Synthesis and biological evaluation of 6-substituted-5-fluorouridine ProTides

Article abstract of DOI:10.1016/j.bmc.2017.11.037

A new family of thirteen phosphoramidate prodrugs (ProTides) of different 6-substituted-5-fluorouridine nucleoside analogues were synthesized and evaluated as potential anticancer agents. In addition, antiviral activity against Chikungunya (CHIKV) virus was evaluated using a cytopathic effect inhibition assay. Although a carboxypeptidase Y assay supported a putative mechanism of activation of ProTides built on 5-fluorouridine with such C6-modifications, the Hint docking studies revealed a compromised substrate-activity for the Hint phosphoramidase-type enzyme that is likely responsible for phosphoramidate bioactivation through P–N bond cleavage and free nucleoside 5′-monophosphate delivery. Our observations may support and explain to some extent the poor in vitro biological activity generally demonstrated by the series of 6-substituted-5-fluorouridine phosphoramidates (ProTides) and will be of guidance for the design of novel phosphoramidate prodrugs.

Full text of DOI:10.1016/j.bmc.2017.11.037

Accepted Manuscript
Synthesis and Biological Evaluation of 6-Substituted-5-Fluorouridine ProTides.
Magdalena Slusarczyk, Salvatore Ferla, Andrea Brancale, Christopher
McGuigan
PII:
S0968-0896(17)31978-8
https://doi.org/10.1016/j.bmc.2017.11.037
BMC 14089
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 October 2017
21 November 2017
23 November 2017
Please cite this article as: Slusarczyk, M., Ferla, S., Brancale, A., McGuigan, C., Synthesis and Biological Evaluation
of 6-Substituted-5-Fluorouridine ProTides., Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/
10.1016/j.bmc.2017.11.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and Biological Evaluation of 6-Substituted-5-
Fluorouridine ProTides.
Magdalena Slusarczyk*1, Salvatore Ferla,1 Andrea Brancale,1 Christopher McGuigan1
1School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, King Edward
VII Avenue, Cardiff CF10 3NB, UK.
*Author for correspondence:
Tel.: +44 02920874551
SlusarczykM1@cardiff.ac.uk
In memory of, and dedicated to Professor Christopher McGuigan (1958-2016).
Abstract
A new family of thirteen phosphoramidate prodrugs (ProTides) of different 6-
substituted-5-fluorouridine nucleoside analogues were synthesized and evaluated as
potential anticancer agents. In addition, antiviral activity against Chikungunya
(CHIKV) virus was evaluated using a cytopathic effect inhibition assay. Although a
carboxypeptidase Y assay supported a putative mechanism of activation of ProTides
built on 5-fluorouridine with such C6-modifications, the Hint docking studies
revealed a compromised substrate-activity for the Hint phosphoramidase-type enzyme
that is likely responsible for phosphoramidate bioactivation through P-N bond
cleavage and free nucleoside 5’-monophosphate delivery. Our observations may
support and explain to some extent the poor in vitro biological activity generally
demonstrated by the series of 6-substituted-5-fluorouridine phosphoramidates
(ProTides) and will be of guidance for the design of novel phosphoramidate prodrugs.
Keywords: Orotidine-5’-monophosphate decarboxylase (ODCase), nucleoside
analogue (NA), phosphoramidate (ProTide) approach, anticancer, human Hint
enzyme
Introduction
Antimetabolites such as nucleoside analogues (NAs) play a crucial role in the
clinical treatment of patients with cancer and viral infections.1-3 Nucleoside analogues

are chemically-modified synthetic compounds that have been developed with the aim
to closely mimic their natural counterparts. Hence, they have a good chance to be able
to be taken-up by cells, metabolized and incorporated into nucleic acids to
subsequently inhibit cellular division and/or viral replication. At the molecular level,
the biological effect of NAs is usually exerted following their metabolic conversion
into corresponding 5’-mono-, di- and tri-phosphate forms. In addition to their
incorporation into newly synthesized DNA or RNA, NAs can target and inhibit
intracellular enzymes like for example viral or human polymerases, ribonucleotide
reductase4 (RNR), thymidylate synthase5 (TS) or orotidine-5’-monophosphate
decarboxylase6a,b (ODCase). All these enzymes are involved either in the DNA or
RNA synthesis, DNA repair or de novo pyrimidine nucleotide synthesis. Because de
novo synthesis of pyrimidine nucleotides is upregulated during abnormal cell growth
or during the replication of cells, when the demand for pyrimidine nucleotides is high,
the ODCase can be considered as one of the potential anticancer targets.7 In fact, in
the last decades a significant interest has been given to ODCase as a drug target for a
number of modified nucleos(t)ide analogues, in particular C6-substituted UMP
derivatives also in the antiviral and antimalarial arena.7-10 This is due to a pivotal role
that ODCase plays in the de novo synthesis of pyrimidine nucleotides such as uridine-
5’-O-monophosphate (UMP, 2) from orotidine-5’-O-monophosphate (OMP, 1, Figure
1) via decarboxylation6a and its extraordinary reaction rate enhancement (over 17
orders of magnitude) in comparison with spontaneous uncatalyzed decarboxylation of
OMP observed in water at neutral pH and ambient temperature.11a,b Present in most
species except viruses, ODCase exists as a monofunctional enzyme in bacteria and
parasites and as a part of the bifunctional enzyme UMP synthase in human and other
high-developed organisms.12a,b A number of nucleoside-like and non-nucleoside
ODCase inhibitors have been developed and reported in the literature13 with 6-
azauridine14a-c (3) and pyrazofurin15 (4) as representative examples of the nucleoside
class of compounds being effective at the monophosphate level (Figure 2). In 2009,
Kotra and co-workers reported in cell-based assays a variety of modified 5-
fluorouridine (Figure 2) nucleoside derivatives bearing at the 6-position of the
pyrimidine nucleobase small groups like iodo, azido, amino, or ethyl as potent
anticancer agents, and their corresponding monophosphate analogues as inhibitors of
human (Hs) and Methanobacterium thermoautotrophicium (Mt) ODCase.16 Later, the

same group disclosed novel N-modified cytidine-based (CMP) orotidine-5’-
monophosphate decarboxylase inhibitors with anti-parasitic activity, improved
inhibition of the catalytic enzyme activity and binding conformations studies.17
Figure 1. De novo synthetic pathway of OMP (1) to UMP (2) catalysed by ODCase.
Nucleoside analogues, including the above-mentioned 6-substituted-5-fluorouridines
are biologically effective after being phosphorylated into their 5’-monophosphate and
further to their 5’-di- and 5’-triphosphate forms. The effectiveness of NAs can be
impaired as a consequence of limited cellular uptake via nucleoside-specific
transporters, down-regulation of nucleoside kinases responsible for the
phosphorylation step (activation) and up-regulation of deactivating enzymes (i.e
nucleo(s)(t)ide deaminases, purine/pyrimidine nucleoside phosphorylase…).18 To
overcome these limitations, and thus improving their effectiveness, several prodrug
approaches have been developed and reviewed over the last years. Notably, phosphate
and phosphonate prodrugs such as phosphodiesters (HepDirect, CycloSal, SATE) or
phosporamidates in which a nucleoside analogue is linked to the prodrug entity by
either a phosphorus-oxygen or a phosphorus-nitrogen bond, respectively, have been
extensively studied.19-21 One of the prominent strategies applied in the modulation of
the activity of many nucleoside analogues and potentially overcoming the NA
limitations accounting on both innate and acquired resistance of cancer cells to
nucleoside analogues, is the ProTide technology pioneered by McGuigan and
colleagues.22 Designed to mask the negative charges in a “monophosphate moiety” of
a ProTide template, the ProTide approach introduces an amino acid ester and aryloxy
entities as two lipophilic and biolabile groups linked to the phosphate part of the
molecule. These two groups increase membrane permeability thereby circumventing
the need for nucleoside-transporters and after their intracellular metabolism delivering
a nucleoside monophosphate form suitable for further phosphorylation and exertion of
its eventual biological activity.

Figure 2. Examples of anticancer nucleoside analogues (3-11) targeting different
intracellular enzymes to exert their eventual biological activity.
The phosphoramidate (ProTide) technology has now an established position in the
nucleotide prodrug field and led to the discovery of clinically successful drugs such as
Sofosbuvir23 (FDA approval in 2013 for the treatment of HCV, 12) and others being
subject of currently ongoing clinical trials as exemplified in Figure 3 for antiviral (i.e.
stampidine24 (phase I for HIV treatment, 13), and anticancer (i.e. thymectacin)25
(phase I/II, 14), NUC-103126 (phase II, 15) and NUC-337327 (phase I, 16) activity. In
our laboratory we have extensively investigated and applied the ProTide technology
to a number of antiviral28-30 and anticancer agents such as for example 5-fluoro-2’-
deoxyuridine (FdUrd),31 and gemcitabine.32 As part of our anticancer program and
driven by our continuous interests in the discovery of novel anticancer agents we
decided to apply the ProTide approach also to 6-substituted-5-fluorouridine
analogues. In addition, a new class of 2’-fluoro-6-substituted uridine derivatives
reported in the literature7 as potential inhibitors of ODCase revealed the lack of

cellular anticancer activities most likely due to their poor activation to the
corresponding 5’-monophosphate forms. Herein, we report the ProTide technology
approach employed to 6-substituted-5-fluorouridine analogues (5-8) to design novel
nucleoside phosphoramidates (25-37) as potential anticancer agents. These
compounds were prepared with the aim to improve cellular uptake of their parent
nucleoside analogues and intracellular delivery of their corresponding monophosphate
forms. All compounds were preliminary tested for their IC50 activity in a panel of
cancer cell lines including tumor cell lines of hematopoietic origin as these cell lines
were found to be strongly inhibited by 6-substituted-5-fluorouridine analogues.16
Figure 3. Examples of antiviral and anticancer phosporamidate-type prodrugs.
Chemistry
The 6-modified nucleosides 5-8 were prepared according to previously
reported methods16 starting from a two-steps full protection of 5-fluorouridine (17) to
give an intermediate (19) as shown in Scheme 1. The treatment of compound 19 with
LDA followed by iodination with I2 led to the formation of the key nucleoside 20 that
was further deprotected under acidic conditions to yield 6-I-FUR (5). In addition the
iodo-derivative 20 was used to form three other 6-substituted nucleoside analogues,
such as 6-azido-, 6-methyl- and 6-ethyl-FUR (21-23). The 6-azido-derivative 21 was

prepared by treating 20 with NaN3 followed by removal of its silyl and isopropylidine
moieties to give compound 6. The 6-methyl (22) and 6-ethyl (23) derivatives were
successfully prepared using a modified procedure16 in one-pot reaction upon the
treatment of 19 with LDA and subsequent addition of 2.0 eq. of CH3I. Interestingly,
both derivatives 22 and 23, the latter compound obtained as a side product upon the
additional methylation of monomethylated derivative 22, were isolated and further
submitted to the standard acidic deprotection conditions to furnish nucleosides 7 and
8, respectively. Next, the ProTide technology was applied to the four nucleoside
analogues 5-8 leading to the formation of 6-substitited-5-fluorouridine ProTides 25-
37 in moderate yields. The key reagents used in the synthesis of ProTides are the
arylaminoacyl phosphorochloridates represented by the general structure 24, formed
by allowing the aryl phosphorodichloridates, either phenyl or 1-naphthyl, to react with
L-alanine esters in the presence of triethylamine at low temperature (Scheme 2). The
formation of phosphorochloridates 24, each obtained as a pair of diastereoisomers at
31
the phosphate centre (1:1 mixture), was monitored and confirmed by P NMR. Due
to their limited stability, 24 were used in the ProTide syntheses as crude materials or
after rapid silica gel chromatography. Finally, the four 6-modified nucleosides 5-8
were reacted with the key reagents 24 in the presence of NMI to give a number of
ProTides 25-37 isolated as diastereisomeric mixtures (4-17% yield), as evidenced by
1
13
31P NMR, HPLC (two peaks), H NMR, and C NMR (splitting of many nucleoside
signals). Given the low yield of ProTides formation and requirement for an extensive
and repeated chromatographic purification, the final compounds 25-37 were
submitted to in vitro evaluation as diastereoisomeric mixtures. Because a primary
goal of the following study was to establish preliminary biological activity, at this
stage a coupling reaction conditions and methods of isolation of final ProTides as two
separate diastereoisomeres were not optimized in the present report.
Scheme 1. Synthesis of 6-substituted-5-fluorouridine nucleoside analogues 5-8a.

aReagents and conditions: (a) acetone, H2SO4, 0 C, 2 h, 96%; (b) TBDMSCl, imidazole,
DCM, 0 C then room temp 3 h, 90%; (c) LDA, I2, THF, –78 C, 7 h, 54%; (d) LDA, CH3I,
THF, –78 C, 5 h, 20-42%; (e) 1, NaN3, DMF, room temp, 3 h, 74%; (f) 50% TFA/H2O, 0 C
to room temp, 2 h, 80-92%.
Scheme 2. General synthesis of 6-modified ProTides 25-37a.
aReagents and conditions: (a*) for the synthesis of 1-naphthyl phosphorodichloridate POCl3,
Et3N, anhydrous Et2O, –78 C for 1 h, then room temp for 1 h, 91%; phenyl
phosphorodichloridate
commercially
available;
(b)
phenyl
or
1-naphthyl
phosphorodichloridate, Et3N, anhydrous DCM, –78 C for 1 h, then room temp for 1 – 3 h,
70-90%; (c) NMI, anhydrous THF, room temp, 16 h, 4-17%.

Figure 4. Four classes of 6-substituted-5-fluorouridine target ProTides (25-37).
Biology
The cytostatic activities of 6-substitited-FUR ProTides 25-37 (Figure 4) were
evaluated against various cancer cell lines including human breast adenocarcinoma
(MCF-7), colon (SW620), pancreatic (Mia-Pa-Ca), acute myeloid leukaemia (MV4-
11), erythroleukaemia (HEL92.1.7), non-Hodgkin’s lymphoma (RL) and Hodgkin’s
lymphoma (HS445). The L-alanine-Bn-Naph 6-I-FUR prodrug 27 was among the
most active in the series of all 6-modified-FUR ProTides with submicromolar IC50
values ranging between 0.08 M (MV4-11) and 3.9 M in (MCF-7) cell cultures. The
ProTide 27 proved to be equipotent as a cytostatic agent as its parent nucleoside 5 in
SW620 cell cultures (0.52 vs 0.50 M) and 1.8–5.8-fold less potent against HS445
(0.71 vs 0.38 M) and MCF-7 cell cultures (3.9 vs 0.67 M), respectively. The loss of

inhibitory activity of the prodrug versus 5 was more significant for two other L-
alanine 6-I-FUR ProTides, the phenyl-pentyl 25 and phenyl-cyclohexyl 26, and the
activity loss ranged between 2–65-fold and 9–228-fold, respectively.
In the 6-N3-FUR series, the cytostatic activities for nucleoside analogue 6 were in the
lower micromolar range (6–14 M), whereas for three 6-N3-FUR ProTides (30, 31,
32) the IC50 values ranged between 6.0 and 44 M. The ProTides 31 and 32 turned
out to be relatively equipotent in three cell lines of hematopoietic origin (MV4-11, RL
and HS445) with a 2.3-fold boost in activity for 31 versus the parent nucleoside 6 (6.0
M vs 14.0 M in the HS445 cell line). In general, the solid tumour cell lines were
markedly less sensitive to the 6-N3-FUR ProTides in particular to the L-alanine
phenyl hexyl (29) and phenyl ethyl (28) derivatives with the latter compound even
being inactive up to 50 M in six out of seven cancer cell lines.
Similar to nucleoside 6, the 6-Me-FUR nucleoside 7 exerted micromolar activity
across all tumor cell lines tested ranging between 2.0 M (MV4-11) and 16 M (RL).
In the 6-Me-FUR series, no cytostatic activity was detected for ProTides 33-36 up to
50 M in the panel of cancer cell lines. A somewhat similar trend was noted for 6-Et-
FUR (8) versus its ProTide derivative 37, although the latter compound showed
micromolar activity in three out of five tumour cell lines (IC50’s for SW620 10 M;
for RL 27 M; and for HS445 10 M).
a
Table 1. Cytotoxicity of 6-modifed-FUR ProTides 25-37 reported as IC50 (M)
Values.

a
Cytotoxicity data reported as IC50 (M) values (50% inhibitory concentration of cell
viability). The compounds were added to the cell in duplicate and tested in 9 serial
concentrations from 198 M to 0.0199 M.
The screening of the compounds was performed on a broad variety of at least 6
different cancer cell lines to cover different metabolic properties that may exist
between different types of cancer cells that may result in a different outcome of the
eventual cytostatic activity of the compounds. In fact, it was observed that the
cytostatic activity for some of the 6-substituted-FUR derivatives and their
corresponding prodrugs (i.e. 6-I-FUR, 6-Me-FUR) may significantly differ depending
on the nature of the prodrug part in the molecule and/or the nature of the tumor cell
line investigated. Although the molecular basis of the observed differences are not
further investigated in detail yet, the cytostatic differences may be most likely due to
i) differences in drug uptake (i.e. depending on the presence and activity of different
nucleoside carriers in the tumor cell membrane) and/or efflux of the drugs by the
different tumor cell lines, ii) differences in levels and activity of metabolic enzymes
that intracellularly convert the particular nucleotide prodrug to the parent nucleotide

5’-monophosphate, and/or iii) differences in nucleotide kinases and other enzymes
that may convert the nucleoside 5’-monophosphate to the eventual 5’-triphosphate (i.e.
nucleoside 5'-monophosphate kinases and nucleoside 5’-diphosphate kinases) or to the
free nucleoside or nucleobase (i.e. 5’-nucleotidases, thymidine phosphorylase). Thus,
a complex interplay of a broad variety of enzymes in the particular tumor cell lines
will play a determining role in the eventual cytostatic activity of the nucleotide
prodrugs. It would therefore be of importance to identify these different factors to
clarify and understand the underlying metabolic processes that lead to the cytostatic
activity of the different ProTides.
Moreover, it has been suggested that anticancer activity of 6-substituted-FUR
analogues might be exerted via inhibition of thymidylate synthase (TS). These 6-
modified nucleosides would be intracellularly degraded to their nucleic bases by
thymidine phosphorylase (TPase) and further transformed to the corresponding 2’-
deoxynucleosides as acceptable substrates for TS.16 In the studies of 5-fluoro-2’-
deoxyuridine (FdUrd) ProTides, we have previously reported that FdUrd ProTides are
completely stable in the presence of TP and uridine phosphorylase (UP).33 In this
view, it might be speculated that the ProTide promoiety introduced into the 6-
substituted-5-fluorouridine analogues can potentially compromise activity of such
compounds as it would prevent their conversion to 6-substituted-5-fluorouracil. Thus,
a formation of 6-substituted-2’-deoxy-5-fluorouridine derivatives and their
corresponding monophosphate forms would be impaired resulting in potential lack of
TS inhibition.
Antiviral Activity In vitro
ODCase has been considered as a potential target for agents directed against RNA
viruses such as flaviviruses, and togaviruses.34 Numerous pyrimidine-nucleosides and
their derivatives including the phosphoramidate prodrug 6-aza-uridine-5’-(ethyl-
methoxyalaninyl)phosphate were shown to exhibit antiviral (RNA) activity in vitro
and in vivo.14c In addition, 6-azauridine was also reported as an in vitro inhibitor of
Chikungunya virus (CHIKV) via inhibition of host ODCase rather than by inhibiting
viral specific enzymes.35a,b CHIKV as a re-emerging RNA virus for which currently
there is no approved treatment or vaccination,36 is considered as a global health
concern. Although, the recent expanding knowledge about the CHIKV genome allows

design of inhibitors that would target individual viral enzymes,37a-c there is continuous
need for the discovery of novel anti-CHIKV agents. In this view, we evaluated
selected nucleosides (6, 7) and their ProTides 32, 33, 35-37 against Chikungunya
virus in cell culture. Most of the compounds tested in the cytopathicity (CPE)-based
assay were devoid of antiviral activity (EC50 of >200 M in comparison with the
control 6-azauridine EC50 = 0.468 M). However, five compounds (6, 32, 7, 35 and
37) proved to be cytotoxic in a CC50 range of 9.4-82 M. A significant difference in
cytotoxicity was observed for the 6-alkylated nucleoside analogues 6-methyl-FUR (7,
9.0 M) versus 6-ethyl-FUR (8, >200 M) as well as their corresponding ProTides, in
particular 36 (>200 M) and 37 (53 M), respectively.
Table 2. Antiviral activity and cytotoxicity in Vero cells of nucleosides 6, 7 and 8 and
ProTides 32, 33, 35-37.
EC50 or compound concentration required to inhibit the Chikungunya virus-induced
cytopathic effect of 50%. The compounds were added to the cells in triplicate and
tested in 6 serial concentrations from 198 M to 0.0196 M.

Prodrugs such as phosphoramidates exert their biological activity after metabolic
activation38 and intracellular release of the monophosphate form, which would next
be phosphorylated to their di- and -triphosphates. In general, the first step in the
activation pathway for ProTides is believed to be a hydrolysis of an ester moiety in
the amino acid part of the prodrug to form the intermediate 29-A (Scheme 3). This
step is mediated by a carboxyesterase-type enzyme and is followed by a spontaneous
cyclization leading to a displacement of an aryl moiety via an internal nucleophilic
attack of the carboxylate residue on the phosphorus to yield an unstable cyclic
intermediate 29-B. In the following two final steps, the cyclic anhydride 29-B is
hydrolyzed to the intermediate 29-C, which further give rise to the corresponding
monophosphate 29-D upon P-N bond cleavage mediated by the phosphoramidase-
type enzyme.39 In order to assess whether also 6-substituted-FUR ProTides act as a
good substrate for the carboxyesterase-type enzyme and hence would be activated by
the same common pathway, we performed the carboxypeptidase Y assay on one of
the 6-N3-FUR ProTide, the compound 29 using a reported assay procedure.40 Thus,
compound 29 dissolved in acetone-d6 in the presence of Trizma buffer (pH 7.6) was
treated with carboxypeptidase Y and submitted to 31P NMR analysis over 14 h (Figure
5). Two peaks recorded in the blank spectrum at P 3.64 and 3.82 ppm, correlate to the
diastereoisomers of the parent ProTide 29. Within the first 10 min of the experiment
the prodrug 29 was rapidly hydrolysed to the first metabolite 29-A, lacking the ester
moiety (represented as two signals at P 4.68 and 4.87 ppm), which further was
processed to the corresponding metabolite 29-C (single peak at P 7.17 ppm). A
complete conversion of the ProTide 29 to 29-C occurred within approximately 60 min
with an estimated half-life of less than 5 min.
Scheme 3. Proposed Activation Route of 6-N3-FUR ProTide 29.

29
29-A
4.87 and 4.68 ppm
29-C
7.17 ppm
3.82 and 3.64 ppm
C
59 mins
45 mins
31 mins
17 mins
10 mins
Blank
B
A
Figure 5. Carboxypeptidase Y-mediated cleavage of ProTide 29 as monitored by 31P
NMR.

Docking Studies: Human Hint Enzyme
The last enzymatic step required for the activation of phosphoramidate prodrugs is the
cleavage of the P-N bond catalysed by the phosphoramidase-type enzyme, which
belongs to the human histidine triad nucleotide-binding protein (Hint).41 The
efficiency of this step determines the eventual activity of ProTides since during this
step the free nucleoside monophosphate form is intracellularly delivered. In order to
investigate the potential interaction between Hint protein and metabolite 29-C and to
asses its potentiality as a substrate of this enzyme, a series of molecular docking
studies using the human Hint enzyme (PDB id: 1KPF)42 were performed. The
catalytic active-site is well-defined by the co-crystallized adenosine monophosphate
(AMP), with the three histidine residues interacting with the substrate and Ser107, an
important amino acid reported as playing a central role in catalysing the P-N bond
cleavage through an acid-base catalysis,43 making an interaction with the phosphate
head. Figure 6 shows the metabolite 29-C proposed binding mode into the Hint
enzyme active pocket. The nucleobase and the sugar are oriented in a different
manner if compared to the AMP substrate thus forcing the phosphate moiety in a not-
ideal position for the cleavage of the P-N bond (lacking direct interaction with
Ser107). This results in a binding that could be considered as being not optimal for
the proper enzyme catalytic activity. The docking results seem to suggest that 29-C
and, as a consequence, the other members of this new ProTide series, might not be
optimal substrates for Hint. Therefore, the release of the monophosphate form could
be drastically reduced or even completely impeded. These findings are in line with
previously reported data in which the Hint enzyme has been found to have a lower
affinity for pyrimidine than purine derivatives43,44 and could potentially explain the
substantial reduction or even the total lack of cytostatic activity of 6-substituted-FUR
ProTides in general when compared with the parent nucleoside.

Figure 6. Proposed binding mode of metabolite 29-C (carbon atoms in orange) in the
Hint-1 enzyme. The nucleobase and the sugar occupy the binding site in a different
manner when compared with AMP (carbon atoms in green), forcing the P-N bond
away from the catalytic Ser107.
Conclusion
In conclusion, we report on the application of the ProTide approach to 6-
substituted-5-fluorouridine nucleoside analogues bearing at the C6-position of the 5-
fluorouridine scaffold small substituents such as iodo, azido, methyl and ethyl with
the aim to prepare potential anticancer and anti-viral agents. It is worth mentioning
that during the one-pot synthesis of the 6-alkyl substituted-5-fluorouridine analogues
using methyl iodide and LDA, two derivatives were formed and isolated, being the
target 6-methyl- and 6-ethyl-5-fluorouridine (as a side-product), respectively. In
general, in our in vitro studies, 6-substituted-5-fluorouridine ProTides revealed to be
less active in comparison with the parent nucleosides. Among four different 6-
substituted-5-fluorouridine ProTide series, only the 6-iodo- (27) and 6-azido- (31 and
32) compounds were equipotent to the parent nucleosides 5 and 6. The differences in
and/or lack of anticancer and anti-CHIKV activity in most cases may indicate either
differences in drug uptake depending on the tumor cell, differences in levels and
activity of metabolic enzymes and/or poor bioactivation and thus inefficient delivery
of a free nucleoside 5’-monophosphate form, respectively. Although carboxypeptidase

Y was able to efficiently activate these phosphoramidate prodrugs to their nucleoside
aminoacid phosphate key metabolite, the ProTides seem to be poor, if any substrate
for the Hint enzyme. The HINT docking studies performed with 6-azido-flurouridine
metabolite indeed showed that structures bearing modifications at the C6-position in a
nucleobase moiety are not recognized as good substrates for the phosphoramidase-
type Hint enzyme. Overall, the application of the phosphoramidate approach to 6-
substituted-5-fluorouridine nucleosides was only modestly successful since none of
the ProTides tested showed a significant boost in cytostatic activity against a broad
panel of cancer cell lines in comparison with the parent nucleoside analogues. These
finding should be kept in mind for the design of novel phosphoramidate ProTides.
Experimental Section
MTS Cell Viability Assay. The assay was contracted and carried-out by
WuXi AppTec (Shanghai) Co., Ltd. The tumour cell lines MCF-7, SW620, Mia-Pa-
Ca, MV4-11, HEL92.1.7, RL and HS445 were seeded at cell densities of 0.5 to 100 x
103 cells/well in a 96-well plate the day before drug incubation. Then the plates were
incubated for 72 hours with the different concentrations of compound to be tested.
After the incubation period, 50 L of MTS was added and the tumour cells were
incubated for 4 h at 37 C. The data were read and collected by a Spectra Max 340
Absorbance Microplate Reader. The compounds were tested in duplicate with 9 serial
concentrations (3.16-fold titrations with 198 μM as the highest concentration), and the
data were analyzed by XL-fit software.
Chikungunya CPE-based EC50 Assay. The assay was contracted and carried-
out by IBT Bioservices. Vero cells were seeded in 96-well plates and incubated
overnight. The next day 6 serial dilutions (starting at 198 μM with 6.32-fold dilutions)
of the test compounds and a control compound (6-azauridine) were prepared in
culture medium. The growth medium was aspirated from the cells and the compound
dilutions were added to the cells in triplicate for a one-hour incubation period.
Thereafter, the virus was added at a predetermined MOI (0.01) and the cells were
incubated for 3 days. The cell cultures were then fixed and stained with crystal violet
in glutaric dialdehyde solution. The optical density was determined and the EC50’s
were calculated using the uninfected (cell only) control as 0% CPE and the controls
without compound (virus only) as 100% CPE using a 4-PL curve fit of the OD.

Cytotoxicity Assay. The assay was contracted and carried-out by IBT
Bioservices. Vero cells were seeded in 96-well plates and incubated overnight. The
next day serial dilutions of the test compounds and a control compound (6-azauridine)
were prepared. The growth medium was aspirated from the cells and the compound
dilutions were added in triplicate. Cells that were incubated with medium only were
used for generating the 0% cytotoxicity data. Medium was aspirated and cells were
lysed for evaluation of the ATP content using Promega’s CelltiterGlo kit on day 3.
The resulting luciferase luminescence was quantified and used to calculate the CC50
using a 4-PL curve fit of the OD.
Carboxypeptidase Y (EC 3.4.16.1) Assay. The experiment was carried-out
by dissolving ProTide 29 (5.0 mg) in acetone-d6 (0.15 mL) followed by addition of
31
0.30 mL of Trizma buffer (pH 7.6). After recording the control P NMR at 25 C, a
previously defrosted carboxypeptidase Y (0.1 mg dissolved in 0.15 mL of Trizma)
was added to the sample, which was then immediately submitted to the 31P NMR
analyses (at 25 C). The spectra were recorded every 7 minutes and followed 14
31
hours. P NMR recorded data were processed and analyzed with the Bruker Topspin
2.1 program.
Chemistry. General Procedures. Solvents and Reagents. The following anhydrous
solvents were purchased from Sigma-Aldrich: dichloromethane (CH2Cl2), diethyl
ether (Et2O), tetrahydrofuran (THF), dimethylformamide (DMF), and any other
reagents used. Amino acid esters commercially available were purchased from
Novabiochem. All reagents commercially available were used without further
purification.
Thin Layer Chromatography (TLC).
Precoated aluminum backed plates (60 F254, 0.2 mm thickness, Merck) were
visualized under both short- and long-wave ultraviolet light (254 and 366 nm).
Preparative TLC plates (20 cm × 20 cm, 500-2000 μm) were purchased from Merck.
Flash Column Chromatography. Flash column chromatography was carried-out
using silica gel supplied by Fisher (60A, 35-70 μm). Glass columns were slurry-
packed using the appropriate eluent with the sample being loaded as a concentrated
solution in the same eluent or preadsorbed onto silica gel. Fractions containing the
product were identified by TLC, pooled and the solvent was removed in vacuo.

High Performance Liquid Chromatography (HPLC). The purity of the final
compounds was verified to be >95% by HPLC analysis using either i)
ThermoSCIENTIFIC, SPECTRA SYSTEM P4000, detector SPECTRA SYSTEM
UV2000, Varian Pursuit XRs 5 C18, 150 x 4.6 mm (as an analytical column) or ii)
Varian Prostar (LC Workstation-Varian Prostar 335 LC detector), Thermo
SCIENTIFIC Hypersil Gold C18, 5, 150 x 4.6 mm (as an analytical column). For the
method of elution, see the experimental part.
1
13
Nuclear Magnetic Resonance (NMR). H NMR (500 MHz), C NMR (125 MHz),
19
31P NMR (202 MHz) and F NMR (470 MHz) were recorded on a Bruker Avance
500 MHz spectrometer at 25 ºC. Chemical shifts (δ) are quoted in parts per million
(ppm) relative to internal MeOH-d4 (δ 3.34 1H-NMR, δ 49.86 13C-NMR) and CHCl3-
1
13
31
d4 (δ 7.26 H NMR, δ 77.36 C NMR) or external 85 % H3PO4 (δ 0.00 P NMR).
Coupling constants (J) are measured in Hertz. The following abbreviations are used in
the assignment of the NMR signals: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), bs (broad singlet), dd (doublet of doublet), dt (doublet of triplet), app
1
13
(apparent). The assignment of the signals in H NMR and C NMR was done based
on the analysis of coupling constants and additional two-dimensional experiments
(COSY, HSQC, HMBC, PENDANT).
Mass spectromethry (MS). Low resolution mass spectra were performed on Bruker
Daltonics microTof-LC, (atmospheric pressure ionization, electron spray mass
spectroscopy) in either positive or negative mode.
Purity of final compounds. The ≥95% purity of all the final compounds was
confirmed using HPLC analysis.
The following compounds 2’,3’-O-isopropylidene-5-fluoro-uridine (18), 5’-O-
(t-butyldimethylsilyl)-2’,3’-O-isopropylidene-5-fluoro-uridine (19), fully protected 6-
substituted nucleosides 20-23 and their deprotected analogues 5-8 were prepared
according to the slightly modified procedures previously reported in the literature and
the experimental data are in agreement with the data reported.16
5’-O-(t-Butyldimethylsilyl)-2’,3’-O-isopropylidene-5-fluoro-uridine (19). To a
stirred suspension of 5-fluorouridine (3.0 g, 11.4 mmol) in anhydrous acetone (120
mL) H2SO4 (1.5 mL) was added dropwise at 0C. The reaction mixture was stirred for
2 h at room temperature. The mixture was then neutralized with 6N NH4OH and

evaporated to afford crude product which was purified by column chromatography
with gradient of MeOH in DCM (5 to 8%) to yield 2’,3’-O-isopropylidene-5-fluoro-
1
uridine (18) as a white solid (3.30 g, 96%). H NMR (500 MHz, MeOD): δ 8.18 (1H,
d, J = 7.0 Hz, H-6), 5.92 (1H, d, J = 2.5 Hz, H-1’), 4.89 (1H, dd, J = 6.5, 2.5 Hz, H-
2’), 4.87 – 4.85 (1H, apparent m, H-3’), 4.25 (1H, apparent q, J = 3.0 Hz H-4’) 3.83
(1H, dd, J = 12.0, 3.0 Hz, 1 x H-5’), 3.75 (1H, dd, J = 12.0, 3.0 Hz, 1 x H-5’), 1.56
(3H, s, CH3), 1.37 (3H, s, CH3). A solution of 18 (3.30 g, 10.91 mmol) in anhydrous
DCM (130 mL) was treated with imidazole (1.48 g, 21.83 mmol), and TBDMSCl
(1.64 g, 10.91 mmol) at 0C. The reaction mixture was allowed to warm to room
temperature and was stirred for 3 h. After solvent evaporation, the crude residue was
re-dissolved in EtOAc (60 mL), washed with water (30 mL), brine (30 mL), and dried
with Na2SO4. Evaporation of the solvent followed by purification by column
chromatography with EtOAc/Hexane (3:7) gave 5’-O-(t-butyldimethylsilyl)-2’,3’-O-
1
isopropylidene-5-fluoro-uridine as a foam (4.08 g, 90%). H NMR (500 MHz,
CDCl3): δ 7.99 (1H, d, J = 7.0 Hz, H-6), 5.87 (1H, d, J = 1.5 Hz, H-1’), 4.84 – 4.79
(2H, m, H-2’, H-3’), 4.36 (1H, apparent q, J = 3.0 Hz H-4’), 3.98 (1H, d, J = 11.5, 3.0
Hz, 1 x H-5’), 3.87 (1H, d, J = 11.5, 3.0 Hz, 1 x H-5’), 1.56 (3H, s, CH3), 1.37 (3H, s,
CH3), 0.94 (s, 9H, C(CH3)3), 0.14 (6H, s, Si(CH3)2); 13C NMR (125 MHz, MeOD): δC
1
158.27 (d, 2JC-F = 23.5 Hz, C-4), 149.40 (C-2), 140.30 (d, JC-F = 231.0 Hz, C-5),
125.31 (d, 2JC-F = 34.5 Hz, C-6), 113.45 (C(CH3)3), 91.81 (C-1’), 87.23 (C-4’), 85.17
(C-2’), 80.72 (C-3’), 63.27 (C-5’), 26.08 (CH3), 24.96 (C(CH3)3), 24.07 (CH3), -6.81
(Si(CH3)2).
5’-O-(t-Butyldimethylsilyl)-2’,3’-O-isopropylidene-5-fluoro-6-iodouridine (20). 5’-
O-(t-Butyldimethylsilyl)-2’,3’-O-isopropylidene-5-fluoro-uridine 19 (3.1 g, 7.44
mmol) was dissolved in anhydrous THF (40 mL) and at -78 C was treated with
dropwise addition of LDA (11.16 mL, 22.32 mmol, 2.0 M solution in THF). After
stirring for 1 h, iodine (2.83 g, 11.2 mmol) dissolved in anhydrous THF (50 mL) was
added slowly at -78 C and the resulting mixture was stirred for an additional 6 h in
dark. The reaction was quenched with water and brought to room temperature and
diluted with EtOAc (80 mL). The organic layer was washed with water (40 mL),
brine (40 mL), and dried over Na2SO4. The solvent was evaporated and further
purified by column chromatography using EtOAc/Hexane (3:7) as an eluent to afford
1
the product as an yellow foam (2.17 g, 54%). H NMR (500 MHz, CDCl3): δ 8.87

(1H, bs, NH), 6.10 (1H, d, J = 1.5 Hz, H-1’), 5.22 (1H, dd, J = 6.5, 1.5 Hz, H-2’), 4.83
(1H, dd, J = 6.0, 4.0 Hz, H-3’), 4.21 – 4.18 (1H, m, H-4’) 3.84 – 3.77 (2H, m, 2 x H-
5’), 1.58 (3H, s, CH3), 1.37 (3H, s, CH3), 0.90 (s, 9H, C(CH3)3), 0.07 (6H, s,
Si(CH3)2).
5-Fluoro-6-Iodouridine (5) A stirred solution of 5’-O-(t-butyldimethylsilyl)-2’,3’-O-
isopropylidene-5-fluoro-6-iodouridine 20 (2.0 g, 3.68 mmol) in water (6 mL) was
treated with 50% aqueous (6 mL) at 0C, brought to room temperature, and stirred for
an additional 2 h in dark. The reaction mixture was concentrated under vacuum and
was purified by column chromatography with a gradient of MeOH in DCM (5% to
10%) to give a product as a yellowish solid (1.24 g, 87%). 1H NMR (500 MHz,
MeOD): δ 5.96 (1H, d, J = 3.5 Hz, H-1’), 4.76 (1H, dd, J = 6.5, 3.5 Hz, H-2’), 4.36
(1H, t, J = 6.5 Hz, H-3’), 3.92 (1H, td, J = 6.0, 3.0 Hz, H-4’) 3.83 (1H, dd, J = 12.0,
3.0 Hz, 1 x H-5’), 3.70 (1H, dd, J = 12.0, 6.0 Hz, 1 x H-5’); 13C NMR (125 MHz,
1
MeOD): δC 156.05 (d, 2JC-F = 29.0 Hz, C-4), 148.74 (C-2), 145.29 (d, JC-F = 228.0
2
Hz, C-5), 105.17 (d, JC-F = 39.0 Hz, C-6), 103.16 (C-1’), 86.29 (C-4’), 73.42 (C-2’),
71.32 (C-3’), 63.71 (C-5’); MS (ES+) m/z: 387.96 (M + Na+, 100%), Accurate mass:
C9H10FIN2O6 required 387.96 found 410 (M + Na+).
5’-O-(t-Butyldimethylsilyl)-2’,3’-O-isopropylidene-5-fluoro-6-azidouridine (21).
The compound 20 (1.86 g, 3.43 mmol) dissolved in anhydrous DMF (15 mL) was
treated with NaN3 (0.22 g, 3.43 mmol). The reaction mixture was stirred for 3 h in
dark. After that time, the solvent was evaporated, and the residue was re-dissolved in
EtOAc (30 mL), washed with brine, and dried over Na2SO4. The combined organic
layers were evaporated and to afford a yellowish residue which was purified by
column chromatography using DCM/MeOH (99:1) as an eluent to give compound 2
1
as a light-yellow solid (1.16 g, 74%). H NMR (500 MHz, CDCl3): δ 9.40 (1H, s,
NH), 6.08 (1H, d, J = 1.5 Hz, H-1’), 5.15 (1H, dd, J = 6.5, 1.5 Hz, H-2’), 4.80 (1H, dd,
J = 6.5, 4.5 Hz, H-3’), 4.16 – 4.12 (1H, m, H-4’), 3.84 – 3.77 (2H, m, 2 x H-5’), 1.56
(3H, s, CH3), 1.35 (3H, s, CH3), 0.90 (s, 9H, C(CH3)3), 0.07 (6H, s, Si(CH3)2). MS
(ES+) m/z: 480.2 (M + Na+, 20%), Accurate mass: C18H28FN5O6Si required 457.52
found 480.2 (M + Na+), 937.4 (2 x M + Na+).
5-Fluoro-6-azidouridine (6) A stirred solution of 5’-O-(t-butyldimethylsilyl)-2’,3’-O-
isopropylidene-5-fluoro-6-azidouridine 21 (1.10 g, 2.18 mmol) in water (10 mL) was
treated with 50% aqueous (10 mL) at 0C, brought to room temperature, and stirred

for an additional 2 h in dark. The reaction mixture was concentrated under vacuum
and was purified by column chromatography with a gradient of MeOH in DCM (6%
1
to 12%) to give a product as a yellowish solid (0.56 g, 85%). H NMR (500 MHz,
MeOD): δ 5.95 (1H, d, J = 3.5 Hz, H-1’), 4.65 (1H, dd, J = 6.5, 3.5 Hz, H-2’), 4.32
(1H, t, J = 6.5 Hz, H-3’), 3.86 – 3.81 (2H, m, H-4’, 1 x H-5’), 3.70 – 3.67 (1H, m, 1 x
H-5’); 13C NMR (125 MHz, MeOD): δC 156.54 (d, 2JC-F = 26.0 Hz, C-4), 148.40 (C-
1
2
2), 137.20 (d, JC-F = 213.0 Hz, C-5), 132.8 (d, JC-F = 24.0 Hz, C-6), 91.60 (C-1’),
86.29 (C-4’), 84.50 (C-2’), 71.94 (C-3’), 69.0 (C-5’); MS (ES+) m/z: 326.20 (M + Na+,
100%), Accurate mass: C9H10FN5O6 required 303.06 found 326.20 (M + Na+).
5’-O-(t-Butyldimethylsilyl)-2’,3’-O-isopropylidene-5-fluoro-6-methyluridine (22)
and
5’-O-(t-Butyldimethylsilyl)-2’,3’-O-isopropylidene-5-fluoro-6-ethyluridine
(23). 5’-O-(t-Butyldimethylsilyl)-2’,3’-O-isopropylidene-5-fluoro-uridine 19 (3.5 g,
8.40 mmol) was dissolved in anhydrous THF (40 mL) and at -78 C was treated with
dropwise addition of LDA (12.6 mL, 25.20 mmol, 2.0 M solution in THF). After
stirring for 1 h, CH3I (1.04 mL, 16.80 mmol) dissolved in anhydrous THF (5 mL) was
added and, and the mixture was stirred for 5 h at -78 C. The reaction was quenched
with water (7 mL) and allowed to warm to rt and then dissolved in ethyl acetate (100
mL). The organic layer was washed with water (40 mL), brine (40 mL), and dried
over NaSO4. The solvent was evaporated and the residue was purified by column
chromatography using hexane/EtOAc (7:3) as an eluent to give the product 23 (fast
eluting fraction, 0.75 g, 20%), and the product 22 (slow eluting fraction, 1.53 g, 42%),
both as a white foam. 1H NMR (500 MHz, CDCl3): δ 10.40 (1H, s, NH), 5.63 (1H, d,
J = 1.0 Hz, H-1’), 5.18 (1H, dd, J = 6.0, 1.0 Hz, H-2’), 4.78 (1H, dd, J = 6.0, 4.5 Hz,
H-3’), 4.13 – 4.10 (1H, m, H-4’), 3.80 – 3.74 (2H, m, 2 x H-5’), 2.31 (3H, d, JH-F = 3.5
Hz, C-6-CH3), 1.50 (3H, s, CH3), 1.30 (3H, s, CH3), 0.83 (s, 9H, C(CH3)3), 0.02 (6H,
s, Si(CH3)2). 13C NMR (125 MHz, CDCl3): δC 156.70 (d, 2JC-F = 28.0 Hz, C-4), 149.20
1
(C-2), 138.40 (d, JC-F = 228.0 Hz, C-5), 137.85 (d, 2JC-F = 24.3 Hz, C-6), 113.78
(C(CH3)3), 91.89 (C-1’), 89.58 (C-4’), 84.12 (C-2’), 81.70 (C-3’), 64.10 (C-5’), 27.20
(CH3), 25.87 (C(CH3)3), 25.31 (CH3), 11.92 (d, JC-F = 2.9 Hz, C-6-CH3), -5.31
(Si(CH3)2).
5’-O-(t-Butyldimethylsilyl)-2’,3’-O-isopropylidene-5-fluoro-6-ethyluridine (23). δ
9.70 (1H, s, NH), 5.57 (1H, d, J = 1.5 Hz, H-1’), 5.16 (1H, dd, J = 6.5, 1.5 Hz, H-2’),
4.77 (1H, dd, J = 6.5, 4.5 Hz, H-3’), 4.14 – 4.10 (1H, m, H-4’), 3.81 – 3.75 (2H, m, 2

x H-5’), 2.82 – 2.65 (2H, m, C-6-CH2CH3), 1.51 (3H, s, CH3), 1.30 (3H, s, CH3), 1.27
13
(3H, t, J = 8.0 Hz, C-6-CH2CH3), 0.83 (9H, s, C(CH3)3), 0.04 (6H, s, Si(CH3)2); C
NMR (125 MHz, CDCl3): δC 156.90 (d, 2JC-F = 27.8 Hz, C-4), 149.28 (C-2), 143.00
1
(d, 2JC-F = 23.9 Hz, C-6), 137.64 (d, JC-F = 229.3 Hz, C-5), 113.91 (C(CH3)3), 91.88
(C-1’), 89.68 (C-4’), 84.26 (C-2’), 81.93 (C-3’), 64.19 (C-5’), 27.24 (CH3), 25.92
(C(CH3)3), 25.31 (CH3), 18.79 (C-6-CH2CH3), 12.44 (C-6-CH2CH3), -5.23
(Si(CH3)2).
5-Fluoro-6-methyluridine (7). A stirred solution of 5’-O-(t-butyldimethylsilyl)-2’,3’-
O-isopropylidene-5-fluoro-6-methyluridine 22 (1.43 g, 3.32 mmol) in water (10 mL)
was treated with 50% aqueous (10 mL) at 0C, brought to room temperature, and
stirred for an additional 2 h. The reaction mixture was concentrated under vacuum
and was purified by column chromatography with a gradient of MeOH in DCM (6%
to 10%) to give a product as a yellowish solid (0.84 g, 92%). 1H NMR (500 MHz,
MeOD): δ 5.53 (1H, d, J = 4.0 Hz, H-1’), 4.78 (1H, dd, J = 6.5, 4.0 Hz, H-2’), 4.33
(1H, t, J = 6.5 Hz, H-3’), 3.93 – 3.90 (1H, m, H-4’), 3.82 (1H, dd, J = 12.0, 3.0 Hz, 1
x H-5’), 3.69 (1H, dd, J = 12.0, 5.5 Hz, 1 x H-5’), 2.38 (3H, d, JH-F = 4.0 Hz, C-6-
13
CH3); C NMR (125 MHz, MeOD): δC 158.75 (d, 2JC-F = 27.8 Hz, C-4), 150.77 (C-
1
2), 140.20 (d, 2JC-F = 24.5 Hz, C-6), 139.71 (d, JC-F = 225.5 Hz, C-5), 94.12 (C-1’),
3
86.32 (C-4’), 72.81 (C-2’), 71.40 (C-3’), 63.62 (C-5’), 12.02 (d, JC-F = 3.80 Hz, C-6-
CH3); MS (ES+) m/z: 299.2 (M + Na+, 100%), Accurate mass: C10H13FN2O6 required
276.22 found 299 (M + Na+). Reverse-phase HPLC, eluting with H2O/AcCN from
100/0 to 0/100 in 35 min, F = 1 mL/min,  = 254, tR = 5.01 min.
5-Fluoro-6-ethyluridine (8). A stirred solution of 5’-O-(t-butyldimethylsilyl)-2’,3’-O-
isopropylidene-5-fluoro-6-ethyluridine 23 (0.75 g, 1.68 mmol) in water (10 mL) was
treated with 50% aqueous (10 mL) at 0C, brought to room temperature, and stirred
for an additional 2 h. The reaction mixture was concentrated under vacuum and was
purified by column chromatography with a gradient of MeOH in DCM (6% to 10%)
to give a product as a yellowish solid (0.38 g, 80%). 1H NMR (500 MHz, MeOD): δ
5.34 (1H, d, J = 4.0 Hz, H-1’), 4.64 (1H, dd, J = 6.5, 4.0 Hz, H-2’), 4.19 (1H, t, J = 6.5
Hz, H-3’), 3.79 – 3.76 (1H, m, H-4’), 3.67 (1H, dd, J = 12.0, 3.0 Hz, 1 x H-5’), 3.59
(1H, dd, J = 12.0, 5.5 Hz, 1 x H-5’), 2.73 – 2.60 (2H, m, C-6-CH2CH3), 1.18 (3H, t, J
= 7.5 Hz, C-6-CH2CH3); 13C NMR (125 MHz, MeOD): δC 157.50 (d, 2JC-F = 28.8 Hz,
1
C-4), 149.52 (C-2), 143.68 (d, 2JC-F = 23.8 Hz, C-6), 137.62 (d, JC-F = 225.0 Hz, C-

5), 92.71 (C-1’), 85.01 (C-4’), 71.38 (C-2’), 70.06 (C-3’), 62.38 (C-5’), 18.25 (d, 3JC-F
= 2.5 Hz, C-6-CH2CH3), 11.31 (C-6-CH2CH3); MS (ES+) m/z: 313.25 (M + Na+,
100%), Accurate mass: C11H15FN2O6 required 290 found 313 (M + Na+). Reverse-
phase HPLC, eluting with H2O/AcCN from 100/0 to 0/100 in 35 min, F = 1 mL/min,
 = 254, tR = 6.05 min.
General Method for the Preparation of phosphorochloridates (24).45 Anhydrous
triethylamine (2.0 mol eq.) was added dropwise at -78 ºC to a stirred solution of the
appropriate aryl dichlorophosphate (1.0 mol eq.) and an appropriate amino acid ester
(1.0 mol eq.) in anhydrous DCM under argon atmosphere. Following the addition, the
reaction mixture was allowed to slowly warm to room temperature and stirred for 1-2
31
hours. A formation of a desired compound was monitored by P NMR. After the
reaction was completed, the solvent was evaporated under reduced pressure and the
resulting residue was re-dissolved in anhydrous Et2O and filtered. The filtrate was
reduced to dryness to give a crude product as an oil, which was in some cases used
without further purification in the next step. Most of aryl phosphorochloridates, in
particular those obtained from the amino acid tosylate salt were purified by flash
column chromatography using EtOAc/Hexane (7:3) as an eluent.
General method for the preparation of phosphoramidates.
To a stirring solution of nucleoside analogue (1.0 mol/eq.) in anhydrous THF, an
appropriate phosphorochloridate (3.0 mol/eq.) dissolved in anhydrous THF was added
dropwise under an argon atmosphere. To that reaction mixture NMI (5.0 mol/eq.) was
added dropwise over 5 minutes at –78°C under an argon atmosphere. After 15
minutes, the reaction mixture was let to rise to room temperature and stirred overnight
(16 - 18h). The solvent was removed under reduced pressure and the residue was re-
dissolved in DCM and washed with 0.5 M HCl (3 x 3 mL). The organic layer was
dried over MgSO4, filtered, reduced to dryness and purified by column
chromatography with gradient of eluent (DCM/MeOH 99:1 to 97:3 to 95:5) followed
by preparative TLC purification (DCM/MeOH 95:5).
5-Fluoro-6-iodouridine-5’-O-[phenyl-(pentoxy-L-alaninyl)]
phosphate
(25).
Prepared according to the standard procedure from 5-fluoro-6-iodouridine 5 (0.20 g,
0.51 mmol), NMI (0.20 mL, 2.57 mmol), phenyl-(pentoxy-L-alaninyl)-

phosphorochloridate (0.43 g, 1.03 mmol). After column purification on silica gel 25
was obtained as a yellowish solid (0.021 g, 6%). 31P NMR (202 MHz, MeOD): δP
3.52, 3.46; 1H NMR (500 MHz, MeOD): δH 7.36 – 7.33 (2H, m, H-Ar), 7.24 – 7.22
(2H, m, H-Ar), 7.19 – 7.16 (1H, m, H-Ar), 5.96 (1H, apparent t, J = 3.0 Hz, H-1’),
4.73, 4.70 (1H, 2 x dd, J = 6.0, 2.5 Hz, H-2’), 4.53 – 4.34 (2H, m, H-3’, 1 x H-5’),
4.27 – 4.22 (1H, m, 1 x H-5’), 4.18 – 4.02 (2H, m, OCH2), 3.98 – 3.94 (1H, m, H-4’),
3.64 – 3.59 (1H, m, NHCHCH3), 1.71 – 1.60 (6H, m, 3 x CH2 ester), 1.39 – 1.32 (3H,
m, NHCHCH3), 0.96 – 0.91 (3H, m, CH3); 13C NMR (125 MHz, MeOD): δC 173.59,
172.90 (C=O ester), 156.56 (C-4), 151.75, 151.26 (C-2), 149.19 (C-Ar), 145.28 (d,
1JC-F = 205.0 Hz, C-5), 129.28, 127.57, 124.61, 120.12 (CH-Ar), 108.98 (C-6), 102.19
(C-1’), 83.02, 82.51 (C-4’), 72.29 (C-2’), 70.59, 69.89 (C-3’), 65.08 (C-5’), 64.24
(OCH2), 50.06 (NHCHCH3), 28.00, 21.96, 21.06 (CH2), 19.37 (NHCHCH3), 12.90
(O(CH2)4CH3); MS (ES+) m/z: 708.0 (M + Na+, 100%), Accurate mass:
C23H30FIN3O10P required 685.38 found 708.0 (M + Na+); Reverse-phase HPLC,
eluting with H2O/AcCN from 100/0 to 0/100 in 35 min, F = 1 mL/min,  = 254, two
peaks for two diastereoisomers with tR = 17.11, 17.43 min.
5-Fluoro-6-iodouridine-5’-O-[phenyl-(cyclohexoxy-L-alaninyl)] phosphate (26).
Prepared according to the standard procedure from 5-fluoro-6-iodouridine 5 (0.23 g,
0.59 mmol), NMI (0.23 mL, 2.96 mmol), phenyl-(cyclohexoxy-L-alaninyl)-
phosphorochloridate (0.40 g, 1.18 mmol). After column purification on silica gel 26
was obtained as a yellowish solid (0.028 g, 7%). 31P NMR (202 MHz, MeOD): δ
1
3.68, 3.54; H NMR (500 MHz, MeOD): δ 7.37 – 7.33 (2H, m, H-Ar), 7.24 – 7.23
(2H, m, H-Ar), 7.20 – 7.17 (1H, m, H-Ar), 5.97 – 5.95 (1H, m, H-1’), 4.51 – 4.34 (2H,
m, H-2’, OCH ester), 4.51 – 4.34 (2H, m, H-3’, 1 x H-5’), 4.28 – 4.23 (1H, m, 1 x H-
5’), 4.05 – 4.01 (1H, m, H-4’), 3.96 – 3.89 (1H, m, NHCHCH3), 1.83 – 1.73 (5H, m,
CH2 ester), 1.58 – 1.54 (1H, m, CH2 ester), 1.49 – 1.38 (4H, m, CH2 ester), 1.37, 1.32
13
(3H, 2 x dd, J = 7.0, 1.0 Hz, NHCHCH3); C NMR (125 MHz, MeOD): δC 174.52,
3
174.4 (2 x d, JC-P = 5.4 Hz, C=O ester), 156.40 (d, 2JC-F = 32.0 Hz, C-4), 152.30,
4
152.25 (2 x d, JC-F = 4.70 Hz, C-2), 148.67, 148.52 (C-Ar), 145.36, 145.30 (2 x d,
3
1JC-F = 228.0 Hz, C-5), 130.69, 129.34, 126.03 (CH-Ar), 121.68 (d, JC-P = 4.6 Hz,
2
CH-Ar), 104.94, 104.78 (2 x d, JC-F = 39.0 Hz, C-6), 103.58, 103.43 (C-1’), 84.01,
3
83.55 (d, JC-P = 7.10 Hz, C-4’), 75.01, 74.99 (OCH), 73.70, 73.55 (C-2’), 71.28,

2
2
70.69 (C-3’), 68.52, 67.48 (2 x d, JC-P = 5.5 Hz, C-5’), 51.63 (d, JC-P = 5.5 Hz,
NHCHCH3), 32.49, 32.47, 32.39, 26.45, 26.43, 26.41, 24.69, 24.62 (CH2), 20.85,
3
20.76 (2 x d, JC-P = 5.75 Hz, NHCHCH3); MS (ES+) m/z: 720.1 (M + Na+, 100%),
Accurate mass: C24H30FIN3O10P required 697.39 found 720.1 (M + Na+); Reverse-
phase HPLC, eluting with H2O/AcCN from 100/0 to 0/100 in 35 min, F = 1 mL/min,
 = 254, two peaks for two diastereoisomers with tR = 17.60, 17.64 min.
5-Fluoro-6-iodouridine-5’-O-[1-naphthyl-(benzoxy-L-alaninyl)] phosphate (27).
Prepared according to the general procedure from 5-fluoro-6-iodouridine 5 (0.18 g,
0.46 mmol), NMI (0.18 mL, 2.32 mmol), 1-naphthyl-(benzoxy-L-alaninyl)-
phosphorochloridate (0.37 g, 0.93 mmol). After column purification on silica gel 27
31
was obtained as a yellowish solid (0.017 g, 5%). P NMR (202 MHz, MeOD): δ
1
3.89, 3.74; H NMR (500 MHz, MeOD): δ 8.19 – 8.16 (1H, m, H-Ar), 7.89 – 7.85
(1H, m, H-Ar), 7.69 – 7.65 (1H, m, H-Ar), 7.54 – 7.47 (3H, m, H-Ar), 7.41 – 7.36
(1H, m, H-Ar), 7.33 – 7.25 (5H, m, H-Ar), 5.96 – 5.94 (1H, m, H-1’), 5.01 – 4.96 (2H,
m, CH2Ph), 4.73, 4.68 (1H, 2 x dd, J = 6.5, 3.0 Hz, H-2’), 4.54 – 4.41 (2H, m, H-3’, 1
x H-5’), 4.33 – 4.27 (1H, m, 1 x H-5’), 4.12 – 4.02 (2H, m, H-4’, NHCHCH3), 1.35,
1.30 (3H, 2 x dd, J = 7.0, 0.5 Hz, NHCHCH3); 13C NMR (125 MHz, MeOD): δC
174.40, 174.26 (C=O, ester), 156.50 (C-4), 152.40, 152.20 (C-2), 148.67 (C-Ar),
3
1
137.20 (d, JC-P = 4.5 Hz, C-Ar), 136.10 (C-Ar), 135.30, 135.22 (2 × d, JC-F = 225.0
Hz, C-5), 129.68, 129.58, 129.36, 129.25, 129.26, 128.76, 128.69, 127.75, 127.71,
127.48, 127.46, 126.57, 126.54, 125.90, 122.90, 122.75 (CH-Ar), 116.30, 116.21 (2 x
3
d, JC-P = 3.4 Hz CH-Ar), 108.74 (C-6), 103.63, 103.44 (C-1’), 84.00, 83.64 (2 × d,
2
3JC-P = 7.25 Hz, C-4’), 74.05, 73.88 (C-2’), 71.28, 70.72 (C-3’), 68.52 (d, JC-P = 5.8
Hz, C-5’), 67.97, 67.85 (OCH2Ph), 67.71 (d, 2JC-P = 5.4 Hz, C-5’), 51.70 (NHCHCH3),
3
20.86, 20.74 (2 × d, JC-P = 6.4 Hz, NHCHCH3); MS (ES+) m/z: 755.42 (M + Na+,
100%), Accurate mass: C29H28FIN3O10P required 755.42 found 778.0 (M + Na+);
Reverse-phase HPLC, eluting with H2O/AcCN from 100/0 to 0/100 in 35 min, F = 1
mL/min,  = 254, two peaks for two diastereoisomers with tR = 17.21, 17.37 min.
5-Fluoro-6-azidouridine-5’-O-[phenyl-(ethoxy-L-alaninyl)] phosphate (28).
Prepared according to the general procedure from 5-fluoro-6-azidouridine 6 (0.15 g,
0.49 mmol), NMI (0.19 mL, 2.47 mmol), phenyl-(ethoxy-L-alaninyl)-
phosphorochloridate (0.28 g, 0.99 mmol). After column purification on silica gel 28
was obtained as a yellowish solid (0.014 g, 5%). 31P NMR (202 MHz, MeOD): δP

3.69, 3.53; 1H NMR (500 MHz, MeOD): δH 7.38 – 7.35 (2H, m, H-Ar), 7.25 – 7.18
(3H, m, H-Ar), 5.95, 5.92 (1H, 2 × d, J = 2.80 Hz, H-1’), 4.67 – 4.62 (1H, m, H-2’),
4.47 – 4.34 (2H, m, H-3’, 1 x H-5’), 4.28 – 4.22 (1H, m, 1 x H-5’), 4.18 – 4.10 (2H, m,
OCH2CH3), 4.00-3.92 (2H, m, NHCHCH3, H-4’), 1.36, 1.32 (3H, 2 × d, J = 7.17 Hz,
NHCHCH3), 1.27 – 1.23 (m, 3H, OCH2CH3); 13C NMR (125 MHz, MeOD): δC
3
2
175.11, 174.97 (2 × d, JC-P = 5.60 Hz, C=O, ester), 157.85 (d, JC-F = 24.1 Hz, C-4),
152.25 (apparent t, 2JC-P = 12.75 Hz, O-C-Ar), 149.32, 149.22 (C-2), 139.36 (d, 2JC-F
=
1
21.3 Hz, C-6), 136.26, 134.45 (2 × d, JC-F = 235 Hz, C-5), 130.0, 121.47, 121.43,
121.56, 121.53, 121.47, 121.43 (CH-Ar), 92.96, 92.84 (C-1’), 83.60, 83.33 (2 × d, 3JC-
2
P = 7.12 Hz, C-4’), 73.41, 73.30 (C-2’), 70.97, 70.50 (C-3’), 68.22, 67.38 (2 × d, JC-P
2
= 6.10 Hz, C-5’), 62.41, 61.37 (OCH2CH3), 51.50 (d, JC-P = 5.17 Hz, NHCHCH3),
3
20.15, 20.53 (2 × d, JC-P = 6.39 Hz, NHCHCH3), 14.97 (OCH2CH3); MS (ES+) m/z:
581.1 (M + Na+, 100%), Accurate mass: C20H24FN6O10P required 558.1 found 581.1
(M + Na+); Reverse-phase HPLC, eluting with H2O/AcCN from 100/0 to 0/100 in 35
min, F = 1 mL/min,  = 254, two peaks for two diastereoisomers with tR = 14.68,
14.95 min.
5-Fluoro-6-azidouridine-5’-O-[phenyl-(hexoxy-L-alaninyl)] phosphate (29).
Prepared according to the general procedure from 5-fluoro-6-azidouridine 6 (0.10 g,
0.33 mmol), NMI (0.13 mL, 1.65 mmol), phenyl-(hexoxy-L-alaninyl)-
phosphorochloridate (0.23 g, 0.66 mmol). After column purification on silica gel 29
was obtained as a yellowish solid (0.008 g, 4%). 31P NMR (202 MHz, MeOD): δP
3.67, 3.51; 1H NMR (500 MHz, MeOD): δH 7.38 – 7.35 (2H, m, H-Ar), 7.25 – 7.18
(3H, m, H-Ar), 5.95, 5.93 (1H, 2 × d, J = 2.75 Hz, H-1’), 4.67 – 4.62 (1H, m, H-2’),
4.47 – 4.34 (2H, m, H-3’, 1 x H-5’), 4.28 – 4.22 (1H, m, 1 x H-5’), 4.14 – 4.05 (2H, m,
OCH2), 4.00 – 3.93 (2H, m, NHCHCH3, H-4’), 1.66 – 1.60 (2H, m,
OCH2CH2(CH2)3CH3), 1.40 – 1.31 (9H, m, 3 x CH2, NHCHCH3), 0.93 – 0.90 (3H, m,
3
CH3);13C NMR (125 MHz, MeOD): δC 175.17, 175.03 (2 × d, JC-P = 5.90 Hz, C=O,
2
2
ester), 157.80 (d, JC-F = 23.7 Hz, C-4), 152.25 (apparent t, JC-P = 12.9 Hz, O-C-Ar),
1
149.42, 149.37 (C-2), 139.35 (d, 2JC-F = 20.92 Hz, C-6), 136.27, 134.47 (2 × d, JC-F
=
232.9 Hz, C-5), 130.70, 126.08, 126.04, 121.58, 121.54, 121.47, 121.43 (CH-Ar),
3
92.97, 92.84 (C-1’), 83.65, 83.33 (2 × d, JC-P = 7.11 Hz, C-4’), 73.42, 73.29 (C-2’),
2
70.96, 70.50 (C-3’), 68.24, 67.39 (2 × d, JC-P = 5.44 Hz, C-5’), 66.49, 66.45 (OCH2),
49.87 (NHCHCH3), 32.88, 32.57, (OCH2CH2), 29.66, 26.63, 26.60, 23.59, 23.58

3
(CH2), 20.70, 20.59 (2 × d, JC-P = 6.9 Hz, NHCHCH3), 14.34 (O(CH2)5CH3); MS
(ES+) m/z: 581.1 (M + Na+, 100%), Accurate mass: C24H32FN6O10P required 614.52
found 637.2 (M + Na+); Reverse-phase HPLC, eluting with H2O/AcCN from 100/0 to
0/100 in 35 min, F = 1 mL/min,  = 254, two peaks for two diastereoisomers with tR =
18.99, 19.23 min.
5-Fluoro-6-azidouridine-5’-O-[phenyl-(benzoxy-L-alaninyl)] phosphate (30).
Prepared according to the general procedure from 5-fluoro-6-azidouridine 6 (0.18 g,
0.59 mmol), NMI (0.24 mL, 2.90 mmol), phenyl-(benzoxy-L-alaninyl)-
phosphorochloridate (0.42 g, 1.18 mmol). After column purification on silica gel 30
was obtained as a yellowish solid (0.026 g, 7%). 31P NMR (202 MHz, MeOD): δP
3.71, 3.45; 1H NMR (500 MHz, MeOD): δH 7.37 – 7.32 (7H, m, H-Ar), 7.22 – 7.16
(3H, m, H-Ar), 5.96 (1H, 2 x d, J = 3.0 Hz, H-1’), 5.17 – 5.11 (2H, m, CH2Ph), 4.65,
4.62 (1H, 2 x dd, J = 6.5, 3.0 Hz, H-2’), 4.45 – 4.33 (2H, m, H-3’, 1 x H-5’), 4.26 –
4.21 (1H, m, 1 x H-5’), 4.04 – 3.95 (2H, m, NHCHCH3, H-4’), 1.37, 1.33 (3H, dd, J =
13
7.0, 1.0 Hz, NHCHCH3); C NMR (125 MHz, MeOD): δC 173.39, 173.26 (2 × d,
2
3JC-P = 4.75 Hz, C=O, ester), 157.76 (d, JC-F = 24.8 Hz, C-4), 149.26, 149.15 (C-2),
152.36 (C-Ar), 139.31 (C-6), 136.18 (C-Ar), 135.30, 135.21 (2 × d, 1JC-F = 224.0 Hz,
C-5), 130.10, 130.04, 129.58, 129.52, 128.86, 128.79, 127.65, 127.55 (CH-Ar),
120.06, 120.0 (CH-Ar), 92.90, 92.81 (C-1’), 83.45, 83.25 (C-4’), 73.45, 73.28 (C-2’),
70.94, 70.50 (C-3’), 68.50 (d, 2JC-P = 5.4 Hz, C-5’), 68.00, 67.80 (OCH2Ph), 67.70 (d,
3
2JC-P = 5.4 Hz, C-5’), 51.70 (NHCHCH3), 20.70, 20.55 (2 × d, JC-P = 6.4 Hz,
NHCHCH3); MS (ES+) m/z: 643.0 (M + Na+, 100%), Accurate mass: C25H26FN6O10P
required 620.48 found 643.0 (M + Na+); Reverse-phase HPLC, eluting with
H2O/AcCN from 100/0 to 0/100 in 35 min, F = 1 mL/min,  = 254, two peaks for two
diastereoisomers with tR = 17.60, 17.76 min.
5-Fluoro-6-azidouridine-5’-O-[1-naphthyl-(hexoxy-L-alaninyl)] phosphate (31).
Prepared according to the general procedure from 5-fluoro-6-azidouridine 6 (0.18 g,
0.59 mmol), NMI (0.23 mL, 2.97 mmol), 1-naphthyl-(hexoxy-L-alaninyl)-
phosphorochloridate (0.47 g, 1.19 mmol). After column purification on silica gel 31
31
was obtained as a yellowish solid (0.019 g, 5%). P NMR (202 MHz, MeOD): δP
3.89;1H NMR (500 MHz, MeOD): δH 8.08 – 8.06 (1H, m, H-Ar), 7.78 – 7.74 (1H, m,
H-Ar), 7.59 – 7.57 (1H, m, H-Ar), 7.45 – 7.30 (3H, m, H-Ar), 7.32 – 7.28 (1H, m, H-
Ar), 5.81, 5.79 (1H, 2 × d, J = 3.0 Hz, H-1’), 4.53, 4.50 (1H, 2 x dd, J = 6.5, 3.0 Hz,

H-2’), 4.38 – 4.26 (2H, m, H-3’, 1 x H-5’), 4.23 – 4.17 (1H, m, 1 x H-5’), 3.93 – 3.85
(4H, m, OCH2, NHCHCH3, H-4’), 1.45 – 1.38 (2H, m, OCH2CH2(CH2)3CH3), 1.24,
1.20 (3H, 2 x dd, J = 7.0, 0.5 Hz, NHCHCH3), 1.17 – 1.10 (6H, m, 3 x CH2), 0.78 –
3
0.73 (3H, m, CH3); 13C NMR (125 MHz, MeOD): δC 175.18, 175.0 (2 × d, JC-P
=
2
4.50 Hz, C=O, ester), 157.75 (d, JC-F = 24.0 Hz, C-4), 149.25, 149.17 (C-2), 148.10
(d, 2JC-P = 7.0 Hz, O-C-Ar), 139.30 (d, 2JC-F = 19.5 Hz, C-6), 136.29 (d, 3JC-P = 2.0 Hz,
1
C-Ar), 135.32, 135.29 (2 × d, JC-F = 225.0 Hz, C-5),128.87, 128.79, 127.75, 127.73,
3
127.42, 127.38 (CH-Ar), 126.54, 126.52 (2 x d, JC-P = 1.5 Hz, CH-Ar), 125.83,
122.95, 122.78, 116.23, 116.20, 116.19, 116.17 (CH-Ar), 92.98, 92.85 (C-1’), 83.68,
3
83.36 (2 × d, JC-P = 7.0 Hz, C-4’), 73.46, 73.31 (C-2’), 70.95, 70.52 (C-3’), 68.51,
2
67.66 (2 × d, JC-P = 5.5 Hz, C-5’), 66.52, 66.45 (OCH2), 51.64 (NHCHCH3), 32.57,
32.54 (OCH2CH2), 29.60, 26.60, 26.57, 23.57, 23.55 (CH2), 20.78, 20.64 (2 × d, 3JC-P
= 6.5 Hz, NHCHCH3), 14.35, 14.33 (O(CH2)5CH3); MS (ES+) m/z: 687.58 (M + Na+,
10%), Accurate mass: C28H34FN6O10P required 664.58 found 687.2 (M + Na+),
1351.4 (2 x M + Na+), Reverse-phase HPLC, eluting with H2O/AcCN from 100/0 to
0/100 in 35 min, F = 1 mL/min,  = 254, two peaks for two diastereoisomers with tR =
23.08, 23.51 min.
5-Fluoro-6-azidouridine-5’-O-[1-naphthyl-(benzoxy-L-alaninyl)] phosphate (32).
Prepared according to the general procedure from 5-fluoro-6-azidouridine 6 (0.17 g,
0.56 mmol), NMI (0.23 mL, 2.80 mmol), 1-naphthyl-(benzoxy-L-alaninyl)-
phosphorochloridate (0.45 g, 1.12 mmol). After column purification on silica gel 32
was obtained as a yellowish solid (0.029 g, 8%). 31P NMR (202 MHz, MeOD): δP
3.95, 3.79; 1H NMR (500 MHz, MeOD): δH 8.19 – 8.17 (1H, m, H-Ar), 7.89 – 7.86
(1H, m, H-Ar), 7.70 (1H, apparent d, J = 12.5 Hz, H-Ar), 7.55 – 7.48 (3H, m, H-Ar),
7.43 – 7.38 (1H, m, H-Ar), 7.32 – 7.29 (5H, m, H-Ar), 5.91, 5.90 (1H, 2 x d, J = 3.0
Hz, H-1’), 5.09 – 5.02 (2H, m, CH2Ph), 4.65, 4.60 (1H, 2 x dd, J = 6.0, 3.0 Hz, H-2’),
4.48 – 4.37 (2H, m, H-3’, 1 x H-5’), 4.33 – 4.28 (1H, m, 1 x H-5’), 4.13 – 4.05 (1H, m,
NHCHCH3), 4.02 – 3.98 (1H, m, H-4’), 1.35, 1.32 (3H, 2 x dd, J = 7.0, 1.0 Hz,
3
NHCHCH3); 13C NMR (125 MHz, MeOD): δC 173.39, 173.26 (2 × d, JC-P = 4.75
2
Hz, C=O, ester), 157.76 (d, JC-F = 24.8 Hz, C-4), 149.26, 149.15 (C-2), 148.07 (d,
2JC-P = 7.3 Hz, O-C-Ar), 139.31, 139.26 (2 x d, 2JC-F = 19.1 Hz, C-6), 137.18 (d, 3JC-P
= 4.5 Hz, C-Ar), 136.28 (C-Ar), 135.28, 135.25 (2 × d, 1JC-F = 224.8 Hz, C-5), 129.58,
129.54, 129.31, 129.23, 129.18, 128.86, 128.79, 127.75, 127.73, 127.46, 127.41,