NMR-based conformational analysis of 2′,6-disubstituted uridines and antiviral evaluation of new phosphoramidate prodrugs

Article abstract of DOI:10.1016/j.bmc.2015.07.003

Six novel phosphoramidate prodrugs of uridine analogues, with structural modifications introduced at the 6- and 2′,6-positions, have been prepared and evaluated for selective antiviral activity against hepatitis C virus, as well as other positive-stranded RNA viruses. An analysis of the conformational properties of the parent nucleosides was carried out using two-dimensional NMR spectroscopy based experiments, highlighting a 3′-endo (North) sugar puckering preference and syn orientation.

Full text of DOI:10.1016/j.bmc.2015.07.003

Accepted Manuscript
NMR-based conformational analysis of 2’,6-disubstituted uridines and antiviral
evaluation of new phosphoramidate prodrugs
Fábio da Paixão Soares, Elisabetta Groaz, Eveline Lescrinier, Johan Neyts,
Pieter Leyssen, Piet Herdewijn
PII:
S0968-0896(15)00570-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.003
BMC 12432
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 April 2015
1 July 2015
3 July 2015
Please cite this article as: da Paixão Soares, F., Groaz, E., Lescrinier, E., Neyts, J., Leyssen, P., Herdewijn, P., NMR-
based conformational analysis of 2’,6-disubstituted uridines and antiviral evaluation of new phosphoramidate
prodrugs, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.003
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Graphical Abstract
Leave this area blank for abstract info.
NMR-based conformational analysis of 2’,6-di-
substituted uridines and antiviral evaluation of new
phosphoramidate prodrugs.
Fábio da Paixão Soares,a Elisabetta Groaz,a Eveline Lescrinier,a Johan Neyts,b Pieter Leyssenb and Piet
Herdewijn.a*
a Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
b Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
* Tel. +32 16 337387; Email: Piet.Herdewijn@rega.kuleuven.be
O
O
HN
HN
O
O
O
N
R2
N
R2
H
N
O
P
O
O
O
HO
O
O
R1
OH
R1
OH
HO
HO
R1 = H, CH3, R2 = CH3, I, N3, CN

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
NMR-based conformational analysis of 2’,6-disubstituted uridines and antiviral
evaluation of new phosphoramidate prodrugs.
Fábio da Paixão Soares,a Elisabetta Groaz,a Eveline Lescrinier,a Johan Neyts,b Pieter Leyssenb and Piet
Herdewijn.a*
a Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
b Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
* Tel. +32 16 337387; Email: Piet.Herdewijn@rega.kuleuven.be
ARTICLE INFO
ABSTRACT
Article history:
Received
Six novel phosphoramidate prodrugs of uridine analogues, with structural modifications
introduced at the 6- and 2’,6-positions, have been prepared and evaluated for selective antiviral
activity against hepatitis C virus, as well as other positive-stranded RNA viruses. An analysis of
the conformational properties of the parent nucleosides was carried out using two-dimensional
Received in revised form
Accepted
Available online
NMR spectroscopy based experiments, highlighting a 3′-endo (North) sugar puckering
preference and syn orientation.
Keywords:
Nucleosides
Prodrugs
2009 Elsevier Ltd. All rights reserved.
HCV
Conformation
Phosphoramidates
1. Introduction
A well-known concept in the development of new biologically
active nucleoside derivatives is the importance of their
conformational preference in view of their possible interaction
with target enzymes. The introduction of a substituent at the 6-
position of a pyrimidine base is described in the literature as
giving rise to syn-conformationally constrained ribonucleosides,
some of which have shown interesting anti-plasmodial11-13 and
anti-cancer14 activities. Their scope as antivirals has largely
remained uninvestigated and it is deemed important to provide
information on the tolerability of the syn-type conformation of a
pyrimidine base at the level of viral kinases and polymerases.
Hepatitis C virus (HCV) is a single-stranded, positive-sense
RNA virus of which the replication is exclusively located in
membrane-associated replication complexes in the cytoplasm of
infected cells.1, In contrast to HIV and HBV, for which the
2
chronic infection is caused respectively by integration of the viral
genome into the host cell DNA3 or the establishment of the viral
extrachromosomal cccDNA,4 antiviral treatment of a chronic
HCV infection can successfully result in a sustained virological
response (SVR). In the past, the immunotherapeutic regimen with
IFN/RBV allowed to achieve an SVR of 50% (genotype 1) to 70-
90% (genotypes 2 and 3). Recently, SVR’s have significantly
improved with the clinical approval of combination regimens
with selective antiviral inhibitors (Harvoni®, Gilead; Viekira
Pak®, AbbVie).
The present study was designed to evaluate the antiviral
activity of a series of 6-substituted uridine analogues, with a
particular interest in combining such modification with the
familiar 2’-C-methylribose sugar pharmacophore, and thus to
investigate whether compounds exhibiting both structural motifs
would maintain the preferred conformation and biological profile
of their parent ribonucleosides.
The introduction of structural variation at multiple positions of
both the base and sugar moieties of natural ribonucleos(t)ides has
yielded numerous analogues with the potential to interfere with
viral replication, i.e. by competitive binding to the RNA-
dependent RNA polymerase (RdRp) catalytic site and/or by
termination of the growing RNA chain.5-7 The insight into the
SAR of these compounds has significantly increased, for
example allowing to conclude that the presence of a methyl
group at the 2’-β-position on the ribose ring is an important
structural element necessary to achieve good inhibitory activity
on the HCV RNA polymerase activity without interfering with
the function of host cell RNA-dependent polymerases (and thus
less cytotoxicity).8-10
Furthermore, a 5’-monophosphate prodrug strategy was
pursued as many synthetic nucleotides that prove to be active in
ex cellulo enzymatic assays, often fail to inhibit viral RNA
replication in cell culture due to poor phosphorylation in the first
rate-limiting step required for intracellular triphosphate activation
by kinases.15 In this context, the introduction of aryl
phosphoramidates to enhance the pharmacokinetic properties of
nucleotides has shown fruitful in anti-HCV drug discovery,16
eventually leading to the FDA approval of Sufosbuvir for the
treatment of chronic hepatitis C.

2. 2. Results and discussion
Steric factors linked to the presence of the 2’-methyl group on the
sugar ring might be accountable for the absence of the formation
of mixed aggregate derived stabilizing the intermediate species.18
2.1. Chemical synthesis
Standard preparative routes were followed for the synthesis of
variety of 6-substituted and 2’,6-disubstituted uridine
analogues, which were then used as platforms for the generation
of an entire novel set of phosphoramidate prodrugs.
a
O
N
O
N
O
N
HN
HN
HN
a,b
O
c
O
I
O
O
O
(81%)
(55%)
O
TBDMSO
TBDMSO
Methyl-substituted uridine analogues 3 and 4 were readily
obtained in two steps from commercially available 6-
methyluracil and peracetylated ribofuranose or 1,2,3,5-tetra-O-
HO
O
O
O
d
O
HO
OH
5
6
benzoyl-2’methyl-β-D-ribofuranose,
respectively,
under
(90%)
e
optimized Vorbruggen condensation reaction conditions (Scheme
1), followed by total deprotection of intermediates 1 and 2. In
both cases, the outcome of the glycosylation step showed to be
highly dependent on the reaction conditions, since the undesired
N1,N3-bis-riboside was preferentially formed at room
temperature. At lower temperatures, however, the exclusive
formation of a N1-glycosidic bond rather that a N3-bond was
confirmed by NOESY experiments, as selective irradiation of the
6-methyl group of compounds 3 and 4 showed a strong
interaction with H-1’. These results were found to be in
agreement with 1H correlations to C2 and C6 observed in HMBC
spectra.
O
N
O
N
O
HN
NH
HN
O
R
f
R
O
O
N
I
O
O
O
TBDMSO
HO
HO
O
O
HO
HO
OH
OH
R = CN 10 (48%)
R = N3 11 (70%)
R = CN
R = N3
8
7
9
Scheme 2. (a) Acetone, H2SO4, 3 h; (b) TBDMSCl, Im, dry
DMF, overnight; (c) LDA, I2, AcOH, dry THF, 5 h; (c) 50%
aq. TFA; (e) (Et)4NCN, dry DMF, overnight or NaN3, dry
O
O
DMF, overnight; (f) 50% aq. TFA.
O
HN
HN
a
b
HN
O
O
N
O
N
CH3
O
N
CH3
O
O
O
HN
HN
O
N
H
CH3
R1O
HO
R
R2
OR1
a
b
R2
OH
HN
O
O
N
(75%)
(88%)
O
O
R1O
HO
O
N
H
BzO
HO
CH3
OBz
CH3
OH
R1 = OAc, R2 = H
1 (81%)
R2 = H
3 (75%)
BzO
HO
R1 = OBz, R2 = CH3 2 (89%)
R2 = CH3 4 (84%)
R = H 12
R = I 13
14
15
Scheme 1. (a) BSA, sugar, TMSOTf, -20 oC to 10-15 oC, 6-48
h; (b) 7N NH3 in MeOH, 5-48 h.
e,f (68%)
c,d (70%)
O
O
O
N
HN
I
HN
HN
Initial attempts to form analogues starting directly from
nucleobases substituted with a 6-functional group other than
methyl by a similar condensation approach were unsuccessful,
leading to the preferential formation of a glycosidic bond at the
N3-position (HMBC). Thus, a literature route was pursued to
access 6-modified ribouridines, which relies upon the initial
introduction of an iodine atom at the 6-position of the base,
followed by an addition-elimination step. As shown in Scheme 2,
the hydroxyl groups at the 2’- and 3’-positions of uridine were
simultaneously protected with acetone in the presence of a
catalytic amount of sulfuric acid, followed by conventional
silylation at the 5’-position. The resulting protected compound 5
was then treated with LDA and iodine at low temperature under
dry conditions to give compound 6 in 55% yield.11 Removal of
the TBDMS and isopropylidene groups in the presence of TFA
proceeded in excellent yield to afford 6-iodouridine 7. In
addition, treatment of compound 6 with (Et)4NCN or NaN3 in dry
DMF,17 afforded the corresponding 6-cyanouridine 10 and 6-
azidouridine 11 in moderate yields, after a deprotection step.
O
N
O
g
O
N
O
CN
O
O
O
(89%)
TBDMSO
HO
HO
CH3
CH3
OH
CH3
OH
O
HO
HO
17
18
16
Scheme 3. (a) BSA, sugar, TMSOTf, -20 oC to 70 oC, 3 h; (b)
7N NH3 in MeOH, 5-48 h; (c) Acetone, H2SO4, 3 h; (d)
TBDMSCl, Im, dry DMF, overnight; (e) BSA, sugar, SnCl4,
CH2Cl2, rt; (f) 7N NH3 in MeOH; (g) (i) TBDMSCl, Im, dry
DMF, overnight, (ii) (Et)4 NCN, dry DMF, overnight, (iii)
TBAF in THF.
An alternative route, however, provided an additional 2’,6-
modified substrate, compound 18. The formation of 2’-methyl-5-
iodouridine 17 was readily realized as reported in the literature19,
followed by an addition-elimination reaction which converted
compound 17 to 2’-methyl-6-cyanouridine 18 in 89% yield after
a final deprotection step using TBAF.20 The presence of the new
chemical bond C6-C7 (CSp2-CSp) was confirmed by HMBC
correlation.
In a similar fashion, we aimed to generate another group of
compounds that incorporate in the sugar moiety the key feature
necessary for HCV inhibitory activity. Glycosylation of 2’-
methyl substituted ribose with uracil proceeded selectively at the
N1-position to give 14, which, after debenzoylation, underwent
the same protecting steps previously described for uridine.
However, it was found that, when compound 16 was subjected to
the iodination reaction, no product was formed. This result was
attributed to the poor stabilization of the anionic intermediate
forming at the 6-position under the applied reaction conditions.
The substitution of the phosphate function with an amino acid
ester residue and a lipophilic aromatic group to mask the negative
charge allows hepatic delivery of higher concentrations of
monophosphate nucleosides, which are then liberated following
an intracellular cascade of metabolic steps and further converted
into their active triphosphate forms.16 The conversion of all of the
prepared nucleosides to their corresponding 5’-phosphoramidates
was achieved
by
a
common
methodology.
The

Figure 1. NOESY spectra for 6-dimethyluridine 3 and 2’,6-dimethyluridine 4, run in DMSO-d6 on Bruker Avance II 500 MHz spectrometer with a TXI HCP
probe.
phosphoramidating reagent was readily prepared by reacting
alanine isopropyl ester hydrochloride and phenyl
nucleosides in the pseudorotation cycle is commonly confined to
either of two antipodal regions, C3′-endo or “North” and C2′-
endo or “South”.22 An estimated ratio of North and South
conformers in solution can be determined on the basis of the
values of vicinal homonuclear coupling constants,23, 24 and in our
specific case exclusively from the coupling between H3’ and H4’
protons.
dichlorophosphate in the presence of triethylamine at low
temperature. Addition of the so obtained reagent mixture to each
of the modified nucleosides provided the relative
phosphoramidates as diastereomeric mixtures, with moderate to
good yields.
3
For compound 4, a JH3’-H4’ = 9.06 Hz was measured in D2O,
O
N
O
N
which is indicative for
a predominant North-type sugar
conformation,25 further corroborated by the presence of a
significant NOE enhancement between 2'-OH and H4' and the
absence of any interaction between 2'-Me and H5' protons
(Figure 1). For comparison, the conformational analysis was also
applied to 6-methyluridine 3 and results were consistent with
literature report.14
HN
HN
O
O
R2
O
R2
H
N
O
P
a
O
O
O
O
HO
O
R1
OH
R1
OH
HO
HO
R1
=
H, R2
=
CH3
3
4
7
R1 = H, R2 = CH3
19 (60%)
R1 = CH3, R2 = CH3
R1 = CH3, R2 = CH3 20 (55%)
R1
R1
R1
=
H, R2
H, R2
H, R2
=
I
R1 = H, R2 = I
21 (39%)
22 (42%)
23 (22%)
3
The JH3’-H4’ values measured at five different temperatures
=
=
CN 10
R1 = H, R2 = CN
R1 = H, R2 = N3
=
= N3 11
indicates that compound 4 does not undergo a significant
temperature dependent conformational change, as shown in Table
1. The percentage of preferred C3′-endo ring conformer was
obtained by performing a pseudorotational analysis with the aid
of a Matlab interface.26
R1 = CH3, R2 = CN 18
R1 = CH3, R2 = CN 24 (60%)
Scheme 4. (a) (i) PhOP(O)Cl2, L-Alanine isopropyl ester
hydrochloride, dry CH3CN, -40 oC, TEA, (ii) Nucleoside, N-
methylimidazole, 5 h.
2.2. Conformational analysis
Table 1. Pseudorotational analysis data for compound 4.
Subsequently,
the
predominant
conformation
of
T oC
peak 1
peak 1
% N-
conformer
92.24
3JH3’-H4’
unprecedented 2’,6-dimethylsubstituted uridine analogue 4 in
solution was determined by NMR spectroscopy techniques and
compared with that of 6-dimethyluridine 3. An anti-relationship
across the glycosidic bond is common in most pyrimidine
nucleosides as a result of repulsive interactions between the sugar
ring and the carbonyl group at the 2-position. Nevertheless, the
insertion of a bulky substituent at the 6-position sterically
restrains the molecule into a syn-conformation, as described for
6-dimethyluridine.21 We were interested in studying the effect
played by the additional presence of a methyl group at the 2’-
beta-position on both the glycosidic bond orientation and sugar
puckering.
5
2439.1682
2474.9359
2510.2236
2544.1909
2577.1981
2430.0462
2465.814
2501.1616
2535.129
2568.1361
9.122
9.1219
9.062
10
15
20
25
92.24
91.36
9.0619
9.062
91.36
91.36
The determination of the prevalent orientation state, derived
from the relative positions of sugar and base around the
glycosidic bond, was based upon the values of 3JH1’C6 and 3JH1’C2
the combination of which represents a direct correlation to the
,
syn- or anti-range for the glycosidic torsion angle χ.27 Thus, in a
Although an infinite number of conformations is theoretically
possible, the pucker position of five-membered rings of
1
1D 13C NMR spectrum where no H decoupling was applied
during signal detection, compound 4 exhibited JH1’C6 = 4.78 Hz
3

and 3JH1’C2 = 6.49 Hz at 600 MHz in D2O. Since 3JH1’C2 > 3JH1’C6
it can be concluded that the glycosidic torsion angle χ is in the
syn domain.
,
(FWHM) using leucine enkephalin as lock mass. Thin-layer
chromatography (TLC) was performed on Alugram silica gel
UV254 mesh 60, 0.20 mm (Macherey-Nagel).
5.1.1. 1-(2’,3’,5’-Tri-O-acetyl-β-D-ribofuranosyl)
-6-methyluracil (1).
The observed syn-relationship of the nucleobase relative to the
sugar ring was supported by analysis of NOE interactions.
Selective irradiation of the group at the 6-position showed the
presence of strong cross peaks between R-6 and H-1’ in all
compounds, whilst any correlation of R-6 to H5’ and H5” was
absent.
To a solution of 6-methyluracil (604.0 mg, 4.80 mmol) in dry
CH3CN (10 mL), was added N,O-bistrimethylsilylacetamide (2.4
mL, 14.0 mmol) and the mixture was stirred for 20 min at room
temperature. To this mixture, a solution of 1,2,3,5-tetra-O-acetyl-
β-D-ribofuranose (1.27 g, 4.00 mmol) in dry CH3CN (15 mL)
was then added. After cooling to -20 °C, TMSOTf (2.4 mL, 4.4
mmol) was added and the reaction mixture was allowed to warm
slowly to 15-20 °C and stirred for 6 h. The mixture was then
poured into CH2Cl2 and washed with saturated aq. NaHCO3. The
water layer was extracted with CH2Cl2. The organic extracts were
combined and dried over Na2SO4, filtered and evaporated in
vacuo. The crude residue was purified by column
chromatography (CH2Cl2/MeOH 49:1) on silica gel to give
product 1 (1.50 g) as an anomeric mixture. The β-anomer was
isolated in 81% yield (1.24 g) and the α-anomer in 11% yield
(168.0 mg). The undesired N1,N3-bis-riboside was isolated in 11.2
% yield (173.0 mg). All products were oils. Data for 1 : 1H-NMR
(600MHz, DMSO-d6): δ 11.39 (s, 1H, NH), 5.57 (d, 1H, J = 2.2
Hz, H-2’), 5.57-5.55 (m, 2H, H-5 and H-1’), 5.42 (dd, 1H, J = 2.2
Hz and J = 6.6 Hz, H-3’), 4.32 (m, 1H, H-5’), 4.15-4.12 (m, 1H,
H-4’), 4.03 (m, 1H, H-5’’), 2.25 (s, 3H, 6-CH3), 2.11 (s, 3H,
CH3), 2.10 (s, 3H, CH3), 2.09 (s, 3H, CH3); 13C-NMR (150MHz,
DMSO-d6): δ 170.2 (2 x CO), 169.5 (CO), 162.4 (4-C), 153.0 (2-
C), 150.6 (6-C), 102.5 (5-CH), 90.1 (1’-CH), 78.2 (4’-CH), 73.0
(2’-CH), 69.7 (3’-CH), 62.9 (5’-CH2), 20.6 (CH3), 20.5 (CH3),
20.3 (CH3), 19.5 (6-CH3); HRMS for C16H20N2O9 [M–H]- Calc.:
383.1095, found: 383.1103.
Showing a 3JH3’-H4’ = 8.65 Hz (D2O) and similar interactions by
NOE analysis, we could conclude that 2’-methyl-6-cyano uridine
18 also adopted a preferential “North” puckered conformation.
3
Unfortunately, the JH1’C2 and JH1’C6 values for this compound
could not be extrapolated, as C6 appeared as a broad signal and
no splitting occurred in the corresponding non-decoupled 1D 13
NMR spectrum.
C
3. Biological evaluation
Initially, the synthetized nucleoside phosphoramidates 19-24
were evaluated for selective antiviral activity against hepatitis C
virus (type 1b, Con1 strain) in the Huh 5-2 replicon system, but
no selective antiviral activity could be observed. Because of the
high conservation of the catalytic amino acid motifs across RNA-
dependent RNA polymerases of positive-strand RNA viruses,28, 29
the compounds were subsequently also evaluated against
chikungunya virus and also various species belonging to the
genus Enteroviruses, for instance Coxsackie virus (type B3,
Nancy strain) in Vero cells (subtype A), Coxsackievirus (type
B4, E2 Edwards strain) in Vero cells (subtype A), Poliovirus
(type 1, Sabin strain) in BGM cells, enterovirus 71, and
rhinovirus (type 14) in Hela cells (subtype Rh). Unfortunately, no
selective antiviral activity could be observed either.
5.1.2. 1-(2’,3’,5’-Tri-O-benzoyl-2’-methyl-β-D-
ribofuranosyl)-6-methyluracil (2).
To a solution of 6-methyluracil (302.0 mg, 2.40 mmol) in dry
CH3CN (5 mL), was added N,O-bistrimethylsilylacetamide (1.18
mL, 7.00 mmol) and the mixture was stirred for 20 min at room
temperature. To this mixture, a solution of 1,2,3,5-tetra-O-
benzoyl-2’-methyl-β-D-ribofuranose (1.16 g, 2.00 mmol) in dry
CH3CN (10 mL) was added, followed by dropwise addition of
TMSOTf (1.4 mL) at -20 °C. The reaction mixture was then
allowed to warm slowly to 10-15 °C and stirred for 6 h. The
mixture was then poured into CH2Cl2 and washed with saturated
aq. NaHCO3. The organic layer was combined, dried over
Na2SO4 and filtered. After removal of all the volatiles, the residue
was purified by column chromatography (hexane/ethyl acetate
3:1ꢀ3:2), to give product 2 (1.08 g) as an oil in 88.5% yield. The
undesired N1,N3-bis-riboside (66.0 mg) was obtained as an oil in
4. Conclusion
A new set of phosphoramidate nucleosides, derived from 6-
and 2’,6-modified uridines, were prepared but found to be devoid
of selective antiviral activity on the replication of several single-
stranded, positive-sense RNA viruses. Spectroscopic analysis of
the conformational properties of the parent nucleosides showed a
syn/N preference, indicating that the 6- and 2’,6-substitution
patterns in combination with a syn oriented base are incompatible
with recognition by viral replication enzymes and as such
provides an explanation for the lack of antiviral activity.
1
5.7% yield. H-NMR (300MHz, DMSO-d6): δ 11.56 (s, 1H, 3-
NH), 8.07–7.35 (m, 15H, aromatic), 6.34 (s, 1H, H-1’), 6.31 (d,
1H, J = 8.7 Hz, 3’- H), 5.66 (s, 1H, H-5), 4.73-4.66 (m, 1H, H-
4’), 4.61–4.59 (m, 2H, H-5’), 2.39 (s, 3H, 6-CH3), 1.76 (s, 3H,
2’-CH3); 13C-NMR (75MHz, DMSO-d6): δ 165.6 (5’’-C=O),
165.1 (2’’-C=O), 164.9 (3’’-C=O), 162.4 (4-C), 154.1 (2-C),
150.9 (6-C), 134.0–128.7 (15 x C-aromatic), 102.9 (5-CH), 91.3
(1’-CH), 88.0 (4’-CH), 77.4 (2’-CH), 77.1 (3’-CH), 64.5 (5’-
CH2), 19.7 (7-CH3), 18.1 (6’-CH3); HRMS for C32H28N2O9
[M+H]+ Calc.:585.1867, found: 585.1897.
5. Experimental section
5.1. General
NMR spectra were recorded on Bruker Avance I 300 MHz
(BBI probe), Avance II 500 MHz (TXI HCP probe) and 600
MHz (TCI HCN cryoprobe) spectrometers using a TXI HCP
probe. Chemical shifts (δ) are expressed in parts per million
(ppm). 1H and 13C NMR chemical shifts are referenced relative
to TMS (δ = 0.00 ppm). Mass spectra were acquired on a
quadrupole orthogonal acceleration time-of-flight mass
spectrometer (Synapt G2 HDMS, Waters, Milford, MA).
Samples were infused at 3uL/min and spectra were obtained in
positive (or negative) ionization mode with a resolution of 15000

5.1.3. 6-Methyluridine (3).
Na2SO4, filtered and concentrated in vacuo. The residue was then
treated with 50% aq. TFA (10 mL) at 0 C and stirred at room
o
temperature overnight in the dark. The solvent was evaporated
under reduced pressure and the crude product was purified by
column chromatography (MeOH/CH2Cl2, 10:100) on silica gel to
give product 11 as a yellow solid (146 mg, 90%). 1H-NMR
(300MHz, D2O): δ 5.97 (d, 1H, J = 3.51 Hz, H-1’), 5.63 (s, 1H,
H-5), 4.65 (q, 1H, J = 3.6 Hz, H-2’), 4.32 (t, 1H, J = 6.69 Hz, H-
3’), 3.85-3.79 (m, 2H, H-5’), 3.70-3.64 (m, 1H, H-4’); 13C-NMR
(75MHz, D2O): δ 163.9 (4-C), 153.4 (2-C), 150.1 (6-C), 89.6 (5-
CH), 88.3 (1’-CH), 83.3 (4’-CH), 71.3 (2’-CH), 68.8 (3’-CH),
61.1 (5’-CH2).
A dried 50 mL round-bottomed flask was charged with 1
(550.0 mg, 1.43 mmol) and 20 mL of a 7N solution of NH3 in
MeOH was then added at 0 °C. The reaction was allowed to
warm slowly to room temperature and stirred for 5 h to give a
yellow solution. The solvent was then removed in vacuo and the
resulting residue was purified by column chromatography
(CH2Cl2/ MeOH 9:1) on silica gel to give product 3 (268 mg) as a
white solid in 75% yield. UV λmax (MeOH) = 269 nm (ε = 5 179
mol-1 cm-1); 1H-NMR (500MHz, DMSO-d6): δ 11.10 (s, 1H, NH),
6.03 (d, 1H, J = 3.7 Hz, H-1’), 5.45 (s, 1H, H-5), 4.99 (d, 1H, J =
4.8 Hz, OH-2’), 4.82 (d, 1H, J = 6.3 Hz, OH-3’), 4.55 (t, 1H, J =
5.8 Hz, H-2’), 4.46 (d, 1H, J = 5.35 Hz, OH-5’), 4.08 (q, 1H, J =
6.0 Hz, H-3’), 3.69-3.55 (m, 2H, H-5’), 3.68-3.65 (m, 1H, H-4’),
2.02 (s, 3H, 6-CH3); 13C-NMR (125MHz, DMSO-d6): δ 162.9 (4-
C), 152.4 (2-C), 151.1 (6-C), 98.7 (5-CH), 87.3 (1’-CH), 84.4
(4’-CH), 71.0 (2’-CH), 70.3 (3’-CH), 62.5 (5’-CH2), 18.2 (7-
CH3); HRMS for C10H14N2O6 [M+H]+ Calc.: 259.0924, found:
259.0931.
5.1.7. 1-(2,3,5-Tri-O-benzoyl-2’-methyl-β-D-
ribofuranosyl)-uridine (14).
To a solution of uracil 12 (250.0 mg, 2.20 mmol) in dry
CH3CN (5 mL), was added N,O-bistrimethylsilylacetamide (1.18
mL, 7.00 mmol) and the mixture was stirred for 20 min at room
temperature. To this mixture, a solution of 1,2,3,5-tetra-O-
benzoyl-2’-methyl-β-D-ribofuranosyl (1.16 g, 2.00 mmol) in dry
CH3CN (10 mL) was added. After cooling to -20 ˚C, TMSOTf
(1.4 mL) was added and the reaction mixture was allowed to
warm slowly to room temperature and then stirred at 70 °C for 2
h. The mixture was then poured into ethyl acetate and washed
with saturated aq. NaHCO3. The water layer was again extracted
with ethyl acetate. The organic extracts were combined, dried
over Na2SO4 and filtered. After removal of all the volatiles in
vacuo, the residue was purified by column chromatography
(CH2Cl2/ MeOH 49:1) on silica gel to give product 14 (983.0 mg)
as an anomeric mixture. The β-anomer (946.0 mg) was isolated
5.1.4. 2’,6-Dimethyluridine (4).
A dried 50 mL round-bottomed flask was charged with 2 (1.20
g, 4.41 mmol) and 20 mL of a 7N solution of NH3 in MeOH was
then added at 0 °C. The reaction was allowed to warm slowly to
room temperature and stirred for two days. The solvent was then
removed in vacuo and the residue was purified by column
chromatography (CH2Cl2/MeOH 100:7) on silica gel to give
1
product 4 (459.0 mg) as a white solid in 84% yield. H-NMR
(300MHz, D2O): δ 5.67 (s, 2H, H-1’ and H-5), 4.22-4.19 (m, 1H,
H-3’), 3.89-3.76 (m, 3H, H-4’ and H-5’), 2.30 (s, 3H, 6-CH3),
1.21 (s, 3H, 2’-CH3); 13C-NMR (75 MHz, D2O): δ 165.1 (4-CO),
155.6 (2-CO), 150.7 (5-CH), 102.3 (5-CH), 94.3 (1’-CH), 82.4
(4’-CH), 79.0 (2’-CH), 73.9 (3’-CH), 61.4 (5’-CH2), 20.0 (7-
CH3), 19.0 ( 6’-CH3). MS for C32H28N2O9 [M+H]+ Calcd.:
273.1089, found: 273.1089.
1
as an oil in 75% yield. H-NMR (300MHz, DMSO-d6): δ 9.7 (s,
1H, 3-NH), 8.20-7.26 (m, 16H, H-6 and 3 x aromatic), 6.55 (s,
1H, H-1’), 5.79 (d, 1H, J = 5.3 Hz, H-3’), 5.75 (d, 1H, J = 8.19
Hz, H-5), 4.67-4.63 (m, 1H, 4’- H), 4.93-4.80 (m, 2H, 2H-5’),
1.78 (s, 3H, CH3); 13C-NMR (75MHz, DMSO-d6): δ 165.9 (2’’-
C=O), 164.97 and 164.94 (2’’ and 3’’-C=O), 162.8 (4-C), 149.7
(2-C), 140.6 (6-C), 133.32-128.09 (aromatic), 102.1 (5-CH), 84.2
(1’-CH), 80.1 (4’-CH), 75.2 (2’-CH), 63.0 (3’-CH), 60.1 (5’-
CH2), 17.7 (CH3); HRMS for C31H26N2O9 [M+Na]+ Calc.:
593.1530, found: 593.1541.
5.1.5. 6-Cyanouridine (10).
A dried 100 mL, round-bottomed flask was charged with 5-O-
(tert-butyldimethylsilyl)-6-iodo-2’,3’-O-isopropylideneuridine 6
(200.0 mg, 0.38 mmol) in anhydrous dimethylformamide (4 mL)
and then (Et)4NCN (150.0 mg, 1.00 mmol) was added at room
temperature. The reaction mixture was stirred overnight, poured
into water (20 mL) and extracted with EtOAc (100 mL). The
organic layer was dried over Na2SO4, filtered and concentrated in
vacuo. The residue was treated with 50% aq. TFA (10 mL) at 0
oC and then stirred at room temperature overnight in the dark.
The solvent was evaporated and the crude residue was purified
by column chromatography (MeOH/CH2Cl2, 10:100) on silica gel
5.1.8. 2’-Methyluridine (15).
A dried 50 mL round-bottomed flask was charged with 14
(3.00 g, 5.26 mmol) and 50mL of a 7N solution of NH3 in MeOH
was then added at 0 oC. The reaction was stirred at room
temperature for 48 h. The solvent was then removed in vacuo and
the residue was purified by column chromatography (ethyl
acetate/ MeOH 100:5ꢀ100:8) on silica gel to give product 15
(1.19 g) as a white solid in 88% yield. 1H-NMR (300MHz,
DMSO-d6): δ 11.34 (s, 1H, 3-NH), 8.05 (d, 1H, J = 8.07 Hz, H-
6), 5.80 (s, 1H, H-1’), 5.61 (d, 1H, J = 8.07 Hz, H-5), ), 5.18 (d,
2H, J = 4.08 Hz, H-3’, OH-3’), 5.10 (s, 1H, OH-2’), 4.06-3.59
(m, 4H, 2H-5’, H-4’, OH-5’), 1.01 (s, 3H, CH3); 13C-NMR
(75MHz, DMSO-d6): δ 163.1 (4-C), 150.8 (2-C), 140.5 (6-C),
101.5 (5-CH), 91.1 (1’-CH), 82.3 (4’-CH), 78.4 (2’-CH), 71.7
(3’-CH), 58.8 (5’-CH2), 19.93 (CH3); HRMS for C10H14N2O6
[M+H]+ Calcd.: 259.0924, found: 259.0929.
1
to give product 10 as a brown solid (50.0 mg) in 48% yield. H-
NMR (300MHz, D2O): δ 6.66 (s, 1H, H-5), 5.97 (d, 1H, J = 4.56
Hz, H-1’), 4.73 (q, 1H, J = 4.59, 6.6 Hz, H-2’), 4.34 (t, 1H, J =
6.6 Hz, H-3’), 4.07-4.01 (m, 1H, H-4’), 3.95-3.81 (m, 2H, H-5’);
13C-NMR (75MHz, D2O): δ 162.7 (4-C), 149.7 (2-C), 126.8 (7-
CN) 114.1 (6-C), 110.7 (5-CH), 92.3 (1’-CH), 84.0 (4’-CH), 71.4
(2’-CH), 68.5 (3’-CH), 60.8 (5’-CH2).
5.1.6. 6-Azidouridine (11).
5’-O-(tert-Butyldimethylsilyl)-6-iodo-2’,3’-O-
5.1.9. 2’-Methyl-6-cyanouridine (18).
isopropylideneuridine 6 (300.0 g, 0.57 mmol) in anhydrous DMF
(3.4 mL) was treated with sodium azide (60.0 mg, 0.9 mmol).
The reaction mixture was stirred overnight at room temperature,
concentrated, and redissolved in EtOAc (30 mL). The organic
layer was washed with water (20 mL), brine (20 mL), dried over
A dried 100 mL, round-bottomed flask was charged with
2’,3’,5’-tri-O-tert-butyldimethylsilyl-5-iodo-uridine 17 (200.0
mg, 0.275 mmol) in anhydrous dimethylformamide (4 mL) and
then (Et)4NCN (75.0 mg, 0.5 mmol) was added at room

temperature. The reaction mixture was stirred overnight, it was
then poured into NaHCO3 (20 mL) and extracted with EtOAc
(100 mL). The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was treated with TBAF in
THF (3 mL) at room temperature overnight. The solvent was
evaporated and the crude residue was purified by column
chromatography (MeOH/CH2Cl2, 10:100) on silica gel to give
(CH3), 20.4 (CH3), 13.9 (6’-CH3). 31P-NMR (200MHz, DMSO-
d6): δ 3.75; HRMS for C23H32N3O10P[M+H]+ Calcd.: 542.1897,
found: 542.1897.
5.1.13. (2S)-Isopropyl 2-(2’R,3’R,4’R) (((3’,4’-
dihydroxy-5-(6-iodo-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphorylamino)propanoa
te (21)
1
product 18 as a brown solid (69 mg, 89%). H-NMR (300 MHz,
MeOD): δ 6.47 (s, 1H, H-5), 5.97 (s, 1H, H-1’), 4.03 (br s, 1H,
H-3’), 3.95-3.91 (m, 2H, H-5’, H-5’’), 3.89-3.79 (m, 1H, H-4’),
1.26 (s, 3H, CH3); 13C-NMR (75MHz, MeOD): δ 161.2 (4-C),
149.0 (2-C), 126.7 (7-CN) 113.1 (6-C), 111.2 (5-CH), 96.8 (1’-
CH), 83.4 (4’-CH), 77.7 (2’-CH), 74.0 (3’-CH), 61.4 (5’-CH2),
13.9 (2’-CH3). HRMS for C11H13N3O6 [M+Na+] Calcd.:
306.0696, found: 306.0698.
Yellow oil (50.3 mg, 39% yield). 1H-NMR (300MHz, DMSO-
d6): δ 11.54 (s, 1H, NH), 7.65-7.15 (m, 10H, aromatics), 6.35 (s,
1H, H-5), 5.76 (d, 1H, J = 2.31 Hz, H-1’), 5.35 (t, 1H, J = 4.38
Hz), 5.16 (d, 1H, J = 6.81 Hz), 4.88-4.80 (m, 1H), 4.51 (s, 1H),
4.51-3.72 (m, 5H), 1.23-1.13 (m, 18H, 6 x CH3); 13C-NMR
(125MHz, DMSO-d6): δ 172.7 (=CO), 161.6 (4=CO), 150.8
2
5.1.10. General procedure for phosphoramidates
synthesis
(2=CO), 147.3 (5-C sp ), 132.1-120.2 (aromatics), 118.4 (6-C),
115.4 (1’-CH), 82.4 (4’-CH), 71.6 (2’-CH), 69.8 (3’-CH), 68.0
(5’-CH2), 49.9 (CH), 49.8 (CH), 29.1 (CH3), 21.5 (CH3), 21.4
(CH3); 31P-NMR (121MHz, CDCl3): δ 2.86 and 2.75; HRMS for
C21H27IN3O10P[M+H]+Calcd.: 640.0553, found: 640.0552.
A dried 50 mL, round-bottomed flask was charged with
phosphate dichloride phenol (1 mmol) and alanine isopropyl
hydrochloric ester (1 mmol) in dry CH3CN under inert
o
atmosphere. After cooling to -40 C, Et3N (2 mmol) was added
dropwise and stirred overnight. A portion of this solution was
then removed under inert atmosphere and added to another
round-bottomed flask containing the relevant nucleoside (0.35
mmol). The mixture was stirred until complete solubilization,
then 1-methylimidazole (2 mmol) was added dropwise over 5
min and the stirring was continued for 3 h. The solvent was then
removed in vacuo and the residue was purified by column
chromatography (DCM/ ethanol/pyridine (50/3/0.2) on silica gel.
5.1.14. (2S)-Isopropyl 2-(2’R,3’R,4’R) ((((5-(6-
cyano-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
3,4-dihydroxytetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphorylamino)propanoa
te (22)
Yellow oil (39.5 mg, 42% yield). 1H-NMR (300MHz, DMSO-
d6): δ 9.51 (s, 1H, NH), 7.30-7.16 (m, 10H, aromatics), 6.43 (s,
1H, H-5), 5.91 (s, 1H, H-1’), 5.30 (s, 1H), 5.05-4.94 (m, 2H),
4.44-3.96 (m, 5H), 3.94-3.59 (m, 4H), 1.23 (s, 9H, 3 x CH3); 13C-
NMR (125MHz, CDCl3): δ 173.1 (=CO), 150.5 (2=CO), 160.8
(4=CO), 147.3 (5-Csp2), 129. 8-129.6 (aromatic), 125.1 (6-CN),
117.1 (6-C), 113.7 (1’-CH), 82.5 (4’-CH), 73.5 (2’-CH), 70.0 (3’-
CH), 69.5 (5’-CH2), 66.5 (CH), 50.2 (CH), 29.7 (CH3), 21.7
(CH3), 21.6 (CH3); 31P-NMR (121MHz, DMSO-d6): δ 2.91 and
2.81; HRMS for C23H29N4O10P1 [M+Na]+ Calcd.: 575.1513,
found: 575.1516.
5.1.11. (2S)-Isopropyl 2-(2’R,3’R,4’R) (((3’,4’-
dihydroxy-5-(6-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-
)methoxy)(phenoxy)phosphorylamino)propanoate
(19)
Yellow oil (54.1 mg, 60% yield). 1H-NMR (300MHz, DMSO-
d6): δ 11.24 (s, 1H, NH), 7.34-7.17 (m, 10H, aromatics), 5.91 (d,
1H, H-1’), 5.57 (s, 1H, H-5), 5.44 (s, 1H, OH-2’), 5.33 (s, 1H,
OH-3’), 5.12 (d, 1H, J = 6.6 Hz, H-2’), 4.87-4.81 (m, 1H, H from
alanine), 4.22-4.12 (m, 1H, H-3’), 3.91-3.88 (m, 1H, H-4’), 3.76-
3.70 (m, 2H, H-5’), 2.24 (s, 3H, 7-CH3); 13C-NMR (75MHz,
CDCl3): δ 172.9 (=CO), 162.6 ( 4=CO), 152.7 (2=CO), 150.3 (5-
C sp2), 102.6 (6-CH), 138.3-119.8 (aromatics), 92.9 (1’-CH), 82.0
(4’-CH), 72.5 (2’-CH), 70.14 (3’-CH), 69.4 (5’-CH2), 69.4 (CH),
49.9 (CH), 31.6 (CH3), 21.3 (CH3), 21.2 (CH3), 20.2 (7-CH3).
5.1.15. (2S)-Isopropyl 2-(2’R,3’R,4’R) (((5-(6-
azido-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
3,4-dihydroxytetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphorylamino)propanoa
te (23)
Pale yellow oil (42.8 mg, 22% yield). 1H-NMR (500MHz,
CDCl3): δ 9.50 (s, 1H, NH), 7.35-7.17 (m, 5H, aromatics), 5.93
(s, 1H, H-5), 5.50-5.49 (d, J = 4.05 Hz, 1H, H-1’), 5.02-4.98 (m,
1H), 4.75-4.74 (d, J = 7.25 Hz, 1H), 4.67-4.65, (d, J = 10.5 Hz ,
1H), 4.42-4.40 (m, 1H), 4.32 (s, 1H), 4.16-4.13 (m, 1H), 4.94-
4.02 (m, 1H), 1.22-1.24 (m, 9H, 3x CH3); 13C-NMR (200MHz,
CDCl3): δ 173.2 (=CO), 161.5 (4=CO), 152.3 (2=CO), 150.6 (5-
31P-NMR (121MHz, DMSO-d6):
δ
3.55; HRMS for
C22H30N3O10P[M+H]+ Calcd.: 528.1741, found: 528.1754.
5.1.12. (2S)-Isopropyl 2-(2’R,3’R,4’R)(((3',4'-
dihydroxy-2'-methyl-5-(6-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphorylamino)propanoa
te (20)
Yellow oil (52.3 mg, 55% yield). 1H-NMR (500MHz, DMSO-
d6): δ 11.19 (s, 1H, 3-NH), 7.32-7.09 (m, 5H, aromatic), 5.47 (s,
1H, H-1’), 5.55 (s, 1H, H-5), 5.12 (d, 1H, OH-2’), 4.92 (s, 1H,
OH-3’), 4.89-4.80 (m, 1H, 1H), 4.48 (t, 1H, J = 5.0 Hz, H-3’),
4.07 (t, 1H, J = 5.1 Hz H-4’), 3.86-3.62 (m, 3H, 2H-5’overlapped
with 1H, CH), 2.25 (s, 3H, 6-CH3), 1.23 (s, 6H, 2x CH3), 1.16 (d,
3H, J = 6.15 Hz, CH3), 1.09 (s, 3H, 2’-CH3); 13C-NMR
(125MHz, DMSO-d6): δ 173.4 (CO), 162.5 (4=CO), 153.5
(2=CO), 150.4 (5-CH), 129.3-120.6 (aromatics), 102.3 (6-CH),
94.9 (1’-CH), 82.1 (4’-CH), 77.9 (2’-CH), 74.9 (3’-CH), 68.8
(5’-CH), 67.8 (CH), 49.9 (CH), 29.3 (CH3), 20.5 (7-CH3), 20.5
C
2), 149.5 (2=CO), 129.7 (aromatic), 125.0 (aromatic), 120.2
sp
(aromatic), 120.1 (aromatic), 91.0 (6-C), 88.8 (1’-CH), 82.3 (4’-
CH), 73.1 (2’-CH), 70.5 (3’-CH), 69.5 (5’-CH2), 66.7 (CH), 63.1
(CH), 50.3 (CH), 29.7 (CH3), 21.6 (CH3), 21.6 (CH3); 31P-NMR
(75MHz, CDCl3): δ 2.99 and 2.84; HRMS for C21H27N6O10P1
[M+H]+ calcd.:555.1598, found: 555.1595.
5.1.16. (2S)-Isopropyl 2-(2’R,3’R,4’R)(((3',4'-
dihydroxy-2'-methyl-5-(6-cyano-2,4-dioxo-3, 4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphorylamino)propanoa
te (24)

19
Yellow oil (33.0 mg, 60% yield). 1H-NMR (500 MHz,
DMSO): δ 10.36 (s, 1H, H-3), 7.35-7.11 (m, 5H, aromatic), 6.31
(s, 1H, H-5), 5.96 (s, 1H, H-1’), 5.03-4.95 (q, 1H, J = 6.24, H
amino ester), 4.54-4.43 (m, 3H), 4.35-4.20 (m, 2H), 4.18-4.11
(m, 2H), 1.37-1.20 (m, 12H, 4 x -CH3); 13C-NMR (125MHz,
CDCl3): δ 172.8 (-CO), 160.0 (4-C), 150.3 (2-C), 148.5 (5-C sp2),
129.4 (aromatic), 124.8 (aromatic), 129.7-119.9 (aromatic), 125.9
(7-CN), 113.1 (6-C), 110.6 (5-CH), 97.5 (1’-CH), 82.1 (4’-CH),
74.5 (2’-CH), 69.9 (3’-CH), 66.7 (-CH), 64.8 (5’-CH2), 50.0
(CH), 29.4 (CH3), 21.3 (CH3), 21.3 (CH3), 13.8 (CH3); 31P-NMR
(75 MHz, DMSO-d6): δ 3.05 and 2.79; HRMS for C23H29N4O10P1
[M+Na+] Calcd.: 575.1513, found: 575.1516.
P. Januszczyk, J. Fogt, J. Boryski, K. Izawa, T. Onishi, J. Neyts,
E. De Clercq, Nucleosides Nucleotides Nucleic Acids, 2009 28, 713-
723.
20
Y.-C. Shih, Y.-Y. Yang, C.-C. Lin, T.-C. Chien, J. Org. Chem.,
2013 78, 4027-4036.
21
M. Schweizer, J.T. Witkowski, R.K. Robins, J. Am. Chem. Soc.,
1971 93, 277-279.
C. Altona, M. Sundaralingam, J. Am. Chem. Soc., 1972 94,
22
8205-8212.
23
C. Altona, Sundaral.M, J. Am. Chem. Soc., 1973 95, 2333-2344.
F. Deleeuw, C. Altona, J. Chem. Soc., Perkin Trans. 2, 1982
24
375-384.
25
S.S. Wijmenga, M.M.W. Mooren, C.W. Hilbers, NMR of
macromolecules: a practical approach, IRL Press at Oxford
University Press, Oxford, 1993.
P.M.S. Hendrickx, J.C. Martins, Chem. Cent. J., 2008 2.
26
Acknowledgments
27
S.S. Wijmenga, B.N.M. van Buuren, Prog. Nucl. Mag. Res. Sp.,
1998 32, 287-387.
28
The authors wish to thank Luc Baudemprez for recording the
NMR spectra.
E.V. Koonin, J. Gen. Virol., 1991 72, 2197-2206.
J. Deval, J.A. Symons, L. Beigelman, Curr. Opin. Virol., 2014
29
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