The DiPPro approach: Synthesis, hydrolysis, and antiviral activity of lipophilic d4T diphosphate prodrugs

Article abstract of DOI:10.1002/cmdc.201300500

Bioreversible protection of the β-phosphate group of nucleoside diphosphates (NDPs) as bis(acyloxybenzyl)phosphate esters is presented. To investigate the structure-activity relationship of these potential NDP prodrugs (DiPPro drugs) a series of DiPPro compounds was synthesized bearing fatty acids of various lengths and d4T as a model nucleoside. For synthesis of the lipophilically modified diphosphate group, preformed phosphoramidites were allowed to react with nucleotides, and the β-P^III moiety was subsequently oxidized. The chemical and enzymatic stability of these prodrugs was studied in different media such as phosphate buffer (pH 7.3) or CEM cell extracts. In all media, the hydrolysis rate was clearly dependent on the acyl moiety and decreased with increasing alkyl chain length. The compounds showed a markedly lower half-life in cell extracts than in pH 7.3 phosphate buffer due to the presence of enzyme-catalyzed cleavage. In all media, the DiPPro compounds released d4T diphosphate (d4TDP) as the main product beside d4TMP. In antiviral assays, the compounds proved to be at least as potent as d4T against HIV-1 and 2 in wild-type CEM/0 cells. As a proof of concept, compounds with longer acyl residues showed very good anti-HIV activities in thymidine-kinase-deficient cells (CEM/TK^-), indicating their ability to penetrate cell membranes and the delivery of phosphorylated metabolites. NDPs delivered! A structure-activity relationship of bioreversibly protected DiPPro-d4TDP was performed. The stability of the compounds was studied in various media such as cell extracts. Stability increased with increasing lipophilicity of the acyl chain. D4TDP was released as main product. Compounds with long acyl residues showed good anti-HIV activities in TK-deficient cells, proving intracellular uptake of the compounds.

Full text of DOI:10.1002/cmdc.201300500

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DOI: 10.1002/cmdc.201300500
The DiPPro Approach: Synthesis, Hydrolysis, and Antiviral
Activity of Lipophilic d4T Diphosphate Prodrugs
Tilmann Schulz,[a] Jan Balzarini,[b] and Chris Meier*[a]
Dedicated to Prof. Dr. Joachim Engels on the occasion of his 70th birthday
Bioreversible protection of the b-phosphate group of nucleo-
side diphosphates (NDPs) as bis(acyloxybenzyl)phosphate
esters is presented. To investigate the structure–activity rela-
tionship of these potential NDP prodrugs (DiPPro drugs)
a series of DiPPro compounds was synthesized bearing fatty
acids of various lengths and d4T as a model nucleoside. For
synthesis of the lipophilically modified diphosphate group, pre-
formed phosphoramidites were allowed to react with nucleo-
tides, and the b-PIII moiety was subsequently oxidized. The
chemical and enzymatic stability of these prodrugs was stud-
ied in different media such as phosphate buffer (pH 7.3) or
CEM cell extracts. In all media, the hydrolysis rate was clearly
dependent on the acyl moiety and decreased with increasing
alkyl chain length. The compounds showed a markedly lower
half-life in cell extracts than in pH 7.3 phosphate buffer due to
the presence of enzyme-catalyzed cleavage. In all media, the
DiPPro compounds released d4T diphosphate (d4TDP) as the
main product beside d4TMP. In antiviral assays, the compounds
proved to be at least as potent as d4T against HIV-1 and 2 in
wild-type CEM/0 cells. As a proof of concept, compounds with
longer acyl residues showed very good anti-HIV activities in
thymidine-kinase-deficient cells (CEM/TKꢀ), indicating their abil-
ity to penetrate cell membranes and the delivery of phos-
phorylated metabolites.
Introduction
Nucleoside analogues are commonly used as antiviral and anti-
tumor agents and are the backbone of current anti-HIV thera-
pies.[1–3] The antiviral efficacy of many nucleoside analogues,
such as 3’-deoxy-2’,3’-dehydrothymidine (d4T, 1, Scheme 1)
and 3’-deoxy-3’-azidothymidine (AZT), depends on their con-
version into the ultimately bioactive nucleoside triphosphates
(NTPs) via the mono- and diphosphate forms by cellular kinas-
es. However, cellular kinases often insufficiently catalyze the
phosphorylation of nucleoside analogues. In the case of d4T 1,
the metabolism-limiting step in human cells is the initial phos-
phorylation event catalyzed by thymidine kinase (TK).[1,3] If this
is the case, antiviral activity can be enhanced by using pro-
drugs of the phosphorylated parent nucleosides (masked nu-
cleoside monophosphates; NMPs) that are able to penetrate
cell membranes and deliver the nucleotide intracellularly, thus
bypassing the limiting step and leading to increased intracellu-
lar levels of the active metabolites. In some cases drug resist-
ance could be overcome, and the application spectrum of nu-
cleosides was extended to cover different viruses.[4–7] Several
[a] Dr. T. Schulz, 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)
Scheme 1. Reagents and conditions: Method A: 1) N,N-diisopropylamine,
Et2O, ꢀ208C, 2 h. Method B: 1) THF, TEA, ꢀ788C, 1–2 h; 2) RT, 16–24 h.
Method C: 1) CH3CN, DCI, RT; 2) tBuOOH, ꢀ258C, 15 min; 3) RP-18 chroma-
tography.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300500.
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NMP prodrugs[8] have been designed over the past 20 years,
for example, cycloSal technology.[9] In contrast, it was shown
that the phosphorylation of AZT by cellular TK-1 proceeded ef-
ficiently, but the second step catalyzed by thymidylate kinase
(TMPK) was metabolism-limiting, which led to an intracellular
accumulation of AZT monophosphate (AZTMP).[10,11] Beside
anemia and neutropenia, the latter is the main cause of the
severe side effects associated with AZT treatment. Additionally,
AZTMP interferes with lipid glycosylation.[12–14] Therefore, pro-
drug systems that deliver nucleoside diphosphates (NDPs) into
intact cells may overcome metabolic hurdles and potential
side effects caused by the parent nucleoside or its monophos-
phate.
tion at the pyrophosphate unit is necessary for delivery. In our
work we focused on the use of esterases/lipases for intracellu-
lar activation because of their high intracellular concentration.
The DiPPro approach is based on two identical acceptor-substi-
tuted benzyl esters linked to the b-phosphate group of the nu-
cleoside diphosphate (Figure 1). The a-phosphate remained
unmasked, because a negatively charged a-phosphate led to
a considerable stabilization of the pyrophosphate unit. Upon
enzymatic cleavage of the phenolic acyl ester, an Umpolung
took place, which converted the acceptor group (ester) into
a strong donor substituent (hydroxy group). As a consequence,
spontaneous cleavage of the benzyl bond occurred in a 1,6-
elimination reaction resulting in the formation of mono-
masked acyloxybenzyl-NDP 3. Repetition of this process re-
leased the NDP.[23–25]
Only very few attempts to design NDP prodrugs have been
published so far. Nucleoside pyrophosphate diesters based on
glycerides have been reported by Hostetler and co-workers;
however, these compounds do not serve as diphosphate pro-
drugs, as they released the corresponding NMPs due to cleav-
age of the pyrophosphate group.[15–18] A second approach was
published by Dinh and co-workers,[19] in which an acyl group
was attached to the b-phosphate of the pyrophosphate, form-
ing a mixed anhydride. The concept was based on a rapid and
selective cleavage of the mixed anhydride rather than cleavage
of the phosphate anhydride bond.[20] The idea was proven in
hydrolysis studies in aqueous buffer. However, no differences
in antiviral activity between the investigated nucleotide di-
phosphate–carboxylic acids and the parent nucleosides were
observed.[21]
However, DiPPro-d4TDP compounds 4a,b with short or
branched acyl moieties (R) did not show antiviral activity in TK-
deficient CEM cells (CEM/TKꢀ).[22] A possible explanation for
this lack of activity is that the remaining polarity of these com-
pounds due to the negatively charged a-phosphate is still too
high and prevents effective uptake into cells. One option to
solve this problem is to increase the lipophilicity of the DiPPro
compounds. To achieve this, longer chains in the acyl moieties
were investigated. Lipophilic aliphatic esters have been exten-
sively used for prodrugs and are known to be taken up by the
mononuclear phagocyte system (MPS). Besides CD4+ T-lym-
phocytes, cells of the MPS play an important role in the patho-
genesis of AIDS and are considered as reservoirs for HIV.[26–28]
Long aliphatic acid esters are cleaved by esterases/lipases and
therefore may enable an efficient intracellular release of the
NDP.
The reason for difficulties in the development of lipophilic
NDP prodrugs is due to chemical instability of the pyrophos-
phate bond, because this bond is energetically rich (as evi-
denced by the ubiquitous use of ATP as “fuel” for metabolic
processes). However, this bond is kinetically stable due to the
negative charges that prevent cleavage by nucleophiles. Thus,
if the charged oxygen atoms of the NDPs are masked/esterified
and therefore neutralized, a nu-
Herein we report the synthesis, hydrolysis properties, and
structure–activity relationships in the antiviral evaluation of lip-
ophilic DiPPro-d4TDPs 4 bearing various fatty acyl moieties (R).
The nucleoside analogue d4T was used in this study because
cleophilic attack at the a- or b-
phosphate groups is possible,
and the chemical hydrolysis of
the anhydride bond is signifi-
cantly increased. We have re-
ported on a new concept of
lipophilic masking of nucleoside
diphosphates shown in bis(acyl-
oxybenzyl)nucleoside
diphos-
phates 2 (DiPPro compounds;
Figure 1) endowed with high
chemical stability but rapid and
efficient conversion into the cor-
responding NDP in CEM cell ex-
tracts.[22] In this approach the re-
lease of NDP is driven chemically
or enzymatically by cleavage of
the phenyl ester moiety within
the masking group that initiates
a spontaneous second chemical
reaction. Thus, no chemical reac- Figure 1. General structure and proposed hydrolysis pathway of DiPPro compounds 2.
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d4T is well known as a model system for nucleoside mono-
phosphate prodrug systems. For a systematic characterization,
this nucleoside analogue is also suitable for the development
of nucleoside diphosphate prodrugs.
tylhydroperoxide was added after completion of the reaction
to yield target compounds 4. The solvent was removed, and
purification of the crude product was achieved by RP-18 chro-
matography using gradients of methanol and water. Aqueous
extraction was avoided because of the amphiphilic character
of the compounds. DiPPro compounds 4 were obtained in
yields of 32–67% (Scheme 1, Table 1). The amount of product
was sometimes considerably decreased during the purification
because of difficulties in separating the prodrugs 4 from bis-
(acyloxybenzyl) phosphate 10 formed as by-product. For this
reason, we tried various purification methods—Sephadex LH-
20 column chromatography, crystallization/precipitation, prep-
arative ion-pair HPLC—all of which led to unsatisfactory re-
sults. In the case of 4 f, the use of only 1.1 equivalents of phos-
phoramidite for the reaction led to a better result during chro-
matography and thus to an increased yield. Unfortunately, this
result could not be confirmed in all cases. Finally, it could not
be concluded whether corresponding phosphodiesters 10
were formed through hydrolysis and oxidation of unreacted
phosphoramidites 7 or by partial hydrolysis of 4 during chro-
matography.
Results and Discussion
Chemistry
For the synthesis of DiPPro-d4TDP prodrugs 4a–j a convergent
strategy was chosen to build the pyrophosphate moiety, as re-
ported earlier.[22] Briefly, d4T monophosphate (d4TMP) 6 was
coupled with phosphoramidites 7 containing the lipophilic
acyl-modified bis(acyloxybenzyl) masking groups. The inter-
mediately formed PIII–PV intermediate was then oxidized to
give the pyrophosphate moiety (Scheme 1). D4TMP 6, needed
as starting material, was prepared by the method of Sowa and
Ouchi.[29] The obtained d4TMP was protonated using a Dowex
50WX8 (H+ form), column and was titrated with tetra-n-butyl-
ammonium hydroxide solution and freeze-dried. The di-
(nBu4N)+ salt of d4TMP 6 was highly hygroscopic. Consequent-
ly, this compound was rigorously dried prior to reaction. A reli-
able procedure for the removal of traces of water was to store
d4TMP 6 in an acetonitrile solution over molecular sieves (3 ꢁ)
directly after the freeze-drying procedure. For the reactions, ali-
quots containing the calculated amount of 6 were taken, and
the solvent was removed in vacuum. After co-evaporating, the
material was kept under an inert gas atmosphere as a pale-
yellow foam. The preparation of bis(4-acyloxybenzyl)-N,N-diiso-
propylphosphoramidites 7a–j was carried out by reaction of
PIII reagent 8 with the corresponding benzyl alcohols 9 in the
presence of triethylamine. The formed triethylamine hydrochlo-
ride was removed by filtration, and the crude materials were
purified by preparative thin-layer chromatography on a chro-
matotron. Phosphoramidites 7 were obtained in high yields
(Table 1).
In addition to DiPPro-d4TDPs 4a–j, attempts to prepare the
mono-masked acyloxybenzyl-d4TDP intermediates 5 were con-
ducted (Figure 2). In oligonucleotide chemistry the b-cyano-
Next, dried d4TMP 6 was dissolved in acetonitrile and al-
lowed to react with 1.6–1.7 equivalents of phosphoramidites 7
in the presence of 1.7 equivalents dicyanoimidazole (DCI). In
some cases, 0.2–0.4 equivalents of phosphoramidite 7 and DCI
were added to the reaction mixture after several hours. tert-Bu-
Figure 2. Proposed hydrolysis pathway for 4 under chemical conditions:
Route A: release of d4TDP 13 via intermediate acyloxybenzyl (AB)-d4TDP 5;
Route B: cleavage of anhydride bond. AB-d4TDP 5a: R=Me, Me-DiPPro-
d4TDP; 5b: R=tBu, tBu-DiPPro-d4TDP; 5c: R=C4H9, C4-DiPPro-d4TDP; 5d:
R=C6H13, C6-DiPPro-d4TDP; 5e: R=C7H15, C7-DiPPro-d4TDP; 5 f: R=C9H19,
C9-DiPPro-d4TDP; 5g: R=C11H23, C11-DiPPro-d4TDP; 5h: R=C13H27, C13-
DiPPro-d4TDP; 5i: R=C15H31, C15-DiPPro-d4TDP; 5j: R=C17H33, 8Z-C17-
DiPPro-d4TDP; t1/2(1): half-life of the first step (half-life of DiPPro-d4TDP 4);
t1/2(2): half-life of the second step (half-life of acyloxybenzyl-d4TDP 5).
Table 1. Phosphoramidites 7 and DiPPro-d4TDPs 4.
Phosphoramidites 7
DiPPro-d4TDPs 4
Compd
R
Yield [%]
Compd
R
Yield [%]
7a[a]
7b[a]
7c
7d
7e[a]
7 f
7g
7h
7i
Me
tBu
C4H9
C6H13
C7H15
C9H19
C11H23
C13H27
C15H31
C17H33 (8Z)
75
67
90
49
52
56
58
34
80
16
4a[a]
4b[a]
4c
4d
4e
4 f
4g
4h
4i
Me
tBu
C4H9
C6H13
C7H15
C9H19
C11H23
C13H27
C15H31
C17H33 (8Z)
10
46
32
40
65
62
63
67
49
39
ethoxy group is usually used as a phosphate protecting group.
Therefore, this group was also considered here as a suitable
substituent which should be easily cleaved with a non-nucleo-
philic base such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). In
a first attempt, b-cyanoethoxy-(4-decanoyloxybenzyl)-N,N-diiso-
propylaminophosphoramidite 12 was synthesized from com-
mercially available b-cyanoethyl-N,N-diisopropylchlorophos-
phoramidite and the corresponding benzyl alcohol 9 f. After
7j
4j
[a] Taken from ref. [22].
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d4TDPs 5 (Figure 2). However, as discussed blow, d4TMP was
also consistently formed in the hydrolysis studies. This means
that the half-lives given are the sum of two hydrolysis process-
es: formation of d4TDP and d4TMP. In all cases for prodrugs 4
bearing linear alkyl residues, the amount of d4TDP was higher
than the amount of d4TMP. As expected, the chemical stability
of prodrugs increased with the length of the acyl moieties,
except for 8Z-C17-DiPPro-d4TDP 4j (t1/2 =73 h). For com-
pounds 4b–f the half-lives of the second hydrolysis step (t1/2),
reflecting the stability of the intermediate acyloxybenzyl-
d4TDPs 5b–f, are also listed in Table 2. These were determined
after full conversion of the DiPPro-d4TDPs 4b–f to intermedi-
ates 5b–f. The ratio of d4TDP and d4TMP formation was deter-
mined, and values are given in Table 2.
Generally, in all cases the second hydrolysis step proceeded
much more slowly than the first one, which became evident
by the appearance of intermediates 5b–f. As an example, the
hydrolysis of C9-DiPPro compound 4 f is shown in Figure 3.
This is due to the formation of a second negative charge at
the pyrophosphate unit in intermediates 5 generating repul-
sive interactions with the nucleophile. It was shown that tBu-,
C4-, C6-, C7- and C9-DiPPro-d4TDP (4b–f, respectively) were
hydrolyzed via acyloxybenzyl intermediates 5c–f, which were
eight- to ninefold more stable than the corresponding parent
compounds 4.
Scheme 2. Reagents and conditions: Method A: 1) CH3CN, DCI, RT;
2) tBuOOH, ꢀ258C, 15 min; 3) RP-18 chromatography. Method B: 1) CH3CN,
DBU, RT, 15 min.
RP-18 chromatography, b-cyanoethoxy-(4-decanoyloxybenzyl)-
d4TDP 13 was obtained as a mixture of two diastereomers in
40% yield (Scheme 2). Unfortunately, the synthesis of (4-dec-
anoyloxybenzyl)-d4TDP 5 f by elimination of the cyanoethyl
group with DBU failed. If traces of water are present during
synthesis or workup, the acyl moiety is cleaved rapidly due to
the highly basic DBU; decanoylic acid, 4-hydroxybenzyl alco-
hol, and d4TDP 11 were then detected in the crude mixture
based on 1H and 31P NMR spectroscopy.
The branched intermediate 5b proved to be 25-fold more
stable than the parent prodrug 4b (Table 3). In contrast, only
very small amounts of C11-, C13-, C15- and 8Z-C17-acyloxyben-
zyl intermediates 5g–j were detected (Supporting Information
Figure S4). Half-lives of these intermediates could not be deter-
mined. The high lipophilicity of prodrugs 4g–j in the incuba-
tion media (PBS/DMSO) may result in a slow attack by nucleo-
philes, while the nucleophile accessed the second acyl moiety
of the more polar intermediates 5a–f more easily. One might
speculate that prodrugs 4 form micelles in the incubation
However, selective hydrolysis of C9-DiPPro-d4TDP 4 f in a 1:1
mixture of [D6]DMSO and 50 mm PBS (pH 7.3) led to (4-dec-
anoyloxybenzyl)-d4TDP 5 f. The reaction was fol-
lowed by 31P NMR spectroscopy and stopped by
freeze-drying after a considerable amount of 5 f was
Table 2. Hydrolysis data of DiPPro compounds 4, intermediate 5 f, and the mixed b-
cyanoethyl-C9-DiPPro compound 13.
formed. Compound 5 f was isolated by RP-18 chro-
matography and used as reference compound for
the HPLC experiments described below. Characteriza-
tion of DiPPro-d4TDP prodrugs 4a–j and CE-decanoyl-
PBS (pH 7.3)
tR [min][b] 4: t1/2(1) [h][c] 5: t1/2(2) [h][d] d4TDP/d4TMP[e] t1/2(1) [h]
CE[a]
Compd
R
1
oxybenzyl-d4TDP 13 was performed by H, 13C, and
4a
4b
4c
4d
4e
4 f
4g
4h
4i
C1[f]
tBu
C4
C6
C7
9.41
11.44
11.57
13.2
14.05
15.88
17.76
19.23
20.54
19.86
11.0
10
55
40
36
43
ND[g]
1390
330
330
390
ND[g]
0.6
3
4
2
1.5
1.5
2
1.5
ND[g]
NA[h]
3
ND[g]
1
1
1
1
1
1
1
1
0.05
6.1
0.6
3.6
1.6
7.6
4
31P NMR spectroscopy as well as high-resolution mass
spectrometry.
C9
63
280
Chemical stability
C11B
C13
C15
8Z-C17
C9
100
179
258
73
247
11
NA[h]
NA[h]
NA[h]
NA[h]
NA[h]
280
13
21
Stability studies of DiPPro-d4TDP prodrugs 4 and CE-
decanoyloxybenzyl-d4TDP 13 were conducted in
aqueous 25 mm phosphate buffer (PBS, pH 7.3). The
hydrolysis products were detected by analytical RP-
18 HPLC.
4j
5 f
13
ND[g]
NA[h]
1
9.5
ND[g]
0.46
CE
12.09
[a] 33% CEM cell extract in PBS, pH 6.8. [b] HPLC retention time, HPLC analysis
method 1. [c] Half-life of first step (4!5). [d] Half-life of second step (5!d4TDP 11).
[e] Ratio of d4TDP/d4TMP after full conversion of DiPPro-d4TDPs and intermediates.
[f] See ref. [22]. [g] Not determined. [h] Not applicable.
The chemical hydrolysis half-lives t1/2(1) of com-
pounds 4a–j listed in Table 2 mainly reflect the initial
hydrolysis step of DiPPro-d4TDPs 4 to acyloxybenzyl-
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^
Figure 4. Hydrolysis of C4-DiPPro-d4TDP 4c ( , exponential fit), formation
&
and hydrolysis of intermediate acyloxybenzyl-d4TDP 5c ( , manual), forma-
~
*
tion of d4TMP ( , manual) and d4TDP ( , manual) in PBS, pH 7.3.
Figure 3. HPLC profile for C9-DiPPro-d4TDP 4 f after incubation in PBS,
pH 7.3 (0–48 days). Peaks were attributed by co-injection and/or tR of refer-
ence compounds (Supporting Information Figures S1, S2, S3); HPLC analysis
method A.
heptanoyloxybenzyl-d4TDP 5d, yet small amounts of d4TMP 6
and phosphate diester 10 were also observed after 12 days.
Formation of d4TMP and phosphate diester 10 occurred as
a result of cleavage of the anhydride bond (Figure 2; pathway
B), but it was clearly proven that d4TDP 11 was formed with
marked selectivity via intermediate 5d (42–68 days of hydroly-
sis).
Table 3. Hydrolysis half-lives of C6- and C15-DiPPro-d4TDP 4d,i and CE-
C9-decanoyloxybenzyl-d4TDP 13 in various biological media.
Hydrolysis of the mixed acyloxybenzyl-b-cyanoethyl prodrug
13 was also studied at different pH values (PBS, pH 7.3 or 8.7).
This compound released d4TDP via intermediate C9-acyloxy-
benzyl-d4TDP 5 f, as proven by co-injection (Supporting Infor-
mation Figures S7 and S8). Cleavage of the b-cyanoethyl group
proceeded with a surprisingly short half-life (t1/2(1) =11 h ;
pH 7.3), followed by hydrolysis of the decanoyloxybenzyl
group with t1/2(2) ~270 h. As expected, stabilities decreased at
pH 8.7 to half-lives of t1/2(1) =6.5 and t1/2(2) =133 h, respectively.
The d4TDP/d4TMP ratio was found to be 4:1 at pH 7.3 and 8.7.
t
1/2(1) [h][a]
Compd
R
CE[b]
HS[c]
RPMI/FCS[d]
FCS[e]
PBS
(pH 6.8)
4d
4i
13
C6
C15
CE-C9
3.6
21
0.46
5.0
25
2.6
5.5
28
1.5
5.5
70
ND
59
407
5.5
[a] Hydrolysis half-life. [b] 33% CEM cell extract in PBS, pH 6.8; [c] 20%
human serum in PBS, pH 6.8. [d] RPMI/10% heat-inactivated FCS. [e] 10%
FCS in PBS, pH 6.8.
media, whereas intermediates 5 are unable to form such
aggregates.
Hydrolysis in biological media
A further example of the chemical hydrolysis is shown
below in Figure 5. While C4-DiPPro-d4TDP 4c is hydrolyzed to
C4-acyloxybenzyl-d4TDP 5c, some concomitant cleavage to
d4TMP occurred as well. C4-DiPPro-d4TDP 4c was completely
hydrolyzed at pH 7.3 within 250 h (t1/2 ~40 h). The main prod-
uct after 151 h was intermediate 5c and not d4TMP, indicating
that formation of intermediate 5c proceeds at a higher rate
than cleavage of the anhydride bond. Additionally, the peak
amount of d4TMP was observed at this point. Moreover, the
formation of d4TMP was clearly dependent on the hydrolysis
half-life of the parent d4TDP prodrug 4; the higher the half-life
of the initial cleavage, the higher the amount of d4TMP
formed. However, intermediates 5c–f released exclusively
d4TDP with half-lives of ~280–390 h at pH 7.3 (Table 2). This
was proven by incubation of intermediate 5 f (t1/2 ~250 h; Fig-
ures 3, 4 and Supporting Information Figure S5).
The cleavage of compounds 4 and 13 was further investigated
in T-lymphocyte CEM cell extracts (Table 2). Moreover, C6-
DiPPro-d4TDP 4d and C15-DiPPro-d4TDP 4i were hydrolyzed
in human serum (HS), RPMI-1640 culture medium containing
10% heat-inactivated fetal calf serum (RPMI/FCS) as well as in
FCS (Table 3). The rapid cleavage of 5a in CEM cell extracts
was described previously.[22] Cell extracts were diluted with
phosphate buffer to decrease enzyme-mediated degradation
of d4TDP formed (33% CE in PBS, pH 6.8). Half-lives of pro-
drugs 5b–j were in the range of 1–21 h. The cleavage was 10-
to 67-fold faster in CE than chemical hydrolysis, indicating a sig-
nificant contribution by enzymatic cleavage (Table 2). The for-
mation and further hydrolysis of intermediates 6b–f were also
observed. Again, a predominant formation of d4TDP was ob-
served from DiPPro-d4TDPs, as proven by HPLC experiments
(4 f as an example, Figure 5). Unfortunately, it was not possible
to quantify the released amount of d4TDP because it was still
prone to some enzymatic degradation. In line with data from
the chemical hydrolysis studies, rates of hydrolysis decreased
with increasing length of the aliphatic chains.
Additionally, C6-DiPPro-d4TDP 4d was hydrolyzed in a 1:1
mixture of [D6]DMSO and 50 mm PBS (pH 7.3) to follow the re-
action by 31P NMR (Supporting Information Figure S6). The ma-
jority of C6-DiPPro-d4TDP 4d hydrolyzed to the intermediate
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drolysis may be advantageous for a possible intracellular “lock-
in” of the intermediate 5.[30,31]
Antiviral evaluation
All compounds were evaluated for their anti-HIV activity in
wild-type CEM/0 and mutant thymidine-kinase-deficient cells
(CEM/TKꢀ; Table 4). Unfortunately, cells deficient in thymidine
monophosphate kinase (TMPK) are not available because
TMPK is crucial for supplying thymidine building blocks for
DNA synthesis. Deletion of this enzyme would result in nonvia-
ble cells. The parent nucleoside d4T 1 was used as reference
compound. Antiviral activity in TK-deficient cells is an indica-
tion that the intact d4TDP prodrug is able to enter the cell by
diffusion, because d4T is only very poorly monophosphorylat-
ed in this cell line (Table 4, last entry). The majority of tested
compounds proved to be at least equipotent to or slightly less
potent (4h,i) than d4T against HIV-1 and HIV-2 in wild-type
CEM/0 cells. All compounds with acyl residues RꢁC6H13 (4d–j)
Figure 5. HPLC profile for C9-DiPPro-d4TDP 4 f after incubation in cell ex-
tracts for the time periods shown and reference traces of d4TDP and
d4TMP; degradation products of the extracts are highlighted in the box.
HPLC analysis method A.
Additionally, C6-DiPPro-d4TDP 4d and C15-DiPPro-
d4TDP 4i were incubated in 20% human serum in
PBS (pH 6.8) and the determined half-lives were
Table 4. Antiviral activity and cytotoxicity of compounds 4a–j and 13.
DiPPro
EC50 [mm][a]
CC50 [mm][b]
found to be slightly higher than in CE (Table 3). The
intermediate C6-acyloxybenzyl-d4TDP 5d accumulat-
ed, but its stability could not be determined due to
the relatively short incubation time used (9–10 h;
longer incubation was not relevant because of the
occurring protein denaturation).
CEM/0
CEM/TKꢀ
HIV-2
HIV-1
HIV-2
4a[c,d]
4b[c,d]
4c
1.05ꢂ0.92
0.78ꢂ0.80
2.0ꢂ1.8
1.5ꢂ0.7
21.0ꢂ5.7
14ꢂ5
>125
0.90ꢂ0.16
2.9ꢂ2.8
66ꢂ1.4
80ꢂ0.71
85ꢂ2.1
81.1ꢂ1.4
61ꢂ11
15ꢂ7.1
4d
0.47ꢂ0.28
0.90ꢂ0.44
0.13ꢂ0.087
1.1ꢂ0.14
1.3ꢂ0.39
0.20ꢂ0.006
0.32ꢂ0.12
0.35ꢂ0.078
2.0ꢂ1.2
1.2ꢂ0.28
2.6ꢂ1.9
4e[d]
4 f[d]
4 f
Additionally, the half-lives of 4d,i in the incubation
medium used for the anti-HIV tests (RPMI-1640 with
10% heat-inactivated FCS) were determined. Relative
to their chemical stabilities, lower stability in this
medium was clear (Table 3). A possible explanation
may be residual esterase activity in FCS, as previously
observed by our group.[30] Moreover, compounds
4d,i were incubated in 10% FCS in PBS, pH 6.8. Inter-
estingly, an 11-fold shorter half-life for the first hy-
drolysis step was observed for 4d, and a sixfold re-
duced half-life was found for 4i relative to chemical
hydrolysis in PBS, pH 6.8. Aminolysis or other side re-
actions may also occur in the culture medium due to
the presence of amino acids, proteins, and other nu-
0.15ꢂ0.042
0.11ꢂ0.042
0.23ꢂ0.064
1.2ꢂ0.030
0.99ꢂ0.014
0.65ꢂ0.35
2.8ꢂ2.1
0.080ꢂ0.042
0.16ꢂ0.042
1.8ꢂ1.6
62ꢂ6.4
39ꢂ0.71
134ꢂ21
72ꢂ9.9
100ꢂ3.1
86ꢂ4.9
>250
4g
4h
4i
4j
13
d4T
2.0ꢂ1.8
1.7ꢂ0.42
1.8ꢂ1.5
1.1ꢂ0.29
0.99ꢂ0.16
0.86ꢂ0.45
5.0ꢂ2.5
2.3ꢂ2.4
173ꢂ70
[a] Antiviral activity in CD4+ T-lymphocytes: 50% effective concentration (values are
the meanꢂSD of two to three independent experiments). [b] Cytotoxicity: 50% cyto-
static concentration or compound concentration required to inhibit CEM cell prolifera-
tion by 50% (values are the meanꢂSD of two to three independent experiments).
[c] Previously reported results.[22] [d] Tested as ammonium salt.
cleophiles such as inorganic salts. As before, a longer alkyl
chain led to increased stability.
showed very pronounced anti-HIV activities in CEM/TKꢀ cells,
indicating their ability to diffuse across the cell membrane and
release phosphorylated d4T metabolites. None of the com-
pounds displayed significant cytotoxicity.
The hydrolysis of CE-C9-DiPPro-d4TDP 13 was studied in bio-
logical media (CE, HS, RPMI/FCS, Table 3) as well. Again, in
comparison with the chemical stabilities in PBS (pH 6.8),
a marked decrease in half-lives was observed in these media.
In all media, the b-cyanoethyl group was cleaved first, leading
to decanoyloxybenzyl-d4TDP 5 f, which was identified by
HPLC. Interestingly, hydrolysis of the b-cyanoethyl group of
DiPPro-d4TDP 13 (t1/2(1) =0.46 h) was 16-fold faster than cleav-
age of one decanoyloxybenzyl moiety of C9-DiPPro-d4TDP (t1/
2(1) =7.6 h) in CEM cell extract. This marked acceleration in hy-
In particular, C9-DiPPro-d4TDP 4 f demonstrated the good
potential of the DiPPro concept, with 1570-fold higher antiviral
activity in TK-deficient CEM cells and antiviral activity one
order of magnitude higher in infected wild-type CEM/0 cells
relative to the parent nucleoside d4T. We tested both the am-
monium and the tetra-n-butylammonium salts of C9-DiPPro-
d4TDP 4 f to evaluate the possible influence of the counterion.
No effects on antiviral activity or cytotoxicity were observed.
These results demonstrate that the DiPPro approach has been
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tained from Sigma–Aldrich or VWR (CH3CN, HPLC grade). The soft-
ware used was EzChromElite. Method A: 0–18 min: TBAH ion
buffer/CH3CN gradient (5–95%); 18–26 min: buffer/CH3CN (95%);
26–30 min: buffer/CH3CN gradient (95–5%); 30–35 min: buffer/
CH3CN (5%); flow: 1 mLminꢀ1. Method B: 0–24 min: TBAH ion
buffer/CH3CN gradient (5–90%); 24–28 min: buffer/CH3CN (90%);
28–30 min: buffer/CH3CN gradient (90–10%); 30–35 min: buffer/
CH3CN (5%); flow: 1 mLminꢀ1. TBAH ion buffer: 0.55 mm tetra-n-
butylammonium hydroxide in H2O. Method C: 0–20 min: TBAH ion
buffer/CH3CN gradient (5–90%); 20–24 min: buffer/CH3CN (90%);
24–29 min: buffer/CH3CN gradient (90–5%); 29–36 min: buffer/
CH3CN (5%); flow: 1 mLminꢀ1. Method D: 0–15 min: TBAH ion
buffer/CH3CN gradient (5–70%); 15–18 min: buffer/CH3CN (70%);
18–21 min: buffer/CH3CN gradient (70–5%); 21–27 min: buffer/
CH3CN (5%).
successfully optimized by increasing the lipophilicity of the
prodrugs relative to earlier prepared compounds.[22]
Conclusions
In summary, DiPPro-d4TDP prodrugs 4c–j were synthesized by
using a convergent approach and were obtained with high
purity and in yields of up to 67%. Acyl moieties of varying
chain length were introduced to study their influence on the
hydrolysis mechanism, stability, and antiviral activity of the
DiPPro-d4TDP prodrugs. It was proven that tBu-, C4-, C6-, C7-
and C9-DiPPro-d4TDP 4b–f hydrolyzed predominantly via acy-
loxybenzyl intermediates 5b–f to give d4TDP. The ratio of
d4TDP and d4TMP formation was determined and correlated
with the stability of the compounds; the higher the half-life of
the cleavage of the first masking acyloxybenzyl groups, the
higher the amount of d4TMP formed. Interestingly, intermedi-
ates 5 did not release d4TMP, but exclusively d4TDP. In the
mixed b-cyanoethyl-C9-DiPPro-d4TDP 13, the b-cyanoethyl
group was cleaved markedly faster by b-elimination than the
decanoyloxybenzyl group, leading to a high amount of d4TDP.
In in vitro anti-HIV tests, compounds with acyl residues Rꢁ
C6H13 were found to be highly active in the CEM/TKꢀ cell assay,
proving that DiPPro-NDP prodrugs are the most effective bio-
reversibly protected nucleoside diphosphates reported thus
far. Using these insights, NDP prodrugs of other nucleoside an-
alogues, such as AZT and ddU, will be synthesized and investi-
gated.
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
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
recorded at room temperature in automation mode.
MS: Mass spectra were obtained with a VG Analytical VG/70–250 F
[FAB, (double focusing), matrix: m-nitrobenzyl alcohol], ESI mass
spectra were recorded with a VG Analytical Finnigan ThermoQuest
MAT 95 XL spectrometer or with an Agilent 6224 LC–MS-TOF or
with a Bruker maXis spectrometer.
IR and UV spectroscopy: IR spectra were recorded on a Bruker
Alpha P FT-IR spectrometer at RT in a range of 400–4000 cmꢀ1. UV
spectra of final compounds 4 were determined using the diode
array detector during analytical HPLC. Purity of Final Compounds:
The ꢁ95% purity of the final compounds 4, 5 f and 13 was con-
firmed using HPLC analysis. Melting points are uncorrected.
Experimental Section
Chemistry
3’-Deoxy-2’,3’-didehydrothymidinemonophosphate (d4TMP 6) as
ammonia salt: The reaction was carried out under nitrogen atmos-
phere with dried solvents. To a solution of 2.85 mL (30.5 mmol,
4.4 equiv) of distilled phosphoryl chloride in 14 mL CH3CN at 08C
2.4 mL (2.41 g, 30.5 mmol, 4.4 equiv) of dry pyridine and 0.27 mL
(0.27 g, 15.3 mmol, 2.2 equiv) of H2O were added. After 10 min
1.55 g (6.94 mmol, 1.0 equiv) of d4T were added. After 4 h at RT
the reaction was quenched by addition of ice/H2O and stirred at
48C for 1 h. By careful addition of solid ammonium bicarbonate
the solution was adjusted to pH 8. Solvents were removed by
freeze-drying. The residue was dissolved in H2O and purified by
RP-18 gradient chromatography (H2O/CH3CN 0–10%). The pure
product was obtained after freeze-drying. Yield: 2.21 g (6.53 mmol,
General: Diethyl ether was dried over sodium/benzophenone and
distilled under nitrogen. Tetrahydrofuran (THF) was dried over pot-
assium/benzophenone and distilled under nitrogen. Pyridine, di-
chloromethane, and CH3CN were distilled from calcium hydride
under nitrogen. N,N-Diisopropylethylamine and TEA were distilled
from sodium prior to use. Phosphoryl chloride was distilled under
nitrogen prior to use. All commercially available reagents were
used without further purification. 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 by Macherey &
Nagel Xtra Sil UV254 were used; sugar-containing compounds were
visualized with sugar spray reagent (0.5 mL 4-methoxybenzalde-
hyde, 9 mL EtOH, 0.5 mL conc. sulfuric acid and 0.1 mL glacial
acetic acid). Preparative chromatography: All preparative TLC was
performed on a chromatotron (Harrison Research, Model 7924T)
using glass plates coated with 1, 2, or 4 mm 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 and pooled, and the solvent was removed in vacuo.
Water as eluent was removed by freeze drying.
1
94%) of a colorless fluffy powder. H NMR (400 MHz, D2O) d=7.58
3/4
(d, 1H, 4JHH =1.1 Hz, H-6), 6.93 (ddd, 1H, 3JHH =3.4 Hz,
J =
HH
4
3
4
2.0 Hz, JHH =1.5 Hz, H-1’), 6.45 (ddd, 1H, JHH =6.1 Hz, JHH =1.7 Hz,
3
4
4JHH =1.7 Hz, H-3’), 5.88 (ddd, 1H, JHH =6.1 Hz, 3/4JHH =2.3 Hz, JHH
=
1.4 Hz, H-2’), 5.07–5.02 (m, 1H, H-4’), 4.00–3.91 (m, H-5’), 1.85 ppm
4
(d, 1H, JHH =1.1 Hz, Me); 13C NMR (101 MHz, D2O) d=166.7 (C-4),
152.3 (C-2), 138.2 (C-6), 134.2 (C-3’), 125.2 (C-2’), 111.4 (C-5), 89.9 (C-
3
2
1’), 85.9 (d, JCP =8.6 Hz, C-4’), 65.5 (d, JCP =4.8 Hz, C-5’), 11.41 ppm
(Me); 31P NMR (162 MHz, H-decoupled, D2O) d=0.42 ppm; IR: n˜ =
1
2819, 1661, 1468, 1223, 1164, 1038, 908, 642, 489 cmꢀ1; UV (HPLC):
lmax [nm]=265, HPLC: tR [min]=6.31, method 1; Rf: 0.38 (iPrOH/
H2O/NH3 (25% in H2O) 14:7:1 v/v); MS (ESI+): calcd 302.03 [M]+,
found 302.99; Mr [Da]: 338.25; C10H19N4O7P.
High-performance liquid chromatography (HPLC): analytical HPLC
was performed on a VWR-Hitachi LaChromElite HPLC system
(L-2130, L-2200, L-2455) equipped with a EcoCART 125–3 column
containing reversed-phase silica gel Lichrospher 100 RP-18 (5 mm;
VWR-Merck, Darmstadt, Germany). The solvents for HPLC were ob-
Bis(tetra-n-butyl)-ammonia-d4TMP 6: 133 mg (0.39 mmol) ammo-
nium salt of d4TMP was dissolved in H2O and eluted over a proton-
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3
ated ion-exchange resin (DOWEX 50WX8). The obtained acidic so-
lution was titrated with 511 mg (2.0 equiv) of tetra-n-butylammoni-
um hydroxide solution (40% in H2O w/w). The resulting slightly
basic solution was lyophilized and the resulting material dissolved
in dry CH3CN and stored over molecular sieves 3 ꢁ. Yield: 260 mg
5.7 Hz, OH), 4.48 (d, 2H, 3JHH =5.7 Hz, H-Bn), 2.56 (dd, 2H, JHH
=
3
3
3
7.4 Hz, JHH =7.4 Hz, H-b), 1.63 (tt, 2H, JHH =7.4 Hz, JHH =7.4 Hz, H-
c), 1.40–1.24 (m, 6H, H-d, H-e, H-f), 0.87 ppm (t, 1H, JHH =6.9 Hz,
3
H-g); 13C NMR (101 MHz, [D6]DMSO) d=171.7 (C-a), 149.1 (C-1),
140.0 (C-4), 127.4 (2ꢂC-3), 121.2 (2ꢂC-2), 62.3 (C-Bn), 33.4 (C-b),
24.2 (C-c), 30.7, 27.9, 21.7 (C-d, C-e, C-f), 13.7 ppm (C-g); IR: n˜ =
3353, 2955, 2929, 2859, 1755, 1507, 1195, 1163, 1140, 1101, 1014,
503 cmꢀ1; Rf: 0.28 (PE 50–70/EtOAc 3:1 v/v); MS (FAB): calcd 219.14
[MꢀOH]+, 236.14 [M], 237.14 [M+H]+; found 219.2, 237.2; Mr [Da]:
236.30; C14H20O3.
1
(0.33 mmol, 85%) hygroscopic solid. H NMR (400 MHz, CD3OD) d=
4
7.77 (d, 1H, 4JHH =1.2 Hz, H-6), 6.95 (ddd, 1H, 3JHH =3.4 Hz, JHH
=
=
=
3/4
1.7 Hz, 4JHH =1.7 Hz, H-1’), 6.56 (ddd, 1H, 3JHH =6.0 Hz,
1.8 Hz, 3/4JHH =1.8 Hz, H-3’), 5.80 (ddd, 1H, 3JHH =6.0 Hz,
J
J
HH
3/4
HH
2.3 Hz, 4JHH =1.3 Hz, H-2’), 4.13–4.05 (m, 1H, H-4’), 4.01–3.94 (m,
4
1H, H-5’), 3.28–3.17 (m, 16H, H-A), 1.93 (d, 3H, JHH =1.6 Hz, Me),
1.72–1.60 (m, 16H, H-B), 1.42 (sext., 3JHH =7.4 Hz, 16H, H-C),
4-(Hydroxymethyl)phenyldecanoate 9 f: General procedure
A
1.02 ppm (t, JHH =7.4 Hz, 24H, H-D); 13C NMR (101 MHz, [D6]DMSO)
3
with 8.0 g 4-hydroxybenzyl alcohol (64 mmol, 1 equiv), 8.92 mL TEA
(6.52 g, 64.4 mmol, 1.0 equiv) dissolved in 100 mL THF and drop-
wise addition of 13.4 mL decanoyl chloride (12.3 g, 64.4 mmol,
1.0 equiv) dissolved in 80 mL THF at 08C. Reaction time: 20 h at RT.
Column chromatography (gradient of PE 50–70/EtOAc 5:1 to 5:2,
v/v). Yield: 10.19 g (36.6 mmol, 57%) colorless solid. 1H NMR
(400 MHz, [D6]DMSO) d=7.35–7.31 (m, 2H, H-3), 7.06–7.01 (m, 2H,
d=167.4 (C-4), 153.0 (C-2), 135.1–134.9 (m, C-3’), 134.2 (C-6), 126.3
(C-2’), 109.5 (C-5), 88.7 (C-1’), 85.4 (d, J=7.6 Hz, C-4’), 64.9 (d, J=
5.0 Hz, C-5’), 57.4 (C-a), 23.0 (C-b), 19.1 (C-c), 13.4 (C-d), 13.0 ppm
(Me); 31P NMR (162 MHz, 1H-decoupled, CD3OD) d=3.57 ppm; UV
(HPLC): lmax [nm]=265, HPLC: tR [min]=6.31, method A; Mr [Da]:
787.10; C42H83N4O7P.
3
3
H-2), 5.21 (t, 1H, JHH =5.7 Hz, OH), 4.47 (d, 2H, JHH =5.7 Hz, H-Bn),
3
2.55 (t, 2H, 3JHH =7.3 Hz, H-b), 1.64 (tt, 2H, 3JHH =7.3 Hz, JHH
=
General procedure A: Preparation of 4-acyloxybenzyl alcohols 9:
All reactions were carried out under nitrogen atmosphere with dry
solvents and reagents. To an ice-cold solution of 4-hydroxybenzyl
alcohol (1.0 equiv) and TEA (1.1 equiv) in THF or diethyl ether the
acyl chlorides (1.1 equiv) were added over a period of 20 min. The
mixtures were stirred 2 h at 08C followed by stirring overnight at
room temperature in some cases. The precipitate was removed by
filtration and the solvent was evaporated. The crude products
were diluted with EtOAc, washed twice with saturated sodium car-
bonate solution and twice with H2O. The organic layers were dried
(sodium sulfate) and concentrated. The crude products were puri-
fied either by column chromatography or by crystallization to give
compounds 9.
7.3 Hz, H-c), 1.40–1.18 (m, 12H, H-d, H-e, H-f, H-g, H-h, H-i),
0.85 ppm (t, 3H, 3JHH =6.9 Hz, H-j); 13C NMR (101 MHz, [D6]DMSO)
d=171.8 (C-a), 149.1 (C-1), 139.9 (C-4), 127.4 (C-3), 121.3 (C-2), 62.3
(C-Bn), 33.4 (C-b), 24.3 (C-c), 31.2, 28.8, 28.6, 28.5, 28.3, 22.0 (C-d, C-
e, C-f, C-g, C-h, C-i), 13.8 ppm (C-j); IR: n˜ =3326, 2954, 2916, 2848,
1748, 1604, 1211, 1143, 846, 719, 579, 513 cmꢀ1; mp: 45–488C, Rf:
0.34 (PE 50–70/EtOAc, 3:1 v/v); MS (FAB): calcd 278.19 [M], 279.19
[M+H]+; found 279.2 [M+H]+; Mr [Da]: 278.38; C17H26O3.
4-(Hydroxymethyl)phenyldodecanoate 9g: General procedure A
with 6.01 g 4-hydroxybenzyl alcohol (48.5 mmol, 1 equiv), 8.08 mL
TEA (5.87 g, 58.0 mmol, 1.2 equiv) dissolved in 30 mL THF and
dropwise addition of 13.4 mL dodecanoyl chloride (12.7 g,
58.0 mmol, 1.2 equiv) dissolved in 20 mL THF at 08C. Reaction
time: 20 h at RT. Column chromatography (PE 50–70/EtOAc 10:1
followed by 3:1, v/v). Yield: 5.81 g (18.9 mmol, 40%) colorless solid.
The syntheses of 4-(hydroxymethyl)phenylacetate 9a, 4-(hydroxy-
methyl)phenyl-tert-butanoate 9b and 4-(hydroxymethyl)phenyloc-
tanoate 9e were described previously.[22] The numbering scheme is
shown in Supporting Information Figure S9.
1H NMR (500 MHz, [D6]DMSO) d=7.37–7.28 (m, 2H, H-3), 7.07–6.98
3
(m, 2H, H-2), 5.20 (t, 1H, 3JHH =5.6 Hz, OH), 4.48 (d, 2H, JHH
=
=
3
5.6 Hz, H-Bn), 2.55 (t, 2H, 3JHH =7.4 Hz, H-b), 1.63 (tt, 2H, JHH
4-(Hydroxymethyl)phenylpentanoate 9c: General procedure A
with 6.0 g 4-hydroxybenzyl alcohol (48 mmol, 1 equiv), 8.08 mL TEA
(5.86 g, 58 mmol, 1 equiv) dissolved in 30 mL THF and dropwise ad-
dition of 6.99 mL butyryl chloride (2.96 g, 32 mmol, 1 equiv) at
08C. Reaction time: 23 h at RT. Column chromatography (PE 50–
70/EtOAc 3:1, v/v). Yield: 4.98 g (23.0 mmol, 46%) colorless oil.
3
7.2 Hz, JHH =7.2 Hz, H-c), 1.40–1.16 (m, 18H, H-d, H-e, H-f, H-g, H-h,
H-i, H-j, H-k), 0.85 ppm (t, 3H, JHH =6.7 Hz, H-l); 13C NMR (126 MHz,
3
[D6]DMSO) d=171.8 (C-a), 149.2 (C-1), 140.04 (C-4), 127.4 (2ꢂC-3),
121.3 (2ꢂC-2), 62.4 (C-Bn), 33.4 (C-b), 24.3 (C-c), 31.3, 28.9, 28.9,
28.6, 28.4, 28.3, 22.2 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k), 13.9 ppm
(C-l); IR: n˜ =3332, 2995, 2915, 2848, 1748, 1605, 1509, 1464, 1411,
1H NMR (400 MHz, [D6]DMSO) d=7.44–7.25 (m, 2H, H-3), 7.12–6.96
3
(m, 2H, H-2), 5.21 (t, 1H, 3JHH =5.6 Hz, OH), 4.49 (d, 2H, JHH
=
1384, 1328, 1297, 1267, 1235, 1035, 1013, 719 cmꢀ1; mp: 588C; Rf:
7.4 Hz, H-Bn), 2.56 (dd, 2H, 3JHH =7.4 Hz, 3JHH =7.4 Hz, H-b), 1.62
(quint., 2H, 3JHH =7.4 Hz, H-c), 1.39 (sext., 2H, 3JHH =7.4 Hz, H-d),
0.92 ppm (t, 3H, JHH =7.4 Hz, H-e); 13C NMR (101 MHz, [D6]DMSO)
C
+
0.28 (PE 50–70/EtOAc 3:1, v/v); MS (FAB): calcd 183.17 [C12H23O ] ,
289.22 [MꢀOH]+, 306.22 [M]+, 307.23 [M+H]+; found 183.2
3
+
+
+
+
C
[C12H23O ] , 289.3 [MꢀOH] , 307.3 [M+H] , 329.3 [M+Na] ; Mr
d=171.8 (C-a), 149.2 (C-4), 140.0 (C-1), 127.4 (2ꢂC-3), 121.3 (2ꢂC-
2), 62.4 (C-Bn), 33.2 (C-b), 26.5 (C-c), 21.6 (C-d), 13.5 ppm (C-e); IR:
n˜ =3361, 2959, 2933, 2873, 1754, 1507, 1465, 1417 1366, 1198,
1045, 1014, 811 cmꢀ1; Rf: 0.22 (PE 50–70/EtOAc 3:1, v/v); MS (FAB):
calcd 191.12 [MꢀOH]+, 208.10 [M], 209.12 [M+H]+, 231.09
[M+Na]+; found 191.1, 208.1, 231.1; Mr [Da]: 208.25; C12H16O3.
[Da]: 306.44; C19H30O3.
4-(Hydroxymethyl)phenyltetradecanoate 9h: General procedure
A with 8.0 g 4-hydroxybenzyl alcohol (64 mmol, 1 equiv), 8.92 mL
TEA (6.52 g, 64.4 mmol, 1.0 equiv) dissolved in 100 mL THF and
dropwise addition of 17.5 mL tetradecanoyl chloride (15.9 g,
64.4 mmol, 1.0 equiv) dissolved in 80 mL THF at 08C. Reaction
time: 16 h at RT. Purification was achieved by crystallization (PE
50–70/EtOAc). Yield: 21.5 g (64.3 mmol, 70%) colorless solid.
4-(Hydroxymethyl)phenylheptanoate 9d: General procedure A
with 8.0 g 4-hydroxybenzyl alcohol (64 mmol, 1 equiv), 10.7 mL TEA
(7.82 g, 77.3 mmol, 1.2 equiv) dissolved in 50 mL THF and dropwise
addition of 12.0 mL heptanoyl chloride (11.5 g, 77.3 mmol,
1.2 equiv) at 08C. Reaction time: 20 h at RT. Column chromatogra-
phy (gradient of PE 50–70/EtOAc 8:1 to 4:1, v/v). Yield: 5.50 g
1H NMR (400 MHz, [D6]DMSO) d=7.36–7.30 (m, 2H, H-3), 7.06–7.01
3
(m, 2H, H-2), 5.22 (t, 1H, 3JHH =5.4 Hz, OH), 4.47 (d, 2H, JHH
=
=
3
5.0 Hz, H-Bn), 2.54 (t, 2H, 3JHH =7.4 Hz, H-b), 1.61 (tt, 2H, JHH
3
7.2 Hz, JHH =7.2 Hz, H-c), 1.38–1.17 (m, 20H, H-d, H-e, H-f, H-g, H-h,
H-i, H-j, H-k, H-l, H-m), 0.85 ppm (t, 3H, JHH =6.4 Hz, H-n); 13C NMR
(101 MHz, [D6]DMSO) d=171.8 (C-a), 149.1 (C-1), 139.9 (2ꢂC-4),
(23.3 mmol, 56%) yellowish oil. 1H NMR (400 MHz, [D6]DMSO) d=
3
3
7.36–7.31 (m, 2H, H-3), 7.06–7.02 (m, 2H, H-2), 5.22 (t, 1H, JHH
=
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127.4 (2ꢂC-3), 121.3 (C-2), 62.3 (C-Bn), 33.4 (C-b), 24.3 (C-c), 31.3,
29.0, 28.9, 28.8, 28.7, 28.6, 28.5, 28.3, 22.0 (C-d, C-e, C-f, C-g, C-h, C-
i, C-j, C-k, C-l, C-m), 13.8 ppm (C-n); IR: n˜ =2955, 2914, 2847, 1750,
1508, 1252, 1214, 1165, 1014, 924, 719, 496 cmꢀ1; mp: 618C; Rf:
0.33 (PE 50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 334.25 [M], 335.26
[M+H], found 335.3; Mr [Da]: 334.49; C21H34O3.
midite 7e were described previously.[22] The numbering scheme is
shown Supporting Information Figure S10.
Bis(4-pentanoyloxybenzyl)-N,N-diisopropylphosphoramidite 7c:
General procedure B with 1.997 g 4-(hydroxymethyl)phenylpenta-
noate (9.60 mmol, 1.9 equiv), 1.4 mL TEA (1.0 g, 10 mmol, 2.3 equiv)
dissolved in 25 mL THF and 0.882 g dichloro-N,N-diisopropylphos-
phoramidite (4.36 mmol, 1.0 equiv) dissolved in 25 mL THF. Purifica-
tion by Chromatotron (PE 50–70, 5% TEA). Yield: 2.13 g
4-(Hydroxymethyl)phenylhexadecanoate 9i: General procedure A
with 8.0 g 4-hydroxybenzyl alcohol (64 mmol, 1 equiv), 9.0 mL TEA
(6.5 g, 65.0 mmol, 1.0 equiv) dissolved in 50 mL THF and dropwise
addition of 19.6 mL hexadecanoyl chloride (17.7 g, 64.4 mmol,
1.0 equiv) dissolved in 20 mL THF at 08C. Reaction time: 18 h at RT.
Purification was achieved by crystallization (PE 50–70/EtOAc) fol-
lowed by column chromatography (PE 50–70/EtOAc, 3:1 v/v). Yield:
8.44 g (23.3 mmol, 36%) colorless solid. 1H NMR (500 MHz,
[D6]DMSO) d=7.36–7.30 (m, 2H, H-3), 7.06–7.00 (m, 2H, H-2), 5.20
(t, 1H, 3JHH =5.7 Hz, OH), 4.47 (d, 2H, 3JHH =5.7 Hz, H-Bn), 2.55 (t,
1
(3.91 mmol, 90%) colorless oil. H NMR (400 MHz, CDCl3) d=7.36–
2
7.32 (m, 4H, H-2), 7.06–7.00 (m, 4H, H-3), 4.74 (dd, 2H, JHH
=
=
3
12.6 Hz, 3JHP =8.1 Hz, H-Bn), 4.66 (dd, 2H, 2JHH =12.6 Hz, JHP
3
8.1 Hz, H-Bn), 3.75–3.61 (m, 2H, NC-H), 2.54 (t, 4H, JHH =7.5 Hz, H-
b), 1.74 (tt, 4H, 3JHH =7.5 Hz, 3JHH =7.5 Hz, H-c), 1.44 (sext., 4H,
3JHH =7.5 Hz, H-d), 1.19 (d, 12H, JHH =6.7 Hz, Me), 0.96 ppm (t, 6H,
3
3JHH =7.3 Hz, H-e); 13C NMR (101 MHz, CDCl3) d=172.4 (2ꢂC-a),
150.0 (2ꢂC-4), 137.2 (2ꢂC-1), 128.1 (4ꢂC-2), 121.5 (4ꢂC-3), 64.9 (d,
2JCP =18.5 Hz, 2ꢂC-Bn), 43.2 (d, 2JCP =12.9 Hz, 2ꢂC-N), 34.3, 27.2,
24.7, 24.7, 22.4 (C-b, C-c, C-d, 2ꢂMe), 13.8 ppm (C-e); 31P NMR
3
3
3
2H, JHH =7.4 Hz, H-b), 1.62 (tt, 2H, JHH =7.3 Hz, JHH =7.2 Hz, H-c),
1.38–1.17 (m, 24H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-
n, H-o), 0.85 ppm (t, 3H, 3JHH =6.8 Hz, H-p); 13C NMR (101 MHz,
[D6]DMSO) d=171.5 (C-a), 149.4 (C-1), 137.4 (2ꢂC-4), 128.5 (2ꢂC-
3), 120.7 (C-2), 63.9 (C-Bn), 33.5 (C-b), 24.1 (C-c), 31.1, 28.7, 28.6,
28.5, 28.4, 28.4, 28.3, 21.7 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-
m, C-n, C-o), 13.2 ppm (C-p); IR: n˜ =3396, 3073, 2916, 1758, 1509,
1
(162 MHz, H-decoupled, CDCl3) d=147.86 ppm, IR: n˜ =2963, 2931,
2871, 1757, 1507, 1460, 1417, 1364, 1198, 1100, 1026, 851 cmꢀ1; Rf:
0.69 (PE 50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 545.3 [M]+, 546.3
[M+H]+; found 544.3 [MꢀH]+, 546.4 [M+H]+; Mr [Da]: 545.64;
C30H44NO6P.
1471, 1220, 1152, 1220, 1388, 1471 cmꢀ1; mp: 758C; Rf: 0.53 (PE
Bis(4-heptanoyloxybenzyl)-N,N-diisopropylphosphoramidite 7d:
General procedure B with 1.8 g 4-(hydroxymethyl)phenylhepta-
noate (7.6 mmol, 2.2 equiv), 1.11 mL TEA (0.78 g, 7.96 mmol,
2.3 equiv) dissolved in 10 mL THF and 0.69 g dichloro-N,N-diisopro-
pylphosphoramidite (0.64 mL, 3.46 mmol, 1.0 equiv) dissolved in
10 mL THF. Purification by Chromatotron (PE 50–70 and EtOAc gra-
dient 0–50%, 5% TEA). Yield: 984 mg (1.66 mmol, 49%) colorless
+
C
50–70/EtOAc, 1:1 v/v); MS (FAB): calcd 239.23 [C16H31O ] , 345.27
+
[C23H37O2 ] [MꢀOH]+, 363.28 [M+H]+; found 239.3, 345.3, 363.3;
C
Mr [Da]: 362.27; C23H38O3.
4-(Hydroxymethyl)phenyl-Z-octadec-9-enoate 9j: General proce-
dure A with 8.0 g 4-hydroxybenzyl alcohol (64 mmol, 1 equiv),
10.8 mL TEA (7.82 g, 77.3 mmol, 1.2 equiv) dissolved in 50 mL THF
and dropwise addition of 25.5 mL Z-octadec-9-enoyl chloride
(23.3 g, 77.3 mmol, 1.2 equiv) dissolved in 20 mL THF at 08C. Reac-
tion time: 3 h at RT. Purification was achieved by crystallization (PE
50–70/EtOAc) followed by column chromatography (PE 50–70/
EtOAc, 5:1 v/v). Yield: 13.6 g (36.5 mmol, 57%) colorless oil.
1H NMR (400 MHz, CDCl3) d=7.41–7.33 (m, 2H, H-2), 7.10–7.01 (m,
1
oil. H NMR (400 MHz, CDCl3) d=7.37–7.32 (m, 4H, H-2), 7.05–7.00
2
3
(m, 4H, H-3), 4.74 (dd, 2H, JHH =12.6 Hz, JHP =8.0 Hz, H-Bn), 4.66
2
3
(dd, 2H, JHH =12.6 Hz, JHP =8.0 Hz, H-Bn), 3.74–3.63 (m, 2H, NC-H),
2.54 (t, 4H, 3JHH =7.4 Hz, H-b), 1.75 (tt, 4H, 3JHH =7.4 Hz, JHH
7.4 Hz, H-c), 1.45–1.22 (m, 12H, H-d, H-e, H-f), 1.19 (d, 12H, JHH
3
=
=
3
6.7 Hz, Me), 0.90 ppm (t, 6H, JHH =7.0 Hz, H-g); 13C NMR (101 MHz,
CDCl3) d=173.7 (2ꢂC-a), 154.4 (C-4), 154.3 (C-4), 130.3 (2ꢂC-1),
129.8 (4ꢂC-2), 120.0 (4 C-3), 119.9 (2ꢂC-3), 65.7 (2ꢂC-Bn), 44.1 (d,
2JCP =13.7 Hz, 2ꢂ-N), 34.2, 27.1, 24.5, 24.5, 22.3 (C-b, C-c, C-d, C-e,
C-f, Me), 13.7 ppm (C-g); 31P NMR (162 MHz, 1H-decoupled, CDCl3)
d=147.92 ppm; IR (film): n˜ =2962, 2930, 2860, 1759, 1608, 1459,
1417, 1199, 1142, 1027, 975, 853, 754 cmꢀ1; Rf: 0.55 (PE 50–70/
EtOAc/TEA, 4:1:0.1 v/v/v); MS (FAB): calcd 601.35 [M], 602.36
[M+H]+ found 602.4; Mr [Da]: 601.75; C34H52NO6P.
3
2H, H-3), 5.43–5.31 (m, 2H, H-i, H-j), 4.68 (s, 2H, H-Bn), 2.55 (t, 2H,
3
3JHH =7.5 Hz, H-b), 2.12–1.93 (m, 4H, H-h, H-k), 1.75 (tt, 2H, JHH
=
3
7.4 Hz, JHH =7.4 Hz, H-c), 1.46–1.21 (m, 20H, H-d, H-e, H-f, H-g, H-l,
H-m, H-n, H-o, H-p, H-q), 0.87 ppm (t, 3H, 3JHH =6.8 Hz, H-r);
13C NMR (101 MHz, CDCl3) d=172.1 (C-a), 150.1 (C-4), 138.1 (C-1),
129.5 (C-i, C-j), 127.8 (C-2), 121.5 (C-3), 64.6 (C-Bn), 34.1 (C-b), 29.7,
29.6, 29.5, 29.3, 29.1, 29.1, 24.8, 22.6 (C-c, C-d, C-e, C-f, C-g, C-l, C-
m, C-n, C-o, C-p, C-q), 27.2 (C-h, C-k), 13.7 ppm (C-r); IR: n˜ =2922,
2853, 1739, 1609, 1509, 1463, 1199, 1162, 851, 723 cmꢀ1; Rf: 0.38
(PE 50–70/EtOAc, 3:1 v/v); MS (FAB): calcd 343.26 [MꢀCH3OH],
374.28 [M], found 343.3, 371.3; Mr [Da]: 374.55; C24H38O3.
Bis(4-decanoyloxybenzyl)-N,N-diisopropylphosphoramidite 7 f:
General procedure B with 3.99 g 4-(hydroxymethyl)phenyldeca-
noate (14.4 mmol, 2.2 equiv), 2.1 mL TEA (1.5 g, 14.6 mmol,
2.2 equiv) dissolved in 10 mL THF and 1.31 g dichloro-N,N-diisopro-
pylphosphoramidite (6.47 mmol, 1.0 equiv) dissolved in 30 mL THF.
Purification by Chromatotron (PE 50–70/EtOAc, 9:1 v/v, 5% TEA).
Yield: 2.48 g (3.62 mmol, 56%) colorless oil. 1H NMR (400 MHz,
CDCl3) d=7.37–7.32 (m, 4H, H-2), 7.05–7.00 (m, 4H, H-3), 4.74 (dd,
General procedure B: Preparation of bis-(4-acyloxybenzyl)-N,N-
diisopropylphosphoramidites 7: All reactions were carried out
under nitrogen atmosphere with dry solvents and reagents. A solu-
tion of dichloro-N,N-diisopropylphosphoramidite (1.0 equiv) in THF
was cooled to ꢀ788C. Over a period of 1–2 h a solution of TEA
2
3
2
2H, JHH =12.6 Hz, JHP =8.0 Hz, H-Bn), 4.66 (dd, 2H, JHH =12.6 Hz,
(2.3 equiv) and corresponding 4-acyloxybenzyl alcohols
9
3JHP =8.0 Hz, H-Bn), 3.75–3.64 (m, 2H, NC-H), 2.54 (t, 4H, JHH
=
3
(2.2 equiv) in THF was added dropwise. The reaction mixture was
allowed to warm to room temperature and stirred for 16 to 24 h.
The precipitate was removed by filtration and the filtrate was con-
centrated. The crude products were purified by preparative TLC
(Chromatotron) to give compounds 7.
3
3
7.5 Hz, H-b), 1.75 (tt, 4H, JHH =7.4 Hz, JHH =7.4 Hz, H-c), 1.45–1.23
(m, 24H, H-d, H-e, H-f, H-g, H-h, H-i), 1.21 (d, 12H, 3JHH =6.7 Hz,
Me), 0.88 ppm (t, 6H, JHH =6.7 Hz, H-j); 13C NMR (101 MHz, CDCl3)
3
d=172.4 (2ꢂC-a), 150.0 (2ꢂC-4), 137.1 (d, 3JCP =7.3 Hz, 2ꢂC-1),
2
128.1 (4ꢂC-2), 121.5 (4ꢂC-3), 64.9 (d, JCP =18.6 Hz, 2ꢂC-Bn), 43.2
(d, JCP =13.0 Hz, 2ꢂC-N), 34.5, 31.9, 29.5, 29.4, 29.2, 26.3, 25.1, 22.7
2
The syntheses of bis(4-acetyloxybenzyl)-N,N-diisopropylphosphora-
midite 7a, bis(4-tert-butyloxybenzyl)-N,N-diisopropylphosphorami-
dite 7b, and bis(4-octanoyloxybenzyl)-N,N-diisopropylphosphora-
(C-b, C-c, C-d, C-e, C-f, C-g, C-h, C-i), 24.8 (Me), 24.6 (Me), 14.2 ppm
1
(C-j); 31P NMR (162 MHz, H-decoupled, CDCl3) d=147.94 ppm; IR:
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3
n˜ =2955, 2916, 2848, 1749, 1508, 1210, 1143, 1120, 719, 579,
514 cmꢀ1; Rf: 0.53 (PE 50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 685.44
[M], 686.45 [M+H]+, found 686.5; Mr [Da]: 685.91; C40H64NO6P.
(t, 6H, JHH =6.7 Hz, H-p); 13C NMR (101 MHz, CDCl3) d=172.5 (2ꢂ
C-a), 150.1 (2ꢂC-4), 137.1 (d, 3JCP =7.4 Hz, 2ꢂC-1), 128.1 (4ꢂC-2),
121.5 (4ꢂC-3), 64.9 (d, 2JCP =18.3 Hz, 2ꢂC-Bn), 43.3 (d, JCP
2
=
13.0 Hz, 2ꢂC-N), 34.5, 32.4, 32.1, 29.8, 29.7, 29.7, 29.5, 29.4, 29.3,
29.3, 25.1, 22.4 (C-b, C-c, C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-
m, C-n, C-o), 24.7 (Me), 24.6 (Me), 14.3 ppm (C-p); 31P NMR
Bis(4-dodecanoyloxybenzyl)-N,N-diisopropylphosphoramidite
7g: General procedure B with 3.01 g 4-(hydroxymethyl)phenyldo-
decanoate (9.82 mmol, 2.2 equiv), 1.43 mL TEA (1.01 g, 10.3 mmol,
2.3 equiv) dissolved in 25 mL THF and 0.902 g dichloro-N,N-diiso-
propylphosphoramidite (0.821 mL, 4.45 mmol, 1.0 equiv) dissolved
in 25 mL THF. Purification by Chromatotron (PE 50–70 and EtOAc
gradient 0–50%, 5% TEA). Yield: 1.92 g (2.59 mmol, 58%) colorless
solid. 1H NMR (400 MHz, CDCl3) d=7.36–7.31 (m, 4H, H-2), 7.05–
1
(162 MHz, H-decoupled, CDCl3) d=147.94 ppm; IR: n˜ =2916, 2848,
1754, 1223, 1153, 971, 849, 819, 763 cmꢀ1; mp: 538C; Rf: 0.70 (PE
50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 853.63 [M], 854.64 [M+H]+,
found 854.9; Mr [Da]: 854.24; C52H88O6NP.
Bis(4-Z-octadec-9-enoyloxybenzyl)-N,N-diisopropylphosphorami-
dite 7j: General procedure B with 2.00 g 4-(hydroxymethyl)phenyl-
Z-octadec-9-enoate (5.33 mmol, 2.1 equiv), 0.69 mL TEA (0.48 g,
4.81 mmol, 1.9 equiv) dissolved in 25 mL diethyl ether and
2
3
7.00 (m, 4H, H-3), 4.74 (dd, 2H, JHH =12.8 Hz, JHP =8.0 Hz, H-Bn),
2
3
4.66 (dd, 2H, JHH =12.8 Hz, JHP =8.0 Hz, H-Bn), 3.74–3.63 (m, 2H,
NC-H), 2.54 (t, 4H, 3JHH =7.4 Hz, H-b), 1.75 (tt, 4H, 3JHH =7.5 Hz,
3JHH =7.5 Hz, H-c), 1.45–1.22 (m, 32H, H-d, H-e, H-f, H-g, H-h, H-i, H-
0.455 mL
dichloro-N,N-diisopropylphosphoramidite
(0.499 g,
3
j, H-k), 1.19 (d, 12H, 3JHH =6.7 Hz, Me), 0.87 ppm (t, 6H, JHH
=
2.47 mmol, 1.0 equiv) dissolved in 15 mL diethyl ether. Purification
by Chromatotron (PE 50–70 and EtOAc gradient 0–50%, 5% TEA).
6.7 Hz, H-l); 13C NMR (101 MHz, CDCl3) d=172.4 (2ꢂC-a), 150.0 (2ꢂ
C-4), 137.1 (d, JCP =7.5 Hz, 2ꢂC-1), 128.1 (4x C-2), 121.5 (4x C-3),
3
1
Yield: 795 mg (0.877 mmol, 16%) colorless solid. H NMR (400 MHz,
2
2
64.9 (d, JCP =18.5 Hz, 2ꢂC-Bn), 43.2 (d, JCP =13.0 Hz, 2ꢂC-N), 34.5,
32.0, 29.5, 29.5, 29.5, 29.4, 29.2, 25.1, 22.7 (C-b, C-c, C-d, C-e, C-f, C-
g, C-h, C-i, C-j), 24.7, 24.6 (Me), 14.2 ppm (C-k); 31P NMR (162 MHz,
1H-decoupled, CDCl3) d=147.88 ppm; IR: n˜ =2958, 2915, 2849,
1753, 1508, 1463, 1412, 1387, 1362, 1328, 1298, 1267, 1236, 1083,
1017, 728 cmꢀ1; mp: 478C; Rf: 0.87 (PE 50–70/EtOAc, 5:1 v/v); MS
(FAB): calcd 741.50 [M], 780.47 [M+K]+; found 780.6 [M+K]+; Mr
[Da]: 742.02; C44H72NO6P.
CDCl3) d=7.36–7.32 (m, 4H, H-2), 7.05–7.01 (m, 4H, H-3), 5.43–5.28
(m, 4H, H-i, H-j), 4.75 (dd, 2H, 2JHH =12.8 Hz, 3JHP =8.2 Hz, H-Bn),
2
3
4.67 (dd, 2H, JHH =12.8 Hz, JHP =8.2 Hz, H-Bn), 3.74–3.63 (m, 2H,
3
NC-H), 2.54 (t, 4H, JHH =7.4 Hz, H-b), 2.09–1.93 (m, 8H, H-h, H-k),
1.75 (tt, 4H, JHH =7.5 Hz, JHH =7.5 Hz, H-c), 1.46–1.23 (m, 40H, H-d,
H-e, H-f, H-g, H-l, H-m, H-n, H-o, H-p, H-q), 1.19 (d, 12H, JHH
6.7 Hz, Me), 0.87 ppm (m, 6H, H-r); 13C NMR (101 MHz, CDCl3) d=
172.5 (2ꢂC-a), 150.1 (2ꢂC-4), 137.1 (d, JCP =8.0 Hz, 2ꢂC-1), 130.2,
129.8 (2ꢂC-i, 2ꢂC-j), 128.1 (4ꢂC-2), 121.4 (4ꢂC-3), 64.9 (d, JCP
3
3
3
=
3
2
=
18.5 Hz, 2ꢂC-Bn), 43.3 (d, 2JCP =12.6 Hz, 2ꢂC-N), 34.5 (C-b), 32.1,
29.8, 29.8, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 29.1, 27.4, 27.3,
22.7 (C-c, C-d, C-e, C-f, C-g, C-l, C-m, C-n, C-o, C-p, C-q), 25.1 (C-h,
C-k), 24.7 (d, 3JCP =7.5 Hz, 4x Me), 14.8 ppm (C-r); 31P NMR
Bis(4-tetradecanoyloxybenzyl)-N,N-diisopropylphosphoramidite
7h: General procedure B with 2.00 g 4-(hydroxymethyl)phenyl-
tetradecanoate (5.98 mmol, 2.0 equiv), 0.84 mL TEA (0.59 g,
5.98 mmol, 2.0 equiv) dissolved in 15 mL diethyl ether and 603 mg
dichloro-N,N-diisopropylphosphoramidite ((0.549 mL, 2.99 mmol,
1.0 equiv) dissolved in 30 mL diethyl ether. Purification by Chroma-
totron (PE 50–70 and EtOAc gradient 0–50%, 5% TEA). Yield:
1
(162 MHz, H-decoupled, CDCl3) d=147.91 ppm; IR: n˜ =2923, 2853,
1761, 1198, 1164, 1129, 1051, 1026, 1008, 974, 915, 754, 553 cmꢀ1
;
Rf: 0.94 (PE 50–70/EtOAc, 5:1 v/v), MS (FAB): calcd 905.66 [M],
906.67 [M+H]+ found 906.4; Mr [Da]: 906.31; C56H92NO6P.
1
811 mg (1.01 mmol, 34%) colorless solid. H NMR (400 MHz, CDCl3)
d=7.38–7.32 (m, 4H, H-2), 7.05–7.00 (m, 4H, H-3), 4.74 (dd, 2H,
3
2
3
2JHH =12.5 Hz, JHP =8.0 Hz, H-Bn), 4.66 (dd, 2H, JHH =12.5 Hz, JHP
=
2-Cyanethoxy-(4-decanoyloxybenzyl)-N,N-diisopropylphosphora-
midite 12: General procedure B with 113 mg 4-(hydroxymethyl)-
phenyldecanoate (0.405 mmol, 0.9 equiv), 70.7 mL diisopropylethyl-
amine 52.3 mg, 0.405 mmol, 0.9 equiv) dissolved in 10 mL THF and
100 mg chloro-(2-cyanoethyloxy)-N,N-diisopropylphosphoramidite
(0.449 mmol, 1.1 equiv) dissolved in 10 mL THF. The mixture was
stirred at RT for 90 min. Yield: 150 mg (0.313 mmol, 78%) colorless
oil. 1H NMR (400 MHz, CDCl3) d=7.39–7.31 (m, 2H, Haryl-3), 7.08–
3
8.0 Hz, H-Bn), 3.74–3.63 (m, 2H, NC-H), 2.54 (t, 4H, JHH =7.4 Hz, H-
b), 1.74 (tt, 4H, JHH =7.5 Hz, JHH =7.5 Hz, H-c), 1.45–1.22 (m, 40H,
H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m), 1.19 (d, 12H, JHH
6.7 Hz, Me), 0.88 ppm (t, 6H, JHH =6.7 Hz, H-o); 13C NMR (101 MHz,
CDCl3) d=172.5 (2ꢂC-a), 150.1 (2ꢂC-4), 137.1 (d, JCP =7.6 Hz, 2ꢂ
C-1), 128.1 (4ꢂC-2), 121.5 (4ꢂC-3), 64.5 (d, JCP =18.1 Hz, 2ꢂC-Bn),
43.3 (d, JCP =13.0 Hz, 2ꢂC-N), 34.5, 34.4, 32.1, 29.7, 29.6, 29.5, 29.4,
3
3
3
=
3
3
2
2
2
3
29.3, 29.2, 22.7 (C-b, C-c, C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-
7.02 (m, 2H, Haryl-2), 4.74 (dd, 2H, JHH =12.6 Hz, JHP =8.0 Hz, H-Bn),
1
2
3
m), 25.1 (2ꢂMe), 14.3 ppm (C-n); 31P NMR (162 MHz, H-decoupled,
4.66 (dd, 2H, JHH =12.6 Hz, JHP =8.0 Hz, H-Bn), 3.89–3.78 (m, 2H,
3
CDCl3) d=147.84 ppm; IR: n˜ =2956, 2914, 2847, 1752, 1508, 1463,
1361, 1218, 1197, 1091, 968, 851, 819, 512 cmꢀ1; mp: 478C; Rf: 0.82
(PE 50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 797.57 [M], 798.57
[M+H]+, found 798.5; Mr [Da]: 798.12; C48H80NO6P.
H-1’), 3.70–3.59 (m, 2H, NC-H), 2.62 (t, 2H, JHH =6.5 Hz, H-2’), 2.54
3
3
3
(t, 2H, JHH =7.5 Hz, H-b), 1.75 (tt, 2H, JHH =7.4 Hz, JHH =7.4 Hz, H-
c), 1.45–1.23 (m, 12H, H-d, H-e, H-f, H-g, H-h, H-i), 1.19 (d, 12H,
3JHH =5.2 Hz, Me), 0.87 ppm (t, 3H, 3JHH =6.8 Hz, H-j); 31P NMR
(162 MHz, 1H-decoupled, CDCl3) d=148.54 ppm; Rf: 0.93, (PE 50–
70/EtOAc, 1:1 v/v); Mr [Da]: 478.60; C26H43N2O4P.
Bis(4-hexadecanoyloxybenzyl)-N,N-diisopropylphosphoramidite
7i: General procedure B with 4.018 g 4-(hydroxymethyl)phenyl-
hexadecanoate (11.07 mmol, 2.2 equiv), 1.61 mL TEA (1.17 g,
11.5 mmol, 2.3 equiv) dissolved in 10 mL THF and 0.92 mL dichloro-
N,N-diisopropylphosphoramidite (1.01 g, 4.99 mmol, 1.0 equiv) dis-
solved in 10 mL THF. Purification by Chromatotron (PE 50–70 and
EtOAc gradient 0–50%, 5% TEA). Yield: 3.43 g (4.15 mmol, 80%)
General procedure C: Preparation of DiPPro-d4T-diphosphates
4: All reactions were carried out under nitrogen atmosphere with
dry solvents and reagents. D4T monophosphate 6 was dried in
stock solution over molecular sieves before starting the reactions.
D4T monophosphate (1.0 equiv), phosphoramidites
7
(1.1–
1
colorless solid. H NMR (400 MHz, CDCl3) d=7.37–7.32 (m, 4H, H-2),
2.3 equiv) and DCI (1.1–2.3 equiv) were dissolved in up to 2.5 mL
CH3CN. The reaction mixture was stirred for several hours and then
cooled to ꢀ258C. Oxidation was achieved by addition of tert-butyl-
hydroperoxide (TBHP, 5.5m in n-decane; 1.1–2.3 equiv). The mix-
ture was allowed to warm to room temperature. After 15 min the
solvents were removed in vacuo. The crude products were purified
2
3
7.05–7.00 (m, 4H, H-3), 4.74 (dd, 2H, JHH =12.8 Hz, JHP =8.1 Hz, H-
2
3
Bn), 4.66 (dd, 2H, JHH =12.8 Hz, JHP =8.1 Hz, H-Bn), 3.75–3.62 (m,
2H, NC-H), 2.54 (t, 4H, JHH =7.4 Hz, H-b), 1.74 (tt, 4H, JHH =7.5 Hz,
3JHH =7.5 Hz, H-c), 1.45–1.22 (m, 48H, H-d, H-e, H-f, H-g, H-h, H-i, H-
3
3
3
j, H-k, H-l, H-m, H-n, H-o), 1.19 (d, 12H, JHH =6.7 Hz, Me), 0.88 ppm
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 762 – 775 771

CHEMMEDCHEM
FULL PAPERS
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by RP-18 flash chromatography and purified by RP-18 flash chro-
matography a second and third time if necessary (with different
H2O/MeOH gradients). Product containing fractions were pooled
and MeOH was evaporated. The remaining aqueous solutions were
freeze-dried and the products were obtained as brown syrups.
(101 MHz, CD3OD) d=173.6 (2ꢂC-a), 166.5 (Chet-4), 152.7 (Chet-2),
3
152.4 (C-4), 152.4 (C-4), 138.6 (Chet-6), 135.3 (C-3’), 134.8 (d, JCP
7.3 Hz, C-1), 134.8 (d, JCP =7.3 Hz, C-1), 130.4 (d, JCP =2.7 Hz, 4ꢂC-
2), 127.5 (C-2’), 122.8 (4ꢂC-3), 112.1 (Chet-5), 90.7 (C-1’), 86.9 (d,
=
3
4
2
2
3JCP =8.1 Hz, C-4’), 70.2 (d, JCP =5.8 Hz, C-Bn), 70.2 (d, JCP =5.8 Hz,
C-Bn), 68.0 (d, 2JCP =6.2 Hz, C-5’), 59.5 (pt, 4ꢂC-A), 35.0 (2ꢂC-b),
32.5, 29.7, 25.8, 24.7, 23.5, 20.6, 19.3 (2ꢂC-c, 2ꢂC-d, 2ꢂC-e, 2ꢂC-f,
4ꢂC-B), 14.4 (2ꢂC-g), 13.8 (4ꢂC-D), 12.5 ppm (Mehet); 31P NMR
The syntheses of ammonium bis-(4-acetyloxybenzyl)-d4TDP 4a,
bis-(4-pivaloyloxybenzyl)-d4TDP 4b, and ammonium bis-(4-octa-
noyloxybenzyl)-d4TDP 4e were described previously.[22] The num-
bering scheme is shown in Supporting Information Figure S11.
1
2
(162 MHz, H-decoupled, CD3OD) d=ꢀ12.11 (d, 1P, JPP =20.3 Hz, P-
2
a), ꢀ12.92 ppm (d, 1P, JPP =21.4 Hz, P-b); IR: n˜ =2959, 2931, 2873,
1755, 1688, 1508, 1465, 1262, 1007, 910, 836, 804, 502 cmꢀ1; UV
(HPLC): lmax =265 nm, HPLC: tR =13.2 min, method A; Rf: 0.80
(EtOAc/MeOH, 7:3 v/v); HRMS (ESIꢀ): calcd 820.2737 [M]ꢀ,
819.2665 [MꢀH]ꢀ, found 819.2665; Mr [Da]: 1062.21; C54H85N3O14P2.
(N[nBu]4)-Bis-(4-pentanoyloxybenzyl)-d4TDP 4c: General proce-
dure
C
with 115 mg bis-(tetra-n-butylammonium)-d4TMP
(0.146 mmol, 1 equiv), 129 mg bis-(4-pentanoyloxybenzyl)-N,N-dii-
sopropylphosphoramidite (0.235 mmol, 1.6 equiv) and 27.7 mg DCI
(0.235 mmol, 1.6 equiv) in 4 mL CH3CN at 08C. After 2 h stirring at
RT 32 mg amidite (0.058 mmol, 0.4 equiv) and 6.9 mg DCI
(0.058 mmol, 0.4 equiv) were added. The reaction mixture was
stirred for 23 h and cooled to ꢀ258C followed by oxidation with
53 mL TBHP (0.28 mmol, 1.8 equiv). The crude product was purified
by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1 v/v, final-
ly MeOH). Yield: 47 mg (0.0467 mmol, 32%) colorless syrup.
1H NMR (400 MHz, CD3OD) d=7.65 (d, 1H, 4JHH =1.3 Hz, Hhet-6),
7.43–7.35 (m, 4H, H-2), 7.09–7.02 (m, 4H, H-3), 6.94 (ddd, 1H,
3JHH =3.5 Hz, 3/4JHH =1.7 Hz, 3/4JHH =1.7 Hz, H-1’), 6.37 (ddd, 1H,
(N[nBu]4)-Bis-(4-decanoyloxybenzyl)-d4TDP 4 f: General proce-
dure
C
with 148 mg bis-(tetra-n-butylammonium)-d4TMP
(0.271 mmol, 1 equiv), 208 mg bis-(4-decanoyloxybenzyl)N,N-diiso-
propylphosphoramidite (0.303 mmol, 1.1 equiv) and 37.0 mg DCI
(0.313 mmol, 1.2 equiv) in 4 mL CH3CN. The reaction mixture was
stirred for 3 h and cooled to ꢀ258C followed by oxidation with
53 mL TBHP (0.28 mmol, 1.0 equiv). The crude product was purified
by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1 v/v, final-
ly MeOH). Yield: 121 mg (0.105 mmol, 39%) colorless syrup.
1H NMR (400 MHz, CD3OD) d=7.65 (d, 1H, 4JHH =1.2 Hz, Hhet-6),
7.42–7.34 (m, 4H, H-2), 7.07–7.02 (m, 4H, H-3), 6.95 (ddd, 1H,
3JHH =3.5 Hz, 3/4JHH =1.7 Hz, 3/4JHH =1.7 Hz, H-1’), 6.38 (ddd, 1H,
4
4
3
3JHH =6.1 Hz, JHH =1.7 Hz, JHH =1.7 Hz, H-3’), 5.83 (ddd, 1H, JHH
=
6.0 Hz, 3/4JHH =2.2 Hz, JHH =1.5 Hz, H-2’), 5.01 (dd, 4H, JHH =8.2 Hz,
4
2
3JHP =6.3 Hz, H-Bn), 4.97–4.92 (m, 1H, H-4’), 4.25–4.12 (m, 2H, H-5’),
4
4
3
3
3JHH =6.0 Hz, JHH =1.6 Hz, JHH =1.6 Hz, H-3’), 5.84 (ddd, 1H, JHH
=
3.26–3.19 (m, 8H, H-A), 2.53 (t, 4H, JHH =7.2 Hz, H-b), 1.89 (d, 3H,
6.0 Hz, JHH =1.6 Hz, 3/4JHH =2.0 Hz, H-2’), 5.10 (dd, 4H, JHH =8.5 Hz,
3JHP =6.2 Hz, H-Bn), 4.97–4.92 (m, 1H, H-4’), 4.25–4.15 (m, 2H, H-5’),
4
2
4JHH =1.3 Hz, Mehet), 1.76–1.60 (m, 12H, H-B, H-c), 1.51–1.36 (m,
12H, H-C, H-d), 1.05–0.94 ppm (m, 16H, H-D, H-e); 13C NMR
(126 MHz, CD3OD) d=173.6 (2ꢂC-a), 166.5 (Chet-4), 152.7 (Chet-2),
3
3.25–3.17 (m, 8H, H-A), 2.57 (t, 4H, JHH =7.4 Hz, H-b), 1.89 (d, 3H,
4JHH =1.0 Hz, Mehet), 1.77 (tt, 4H, 3JHH =7.5 Hz, 3JHH =7.5 Hz, H-c),
3
152.4 (2ꢂC-4), 138.6 (Chet-6), 135.3 (C-3’), 134.8 (d, JCP =7.0 Hz, C-
3
4
1.68–1.58 (m, 8H, H-B), 1.46–1.25 (m, 32H, H-d, H-e, H-f, H-g, H-h,
1), 134.8 (d, JCP =7.0 Hz, C-1), 130.4 (d, JCP =3.5 Hz, 4ꢂC-2), 127.5
3
3
H-i, H-C), 1.01 (t, 12H, 3JHH =7.3 Hz, H-D), 0.90 ppm (t, 6H, JHH
=
(C-2’), 122.8 (4ꢂC-3), 112.1 (Chet-5), 90.7 (C-1’), 86.9 (d, JCP =8.1 Hz,
6.9 Hz, H-j); 13C NMR (101 MHz, CD3OD) d=173.6 (2ꢂC-a), 166.5
C-4’), 70.2 (d, 2JCP =5.5 Hz, C-Bn), 70.2 (d, 2JCP =5.5 Hz, C-Bn), 68.0
2
(Chet-4), 152.7 (Chet-2), 152.4 (C-4), 152.4 (C-4), 138.5 (Chet-6), 135.3
(d, JCP =6.0 Hz, C-5’), 59.5 (pt, 4ꢂC-A), 34.6 (2ꢂC-b), 28.1 (2ꢂC-c),
3
3
(C-3’), 134.8 (d, JCP =7.4 Hz, C-1), 134.8 (d, JCP =7.4 Hz, C-1), 130.4
(d, 4JCP =2.7 Hz, 4ꢂC-2), 127.5 (C-2’), 122.5 (4ꢂC-3), 112.1 (Chet-5),
90.7 (C-1’), 86.9 (d, 3JCP =9.0 Hz, C-4’), 70.2 (d, JCP =5.8 Hz, C-Bn),
24.7 (4ꢂC-B), 23.2 (2ꢂC-d), 20.6 (4ꢂC-C), 14.1 (2ꢂC-e), 13.8 (4ꢂC-
1
D), 12.5 ppm (Mehet); 31P NMR (162 MHz, H-decoupled, CD3OD) d=
2
2
ꢀ12.08 (d, 1P, JPP =20.6 Hz, P-a), ꢀ12.96 ppm (d, 1P, JPP =21.3 Hz,
P-b); UV (HPLC): lmax =265 nm, HPLC: tR =11.57 min, method A; Rf:
0.35 (EtOAc/MeOH, 7:3 v/v); HRMS (ESIꢀ): calcd 820.2737 [M]ꢀ,
819.2665 [MꢀH]ꢀ, found 819.2665; Mr [Da]: 1006.11; C50H77N3O14P2.
2
70.2 (d, JCP =5.8 Hz, C-Bn), 68.0 (d, JCP =6.3 Hz, C-5’), 59.5 (pt, 4ꢂC-
A), 35.0 (2ꢂC-b), 33.0, 30.5, 30.4, 30.3, 30.1, 25.9, 24.7, 23.6, 20.6,
19.3 (2ꢂC-c, 2ꢂC-d, 2ꢂC-e, 2ꢂC-f, 2ꢂC-g, 2ꢂC-h, 2ꢂC-i, 4ꢂC-B,
4ꢂC-C), 14.4 (2ꢂC-j), 13.8 (4ꢂC-D), 12.5 ppm (Mehet); 31P NMR
(162 MHz, 1H-decoupled, CD3OD) d=ꢀ12.15 (d, 1P, 2JPP =20.6 Hz,
P-a), ꢀ12.93 ppm (d, 1P, 2JPP =20.5 Hz, P-b); IR: n˜ =2926, 2855,
1758, 1691, 1509, 1465, 1379, 1200, 1167, 1140, 1008, 981, 838,
505 cmꢀ1; UV (HPLC): lmax =265 nm, HPLC: tR =15.88 min, Method
A; Rf: 0.44 (EtOAc/MeOH, 7:3 v/v); HRMS (ESIꢀ): calcd 903.3604
[MꢀH]ꢀ, found 903.36035; Mr [Da]: 1146.37; C60H97N3O14P2.
(N[nBu]4)-Bis-(4-heptanoyloxybenzyl)-d4TDP 4d: General proce-
dure
C
with 190 mg bis-(tetra-n-butylammonium)-d4TMP
(0.348 mmol, 1 equiv), 251 mg bis-(4-heptanoyloxy-benzyl)N,N-dii-
sopropylphosphoramidite (0.417 mmol, 1.1 equiv) and 49.0 mg DCI
(0.418 mmol, 1.2 equiv) in 3 mL CH3CN. The reaction mixture was
stirred for 1 h and cooled to ꢀ258C followed by oxidation with
76 mL TBHP (0.42 mmol, 1.1 equiv). The crude product was purified
by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1 v/v, final-
ly MeOH). This first chromatography yielded 53 mg (0.049 mmol)
pure product. After a second chromatography (MeOH/H2O, 5:1 v/v)
another 96 mg (0.090 mmol) product were isolated. Yield: 149 mg
(N[nBu]4)-Bis-(4-dodecanoyloxybenzyl)-d4TDP 4g: General proce-
dure
C
with 153 mg bis-(tetra-n-butylammonium)-d4TMP
(0.280 mmol, 1 equiv), 228 mg bis-(4-dodecanoyloxybenzyl)N,N-dii-
sopropylphosphoramidite (0.319 mmol, 1.1 equiv) and 40.6 mg DCI
(0.344 mmol, 1.2 equiv) in 3 mL CH3CN. The reaction mixture was
stirred for 2 h and cooled to ꢀ258C followed by oxidation with
58 mL TBHP (0.32 mmol, 1.0 equiv). The crude product was purified
by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1 v/v).
Yield: 211 mg (0.175 mmol, 63%) colorless oil. 1H NMR (400 MHz,
1
(0.140 mmol, 40%) colorless syrup. H NMR (400 MHz, CD3OD) d=
4
7.65 (d, 1H, JHH =1.2 Hz, Hhet-6), 7.42–7.35 (m, 4H, H-2), 7.07–7.02
3/4
(m, 4H, H-3), 6.95 (ddd, 1H, 3JHH =3.5 Hz, 3/4JHH =1.9 Hz,
J =
HH
3
4
4
1.9 Hz, H-1’), 6.37 (ddd, 1H, JHH =6.5 Hz, JHH =1.9 Hz, JHH =1.9 Hz,
H-3’), 5.83 (ddd, 1H, JHH =6.2 Hz, JHH =1.3 Hz, 3/4JHH =2.3 Hz, H-2’),
3
4
2
3
4
5.01 (dd, 4H, JHH =8.5 Hz, JHP =6.2 Hz, H-Bn), 4.97–4.92 (m, 1H, H-
4’), 4.25–4.13 (m, 2H, H-5’), 3.25–3.17 (m, 8H, H-A), 2.57 (t, 4H,
3JHH =7.4 Hz, H-b), 1.88 (d, 3H, 4JHH =1.3 Hz, Mehet), 1.76–1.59 (m,
12H, H-B, H-c), 1.48–1.32 (m, 20H, H-C, H-d, H-e, H-f), 1.02 (t, 12H,
3JHH =7.3 Hz, H-D), 0.92 ppm (t, 6H, 3JHH =6.9 Hz, H-g); 13C NMR
CD3OD) d=7.65 (d, 1H, JHH =1.2 Hz, Hhet-6), 7.39–7.35 (m, 4H, H-
3/4
2), 7.06–7.02 (m, 4H, H-3), 6.95 (ddd, 1H, 3JHH =3.4 Hz,
J
=
=
=
HH
4
1.7 Hz, 3/4JHH =1.7 Hz, H-1’), 6.38 (ddd, 1H, 3JHH =6.0 Hz, JHH
3/4
1.7 Hz, 4JHH =1.7 Hz, H-3’), 5.84 (ddd, 1H, 3JHH =6.0 Hz,
J
HH
2.3 Hz, 4JHH =1.4 Hz, H-2’), 5.09 (dd, 4H, JHH =8.4 Hz, 3JHP =6.4 Hz,
2
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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H-Bn), 4.97–4.93 (m, 1H, H-4’), 4.25–4.15 (m, 2H, H-5’), 3.25–3.19
1.5 equiv). The crude product was purified by RP-18 flash chroma-
tography (MeOH/H2O, 1:1, than 5:1, than 6:1, finally 15:1 v/v).
Yield: 132 mg (0.100 mmol, 49%) colorless fluffy solid. 1H NMR
(400 MHz, CD3OD) d=7.66 (d, 1H, 4JHH =1.2 Hz, Hhet-6), 7.39–7.34
(m, 4H, H-2), 7.06–7.02 (m, 4H, H-3), 6.95 (ddd, 1H, 3JHH =3.4 Hz,
3/4JHH =1.7 Hz, 3/4JHH =1.7 Hz, H-1’), 6.38 (ddd, 1H, 3JHH =6.0 Hz,
4
(m, 8H, H-A), 2.57 (t, 4H, 3JHH =7.4 Hz, H-b), 1.89 (d, 3H, JHH
=
1.2 Hz, Mehet), 1.77–1.61 (m, 12H, H-c, H-B), 1.46–1.26 (m, 40H, H-d,
3
H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-C), 1.01 (t, 12H, JHH =7.4 Hz, H-
3
D), 0.89 ppm (t, 6H, JHH =6.9 Hz, H-l); 13C NMR (101 MHz, CD3OD)
d=173.6 (2ꢂC-a), 166.5 (Chet-4), 152.7 (Chet-2), 152.4 (2ꢂC-4), 138.5
3
4
4
3
4
(Chet-6), 135.3 (C-3’), 134.8 (d, JCP =5.4 Hz, 2ꢂC-1), 130.4 (d, JCP
=
4JHH =1.7 Hz, JHH =1.7 Hz, H-3’), 5.84 (ddd, 1H, JHH =5.9 Hz, JHH
=
2
3
2.7 Hz, 4ꢂC-2), 127.5 (C-2’), 122.8 (4ꢂC-3), 112.1 (Chet-5), 90.7 (C-1’),
1.3 Hz, 3/4JHH =2.2 Hz, H-2’), 5.08 (dd, 4H, JHH =8.5 Hz, JHP =6.4 Hz,
3
86.9 (d, JCP =9.3 Hz, C-4’), 70.2 (d, JCP =5.2 Hz, C-Bn), 70.2 (d, JCP
=
H-Bn), 4.98–4.92 (m, 1H, H-4’), 4.27–4.12 (m, 2H, H-5’), 3.27–3.18
2
4
5.2 Hz, C-Bn), 68.1 (d, JCP =6.3 Hz, C-5’), 59.5 (pt, 4ꢂC-A), 35.0 (2ꢂ
C-b), 33.1, 30.6, 30.5, 30.4, 30.4, 30.2, 25.9, 24.7, 23.4, 20.6 (2ꢂC-c,
2ꢂC-d, 2ꢂC-e, 2ꢂC-f, 2ꢂC-g, 2ꢂC-h, 2ꢂC-i, 2ꢂC-j, 2ꢂC-k, 4ꢂC-B,
4ꢂC-C), 14.4 (2ꢂC-l), 13.8 (4ꢂC-D), 12.5 ppm (Mehet); 31P NMR
(162 MHz, 1H-decoupled, CD3OD) d=ꢀ12.10 (d, 1P, 2JPP =20.4 Hz,
P-a), ꢀ12.94 ppm (d, 1P, 2JPP =20.9 Hz, P-b); IR: n˜ =2926, 2855,
(m, 8H, H-A), 2.56 (t, 4H, 3JHH =7.4 Hz, H-b), 1.89 (d, 3H, JHH
=
1.1 Hz, Mehet), 1.77–1.61 (m, 12H, H-c, H-B), 1.46–1.25 (m, 56H, H-d,
H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-n, H-o, H-C), 1.02 (t,
12H, 3JHH =7.5 Hz, H-D), 0.89 ppm (t, 6H, 3JHH =6.7 Hz, H-p);
13C NMR (101 MHz, CD3OD) d=173.6 (2ꢂC-a), 166.5 (Chet-4), 152.7
(Chet-2), 152.4 (2ꢂC-4), 138.6 (Chet-6), 135.3 (C-3’), 134.8 (2ꢂC-1),
130.4 (d, 4JCP =3.3 Hz, 4ꢂC-2), 127.5 (C-2’), 122.8 (4ꢂC-3), 112.1
1758, 1691, 1509, 1379, 1200, 1140, 1008, 981, 914, 838, 505 cmꢀ1
;
3
UV (HPLC): lmax =265 nm, HPLC: tR =17.76 min, Method A; Rf: 0.46
(EtOAc/MeOH, 7:3 v/v); HRMS (ESIꢀ): calcd 959.4224 [MꢀH]ꢀ,
found 959.42295; Mr [Da]: 1202.48; C64H105N3O14P2.
(Chet-5), 90.7 (C-1’), 86.9 (d, JCP =9.4 Hz, C-4’), 70.2 (d, JCP =5.6 Hz,
C-Bn), 70.2 (d, JCP =5.6 Hz, C-Bn), 68.0 (d, 2JCP =6.1 Hz, C-5’), 59.5
(pt, 4ꢂC-A), 35.0 (2ꢂC-b), 25.9 (2ꢂC-c), 24.7 (4ꢂC-B), 33.0, 30.7,
30.6, 30.6, 30.5, 30.4, 30.4, 30.2, 23.6, 20.6, 19.3 (2ꢂC-d, 2ꢂC-e, 2ꢂ
C-f, 2ꢂC-g, 2ꢂC-h, 2ꢂC-i, 2ꢂC-j, 2ꢂC-k, 2ꢂC-l, 2ꢂC-m, 2ꢂC-n, 2ꢂ
C-o, 4ꢂC-C), 14.4 (2ꢂC-p), 13.8 (4ꢂC-D), 12.4 ppm (Mehet); 31P NMR
(N[nBu]4)-Bis-(4-tetradecanoyloxybenzyl)-d4TDP 4h: General pro-
cedure
C
with 149 mg bis-(tetra-n-butylammonium)-d4TMP
1
2
(162 MHz, H-decoupled, CD3OD) d=ꢀ12.08 (d, 1P, JPP =20.07 Hz,
(0.189 mmol, 1 equiv), 242 mg bis-(4-tetradecanoyloxybenzyl)N,N-
diisopropylphosphoramidite (0.304 mmol, 1.5 equiv) and 35.8 mg
DCI (0.344 mmol, 1.2 equiv) in 3 mL CH3CN giving a suspension
due to the more or less insoluble amidite. The reaction mixture
was stirred for 91.5 h and cooled to ꢀ358C followed by oxidation
with 56 mL TBHP (0.31 mmol, 1.5 equiv). The crude product was pu-
rified by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1,
than 6:1, finally 8:1 v/v). Yield: 159.9 mg (154.6 mmol, 67%) color-
P-a), ꢀ12.92 ppm (d, 1P, 2JPP =20.21 Hz, P-b); IR: n˜ =2915, 2848,
1757, 1687, 1466, 1143, 979, 915, 878, 780, 764, 721, 574, 503 cmꢀ1
;
UV (HPLC): lmax =265 nm, HPLC: tR =20.54 min, Method A; Rf: 0.61
(EtOAc/MeOH, 7:3 v/v); HRMS (ESIꢀ): calcd 1072.555 [M]ꢀ,
1071.5478 [MꢀH]ꢀ, found 1071.5470 [MꢀH]ꢀ; Mr [Da]: 1314.69;
C72H121N3O14P2.
1
less fluffy solid. H NMR (400 MHz, CD3OD) d [ppm]=7.66 (d, 1H,
(N[nBu]4)-Bis-(4-(Z)-octadec-9-enoyloxybenzyl)-d4TDP 4j: General
4JHH =1.2 Hz, Hhet-6), 7.40–7.33 (m, 4H, H-2), 7.06–7.02 (m, 4H, H-3),
procedure
C with 144 mg bis-(tetra-n-butylammonium)-d4TMP
3
6.95 (ddd, 1H, JHH =3.5 Hz, 3/4JHH =1.8 Hz, 3/4JHH =1.8 Hz, H-1’), 6.38
(0.264 mmol, 1 equiv), 263 mg bis-(4-(Z)-octadec-9-enoyloxyben-
zyl)-N,N-diisopropylphosphoramidite (0.290 mmol, 1.1 equiv) and
37.1 mg DCI (0.314 mmol, 1.2 equiv) in 2 mL CH3CN and 2 mL di-
chloromethane. The reaction mixture was stirred for 16 h and
cooled to ꢀ308C followed by oxidation with 58 mL TBHP
(0.26 mmol, 1.0 equiv). The crude product was purified by RP-18
flash chromatography (MeOH/H2O, 1:1, than 10:1 v/v). Yield:
105 mg (76.0 mmol, 29%) colorless oil. 1H NMR (400 MHz, CD3OD)
3
4
4
(ddd, 1H, JHH =6.0 Hz, JHH =1.6 Hz, JHH =1.6 Hz, H-3’), 5.84 (ddd,
1H, 3JHH =5.9 Hz, 3/4JHH =2.2 Hz, 4JHH =1.3 Hz, H-2’), 5.08 (dd, 4H,
3
2JHH =8.5 Hz, JHP =6.5 Hz, H-Bn), 4.97–4.93 (m, 1H, H-4’), 4.25–4.13
3
(m, 2H, H-5’), 3.26–3.19 (m, 8H, H-A), 2.57 (t, 4H, JHH =7.4 Hz, H-b),
4
1.89 (d, 3H, JHH =1.2 Hz, Mehet), 1.77–1.61 (m, 12H, H-c, H-B), 1.46–
1.25 (m, 48H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-C),
1.02 (t, 12H, 3JHH =7.3 Hz, H-D), 0.89 (t, 6H, 3JHH =6.7 Hz, H-n);
13C NMR (126 MHz, CD3OD, CH2Cl2) d=173.7 (2ꢂC-a), 166.5 (Chet-4),
152.7 (Chet-2), 152.4 (2ꢂC-4), 138.5 (Chet-6), 135.3 (C-3’), 134.8 (2ꢂC-
4
d=7.65 (d, 1H, JHH =1.1 Hz, Hhet-6), 7.40–7.33 (m, 4H, H-2), 7.06–
7.00
(m,
4H,
H-3),
6.95
(ddd,
1H,
3JHH =3.4 Hz,
4
3/4JHH =1.7 Hz, 3/4JHH =1.7 Hz, H-1’), 6.38 (ddd, 1H, 3JHH =7.8 Hz,
1), 130.4 (d, JCP =4.3 Hz, 4ꢂC-2), 127.5 (C-2’), 122.8 (4ꢂC-3), 112.1
3
2
4JHH =1.8 Hz, JHH =1.8 Hz, H-3’), 5.84 (ddd, 1H, JHH =6.0 Hz,
J =
HH
4
3
3/4
(Chet-5), 90.7 (C-1’), 86.9 (d, JCP =9.3 Hz, C-4’), 70.2 (d, JCP =5.2 Hz,
C-Bn), 70.2 (d, 2JCP =5.2 Hz, C-Bn), 68.1 (d, 2JCP =6.3 Hz, C-5’), 59.5
(pt, 4ꢂC-A), 54.7 (CH2Cl2), 35.0 (2ꢂC-b), 25.9 (2ꢂC-c), 24.7 (4ꢂC-B),
33.1, 30.7, 30.7, 30.6, 30.5, 30.5, 30.4, 30.2, 23.6, 20.6, 19.3 (2ꢂC-d,
2ꢂC-e, 2ꢂC-f, 2ꢂC-g, 2ꢂC-h, 2ꢂC-i, 2ꢂC-j, 2ꢂC-k, 2ꢂC-l, 2ꢂC-m,
4ꢂC-C), 14.4 (2ꢂC-n), 13.8 (4ꢂC-D), 12.5 ppm (Mehet); 31P NMR
(162 MHz, 1H-decoupled, CD3OD) d=ꢀ12.09 (d, 1P, 2JPP =20.9 Hz,
P-a), ꢀ12.91 ppm (d, 1P, 2JPP =19.9 Hz, P-b); IR: n˜ =2916, 2849,
1756, 1687, 1508, 1466, 1252, 1142, 1111, 1003, 974, 914, 836,
503 cmꢀ1; UV (HPLC): lmax =265 nm, HPLC: tR =19.23 min, Method
A; Rf: 0.73 (EtOAc/MeOH, 7:3 v/v); MS (FAB): calcd 1016.48 [M],
1039.47 [M+Na]+, 1061.45 [MꢀH+ +2Na+] found 1039.5, 1061.5;
Mr [Da]: 1258.58; C68H113N3O14P2.
4
2.2 Hz, JHH =1.4 Hz, H-2’), 5.39–5.34 (m, 4H, H-i, H-j), 5.08 (dd, 4H,
2JHH =8.5 Hz, JHP =6.1 Hz, H-Bn), 4.97–4.93 (m, 1H, H-4’), 4.25–4.13
3
3
(m, 2H, H-5’), 3.25–3.18 (m, 8H, H-A), 2.56 (t, 4H, JHH =7.4 Hz, H-b),
2.13–1.98 (m, 8H, H-h, H-k), 1.89 (s, 3H, Mehet), 1.77–1.61 (m, 12H,
H-c, H-B), 1.47–1.25 (m, 48H, H-d, H-e, H-f, H-g, H-l, H-m, H-n, H-o,
H-p, H-q, H-C), 1.02 (t, 12H, 3JHH =7.3 Hz, H-D), 0.88 ppm (t, 6H,
3JHH =6.5 Hz, H-r); 13C NMR (101 MHz, CD3OD) d=173.6 (2ꢂC-a),
166.5 (Chet-4), 152.4 (Chet-2), 150.5 (2ꢂC-4), 138.6 (Chet-6), 135.3 (C-
3’), 130.9, 130.7 (2ꢂC-i, 2ꢂC-j), 130.4 (d, 4JCP =3.1 Hz, 4ꢂC-2),
129.6, 127.5 (C-2’), 122.8 (4ꢂC-3), 112.0 (Chet-5, hmbc), 90.7 (C-1’),
3
2
86.9 (d, JCP =9.4 Hz, C-4’), 70.2 (m, 2ꢂC-Bn), 68.7 (d, JCP =5.5 Hz,
C-5’), 59.5 (pt, 4ꢂC-A), 35.0 34.9 (2ꢂC-b), 33.0, 30.7, 30.7, 30.6,
30.5, 30.4, 30.4, 30.3, 30.3, 30.2, 28.1, 28.1, 25.9, 24.7, 23.4, 20.6 (2ꢂ
C-c, 2ꢂC-d, 2ꢂC-e, 2ꢂC-f, 2ꢂC-g, 2ꢂC-h, 2ꢂC-k, 2ꢂC-l, 2ꢂC-m,
2ꢂC-n, 2ꢂC-o, 2ꢂC-p, 2ꢂC-q, 4ꢂC-C, 4ꢂC-B), 14.4 (2ꢂC-r), 13.8
(4ꢂC-D), 12.4 ppm (Mehet); 31P NMR (162 MHz, 1H-decoupled,
(N[nBu]4)-Bis-(4-hexadecanoyloxybenzyl)-d4TDP 4i: General pro-
cedure
C
with 160 mg bis-(tetra-n-butylammonium)-d4TMP
(0.203 mmol, 1 equiv), 280 mg bis-(4-hexadecanoyloxy-benzyl)N,N-
diisopropylphosphoramidite (0.328 mmol, 1.6 equiv) and 50.4 mg
DCI (0.427 mmol, 2.1 equiv) in 3 mL CH3CN and 2 mL dichlorome-
thane. The reaction mixture was stirred for 116 h and cooled to
ꢀ308C followed by oxidation with 60 mL TBHP (0.31 mmol,
2
CD3OD) d=ꢀ12.06 (d, 1P, JPP =20.98 Hz, P-a), ꢀ12.91 ppm (d, 1P,
2JPP =20.24 Hz, P-b); 31P NMR (162 MHz, 1H-decoupled, CDCl3) d=
ꢀ11.73 ppm (s, 2P, P-a, P-b); IR: n˜ =2959, 2925, 2874, 2854, 1755,
1687, 1464, 1244, 1044, 503 cmꢀ1; UV (HPLC): lmax =265 nm; HPLC:
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 762 – 775 773

CHEMMEDCHEM
FULL PAPERS
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tR =19.86 min, Method A; Rf: 0.68 (EtOAc/MeOH, 7:3 v/v); HRMS
(ESIꢀ): calcd 1123.5795 [MꢀH]ꢀ, found 1123.5795 [MꢀH]ꢀ; Mr [Da]:
1366.76; C76H125N3O14P2.
liter and containing appropriate dilutions of the test compounds.
After 4–5 days of incubation at 378C in a CO2-controlled humidified
atmosphere, CEM giant (syncytium) cell formation was examined
microscopically. The EC50 (50% effective concentration) was defined
as the compound concentration required to inhibit HIV-induced
giant cell formation by 50%.
Bioassays
Chemical hydrolysis studies of DiPPro-d4TDP compounds 4b–j and
13: 50 mm DMSO stock solutions of the DiPPro-d4TDP compounds
were prepared; 300 mL of a 1.9 mm hydrolysis solution of the ap-
propriate compound were prepared by dilution of 11 mL of stock
solution in 100 mL Millipore water and 189 mL [D6]DMSO. The reac-
tion was started by addition of 300 mL of 50 mm phosphate buf-
fered saline (PBS, pH 6.8, 7.3, 8.7) at room temperature in order to
reach the final concentrations (0.94 mm of BAB-d4TDP 5, 24.8 mm
buffer salts). This kinetic solution was incubated at 378C. To moni-
tor the reaction, aliquots of 30 mL were taken from the kinetic solu-
tion and frozen (liquid nitrogen); 20 mL of these samples, including
a starting sample directly taken after addition of PBS, were ana-
lyzed by analytical HPLC at 263–265 nm. Calculation of exponential
decay curves with commercially available software (Microsoft
excel) yielded the half-life (t1/2) of the prodrug compounds in
hours. For each compound, two to three determinations of t1/2
were performed and the resulting values were averaged.
Supporting Information
HPLC hydrolysis studies of compounds 4 f, 4i, 5 f, and 13 as well
as an NMR study following the hydrolysis of DiPPro compound 4d.
Acknowledgements
C.M. and T.S. thank Sandra Mꢀhmel and Thiago Dinis de Oliveira
(University of Hamburg, Germany) for practical assistance, and
the Deutsche Forschungsgemeinschaft (DFG) and the University
of Hamburg for financial support of this work. J.B. is grateful to
Ria Van Berwaer (Rega Institute for Medical Research, Belgium)
for technical assistance. Financial support to J.B. was provided by
the KU Leuven (GOA 10/014).
Hydrolysis studies of DiPPro-d4TDP compounds 4b–j and 13 in CEM/
0, FCS, human serum, fetal calf serum and RPMI culture medium: Hy-
drolysis in CEM/0 cell extract: 30 mL of the appropriate 50 mm
DMSO stock solution was diluted to 6.0 mm hydrolysis solution by
addition of 220 mL DMSO. Human CEM/0 cell extract (100 mL, 33%
cell extract in PBS, pH 6.8) were mixed with 20 mL of a 70 mm
aqueous magnesium chloride solution. The reaction was started by
addition of 20 mL of 6.0 mm hydrolysis solution of the appropriate
compound 4 to the cell extract solution; 6–8 different samples of
the above mentioned mixture were incubated at 378C for different
periods of hydrolysis (depending on rate of hydrolysis). The reac-
tion was stopped by addition of 300 mL MeOH and the solutions
were kept on ice for 5 min followed by ultrasonification for 10–
15 min. After centrifugation the supernatant was filtered (Schleich-
er & Schuell Spartan 13/30, 0.2 mm) and stored in liquid nitrogen.
Samples were defrosted and 90 mL were subject of HPLC analysis.
The calculation of t(1/2) was performed analogously to that for the
chemical hydrolysis studies using the absolute integral values.
Identification of the hydrolysis products was based on the reten-
tion times of the reference compounds under identical analytical
conditions or by co-injection. Hydrolysis studies in FCS, human
serum (obtained from pooled blood samples from the University
Medical Center (UKE) in Hamburg), and RPMI culture medium were
done as described for the hydrolysis of compounds 4b--j and 13
in CEM/0. FCS (10%) and human serum (20%) were diluted in PBS
(pH 6.8).
Keywords: antivirals · bioreversible protection · nucleoside
diphosphates · prodrugs · pronucleotides
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Received: December 2, 2013
Published online on March 11, 2014
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 762 – 775 775