3H), 1.29 (brs, 2H), 1.25 –
1.20 (m, 4H). 19F NMR (MeOD, 376 MHz) δ -124.46 (m). 31P
NMR (MeOD, 162 MHz) δ 3.60, 3.53. 13C NMR (100 MHz,
MeOD): δ 171.5, 165.5, 155.1, 150.2, 143.8, 130.1, 121.3,
120.3, 106.1, 98.1, 94.1, 78.4, 76.7, 69.5, 63.0, 46.9, 21.6, 19.1.
MS (HR-ESI) for C21H28BrFN4O8P [(M+H)+], Calcd: m/z
593.0812, Found: m/z 593.0813.
4.2.28 Isopropyl ((((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-
1(2H)-yl)-4-chloro-4-fluoro-3-hydroxytetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphoryl)-L-alaninate,
diastereomeric
mixture (35)
Compound 34 (67 mg, 0.1 mmol) was treated with a 4 M
solution of HCl in dioxane for 2 h at rt. Solvents were removed
under vacuum and the residue was purified by flash column
chromatography on silica gel (CH2Cl2/MeOH 10:1) to afford
compound 35 as a diastereomeric mixture of Rp/Sp (~1:1) (28 mg,
50%). 1H NMR (400 MHz, MeOD): δ 7.64 & 7.56 (each dd, J =
7.6 Hz, J = 2.3 Hz, 1H), 7.34-7.40 (m, 2H), 7.20-7.27 (m, 3H),
6.44-6.49 (m, 1H), 5.85 & 5.89 (each d, J = 7.6 Hz, 1H), 4.96-
5.00 (m, 1H), 4.35-4.43 (m, 2H), 4.30 (m, 1H), 4.10 (m, 1H),
3.91 (m, 1H), 1.34 (m, 3H), 1.22 (m, 6H). 19F NMR (376 MHz,
MeOD): -124.83 (s). 31P NMR (162 MHz, MeOD): 3.60, 3.54.
13C NMR (100 MHz, MeOD): δ 174.5, 167.6, 157.9, 152.1,
142.8, 130.8, 126.3, 121.4, 115.8 (d, J = 257 Hz), 96.5, 89.1,
81.4, 74.9, 70.2, 65.9, 51.8, 21.9, 20.5. MS (HR-ESI) for
C21H28ClFN4O8P [(M+H)+], Calcd: m/z 549.1317, Found: m/z
549.1313.
4.2.26 (2R,3R,4R,5R)-5-(4-Amino-2-oxopyrimidin-1(2H)-yl)-
4-chloro-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl
butyl carbonate (33)
tert-
4.2.29
6-bis(tert-Butoxycarbonyl)amino-9-(3,5-di-O-tert-
butyldimethylsilyl-2’-deoxy-2’-chloro-2’-fluoro-D-
To a solution of compound 26 (109 mg, 0.39 mmol) and di-
tert-butyl dicarbonate (85 mg, 0.39 mmol) in dioxane (8 mL) was
added a solution of Na2CO3 (207 mg, 1.9 mmol) in water (2 mL).
The reaction mixture was stirred at rt for 48 h and diluted with
water (4 mL). The product was extracted with ethyl acetate (5
mL), washed with water, brine, dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel
ribofuranosyl)purine (36)
To a solution of compound 11 (0.5 g, 1.2 mmol), 6-bis(tert-
butoxycarbonyl)amino-9H-purine (0.2 g, 1.29 mmol),
triphenylphosphine (0.47 g, 1.79 mmol) in THF (10 mL) was
added DIAD (0.36 mL, 1.79 mmol) dropwise at 0 °C. The
reaction mixture was heated at 70 °C for 1 h, and then diluted
with ethyl acetate. The organic layer was washed with water and
brine, dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure. The residue was purified by flash
column chromatography on silica gel (hexanes/ethyl acetate 9:1)
to afford 36a (236 mg, 27%) and 36b (59 mg, 7%). 36a: 1H
NMR (400 MHz, CDCl3): δ 8.77 (s, 1H), 8.45 (s, 1H), 6.67 (d, J
= 9.0 Hz, 1H), 4.77 (dd, J = 13.0 Hz, 4.8 Hz, 1H), 4.36 (m, 1H),
3.81 (m, 2H), 1.44 (s, 18H), 0.92 (two s, 18H), 0.10 (four s,
1
(CH2Cl2/MeOH 10:1) to afford compound 33 (44 mg, 30%). H
NMR (400 MHz, MeOD): δ 7.80 (dd, J = 7.6 Hz, J = 2.8 Hz,
1H), 6.45 (d, J = 6.4 Hz, 1H), 5.93 (d, J = 7.9 Hz, 1H), 5.31 (dd,
J = 16.9 Hz, J = 6.2 Hz, 1H), 4.10 (m, 1H), 3.92 (m, 1H), 3.78
(m, 1H), 1.50 (s, 9H). 19F NMR (376 MHz, MeOD): -124.07 (t, J
= 14.7 Hz). 13C NMR (100 MHz, MeOD): δ 167.6, 157.8, 153.2,

12H). 19F NMR (MHz, CDCl3): -116.34 (t, J = 11.2 Hz, 1F). 13
C
4.2.32 6-bis(tert-Butoxycarbonyl)amino-9-(2’-deoxy-2’-bromo-
NMR (100 MHz, CDCl3): δ 152.6, 151.8, 151.34, 144.1, 131.5,
116.9 (d, J = 251 Hz), 87.6, 86.5, 74.9, 61.5, 26.0, 25.7, 18.5,
18.2, -4.6, -4.8, -5.3, -5.4. MS (HR-ESI) for C32H56ClFN5O7Si2
2’-fluoro-β-D-ribofuranosyl)purine (39)
To a solution of compound 37 (190 mg, 0.14 mmol) in
anhydrous THF (10 mL) was added TBAF (610 µL, 0.61 mmol,
1 M in THF). The reaction mixture was stirred for 1 h at rt and
quenched with a saturated solution of NH4Cl (10 mL). The
aqueous layer was extracted with ethyl acetate (3 x 10 mL) and
the combined organic layers were dried over anhydrous Na2SO4,
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel (CH2Cl2/MeOH 10:1)
1
[(M+H)+], Calcd: m/z 732.3391, Found: m/z 732.3394. 36b: H
NMR (400 MHz, CDCl3): δ 8.89(s, 1H), 8.44 (s, 1H), 6.56 (d, J
= 10.6 Hz, 1H), 4.63 (dd, J = 15.8 Hz, 5.5 Hz, 1H), 4.03 (m, 1H),
3.97 (m, 1H), 3.87 (m, 1H), 1.44 (s, 18H), 0.95 (two s, 18H),
0.14 (four s, 12H). 19F NMR (MHz, CDCl3): -123.24 (t, J = 12.5
Hz, 1F). 13C NMR (100 MHz, CDCl3): δ 153.4,152.7, 150.7,
150.4, 143.0, 128.7, 114.1 (d, J = 262 Hz), 87.3, 83.9, 83,7, 74.1,
73.8, 61.1, 27.9, 26.1, 25.4, 18.6, 18.2, -3.4, -4.5, -4.8, -5.2. MS
1
to afford compound 39 (93 mg, 70%). H NMR (400 MHz,
CDCl3): δ 8.89 (s, 1H), 8.49 (d, J = 1.6 Hz, 1H), 6.69 (d, J = 9.5
Hz, 1H), 4.71 (dd, J = 15.7, 5.9 Hz, 1H), 4.19 (dt, J = 6.0, 3.0 Hz,
1H), 4.09 (d, J = 12.7 Hz, 1H), 3.98 (d, J = 12.4 Hz, 2H), 1.43 (s,
18H). 19F NMR (MHz, CDCl3): -121.02 (dd, J = 14.7 Hz, J = 8.8
Hz). 13C NMR (101 MHz, CDCl3) δ 153.1, 152.5, 150.7, 150.3,
143.4, 128.9, 111.8, 89.7, 89.5, 84.2, 83.1, 83.1, 73.5, 73.3, 60.6,
27.7. MS (HR-ESI) for C20H28BrFN5O7 [(M+H)+], Calcd: m/z
548.1156, Found: m/z 548.1132.
(HR-ESI) for C32H56ClFN5O7Si2 [(M+H)+], Calcd:
732.3391, Found: m/z 732.3390.
m/z
4.2.30
6-bis(tert-Butoxycarbonyl)amino-9-(3,5-di-O-tert-
butyldimethylsilyl-2’-deoxy-2’-bromo-2’-fluoro-D-
ribofuranosyl)purine (37a and 37b)
To a solution of compound 12 (1.0 g, 2.17 mmol), 6-bis(tert-
butoxycarbonyl)amino-9H-purine (0.80 g, 2.39 mmol) and
triphenylphosphine (0.85 g, 0.33 mmol) in THF (25 mL) was
added DIAD (0.64 mL, 0.33 mmol) dropwise at 0 °C. The
reaction mixture was heated at 70 °C for 1 h, and then diluted
with ethyl acetate, washed with water and brine, dried over
anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by flash column
chromatography on silica gel (hexanes/ethyl acetate 9:1) to afford
4.2.33
(2R,3R,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-chloro-4-
fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (40)
Compound 38 (0.06 g, 0.12 mmol) was dissolved in a 4 M
solution of HCl in dioxane (2 mL). The reaction mixture was
stirred for 1 h at rt. Volatiles were removed under vacuum and
the residue was purified by flash column chromatography on
silica gel (CH2Cl2/MeOH 4:1) to afford compound 40 (21 mg,
61%). 1H NMR (400 MHz, MeOD): δ 8.41 (s, 1H), 8.23 (s, 1H),
6.43 (d, J = 11.2 Hz, 1H), 4.57 (dd, J = 16.7 Hz, J = 6.1 Hz, 1H),
4.1 (m, 1H), 3.98 (m, 1H), 3.88 (m, 1H). 19F NMR (MHz,
MeOD): -123.99 (t, J = 14.2 Hz). 13C NMR (100 MHz, MeOD):
δ 157.0, 154.2, 150.3, 141.2, 119.32 115.2 (d, J = 258 Hz), 88.5,
84.3, 74.3, 61.2. MS (HR-ESI) for C10H12ClFN5O3 [(M+H)+],
Calcd: m/z 304.0613, Found: m/z 304.0605.
1
37a (756 mg, 44%) and 37b (190 mg, 12%). 37a: H NMR (400
MHz, CDCl3): δ 8.87 (s, 1H), 8.41 (s, 1H), 6.65 (d, J = 9.6 Hz,
1H), 4.70 (dd, J = 13.5, 5.2 Hz, 1H), 4.46 – 4.26 (m, 1H), 3.91 –
3.73 (m, 2H), 1.41 (s, 18H), 0.93 (two s, 18H), 0.31 – 0.02 (m,
12H). 19F NMR (MHz, CDCl3): -113.87 (t, J = 11.2 Hz, 1F). 13
C
NMR (101 MHz, CDCl3): δ 153.1, 152.5, 150.6, 150.2, 143.2,
128.8, 88.1, 87.7, 85.7, 83.8, 75.2, 75.0, 61.4, 31.6, 27.8, 25.9,
25.7, 22.7, 18.4, 18.1. -4.5, -4.8, -5.3, -5.4. MS (HR-ESI) for
C32H55BrFN5O7Si2 [(M+H)+], Calcd: m/z 776.2885, Found: m/z
776.2883. 37b: 1H NMR (400 MHz, CDCl3): δ 8.91 (s, 1H),
8.43 (s, 1H), 6.74 (d, J = 9.2 Hz, 1H), 4.55 (dd, J = 13.5, 5.2 Hz,
1H), 4.07 – 4.04 (m, 1H), 4.02 – 3.88 (m, 2H), 1.45 (s, 18H),
0.97 (s, 18H), 0.20 – 0.15 (m, 12H). 19F NMR (MHz, CDCl3): -
122.96 (m, 1F). 13C NMR (101 MHz, CDCl3) : δ 153.2, 152.5,
150.5, 150.2, 143.9, 142.8, 128.5, 111.5, 108.6, 88.3, 88.1, 83.8,
83,2, 74.1, 73.8, 60.9, 27.7, 26.0, 25.6, 18.5, 18.0, -4.4, -4.8, -5.3,
-5.4. MS (HR-ESI) for C32H55BrFN5O7Si2 [(M+H)+], Calcd: m/z
776.2885, Found: m/z 776.2883.
4.2.34
(2R,3R,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-bromo-4-
fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (41)
Compound 39 (0.014 g, 0.026 mmol) was dissolved in a 4 M
solution of HCl in dioxane (1 mL). The reaction mixture was
stirred for 1 h at rt. Volatiles were removed under vacuum and
the residue was purified by flash column chromatography on
silica gel (CH2Cl2/MeOH 4:1) to afford compound 41 (5.2 mg,
1
60%). H NMR (400 MHz, MeOD) δ 8.34 (s, 1H), 8.13 (s, 1H),
6.51 (d, J = 10.5 Hz, 1H), 4.40 (dd, J = 16.7, 6.1 Hz, 1H), 3.94
(m, 1H), 3.86-3.73 (m, 1H), 3.63-3.54 (d, J = 4.8 Hz, 1H). 19F
NMR (MHz, MeOD): -122.50 (dd, J = 12.12 Hz, J = 12.0 Hz ).
13C NMR (101 MHz, MeOD) δ 156.0, 152.8, 149.4, 139.7, 139.7,
118.3, 112.0, 88.3, 88.1, 83.1, 83.1, 73.4, 73.1, 59.9. MS (HR-
ESI) for C10H12BrFN5O3 [(M+H)+], Calcd: m/z 348.0107, Found:
m/z 348.0107.
4.2.31 6-bis(tert-Butoxycarbonyl)amino-9-(2’-deoxy-2’-chloro-
2’-fluoro-β-D-ribofuranosyl)purine (38)
To a solution of compound 36 (103 mg, 0.14 mmol) in
anhydrous THF (5 mL) was added TBAF (350 µL, 0.35 mmol, 1
M in THF). The reaction mixture was stirred for 1 h at rt and
quenched with a saturated solution of NH4Cl (5 mL). The
aqueous layer was extracted with ethyl acetate and the combined
organic layers were dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The residue was purified by flash column
4.2.35 Isopropyl ((((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-
chloro-4-fluoro-3-hydroxytetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphoryl)-L-alaninate,
diastereomeric
mixture (42)
chromatography on silica gel (CH2Cl2/MeOH 10:1) to afford
To a solution of compound 38 (50 mg, 0.099 mmol) in THF (2
mL) was added tert-butylmagnesium chloride (248 µL, 0.248
mmol, 1 M in THF) The reaction mixture was stirred for 20
1
compound 38 (70 mg, 99%). H NMR (400 MHz, CDCl3):
δ
8.87 (s, 1H), 8.62 (s, 1H), 6.54 (d, J = 8.7 Hz, 1H), 5.19 (br s,
1H), 4.73 (dd, J = 15.4 Hz, 6.0 Hz, 1H), 4.37 (br s, 1H), 4.13 (m,
1H), 4.03 (m, 1H), 3.93 (m, 1H), 1.38 (s, 18H). 19F NMR (MHz,
CDCl3): -123.52 (dd, J = 14.7 Hz, J = 8.8 Hz). 13C NMR (100
MHz, CDCl3): δ 153.2, 152.7, 150.4, 144.0, 128.8, 114.3 (d, J =
253.6 Hz), 88.3, 84.7, 83.4, 73.0, 72.7, 60.4, 27.8. MS (HR-ESI)
for C20H28ClFN5O7 [(M+H)+], Calcd: m/z 504.1661, Found: m/z
504.1658.
minutes at
0 °C before addition of L-isopropylalaninyl
phosphorochloridate 21 (90 mg, 0.29 mmol) in THF (0.29 mL).
The reaction mixture was stirred 1 h at rt. The volatiles were
evaporated and the residue was directly treated, without further
purification, with a 4 M solution of HCl in dioxane (2 mL). The
reaction mixture was stirred 1 h at rt. Solvents were removed
under vacuum and the residue was purified by flash column

chromatography on silica gel (CH2Cl2/MeOH 4:1) to afford
766.2998. 44b: 1H NMR (400 MHz, CDCl3): δ 8.52 (s, 1H),
6.49 (d, J = 9.0 Hz, 1H), 4.60 (dd, J = 13.0 Hz, 4.8 Hz, 1H), 4.00
(m, 2H), 3.88 (m, 1H), 1.41 (s, 18H), 0.99 (two s, 18H), 0.15 (s,
compound 42 as a diastereomeric mixture of Rp/Sp (~1:1) (16 mg,
1
29% over two steps). H NMR (400 MHz, CDCl3): δ 8.37 (s,
1H), 8.07 & 7.98 (each s, 1H), 7.28-7.34 (m, 2H), 7.1-7.23 (m,
3H), 6.47 (dd, J = 12.4 Hz, J = 2.8 Hz, 1H), 6.04 (d, J = 11.6 Hz,
2H), 5.59 (br s, 1H), 4.94-5.01 (m, 1H), 4.55-4.68 (m, 1H), 4.38-
4.48 (m, 2H), 4.09-4.28 (m, 2H), 3.92-4.04 (m, 1H), 1.33-1.38
12H). 19F NMR (MHz, CDCl3): -123.58 (t, J = 11.8 Hz, 1F). 13
C
NMR (100 MHz, CDCl3): δ 152.7, 151.5, 150.4, 144.3, 129.9,
113.9 (d, J = 258 Hz), 87.4, 83.8, 73.6, 61.0, 27.9, 26.1, 25.7,
18.8, 18.1, -4.5, -4.9, -5.28, -5.31. MS (HR-ESI) for
C32H55Cl2FN5O7Si2 [(M+H)+], Calcd: m/z 766.3001, Found: m/z
766.3001.
(two s, 3H), 1.16-1.22 (m, 6H). 19F NMR (MHz, CDCl3):
-
123.44 (t, J = 14.7 Hz), -124.17 (t, J = 14.7 Hz). 31P NMR (MHz,
CDCl3): 3.01, 2.88. 13C NMR (100 MHz, CDCl3): δ 173.2,
155.7, 153,7, 150.6, 150.3, 139.4, 129.9, 125.4, 120.3, 119.1,
113.5 (d, J = 256 Hz), 86.8, 81.1, 74.1, 69.7, 64.9, 60.6, 50.6,
21.7, 20.9. MS (HR-ESI) for C22H28ClFN6O7P [(M+H)+], Calcd:
m/z 573.1430, Found: m/z 573.1424.
4.2.38
2-bis(tert-Butoxycarbonyl)amino-6-chloro-9-(3,5-di-O-
tert-butyldimethylsilyl-2’-deoxy-2’-bromo-2’-fluoro-D-
ribofuranosyl)purine (45a and 45b)
To a solution of compound 12 (2.60 g, 5.66 mmol), 2-bis(tert-
butoxycarbonyl)amino-6-chloro-9H-purine (2.29 g, 6.23 mmol),
triphenylphosphine (2.19 g, 8.49 mmol) in THF (50 mL), DIAD
(1.67 mL, 8.49 mmol) was added dropwise at 0 °C. The reaction
mixture was heated at 70 °C for 1 h, and then diluted with ethyl
acetate (50 mL). The organic layer was washed with water, and
brine, dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure. The residue was purified by flash
column chromatography on silica gel (hexanes/ethyl acetate 9:1)
4.2.36 Isopropyl ((((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-
bromo-4-fluoro-3-hydroxytetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphoryl)-L-alaninate,
diastereomeric
mixture (43)
To a solution of compound 39 (70 mg, 0.128 mmol) in THF (2
mL) tert-butylmagnesium chloride (319 µL, 0.319 mmol, 1 M in
THF) was added and the reaction mixture was stirred for 20
minutes at 0 °C. A solution of the phosphorochloridate 21 (46
mg, 0.153 mmol) in THF (0.153 mL) was then added and the
reaction mixture was stirred for 1 h at rt. The volatiles were
removed under vacuum and the residue was directly treated,
without further purification, with a 4 M solution of HCl in
dioxane (2 mL). The reaction mixture was stirred 1 h at rt.
Solvents were removed under vacuum and the residue was
purified by flash column chromatography on silica gel
(CH2Cl2/MeOH 4:1) to afford compound 43 as a diastereomeric
1
to afford 45a (0.95 g, 20%) and 45b (0.26 g, 6%). 45a: H NMR
(400 MHz, CDCl3) δ 8.48 (s, 1H), 6.58 (d, J = 9.7 Hz, 1H), 4.69
(dd, J = 13.5, 5.1 Hz, 1H), 4.41 – 4.31 (m, 1H), 3.83 (m, 2H),
1.43 (s, 18H), 0.94 (d, J = 11.1 Hz, 18H), 0.21-0.10 (m, 12H). 19
F
NMR (MHz, CDCl3): -113.37 (t, J = 10.9 Hz, 1F). 13C NMR
(101 MHz, CDCl3) δ 150.25, 144.56, 129.80, 88.34, 87.97, 85.84,
83.66, 61.28, 60.40, 28.22, 27.83, 25.99, 25.86, 25.65, 25.60,
21.05, 18.34, 18.08, 14.20, -4.60, -4.87, -5.40, -5.44. [(M+H)+]
for C32H54BrClFN507Si2. Calcd: m/z 810.2496, Found: m/z
810.2495. 45b: 1H NMR (400 MHz, CDCl3): δ 8.55 (s, 1H),
6.65 (d, J = 8.4 Hz, 1H), 4.49 (dd, J = 15.3, 5.7 Hz, 1H), 4.06-
3.89 (m, 1H), 3.86 (m, 1H), 1.41 (s, 18H), 0.95 (d, J = 8.6 Hz,
18H), 0.15 (m, 12H). 19F NMR (MHz, CDCl3): -121.88 (t, J =
11.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 152.6, 150.2, 144.9,
88.4, 83.7, 83.3, 73.8, 73.5, 60.8, 30.9, 27.8, 26.0, 25.6, 21.0,
18.5, 18.0, 14.2, -4.4, -4.7, -5.2, -5.3. [(M+H)+] for
1
mixture of Rp/Sp (~1:1) (16 mg, 20% over two steps). H NMR
(400 MHz, CDCl3) δ 8.38 (d, J = 5.1 Hz, 1H), 8.05 (d, J = 2.8
Hz, 1H), 7.99 – 7.91 (m, 1H), 7.38 – 7.28 (m, 2H), 7.25 – 7.14
(m, 3H), 6.67 – 6.59 (m, 1H), 5.85 (s, 2H), 5.00 (q, J = 7.2, 6.7
Hz, 2H), 4.57 (d, J = 4.7 Hz, 1H), 4.48 – 4.39 (m, 3H), 4.25 (d, J
= 5.0 Hz, 1H), 4.09 – 3.93 (m, 3H), 3.79 – 3.71 (m, 1H), 1.37
(dd, J = 6.9, 2.6 Hz, 4H), 1.30 – 1.15 (m, 14H). 19F NMR (MHz,
CDCl3): -121.51 (t, J = 14.7 Hz), -122.53 (t, J = 14.7 Hz). 31P
NMR (MHz, CDCl3): 3.42, 3.07. 13C NMR (101 MHz, CDCl3) δ
173.0, 155.4, 153.5, 150.5, 150.2, 139.4, 129.8, 129.7, 125.4,
125.3, 120.1, 120.1, 87.6, 69.6, 50.5, 29.7, 21.7, 21.6, 20.9, 20.8.
MS (HR-ESI) for C22H28BrFN6O7P [(M+H)+], Calcd: m/z
617.0924, Found: m/z 617.0925.
C32H54BrClFN507Si2. Calcd:
810.2495.
m/z 810.2496, Found:
m/z
4.2.39 2-bis(tert-Butoxycarbonyl)amino-6-chloro-9-(2’-deoxy-2’-
chloro-2’-fluoro-β-D-ribofuranosyl)purine (46)
To a solution of compound 44 (0.3 g, 0.39 mmol) in
anhydrous THF (10 mL) was added TBAF (0.97 mL, 0.98 mmol,
1 M in THF). The reaction mixture was stirred for 1 h at rt and
quenched with a saturated solution of NH4Cl (10 mL). The
aqueous layer was extracted with ethyl acetate (10 mL) and the
combined organic layers were dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel
(CH2Cl2/MeOH 10:1) to afford compound 46 (188 mg, 89%). 1H
NMR (400 MHz, CDCl3): δ 8.74 (s, 1H), 6.49 (d, J = 7.2 Hz,
1H), 4.80 (dd, J = 14.8 Hz, J = 6.03 Hz, 1H), 4.68 (br s, 1H),
4.14 (m, 1H), 4.07 (m, 1H), 3.97 (m, 1H), 3.86 (br s, 1H), 1.39 (s,
18H). 19F NMR (MHz, CDCl3): -123.64 (dd, J = 13.88 Hz, J =
6.4 Hz). 13C NMR (100 MHz, CDCl3): δ 152.5, 152.3, 151.7,
150.5, 145.1, 130.0, 114.2 (d, J = 260 Hz), 88.5, 84.4, 83.2, 72.5,
60.4, 27.9. MS (HR-ESI) for C20H26Cl2FN5NaO7 [(M+Na)+],
Calcd: m/z 560.1091, Found: m/z 560.1084.
4.2.37
2-bis(tert-Butoxycarbonyl)amino-6-chloro-9-(3,5-di-O-
tert-butyldimethylsilyl-2’-deoxy-2’-chloro-2’-fluoro-D-
ribofuranosyl)purine (44a and 44b)
To a solution of compound 11 (3.0 g, 7.22 mmol), 2-bis(tert-
butoxycarbonyl)amino-6-chloro-9H-purine (2.94 g, 7.95 mmol)
and triphenylphosphine (2.84 g, 10.83 mmol) in THF (50 mL)
was added DIAD (2.13 mL, 10.83 mmol) dropwise at 0 °C. The
reaction mixture was heated at 70 °C for 1 h, and then diluted
with ethyl acetate (50 mL). The organic layer was washed with
water, and brine, dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (hexanes/ethyl acetate
9:1) to afford 44a (3.0 g, 54%) and 44b (0.67 g, 12%). 44a: 1H
NMR (400 MHz, CDCl3): δ 8.45 (s, 1H), 6.60 (d, J = 9.0 Hz,
1H), 4.74(dd, J = 12.9 Hz, J = 4.4 Hz, 1H), 4.37 (m, 2H), 3.82
(m, 1H), 1.41 (s, 18H), 0.93 (two s, 18H), 0.15 (four s, 12H). 19
F
4.2.40 2-bis(tert-Butoxycarbonyl)amino-6-chloro-9-(2’-deoxy-2’-
NMR (MHz, CDCl3): -115.58 (t, J = 10.9 Hz, 1F). 13C NMR
(100 MHz, CDCl3): δ 152.6, 151.4, 150.4, 144.8, 129.8, 117.1
(d, J = 251 Hz), 87.8, 85.6, 83.8, 75.0, 61.5, 28.0, 26.0, 25.7,
18.5, 18.2, -4.5, -4.9, -5.28, -5.31. MS (HR-ESI) for
C32H55Cl2FN5O7Si2 [(M+H)+], Calcd: m/z 766.3001, Found: m/z
bromo-2’-fluoro-β-D-ribofuranosyl)purine (47)
To a solution of compound 45 (0.24 g, 0.296 mmol) in
anhydrous THF (10 mL) was added TBAF (0.74 mL, 0.741
mmol, 1 M in THF). The reaction mixture was stirred for 1 h at

rt and quenched with a saturated solution of NH4Cl (10 mL). The
aqueous layer was extracted with ethyl acetate (3x10 mL) and the
combined organic layers were dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel
(CH2Cl2/MeOH 10:1) to afford compound 47 (100 mg, 60%). 1H
NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 6.66 (d, J = 7.5 Hz, 1H),
4.70 (dd, J = 15.7, 6.6 Hz, 1H), 4.54 (br s, 1H), 4.14 (m, 1H),
4.09 (m, 1H), 3.99 (m, 1H), 1.40 (s, 18H). 19F NMR (MHz,
CDCl3): -121.55 (dd, J = 13.88 Hz, J = 6.4 Hz). 13C NMR (101
MHz, CDCl3) δ 152.4, 152.1, 151.6, 150.3, 144.9, 129.9, 111.5,
108.8, 89.6, 89.4, 84.3, 82.8, 82.8, 72.8, 72.5, 60.2, 53.0, 27.8.
MS (HR-ESI) for C20H26BrClFN5O7 [(M+H)+], Calcd: m/z
582.0766. Found: m/z 582.0761.
4.93 (m, 2H), 4.60-4.50 (m, 1H), 4.48-4.39 (m, 2H), 4.20-4.13
(m, 1H), 3.93-3.86 (m, 1H), 1.32 (m, 3H), 1.22- 1.18 (m, 6H). 19
NMR (MHz, CDCl3): -124.55 (t, J = 12.9 Hz, 1F). 31P NMR
(MHz, CDCl3): 3.66, 3.59. 13C NMR (100 MHz, CDCl3):
174.5, 159.31, 155.8, 153.4, 152.1, 137.8, 130.8, 126.2, 121.4,
117.1, 115.3 (d, J = 255 Hz), 88.0, 82.4, 74.9, 70.2, 66.3, 51.7,
21.9, 20.4. MS (HR-ESI) for C22H28ClFN6O8P [(M+H)+], Calcd:
m/z 589.1379, Found: m/z 589.1372.
F
δ
4.2.44
Isopropyl
((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-
dihydro-9H-purin-9-yl)-4-bromo-4-fluoro-3-
hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-
alaninate, diastereomeric mixture (51)
To a solution of compound 47 (50 mg, 0.086 mmol) in THF (2
mL) tert-butylmagnesium chloride (215 µL, 0.215 mmol, 1 M in
THF) was added and the reaction mixture was stirred for 20
minutes at 0 °C. Then (S)-2-[(S)-(2,3,4,5,6-pentafluorophenoxy)-
phenoxyphosphorylamino] propionic acid isopropyl ester, 52 (58
mg, 0.120 mmol) 1M in THF (0.120 mL) was added and the
reaction mixture was stirred 2 h at rt. The solvent was evaporated
and the residue was directly treated, without further purification,
with a mixture of formic acid/H2O (4:1) (2 mL). The reaction
mixture was stirred 4 h at 60 °C. Solvents were removed under
vacuum and the residue was purified by flash column
chromatography on silica gel (CH2Cl2/MeOH 4:1) to afford
4.2.41 2-Amino-9-((2R,3R,4R,5R)-3-chloro-3-fluoro-4-hydroxy-
5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-
one (48)
Compound 46 (40 mg, 0.074 mmol) was dissolved in a
mixture of formic acid/H2O (4:1) and stirred at 60 °C for 16 h.
Volatiles were removed under reduced pressure and the residue
was purified by flash column chromatography on silica gel
1
(CH2Cl2/MeOH 8:2) to afford compound 48 (12.8 mg, 54%). H
NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 6.27 (d, J = 10.8 Hz,
1H), 4.53 (dd, J = 16.5 Hz, J = 6.04 Hz, 1H), 3.96 (m, 1H), 3.91
(m, 1H), 3.81 (m, 1H). 19F NMR (MHz, CDCl3): -124.85 (t, J =
12.9 Hz, 1F). 13C NMR (100 MHz, CDCl3): δ 159.3, 155.8,
153.4, 137.8, 117.04, 115.82 (d, J = 258 Hz), 88.1, 84.3, 74.3,
61.3. MS (HR-ESI) for C10H12ClFN5O4 [(M+H)+], Calcd: m/z
320.0562, Found: m/z 320.0554.
1
compound 51 as a Sp isomer (11.9 mg, 22%). H NMR (400
MHz, MeOD δ 7.72 (dd, J = 12.3, 2.9 Hz, 1H), 7.35-7.27 (m,
2H), 7.19 – 7.03 (m, 3H), 6.32 (m 1H), 4.73 – 4.25 (m, 4H), 4.08
– 4.03 (m, 1H), 3.82 – 3.77 (m, 1H), 3.70 – 3.50 (m, 1H), 1.35 –
1.03 (m, 10H). 19F NMR (MHz, MeOD): -122.80 (d, J = 14.6
Hz, 1F). 31P NMR (MHz, MeOD): 3.84. 13C NMR (100 MHz,
MeOD): δ 173.2, 157.8, 154.3, 152.0, 150.8, 136.4, 129.4,
124.8, 120.0, 115.7, 111.3, 87.6, 80.9, 74.0, 68.7, 65.4, 510.3,
20.5, 19.0. MS (HR-ESI) for C22H28BrFN6O8P [(M+H)+], Calcd:
m/z 633.0873, Found: m/z 633.0874.
4.2.42 2-amino-9-((2R,3R,4R,5R)-3-bromo-3-fluoro-4-hydroxy-
5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-
one (49)
Compound 47 (50 mg, 0.086 mmol) was dissolved in a
mixture of formic acid/H2O (4:1) (2 mL) and stirred at 60 °C for
16 h. Volatiles were removed under reduced pressure and the
residue was purified by flash column chromatography on silica
gel (CH2Cl2/MeOH 8:2) to afford compound 49 (15.6 mg, 50%).
1H NMR (400 MHz, DMSO-d6) δ 10.73 (br s, 1H), 7.94 (s, 1H),
6.72 (d, J = 5.6 Hz, 1H), 6.62 (br s, 1H), 6.27 (d, J = 9.2 Hz, 1H),
5.26 (t, J = 5.5 Hz, 1H), 4.34 (dd, J = 16.2, 5.6 Hz, 1H), 3.83 (m,
1H), 3.73 – 3.58 (m, 2H). 19F NMR (MHz, DMSO-d6): -121.02
(t, J = 12.9 Hz, 1F). 13C NMR (101 MHz, DMSO-d6) δ 157.0,
154.6, 151.8, 135.6, 116.4, 87.2, 87.0, 59.9. MS (HR-ESI) for
C10H12BrFN5O4 [(M+H)+], Calcd: m/z 364.0056, Found: m/z
364.0054.
Acknowledgements
This work was supported in part by NIH grant 5P30-AI-50409
(CFAR). Dr. Schinazi is the Chairman and a major shareholder of
Cocrystal Pharma, Inc. Emory received no funding from
Cocrystal Pharma, Inc. to perform this work and vice versa.
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