Bis(benzoyloxybenzyl)-DiPPro nucleoside diphosphates of anti-HIV active nucleoside analogues

Article abstract of DOI:10.1002/cmdc.201500063

Nucleoside analogues are extensively used as antiviral and anticancer agents. Their efficiency is dependent on their metabolism into the ultimately active nucleoside triphosphates. Often one step or even more in the metabolism of the nucleoside to the tri

Full text of DOI:10.1002/cmdc.201500063

DOI: 10.1002/cmdc.201500063
Full Papers
Bis(benzoyloxybenzyl)-DiPPro Nucleoside Diphosphates of
Anti-HIV Active Nucleoside Analogues
Lina Weinschenk,[a] Tristan Gollnest,[a] Dominique Schols,[b] Jan Balzarini,[b] and Chris Meier*[a]
Nucleoside analogues are extensively used as antiviral and an-
ticancer agents. Their efficiency is dependent on their metabo-
lism into the ultimately active nucleoside triphosphates. Often
one step or even more in the metabolism of the nucleoside to
the triphosphate is inefficient. To overcome this hurdle, pro-
drugs of the nucleotides are needed. Bis(acyloxybenzyl)nucleo-
side diphosphates have been reported by us as a first example
of an efficient nucleoside diphosphate prodrug (DiPPro nucleo-
tides). Here, the synthesis and the properties of bis(benzoylox-
ybenzyl)nucleoside diphosphates of the nucleoside analogues
d4T and AZT are disclosed. The synthesis was achieved by
using a phosphoramidite/oxidation route. In chemical hydroly-
sis studies, most of the compounds formed a nucleoside di-
phosphate. This was confirmed in CEM cell extracts, although
the prodrug stability in extracts was lower than in phosphate
buffer. Furthermore, the stability and the amount of nucleoside
diphosphate formed were dependent on the substituent in
the benzoyl moiety. Some of the compounds were more active
against HIV in thymidine kinase-deficient CEM/TKÀ cells than
were d4T or AZT.
Introduction
Nucleoside analogues are widely used as anticancer and antivi-
ral agents. However, their activity depends on their efficient
phosphorylation by kinases. The metabolism leads via the nu-
cleoside monophosphate (NMP) and the nucleoside diphos-
phate (NDP) to the ultimately antivirally active nucleoside tri-
phosphate (NTP). Former studies have shown that cellular kin-
ases often catalyze this biotransformation insufficiently, and
this results in a loss in antiviral activity or adverse effects can
occur.[1] This hurdle can be overcome by using lipophilic pro-
drugs of the phosphorylated parent nucleosides (pronucleoti-
des), which are able to bypass the rate-limiting, kinase-cata-
lyzed conversion steps. This aim has been successfully ach-
ieved in the past for NMP with prodrugs such as the cycloSal,
bisSate, bisPOM, and phosphoramidate nucleotides.[2]
ical stability of the pyrophosphate moiety, and this results in
a cleavage of this part of the molecule. As a consequence, for
chemical reasons, the design of NDP prodrugs is more de-
manding than the development of NMP prodrugs.
Hostetler et al. synthesized different NDP diglycerides as po-
tential NDP prodrugs.[6] However, the hydrolysis of these com-
pounds delivered the monophosphates instead of the diphos-
phates due to cleavage of the diphosphate moiety. Another
approach was reported by Huynh-Dinh et al., who acylated the
b-phosphate with fatty acids to give a mixed anhydride
bond.[7] In chemical hydrolysis studies, the NDP was indeed
formed; however, in cell extracts, an undefined decomposition
of the compounds was observed.
Recently, we reported a first successful NDP prodrug ap-
proach. In our approach, the b-phosphate of the NDP was es-
terified with two acyloxybenzyl moieties; this neutralized the
two charges on this phosphate group (DiPPro approach).[8,9]
Because this approach is based on an enzymatically triggered
delivery mechanism, the diphosphate is released preferentially
inside cells, and the released NDP is trapped due to the highly
charged pyrophosphate. The chemical stability at physiological
pH was found to be high. The general principle of the hydroly-
sis of these DiPPro nucleotides is summarized in Scheme 1. En-
zymatic cleavage of the acyl group attached to the phenol res-
idue destabilizes the benzyl phosphate ester bond. As a result,
a spontaneous 1,6-elimination gives the mono-masked NDP in-
termediate 3. Repetition of this process eventually delivers the
nucleoside diphosphate.
In the case of the anti-HIV-active 3’-azido-3’-deoxythymidine
(AZT), the rate-limiting step is the conversion of AZT mono-
phosphate (AZTMP) to AZT diphosphate (AZTDP).[3] The result
is high intracellular concentrations of AZTMP, which lead to
several side effects.[4] Therefore, the use of an AZTDP prodrug
could overcome this limitation. However, reports on such NDP
delivery systems have been rare.[5] In the case of NDPs, com-
plete lipophilic modification of the highly polar diphosphate
group in particular can lead to a marked decrease in the chem-
[a] L. Weinschenk, T. Gollnest, Prof. Dr. C. Meier
Organic Chemistry, Department of Chemistry
Faculty of Sciences, University of Hamburg
Martin-Luther-King-Platz 6, 20146 Hamburg (Germany)
and
German Center of Infection Research (DZIF)
E-mail: chris.meier@chemie.uni-hamburg.de
We have reported DiPPro compounds that bear short-chain
carboxylic acids and fatty acids of different lengths in the acy-
loxybenzyl masking unit.[8,9] In that report, 3’-deoxy-2’,3’-dide-
hydrothymidine (d4T) was used as a model nucleoside. The
corresponding DiPPro compounds were synthesized and ana-
[b] Prof. Dr. D. Schols, Prof. Dr. J. Balzarini
Rega Institute for Medical Research
Katholieke Universiteit Leuven (KU Leuven)
Minderbroedersstraat 10, 3000 Leuven (Belgium)
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Scheme 1. General principle of the hydrolysis of DiPPro nucleotides 1.
lyzed to investigate the relationship between chain length, hy-
drolysis behavior, and antiviral activity. It was proven that the
stability of these compounds increased with increasing chain
length. Up to an alkyl chain length of C9 (Scheme 1, R=C9H19),
the nucleoside diphosphate was preferentially released in
phosphate buffer as well as in cell extract. A further increase in
the chain length was associated with a further increase in
chemical stability and, at the same time, led to a side reaction:
nucleophilic attack of, for example, water at the b-phosphorus
atom favored cleavage of the diphosphate moiety and thus
formation of unwanted NMP in addition to the NDP.
Among the DiPPro compounds published earlier were the
benzoyl-protected derivatives bis(benzoyloxybenzyl)-d4TDP
(4c; X=H; Scheme 2) and the corresponding AZTDP derivative
5c.[8] The benzoyl modification offers the possibility to fine-
tune the hydrolysis behavior by adding different substituents
without varying the lipophilicity to any considerable degree.
This was not possible with the previously described acyloxy-
benzyl counterparts. In this report, we disclose a series of
DiPPro nucleoside diphosphates bearing lipophilic and enzy-
matically cleavable benzoyloxybenzyl moieties. DiPPro-d4TDP
derivatives 4 with different donor and acceptor substituents in
the 4-position of the benzoyl moiety were synthesized
(Scheme 2). The hydrolysis behavior was investigated with
regard to chemical stability in phosphate buffer. Incubation
with T-lymphocyte CEM cell extracts provided information
about the properties in biological media. A second series of ac-
ceptor-substituted bis(benzoyloxybenzyl)-AZTDP prodrugs 5 of
the nucleoside analogue AZT was also synthesized.
Scheme 2. Synthesized bis(benzoyloxybenzyl)-NDP prodrugs 4 and 5 (letter-
ing shown in compounds 5 is used for NMR assignment in the Experimental
Section).
zoylchlorides 11 at low temperatures (up to 75% yield). Subse-
quent reaction with (N,N-diisopropyl)dichlorophosphine (12)
gave phosphoramidites 9 in yields of up to 90%. Nucleotides 6
and 7 were synthesized in yields of 83 and 61%, respectively,
by following the Sowa–Ouchi protocol.[10] The final synthesis of
DiPPro nucleoside diphosphates 4 and 5 was successfully ach-
ieved by dicyanoimidazole-mediated coupling and subsequent
oxidation with tert-butylhydroperoxide, which gave the target
products in yields of 30–66% over the two final steps.
Results and Discussion
The synthesis route towards DiPPro-NDPs 4 and 5, based on
phosphoramidite chemistry, is summarized in Scheme 3. Corre-
sponding phosphoramidites 8 bearing two masking units were
coupled with d4TMP (6) or AZTMP (7) to form the target pro-
nucleotide after oxidation. This convergent approach offered
high flexibility for combining different bis(benzoyloxybenzyl)-
phosphoramidites with nucleotides. Briefly, different 4-benzoyl-
benzyl alcohols (9) were obtained in good yield by selective
benzoylation of 4-hydroxybenzyl alcohol (10) by using ben-
The X substituents in the benzoyl moiety of d4TDP prodrugs
4a, b, e–g ranged from strong donors to strong acceptor
groups. The masks that gave the highest selectivity for the de-
livery of d4TDP in the hydrolysis studies were also applied to
AZTMP 7; this led to compounds 5e–g.
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Table 1. Hydrolysis half-lives (t1/2) of bis(benzoyloxylbenzyl)-DiPPro-NDPs
in PBS (pH 7.3) and CEM cell extracts as well as retention times.
Compound
t1/2 [h]
tR [min]
4 or 5
X
PBS (7.3)
CEM/0
(RP-HPLC)
4a
4b
4c
4d
4e
4 f
4g
5c
5e
5 f
5g
OMe
Me
H[8]
Cl
CF3
CN
NO2
H[8]
CF3
CN
480
80
82
13
20
7
9
3
7
7
5.5
4
1.25
0.5
3.5
4
14.8[a]
15.8[a]
9.41
16.1[a]
15.7[a]
13.2[a]
14.8[a]
10.32
18.9[b]
15.9[b]
16.7[b]
4
82
12
6.5
4.5
NO2
1.5
[a] Gradient A with CH3CN/[N(C4H9)4] phosphate (0.55 mm). [b] Gradient B
with CH3CN/[N(C4H9)4] acetate (2 mm).
In the case of the strongest donor substituent (X=OCH3),
the stability increased markedly; at the same time, almost no
d4TDP could be detected. Surprisingly, Me-Ph-DiPPro-d4TDP
4b showed relatively low stability in the cell extracts. For some
unknown reason, this compound seemed to be a good sub-
strate for the esterases present in the cell extracts.
The studies on DiPPro-d4TDPs 4 revealed a direct correlation
between the stability of the compounds and the amount of
d4TDP released, also when using CEM cell extracts. In the case
of the strong acceptor-substituted DiPPro-d4TDPs 4e–g,
mainly d4TDP, but also small amounts d4TMP (6) were detect-
ed. However, the formation of the d4TMP can also be a result
of dephosphorylation of d4TDP by phosphatases present in
the cell extracts. In contrast, the enzymatic hydrolysis of MeO-
Ph-DiPPro-d4TDP (4a) did not deliver d4TDP, only d4TMP 6
was detected. In addition, only very small amounts of inter-
mediate 3 were observed in the chromatograms. In this case, it
is most probable that d4TMP (6) was formed as a result of an-
hydride bond cleavage.
Scheme 3. Synthesis of benzoyl ester-bearing DiPPro-NDPs 4 and 5.
a) 1 equiv benzoylchloride 11, 1.1 equiv 4-hydroxybenzylalcohol 10, 1 equiv
TEA, THF, 2 h, 08C; b) 1 equiv PIII-reagent 12, 2.2 equiv benzyl ester 9
2.3 equiv TEA, THF, 16–20 h, RT; c) 1 equiv [N(nBu)4]2NMP, 1.5–1.8 equiv phos-
phoramidite 8, 1.5–1.75 equiv 4,5-dicyanoimidazole, 16–20 h, RT; d) tBuOOH,
RT, 20 min.
In Scheme 4 the different possible hydrolysis routes are sum-
marized. In addition to the benzoyl ester hydrolysis (path-
way A), cleavage of the phosphate anhydride occurred as
a side reaction (pathway B). The latter led to the formation of
d4TMP, as described above.
Next, DiPPro compounds 4 and 5 were studied with regard
to their chemical stabilities in aqueous phosphate buffered
saline (PBS, pH 7.3) and in CEM cell extracts. The latter was
used to simulate the biological environment and to study the
contribution of enzymes present in the extracts to the hydroly-
sis process. In both cases, the studies were monitored by ana-
lytical RP-HPLC. Generally, the half-lives—measured by the de-
crease in the corresponding peak of the pronucleotide 4 or
5—in PBS were significantly higher than those in cell extracts
(Table 1), and the half-lives increased with the increasing elec-
tron-donating effect of the substituent.
On the other hand, the faster the first hydrolysis of the pro-
drug to the mono-masked NDP intermediate 3, the higher the
amount of nucleoside diphosphate formed. It was observed
that only very small amounts—if any—of NMP were formed in
the hydrolysis of the intermediate 3. Therefore, the phosphate
anhydride bond was most likely cleaved by nucleophilic attack
of water or hydroxide at the b-phosphorus atom of the doubly
masked starting material 4 or 5. The second charge at the b-
phosphorus atom present in the mono-masked NDP intermedi-
ate 3 prevented a nucleophilic attack at the pyrophosphate
moiety. This explains why d4TDP was formed predominantly in
both media from DiPPro compounds bearing acceptor-substi-
tuted masking units (X=CF3, CN, NO2; Figure 1).
Detailed studies showed that from acceptor-substituted
compounds 4e–g, d4TDP was formed in phosphate buffer as
well as in cell extracts either exclusively (4g) or at least pre-
dominantly (4e, f). However, when the electron-withdrawing
effect was reduced by introducing donor substituents, the cor-
responding NMP 6 was also formed.
Because of the favorable properties of the masks bearing
strong electron-withdrawing groups in the benzoyl moiety,
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found to be more labile than the corresponding d4TDP deriva-
tive 4e (Table 1).
As compared to bis(acyloxybenzyl)-DiPPro-d4TDPs, the
chemical stability is markedly lower for all acceptor-substituted
bis(benzoyloxybenzyl)-DiPPro-NDPs, except for R=CH3 (t1/2
=
10 h).[8,9] As a consequence, the amount of d4TDP formed from
the prodrug was also higher. Additionally, it was observed that
DiPPro compounds with short aliphatic chains seemed to be
better substrates for the enzymes present in cell extracts (R=
CH3, t1/2 =0.05 h; R=C4H9, t1/2 =0.6 h).[9]
The lipophilicity of the bis(benzoyloxybenzyl)-DiPPro com-
pounds was estimated from the retention times detected by
RP-HPLC. As seen in Table 1, the values were all in the same
range, so changing the substituent X had only a small influ-
ence on the lipophilicity of the pronucleotide. However, as can
also be seen in Table 1, the substituents had a significant effect
on the stability, for example, CF3-Ph-DiPPro-d4TDP 5e was
beyond the most lipophilic compounds, it showed a low stabil-
ity in PBS as well as in the cell extract. This behavior contrasted
with that of the recently reported bis(acyl)-DiPPro com-
pounds.[9]
Antiviral activity
All compounds were evaluated for their potency in inhibiting
HIV replication in HIV-1- and -2-infected wild-type CEM/0 cells
and in HIV-2-infected mutant thymidine kinase-deficient CEM/
TKÀ cells. Table 2 summarizes the antiviral and cytotoxic data
of the potential prodrugs, with the parent nucleoside ana-
logues d4T and AZT as reference compounds.
Figure 1. HPL chromatograms of the hydrolysis of NO2-Ph-DiPPro-d4TDP
(4g) in phosphate buffer at pH 7.3 (top) and in CEM cell extracts (bottom).
Table 2. Anti-HIV activity of DiPPro-NDPs in wild-type and mutant CEM
cells.
Compd
EC50[a] [mm]
CEM/0
CC50[b] [mm]
CEM/0
CEM/TKÀ
HIV-2
HIV-1
HIV-2
4a
0.69Æ0.41
0.80Æ0.23
0.40
1.1Æ0.29
0.91Æ0.28
0.30
3.9Æ2.3
ꢀ2
0.85
98Æ5.7
41Æ0.0
36Æ5
4b
4c[8]
4d
4e
4 f
4g
5e
0.71Æ0.24
0.97Æ0.49
0.24Æ0.16
1.0Æ0.64
1.0Æ0.51
4.8Æ2.5
2.9Æ2.3
38Æ20
51Æ2.8
193Æ45
108Æ6.4
101Æ3.5
108Æ6
95Æ7
1.4Æ0.61
2.0Æ1.2
14Æ7.9
13Æ5.2
7.7Æ2.1
0.042Æ0.015
0.021Æ0.0046
0.008Æ0.003
0.86Æ0.45
0.052Æ0.0057
0.071Æ0.052
0.28Æ0.20
2.3Æ2.4
5 f
ꢀ10
5g
d4T
AZT
ꢀ10
110Æ8
>250
>250
173Æ70
>250
0.0066Æ0.0048 0.021Æ0.018
[a] Antiviral activity in T-lymphocytes: 50% effective concentration. [b] Cy-
totoxic activity: 50% cytotoxic concentration to decrease cell viability.
Scheme 4. Two different hydrolysis pathways of bis(benzoyloxybenzyl)-
DiPPro-nucleoside diphosphates 4 and 5.
All compounds showed antiviral activity in CEM/0 cells. The
antiviral activity against HIV-1 and HIV-2 were in all cases in the
same range as for the corresponding reference compounds
d4T and AZT. Furthermore, in thymidine kinase-deficient CEM/
TKÀ cells, the activity of the donor-benzoyl-DiPPro compounds
was up to 60 times higher than the activity of the parent nu-
these were also applied to achieve AZTDP delivery. Interesting-
ly, the stabilities and the product distribution in PBS as well as
in cell extracts from DiPPro-AZTDPs 5e–g were comparable
with the observations made with the corresponding DiPPro-
d4TDP compounds. However, CF3-Ph-DiPPro-AZTDP 5e was
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cleosides. This is an indication that the intact pronucleotides
were taken up by the cells and, once inside the cells, delivered
a phosphorylated compound.
NDPs were too unstable and/or too low in lipophilicity to ach-
ieve a rapid uptake of intact DiPPro compounds into the cells.
In comparison to the parent nucleosides, the most active
prodrugs, 4b–d, showed a slightly increased cytotoxicity. The
reason for this remains unclear. It has often been observed,
that in the case of highly active nucleotide prodrugs, such as
the phosphoramidate compounds or the cycloSal-phosphate
triesters, the antiviral activity is always associated with an in-
crease in the CC50 values. However, in the case of the cycloSal
compounds, we have clearly proven that, in comparison to 3-
methyl-cycloSal-d4TMP, which also showed an increase in the
toxicity value, the corresponding dTMP derivative did not
show an increase in CC50 value, even though the same o-qui-
nonemethide or salicylalcohol was formed during the delivery
of the nucleoside monophosphate. In compounds 4 and 5,
a p-quinonemethide or 4-hydroxybenzylalcohol is formed. Fur-
ther studies have to be performed to answer this question.
Somewhat surprising was the loss of antiviral activity in case
of almost all acceptor-substituted DiPPro pronucleotides 4 and
5 because, as shown in the hydrolysis studies, they released
NDPs almost selectively. This might be the result of a too rapid
hydrolysis preventing the efficient uptake of the intact pronu-
cleotides. As a consequence, the NDP was most likely formed
before membrane passage took place. The phosphorylated hy-
drolysis product is then extracellularly dephosphorylated to
give the parent nucleoside, which is then taken up by the cells
and proved to be active in the wild-type cell line but not in
the TK-deficient cell line. Another reason for the partial loss of
antiviral activity might be insufficient membrane permeation
as a result of insufficient lipophilicity. Alternatively, it can be
a mix of both. However, it should be noted that in all cases,
the prodrugs were endowed with a markedly higher anti-HIV
efficacy in the CEM/TKÀ cell cultures than the parental d4T or
AZT.
Experimental Section
Synthesis
General: All experiments involving water-sensitive compounds
were conducted under absolutely anhydride conditions and under
nitrogen. All solvents were dried by using standard procedures.
Et3N and CH3CN were dried by being heated under reflux over cal-
cium hydride for several days followed by distillation. CH3CN was
stored over 3 ꢁ molecular sieves. THF was dried by being heated
under reflux over potassium followed by distillation. Ethyl acetate,
petroleum ether 50–70, CH2Cl2, and CH3OH for chromatography
were distilled before use. Solvents were evaporated on a rotary
evaporator under reduced pressure or by using a high-vacuum
pump. Column chromatography was performed by using Merck
silica gel 60, 230–400 mesh. Analytical thin-layer chromatography
was performed on Merck precoated aluminum plates 60F254 with
a 0.2 mm layer of silica gel containing a fluorescent indicator;
sugar-containing compounds were visualized with a spray reagent
(0.5 mL 4-methoxybenzaldehyde, 9 mL EtOH, 0.1 mL glacial acetic
acid, 0.5 mL concentrated sulfuric acid) by heating with a fan. For
some separations, a chromatotron (Harrison Research 7924T) with
glass plates coated with 1-, 2-, or 4-mm layers of VWR 60PF254 silica
gel containing a fluorescent indicator (VWR no. 7749) was used.
Instrumentation: 1H NMR spectroscopy was carried out on
a Bruker AMX 400 at 400 MHz, a Bruker DMX 500 at 500 MHz or
a Bruker AV 400 at 400 MHz with CDCl3, [D6]DMSO or CD3OD as in-
ternal standards. 13C NMR spectra were recorded on a Bruker
AMX 400 at 101 MHz or a Bruker AV 400 at 101 MHz (CDCl3
[D6]DMSO or CD3OD as internal standard). 19F NMR spectra were re-
corded on a Bruker AV 600 MHz. 31P NMR spectra were recorded on
a Bruker AMX 400 at 162 MHz (H3PO4 as internal standard). All 13C
and 31P NMR spectra were recorded in the proton-decoupled
mode. Mass spectra were obtained on a VG Analytical VG/70-250F
FAB spectrometer (xenon, matrix was m-nitrobenzyl alcohol) or
a VG70S EI spectrometer. High-resolution ESI mass spectra were re-
corded on an Agilent 6224 ESI-TOF spectrometer in positive or
negative mode.
Conclusions
Analytical HPLC: LiChroCART 125-3 with LiChrospher 100 RP-18
(5 mm) standard gradient. Method A: 5–100% CH3CN in [N(C4H9)4]
phosphate buffer (0.55 mm; 0–27 min), flow rate 0.1 mLminÀ1. UV
detection at 265 nm. Method B: 5–80% CH3CN in [N(C4H9)4] acetate
buffer (2 mm; 0–24 min), flow rate 0.1 mLminÀ1. UV detection at
265 nm. The purity of the DiPPro nucleotides was evaluated by
HPLC and was in all cases ꢀ95%.
General procedure A: Preparation of phenylbenzoates: Benzoyl-
chloride (1.1 equiv) dissolved in THF was added dropwise to
a weak solution of 4-hydroxybenzylalcohol (1 equiv) and Et3N
(1 equiv) in THF at 08C. After the mixture had been stirred for 1.5–
2.5 h at 08C and filtration of triethylammonium chloride, the sol-
vent was removed under reduced pressure. The residue was dis-
solved in CH2Cl2 and washed twice with saturated NaHCO3 solution
and once with water. The combined organic layers were dried over
sodium sulfate and concentrated under reduced pressure. The by-
product was removed by filtration over silica gel in CH2Cl2 or by
crystallization from the product in CH2Cl2.
Bis(benzoyloxybenzyl)-DiPPro-NDPs have been studied as
a new class of nucleoside diphosphate prodrug. Their synthesis
by phosphoramidite chemistry was straightforward and led to
high purities and good chemical yields. Their stability could be
adjusted by the use of different donor or acceptor substituents
at the 4-position of the benzoyl moiety. We were able to show
that, unlike the previously reported bis(acyloxybenzyl)-NDPs,
the chemical or enzymatic stability of our compounds was no
longer dependent on the lipophilicity of the prodrugs. DiPPro-
NDPs with acceptor substituents at the 4-position of the ben-
zoyl group hydrolyzed preferentially or even selectively to give
the nucleoside diphosphate in phosphate buffer as well as in
CEM cell extracts. Although we have shown that the basic idea
of using bis(benzoyloxybenzyl) moieties as biolabile masking
groups worked in principle, the results of the antiviral activity
assay were somewhat unexpected. The observed significant
loss in anti-HIV activity in TK-deficient cells might be due to
the fact that the acceptor-substituted bis(benzoyloxybenzyl)-
General procedure B: Preparation of bis(4-benzoyloxybenzyl)-
N,N-diisopropylaminophosphoramidites: A solution of phenyl-
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benzoate (2.2 equiv) and Et3N (2.2 equiv) in THF was added drop-
wise to dichloro-N,N,diisopropylaminophosphoramidite (1 equiv),
also dissolved in THF. After the mixture had been stirred for 1 to
2 days at RT and filtration of triethylammonium chloride, the sol-
vent was removed under reduced pressure. The crude product was
purified by radial preparative TLC (chromatotron) with petroleum
ether/ethyl acetate and 10% Et3N.
uct (3.96 g, 15.1 mmol, 62%) was obtained as colorless crystals.
Rf =0.54 in CH2Cl2/CH3OH (19:1); H NMR (400 MHz, [D6]DMSO): d=
1
8.13–8.12 (m, 2H; H-2’), 7.69–7.67 (m, 2H; H-3’), 7.41–7.39 (m, 2H;
H-2), 7.24–7.22 (m, 2H; H-3), 5.25 (s, 1H; -OH), 4.53 ppm (s, 2H; Ph-
CH2); 13C NMR (101 MHz, [D6]DMSO): d=163.8 (C=O), 149.1 (C-1),
140.4 (C-4’), 138.9 (C-4), 131.6 (C-2’), 129.1 (C-3’), 127.9 (C-1’), 127.5
(C-3), 121.3 (C-2), 62.3 ppm (Ph-CH2); IR: n˜ =3312, 3211, 3188, 2926,
2872, 1732, 1593, 1509, 1293, 1277, 1075, 850, 752, 682, 581,
504 cmÀ1; MS (FAB): m/z 263.0 [M+H+]+.
General procedure C: Preparation of DiPPro nucleoside diphos-
phate: Phosphoramidite (1.8 equiv) was added to a solution of nu-
cleoside monophosphate (1 equiv) in dry CH3CN. The reaction was
started by the addition of 4,5-dicyanoimidazole (1.6 equiv) at RT.
After being stirred for 2 h, the mixture was oxidized by the addi-
tion of tert-butylhydroperoxide (1.8 equiv, 5.5 molar solution in n-
decane). The solvents were removed under reduced pressure. The
compounds were purified by RP-18 chromatography (CH3CN/
water). In some cases the tetra-n-butylammonia ions were ex-
changed against ammonia by elution over DOWEX 50WX8 (NH4+).
4-(Hydroxymethyl)phenyl-4’-trifluoromethylbenzoate (9e): Gen-
eral procedure A with 4-hydroxybenzylalcohol (1.50 g, 12.1 mmol)
and Et3N (1.70 mL, 12.1 mmol) dissolved in THF (30 mL), and 4-tri-
fluoromethylbenzoyl chloride (1.54 mL, 13.3 mmol) dissolved in
THF (15 mL). The product (2.69 g, 9.07 mmol, 75%) was obtained
as a colorless solid. Rf =0.85 in CH2Cl2/CH3OH (19:1); 1H NMR
(400 MHz, [D6]DMSO): d=8.33–8.32 (m, 2H; H-2’), 7.99–7.97 (m,
2H; H-3’), 7.42–7.41 (m, 2H; H-3), 7.28–7.26 (m, 2H; H-2), 5.26 (t,
3JHH =5.7 Hz, 1H; -OH), 4.53 ppm (d, 3JHH =5.7 Hz, 2H; Ph-CH2);
13C NMR (101 MHz, [D6]DMSO): d=163.7 (C=O), 149.1 (C-1), 140.6
General procedure D: Preparation of DiPPro nucleoside diphos-
phate: Phosphoramidite (1.5 equiv) was added to a solution of nu-
cleoside monophosphate (1 equiv) in CH3CN. The reaction was
started by the addition of 4,5-dicyanoimidazole (0.5 equiv, 0.25m
solution in CH3CN) at RT. Every 5 min a further 0.25 equiv of 4,5-di-
cyanoimidazole were added up to a total of 1.5 or 1.75 equiv. After
being stirred for 5 min after the last addition, the mixture was oxi-
dized by the addition of tert-butylhydroperoxide (1.5 equiv,
5.5 molar solution in n-decane). The solvents were removed under
reduced pressure. The compounds were isolated by automatic
flash RP-18 chromatography (CH3CN/water). In some cases the
tetra-n-butylammonia ions were exchanged against ammonia by
elution over DOWEX 50WX8 (NH4+).
2
(C-4), 133.3 (d, JCF =32 Hz, C-4’), 132.8 (C-1’), 130.7 (C-2’), 127.6 (C-
3), 125.9 (d, 3JCF =3.7 Hz, C-3’), 125.9 (CF3), 121.7 (C-2), 64.9 ppm
(Ph-CH2); 19F NMR (101 MHz, [D6]DMSO): d=61.68 ppm; IR: n˜ =
3345, 2926, 2857, 1736, 1509, 1411, 1375, 1167, 1128, 879, 771,
699 cmÀ1; HRMS (EI): m/z calcd for [C15H11F3O3]+ 296.07 [M]+,
173.02 [C8H4F3O]+, found: 295.9, 172.9.
4-(Hydroxymethyl)phenyl-4’-cyanobenzoate (9 f): General proce-
dure A with 4-hydroxybenzylalcohol (1.37 g, 11.0 mmol) and Et3N
(1.55 mL, 11.0 mmol) dissolved in THF (20 mL), and 4-cyanobenzoyl
chloride (2.00 g, 12.1 mmol) dissolved in THF (10 mL). The product
(1.85 g, 7.31 mmol, 66%) was obtained as a colorless solid. Rf =
0.45 in petroleum ether/ethyl acetate (1:1); 1H NMR (400 MHz,
[D6]DMSO): d=8.29–8.27 (m, 2H; H-2’), 8.10–8.08 (m, 2H; H-3’),
4-(Hydroxymethyl)phenyl-4’-methoxybenzoate (9a): General pro-
cedure A with 4-hydroxybenzyl alcohol (4.00 g, 32.2 mmol) and
Et3N (4.53 mL, 32.2 mmol) dissolved in THF (30 mL), and 4-meth-
oxybenzoyl chloride (4.62 mL, 35.2 mmol) dissolved in THF (15 mL).
The product (4.85 g, 18.8 mmol, 59%) was obtained as a colorless
solid. Rf =0.34 in CH2Cl2/CH3OH (19:1); 1H NMR (400 MHz,
3
7.42–7.40 (m, 2H; H-3), 7.28–7.26 (m, 2H; H-2), 5.25 (t, JHH =5.7 Hz,
1H; -OH), 4.53 ppm (d, 3JHH =5.8 Hz, 2H; Ph-CH2); 13C NMR
(101 MHz, [D6]DMSO): d=163.2 (C=O), 149.2 (C-1), 140.4 (C-4),
133.6 (C-1’), 132.7 (C-3’), 130.7 (C-2’), 128.4 (C-3), 121.7 (C-2), 118.0
(CN), 115.4 (C-4’), 62.3 ppm (Ph-CH2); IR: n˜ =3380, 3096, 2874,
2231, 1732, 1605, 1508, 1407, 1269, 1212, 1161, 1077, 1036, 1015,
995, 862, 807, 760, 685, 545, 504 cmÀ1; MS (FAB): m/z 253.1 [M].
[D6]DMSO): d=8.09–8.07 (m, 2H; H-2’), 7.39–7.38 (m, 2H; H-3),
3
7.20–7.19 (m, 2H; H-2), 7.13–7.11 (m, 2H; H-3’), 5.24 (t, JHH
=
5.4 Hz, 1H; -OH), 4.52 (d, 4JHH =5.3 Hz, 2H; Ph-CH2), 3.87 ppm (s,
3H; -OCH3);13C NMR (101 MHz, [D6]DMSO): d=164.3 (C=O), 163.7
(C-4’), 149.4 (C-1), 140.1 (C-4), 131.9 (C-2’), 127.5 (C-3), 121.5 (C-2),
121.0 (C-1’), 114.2 (C-3’), 62.4 (Ph-CH2), 55.6 ppm (CH3); IR: n˜ =3536,
3324, 3010, 2973, 2938, 2844, 1723, 1703, 1604, 1507, 1254, 1162,
1078, 1002, 842, 761 cmÀ1; MS (FAB): m/z 259.1 [M+H+]+.
4-(Hydroxymethyl)phenyl-4’-nitrobenzoate (9g): General proce-
dure A with 4-hydroxybenzylalcohol (4.00 g, 32.2 mmol) and Et3N
(4.53 mL, 32.2 mmol) dissolved in THF (30 mL), and 4-nitrobenzoyl
chloride (6.58 g, 35.4 mmol) dissolved in THF (30 mL). The product
(5.27 g, 19.3 mmol, 60%) was obtained as yellow crystals. Rf =0.41
in CH2Cl2/CH3OH (19:1); 1H NMR (400 MHz, CDCl3): d=8.38–8.37
(m, 4H; H-2’, H-3’), 7.48–7.46 (m 2H; H-3), 7.24–7.22 (m, 2H; H-2),
4.75 (s, 2H; Ph-CH2), 1.62 ppm (s, 1H; -OH); 13C NMR (101 MHz,
CDCl3): d=163.5 (C=O), 150.1 (C-4’), 149.9 (C-1) 139.1 (C-4), 134.9
(C-1’), 131.3 (C-2’), 128.2 (C-3), 123.7 (C-3’), 121.5 (C-2), 64.7 ppm
(Ph-CH2); IR: n˜ =3675, 3392, 3109, 3075, 2988, 2901, 1731, 1608,
1522, 1507, 1270, 1195, 852, 713, 500 cmÀ1; MS (FAB): m/z 273.1
[M].
4-(Hydroxymethyl)phenyl-4’-methylbenzoate (9b): General pro-
cedure A with 4-hydroxybenzylalcohol (4.00 g, 32.2 mmol) and Et3N
(4.53 mL, 32.2 mmol) dissolved in THF (30 mL), and 4-methylbenzo-
yl chloride (4.66 mL, 35.2 mmol) dissolved in THF (15 mL). The
product (5.46 g, 22.5 mmol, 70%) was obtained as a colorless solid.
1
Rf =0.53 in CH2Cl2/CH3OH (19:1); H NMR (400 MHz, [D6]DMSO): d=
8.03–8.01 (m, 2H; H-2’), 7.42–7.38 (m, 4H; H-3’, H-3), 7.22–7.20 (m,
3
3
2H; H-2), 5.24 (t, JHH =5.7 Hz, 1H; -OH), 4.52 (d, JHH =5.7 Hz, 2H;
Ph-CH2), 2.43 ppm (s, 3H; -CH3); 13C NMR (101 MHz, [D6]DMSO): d=
163.9 (C=O), 149.4 (C-1), 144.1 (C-4’), 140.2 (C-4), 129.6 (C-2’), 127.5
(C-3’), 126.2 (C-3), 121.5 (C-2), 120.3 (C-1’), 62.3 (Ph-CH2), 21.2 ppm
(CH3); IR: n˜ =3398, 2930, 2877, 1726, 1610, 1506, 1269, 1175, 1073,
1003, 882, 747, 482 cmÀ1; MS (FAB): m/z 243.1 [M+H+]+.
Bis[4-(4’-methoxybenzoyloxy)benzyl]-N,N-diisopropylaminophos-
phoramidite (8a): General procedure B with 4-(hydroxymethyl)-
phenyl-4’-methoxybenzoate (7a; 2.36 g, 9.12 mmol) and Et3N
(1.34 mL, 9.54 mmol) dissolved in THF (25 mL), and dichloro-N,N-
diisopropylaminophosphoramidite (838 mg, 4.15 mmol) dissolved
in THF (25 mL). The product (1.60 g, 2.48 mmol, 60%) was obtained
as a beige solid. Rf =0.58 in petroleum ether/ethyl acetate (3:1)
with 10% Et3N; 1H NMR (400 MHz, [D6]DMSO): d=8.09–8.07 (m,
4H; H-2’), 7.43–7.41 (m, 4H; H-3), 7.25–7.23 (m, 4H; H-2), 7.13–7.11
4-(Hydroxymethyl)phenyl-4’-chlorobenzoate (9d): General proce-
dure A with 4-hydroxybenzylalcohol (3.00 g, 24.2 mmol) and Et3N
(3.40 mL, 24.2 mmol) dissolved in THF (30 mL), and 4-chlorobenzoyl
chloride (3.40 mL, 26.6 mmol), dissolved in THF (30 mL). The prod-
&
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(m, 4H; H-1’), 4.79–4.68 (m, 4H; Ph-CH2), 3.87 (s, 6H; OCH3), 3.72–
Bis[4-(4’-cyanobenzoyloxy)benzyl]-N,N-diisopropylaminophos-
phoramidite (8 f): General procedure B with 4-(hydroxymethyl)-
phenyl-4’-cyanolbenzoate (7 f; 1.00 g, 3.95 mmol) and Et3N
(0.58 mL, 4.1 mmol) dissolved in THF (20 mL), and dichloro-N,N-dii-
sopropylaminophosphoramidite (363 mg, 1.80 mmol) dissolved in
THF (15 mL). The product (1.02 g, 1.60 mmol, 89%) was obtained
as a colorless solid. Tm =1548C; Rf =0.48 in petroleum ether/ethyl
acetate (4:1) with 10% Et3N; 1H NMR (400 MHz, CDCl3): d=8.31–
8.29 (m, 4H; H-2’), 7.83–7.81 (m, 4H; H-3’), 7.44–7.42 (m, 4H; H-3),
7.20–7.17 (m, 4H; H-2), 4.83–4.70 (m, 4H; Ph-CH2), 3.77–3.67 (m,
3
3.63 (m, 2H; NC-H), 1.19 ppm (d, JHH =6.8 Hz, 12H; iPr); 13C NMR
(101 MHz, [D6]DMSO): d=164.5 (C=O), 158.3 (C-4’), 150.5 (C-1),
137.5 (C-4), 132.0 (C-3’), 128.0 (C-3), 121.8 (C-2), 121.1 (C-1’), 114.3
(C-2’), 64.3 (Ph-CH2), 55.4 (OCH3), 42.7 (NC), 24.2 ppm (iPr); 31P NMR
(162 MHz, [D6]DMSO, decoupled): d=147.4 ppm; IR: n˜ =3044,
2966, 2927, 2867, 2845, 1728, 1606, 1508, 1451, 1252, 1186, 1026,
964, 840, 759, 511 cmÀ1; MS (FAB): m/z 646.3 [M+H+]+.
Bis[4-(4’-methylbenzoyloxy)benzyl]-N,N-diisopropylaminophos-
phoramidite (8b): General procedure B with 4-(hydroxymethyl)-
phenyl-4’-methylbenzoate (7b; 2.16 g, 8.90 mmol) and Et3N
(1.31 mL, 9.29 mmol), dissolved in THF (20 mL), and dichloro-N,N-
diisopropylaminophosphoramidite (817 mg, 4.04 mmol) dissolved
in THF (25 mL). The product (2.38 g, 3.88 mmol, 44%) was obtained
as a beige solid. Rf =0.79 in petroleum ether/ethyl acetate (4:1)
with 10% Et3N; 1H NMR (400 MHz, [D6]DMSO): d=8.03–8.01 (m,
4H; H-3’), 7.44–7.40 (m, 8H; H-2’, H-3), 7.26–7.24 (m, 4H; H-2),
4.80–4.68 (m, 4H; Ph-CH2), 3.72–3.63 (m, 2H; NC-H), 2.43 (s, 6H;
CH3), 1.19 ppm (d, 3JHH =6.8 Hz, 12H; iPr); 13C NMR (101 MHz,
[D6]DMSO): d=165.1 (C=O), 150.1 (C-1), 145.0 (C-4’), 137.5 (C-4),
130.5 (C-2’), 129.8 (C-3’), 128.3 (C-3), 126.4 (C-1’), 122.3 (C-2), 64.9
(PhCH2), 43.1 (NC), 25.2 (iPr), 22.4 ppm (CH3); 31P NMR (162 MHz,
[D6]DMSO, decoupled): d=147.5 ppm; IR: n˜ =2966, 2927, 2862,
1738, 1614, 1508, 1269, 1200, 1079, 979, 737, 500 cmÀ1; MS (FAB):
m/z 614.3 [M+H+]+.
3
2H; NC-H), 1.23 ppm (d, JHH =6.8 Hz, 12H; iPr); 13C NMR (101 MHz,
CDCl3): d=164.6 (C=O), 149.7 (C-1), 137.7 (C-4), 133.5 (C-1’), 132.4
(C-3’), 130.6 (C-2’), 128.2 (C-3), 121.2 (C-2), 117.8 (CN), 117.0 (C-4’),
2
2
64.8 (d, JCP =18.4 Hz, Ph-CH2), 43.3 (d, JCP =12.3 Hz, NC), 24.7 ppm
(iPr); 31P NMR (162 MHz, CDCl3, decoupled): d=148.2 ppm; IR: n˜ =
3056, 2968, 2929, 2868, 2228, 1740, 1507, 1365, 1260, 1189, 1172,
1072, 1028, 1013, 975, 876, 776, 685, 506, 485 cmÀ1; MS (FAB): m/z
636.3 [M+H+]+.
Bis[4-(4’-nitrobenzoyloxy)benzyl]-N,N-diisopropylaminophos-
phoramidite (8g): General procedure B with 4-(hydroxymethyl)-
phenyl-4’-nitrobenzoate (7g; 1.88 g, 6.89 mmol) and Et3N
(1.01 mL, 7.20 mmol), dissolved in THF (25 mL), and dichloro-N,N-
diisopropylaminophosphoramidite (633 mg, 3.13 mmol) dissolved
in THF (25 mL). The product (1.91 g, 2.83, 90%) was obtained as
a colorless solid. Rf =0.79 in petroleum ether/ethyl acetate (4:1)
with 10% Et3N; 1H NMR (400 MHz, [D6]DMSO): d=8.37–8.37 (m,
8H; H-2’, H-3’), 7.46–7.43 (m, 4H; H-3), 7.22–7.19 (m, 4H; H-2),
4.84–4.71 (m, 4H; Ph-CH2), 3.77–3.68 (m, 2H; NC-H), 1.24 ppm (d,
3JHH =6.8 Hz, 12H; iPr); 13C NMR (101 MHz, [D6]DMSO): d=163.3
(C=O), 150.1 (C-4’), 149.7 (C-1), 137.7 (C-4), 135.0 (C-1’), 131.3 (C-
2’), 128.8 (C-3), 128.2 (C-3’), 123.7 (C-2), 66.6 (Ph-CH2), 43.2 (d,
2JCP =12.3 Hz, NC) 24.7, 24.6 ppm (iPr); 31P NMR (162 MHz,
[D6]DMSO, decoupled): d=147.6 ppm; IR: n˜ =3109, 2968, 2930,
Bis[4-(4’-chlorobenzoyloxy)benzyl]-N,N-diisopropylamino-phos-
phoramidite (8d): General procedure B with 4-(hydroxymethyl)-
phenyl-4’-chlorobenzoate (7d; 2.00 g, 7.61 mmol) and Et3N
(1.12 mL, 7.95 mmol), dissolved in THF (15 mL), and dichloro-N,N-
diisopropylaminophosphoramidite (699 mg, 3.46 mmol) dissolved
in THF (15 mL). The product (1.30 g, 1.99 mmol, 58%) was obtained
as a colorless solid. Rf =0.70 in petroleum ether/ethyl acetate (4:1)
with 10% Et3N; 1H NMR (400 MHz, [D6]DMSO): d=8.14–8.12 (m,
4H; H-2’), 7.69–7.67 (m, 4H; H-3’), 7.44–7.43 (m, 4H; H-2), 7.29–
7.27 (m, 4H; H-3), 4.80–4.69 (m, 4H; Ph-CH2), 3.72–3.63 (m, 2H;
NC-H), 1.19 ppm (d, 3JHH =6.8 Hz, 12H; iPr); 13C NMR (101 MHz,
[D6]DMSO): d=164.5 (C=O), 150.1 (C-1), 139.5 (C-4’), 137.8 (C-4),
132.1 (C-2’), 132.0 (C-1’), 129.6 (C-3’), 128.5 (C-2), 122.2 (C-3), 64.6
(Ph-CH2), 42.8 (NC), 24.2 ppm (iPr); 31P NMR (162 MHz, [D6]DMSO,
decoupled): d=147.6 ppm; IR: n˜ =3675, 3659, 2968, 2931, 2872,
1731, 1592, 1506, 1363, 1092, 875, 750, 520, 507 cmÀ1; MS (FAB):
m/z 654.4 [M].
2864, 1738, 1603.35, 1519, 1346, 1260, 1186, 1072, 713, 500 cmÀ1
MS (FAB): m/z 676.2 [M+H+]+.
;
(N[nBu]4)2-d4TMP (6) and N[nBu]4)2-AZTMP (7) were synthesized
according to the protocol of Sowa and Ouchi.[10] Compound 6 was
obtained in 83% yield, and compound 7 was obtained in 61%
yield. The analytical data agreed with the literature.[8,9]
Ammonium-OMe-Ph-DiPPro-d4TDP (4a): General procedure C
with phosphoramidite 8a (376 mg, 0.583 mmol), d4T monophos-
phate (216 mg, 0.324 mmol), and 4,5-dicyanoimidazole (61.2 mg,
0.518 mmol) in CH3CN (7 mL). Oxidation by the addition of tert-bu-
tylhydroperoxide (5.5 molar solution in n-decane, 106 mL,
0.583 mmol). The product (76 mg, 0.086 mmol, 27%) was obtained
Bis[4-(4’-trifluoromethylbenzoyloxy)benzyl]-N,N-diisopropyl-ami-
nophosphoramidite (8e): General procedure B with 4-(hydroxyme-
thyl)phenyl-4’-trifluoromethylbenzoate (7e; 808 mg, 2.73 mmol)
and Et3N (0.40 mL, 2.9 mmol) dissolved in THF (10 mL), and di-
chloro-N,N-diisopropylaminophosphoramidite (250 mg, 1.24 mmol)
dissolved in THF (10 mL). The product (553 mg, 62%) was obtained
as a colorless solid. Tm =1428C; Rf =0.85 in petroleum ether/ethyl
acetate (4:1) with 10% Et3N; 1H NMR (400 MHz, CDCl3): d=8.33–
8.30 (m, 4H; H-2’), 7.79–7.77 (m, 4H; H-3’), 7.44–7.42 (m 4H; H-3),
7.20–7.18 (m, 4H; H-2), 4.84–4.70 (m, 4H; Ph-CH2), 3.77–3.68 (m,
1
as a colorless solid. H NMR (400 MHz, [D4]MeOH): d=8.10–8.08 (m,
4H; H-h), 7.69 (s, 1H; H-6), 7.44–7.41 (m, 4H; H-c), 7.18–7.16 (m,
4H; H-d), 7.04–7.02 (m, 4H; H-i), 6.97–6.96 (m, 1H; H-1’), 6.42 (d,
3JHH =6.0 Hz, 1H; H-3’), 5.86 (d, 3JHH =5.9 Hz, 1H; H-2’), 5.16–5.13
(m, 4H; H-a), 4.99–4.97 (m, 1H; H-4’), 4.27–4.18 (m, 2H; H-5’), 3.89
(s, 6H; OCH3), 1.92 ppm (s, 3H; H7); 13C NMR (101 MHz, [D4]MeOH):
d=165.0 (C-4), 164.3 (C-f), 152.0 (C-e), 137.4 (C-6), 133.3 (C-3’),
133.2 (C-h), 130.4 (C-i), 127.3 (C-2’), 123.1 (C-c), 122.7 (C-2), 115.1 (C-
3
2H; NC-H), 1.23 ppm (d, JHH =6.8 Hz, 12H; iPr); 13C NMR (101 MHz,
2
d), 110.7 (C-5), 90.1 (C-1’), 85.5 (C-4’), 70.0 (C-a), 68.1 (d, JCP
=
CDCl3): d=164.0 (C=O), 150.0 (C-1), 137.7 (d, 3JCP =7.5 Hz, C-4),
5.4 Hz, C5’), 56.1 (OCH3), 11.1 ppm (C-7); 31P NMR (162 MHz,
135.0 (d, 2JCP =32 Hz C-4’), 133.0 (C-1’), 130.7 (C-2’), 128.4 (C-3),
2
[D4]MeOH, decoupled): d=À12.0 (d, JPP =21.7 Hz, Pb), À12.9 ppm
3
2
2
(d, JPP =21.9 Hz, Pa); HPLC: tR =14.8 min, method A; HRMS (ESIÀ):
125.8 (d, JCP =7.5 Hz, C-3’), 122.2 (CF3), 121.5 (C-2), 64.9 (d, JCP
=
=
18.5 Hz, Ph-CH2), 43.2 (d, 1JCN =12.4 Hz, NC), 24.8 ppm (d, JCN
2
m/z [MÀH+]À calcd for [C40H37N2O16P2]À: 863.1624, found:
7.3 Hz, iPr); 31P NMR (162 MHz, CDCl3, decoupled): d=148.2 ppm;
19F NMR (203 MHz, CDCl3): d=67.12 ppm; IR: n˜ =2972, 2936, 2874,
1736, 1506, 1411, 1324, 1267, 1123, 1082, 1028, 971, 877, 829, 769,
698, 515 cmÀ1; MS (FAB): m/z 722.2 [M+H+]+.
863.1616; NMR assignment: Scheme 2.
(N[nBu]4)2-Me-Ph-DiPPro-d4TDP (4b): General procedure C with
phosphoramidite 8b (265 mg, 0.432 mmol), d4T monophosphate
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(160 mg, 0.240 mmol), and 4,5-dicyanoimidazole (45.4 mg,
0.384 mmol) in CH3CN (5 mL). Oxidation by the addition of tert-bu-
tylhydroperoxide (5.5 molar solution in n-decane, 79 mL,
0.432 mmol). The product (98 mg, 0.091 mmol, 38%) was obtained
as a colorless solid. H NMR (400 MHz, [D4]MeOH): d=8.02–8.00 (m,
4H; H-h), 7.68 (s, 4JHH =1.2 Hz, 1H; H-6), 7.46–7.42 (m, 4H; H-c),
127.6 (C-2’), 126.8 (d, 4JCF =3.4 Hz, C-i), 124.3 (CF3), 122.9 (C-d),
2
112.1 (C-5), 90.8 (C-1’), 87.0 (d, 3JCP =9.4 Hz, C-4’), 70.2 (dd, JCP
=
=
4
4
4.3 Hz, JCP =3.6 Hz, C-a), 68.1 (d, 2JCP =6.0 Hz, C-5’), 59.5 (t, JCN
6.0 Hz, C-A), 24.8 (C-B), 20.7 (C-C), 13.9 (C-D), 12.5 ppm (C-7);
19F NMR (203 MHz, [D4]MeOH): d=64.71 ppm; 31P NMR (203 MHz,
1
2
[D4]MeOH, decoupled): d=À12.0 (d, JPP =20.7 Hz, Pb), À12.9 ppm
3
2
7.33–7.31 (m, 4H; H-i), 7.20–7.18 (m, 4H; H-d), 6.97 (ddd, JHH
=
(d, JPP =20.6 Hz, Pa); HPLC: tR =15.7 min, method A; HRMS (ESIÀ):
4
4
3
3.5 Hz, JHH =1.6 Hz, JHH =1.6 Hz, 1H; H-1’), 6.41 (dd, JHH =6.0 Hz,
m/z [MÀH+]À calcd for [C40H31F6N2O14P2]À: 939.1160, found:
4JHH =1.6 Hz, 1H; H-3’), 5.85 (d, JHH =5.4 Hz, JHH =1.9 Hz, 1H; H-2’),
5.18–5.14 (m, 4H; H-a), 4.98–4.97 (m, 1H; H-4’), 4.28–4.18 (m 2H;
H-5’), 3.21 (t, 3JHH =8.4 Hz, 16H; H-A), 2.42 (s, 6H; CH3), 1.92 (d,
4JHH =0.9 Hz, 3H; H-7), 1.67–1.58 (m, 16H; H-B), 1.43–1.34 (m, 16H;
H-C), 1.01 ppm (t, 3JHH =7.3 Hz, 24H; H-D); 13C NMR (101 MHz,
[D4]MeOH): d=166.5 (C-f), 166.5 (C-4), 152.8 (C-2), 152.5 (C-e),
146.2 (C-9), 138.2 (C-6), 135.3 (C-b), 135.0 (d, 4JCP =7.1 Hz, C-3’),
131.1 (C-h), 130.4 (C-i), 130.4 (C-c), 127.9 (C-g), 127.5 (C-2’), 123.0
939.1163; NMR assignment: Scheme 2.
3
4
Ammonium-CN-Ph-DiPPro-d4TDP (4 f): General procedure D with
phosphoramidite 8 f (66 mg, 0.104 mmol), d4T monophosphate
(43 mg, 0.07 mmol), and 4,5-dicyanoimidazole activator solution
(416 mL, 0.104 mmol) in CH3CN (2 mL). Oxidation by the addition of
tert-butylhydroperoxide (5.5 molar solution in n-decane, 19 mL,
0.104 mmol). The product (38 mg, 0.044 mmol, 63%) was obtained
1
3
as a colorless solid. H NMR (400 MHz, [D4]MeOH): d=8.29–8.28 (m,
(C-d), 112.1 (C-5), 90.1 (C-1’), 87.0 (d, JCP =9.2 Hz, C-4’), 70.3 (C-a),
2
4H; H-h), 7.91–7.90 (m, 4H; H-i), 7.73 (s, 1H; H-6) 7.49–7.47 (m, 4H;
68.1 (d, JCP =6.2 Hz, C-5’), 59.5 (C-A), 24.8 (C-B), 21.7 (CH3), 20.7 (C-
3
3
C), 13.9 (C-D), 12.5 ppm (C-7); 31P NMR (162 MHz, [D4]MeOH, decou-
H-c), 7.24–7.23 (m, 4H; H-d), 6.22 (d, JHH =6.8 Hz, JHH =6.8 Hz, 1H;
H-1’), 5.20–5.17 (m, 4H; H-a), 4.44–4.42 (m, 1H; H-3’), 4.24–4.22 (m,
1H; H-5’a), 4.17–4.14 (m, 1H; H-5’b), 4.06 (brs, 1H; H-4’), 2.37–2.27
(m, 2H; H-2’), 1.90 ppm (s, 3H; H-7); 13C NMR (101 MHz, [D4]MeOH):
d=166.4 (C-4), 164.9 (C-f), 152.3 (C-e), 137.8 (C-6), 134.7 (C-b),
133.7 (C-i), 131.7 (C-h), 130.6 (C-g), 130.6 (C-c), 122.9 (C-d), 118.8 (C-
2
2
pled): d=À12.1 (d, JPP =21.4 Hz, Pb), À12.9 ppm (d, JPP =21.4 Hz,
Pa); HPLC: tR =15.8 min, method A; HRMS (ESIÀ): m/z [MÀH+]À
calcd for [C40H37N2O14P2]À: 831.1726, found: 831.1714; NMR assign-
ment: Scheme 2.
3
j), 118.1 (CN), 112.2 (C-5), 85.8 (C-1’), 84.4 (d, J=9.3 Hz, C-4’), 70.3
(N[nBu]4)2- Cl-Ph-DiPPro-d4TDP (4d): General procedure C with
phosphoramidite 8d (280 mg, 0.428 mmol), d4T monophosphate
(158 mg, 0.238 mmol), and 4,5-dicyanoimidazole (45 mg,
0.381 mmol) in CH3CN (5 mL). Oxidation by the addition of tert-bu-
tylhydroperoxide (5.5 molar solution in n-decane, 78 mL,
0.428 mmol). The product (112 mg, 0.101 mmol, 42%) was ob-
2
2
(d, J=5.4 Hz, C-a), 67.3 (d, J=6.5 Hz, C-5’), 62.5 (C-3’), 38.1 (C-2’),
12.6 ppm (C7); 31P NMR (162 MHz, [D4]MeOH, decoupled): d=
À12.1–12.1 (m, Pb), À12.7 ppm (d, 2JPP =19.6 Hz, Pa); HPLC: tR =
13.2 min, method A; HRMS (ESI+): m/z [M+Na+]+ calcd for
[C40H32N4NaO14P2]+: 877.1282, found: 877.1228; NMR assignment:
Scheme 2.
1
tained as a colorless solid. H NMR (400 MHz, [D4]MeOH): d=8.12–
4
8.10 (m, 4H; H-h), 7.69 (d, JHH =1.2 Hz, 1H; H-6), 7.54–7.53 (m, 4H;
Ammonium-NO2-Ph-DiPPro-d4TDP (4g): General procedure C
with phosphoramidite 8g (584 mg, 0.864 mmol), d4T monophos-
phate (320 mg, 0.480 mmol), and 4,5-dicyanoimidazole (90.7 mg,
0.768 mmol) in CH3CN (10 mL). Oxidation by the addition of tert-
butylhydroperoxide (5.5 molar solution in n-decane, 157 mL,
0.864 mmol). The product (137 g, 0.150 mmol, 31%) was obtained
as a colorless solid. 1H NMR (400 MHz, [D4]MeOH): d=8.36 (brs,
8H; H-h, H-i), 7.68 (m, 1H; H-6), 7.48–7.50 (m, 4H; H-c), 7.25–7.23
(m, 4H; H-d), 6.96 (ddd, 3JHH =3.5 Hz, 4JHH =1.8 Hz, 4JHH =1.7 Hz,
H-i), 7.46–7.41 (m, 4H; H-c), 7.20–7.18 (m, 4H; H-d), 6.97 (ddd,
3
3JHH =3.4 Hz, 4JHH =1.8 Hz, 4JHH =1.7 Hz, 1H; H-1’), 6.42 (dd, JHH
=
4
3
4
6.0 Hz, JHH =1.7 Hz, 1H; H-3’), 5.86 (dd, JHH =5.2 Hz, JHH =2.2 Hz,
1H; H-2’), 5.17–5.13 (m, 4H; H-a), 4.99–4.97 (m, 1H; H-4’), 4.28–
4
4.18 (m, 2H; H-5’), 3.21 (m, 8H; H-A), 1.91 (d, J=1.2 Hz, 3H; H-7),
3
1.74–1.62 (m, 8H; H-B), 1.47–1.37 (m, 8H; H-C), 1.04 ppm (t, JHH
=
7.3 Hz, 12H; H-D); 13C NMR (101 MHz, [D4]MeOH): d=164.9 (C-4),
163.8 (C-f), 152.4 (C-e), 151.4 (C-2), 141.2 (C-g), 138.7 (C-6), 135.4
(C-3’), 132.7 (C-h), 130.5 (C-i), 130.1 (C-c), 129.7 (C-b), 127.6 (C-2’),
3
3
4
1H; H-1’), 6.42 (ddd, JHH =6.0 Hz, JHH =6.0 Hz, JHH =1.6 Hz, 1H; H-
3’), 5.86 (ddd, 3JHH =5.9 Hz, 3JHH =5.2 Hz, 4JHH =2.2 Hz, 1H; H-2’),
5.18–5.15 (m, 4H; H-a), 5.00–4.96 (m, 1H; H-4’), 4.28–4.18 (m, 2H;
H-5’), 1.91 ppm (d, 4JHH =1.2 Hz, 3H; H-7); 13C NMR (101 MHz,
[D4]MeOH): d=165.1 (C-4), 164.7 (C-f), 152.4 (C-2), 152.2 (C-j), 150.6
(C-e), 138.7 (C-6), 136.2 (C-3’), 135.3 (C-9), 132.4 (C-h), 130.6 (d,
3
122.9 (C-d), 122.6 (C-5), 90.9 (C-1’), 87.0 (d, JCP =9.2 Hz, C-4’), 70.3
2
2
(d, JCP =3.8 Hz, C-a), 68.1 (d, JCP =4.8 Hz, C-5’), 59.6 (C-A), 24.8 (C-
B), 20.7 (C-C), 13.9 (C-D), 12.5 ppm (C-7); 31P NMR (162 MHz,
2
[D4]MeOH, decoupled): d=À12.0 (d, JPP =1 Hz, Pb), À12.9 ppm (d,
2JPP =6 Hz, Pa); HPLC: tR =16.1 min, method A; HRMS (ESIÀ): m/z
[MÀH+]À calcd for [C38H31Cl2N2O14P2]À: 871.0633, found: 871.0621;
NMR assignment: Scheme 2.
4JCP =3.8 Hz, C-c), 127.7 (C-2’), 124.8 (C-i), 122.8 (C-d), 112.1 (C-5),
2
90.9 (C-1’), 86.5 (d, 3JCP =9.5 Hz, C-4’), 70.3 (C-a), 67.7 (d, JCP
=
6.4 Hz, C-5’), 12.5 ppm (C-7); 31P NMR (162 MHz, [D4]MeOH, decou-
(N[nBu]4)2-CF3-Ph-DiPPro-d4TDP (4e): General procedure D with
phosphoramidite 8e (110 mg, 0.16 mmol), d4T monophosphate
(64 mg, 0.10 mmol), and 4,5-dicyanoimidazole activator solution
(624 mL, 0.16 mmol) in CH3CN (2 mL). Oxidation by the addition of
tert-butylhydroperoxide (5.5 molar solution in n-decane, 28 mL,
0.16 mmol). The product (69 mg, 58%) was obtained as a colorless
solid. 1H NMR (500 MHz, [D4]MeOH): d=8.32–8.31 (m, 4H; H-h),
7.8–7.83 (m, 4H; H-i), 7.69 (s, 1H; H-6), 7.47–7.44 (m, 4H; H-c),
7.23–7.22 (m, 4H; H-d), 6.97 (brs, 1H; H-1’), 6.43–6.42 (m, 1H; H-
3’), 5.87–5.86 (m, 1H; H-2’), 5.18–5.15 (m 4H; H-a), 4.99 (m, 1H; H-
4’), 4.28–4.22 (m, 2H; H-5’), 3.25–3.22 (m, 9H; H-A), 1.91 (s, 3H; H-
2
2
pled) d=À12.0 (d, JPP =20.6 Hz, Pb), À12.9 ppm (d, JPP =20.2 Hz,
Pa); HPLC: tR =14.8 min, method A; HRMS (ESIÀ): m/z [MÀH+]À
calcd for [C38H31N4O18P2]À: 893.1114, found: 893.1100; NMR assign-
ment: Scheme 2.
Ammonium-CF3-Ph-DiPPro-AZTDP (5e): General procedure D with
phosphoramidite 8e (238 mg, 0.33 mmol), AZT monophosphate
(140 mg, 0.22 mmol), and 4,5-dicyanoimidazole activator solution
(1320 mL, 0.33 mmol) in CH3CN (6 mL). Oxidation by the addition of
tert-butylhydroperoxide (5.5 molar solution in n-decane, 60 mL,
0.33 mmol). The product (117 mg, 53%) was obtained as a colorless
solid after ion exchange. 1H NMR (400 MHz, [D4]MeOH): d=8.32–
8.31 (m, 4H; H-h), 7.85–7.83 (m, 4H; H-i), 7.74 (s, 1H; H-6), 7.49–
3
7), 1.69–1.64 (m, 9H; H-B), 1.45–1.39 (m, 9H; H-C), 1.03 ppm (t, J=
7.3 Hz, 13.5H; H-D); 13C NMR (126 MHz, [D4]MeOH): d=166.6 (C-4),
165.2 (C-f), 152.3 (C-2), 151.2 (C-e), 138.7 (C-6), 135.5 (C-j), 135.4 (C-
7.47 (m, 4H; H-c), 7.25–7.23 (m, 4H; H-d), 6.22 (dd, 3JHH =6.8 Hz,
4
3
3’), 134.4 (C-g), 131.8 (C-h), 130.6 (C-h), 130.6 (d, JCP =5.3 Hz, C-c),
3JHH =6.8 Hz, 1H; H-1’), 5.19–5.18 (m, 4H; C-a), 4.45 (dt, JHH
=
&
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3
6.7 Hz, JHH =3.5 Hz, 1H; H-3’), 4.26–4.23 (m, 1H; H-5’a), 4.19–4.15
Hydrolysis studies in CEM cell extracts: A magnesium chloride solu-
tion (20 mL, 70 mm) was mixed with a solution of the DiPPro-NDP
(20 mL, 6 mm) in DMSO. Hydrolysis was started by the addition of
wild-type CEM cell extract (100 mL). Six to eight of these samples
were prepared and incubated at 378C for different time periods.
Hydrolysis was stopped by quenching with CH3OH (300 mL). After
5 min on ice and centrifugation at 13000 rpm (Heraeus, Biofuge
Pico) the samples were filtered and frozen in liquid nitrogen until
required. Samples (10–40 mL) were analyzed by RP-HPLC. Hydrolysis
was monitored for 10 h.
(m, 1H; H-5’b), 4.08–4.07 (m, 1H; H-4’), 2.39–2.29 (m, 2H; H-2’),
1.91 ppm (s, 3H; H-7); 13C NMR (101 MHz, [D4]MeOH): d=166.6 (C-
4), 165.2 (C-f), 152.4 (C-2), 152.3 (C-e), 137.8 (C-6), 136.0 (C-j), 134.4
4
4
(C-g), 131.8 (C-h), 130.6 (d, JCP =3.1 Hz, C-c), 126.8 (d, JCF =3.4 Hz,
3
C-i), 124.4 (CF3), 122.9 (C-d), 112.3 (C-5), 85.8 (C-1’), 84.2 (d, JCP
=
9.2 Hz, C-4’), 70.4 (d, JCP =5.4 Hz, C-a), 67.1 (d, 2JCP =6.2 Hz, C-5’),
2
62.5 (C3’), 38.1 (C2’), 12.6 ppm (C7); 31P NMR (162 MHz, [D4]MeOH,
decoupled): d=À12.1 (d, 2JPP =21.3 Hz, Pb), À12.7 ppm (d, JPP
=
2
21.4 Hz, Pa); HPLC: tR =18.9 min, method B; HRMS (ESI+): m/z
[M+H+]+ calcd for [C40H34F6N5O14P2]+: 984.1487, found: 984.1457;
NMR assignment: Scheme 2.
Preparation of CEM cell extracts: Human CD4+ T-lymphocyte CEM
cells were grown in RPMI-1640-based cell culture medium to
a final density of about 3ꢂ106 cellsmLÀ1. After centrifugation for
10 min at 1250 rpm (Heraeus, Megafuge 3.0R) and 48C, the pellet
Ammonium-CN-Ph-DiPPro-AZTDP (5 f): General procedure D with
phosphoramidite 8 f (38 mg, 0.06 mmol), AZT monophosphate
(31 mg, 0.05 mmol), and 4,5-dicyanoimidazole activator solution
(240 mL, 0.06 mmol) in CH3CN (2 mL). Oxidation by the addition of
tert-butylhydroperoxide (5.5 molar solution in n-decane, 11 mL,
0.06 mmol). The product (21 mg, 0.023 mml, 46%) was obtained as
a colorless solid. 1H NMR (400 MHz, [D4]MeOH): d=8.29–8.28 (m,
4H; H-h), 7.91–7.90 (m, 4H; H-i), 7.73 (s, 1H; H-6) 7.49–7.47 (m, 4H;
H-c), 7.24–7.23 (m, 4H; H-d), 6.22 (d, JHH =6.8 Hz, JHH =6.8 Hz, 1H;
H-1’), 5.20–5.17 (m, 4H; H-a), 4.44–4.42 (m, 1H; H-3’), 4.24–4.22 (m,
1H; H-5’a), 4.17–4.14 (m, 1H; H-5’b), 4.06 (brs, 1H; H-4’), 2.37–2.27
(m, 2H; H-2’), 1.90 ppm (s, 3H; H-7); 13C NMR (101 MHz, [D4]MeOH):
d=166.4 (C-4), 164.9 (C-f), 152.3 (C-e), 137.8 (C-6), 134.7 (C-b),
133.7 (C-i), 131.7 (C-h), 130.6 (C-g), 130.6 (C-c), 122.9 (C-d), 118.8 (C-
was washed twice with cold PBS, resuspended at 108 cellsmLÀ1
,
and sonicated (3ꢂ10 s). After a second centrifugation of this sus-
pension at 10000 rpm (Heraeus, Megafuge 3.0R), the supernatant
was divided into aliquots and frozen to À808C until used.
Antiviral assay: Inhibition of HIV-1(IIIB) and HIV-2(ROD)-induced cy-
topathicity in wild-type CEM/0 or thymidine kinase-deficient CEM/
TKÀ cell cultures was measured in microtiter 96-well plates contain-
ing ꢁ3ꢂ105 CEM cellsmLÀ1 infected with 100 CCID50 of HIVmLÀ1
and containing appropriate dilutions of the test compounds. After
4–5 days of incubation at 378C under a CO2-controlled humidified
atmosphere, CEM giant (syncytium) cell formation was examined
microscopically. EC50 (50% effective concentration) was defined as
the compound concentration required to inhibit HIV-induced giant
cell formation by 50%.
3
3
3
j), 118.1 (CN), 112.2 (C-5), 85.8 (C-1’), 84.4 (d, J=9.3 Hz, C-4’), 70.3
2
2
(d, J=5.4 Hz, C-a), 67.3 (d, J=6.5 Hz, C-5’), 62.5 (C-3’), 38.1 (C-2’),
12.6 ppm (C7); 31P NMR (162 MHz, [D4]MeOH, decoupled): d=
À12.1–12.1 (m, Pb), À12.7 ppm (d, 2JPP =19.6 Hz, Pa); HPLC: tR =
18.9 min, method B; HRMS (ESI+): m/z [M+H+]+ calcd for
[C40H34N7O14P2]+: 898.1639, found: 898.1640; NMR assignment:
Scheme 2.
Acknowledgements
The authors thank Leen Ingels, Evelyne Van Kerckhove, Sandra
Claes, Ria Van Berwaer and Lizette van Berckelaer for excellent
technical assistance. The biological assays were performed with
the financial support of the KU Leuven (Belgium) (GOA 10/014).
Ammonium-NO2-Ph-DiPPro-AZTDP (5g): General procedure D
with phosphoramidite 8g (160 mg, 0.24 mmol), AZT monophos-
phate (100 mg, 0.16 mmol), and 4,5-dicyanoimidazole activator so-
lution (1120 mL, 0.28 mmol) in CH3CN (4 mL). Oxidation by the addi-
tion of tert-butylhydroperoxide (5.5 molar solution in n-decane,
44 mL, 0.24 mmol). The product (100 mg, 0.105 mmol, 66%) was
Keywords: anti-HIV · antivirals · bioreversible protection · drug
delivery · nucleoside analogues · pronucleotides
1
obtained as a colorless solid after ion exchange. H NMR (400 MHz,
[D4]MeOH): d=8.29 (brs, 8H; H-h, H-i), 7.74 (s, 1H; H-6) 7.50–7.48
[1] a) J. Balzarini, P. Herdewijn, E. De Clercq, J. Biol. Chem. 1989, 264, 6127–
6133; b) J. Balzarini, Pharm. World Sci. 1994, 16, 113–126.
3
(4H; H-c), 7.26–7.24 (m 4H; H-d), 6.23 (dd, 3JHH =6.8 Hz, JHH
=
6.8 Hz, 1H; H-1’), 5.20–5.18 (m, 4H; H-a), 4.45–4.43 (m, 1H; H-3’),
4.26–4.22 (m, 1H; H-5’a), 4.18–4.15 (m, 1H; H-5’b), 4.07–4.06 (m,
1H; H-4’), 2.38–2.29 (m, 2H; H-2’), 1.91 ppm (s 3H; H-7); 13C NMR
(101 MHz, [D4]MeOH): d=166.5 (C-4), 164.7 (C-f), 152.4 (C-j), 152.3
(C-2), 137.7 (C-6), 136.3 (C-g), 135.5 (C-b), 132.4 (C-h), 130.6 (d,
[2] S. J. Hecker, M. D. Erion, J. Med. Chem. 2008, 51, 2328–2345.
[3] a) P. A. Furman, J. A. Fyfe, M. H. St. Clair, K. Weinhold, J. L. Rideout, G. A.
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ther. 1993, 37, 918–920; b) J. P. Sommadossi, R. Carlisle, Z. Zhou, Mol.
Pharmacol. 1989, 36, 9–14.
4JCP =3.8 Hz, C-c), 124.8 (C-i), 122.9 (C-d), 112.3 (C-5), 85.8 (C-1’),
2
84.4 (d, 3J=8.9 Hz, C-4’), 70.3 (d, 2JCP =5.7 Hz, C-a), 67.3 (d, JCP
=
5.7 Hz, C-5’), 62.5 (C-3’), 38.1 (C-2’), 12.6 ppm (C-7); 31P NMR
(162 MHz, [D4]MeOH, decoupled): d=À12.1 (d, 2JPP =20.5 Hz, Pb),
À12.7 ppm (d, 2JPP =20.5 Hz, Pa); HPLC: tR =16.7 min, method B;
HRMS (ESI+): m/z [M+H+]+ calcd for [C38H34N7O18P2]+: 938.1441,
found: 938.1428; NMR assignment: Scheme 2.
[5] U. Pradere, E. C. Garnier-Amblard, S. J. Coats, F. Amblard, R. F. Schinazi,
Chem. Rev. 2014, 114, 9154–9218.
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K. Y. Hostetler, H. van den Bosch, Biochim. Biophys. Acta Lipids Lipid
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van den Bosch, M. F. Gardner, Proc. Natl. Acad. Sci. USA 1993, 90,
11835–11839.
Compound evaluation methods
Hydrolysis studies in phosphate buffer at pH 7.3: Hydrolysis was
started by the addition of PBS (300 mL 50 mm, pH 7.3) to a solution
of the DiPPro-NDP (300 mL, 1.9 mm) in DMSO. The mixture incubat-
ed at 378C. Aliquots were taken at certain points of time and
frozen in liquid nitrogen. Directly after unfreezing, samples were
analyzed by RP-HPLC.
[7] a) D. Bonnaffꢃ, B. Dupraz, J. Ughetto-Monfrin, A. Namane, T. Huynh
Dinh, Tetrahedron Lett. 1995, 36, 531–534; b) D. Bonnaffꢃ, B. Dupraz, J.
Ughetto-Monfrin, A. Namane, T. Huynh Dinh, Nucleos. Nucleot. 1995, 14,
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783–787; c) D. Bonnaffꢃ, B. Dupraz, J. Ughetto-Monfrin, A. Namane, T.
Huynh Dinh, J. Org. Chem. 1996, 61, 895–902.
[10] T. Sowa, S. Ouchi, Bull. Chem. Soc. Jpn. 1975, 48, 2084–2090.
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47, 8719–8722; Angew. Chem. 2008, 120, 8847–8850.
Received: February 9, 2015
[9] T. Schulz, J. Balzarini, C. Meier, ChemMedChem 2014, 9, 762–775.
Published online on && &&, 0000
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FULL PAPERS
Fine-tuning stability: Bis(benzoyloxy-
benzyl) groups were used to mask two
of the negative charges of nucleoside
diphosphates to form DiPPro com-
pounds. Modification by different sub-
stituents in the 4-positions had a strong
impact on the chemical and enzymatic
stability. With strong acceptor substitu-
ents, the NDPs were released almost ex-
clusively in PBS or in cell extracts.
L. Weinschenk, T. Gollnest, D. Schols,
J. Balzarini, C. Meier*
&& – &&
Bis(benzoyloxybenzyl)-DiPPro
Nucleoside Diphosphates of Anti-HIV
Active Nucleoside Analogues
ChemMedChem 0000, 00, 0 – 0
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These are not the final page numbers! ÞÞ