Nucleoside mono- and diphosphate prodrugs of 2′,3′-dideoxyuridine and 2′,3′-dideoxy-2′,3′-didehydrouridine

Article abstract of DOI:10.1002/cmdc.201402295

Despite their close structural similarity to nucleoside analogues such as the anti-HIV drugs AZT and d4T, 2′,3′-dideoxyuridine (ddU) and 2′,3′-dideoxy-2′,3′-didehydrouridine (d4U) are entirely inactive against HIV in their nucleoside form. However, it has been shown that the corresponding triphosphates of these two nucleosides can effectively block HIV reverse transcriptase. Herein we report on two types of nucleotide prodrugs (cyclo-Sal and DiPPro nucleotides) of ddU and d4U to investigate their ability to overcome insufficient intracellular phosphorylation, which may be the reason behind their low anti-HIV activity. The release of the corresponding mono- and diphosphates from these compounds was demonstrated by hydrolysis studies in phosphate buffer (pH 7.3) and human CD₄⁺ T-lymphocyte CEM cell extracts. Surprisingly, however, these compounds showed low or no anti-HIV activity in tests with human CD₄⁺ T-lymphocyte CEM cells. Studies of the conversion of ddUDP and d4UDP into their triphosphate metabolites by nucleoside diphosphate kinase (NDPK) showed nearly no conversion of either diphosphate, which may be the reason for low intracellular triphosphate levels that result in low antiviral activity.

Full text of DOI:10.1002/cmdc.201402295

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DOI: 10.1002/cmdc.201402295
Nucleoside Mono- and Diphosphate Prodrugs of 2’,3’-
Dideoxyuridine and 2’,3’-Dideoxy-2’,3’-didehydrouridine
Florian Pertenbreiter,[a] Jan Balzarini,[b] and Chris Meier*[a]
Despite their close structural similarity to nucleoside analogues
such as the anti-HIV drugs AZT and d4T, 2’,3’-dideoxyuridine
(ddU) and 2’,3’-dideoxy-2’,3’-didehydrouridine (d4U) are entire-
ly inactive against HIV in their nucleoside form. However, it has
been shown that the corresponding triphosphates of these
two nucleosides can effectively block HIV reverse transcriptase.
Herein we report on two types of nucleotide prodrugs (cyclo-
Sal and DiPPro nucleotides) of ddU and d4U to investigate
their ability to overcome insufficient intracellular phosphoryla-
tion, which may be the reason behind their low anti-HIV activi-
ty. The release of the corresponding mono- and diphosphates
from these compounds was demonstrated by hydrolysis stud-
+
ies in phosphate buffer (pH 7.3) and human CD4 T-lympho-
cyte CEM cell extracts. Surprisingly, however, these compounds
+
showed low or no anti-HIV activity in tests with human CD4
T-lymphocyte CEM cells. Studies of the conversion of ddUDP
and d4UDP into their triphosphate metabolites by nucleoside
diphosphate kinase (NDPK) showed nearly no conversion of
either diphosphate, which may be the reason for low intracel-
lular triphosphate levels that result in low antiviral activity.
Introduction
Analogues of natural nucleosides are widely used in antiviral
and anticancer therapy. One major drawback to the use of
these compounds for medical purposes is the requirement for
their intracellular conversion into the corresponding biological-
ly active nucleoside 5’-triphosphates. Owing to the high sub-
strate specificity of the kinases that catalyze the stepwise phos-
phorylation, one or more of these phosphorylation steps can
be insufficient, resulting in low or no biological activity of the
given agent.[1] For example, in the case of the nucleoside HIV
reverse transcriptase inhibitor (NRTI) 2’,3’-dideoxy-2’,3’-didehy-
drothymidine (stavudine, d4T (1); Figure 1) the first phosphory-
lation step (catalyzed by thymidine kinase, TK) is hindered,
whereas for the NRTI 3’-azido-3’-deoxythymidine (zidovudine,
AZT (2)) the second phosphorylation (catalyzed by thymidylate
kinase, TMP-K) is the rate-limiting step in the formation of the
corresponding nucleoside triphosphate.[2,3]
To overcome this hurdle and to provide the active nucleo-
side 5’-triphosphates after in vivo administration, several nu-
cleoside monophosphate prodrug strategies have been devel-
oped based on masking the negative charges of the corre-
sponding nucleoside monophosphate with lipophilic groups
(masking units). These masking groups undergo intracellular
cleavage, releasing the nucleoside monophosphate.
This membrane-permeable prodrug strategy there-
fore circumvents the first phosphorylation step.[4] An
example of this concept is the well-established cyclo-
Sal approach, in which a substituted salicyl alcohol as
the lipophilic masking unit is cleaved intracellularly
by chemically driven hydrolysis, releasing the nucleo-
side monophosphate and the corresponding salicyl
alcohol.[5] More recent developments include “lock-
in” modified[6] and enzymatically activated cycloSal
Figure 1. Active antiviral nucleoside analogues d4T 1 and AZT 2, and inactive nucleoside
analogues d4U 3 and ddU 4.
pronucleotides.[7] We also recently reported on the
diastereoselective synthesis of cycloSal compounds[8]
and applications of the cycloSal concept for the syn-
thesis of phosphorylated biomolecules.[9]
In contrast to the numerous examples of pronucleotide
monophosphate approaches, nucleoside diphosphate pro-
drugs have been reported only rarely thus far. In this context,
we recently reported an efficient nucleoside diphosphate pro-
drug system, which we termed the DiPPro nucleotide ap-
proach.[10] In this approach two 4-acyloxybenzyl groups are
used to protect the b-phosphate group of a nucleoside di-
phosphate, leaving the a-phosphate unprotected to prevent
[a] Dr. F. Pertenbreiter, Prof. Dr. C. Meier
Organic Chemistry, Department of Chemistry
Faculty of Sciences, University of Hamburg
Martin-Luther-King-Platz 6, 20146 Hamburg (Germany)
E-mail: chris.meier@chemie.uni-hamburg.de
[b] Prof. Dr. J. Balzarini
Rega Institute for Medical Research
Katholieke Universiteit Leuven (KU Leuven)
Minderbroedersstraat 10, 3000 Leuven (Belgium)
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hydrolytic cleavage of the anhydride bond. The concept in-
volves cleavage of the acyl ester bond either by enzymatic or
chemical hydrolysis under physiological conditions. As a result,
the polarity of the substituent in the para position to the ben-
zyloxy group changes from an acceptor to a donor group (Um-
polung) which destabilizes the benzyl phosphate ester bond
and induces a 1,6-elimination of the first masking unit. Repeti-
tion of this cleavage mechanism at the second masking unit
leads to the release of the nucleoside diphosphate (Scheme 1).
The intracellular release of d4TDP and AZTDP from the corre-
and the delivery mechanism of the prodrug system on anti-HIV
activity; the results also led us to the synthesis of nucleoside
diphosphate prodrugs (DiPPro compounds) of 3 and 4, hy-
pothesizing that the second phosphorylation step might be
the bottleneck in the formation of the triphosphates of these
two nucleoside analogues. As masking units, 4-acyloxybenzyl
groups with alkyl substituents of various length (isopropyl to
undecyl) were chosen. In addition to the corresponding 3-
methyl-cycloSal phosphate triesters 24 (with d4U) and 25 (with
ddU), the nucleoside monophosphates d4UMP 8 and ddUMP
9, the DiPPro compounds 15–22,
and the nucleoside diphos-
phates d4UDP 26 and ddUDP 27
were synthesized as well.
Results and Discussion
Chemistry
The nucleosides d4U 3 and ddU
4
were synthesized in good
yields by following a modified
version of the protocol reported
by Horwitz et al. (Scheme 2).[16]
Briefly, starting from 2’-deoxyuri-
dine 5 the two hydroxy groups
were mesylated using methane-
sulfonyl chloride in pyridine,
yielding compound 6. Treatment
of dimesylate 6 with aqueous
sodium hydroxide resulted in
Scheme 1. Cleavage mechanisms of cycloSal pronucleotides and DiPPro nucleotides.
the formation of 3’,5’-anhydro-
nucleoside 7, which was convert-
sponding DiPPro nucleotides was proven for compounds bear-
ing various alkyl and alkenyl moieties in the masking unit.[10]
d4T 1 is an anti-HIV nucleoside analogue approved for use
in the clinic. Despite their close structural similarity to d4T 1,
d4U 3 and ddU 4 were found to be entirely inactive against
HIV in their nucleoside form.[11] However, the corresponding 5’-
triphosphates of the latter two nucleosides are highly effective
inhibitors of HIV reverse transcriptase.[12] It was concluded from
earlier metabolism studies that the first phosphorylation to
yield the nucleoside monophosphate is the bottleneck in the
activation of ddU and d4U to the corresponding triphosphates.
Therefore, several nucleoside monophosphate prodrugs of
d4U 3 and ddU 4 were synthesized and evaluated for their an-
tiviral activity, with a particular focus on ddU 4. Although
promising results were reported in a few cases,[13] surprisingly,
in the majority of these approaches the pronucleotides
showed no or only moderate anti-HIV activity.[14] The release of
the monophosphates of both d4U 3 and ddU 4 from phos-
phoramidate prodrugs was demonstrated in incubation studies
using (carboxyl)esterase. However, even this resulted in no (for
d4U 3) or only very moderate (for ddU 4) anti-HIV activity.[15]
These results led us to the synthesis of cycloSal pronucleotides
of 3 and 4 to investigate the potential influence of the type
ed into d4U 3 in 70% yield using potassium tert-butoxide in
DMSO. ddU 4 was then obtained in 85% yield after palladium-
catalyzed hydrogenation of the double bond.[17] The corre-
sponding nucleoside 5’-monophosphates 8 and 9 were synthe-
sized via the method of Sowa and Ouchi using phosphoryl
chloride as phosphorylating agent,[18] followed by cation ex-
change from ammonium to tetra-n-butylammonium in 83 and
80% yields, respectively.
Using a 4,5-dicyanoimidazole (DCI)-mediated coupling reac-
tion of the nucleoside monophosphates 8 or 9 and a phosphor-
amidite 11–14 bearing various acyloxybenzyl masking groups,
followed by oxidation of the b-phosphate with tert-butylhydro-
peroxide, the DiPPro compounds 15–22 were obtained in
yields from 31 to 41% (Scheme 3). The phosphoramidites were
synthesized as published before by starting from 4-hydroxy-
benzyl alcohol.[10] For the coupling reactions, the nucleoside
monophosphates were used as tetra-n-butylammonium salts
in order to increase their reactivity and solubility in acetonitrile.
Thorough drying of these hygroscopic salts and the other reac-
tants was crucial for the success of the coupling reactions.
Monitoring the reactions by TLC was complicated by the large
differences in polarity of the components in the reaction mix-
ture. In contrast, the reactions could be easily followed by RP-
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Both 3-methyl-cycloSal phos-
phate triesters 24 and 25 of the
two nucleosides and the corre-
sponding nucleoside diphos-
phates d4UDP 26 and ddUDP 27
were synthesized as reference
compounds as well. For the syn-
thesis of the 3-methyl-cycloSal
phosphate triesters 24 and 25,
nucleosides 3 and 4 were cou-
pled with 3-methyl saligenyl-
chlorophosphite followed by oxi-
dation with tert-butylhydroper-
oxide (Scheme 4).[5] The diphos-
phates 26 and 27 were obtained
from the corresponding 5-
chloro-cycloSal phosphate triest-
ers 28 and 29 by reaction with
Scheme 2. Synthesis of the nucleosides d4U 3 and ddU 4 and their 5’-monophosphates 8 and 9. Reagents and
conditions: a) MeSO2Cl, pyridine, 08C, 3 h, 85%; b) NaOH in H2O, 1008C, 3 h, 80%; c) KOtBu, DMSO, RT, 70 h, 70%;
d) H2, Pd/C, CH3OH, RT, 70 h, 85%; e) 1. POCl3, pyridine, H2O, CH3CN, RT, 4 h, 2. NH4HCO3, H2O, 3. Dowex H+,
4. (nBu4N)OH.
tetra-n-butylammonium
phos-
phate. The acceptor-substituted
5-chloro-cycloSal triesters 28 and
29 were prepared as described
before.[9c]
Hydrolysis studies
The cycloSal triesters 24 and 25
and DiPPro compounds 15–22
were studied first for their hy-
drolytic stability at physiological
pH 7.3 in aqueous 25 mm phos-
phate buffer. The enzymatic
cleavage was then examined in
+
human CD4 T-lymphocyte CEM
Scheme 3. Synthesis of DiPPro compounds 15–22. Reagents and conditions: a) DCI, CH3CN, RT, 20 h; b) tBuOOH,
ꢀ208C!RT, 30–60 min; c) ion exchange: 1. DOWEX 50WX8 (NH4+), 2. RP-18 chromatography.
cell extracts. Hydrolysis products
were identified by analytical RP-
HPLC, and complete consumption of the corresponding nu-
cleoside monophosphate was confirmed (Figure 2).
The coupling reactions were usually complete within mi-
nutes after the addition of DCI to the mixture of nucleotide 8
or 9 and the phosphoramidite. The subsequent oxidation of
the P-III/P-V intermediate 23 could be followed by HPLC as
well (Figure 2). After completion of the oxidation reaction, the
crude products were purified by RP-18 chromatography using
mixtures of methanol and water as eluent. After an initial pu-
rification to separate the remaining DCI, cations were changed
from tetra-n-butylammonium to ammonium, and further purifi-
cation steps were performed to separate the phosphate diester
and the nucleotide that were supposedly formed by partial
cleavage of the anhydride bond. To determine the point at
which these cleavage products were formed, the coupling re-
actions were monitored for 20 h, showing no re-formation of
the monophosphate. Also, no monophosphate was formed
during oxidation. It was therefore assumed that cleavage of
the anhydride bond occurs during purification.
Scheme 4. Synthesis of cycloSal triesters 24, 25 and 28, 29 and nucleoside
diphosphates 26 and 27. Reagents and conditions: a) 1. 3-methyl saligenyl-
chlorophosphite, DIPEA, CH3CN, ꢀ208C!RT, 2.5 h, 2. tBuOOH, ꢀ208C!RT,
2 h; b) 1. 5-chloro saligenylchlorophosphite, DIPEA, CH3CN, ꢀ208C!RT,
45 min, 2. oxone, ꢀ208C!RT, 15 min; c) tetra-n-butylammonium phos-
phate, DMF, RT, 16–18 h.
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the desired nucleoside diphos-
phate,
with
formation
of
a mono-masked intermediate. In
contrast, for the undecyl-substi-
tuted DiPPro nucleotides 18 and
22 only a small amount of the
corresponding intermediate was
observed. This has been deter-
mined earlier for other DiPPro
nucleotides with long alkyl
chains in the masking unit as
well.[10] The half-life of the inter-
mediate formed was always sig-
nificantly higher than that of the
original prodrugs, supposedly
caused by repulsive interaction
between the negative charges of
the intermediate and the nucleo-
phile. As a result of hydrolytic
cleavage of the anhydride bond,
a significant amount of nucleo-
Figure 2. Monitoring of the synthesis of C6-ddUDP 20 by RP-HPLC.
side
monophosphate
was
formed as well. The fraction of
monophosphate increased with longer half-lives of the starting
prodrug, as we proved that the monophosphates were only
formed from the original prodrug but were not formed from
the intermediate.[10] However, due to partial hydrolysis of the
mono- and diphosphates after long hydrolysis periods, their
exact ratio could not be determined (Figure 3). In contrast, the
hydrolysis of cycloSal compounds 24 and 25 showed, as ex-
pected, selective formation of the corresponding nucleoside
monophosphates via a benzyl phosphate diester intermediate.
HPLC methods. The calculated half-lives refer to the degrada-
1
tion of the starting prodrug (t1 = ) or, in case of the DiPPro
2
1
compounds, of the singly masked intermediate (t2 = ) as well.
2
1
The latter half-lives (t2 = ) were determined after complete con-
2
sumption of the starting DiPPro compound.
Chemical hydrolysis
In general, the hydrolysis behavior of compounds 15–22 was
in good agreement with the data published previously for
DiPPro compounds of d4T 1 and AZT 2.[10] Hydrolysis half-lives
of the potential prodrugs increased with increasing alkyl chain
length, with the exception of isopropyl-substituted com-
pounds 15 and 19 (Table 1). Their half-lives are in the same
range as those of the C6-substituted compounds 16 and 20. In
accordance with the hydrolysis mechanism shown in
Scheme 1, degradation of the prodrugs led to the formation of
Hydrolysis in cell extracts
+
Hydrolysis of the DiPPro compounds in human CD4 T-lympho-
cyte CEM cell extracts led to markedly lower half-lives than the
chemical hydrolysis half-lives due to enzymatic cleavage of the
ester group. This enzymatic cleavage also took place for the
singly masked intermediates that did not accumulate for this
reason (Figure 4). The release of nucleoside mono- and diphos-
phates was proven, although the ratio of these compounds
could not be determined, as they were rapidly hydrolyzed
under these conditions as well. As observed in the chemical
hydrolysis studies, half-lives increased with increasing alkyl
chain length in the masking group.
Table 1. Hydrolysis half-lives of DiPPro nucleotides 15–22 and cycloSal
triesters 24 and 25.
Compd
Nucl
R
PBS, pH 7.3
CE[a]
1
1
1
t1 = [h][b]
t2 = [h][c]
t1 = [h][b]
2
2
2
15
16
17
18
19
20
21
22
24
25
d4U
d4U
d4U
d4U
ddU
ddU
ddU
ddU
d4U
ddU
iPr
C6
C9
C11
iPr
C6
C9
C11
–
40
30
63
76
36
28
66
88
9
275
326
223
NA[d]
255
337
290
NA[d]
NA[d]
NA[d]
0.3
0.4
1.1
1.9
0.3
0.5
0.7
1.7
11
Antiviral evaluation
All DiPPro nucleotides 15–22 as well as the cycloSal com-
pounds 24 and 25 were investigated for their in vitro anti-HIV
activity. As anticipated, the monophosphate-releasing cycloSal
prodrugs 24 and 25 showed no activity. Unexpectedly, al-
though the release of the corresponding nucleoside diphos-
phates was proven by the hydrolysis studies, compounds 15–
22 exhibited very moderate or no antiviral activity as well
(Table 2). This result could be explained by insufficient permea-
–
7
16
[a] CEM/0 cell extract. [b] Half-life of the first hydrolysis step. [c] Half-life
of the second hydrolysis step. [d] Not applicable.
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pound
uridine
diphosphate
(UDP) was very efficiently con-
verted into its 5’-triphosphate
derivative (Figure 5).
We conclude that under con-
ditions in which the vast majori-
ty of UDP has been converted
into UTP in the presence of
NDPK after 10 min (70% conver-
sion reached under equilibrium
reaction conditions), there were
no traces at all on the HPLC
chromatogram to indicate any
conversion of ddUDP or d4UDP
after 60 min under similar exper-
imental conditions. This means
that the compounds must act as
extremely poor substrates for
NDPK (if substrates at all). There-
fore, kcat/KM values could not be
Figure 3. pH-dependent hydrolysis of C6-d4UDP 16 monitored by RP-HPLC.
determined. Moreover, given the
pronounced intracellular levels
of the natural substrate UDP
(and UTP) for NDPK, it is very un-
likely that this enzyme will con-
vert the compounds at a physio-
logically relevant concentration.
However, it should be kept in
mind that enzymes other than
NDPK might still be able to con-
vert the diphosphates of ddU
and d4U into their triphosphate
derivatives.
Given its unique mechanism
of action that does not require
simultaneous binding of accept-
or and donor in its active site,
NDPK is significantly less sub-
strate specific than nucleoside
kinases and nucleoside mono-
phosphate kinases (NMPKs).
Figure 4. Enzymatic hydrolysis of C6-d4UDP 16 monitored by RP-HPLC.
However, the interaction of the
3’-hydroxy group of the acceptor
tion of the prodrugs into the cells, inefficient intracellular re-
lease of the diphosphates, or poor conversion of the diphos-
phates to the corresponding triphosphates.
nucleoside diphosphate with a Mg2+ ion in the active site is
necessary to stabilize the transition state.[19] Therefore, most
probably the lack of a 3’-hydroxy group combined with confor-
mational changes in the glycon due to modifications can result
in a significant decrease in catalytic efficiency of NDPK.
Although other possible reasons for the low antiviral activity
of the tested DiPPro nucleotides cannot be excluded, it can be
assumed that the insufficient formation of the nucleoside tri-
phosphates from the corresponding diphosphates might be
the main reason for the poor, or absent, antiviral activity in cell
culture.
NDPK studies
To investigate the efficiency of the conversion of nucleoside di-
phosphates 26 and 27 to the corresponding triphosphates,
both compounds were subjected to an enzyme assay using
purified nucleoside diphosphate kinase (NDPK). Both d4U and
ddU nucleoside diphosphates showed no conversion into the
corresponding triphosphates under these conditions (using
ATP as the phosphate donor), whereas the reference com-
The anti-HIV activity of other dideoxynucleoside analogues
such as d4T, ddC, and AZT, which are also expected to be poor
NDPK substrates in their diphosphate form, can be much
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tion into the monophosphate metabolite was the
rate-limiting step in their activation into the triphos-
phates. Therefore, for both nucleosides, nucleoside
monophosphate prodrug strategies have been de-
scribed, although with no or very limited success. We
applied our recently reported nucleoside diphos-
phate prodrug system, the DiPPro compounds, as
well as the well-established cycloSal pronucleotide
approach for comparison, to these two nucleoside
analogues to determine if the intracellular delivery of
the nucleoside diphosphates could overcome the
lack of activity. However, from the data reported
herein it became apparent that neither the cycloSal
nor the DiPPro approach was able to solve the prob-
lem; however, we could prove that both systems de-
liver the nucleoside monophosphate and the nucleo-
side diphosphate, respectively. Separate studies using
nucleoside diphosphate kinase showed that this
enzyme is practically unable to form the triphosphate
starting from both nucleoside diphosphates, which
might explain the poor antiviral activity. As a conse-
quence, for these two nucleoside analogues only the
intracellular delivery of the corresponding triphos-
Table 2. Antiviral activity and cytotoxicity of nucleoside analogues 3 and 4, and pro-
drug compounds 15–22 and 24 and 25.
Compd Nucl
R
EC50 [mm][a]
CEM/0
CC50 [mm][b]
CEM/0
CEM/TKꢀ
HIV-2
HIV-1
HIV-2
15
16
17
18
24
d4U
19
20
21
22
d4U iPr
d4U C6
d4U C9
d4U C11
32ꢁ16
35ꢁ2.1
36ꢁ20
23ꢁ1.4
30ꢁ4.2
20ꢁ3.5
ꢂ50
24ꢁ2.8
>10
>50
>10
>250
>50
>50
>50
>50
110ꢁ1.4
109ꢁ2.1
95ꢁ3.5
116ꢁ2.8
93ꢁ7.1
>250
>50
>50
d4U
–
–
–
>50
>250
>50
>50
>250
>50
30ꢁ4.9
42ꢁ12
>50
ddU iPr
ddU C6
ddU C9
ddU C11
>250
26ꢁ9.9
124ꢁ11
99ꢁ3.5
144ꢁ7.1
104ꢁ3.5
>250
ꢂ50
>50
25
ddU
d4T
ddU
–
–
–
–
–
>50
>250
>50
>250
>50
>250
250
0.52ꢁ0.32
0.72ꢁ0.35
>250
+
[a] Antiviral activity in CD4 T-lymphocytes: 50% effective concentration; values are
the meanꢁSD of n=2–3 independent experiments. [b] Cytotoxicity: 50% cytostatic
concentration or compound concentration required to inhibit CEM cell proliferation
by 50%; values are the meanꢁSD of n=2–3 independent experiments.
phate could overcome the metabolic hurdles. We are currently
working on strategies to deliver nucleoside triphosphates from
lipophilic, membrane-permeable precursors. This approach will
then be applied to ddU and d4U.
Experimental Section
Chemistry
General: Pyridine and CH3CN were distilled from CaH2 under nitro-
gen. N,N-Diisopropylethylamine (DIPEA) was distilled from sodium
prior to use. POCl3 was distilled under nitrogen prior to use. All
commercially available reagents were used without further purifica-
tion. Thin-layer chromatography (TLC): Merck pre-coated 60 F254
plates (0.2 mm layer of silica gel), or pre-coated plates with
a 0.2 mm layer of silica gel (Macherey & Nagel Xtra Sil UV254) were
used; sugar-containing compounds were visualized with sugar
spray reagent (0.5 mL 4-methoxybenzaldehyde, 9 mL EtOH, 0.5 mL
conc. H2SO4, and 0.1 mL glacial AcOH). All preparative TLC was per-
formed on a Chromatotron (Harrison Research, Model 7924T) using
glass plates coated with 1-, 2-, or 4-mm-thick layers of Merck
60 PF254 silica gel containing a fluorescent indicator. Flash column
chromatography: Merck silica gel 60, 230–400 mesh or Merck RP-
18 silica gel was used. Glass columns were slurry packed using the
appropriate eluent. Fractions containing the product were identi-
fied by TLC, pooled, and the solvent was removed in vacuo. Water
as eluent was removed by freeze-drying.
Figure 5. Enzyme assay to evaluate the conversion of nucleoside diphos-
phates 26 and 27 by NDPK.
better substrates for the activating enzymes (i.e., thymidine
kinase or dCyd kinase) than ddU or d4U, explaining antiviral ef-
ficacy for these other drugs. It has, in fact, been shown that
AZT is a very good substrate for thymidine kinase, and d4T and
ddC have been shown to have only a hundredfold less sub-
strate affinity for their activating enzymes than the natural sub-
strates.[1,11,12]
Analytical HPLC was performed on a VWR–Hitachi LaChromElite
HPLC system (L-2130, L-2200, L-2455) equipped with an EcoCART
125-3 column containing reversed-phase silica gel Lichrospher 100
RP-18 (5 mm; VWR-Merck, Darmstadt, Germany). The solvents for
HPLC were obtained from Sigma–Aldrich or VWR (CH3CN, HPLC
grade). The software used was EZChromElite. Method A: 0–20 min:
TBAH ion buffer/CH3CN gradient (5!90%); 20–24 min: buffer/
CH3CN (90%); 24–29 min: buffer/CH3CN gradient (90!5%); 30–
35 min: buffer/CH3CN (5%); flow: 1 mLminꢀ1, TBAH ion buffer:
0.55 mm tetra-n-butylammonium hydroxide in H2O. Method B: 0–
Conclusions
In summary we report on the successful synthesis of two dif-
ferent classes of potential nucleotide prodrugs of the two uri-
dine nucleoside analogues d4U and ddU. The significant inhibi-
tory activity of the triphosphates of these nucleosides to block
HIV reverse transcriptase has been reported previously. In con-
trast, the nucleoside analogues themselves proved to be en-
tirely inactive, and the hypothesis was made that phosphoryla-
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25 min: H2O/CH3CN gradient (5!95%); 25–35 min: H2O/CH3CN
were separated and the organic layer was extracted with H2O. The
combined aqueous layers were freeze-dried and the crude product
thus obtained was purified by RP-18 chromatography. The cations
were changed to ammonium ions using a DOWEX 50WX8 cation-
(95%); flow: 0.5 mLminꢀ1
.
NMR spectra were recorded with Bruker AMX 400, AV I 400, AV II
1
400, or Bruker DRX 500 Fourier transform spectrometers. All H and
+
exchange resin (NH4 form).
13C NMR chemical shifts (d) are quoted in parts per million (ppm)
downfield from tetramethylsilane and calibrated on solvent signals.
The 31P NMR chemical shifts (proton decoupled) are quoted in
ppm using H3PO4 as the external reference. The spectra were re-
corded at room temperature in automation mode. Mass spectra
were obtained with a VG Analytical VG/70S Xenon FAB [FAB,
(double focusing), matrix: m-nitrobenzyl alcohol] instrument; ESI
mass spectra were recorded with an Agilent 6224 ESI-TOF spec-
trometer in positive or negative modes. IR spectra were recorded
on a Bruker Alpha P FT-IR spectrometer at room temperature in
the range of 400–4000 cmꢀ1. Melting points are uncorrected, and
ꢂ95% purity for the final compounds 15–22, 24–27 was con-
firmed by HPLC analysis.
General procedure 5: Synthesis of DiPPro nucleotides. The reac-
tion was carried out under nitrogen and with dry solvents and re-
agents. The nucleoside monophosphate (tetra-n-butylammonium
salt) was freeze-dried, co-evaporated with CH3CN and dissolved in
CH3CN. The corresponding bis(acyloxybenzyl)phosphoroamidite
(1.7–3.2 equiv) and DCI (1.8–3.3 equiv) were added, and the reac-
tion mixture was stirred at room temperature for 20 h. The solution
was subsequently cooled to ꢀ208C, and tert-butylhydroperoxide
(5.5m in n-decane, 1.8–3.2 equiv) was added. The solution was
stirred for 30–60 min at room temperature and the solvent was re-
moved under reduced pressure. The crude product thus obtained
was purified by a first RP-18 flash column chromatography (MeOH/
H2O gradient) and the cations were changed to ammonium by elu-
General procedure 1: Synthesis of 3-methyl-cycloSal triesters.
The reaction was carried out under nitrogen and with dry solvents
and reagents. A solution of the nucleoside in CH3CN was cooled to
ꢀ208C and N,N-diisopropylethylamine (1.6 equiv) followed by 3-
methyl saligenylchlorophosphite (2.0 equiv) dissolved in CH3CN
were added. The reaction mixture was stirred at room temperature
until the coupling reaction was completed and subsequently
cooled to ꢀ208C before addition of tert-butylhydroperoxide (5.5m
in n-decane, 3.0 equiv). The solution was stirred at room tempera-
ture until completion of the oxidation reaction, poured into ice
water and immediately extracted with EtOAc. The combined or-
ganic layers were dried over Na2SO4 and the solvent was removed
under reduced pressure. The crude product was purified by prepa-
rative TLC, dissolved in CH3CN and H2O (1:1 v/v) and freeze-dried.
+
tion over Dowex 50WX8 cation-exchange resin (NH4 form). The
ammonium salts of the products were purified a second and third
time if necessary by RP-18 flash column chromatography using dif-
ferent MeOH/H2O gradients. Product containing fractions were
pooled, and the MeOH was removed under reduced pressure. The
remaining aqueous solution was freeze-dried and the product was
obtained as a colorless foam or syrup.
3’,5’-Di-O-methanesulfonyl-2’-deoxyuridine 6. The reaction was
carried out under nitrogen and with dry solvents and reagents. A
suspension of 2’-deoxyuridine 5 (1.00 g, 4.40 mmol) in pyridine
(18 mL) was cooled to 08C, and methanesulfonyl chloride (0.85 mL,
1.3 g, 11 mmol, 2.5 equiv) was added dropwise. The mixture was
stirred at 08C for 3 h, then poured into ice water and stirred for an
additional hour at 08C. Subsequently, the precipitate was filtered,
washed with cold H2O and dried in vacuum. The remaining aque-
ous phase was extracted with EtOAc until no product could be de-
tected in the aqueous phase by TLC. The combined organic layers
were dried over Na2SO4 and the solvent was removed under re-
duced pressure. The obtained solid was combined with the one
obtained by filtration to yield a colorless solid (1.44 g, 3.74 mmol,
General procedure 2: Synthesis of 5-chloro-cycloSal triesters.
The reaction was carried out as described above in general proce-
dure 1, except that for the oxidation, oxone (1.0 equiv) dissolved in
cold H2O was used.
General procedure 3: Synthesis of nucleoside monophosphates
(bis(tetra-n-butylammonium) salts). The reaction was carried out
under nitrogen and with dry solvents and reagents. POCl3
(4.4 equiv) was dissolved in CH3CN and cooled to 08C. Then, pyri-
dine (4.4 equiv) and H2O (2.2 equiv) were carefully added to the
stirred solution. After 10 min the nucleoside was added in portions
and the solution was stirred at room temperature for 4 h. Ice water
was then added, and the solution was stirred for 1 h at 08C. After
neutralization by the addition of solid ammonium hydrogen car-
bonate the solution was freeze-dried and the crude product was
purified by RP-18 chromatography with H2O as eluent. The ammo-
nium salt thus obtained was dissolved in H2O and eluted over
Dowex 50WX8 cation-exchange resin (H+ form). The resulting
acidic solution was titrated with tetra-n-butylammonium hydroxide
solution (40% w/w in H2O) to pH 6–7 and the solvent was removed
by freeze-drying.
1
85%). H NMR (400 MHz, [D6]DMSO): d=11.41 (s, 1H, NH), 7.66 (d,
3
3
3JH,H =8.1 Hz, 1H, H-6), 6.19 (dd, JH,H =7.0 Hz, JH,H =7.0 Hz, 1H, H-
3
4
1’), 5.68 (dd, JH,H =8.1 Hz, JH,H =2.3 Hz, 1H, H-5), 5.31–5.25 (m, 1H,
H-3), 4.50–4.34 (m, 3H, H-4’, H-5’), 3.31, 3.24 (2ꢂs, 2ꢂ3H, 2ꢂCH3),
2.55 ppm (dd, 3JH,H =7.1 Hz, 3JH,H =5.1 Hz, 2H, H-2’); 13C NMR
(101 MHz, [D6]DMSO): d=162.9 (C-4), 150.3 (C-2), 140.6 (C-6), 102.2
(C-5), 84.6 (C-1’), 80.6 (C-4’), 79.2 (C-3’), 68.2 (C-5’), 37.7, 36.8 (2ꢂ
CH3), 36.0 ppm (C-2’); IR: n˜ =3034, 1685, 1328, 1169, 919, 805,
524 cmꢀ1; mp: 1428C; TLC: Rf =0.59 (CH2Cl2/MeOH 9:1 v/v); HRMS
(ESI+): m/z calcd: 407.0189 [M+Na]+, found: 407.0175.
3’,5’-Anhydro-2’-deoxyuridine 7. At room temperature, 3’,5’-di-O-
methanesulfonyl-2’-deoxyuridine 6 (1.44 g, 3.74 mmol) was added
in portions to a solution of NaOH (447 mg, 11.2 mmol, 3.0 equiv) in
H2O (80 mL). The reaction mixture was stirred at reflux for 3 h and
after cooling to room temperature neutralized by addition of 1m
HCl. Half of the solvent was removed under reduced pressure and
the product crystallized over night at 48C. The crystalline product
was filtered, washed with cold H2O and dried under vacuum. The
filtrate was extracted with EtOAc until no product could be detect-
ed in the aqueous phase by TLC. The combined organic layers
were dried over Na2SO4 and the solvent was removed under re-
duced pressure. The obtained solid was combined with the one
obtained by filtration to yield a colorless solid (630 mg, 3.00 mmol,
General procedure 4: Synthesis of nucleoside diphosphates (am-
monium salts). The reaction was carried out under nitrogen and
with dry solvents and reagents. Tetra-n-butyl ammonium phos-
phate (2.0 equiv) was vigorously dried under vacuum, dissolved in
N,N-dimethylformamide and stirred over molecular sieves (4 ꢁ) for
1 h. The corresponding 5-chloro-cycloSal triester was dissolved in
N,N-dimethylformamide and added dropwise over 1 h. The reac-
tion mixture was stirred at room temperature until completion of
the reaction. The solvent was removed under reduced pressure
and the residue was dissolved in H2O and EtOAc (1:1). The layers
1
80%). H NMR (500 MHz, [D6]DMSO): d=11.36 (s, 1H, NH), 8.16 (d,
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3
3
3JH,H =8.0 Hz, 1H, H-6), 6.51 (dd, JH,H =5.2 Hz, JH,H =5.2 Hz, 1H, H-
1’), 5.71 (d, 3JH,H =8.0 Hz, 1H, H-5), 5.50–5.46 (m, 1H, H-3’), 4.91
(ddd, 3JH,H =4.0 Hz, 3JH,H =4.0 Hz, 3JH,H =2.2 Hz, 1H, H-4’), 4.68 (dd,
72 mg, 0.71 mmol, 2.0 equiv) and 3-methylsaligenyl chlorophos-
phite (140 mg, 0.691 mmol, 2.0 equiv) dissolved in CH3CN (2.2 mL)
were stirred for 3 h. tert-butylhydroperoxide (5.5m in n-decane,
190 mL, 1.00 mmol, 2.9 equiv) was added and the solution was
stirred for 2.5 h. The crude product was purified by preparative
TLC (CH2Cl2/MeOH 19:1). The product was obtained as a colorless
foam as a mixture of two diastereomers (1:0.9) (27 mg, 68 mmol,
2JH,H =8.1 Hz, JH,H =4.0 Hz, 1H, H-5’a), 4.01 (dd, JH,H =8.1 Hz, JH,H
=
3
2
3
4.0 Hz, 1H, H-5’b), 2.49–2.45 ppm (m, 2H, H-2’); 13C NMR (126 MHz,
[D6]DMSO): d=163.2 (C-4), 151.2 (C-2), 141.2 (C-6), 102.2 (C-5), 88.5
(C-1’), 86.9 (C-3’), 80.1 (C-4’), 75.2 (C-5’), 37.2 ppm (C-2’); IR: n˜ =
3039, 1714, 1682, 1461, 1269, 1087, 802, 549, 525 cmꢀ1; mp:
1838C; TLC: Rf =0.57 (CH2Cl2/MeOH 9:1 v/v); HRMS (ESI+): m/z
calcd: 233.0533 [M+Na]+, found: 233.0549.
1
19%). H NMR (400 MHz, [D6]DMSO): d=11.28 (s, 2H, 2ꢂNH), 7.55
(d, 3JH,H =8.1 Hz, 1H, 1ꢂH-6), 7.52 (d, 3JH,H =8.1 Hz, 1H, 1ꢂH-6),
7.29–7.22 (m, 2H, 2ꢂH-6’’), 7.13–7.06 (m, 4H, 2ꢂH4’’, 2ꢂH-5’’),
6.00–5.92 (m, 2H, 2ꢂH-1’), 5.54–5.35 (m, 6H, 4ꢂH-7’’, 2ꢂH-5),
4.44–4.14 (m, 6H, 2ꢂH-4’, 4ꢂH-5’), 2.35–2.24 (m, 2H, 2ꢂH-2’a),
2.22 (s, 3H, 1ꢂCH3), 2.20 (s, 3H, 1ꢂCH3), 2.07–1.86 (m, 4H, 2ꢂH-
2’b, 2ꢂH-3’a), 1.84–1.71 ppm (m, 2H, 2ꢂH-3’b); 13C NMR (101 MHz,
[D6]DMSO): d=163.0 (2ꢂC-4), 150.3 (2ꢂC-2), 140.1 (1ꢂC-6), 140.1
(1ꢂC-6), 130.8 (1ꢂC-6’’), 130.8 (1ꢂC-6’’), 126.9 (1ꢂC-2’’), 126.8 (1ꢂ
C-2’’), 123.9 (2ꢂC-5’’), 123.5 (1ꢂC-4’’), 123.5 (1ꢂC-4’’), 120.9 (2ꢂC-
1’’), 101.4 (1ꢂC-5), 101.4 (1ꢂC-5), 85.2 (1ꢂC-1’), 85.1 (1ꢂC-1’), 78.1
(d, 3JC,P =3.8 Hz, 1ꢂC-4’), 78.0 (d, 3JC,P =3.8 Hz, 1ꢂC-4’), 68.8 (d,
2’,3’-Dideoxy-2’,3’-didehydrouridine 3. The reaction was carried
out under nitrogen and with dry solvents and reagents. To a solu-
tion of 3’,5’-anhydro-2’-deoxyuridine 7 (630 mg, 3.00 mmol) in
6 mL DMSO KOtBu (537 mg, 4.80 mmol, 1.6 equiv) was added in
portions. The solution was stirred at room temperature for 70 h
and the solvent was removed under reduced pressure. The residue
was dissolved in H2O, neutralized by addition of 1m HCl and
freeze-dried. The crude product was purified by column chroma-
tography on silica gel (CH2Cl2/MeOH 9:1 v/v) to yield a colorless
solid (441 mg, 2.10 mmol, 70%). The analytical data were identical
to those reported earlier.[15]
2
2
2JC,P =5.2 Hz, 1ꢂC-5’), 68.7 (d, JC,P =5.2 Hz, 1ꢂC-5’), 68.4 (d, JC,P
=
2
7.4 Hz, 1ꢂC-7’’), 68.3 (d, JC,P =7.4 Hz, 1ꢂC-7’’), 30.6 (1ꢂC-2’), 30.5
(1ꢂC-2’), 25.1 (1ꢂC-3’), 25.0 (1ꢂC-3’), 14.8 (1ꢂCH3), 14.8 ppm (1ꢂ
CH3); 31P NMR (162 MHz, [D6]DMSO): d=ꢀ9.05, ꢀ9.21 ppm; IR: n˜ =
1680, 1464, 1377, 1265, 1188, 1090, 993, 937, 771, 720, 522,
422 cmꢀ1; TLC: Rf =0.52 (CH2Cl2/MeOH 9:1 v/v); HRMS (ESI+): m/z
calcd: 417.0822 [M+Na]+, found: 417.0718.
2’,3’-Dideoxyuridine 4. The reaction was carried out with dry sol-
vents and reagents. 2’,3’-Dideoxy-2’,3’-didehydrouridine 3 (1.97 g,
9.37 mmol) was dissolved in MeOH (80 mL) and palladium on
carbon (10% Pd, 100 mg) was added. The solution was stirred
under hydrogen atmosphere at room temperature for 70 h, filtered
and the solvent was removed under reduced pressure. The prod-
uct was obtained as colorless solid (1.70 g, 8.01 mmol, 85%). The
analytical data were identical to those reported earlier.[15]
5-Chloro-cycloSal-2’,3’-dideoxy-2’,3’-didehydrouridine
mono-
phosphate 28. General procedure 2 with 2’,3’-dideoxy-2’,3’-didehy-
drouridine 3 (192 mg, 0.913 mmol) dissolved in CH3CN (12 mL),
N,N-diisopropylethylamine (0.25 mL, 0.19 g, 1.5 mmol, 1.6 equiv)
and 5-chlorosaligenyl chlorophosphite (400 mg, 1.79 mmol,
2.0 equiv) dissolved in CH3CN (5.0 mL) were stirred for 45 min.
Oxone (562 mg, 0.914 mmol, 1.0 equiv) was added and the solu-
tion was stirred for 15 min. The crude product was purified by
preparative TLC (CH2Cl2/MeOH 9:1 + 0.1% vol. glacial AcOH). The
product was obtained as a colorless foam as a mixture of two dia-
stereomers (1:0.9) (237 mg, 0.574 mmol, 63%). 1H NMR (400 MHz,
[D6]DMSO): d=11.35 (s, 2H, 2ꢂNH), 7.47–7.40 (m, 4H, 2ꢂH-6’’, 2ꢂ
3-Methyl-cycloSal-2’,3’-dideoxy-2’,3’-didehydrouridine
mono-
phosphate 24. General procedure 1 with 2’,3’-dideoxy-2’,3’-didehy-
drouridine 3 (200 mg, 952 mmol) dissolved in CH3CN (25 mL), N,N-
diisopropylethylamine (0.30 mL, 0.22 g, 2.2 mmol, 2.3 equiv) and 3-
methylsaligenyl chlorophosphite (474 mg, 2.34 mmol, 2.5 equiv)
dissolved in CH3CN (8 mL) were stirred for 2.5 h. tert-butylhydroper-
oxide (5.5m in n-decane, 500 mL, 2.73 mmol, 2.9 equiv) was added
and the solution was stirred for 2 h. The crude product was puri-
fied by preparative TLC (CH2Cl2/MeOH 0–5%). The product was ob-
tained as a colorless foam as a mixture of two diastereomers (1:1)
3
H-4’’), 7.28 (d, JH,H =8.1 Hz, 1H, 1ꢂH-6), 7.23–7.16 (m, 3H, 1ꢂH-6,
3
2ꢂH-3’’), 6.81–6.75 (m, 2H, H-1’), 6.43 (ddd, 3JH,H =6.1 Hz, JH,H
=
=
3
1.6 Hz, 4JH,H =1.6 Hz, 1H, 1ꢂH-3’), 6.39 (ddd, 3JH,H =6.1 Hz, JH,H
1
(65 mg, 0.17 mmol, 18%). H NMR (400 MHz, [D6]DMSO): d=11.33
(s, 2H, 2ꢂNH), 7.30–7.20 (m, 4H, 2ꢂH-6’’, 2ꢂH-6), 7.13–7.06 (m,
4
1.6 Hz, JH,H =1.6 Hz, 1H, 1ꢂH-3’), 6.05–5.97 (m, 2H, 2ꢂH-2’), 5.55–
3
4
3
5.31 (m, 5H, 4ꢂH-7’’, 1ꢂH-5), 5.26 (dd, JH,H =8.1 Hz, JH,H =2.0 Hz,
1H, 1ꢂH-5), 5.00–4.94 (m, 2H, 2ꢂH-4’), 4.38–4.25 ppm (m, 4H, 4ꢂ
H-5’); 13C NMR (101 MHz, [D6]DMSO): d=163.0 (1ꢂC-4), 163.0 (1ꢂ
C-4), 150.7 (1ꢂC-2), 150.7 (1ꢂC-2), 148.5 (2ꢂC-1’’), 140.2 (1ꢂC-6),
140.1 (1ꢂC-6), 133.1 (2ꢂC-3’), 129.7 (1ꢂC-4’’), 129.6 (1ꢂC-4’’),
4H, 2ꢂH-4’’, 2ꢂH-5’’), 6.80–6.76 (m, 2H, 2ꢂH-1’), 6.43 (ddd, JH,H
=
=
6.1 Hz, 3JH,H =1.6 Hz, 4JH,H =1.6 Hz, 1H, 1ꢂH-3’), 6.39 (ddd, JH,H
3
6.1 Hz, 3JH,H =1.6 Hz, 3JH,H =1.6 Hz, 1H, 1ꢂH-3’), 6.04–5.97 (m, 2H,
3
4
2ꢂH-2’), 5.51–5.32 (m, 4H, 4ꢂH-7’’), 5.29 (dd, JH,H =8.1 Hz, JH,H
=
3
4
2.3 Hz, 1H, 1ꢂH-5), 5.15 (dd, JH,H =8.1 Hz, JH,H =2.1 Hz, 1H, 1ꢂH-
5), 5.00–4.92 (m, 2H, 2ꢂH-4’), 4.36–4.23 (m, 4H, 4ꢂH-5’), 2.21 (s,
3H, 1ꢂCH3), 2.20 ppm (s, 3H, 1ꢂCH3); 13C NMR (101 MHz,
[D6]DMSO): d=163.0 (1ꢂC-4), 162.9 (1ꢂC-4), 150.7 (1ꢂC-2), 150.6
(1ꢂC-2), 140.1 (1ꢂC-6), 140.0 (1ꢂC-6), 133.1 (1ꢂC-3’), 133.1 (1ꢂC-
3’), 131.0 (2ꢂC-6’’), 127.0 (2ꢂC-2’), 126.9 (2ꢂC-2’’), 124.0 (2ꢂC-5’’),
123.6 (2ꢂC-4’’), 121.0 (2ꢂC-1’’), 101.7 (1ꢂC-5), 101.6 (1ꢂC-5), 89.4
(1ꢂC-1’), 89.3 (1ꢂC-1’), 84.3 (d, 3JC,P =5.5 Hz, 1ꢂC-4’), 84.2 (d,
3JC,P =5.8 Hz, 1ꢂC-4’), 68.4–68.1 (m, 2ꢂC-5’, 2ꢂC-7’’), 14.9 (1ꢂCH3),
14.8 ppm (1ꢂCH3); 31P NMR (162 MHz, [D6]DMSO): d=ꢀ8.78,
ꢀ8.97 ppm; IR: n˜ =1685, 1459, 1376, 1287, 1243, 1188, 1086, 994,
940, 844, 720, 654, 433 cmꢀ1; TLC: Rf =0.50 (CH2Cl2/MeOH 9:1 v/v);
HRMS (ESI+): m/z calcd: 415.0666 [M+Na]+, found: 415.0630.
128.4 (1ꢂC-5’’), 128.3 (1ꢂC-5’’), 127.1 (2ꢂC-2’), 126.1 (1ꢂC-6’’),
3
126.0 (1ꢂC-6’’), 123.0 (1ꢂC-2’’), 122.9 (1ꢂC-2’’), 120.2 (d, JC,P
=
2.2 Hz, 1ꢂC-3’’), 120.1 (d, 3JC,P =2.2 Hz, 1ꢂC-3’’), 101.8 (1ꢂC-5),
3
101.7 (1ꢂC-5), 89.4 (2ꢂC-1’), 84.3 (d, JC,P =7.7 Hz, 1ꢂC-4’), 84.2 (d,
3JC,P =7.7 Hz, 1ꢂC-4’), 68.4–68.2 (m, 2ꢂC-5’), 68.0–67.8 ppm (m, 2ꢂ
C-7’’); 31P NMR (162 MHz, [D6]DMSO): d=ꢀ10.05, ꢀ10.08 ppm; IR:
n˜ =1685, 1481, 1244, 1187, 1086, 1027, 995, 941, 816, 440 cmꢀ1
;
TLC: Rf =0.38 (CH2Cl2/MeOH 9:1 v/v); HRMS (ESI+): m/z calcd:
435.0119 [M+Na]+, found: 435.0124.
5-Chloro-cycloSal-2’,3’-dideoxyuridine monophosphate 29. Gen-
eral procedure 2 with 2’,3’-dideoxyuridine 4 (195 mg, 0.918 mmol)
dissolved in CH3CN (12 mL), N,N-diisopropylethylamine (0.25 mL,
0.19 g, 1.5 mmol, 1.6 equiv) and 5-chlorosaligenyl chlorophosphite
(400 mg, 1.79 mmol, 2.0 equiv) dissolved in CH3CN (5.0 mL) were
stirred for 45 min. Oxone (560 mg, 0.911 mmol, 1.0 equiv) was
3-Methyl-cycloSal-2’,3’-dideoxyuridine monophosphate 25. Gen-
eral procedure 1 with 2’,3’-dideoxyuridine 4 (74 mg, 0.35 mmol)
dissolved in CH3CN (10 mL), N,N-diisopropylethylamine (0.10 mL,
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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added and the solution was stirred for 15 min. The crude product
was purified by preparative TLC (CH2Cl2/MeOH 9:1 + 0.1% vol. gla-
cial AcOH). The product was obtained as a colorless foam as a mix-
ture of two diastereomers (1:0.9) (245 mg, 0.591 mmol, 64%).
1H NMR (400 MHz, [D6]DMSO): d=11.28 (s, 2H, 2ꢂNH), 7.56 (d,
790, 521 cmꢀ1; TLC: Rf =0.52 (iPrOH/NH4OAc (1m) 2:1 v/v); HRMS
(ESIꢀ): m/z calcd: 291.0388 [MꢀH]ꢀ, found: 291.0451.
2’,3’-Dideoxy-2’,3’-didehydrouridine-5’-diphosphate (d4UDP, am-
monium salt) 26. General procedure 4 with tetra-n-butylammoni-
um phosphate (1.5ꢂnBu4N+; 619 mg, 1.35 mmol, 2.9 equiv) in
10 mL N,N-dimethylformamide and 5-chloro-cycloSal-2’,3’-dideoxy-
2’,3’-didehydrouridine monophosphate 28 (194 mg, 470 mmol) in
8 mL N,N-dimethylformamide. The reaction mixture was stirred at
room temperature for 18 h. The crude product was purified using
RP-18 flash chromatography (1. H2O; 2. H2O/MeOH 9:1 v/v). The
product was obtained as a hygroscopic, colorless solid (62 mg,
3
3JH,H =8.1 Hz, 1H, 1ꢂH-6), 7.52 (d, JH,H =8.1 Hz, 1H, 1ꢂH-6), 7.47–
7.39 (m, 4H, 2ꢂH-6’’, 2ꢂH-4’’), 7.23–7.16 (m, 2H, 2ꢂH-3’’), 6.00–
5.93 (m, 2H, 2ꢂH-1’), 5.56–5.34 (m, 6H, 4ꢂH-7’’, 2ꢂH-5), 4.42–4.24
(m, 4H, 4ꢂH-5’), 4.22–4.15 (m, 2H, 2ꢂH-4’), 2.34–2.22 (m, 2H, 2ꢂ
H-2’a), 2.05–1.88 (m, 4H, 2ꢂH-2’b, 2ꢂH-3’a), 1.84–1.71 ppm (2ꢂH-
3’b); 13C NMR (101 MHz, [D6]DMSO): d=163.1 (1ꢂC-4), 163.0 (1ꢂC-
4), 150.3 (2ꢂC-2), 148.2 (2ꢂC-1’’), 140.2 (1ꢂC-6), 140.2 (1ꢂC-6),
129.6 (2ꢂC-4’’), 128.3 (2ꢂC-5’’), 126.0 (2ꢂC-6’’), 122.9 (1ꢂC-2’’),
122.8 (1ꢂC-2’’), 120.2 (2ꢂC-3’’), 101.5 (1ꢂC-5), 101.5 (1ꢂC-5), 85.2
(1ꢂC-1’), 85.2 (1ꢂC-1’), 78.1 (d, 3JC,P =6.7 Hz, 1ꢂC-4’), 78.0 (d,
1
3
0.15 mmol, 32%). H NMR (400 MHz, D2O): d=7.84 (d, JH,H =8.0 Hz,
3
1H, H-6), 6.99–6.95 (m, 1H, H-1’), 6.53 (d, JH,H =6.4 Hz, 1H, H-3’),
5.95 (d, 3JH,H =6.0 Hz, 1H, H-2’), 5.89 (d, 3JH,H =8.0 Hz, 1H, H-5),
5.15–5.10 (m, 1H, H-4’), 4.18–4.08 ppm (m, 2H, H-5’); 13C NMR
(101 MHz, D2O): d=166.3 (C-4), 151.8 (C-2), 143.0 (C-6), 134.6 (C-3’),
124.9 (C-2’), 102.3 (C-5), 90.1 (C-1’), 86.1 (d, 3JC,P =8.8 Hz, C-4’),
2
2
3JC,P =6.7 Hz, 1ꢂC-4’), 69.0 (d, JC,P =5.9 Hz, 1ꢂC-5’), 68.9 (d, JC,P
=
2
2
5.9 Hz, 1ꢂC-5’), 68.0 (d, JC,P =7.5 Hz, 1ꢂC-7’’), 67.9 (d, JC,P =7.5 Hz,
1ꢂC-7’’), 30.6 (1ꢂC-2’), 30.5 (1ꢂC-2’), 25.1 (1ꢂC-3’), 25.1 ppm (1ꢂ
C-3’); 31P NMR (162 MHz, [D6]DMSO): d=ꢀ10.20, ꢀ10.31 ppm; IR:
n˜ =1680, 1481, 1460, 1263, 1186, 1028, 994, 939, 865, 810, 718,
435 cmꢀ1; TLC: Rf =0.38 (CH2Cl2/MeOH 9:1 v/v); HRMS (ESI+): m/z
calcd: 437.0270 [M+Na]+, found: 437.0274.
66.0 ppm (d, JC,P =6.0 Hz, C-5’); 31P NMR (162 MHz, D2O): d=ꢀ9.74
2
2
2
(d, JP,P =19.7 Hz), ꢀ11.29 ppm (d, JP,P =21.5 Hz); IR: n˜ =2953, 2927,
1699, 1541, 1250, 1178, 1066, 1037, 963, 902, 833, 776, 702, 668,
515, 419 cmꢀ1; TLC: Rf =0.17 (iPrOH/NH4OAc (1m) 2:1 v/v); HRMS
(ESIꢀ): m/z calcd: 368.9894 [MꢀH]ꢀ, found: 368.9719.
2’,3’-Dideoxy-2’,3’-didehydrouridine-5’-monophosphate (d4UMP,
tetra-n-butylammonium salt) 8. General procedure 3 with 2’,3’-di-
2’,3’-Dideoxyuridine-5’-diphosphate (ddUDP, ammonium salt)
27. General procedure 4 with tetra-n-butylammonium phosphate
(1.5ꢂnBu4N+; 300 mg, 653 mmol, 2.0 equiv) in 5 mL N,N-dimethyl-
formamide and 5-chloro-cycloSal-2’,3’-dideoxy-2’,3’-didehydrouri-
dine monophosphate 28 (137 mg, 330 mmol) in 5 mL N,N-dimethyl-
formamide. The reaction mixture was stirred at room temperature
for 16 h. The crude product was purified using RP-18 flash chroma-
tography (H2O). The product was obtained as a hygroscopic, color-
deoxy-2’,3’-didehydrouridine
3
(245 mg, 1.17 mmol), POCl3
(0.48 mL, 0.81 g, 5.3 mmol, 4.4 equiv), pyridine (0.42 mL, 0.41 g,
5.2 mmol, 4.4 equiv), H2O (48 mL, 48 mg, 2.6 mmol, 2.2 equiv) in
11.5 mL CH3CN. The product was obtained as a hygroscopic, color-
less solid (754 mg, 975 mmol, 83%). 1H NMR (400 MHz, D2O): d=
3
7.88 (d, JH,H =8.1 Hz, 1H, H-6), 6.99–6.96 (m, 1H, H-1’), 6.51 (ddd,
3
4
3
3JH,H =6.2 Hz, JH,H =1.9 Hz, JH,H =1.6 Hz, 1H, H-3’), 5.95 (ddd, JH,H
=
1
less solid (39 mg, 92 mmol, 28%). H NMR (500 MHz, D2O): d=8.00
6.2 Hz, 3JH,H =2.2 Hz, 3JH,H =1.5 Hz, 1H, H-2’), 5.89 (d, 3JH,H =8.1 Hz,
3
3
3
(d, JH,H =8.0 Hz, 1H, H-6), 6.11 (dd, JH,H =6.7 Hz, JH,H =3.5 Hz, 1H,
1H,H-5), 5.13–5.08 (m, 1H, H-4’), 4.01 (dd, 2JH,H =5.5 Hz, JH,H
=
3
3
H-1’), 5.92 (d, JH,H =8.2 Hz, 1H, H-5), 4.43–4.35 (m, 1H, H-4’), 4.28–
3.3 Hz, 2H, H-5’), 3.26–3.17 (m, 16H, H-a), 1.74–1.61 (m, 16H, H-b),
4.21 (m, 1H, H-5’a), 4.11–4.05 (m, 1H, H-5’b), 2.50–2.40 (m, 1H, H-
2’a), 2.21–2.08 (m, 2H, H-2’b, H-3’a), 2.05–1.94 ppm (m, 1H, H-3’b);
3
1.38 (tq, 3JH,H =7.3 Hz, 3JH,H =7.3 Hz, 16H, H-c), 0.96 ppm (t, JH,H
=
7.4 Hz, 24H, H-d); 13C NMR (101 MHz, D2O): d=166.4 (C-4), 152.1
(C-2), 143.0 (C-6), 134.6 (C-3’), 125.0 (C-2’), 102.1 (C-5), 90.1 (C-1’),
86.1 (d, 3JC,P =8.5 Hz, C-4’), 65.3 (d, 2JC,P =5.5 Hz, C-5’), 58.1, (C-a),
23.1 (C-b), 19.1 (C-c), 12.8 ppm (C-d); 31P NMR (162 MHz, D2O): d=
0.29 ppm; IR: n˜ =2959, 2873, 1684, 1458, 1380, 1246, 1175, 1102,
1045, 886, 740, 527, 424 cmꢀ1; TLC: Rf =0.57 (iPrOH/NH4OAc (1m)
2:1 v/v); HRMS (ESIꢀ): m/z calcd: 289.0231 [MꢀH]ꢀ, found:
289.0233.
13C NMR (101 MHz, D2O): d=166.5 (C-4), 151.7 (C-2), 142.2 (C-6),
2
101.8 (C-5), 86.5 (C-1’), 80.6 (d, 3JC,P =8.8 Hz, C-4’), 66.4 (d, JC,P
=
5.1 Hz, C-5’), 31.3 (C-2’), 24.7 ppm (C-3’); 31P NMR (162 MHz, D2O):
d=ꢀ9.54 (bs), ꢀ10.94 ppm (bs); IR: n˜ =2987, 2901, 1666, 1409,
1221, 1055, 900, 809, 715, 523 cmꢀ1; TLC: Rf =0.17 (iPrOH/NH4OAc
(1m) 2:1 v/v); HRMS (ESIꢀ): m/z calcd: 371.0051 [MꢀH]ꢀ, found:
371.0091.
Bis(4-isobutyryloxybenzyl)-d4UDP 15. General procedure 5 with
bis(tetra-n-butylammonium)-d4UMP 8 (107 mg, 138 mmol) in 5 mL
CH3CN, bis(4-isobutyryloxybenzyl)-N,N-diisopropylaminophosphor-
oamidite 11 (129 mg, 249 mmol, 1.8 equiv), DCI (30 mg, 0.25 mmol,
1.8 equiv). The reaction mixture was stirred at room temperature
for 20 h, cooled to ꢀ208C and tert-butylhydroperoxide (5.5m in n-
decane, 45 mL, 0.25 mmol, 1.8 equiv) was added. The mixture was
stirred for 30 min at room temperature. The crude product was pu-
rified by a first RP-18 flash chromatography (MeOH/H2O 1:1 v/v),
2’,3’-Dideoxyuridine-5’-monophosphate (ddUMP, tetra-n-buty-
lammonium salt) 9. General procedure 3 with 2’,3’-dideoxyuridine
4 (130 mg, 613 mmol), POCl3 (0.25 mL, 0.42 g, 2.8 mmol, 4.4 equiv),
pyridine (0.22 mL, 0.21 g, 2.7 mmol, 4.4 equiv), H2O (25 mL, 25 mg,
1.4 mmol, 2.2 equiv) in 6 mL CH3CN. The product was obtained as
1
a hygroscopic, light yellow solid (380 mg, 490 mmol, 80%). H NMR
3
3
(400 MHz, D2O): d=8.09 (d, JH,H =8.1 Hz, 1H, H-6), 6.12 (dd, JH,H
=
3
3
6.7 Hz, JH,H =3.5 Hz, 1H, H-1’), 5.92 (d, JH,H =8.1 Hz, 1H, H-5), 4.41–
4.33 (m, 1H, H-4’), 4.10 (ddd, 2JH,H =11.6 Hz, 3JH,H =4.9 Hz, JH,P
=
3
+
counter-ions were changed to ammonium (Dowex 50WX8, NH4
form) and the product was further purified by RP-18 flash chroma-
tography (MeOH/H2O 3:1 v/v and MeOH/H2O 1:1 v/v). The product
2.9 Hz, 1H, H-5’), 3.97–3.90 (m, 1H, H-5’), 3.26–3.16 (m, 16H, H-a),
2.51–2.40 (m, 1H, H-2’a), 2.22–2.07 (m, 2H, H-2’b, H-3’a), 2.05–1.91
(m, 1H, H-3’b), 1.73–1.60 (m, 16H, H-b), 1.37 (tq, 3JH,H =7.5 Hz,
3JH,H =7.5 Hz, 16H, H-c), 0.96 ppm (t, 3JH,H =7.3 Hz, 24H, H-d);
13C NMR (101 MHz, D2O): d=166.4 (C-4), 151.6 (C-2), 142.4 (C-6),
was obtained as a hygroscopic, colorless solid (33 mg, 45 mmol,
3
33%, 1ꢂNH4+). 1H NMR (400 MHz, CD3OD): d=7.82 (d, JH,H
=
8.1 Hz, 1H, H-6), 7.43–7.37 (m, 4H, H-2’’), 7.08–7.03 (m, 4H, H-3’’),
3
2
101.74 (C-5), 86.5 (C-1’), 81.0 (d, JC,P =8.5 Hz, C-4’), 65.4 (d, JC,P
=
6.94–6.92 (m, 1H, H-1’), 6.39 (ddd, 3JH,H =6.1 Hz, 3JH,H =1.7 Hz,
5.1 Hz, C-5’), 58.1 (C-a), 31.6 (C-2’), 24.8 (C-3’), 23.1 (C-b), 19.1 (C-c),
12.8 ppm (C-d); 31P NMR (162 MHz, D2O): d=2.33 ppm; IR: n˜ =
2959, 2873, 1683, 1521, 1459, 1381, 1268, 1185, 1101, 1056, 886,
3
3
3
4JH,H =1.7 Hz, 1H, H-3’), 5.85 (ddd, JH,H =6.1 Hz, JH,H =2.2 Hz, JH,H
=
=
1.4 Hz, 1H, H-2’), 5.75 (d, 3JH,H =8.1 Hz, 1H, H-5), 5.11 (dd, JH,H
8.3 Hz, JH,P =5.1 Hz, 1H, Bn), 4.98–4.93 (m, 1H, H-4’), 4.22–4.10 (m,
2H, H-5’), 2.82 (sept, JH,H =7.0 Hz, 2H, CH-iPr), 1.30 ppm (d, JH,H
2
3
3
3
=
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3
7.0 Hz, 12H, CH3-iPr); 13C NMR (101 MHz, CD3OD): d=177.1 (2ꢂC=
O), 166.2 (C-4), 152.7 (2ꢂC-4’’), 152.5 (C-2), 143.3 (C-6), 135.5 (C-3’),
0.91 ppm (t, JH,H =6.8 Hz, 6H, H-i); 13C NMR (101 MHz, CD3OD): d=
173.8 (2ꢂC=O), 166.3 (C-4), 152.7 (2ꢂC-4’’), 152.4 (C-2), 143.3 (C-6),
3
4
134.9 (d, 3JC,P =7.7 Hz, 2ꢂC-1’’), 130.4 (d, 4JC,P =2.9 Hz, 4ꢂC-2’’),
135.6 (C-3’), 149.9 (d, JC,P =5.9 Hz, 2ꢂC-1’’), 130.5 (d, JC,P =2.9 Hz,
4x C-2’’), 127.3 (C-2’), 122.9 (4ꢂC-3’’), 103.3 (C-5), 91.0 (C-1’), 87.0
(d, 3JC,P =9.5 Hz, C-4’), 70.3 (dd, 2JC,P =5.9 Hz, 4JC,P =2.9 Hz, 2ꢂBn),
68.0 (d, 2JC,P =5.9 Hz, C-5’), 35.0, 33.1, 30.6, 30.4, 30.4, 30.2, 26.0,
23.7 (C-a, C-b, C-c, C-d, C-e, C-f, C-g, C-h), 14.4 ppm (C-i); 31P NMR
(162 MHz, CD3OD): d=ꢀ12.12 (d, 2JP,P =21.1 Hz), ꢀ12.93 ppm (d,
2JP,P =19.6 Hz); IR: n˜ =2921, 2851, 1754, 1687, 1509, 1464, 1379,
1248, 1105, 1005, 968, 914, 837, 764, 695, 504 cmꢀ1; TLC: Rf =0.38
(EE/MeOH 7:3 v/v); HRMS (ESIꢀ): m/z calcd: 889.3520 [MꢀH]ꢀ,
found: 889.3448.
3
127.3 (C-2’), 122.8 (4ꢂC-3’’), 103.2 (C-5), 91.0 (C-1’), 87.0 (d, JC,P
=
9.6 Hz, C-4’), 70.3 (dd, 2JC,P =5.9 Hz, 4JC,P =2.2 Hz, 2ꢂBn), 68.0 (d,
3JC,P =5.8 Hz, C-5’), 35.2 (2ꢂCH-iPr), 19.2 ppm (4ꢂCH3-iPr); 31P NMR
(162 MHz, CD3OD): d=ꢀ12.10 (d, 2JP,P =20.1 Hz), ꢀ12.88 ppm (d,
2JP,P =20.1 Hz); IR: n˜ =2972, 2877, 1753, 1684, 1509, 1460, 1386,
1244, 1106, 1004, 964, 837, 764, 694, 501 cmꢀ1; TLC: Rf =0.65 (EE/
MeOH 7:3 v/v); HRMS (ESIꢀ): m/z calcd: 721.1569 [MꢀH]ꢀ, found:
721.1798.
Bis(4-heptanoyloxybenzyl)-d4UDP 16. General procedure 5 with
bis(tetra-n-butylammonium)-d4UMP 8 (107 mg, 138 mmol) in 5 mL
CH3CN, bis(4-heptanoyloxybenzyl)-N,N-diisopropylaminophosphor-
oamidite 12 (152 mg, 253 mmol, 1.8 equiv), DCI (30 mg, 0.25 mmol,
1.8 equiv). The reaction mixture was stirred at room temperature
for 20 h, cooled to ꢀ208C and tert-butylhydroperoxide (5.5m in n-
decane, 45 mL, 0.25 mmol, 1.8 equiv) was added. The mixture was
stirred for 30 min at room temperature. The crude product was pu-
rified by a first RP-18 flash chromatography (MeOH/H2O 1:1–5:1 v/
v), counter-ions were changed to ammonium (Dowex 50WX8,
Bis(4-dodecanoyloxybenzyl)-d4UDP 18. General procedure 5 with
bis(tetra-n-butylammonium)-d4UMP 8 (100 mg, 129 mmol) in 5 mL
CH3CN,
bis(4-dodecanoyloxybenzyl)-N,N-diisopropylaminophos-
phoroamidite 14 (190 mg, 0.256 mmol, 2.0 equiv), DCI (30 mg,
0.25 mmol, 1.8 equiv). The reaction mixture was stirred at room
temperature for 24 h, cooled to ꢀ208C and tert-butylhydroperox-
ide (5.5m in n-decane, 45 mL, 0.25 mmol, 1.8 equiv) was added. The
mixture was stirred for 1 h at room temperature. The crude prod-
uct was purified by a first RP-18 flash chromatography (MeOH/H2O
1:1–9:1 v/v), counter-ions were changed to ammonium (Dowex
+
NH4 form) and the product was further purified by RP-18 flash
+
chromatography (MeOH/H2O 3:1 v/v). The product was obtained as
a hygroscopic, colorless solid (47 mg, 57 mmol, 41%, 1ꢂNH4+).
1H NMR (400 MHz, CD3OD): d=7.82 (d, 3JH,H =8.1 Hz, 1H, H-6),
7.42–7.37 (m, 4H, H-2’’), 7.08–7.03 (m, 4H, H-3’’), 6.96–6.93 (m, 1H,
50WX8, NH4 form) and the product was further purified by RP-18
flash chromatography (MeOH/H2O 9:1 v/v). The product was ob-
tained as a hygroscopic, colorless solid (40 mg, 41 mmol, 32%, 1ꢂ
1
3
NH4+). H NMR (400 MHz, CD3OD): d=7.82 (d, JH,H =8.1 Hz, 1H, H-
6), 7.41–7.35 (m, 4H, H-2’’), 7.07–7.03 (m, 4H, H-3’’), 6.96–6.93 (m,
1H, H-1’), 6.39 (ddd, 3JH,H =6.0 Hz, 3JH,H =1.7 Hz, 4JH,H =1.7 Hz, 1H,
3
3
4
H-1’), 6.39 (ddd, JH,H =6.1 Hz, JH,H =1.7 Hz, JH,H =1.7 Hz, 1H, H-3’),
5.87–5.83 (m, 1H, H-2’), 5.75 (d, 3JH,H =8.1 Hz, 1H, H-5), 5.10 (dd,
2JH,H =8.2 Hz, 3JH,P =6.9 Hz, 4H, Bn), 4.97–4.93 (m, 1H, H-4’), 4.21–
H-3’), 5.87–5.84 (m, 1H, H-2’), 5.75 (d, JH,H =8.1 Hz, 1H, H-5), 5.10
3
3
4.11 (m, 2H, H-5’), 2.57 (t, 3JH,H =7.4 Hz, 4H, H-a), 1.73 (tt, JH,H
=
(dd, 2JH,H =8.5 Hz, 3JH,P =5.2 Hz, 4H, Bn), 4.98–4.93 (m, 1H, H-4’),
4.21–4.11 (m, 2H, H-5’), 2.57 (t, 3JH,H =7.4 Hz, 4H, H-a), 1.73 (tt,
3JH,H =7.3 Hz, 3JH,H =7.3 Hz, 4H, H-b), 1.48–1.24 (m, 32H, H-c, H-d,
H-e, H-f, H-g, H-h, H-i, H-j), 0.90 ppm (t, 3JH,H =7.0 Hz, 6H, H-k);
7.4 Hz, 3JH,H =7.4 Hz, 4H, H-b), 1.47–1.33 (m, 12H, H-c, H-d, H-e),
0.93 ppm (t, JH,H =7.0 Hz, 6H, H-f); 13C NMR (101 MHz, CD3OD): d=
3
173.8 (2ꢂC=O), 166.2 (C-4), 152.7 (2ꢂC-4’’), 152.4 (C-2), 143.3 (C-6),
3
4
135.5 (C-3’), 134.9 (d, JC,P =6.9 Hz, 2ꢂC-1’’), 130.5 (d, JC,P =3.7 Hz,
4ꢂC-2’’), 127.3 (C-2’), 122.9 (4ꢂC-3’’), 103.3 (C-5), 91.0 (C-1’), 87.0
(d, 3JC,P =8.7 Hz, C-4’), 70.3 (dd, 2JC,P =5.7 Hz, 4JC,P =2.8 Hz, 2ꢂBn),
13C NMR (101 MHz, CD3OD): d=173.7 (2ꢂC=O), 166.2 (C-4), 152.7
(2ꢂC-4’’), 152.4 (C-2), 143.3 (C-6), 135.5 (C-3’), 134.9 (d, JC,P
3
=
4
6.5 Hz, 2ꢂC-1’’), 130.4 (d, JC,P =2.9 Hz, 4ꢂC-2’’), 127.3 (C-2’), 122.9
(2ꢂC-3’’), 103.3 (C-5), 91.0 (C-1’), 87.0 (d, 3JC,P =8.8 Hz, C-4’), 70.3
(dd, 2JC,P =5.9 Hz, 4JC,P =3.0 Hz, 2ꢂBn), 67.9 (d, 3JC,P =6.5 Hz, C-5’),
35.0, 33.1, 30.7, 30.6, 30.5, 30.4, 30.2, 26.0, 23.7 (C-a, C-b, C-c, C-d,
2
68.0 (d, JC,P =5.6 Hz, C-5’), 35.0, 32.6, 29.8, 25.9, 23.6 (C-a, C-b, C-c,
C-d, C-e), 14.4 ppm (C-f); 31P NMR (162 MHz, CD3OD): d=ꢀ12.11 (d,
2JP,P =19.8 Hz), ꢀ12.92 ppm (d, 2JP,P =21.8 Hz); IR: n˜ =2958, 2873,
1754, 1687, 1508, 1461, 1378, 1198, 1103, 1005, 977, 763, 498 cmꢀ1
;
C-e, C-f, C-g, C-h, C-i, C-j), 14.4 ppm (C-k); 31P NMR (162 MHz,
TLC: Rf =0.29 (EE/MeOH 7:3 v/v); HRMS (ESIꢀ): m/z calcd: 805.2508
[MꢀH]ꢀ, found: 805.2505.
CD3OD): d=ꢀ12.10 (d, 2JP,P =20.8 Hz), ꢀ12.91 ppm (d, JP,P
=
2
19.7 Hz); IR: n˜ =2917, 2849, 1753, 1686, 1509, 1465, 1380, 1221,
1169, 1107, 977, 917, 837, 764, 720, 506 cmꢀ1; TLC: Rf =0.34 (EE/
MeOH 7:3 v/v); HRMS (ESI+): m/z calcd: 947.4219 [M+H]+, found:
947.4212.
Bis(4-decanoyloxybenzyl)-d4UDP 17. General procedure 5 with
bis(tetra-n-butylammonium)-d4UMP 8 (106 mg, 137 mmol) in 5 mL
CH3CN, bis(4-decanoyloxybenzyl)-N,N-diisopropylaminophosphor-
oamidite 13 (171 mg, 249 mmol, 1.8 equiv), DCI (30 mg, 0.25 mmol,
1.8 equiv). The reaction mixture was stirred at room temperature
for 18 h, cooled to ꢀ208C and tert-butylhydroperoxide (5.5m in n-
decane, 45 mL, 0.25 mmol, 1.8 equiv) was added. The mixture was
stirred for 1 h at room temperature. The crude product was puri-
fied by a first RP-18 flash chromatography (MeOH/H2O 1:1–9:1 v/v),
Bis(4-isobutyryloxybenzyl)-ddUDP 19. General procedure 5 with
bis(tetra-n-butylammonium)-ddUMP 9 (66 mg, 85 mmol) in 2.5 mL
CH3CN, bis(4-isobutyryloxybenzyl)-N,N-diisopropylaminophosphor-
oamidite 11 (75 mg, 0.14 mmol, 1.6 equiv), DCI (13 mg, 0.11 mmol,
1.3 equiv) and after 1 h stirring at room temperature additional
bis(4-isobutyryloxybenzyl)-N,N-diisopropylaminophosphoramidite
11 (75 mg, 0.14 mmol, 1.6 equiv) and DCI (20 mg, 0.17 mmol,
2.0 equiv). The reaction mixture was stirred at room temperature
for 20 h, cooled to ꢀ208C and tert-butylhydroperoxide (5.5m in n-
decane, 48 mL, 0.27 mmol, 3.2 equiv) was added. The mixture was
stirred for 45 min at room temperature. The crude product was pu-
rified by a first RP-18 flash chromatography (MeOH/H2O 1:1–5:1 v/
v), counter-ions were changed to ammonium (Dowex 50WX8,
+
counter-ions were changed to ammonium (Dowex 50WX8, NH4
form) and the product was further purified by RP-18 flash chroma-
tography (MeOH/H2O 5:1 v/v). The product was obtained as a hy-
1
groscopic, colorless solid (39 mg, 43 mmol, 31%, 1ꢂNH4+). H NMR
3
(400 MHz, CD3OD): d=7.82 (d, JH,H =8.1 Hz, 1H, H-6), 7.42–7.35 (m,
4H, H-2’’), 7.08–7.03 (m, 4H, H-3’’), 6.96–6.93 (m, 1H, H-1’), 6.39
3
3
4
(ddd, JH,H =6.0 Hz, JH,H =1.7 Hz, JH,H =1.7 Hz, 1H, H-3’), 5.88–5.83
3
2
+
(m, 1H, H-2’), 5.75 (d, JH,H =8.1 Hz, 1H, H-5), 5.10 (dd, JH,H =8.6 Hz,
NH4 form) and the product was further purified by RP-18 flash
3JH,P =5.4 Hz, 4H, Bn), 4.98–4.93 (m, 1H, H-4’), 4.22–4.11 (m, 2H, H-
chromatography (MeOH/H2O 2:1 v/v). The product was obtained as
a hygroscopic, colorless solid (19 mg, 26 mmol, 31%, 1ꢂNH4+).
1H NMR (400 MHz, CD3OD): d=7.97 (d, 3JH,H =8.4 Hz, 1H, H-6),
5’), 2.58 (t, 3JH,H =7.4 Hz, 4H, H-a), 1.73 (tt, 3JH,H =7.5 Hz, JH,H
=
3
7.2 Hz, 4H, H-b), 1.47–1.25 (m, 24H, H-c, H-d, H-e, H-f, H-g, H-h),
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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7.45–7.37 (m, 4H, H-2’’), 7.09–7.02 (m, 4H, H-3’’), 6.05–5.99 (m, 1H,
2.25 (m, 1H, H-2’a), 2.07–1.93 (m, 3H, H-2’b, H-3’), 1.78–1.61 (m,
3
3
H-1’), 5.72 (d, JH,H =8.4 Hz, 1H, H-5), 5.13 (d, JH,H =8.3 Hz, 4H, Bn),
7H, H-b, H-B), 1.47–1.24 (m, 27H, H-c, H-d, H-e, H-f, H-g, H-h, H-C),
3
3
3
4.27–4.19 (m, 2H, H-5’), 4.11–4.04 (m, 1H, H-4’), 2.81 (sept, JH,H
=
1.03 (t, JH,H =7.4 Hz, 5H, H-D), 0.91 ppm (t, JH,H =6.9 Hz, 6H, H-i);
13C NMR (126 MHz, CD3OD): d=173.8 (2ꢂC=O), 152.4 (2ꢂC-4’’),
142.6 (C-6), 134.8 (d, 3JC,P =7.4 Hz, 2ꢂC-1’’), 130.5 (2ꢂC-2’’), 122.9
7.0 Hz, 2H, CH-iPr), 2.38–2.27 (m, 1H, H-2’a), 2.07–1.91 (m, 3H, H-
2’b, H-3’), 1.30 ppm (d, 3JH,H =7.0 Hz, 12H, CH3-iPr); 13C NMR
(101 MHz, CD3OD): d=163.4 (C-4), 152.5 (2ꢂC-4’’), 142.6 (C-6),
3
(2ꢂC-3’’), 102.5 (C-5), 87.5 (C-1’), 81.2 (d, JC,P =9.2 Hz, C-4’), 70.35
134.9 (d, 3JC,P =6.8 Hz, 2ꢂC-1’’), 130.5 (2ꢂC-2’’), 122.8 (2ꢂC-3’’),
(d, JC,P =9.2 Hz, 2ꢂBn), 68.3 (d, JC,P =6.0 Hz, C-5’), 59.5 (C-A), 35.0
(C-a), 33.3 (C-2’), 33.0, 30.6, 30.4, 30.4, 30.2 (C-b, C-c, C-d, C-e, C-f),
2
3
2
102.5 (C-5), 87.6 (C-1’), 81.2 (d, 3JC,P =9.4 Hz, C-4’), 70.3 (d, JC,P
=
2
5.9 Hz, 2ꢂBn), 68.3 (d, JC,P =6.2 Hz, C-5’), 35.3 (2ꢂCH-iPr), 33.3 (C-
26.1 (C-3’), 26.0, 24.7 (C-g, C-h), 23.7 (C-B), 20.7 (C-C), 14.4 (C-i),
2’), 26.1 (C-3’), 19.2 ppm (4ꢂCH3-iPr); 31P NMR (162 MHz, CD3OD):
13.9 ppm (C-D); 31P NMR (162 MHz, CD3OD): d=ꢀ11.67 (d, JP,P
=
2
2
2
d=ꢀ11.66 (d, JP,P =19.7 Hz), ꢀ12.83 ppm (d, JP,P =19.8 Hz); IR: n˜ =
2974, 2878, 1753, 1674, 1509, 1461, 1261, 1202, 1166, 1005, 956,
803, 498 cmꢀ1; TLC: Rf =0.69 (EE/MeOH 7:3 v/v); HRMS (ESIꢀ): m/z
calcd: 723.1726 [MꢀH]ꢀ, found: 723.1736.
20.3 Hz), ꢀ12.86 ppm (d, 2JP,P =20.9 Hz); IR: n˜ =2921, 2851, 1755,
1686, 1509, 1464, 1380, 1264, 1200, 1106, 964, 813, 510 cmꢀ1; TLC:
Rf =0.29 (EE/MeOH 7:3 v/v); HRMS (ESIꢀ): m/z calcd: 891.3676
[MꢀH]ꢀ, found: 891.3592.
Bis(4-heptanoyloxybenzyl)-ddUDP 20. General procedure 5 with
bis(tetra-n-butylammonium)-ddUMP 9 (130 mg, 168 mmol) in 5 mL
CH3CN, bis(4-heptanoyloxybenzyl)-N,N-diisopropylaminophosphor-
oamidite 12 (170 mg, 0.283 mmol, 1.7 equiv), DCI (36 mg,
0.30 mmol, 1.8 equiv). The reaction mixture was stirred at room
temperature for 24 h, cooled to ꢀ208C and tert-butylhydroperox-
ide (5.5m in n-decane, 55 mL, 0.31 mmol, 1.8 equiv) was added. The
mixture was stirred for 1 h at room temperature. The crude prod-
uct was purified by a first RP-18 flash chromatography (MeOH/H2O
1:1–5:1 v/v), counter-ions were changed to ammonium (Dowex
Bis(4-dodecanoyloxybenzyl)-ddUDP 22. General procedure 5 with
bis(tetra-n-butylammonium)-ddUMP 9 (91 mg, 110 mmol) in 5 mL
CH3CN,
bis(4-dodecanoyloxybenzyl)-N,N-diisopropylaminophos-
phoroamidite 14 (160 mg, 216 mmol, 2.0 equiv), DCI (25 mg,
0.21 mmol, 1.9 equiv). The reaction mixture was stirred at room
temperature for 22 h, cooled to ꢀ208C and tert-butylhydroperox-
ide (5.5m in n-decane, 40 mL, 0.22 mmol, 2.0 equiv) was added. The
mixture was stirred for 30 min at room temperature. The crude
product was purified by a first RP-18 flash chromatography
(MeOH/H2O 1:1–10:1 v/v), counter-ions were changed to ammoni-
+
+
50WX8, NH4 form) and the product was further purified by RP-18
um (Dowex 50WX8, NH4 form) and the product was further puri-
flash chromatography (MeOH/H2O 5:1 v/v). The product was ob-
tained as a hygroscopic, colorless solid (48 mg, 58 mmol, 41%, 1ꢂ
fied by RP-18 flash chromatography (MeOH/H2O 9:1 v/v). The prod-
uct was obtained as a colorless oil (37 mg, 34 mmol, 31%, 0.6ꢂ
1
3
1
3
NH4+). H NMR (400 MHz, CD3OD): d=7.96 (d, JH,H =8.2 Hz, 1H, H-
nBu4N+, 0.4ꢂNH4+). H NMR (500 MHz, CD3OD): d=7.97 (d, JH,H
=
3
6), 7.43–7.37 (m, 4H, H-2’’), 7.08–7.01 (m, 4H, H-3’’), 6.02 (dd, JH,H
=
8.1 Hz, 1H, H-6), 7.42–7.37 (m, 4H, H-2’’), 7.07–7.02 (m, 4H, H-3’’),
6.04–6.00 (m, 1H, H-1’), 5.73 (d, 3JH,H =8.1 Hz, 1H, H-5), 5.12 (d,
3JH,H =8.6 Hz, 4H, Bn), 4.27–4.19 (m, 2H, H-5’), 4.11–4.04 (m, 1H, H-
3
3
6.8 Hz, JH,H =3.6 Hz, 1H, H-1’), 5.73 (d, JH,H =8.1 Hz, 1H, H-5), 5.12
(d, JH,H =8.4 Hz, 4H, Bn), 4.27–4.18 (m, 2H, H-5’), 4.11–4.03 (m, 1H,
H-4’), 2.58 (t, JH,H =7.4 Hz, 4H, H-a), 2.38–2.27 (m, 1H, H-2’a), 2.06–
1.93 (m, 3H, H-2’b, H-3’), 1.73 (tt, JH,H =7.5 Hz, JH,H =7.3 Hz, 4H, H-
3
3
3
4’), 3.26–3.20 (m, 5H, H-A), 2.57 (t, JH,H =7.5 Hz, 4H, H-a), 2.37–2.27
3
3
3
(m, 1H, H-2’a), 2.07–1.94 (m, 3H, H-2’b, H-3’), 1.73 (tt, JH,H =7.4 Hz,
3
b), 1.48–1.28 (m, 12H, H-c, H-d, H-e), 0.93 ppm (t, JH,H =6.8 Hz, 6H,
3JH,H =7.4 Hz, 4H, H-b), 1.70–1.61 (m, 5H, H-B), 1.47–1.24 (m, 37H,
H-f); 13C NMR (101 MHz, CD3OD): d=173.7 (2ꢂC=O), 166.3 (C-4),
152.4 (2ꢂC-4’’), 152.3 (C-2), 142.6 (C-6), 134.9 (d, 3JC,P =7.4 Hz, C-
H-c, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-C), 1.03 (t, JH,H =7.3 Hz, 7H,
3
H-D), 0.90 ppm (t, 3JH,H =6.9 Hz, 6H, H-k); 13C NMR (126 MHz,
CD3OD): d=173.7 (2ꢂC=O), 167.8 (C-4), 152.4 (2ꢂC-4’’), 152.3 (C-
2), 142.6 (C-6), 134.9 (d, 3JC,P =6.5 Hz, 2ꢂC-1’’), 130.5 (4ꢂC-2’’),
1’’), 130.5 (4ꢂC-2’’), 122.9 (4ꢂC-3’’), 102.5 (C-5), 87.5 (C-1’), 81.2 (d,
2
3JC,P =9.2 Hz, C-4’), 70.3 (d, 3JC,P =5.5 Hz, 2ꢂBn), 68.3 (d, JC,P
=
3
6.5 Hz, C-5’), 35.0 (C-a), 33.3 (C-2’), 32.6, 29.8 (C-b, C-c), 26.1 (C-3’),
122.9 (4ꢂC-3’’), 102.5 (C-5), 87.5 (C-1’), 81.2 (d, JC,P =9.1 Hz, C-4’),
25.9, 23.6 (C-d, C-e), 14.4 ppm (C-f); 31P NMR (162 MHz, CD3OD):
70.3 (d, JC,P =6.4 Hz, 2ꢂBn), 68.3 (d, JC,P =5.6 Hz, C-5’), 59.5 (C-A),
35.0 (C-a), 33.3 (C-2’), 33.1, 30.7, 30.6, 30.5, 30.4, 30.2 (C-b, C-c, C-d,
C-e, C-f, C-g, C-h), 26.1 (C-3’), 26.0, 24.8 (C-i, C-j), 23.7 (C-B), 20.7 (C-
C), 14.4 (C-k), 13.9 ppm (C-D); 31P NMR (162 MHz, CD3OD): d=
ꢀ13.18 (d, 2JP,P =19.8 Hz), ꢀ14.35 ppm (d, 2JP,P =22.2 Hz); IR: n˜ =
2956, 2916, 2849, 1752, 1685, 1510, 1465, 1382, 1222, 1004, 968,
816, 720, 513 cmꢀ1; TLC: Rf =0.19 (EE/MeOH 7:3 v/v); HRMS (ESIꢀ):
m/z calcd: 947.4230 [MꢀH]ꢀ, found: 947.4225.
2
2
2
2
d=ꢀ11.73 (d, JP,P =17.7 Hz), ꢀ12.86 ppm (d, JP,P =20.1 Hz); IR: n˜ =
2955, 2929, 2858, 1755, 1678, 1509, 1462, 1263, 1198, 1102, 1025,
960, 810, 557 cmꢀ1; TLC: Rf =0.75 (EE/MeOH 7:3 v/v); HRMS (ESIꢀ):
m/z calcd: 807.2737 [MꢀH]ꢀ, found: 807.2652.
Bis(4-decanoyloxybenzyl)-ddUDP 21. General procedure 5 with
bis(tetra-n-butylammonium)-ddUMP 9 (86 mg, 110 mmol) in 5 mL
CH3CN, bis(4-decanoyloxybenzyl)-N,N-diisopropylaminophosphor-
oamidite 13 (135 mg, 197 mmol, 1.8 equiv), DCI (25 mg, 0.21 mmol,
1.9 equiv). The reaction mixture was stirred at room temperature
for 20 h, cooled to ꢀ208C and tert-butylhydroperoxide (5.5m in n-
decane, 36 mL, 0.20 mmol, 1.8 equiv) was added. The mixture was
stirred for 30 min at room temperature. The crude product was pu-
rified by a first RP-18 flash chromatography (MeOH/H2O 1:1–9:1 v/
v), counter-ions were changed to ammonium (Dowex 50WX8,
Chemical hydrolysis of cycloSal and DiPPro compounds 15–22,
24, and 25. Stock solutions (50 mm in [D6]DMSO) of the com-
pounds were prepared. For the hydrolysis solution, 11 mL of the
stock solution were added to 189 mL [D6]DMSO and 100 mL Milli-
pore H2O. In case of the cycloSal compounds 24 and 25, 5 mL of an
AZT 2 solution (37 mm in Millipore H2O) were added as internal
standard. The hydrolysis was started by the addition of 300 mL of
phosphate-buffered saline (PBS, 50 mm, pH 7.3). This solution was
incubated at 378C in a Thermomixer. An initial aliquot (30 mL) was
taken directly after addition of the buffer and further aliquots
(30 mL) were taken to monitor the hydrolysis. Of each aliquot 20 mL
were directly analyzed by analytical RP-HPLC (method A for DiPPro
compounds, method B for cycloSal compounds; 260 nm). Calcula-
tion of exponential decay curves with commercially available soft-
+
NH4 form) and the product was further purified by RP-18 flash
chromatography (MeOH/H2O 5:1 v/v). The product was obtained as
a colorless oil (42 mg, 42 mmol, 38%, 0.4ꢂnBu4N+, 0.6ꢂNH4+).
1H NMR (400 MHz, CD3OD): d=7.97 (d, 3JH,H =8.1 Hz, 1H, H-6),
3
7.43–7.37 (m, 4H, H-2’’), 7.07–7.03 (m, 4H, H-3’’), 6.02 (dd, JH,H
=
3
3
6.4 Hz, JH,H =3.6 Hz, 1H, H-1’), 5.73 (d, JH,H =8.1 Hz, 1H, H-5), 5.12
(d, JH,H =8.2 Hz, 4H, Bn), 4.27–4.19 (m, 2H, H-5’), 4.11–4.04 (m, 1H,
H-4’), 3.26–3.19 (m, 3H, H-A), 2.57 (t, JH,H =7.4 Hz, 4H, H-a), 2.37–
2
3
ware (Microsoft Excel) yielded the half-life (t1= ) of the prodrug
2
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compounds in hours. For each compound, two determinations of
Acknowledgements
t1= were performed, and the resulting values were averaged.
2
We thank Ms. Leen Ingels and Ms. Ria Van Berwaer for dedicated
technical assistance. The biological research was supported by
KU Leuven (grant GOA 10/014).
Bioassays
Keywords: antiviral agents · bioreversible protection · drug
delivery · HIV · nucleosides · prodrugs
Hydrolysis of cycloSal and DiPPro compounds 15–22, 24 and 25
in CD4 T-lymphocyte CEM cell extracts: Stock solutions (4.3 mm
+
in [D6]DMSO) of the compounds were prepared. For the hydrolysis
solution, 10 mL of the stock solution were added to 50 mL of the
cell extract and 10 mL of magnesium chloride solution (70 mm in
Millipore H2O). For every reading a separate solution was prepared
and incubated at 378C in a Thermomixer for various periods (de-
pending on the rate of hydrolysis). The reactions were stopped by
the addition of 200 mL MeOH, the samples were stored on ice for
10 min followed by ultrasonication for 10 min. The samples were
centrifuged at 08C (14000 rpm) for 10 min, the supernatant was fil-
tered using a syringe filter (Schleicher & Schuell Spartan 13/30,
0.2 mm) and stored under liquid nitrogen. For analysis, the samples
were defrosted and aliquots of 90 mL were analyzed by analytical
RP-HPLC (method A for DiPPro compounds, method B for cycloSal
compounds; 260 nm). Calculation of exponential decay curves with
commercially available software (Microsoft Excel) yielded the half-
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life (t1= ) of the prodrug compounds in hours. For each compound,
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ic-Tenter, J. Balzarini, Eur. J. Org. Chem. 2006, 197–206; c) H. J. Jessen, J.
2
two determinations of t1= were performed, and the resulting
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8115–8123.
2
values were averaged.
+
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1659; b) E. H. Rios Morales, C. Arbelo Romꢃn, J. O. Thomann, C. Meier,
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cells were grown in RPMI-1640-based cell culture medium to
a final density of ~3ꢂ106 cellsmLꢀ1. Then, cells were centrifuged
for 10 min at 1250 rpm, 48C, washed twice with cold PBS, and the
pellet was resuspended at 108 cellsmLꢀ1 and sonicated (Hielsecher
Ultrasound Techn., 100% amplitude, 3ꢂ10 s) to destroy cell integ-
rity. The resulting cell suspension was then centrifuged at
10000 rpm to remove cell debris, and the supernatant divided in
aliquots before being frozen at ꢀ808C and used.
Anti-HIV activity assays: Inhibition of HIV-1(IIIB)- and HIV-2(ROD)-
induced cytopathicity in CEM cell cultures was measured in micro-
titer 96-well plates containing ~3ꢂ105 CEM cellsmLꢀ1 infected
with 100 CCID50 of HIV per milliliter and containing appropriate di-
lutions of the test compounds. After 4–5 days of incubation at
378C in a CO2-controlled humidified atmosphere, CEM giant (syn-
cytium) cell formation was examined microscopically. The EC50
(50% effective concentration) was defined as the compound con-
centration required to inhibit HIV-induced giant cell formation by
50%.
[13] a) C. Pꢄrigaud, G. Gosselin, I. Lefebvre, J.-L. Girardet, S. Benzaria, I.
Barber, J.-L. Imbach, Bioorg. Med. Chem. Lett. 1993, 3, 2521–2526; b) J.
´
Romanowska, M. Sobkowski, A. Szymanska-Michalak, K. Kołodziej, A.
NDP kinase assays: Nucleoside diphosphate kinase was derived
and purified from bovine liver (Sigma–Aldrich, St. Louis, MO, USA).
The reaction mixture contained 200 mm ATP in PBS, pH 7.2, 1 mm
MgCl2 (Sigma–Aldrich) and 100 mm UDP, ddUDP or d4UDP. The re-
action was started by adding the enzyme (134 mU), and the reac-
tion was incubated at 378C for 0, 10, 20, 30 or 60 min. At the end
of the incubation period, 100 mL aliquots were added to 200 mL
ice-cold MeOH, kept on ice for 10 min, centrifuged for 10 min at
48C, 13000 rpm (5415R Eppendorf centrifuge) to remove protein
and the cleared supernatants were subjected to anion-exchange
HPLC analysis (Partisphere-SAX, Whatman Int., Maidstone, UK). The
gradient used consisted of 5 mm NH4H2PO4 pH 5.0 (B) and 0.3m
NH4H2PO4 pH 5.0 (C) and was run as follows: 5 min 100% B, 15 min
from 100% B to 100% C, 20 min 100% C, 5 min from 100% C to
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F. Pertenbreiter, J. Balzarini, C. Meier*
&& – &&
Nucleoside Mono- and Diphosphate
Prodrugs of 2’,3’-Dideoxyuridine and
2’,3’-Dideoxy-2’,3’-didehydrouridine
Chemical Trojan horses: Can they
rescue antiviral activity? Nucleoside
mono- and diphosphate prodrugs of
d4U and ddU were prepared by apply-
ing the cycloSal and DiPPro approaches.
These compounds underwent successful
delivery, but surprisingly showed weak
or no antiviral activity. Phosphorylation
studies with nucleoside diphosphate
kinase showed that this enzyme is prac-
tically unable to convert ddUDP and
d4DUP to the triphosphate form.
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ChemMedChem 0000, 00, 1 – 14
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ÞÞ
These are not the final page numbers!