Ketobenzyl-Modified Nucleoside Triphosphate Prodrugs as Potential Antivirals

Article abstract of DOI:10.1021/acs.jmedchem.0c01293

The antiviral activity of nucleoside reverse transcriptase inhibitors is often hampered by insufficient phosphorylation. Nucleoside triphosphate analogues are presented, in which the ?-phosphate was covalently modified by a non-bioreversible, lipophilic 4-alkylketobenzyl moiety. Interestingly, primer extension assays using human immunodeficiency virus reverse transcriptase (HIV-RT) and three DNA-polymerases showed a high selectivity of these ?-modified nucleoside triphosphates to act as substrates for HIV-RT, while they proved to be nonsubstrates for DNA-polymerases α, β, and ?. In contrast to d4TTP, the ?-modified d4TTPs showed a high resistance toward dephosphorylation in cell extracts. A series of acyloxybenzyl-prodrugs of these ?-ketobenzyl nucleoside triphosphates was prepared. The aim was the intracellular delivery of a stable ?-modified nucleoside triphosphate to increase the selectivity of such compounds to act in infected versus noninfected cells. Delivery of ?-ketobenzyl-d4TTPs was proven in T-lymphocyte cell extracts. The prodrugs were potent inhibitors of HIV-1/2 in cultures of infected CEM/0 cells and more importantly in thymidine kinase-deficient CD4+ T-cells.

Full text of DOI:10.1021/acs.jmedchem.0c01293

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Article
γ‑Ketobenzyl-Modified Nucleoside Triphosphate Prodrugs as
Potential Antivirals
Tobias Nack, Thiago Dinis de Oliveira, Stefan Weber, Dominique Schols, Jan Balzarini,
and Chris Meier*
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ABSTRACT: The antiviral activity of nucleoside reverse tran-
scriptase inhibitors is often hampered by insufficient phosphor-
ylation. Nucleoside triphosphate analogues are presented, in which
the γ-phosphate was covalently modified by a non-bioreversible,
lipophilic 4-alkylketobenzyl moiety. Interestingly, primer extension
assays using human immunodeficiency virus reverse transcriptase
(HIV-RT) and three DNA-polymerases showed a high selectivity
of these γ-modified nucleoside triphosphates to act as substrates for
HIV-RT, while they proved to be nonsubstrates for DNA-polymerases α, β, and γ. In contrast to d4TTP, the γ-modified d4TTPs
showed a high resistance toward dephosphorylation in cell extracts. A series of acyloxybenzyl-prodrugs of these γ-ketobenzyl
nucleoside triphosphates was prepared. The aim was the intracellular delivery of a stable γ-modified nucleoside triphosphate to
increase the selectivity of such compounds to act in infected versus noninfected cells. Delivery of γ-ketobenzyl-d4TTPs was proven
in T-lymphocyte cell extracts. The prodrugs were potent inhibitors of HIV-1/2 in cultures of infected CEM/0 cells and more
importantly in thymidine kinase-deficient CD4+ T-cells.
marked selectivity of the corresponding nucleoside triphos-
phate to act as a substrate for a viral polymerase but not for
cellular polymerases. A lack of selectivity may lead to (toxic)
side effects, particularly if the mitochondrial DNA-polymerase
γ is involved.17,18
INTRODUCTION
■
Over the last decades a variety of nucleoside analogues were
applied in antitumor and antiviral therapy, and they still play
an important role to combat human immunodeficiency virus
(HIV), herpes virus, influenza, hepatitis B, hepatitis C, and
most recently SARS-CoV-2 virus infections.1,2 The targets of
these drugs are the virus-encoded DNA- or RNA polymerases,
such as HIV reverse transcriptase (HIV-RT)3,4 or the HCV-
encoded RNA-dependent RNA-polymerase NS5B.5 To date,
several nucleoside analogues have been approved as HIV
reverse transcriptase inhibitors (NRTIs),6 which are still used
as the backbone of the combined antiretroviral therapy.7
However, the antiviral efficacy of nucleoside analogues, such as
3′-deoxy-2′,3′-didehydrothymidine 1 (d4T), is dependent on
the in-vivo phosphorylation into their nucleoside analogue
triphosphate (NTP), for example, d4TTP 2, by host cell
kinases via the nucleoside mono- (3, NMP) and the
diphosphate (4, NDP, Scheme 1).8−10 The stepwise trans-
formation into the corresponding triphosphates often occurs
insufficiently because of the substrate specificity of the involved
kinases. Furthermore, many nucleoside analogues have
limitations such as poor biological half-lives because of
catabolic elimination, variable bioavailability after oral
administration, or selection of drug resistance, which reduce
their clinical efficacy.11,12 To overcome these hurdles the usage
of prodrugs of the phosphorylated metabolites have been
explored in the past.13−16 An important requirement for the
use of nucleoside analogues as antivirals is the need for a
In the case of d4T 1, the first phosphorylation step to yield
its monophosphate 3 catalyzed by the host cell enzyme
thymidine kinase (TK) is metabolism-limiting because of the
rather modest affinity of d4T 1 to TK.9,10,19 However, it is
impossible to use the charged phosphorylated metabolite
because of the high polarity and thereby extremely poor, if any,
membrane permeability. Additionally, phosphorylated metab-
olites are readily dephosphorylated by unspecific phospha-
tases/kinases. Prodrug strategies to circumvent these problems
have resulted in orally administered forms of some antiviral
nucleoside monophosphates or are currently under continuing
development.13−16 Examples of successful nucleoside mono-
phosphate prodrug strategies such as phosphoramidates 5 and
cycloSal-phosphate triesters 6 have been reported in the past
(Scheme 1).20−26 All these approaches deliver the
monophosphor(n)ylated forms of the corresponding nucleo-
Received: July 24, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01293
© XXXX American Chemical Society
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A

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Scheme 1. Metabolism of Nucleoside Analogues Such as d4T 1 and the Corresponding Nucleotide Prodrugs Thereof
Scheme 2. TriPPPro-Nucleoside Triphosphate Prodrugs and the Delivery Pathway
sides which, however, still need further phosphorylation into
the triphosphates by cellular kinases. Recently, we have
reported also on a first approach to deliver NDPs inside cells
using lipophilic but still partially charged NDP prodrugs
(DiPPro-approach 7; Scheme 1).27−31
that the corresponding TriPPPro-compounds showed pro-
nounced anti-HIV activity even in CEM/TK− cell cultures.
The parent nucleoside d4T 1 was virtually inactive in these
cells because of the lack of phosphorylation. The membrane
permeability was achieved by two 4-acyloxybenzyl (AB) esters
covalently attached at the γ-phosphate of the triphosphate
moiety. The enzyme-driven cleavage of the two masks by an
initial cleavage of the phenolic acyl ester and a subsequent
spontaneous cleavage of the remaining part of the mask led, for
example, to the formation of d4TTP 2 from TriPPPro-
compounds 8 (Scheme 2). The cellular uptake of these
TriPPPro-compounds was proven with fluorescent nucleoside
analogues.36
Further studies showed that the γ-dimasked TriPPPro-
compounds were not substrates for polymerases such as HIV-
RT or DNA-pol β. However, a surprising result was obtained
when the monomasked intermediates (Scheme 2) were studied
in primer extension assays as well as in antiviral assays. In
contrast to TriPPPro-compounds 8, the intermediates found to
be substrates for HIV-RT and d4TMP were incorporated into
the DNA primer strand.38 Although to a lesser extent as the
TriPPPro-derivatives, the monomasked intermediates bearing a
The development of NTP prodrugs is still highly desirable
because this would lead to the bypass of all steps of
phosphorylation and would in principle maximize the intra-
cellular concentration of the ultimately bioactive NTP
(Scheme 1).32 However, the challenges are not to be
underestimated: triphosphate is highly negatively charged
which requires appropriate masking, in comparison to only
two charges in a NMP analogue, and one has to cope with the
inherent instability of two phosphate anhydride bonds. It was
also claimed that the development of NTP-prodrugs is
chemically not feasible because of the low stability of such
compounds.33 For this reason, NTPs were very rarely used as
drug platforms because of their expected poor deliverability
and their high sensitivity for enzymatic dephosphorylation.34
Nevertheless, recently we disclosed the first delivery system of
NTPs through a prodrug technology (TriPPPro-approach 8;
Scheme 1).35−37 It was proven for several nucleoside analogues
B
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Scheme 3. Prodrug of γ-Alkylketobenzyl-d4TTPs 9 and the Demasked γ-Ketobenzyl-d4TTP Derivatives 10
Figure 1. Synthesis of alkylketobenzyl alcohol 13 and γ-(AB,kb)-d4TTPs 9. Reagents and conditions: (a) i-PrMgCl·LiCl, THF/dioxane, rt, 16 h;
+
(b) Me(OMe)N·HCl, NEt3, CH2Cl2, rt, 2 h; (c) THF, 0 °C, 2 h; (d) TBAF, THF, rt, 1.5 h; (e) DCI, CH3CN, rt, 1 h; and (f) (1) (n-Bu4N)2 ·
d4TDP 4, DCI, CH3CN, rt, 2 h, and (2) t-BuOOH, CH3CN, rt, 30 min.
lipophilic C17-AB-moiety proved to be antivirally active in the
cell assay using TK-deficient CEM cells (EC50 = 1.55 μM,
unpublished results). Although the test compounds were
vigorously purified to exclude even traces of unmasked d4TTP,
it could not be excluded that the second enzyme-cleavable AB
group was cleaved at least in part while performing the antiviral
assay. This was the motivation to develop a new series of
nucleoside triphosphate derivatives comprising a noncleavable
lipophilic moiety at the γ-phosphate group of the NTP. The
ester functionality is an acceptor substituent in the benzyl
group and also the cleavage site for the (carboxy)esterase
(Scheme 2). Therefore, we replaced one of the bioreversible
masking groups by a non-bioreversible γ-alkylketobenzyl group
(kb-modified NTP prodrugs 9). The second benzyl ester at the
γ-phosphate of 9 was still a bioreversible AB group as in the
original TriPPPro-compounds. Thus, the enzymatic cleavage of
this AB-group should lead to γ-ketobenzyl-NTPs 10 (Scheme
3).
primer extension assays were conducted with compounds 10.
As a nucleoside analogue, d4T 1 was used to enable a
comparison with the TriPPPro-compounds 8. Compounds 8,
comprising two enzyme-cleavable γ-modifications, were de-
signed to deliver entirely unmasked NTPs (Scheme 2).
RESULTS
■
Chemistry. Synthesis of AB Prodrugs of γ-Alkylketo-
benzyl-d4TTPs 9. For the synthesis of the title compounds γ-
(AB,kb)-d4TTPs 9, an analogous procudure to our previously
reported convergent synthesis using phosphoramites was used
here as well. Thus, a DCI-mediated coupling of a nonsym-
metrically substituted γ-(Ab,kb)-phosphoramidite 11 and
d4TDP 4 led to compounds 9 in 34−63% yield (Figure 1,
step f).31 The synthesis route of (AB)-containing diamidites 12
was adapted from a previously described protocol.39 The
alkylketobenzyl groups 13a,b were synthesized involving the
coupling of the Weinreb amide 14 and a Grignard reagent 15
in good yields (65−70%).40 The latter was synthesized starting
from OTBDMS-protected 4-bromo-benzylalcohol 16 using an
improved version of the “turbo Grignard reagent” with dioxane
as an additive to ensure a relatively fast and quantitative
exchange.41 The one-step synthesis of Weinreb amides 14 was
adapted from a literature-known procedure in almost
We report the synthesis of these new nucleoside
triphosphate prodrugs 9 bearing a stable γ-phosphate
modification in combination with a bioreversible AB-group
at the same phosphate moiety as well as their hydrolysis
products 10, their hydrolysis properties in different media and
their anti-HIV activity will be reported as well. In addition,
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Figure 2. Synthesis of γ-kb-modified-NTPs 10 using two routes (d4T as an example). Reagents and conditions: (a) (FmO)P(N(i-Pr)2)2, THF, 0
°C, 2 h; (b) (1) (n-Bu4N)+ ·NDP, DCI, CH3CN, rt, 2 h, (2) t-BuOOH, CH3CN, rt, 30 min, and (3) NEt3, CH3CN, rt, 15−70 min; and (c) PLE,
2
H2O/phosphate buffer, 50 mM pH = 7.3, 11 h.
quantitative yields.42 TBAF-mediated deprotection led to
Table 1. Half-Lives of (γ-AB)-γ-ketobenzyl-d4TTP-
benzyl alcohols 13 in very high yields, which were then
converted into (AB,kb)-phosphoramidites 11 in a DCI-
mediated coupling in yields ranging from 37% to almost
quantitative.
Synthesis of γ-kb-Modified-NTPs 10. As concluded from
previous studies, we expected a suitable access to γ-kb-
modified-d4TTP 10a starting from γ-(AB,kb)-d4TTPs 9ax by
treatment with pig liver esterase (PLE).31 Surprisingly, the
reaction proceeded very slowly, using a scale of 5 mg of γ-
(AB,kb)-d4TTP 9ax, and required the successive addition of
the enzyme to ensure complete transformation (Figure 2, step
c).
prodrugs 9, γ-Ketobenzyl-NTPs 10, and Two TriPPPro-
NTPs 8 in Different Medium
CEM/0 cell
a
PB, pH = 7.3
t1/2
extracts
comp. nucl.
R1
R2
h
t1/2 h
9ax
9az
9by
9bz
10a
10b
10c
10d
d4T
d4T
d4T
d4T
d4T
d4T
T
dA
d4T
d4T
C4H9
C9H19
C9H19
C17H35
C17H35
C9H19
C17H35
C9H19
C9H19
C8H17
C4H9
43
94
109
11
no hydrolysis
no hydrolysis
no hydrolysis
no hydrolysis
52
1.1
7.8
6.9
2.1
>30
>30
>30
n.d.
2.5
3.3
C17H35
C6H13
C17H35
Therefore, a synthetic access using O-fluorenylmethyl (Fm)-
protected phosphoramidite chemistry was developed.43 First,
we synthesized nonsymmetric (kb,Fm)-phosphoramidites 17
in a DCI-mediated coupling of Fm-phosphodiamidites and
benzyl alcohols 13 in up to quantitative yields. As impurities,
phosphoramidite (FmO)2PN(iPr)2, which was formed in the
synthesis of the (FmO)P(N(i-Pr)2)2 reagent and was very
difficult to separate during purification, and dibenzofulvene
were observed. However, both impurities were removed easily
after the next reaction. The final reaction started analogous as
above with a DCI-mediated coupling of phosphoramidites 17
with a NDP such as d4TDP 4, followed by oxidation. Finally,
the Fm-group was cleaved using NEt3 (10% v/v) in CH3CN.
In some cases, for example, for (Fm,kb-C9H19)-TTP, some of
the starting material was re-isolated after RP18 flash column
chromatography and was again treated using the same cleavage
conditions to obtain γ-ketobenzyl-modified-NTPs such as 10
in a yield of about 60−70% (Figure 2, steps a,b).
b
c
8a
C8H17
C17H35
c
8b
50
a
The hydrolysis experiments of γ-modified-NTP 9, 10 were
conducted in aqueous 25 mM phosphate buffer (PB, pH = 7.3).
The hydrolysis products were detected by analytical rp18 HPLC.
b
c
n.d.: not determined. Compounds 8 comprise two AB groups and
the chemical structures are shown in Scheme 2.
between 11 and 109 h without showing a clear trend and
surprisingly decreased for the most lipophilic compound 9bz
(11 h, Table 1). The initial cleavage step in the hydrolysis
mechanism proceeded similarly to the previously published
cleavage pathway for TriPPPro-NTPs 8. As the hydrolysis
product starting from the γ-modified-d4TTP prodrugs 9, a
predominate formation of γ-kb-d4TTPs 10 and a very small
amount of d4TDP 4 was detected. After consumption of the
starting material, no further increase of the amount of d4TDP
was observed. This supports again the hypothesis that only the
γ-modified d4TTP prodrugs 9 were prone to a breakage
between the γ- and β-phosphate. More importantly, and in
contrast to the studies using TriPPPro-NTPs 8 containing a
second cleavable mask (first generation NTP prodrugs),35−37
no d4TTP was detected in these studies starting from
compounds 9. The formed γ-kb-d4TTPs 10 proved to be
entirely stable at pH 7.3 (Table 1). Because of the very long
hydrolysis time periods, also a cleavage of the glycosidic bond
in d4T occurred as proven by the appearance of the nucleobase
thymine. This has been observed before.35 The amount of
thymine doubles every 63 h; however, this aspect has not been
further considered in these investigations because it was
irrelevant in the enzyme or cell extract incubations because of
the much shorter hydrolysis periods and in the case of other
Stability Studies. γ-Modified-d4TTP prodrugs 9 and γ-kb-
modified NTPs 10 were incubated in phosphate buffer (PB, 25
+
mM, pH 7.3), exposed to PLE in PB or to human CD4 T-
lymphocyte cell extracts to study their stability and the product
distribution. The hydrolysis mixtures were analyzed by means
of analytical RP18-HPLC. The calculated half-lives of prodrugs
9 (Table 1, t1/2) reflect the removal of the bioreversible AB-
group to yield γ-kb-d4TTPs 10. In general, three reactions
should be considered in the hydrolysis pathways of nucleoside
triphosphate prodrugs 9. First, the designated pathway that
should lead to γ-ketobenzyl-nucleoside triphosphate. Second, a
reaction involving a nucleophilic reaction at the γ-phosphate to
give d4TDP 4. Optionally, a nucleophilic reaction at the β-
phosphate would lead to d4TMP 3.
Chemical Stability in Phosphate Buffer, pH 7.3. In PB,
the half-lives of prodrugs of γ-kb-d4TTPs 9 were found to be
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nucleoside analogues. The γ-ketobenzyl analogues of thymi-
dine triphosphate (10c) and 2′-deoxyadenosine triphosphate
(10d) were used to prove this effect. No cleavage of the
glycosidic bond occurred in these cases.
As an example, the hydrolysis of compound 9az is
summarized in Figure 3. The hydrolysis was followed over a
Figure 4. HPLC profile of γ-(AB-C17,kb-C9)-d4TTP 9az after
incubation in CEM/0 cell extracts for different time points. “MeOH”
highlights that MeOH was used for the sample workup after
incubation instead of dioxane. * Buffer impurity ** CEM/0
components.
short alkyl ester proved extremely unstable in cell extracts (t1/2
of 1.1 h, respectively). However, the difference to compounds
bearing long lipophilic acyl groups was not very pronounced (2
to 3-fold). In all cases, the γ-kb-d4TTPs 10 were detected as
predominate products. This was in sharp difference to the
studies performed with the TriPPPro-compounds 8.35 There, it
was almost impossible to detect marked amounts of d4TTP 2
because of its fast dephosphorylation in the extracts to form
first d4TDP 4 and finally d4TMP 3 (half-life of d4TTP in cell
extracts: 38 min).
More interestingly, even after very long incubation periods
(up to 98 h), compounds 10a−c proved to be entirely stable,
for example, 98% of the thymidine-bearing compound 10c still
was detectable. The lower values of 89% for the d4T
comprising compounds 10a,b ((kb-C9)-d4TTP and (kb-
C17-d4TTP)) after 98 h of incubation can be explained by
the above-mentioned side reaction with the formation of
thymine (8%) (Figures 5 and 6) and no formation of neither
d4TDP nor d4TTP was detected after 96 h incubation of γ-
ketobenzyl-d4TTPs 10a,b.
Figure 3. Chemical hydrolysis study of γ-(AB-C17,kb-C9)-d4TTP
9az in PB (pH 7.3).
time period of 800 h. Clearly, the starting material disappeared
and the expected γ-kb-d4TTP 10a was formed as well as low
amounts of d4TDP and thymine.
Hydrolysis Using PLE. Next, we examined the enzymatic
stability of prodrugs 9 by incubation with PLE in phosphate
buffer, pH 7.3. The cleavage of the prodrug moieties in 9 led to
the selective formation of γ-ketobenzyl-d4TTPs 10 and
occurred much faster than in PB (t1/2 ∼10 min to 2 h; data
not shown). This was in full agreement with the results from
the studies of TriPPPro-compounds 8 described before and
proved a significant contribution of the enzymatic cleavage.
Moreover, in these enzymatic hydrolysis studies, no cleavage of
the ketobenzyl residue in compounds 9 or 10 was observed.
This proved our initial concept of introducing an esterase-
stable group to the γ-phosphate unit.
Incubation of the Prodrugs of the γ-kb-Modified
d4TTPs 9,10 in Cell Extracts, Comparison with the
TriPPPro-NTPs 8. γ-(AB,kb)-NTPs 9 and γ-kb-NTPs 10 were
further investigated in human CD4+ T-lymphocyte CEM cell
extracts. Half-lives as low as 1.1 h (9ax) to 7.8 h (9az) were
determined, which clearly pointed to an enzymatically
catalyzed cleavage (Table 1). The prodrug 9ax comprising
the shortest acyl group in the prodrug moiety was found to be
the most quickly cleaved compound. As in the chemical
hydrolysis, compound 9bz was surprisingly unstable here as
well (2.1 h, Table 1). In all cases, we observed the predominate
formation of γ-modified NTPs 10, d4TDP 4 was observed in
very low concentrations but no NTP formation was detected in
contrast to the studies using the previously described (AB)2-
TriPPPro-compounds 8; for example, compound 9az showing
a t1/2 of 7.8 h was converted within 48 h into 70% γ-(kb-C9)-
d4TTP and <10% d4TDP (Figure 4). Here, compound 9ax
comprising a pentanoyl ester moiety was found to be the least
stable compound. This is in accordance to our previous results
of the DiPPro- (7) or the TriPPPro-compounds 8. Particularly,
Figure 5. HPLC profile of γ-kb-C9-d4TTP 10a after incubation in
CEM/0 cell extracts at different time points. * Buffer impurity **
CEM/0 components.
Interestingly, although the reported compounds were not
sufficiently characterized and proved inactive in antiviral tests,
methyl or phenyl groups introduced at the γ-phosphate of
nucleoside triphosphates or the replacement of the γ-
phosphate group by a methyl phosphonate moiety led to an
increase in stability of the triphosphate unit in the human
serum (about a 10-fold).44−46 In our case the modification
with a ketobenzyl group led to a very stable compound in
CEM-cell extracts.
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wild-type CEM cells as compared to the other prodrugs, most
probably because of poorer lipophilicity. It should also be
noted that none of the prodrugs 9 were endowed with a
significantly higher cytotoxicity (2-3-fold higher as compared
to d4T 1) than the parent d4T 1 (CC50 = 79 μM; Table 2).
Interestingly, also the γ-kb-modified (10b) was active in CEM/
TK− cells (0.26 μM), which was even better than for the
intermediates formed from the original TriPPPro-compounds,
comprising a C17 aliphatic chain (antiviral activity in the TK-
deficient CEM cells: 1.55 μM). Thus, it may be concluded that
even one lipophilic aliphatic chain provides sufficient lip-
ophilicity to enable at least in some extent a cellular uptake of
the compounds. As expected, compounds 10c and 10d
comprising the natural nucleosides thymidine (T) and 2′-
deoxyadenosine (dA) were found to be inactive. Surprisingly,
intermediate 10c of the natural thymidine showed the highest
toxicity of all tested compounds. In contrast, intermediate 10d
bearing the natural nucleoside dA was found to be 7-fold less
toxic for unknown reasons.
Figure 6. HPLC profile of γ-kb-C9-TTP 10c after incubation in
CEM/0 cell extracts at different time points. * Buffer impurity, **
CEM/0 components.
Antiviral Evaluation. Prodrugs of γ-ketobenzyl-d4TTPs 9
and the γ-ketobenzyl-d4TTPs 10 were evaluated for their
ability to inhibit the replication of HIV. For this purpose, HIV-
1- or HIV-2-infected wild-type CEM/0 as well as mutant TK-
deficient CEM cell cultures (CEM/TK−) were treated with
compounds 9 and 10. For comparison also, two TriPPPro-
derivatives 8 were also included in this series. The results are
summarized in Table 2. As can be seen in Table 2, all
The two TriPPPro-compounds bearing either two C8 chains
(8a) or a mixture of a short C4 and a long C17 alkyl group
(8b) were active in the TK-deficient CEM cells as well. Again,
the compound bearing the C17 chain proved to be more active
as compared to the second one (C8 chain).
Primer Extension Assays. In the antiviral assays, all
compounds described here showed significant antiviral activity
against HIV-1- and HIV-2-infected CEM cells. This activity
was also mainly retained in CEM/TK-deficient cells. Taking
the results from the hydrolysis studies, it can be concluded that
the delivered γ-ketobenzyl-d4TTPs (10) are responsible for
the inhibitory effect because we have proven that no d4TTP
was formed in all the hydrolysis studies. This is a marked
difference to the formerly reported TriPPPro-compounds 8.
To shed more light into these results, primer extension assays
were performed investigating the substrate properties of the γ-
ketobenzyl triphosphates to four different DNA-polymerases:
reverse transcriptase (HIV-RT, a RNA-dependent-DNA-
polymerase), DNA-polymerase α, β, and γ (a mitochondrial
polymerase and often responsible for toxic side effects caused
by NTPs).17,18 It should be added that previously also the
replacement of the γ-phosphate group by a methyl or a phenyl
phosphonate moiety had almost no effect on the substrate
properties toward retroviral RT but abolished their ability to
function toward DNA polymerase α and β.45,46 In contrast to
the compounds reported here, those compounds proved to be
antivirally inactive and were not studied in combination with a
prodrug approach.
Two different setups have been used using HIV-RT. First,
dATP, dCTP, dGTP, and γ-ketobenzyl-TTP or -d4TTP was
used as a mixture (N*). The results obtained from these
experiments were compared to those in which just the T* (γ-
ketobenzyl-TTP or d4TTP; single nucleotide incorporation)
was used. In each experiment, a primer extension without
added polymerase [negative control (−lane)] and an experi-
ment, in which all four canonical NTP were added [positive
control (+lane)], were used as controls.
Studies Using γ-Ketobenzyl-TTP 10c and HIV-RT and
Cellular DNA Polymerases. First, as a proof of concept, γ-
ketobenzyl-TTP 10c was studied for its substrate properties for
both the viral and human DNA polymerases.
Table 2. Antiviral Activity and Cytotoxicity of γ-(AB,kb)-
d4TTP Prodrugs 9 and γ-Ketobenzyl-NTPs 10 in
Comparison to TriPPPro-d4TTPs 8 and the Parent d4T 1
a
EC50 [μM]
CC50
b
CEM/0
CEM/TK−
HIV-2
[μM]
comp.
HIV-1
HIV-2
CEM/0
9ax
9az
9by
9bz
10b
10c
10d
0.37 0.24
0.58 0.32
0.44 0.28
0.42 0.18
0.33 0.11
6.6 1.5
1.2 0.92
0.78 0.51
1.1 0.83
0.83 0.43
0.60 0.00
8.8 1.7
>10
2.5 1.8
1.1 0.75
0.40 0.15
0.38 0.19
0.26 0.20
>10
25
35
34 12
28
13
10
3
6
2
1
1
10
>10
74 12
bis(AB)-
0.31 0.01
0.62 0.30
2.26 1.03
52
1
7
3
d4TTP 8a
bis(AB)-
d4TTP 8b
d4T 1
d4TTP
0.12 0.05
0.10 0.03
0.54 0.41
33
0.43 0.25
1.05 0.35
0.22 0.05
0.40 0.26
150
>100
79
>100
a
Antiviral activity in CD4+ T-lymphocytes: 50% effective concen-
tration; values are the mean
SD of n = 2−3 independent
b
experiments. Cytotoxicity: 50% cytostatic concentration or com-
pound concentration required to inhibit CD4+ T-cell (CEM)
proliferation by 50%; values are the mean
independent experiments.
SD of n = 2−3
compounds showed virtually similar or even slightly better
activities against HIV-1 and HIV-2 as the parent d4T 1.
Because the lipophilicity of compounds 9 is similar because of
the combination of the introduced aliphatic alkyl groups in the
masking unit as well as in the stable γ-modification, the
antiviral activity did not differ much. In addition, and more
importantly, almost all prodrugs 9 were also highly potent in
CEM/TK− cells, whereas d4T 1 lacked any relevant anti-HIV
activity in this TK-deficient cell model (EC50: 150 μM). Only
prodrug 9ax (C4/C9) lost some of the activity found in the
Figure 7 shows the autoradiogram of a primer extension
assay in which the viral enzyme HIV-RT was used. The 25mer
primer has to be fully elongated by the polymerase into a
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Figure 7. Primer extension assay using HIV-RT and the γ-ketobenzyl-
TTP 10c. Lane 1 (−): dATP, dCTP, dGTP, and dTTP without HIV-
RT; lane 2 (+): dATP, dCTP, dGTP, and dTTP with HIV-RT; lane 3
(N*): γ-ketobenzyl-TTP 10c, dATP, dCTP, and dGTP; lane 4: γ-
ketobenzyl-TTP10c. Assay conditions are summarized in the
Experimental Section.
30mer by the addition of the sequence TCTGT. As expected,
without HIV-RT no elongation proceeded (−lane). However,
with HIV-RT and the mixture of all four canonical
triphosphates, full extension to the 30mer occurred (+lane).
In the case of the mixture labeled “N*” (dCTP, dATP, dGTP,
and γ-ketobenzyl-TTP 10c), again a full extension of the
primer to n + 5 (30 nt) was observed. This complete strand
extension implies that HIV-RT recognizes the γ-modified TTP
10c as a substrate and inserts TMP three times efficiently into
the growing DNA strand, without showing marked stalls in
between. In full agreement with this experiment, a single
incorporation of TMP starting from the γ-ketobenzyl-TTP 10c
was observed in the last experiment, and thus, γ-ketobenzyl-
TTP 10c was efficiently used as the substrate by HIV-RT.
Next, the substrate properties of compound 10c were
studied using human DNA polymerases, β, γ (Figure 8), and α
(Figure 11). Autoradiogram A shows the results using human
DNA polymerase β and the γ-ketobenzyl-TTP 10c compared
to the canonical TTP. Obviously TTP showed as expected the
incorporation of TMP, which led to the presence of the n + 1
band (26mer, TTP lane; A). However, when γ-ketobenzyl-
TTP 10c was used, no incorporation was detectable. Identical
results were obtained using human DNA polymerase γ
(fluorescently labeled primer; Figure 8B) and human DNA
polymerase α (fluorescently labeled primer; Figure 11).
Thus, in contrast to HIV-RT, the three human DNA
polymerases did not accept γ-ketobenzyl-TTP 10c as a
substrate.
Figure 8. Primer extension assay using γ-ketobenzyl-TTP 10c and
DNA pol β (A) and DNA pol γ (B). (A): Lane 1 (−): dATP, dCTP,
dGTP, and dTTP without DNA pol β; lane 2 (+): dATP, dCTP,
dGTP, and dTTP with DNA pol β; lane 3: TTP; lane 4: γ-ketobenzyl-
TTP 10c. (B): lane 1 (−): dATP, dCTP, dGTP, and dTTP without
pol γ; lane 2 (+): dATP, dCTP, dGTP, and dTTP with pol γ; lane 3:
TTP; lane 4: γ-ketobenzyl-TTP 10c. Assay conditions are
summarized in the Experimental Section.
Figure 9. Primer extension assay using HIV-RT, d4TTP 2, and the γ-
ketobenzyl-d4TTPs 10a,b. Lane 1 (−): dATP, dCTP, dGTP, and
dTTP without HIV-RT; lane 2 (+): dATP, dCTP, dGTP, and dTTP
with HIV-RT; lane 3: γ-ketobenzyl-d4TTP 10a; lane 4: γ-ketobenzyl-
d4TTP 10b; lane 5: d4TTP. Assay conditions are summarized in the
Experimental Section.
Studies Using the γ-Ketobenzyl-d4TTPs 10a,b and
HIV-RT or Cellular DNA Polymerase β. Next, γ-ketobenzyl-
d4TTPs 10a,b were used with HIV-RT and DNA-polymerase
β. The results are shown in Figure 9 (HIV-RT) and Figure 10
(human DNA-polymerases β).
As shown in Figure 9, d4TTP was a substrate for HIV-RT as
expected. In all cases, the n + 1 (26 nt) band was detected,
showing d4TTP being a substrate for HIV-RT, even when
modified in the γ-phosphate position.
Cellular DNA-polymerase β was incubated in these primer
extension assays with d4TTP 2 as well as the γ-ketobenzyl-
d4TTPs 10a,b to compare the elongation properties of these
three triphosphates. The polymerase assay using a fluorescently
labeled primer (Figure 10) showed that d4TTP 2 was
surprisingly incorporated into the primer strand forming the
26mer.
Figure 10 also shows the results of an identical experiment
but using γ-kb-d4TTP 10a and 10b. In contrast to d4TTP 2,
no insertion was detected for kb-d4TTP 10b, but eventually, a
small incorporation for kb-d4TTP 10a was observed. This
implies that human DNA polymerase β did not recognize the γ-
kb-d4TTP 10b as a substrate and 10a as a poor substrate only.
Consequently, the specificity of the enzyme toward the two
d4TTP derivatives was enhanced in favor of the γ-modified
compound with longer alkyl chains. Thus, a marked difference
in the substrate properties of these TTP analogues has been
observed for a viral versus a cellular polymerase.
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substrates as no incorporation to form the n + 1 26mer was
detected.
CONCLUSIONS
■
In summary, here, we disclosed a second generation of
lipophilic nucleoside triphosphate prodrugs which differ from
the previously described TriPPPro-derivatives in that they
comprise a noncleavable moiety in addition to a biodegradable
prodrug moiety at the γ-phosphate group. An alkylketobenzyl
group was attached in addition to the previously used
bioreversible AB-prodrug group. The synthesis was achieved
in good chemical yields using phosphoramidite chemistries.
We have proven that the prodrug group was selectively cleaved
to give γ-modified nucleoside triphosphates 10 by chemical
hydrolysis (slow process) or by enzymes present in cell extracts
(fast process). The γ-ketobenzyl-nucleoside triphosphates 10
proved to be stable in cell extracts, while the corresponding
nucleoside triphosphates d4TTP or TTP were rapidly
enzymatically dephosphorylated. All compounds showed
marked antiviral activity against HIV-2 in TK deficient cells
(CEM/TK− cells), indicating a successful cell membrane
passage of these compounds and subsequent intracellular
enzymatic delivery of γ-ketobenzyl-d4TTPs, while the parent
nucleoside d4T 1 lacked activity in this cell assay. Thus,
although these second generation nucleoside triphosphate
prodrug compounds are still charged at the phosphate groups,
obviously the introduction of a modification at the γ-phosphate
group by one lipophilic, bioreversible moiety, and the stable γ-
ketobenzyl group gives the molecule sufficient lipophilicity to
penetrate the cell membrane. Most interestingly, the γ-kb-
nucleoside triphosphates showed significantly different sub-
strate properties when incubated with HIV-RT or cellular
DNA polymerases α, β, or γ in primer extension assays. γ-Kb-
TTP 10c was a substrate for HIV-RT, while it proved to be no
substrate for the used three cellular DNA polymerases. The
corresponding γ-kb-d4TTP derivatives were also found to be
substrates for HIV-RT but were still no substrates for all
studied DNA polymerases. Thus, these results point to a
marked increased selectivity of the compounds to different
polymerases with a significant preference to the viral HIV-RT.
Interestingly, the observed higher selectivity between HIV-RT
and the three DNA polymerases studied here did not translate
into lower cytotoxicity of the compounds as compared to the
parent itself. Thus, there may be other reasons for the observed
cytotoxicity than the interaction with the different polymerases,
which is nevertheless an important property of the disclosed
compounds. We are currently working on the question if this
increased selectivity toward the viral enzyme is a general
property of these γ-kb compounds by using different
nucleoside analogues and different viral polymerases. The
second improvement over the first generation TriPPPro
derivatives is that the compounds described here deliver
metabolically stable nucleoside triphosphate derivatives, which
showed no dephosphorylation for at least 24 h. This should
lead to a significant increase in bioactive metabolites inside
cells.
Figure 10. Primer extension assay using DNA pol β, d4TTP 2, and γ-
ketobenzyl-d4TTPs 10a,b. Lane 1 (−): dATP, dCTP, dGTP, and
dTTP without DNA pol β; lane 2 (+): dATP, dCTP, dGTP, and
dTTP with DNA pol β; lane 3: γ-ketobenzyl-d4TTP 10a; lane 4: γ-
ketobenzyl-d4TTP 10b; lane 5: d4TTP. Assay conditions are
summarized in the Experimental Section.
Studies Using the γ-Ketobenzyl-d4TTPs 10a,b and
Cellular DNA Polymerase α or Mitochondrial DNA
Polymerase γ. Human DNA-polymerase α (Figure 11) and
Figure 11. Primer extension assay using DNA pol α and d4TTP 2, γ-
ketobenzyl-d4TTPs 10a,b as well as TTP and γ-ketobenzyl-TTP 10c.
Lane 1 (−): dATP, dCTP, dGTP, and dTTP without DNA pol α;
lane 2 (+):dATP, dCTP, dGTP, and dTTP with DNA pol α; lane 3:
γ-ketobenzyl-d4TTP 10a; lane 4: γ-ketobenzyl-d4TTP 10b; lane 5:
d4TTP 2; lane 6: TTP; lane 7: γ-ketobenzyl-dTTP 10c. Assay
conditions are summarized in the Experimental Section.
human polymerase γ (Figure 12) were incubated in these
primer extension assays with d4TTP as well as the γ-
ketobenzyl-d4TTPs 10a,b to compare the elongation proper-
ties of these three triphosphates. For both polymerases, it was
shown that d4TTP 2 and derivatives 10a and 10b were not
Even to a higher extent, as the TriPPPro approach, this novel
concept seems to be very interesting for application with
nucleoside analogues that show severe limitations in their
activation to give the corresponding nucleoside triphosphates
and/or have a lack of selectivity with respect to the cellular
polymerases at the triphosphate level. Moreover, we are
Figure 12. Primer extension assay using DNA pol γ, d4TTP 2, as well
as γ-ketobenzyl-d4TTPs 10a and 10b. Lane 1 (−): dATP, dCTP,
dGTP, and dTTP without DNA pol γ; lane 2 (+): dATP, dCTP,
dGTP, and dTTP with DNA pol γ; lane 3: γ-ketobenzyl-d4TTP 10a;
lane 4: γ-ketobenzyl-d4TTP 10b; lane 5: d4TTP. Assay conditions
are summarized in the Experimental Section.
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measurements (matrix: 9-aminoacridine [9AA] or 2,5-dihydroxyben-
zoic acid [DHB]) were performed with a Bruker UltrafleXtreme
spectrometer. Infrared spectroscopy (IR): IR spectra were recorded on
a Bruker ALPHA P FT-IR at room temperature in the range of 400−
4000 cm−1. Freeze dryer: ALPHA 2-4 LDPlus freeze dryer from Christ
Co. and a chemistry hydrid pump RC6 from Vacuubrand Co. was
used. Ultrapure water: Arium pro ultrapure water system was used. pH
meter: pH value was tested with a ProLab 300 from Schott Co.
Thermomixer: Eppendorf Thermomixer 5436 and CellMedia
convinced that this approach is not limited to HIV treatment
but can also be used for other viral targets.
Because this second concept should also be generally
applicable to natural nucleosides and a broad variety of
nucleoside analogues, an improved novel option to deliver the
corresponding bioactive triphosphate derivatives without any
need for further enzymatically catalyzed phosphorylation has
been discovered.
Thermoschuttler basic were used. Fluorescent Imager: ChemiDoc
̈
EXPERIMENTAL SECTION
General. All reactions were carried out under dry conditions and
at room temperature.
■
MP Imaging System (170−8280) from Bio-Rad Co. was used for the
primer extension study. Electrophoresis: Thermo Fisher OwlTM S4S
Aluminum-Backed Sequencer System. Power supply: Consort
EV2230 Gel Electrophoresis (1500 V, 300 mA, 150 W).
Solvents and Reagents. CH3CN, tetrahydrofuran (THF), and
DMF were purchased from Acros Organics (extra dry over molecular
sieves) and dried with activated molecular sieves. Triethylamine
(NEt3) and N,N-diisopropylethylamine were refluxed over CaH2 for
three days and distilled under nitrogen. 5-Chlorosaligenylchlorophos-
phite 18 and 5-nitrosaligenylchlorophosphite 19 were synthesized
according to the literature, and 18 freshly distilled prior to use.47 All
further reagents commercially available were used as received. Thin-
layer chromatography (TLC): for TLC Macherey-Nagel pre-coated
TLC sheets, Alugram Xtra SIL G/UV254 were used; compounds
were stained with a solution of vanillin−sulfuric acid (“VSS”, 5.0 g
vanillin, 30 mL conc. H2SO4, 900 mL methanol, and 100 mL glacial
acid) or iodine as marked in the Rf-value section. Phosphomolybdanic
acid (PMA) stain was used and prepared by dissolving 10 g of PMA in
100 mL of ethanol. Sugar-containing compounds were visualized with
a sugar spray reagent (4-methoxybenzaldehyde/EtOH/conc. sulfuric
acid/glacial acetic acid in ratio 5/90/5/0.1 v/v) and phosphate-
containing compounds with an ammonium molybdate solution [1 g
(NH4)6Mo7O24·4H2O in 7 mL semiconcentrated nitric acid and 13
mL water], followed by tin(II)chloride solution (0.1 g SnCl2·2H2O in
20 mL 0.5 mol/L hydrochloric acid). Preparative chromatography: the
preparative TLCs were accomplished with a Chromatotron (Harrison
Research, model 7924T) using glass plates coated with 2 or 4 mm
layers of Merck 60 PF254 silica gel. Column chromatography: normal
phase column chromatography were performed with Macherey-Nagel
silica gel 60 M (0.04−0.063 mm). Automatic RP-18 chromatography:
for reversed phase chromatography an Interchim Puriflash 430 in
combination with Chromabond Flash RS40 C18 ec was used. High-
Performance Liquid Chromatography (HPLC): HPLC was required for
analytical studies and monitoring reactions. A VWR-Hitachi
LaChromElite HPLC system (L-2130, L-2200, and L-2455),
EzChromElite software, and equipped with a Nucleodur 100-5
C18ec (Macherey-Nagel) was available. Acetonitrile for HPLC was
obtained from VWR (HPLC grade) and ultrapure water was
produced by a Sartorius Arium pro (Sartopore 0.2 μm, UV). Tetra-
n-butylammonium acetate solution (TBAA, pH 6.3, 2 mM) or 10 mM
triethylammonium acetate (pH 6.2) were used for buffering. Method
A: Nucleodur 100-5 C18ec; 0−20 min: TBAA buffer/acetonitrile
gradient (5−80%); 20−30 min: buffer/acetonitrile (80%); 30−33
min: buffer/acetonitrile (80−5%); 33−38 min: buffer/acetonitrile
(5%); and flow: 1 mL/min. Method B: Nucleodur 100-5 C18ec; 0−20
min: TBAA buffer/acetonitrile gradient (5−80%); 20−25 min:
buffer/acetonitrile (80−100%); 25−30 min: buffer/acetonitrile
(100%); 30−33 min; buffer/acetonitrile (100−5%); 33−38 min:
buffer/acetonitrile (5%); and flow: 1 mL/min. Final compounds
showed a purity of >95% according to HPLC analysis, using different
gradients as well as judged by NMR spectroscopy.
General Procedures. Syntheses and Characterization. The
syntheses and characterization of 4-(hydroxymethyl)phenylalkanoates
were described previously31,47 as were the syntheses of 4-(((tert-
butyldimethylsilyl)oxy)methyl)phenylbromide48 and (FmO)P(N(i-
Pr)2)2.43 The syntheses of (n-Bu4N)2·d4TDP were performed using
a cycloSal-technique starting from d4T.31,49 Other (n-Bu4N)2·NDPs
were obtained by the cation exchange of commercially available NDP
salts.
General Procedure 1: Syntheses of Ketones (kb) (13). To a
solution of N-methoxy-N-methylalkylamide 14 (1.0 equiv) in THF (5
mL) was added dropwise
a suspension of bis(4-((tert-
butyldimethylsilyl)oxymethyl)phenyl)-magnesium·LiCl 15 (0.91 M
in THF/dioxane 9:1, 1.83 mmol, 1.1 equiv) at −20 °C and stirred for
4 h at −20 °C and 1 h at 0 °C. The reaction was stopped by adding a
solution of ammonium chloride (50%, 20 mL). Ethyl acetate was
added (20 mL), the phases separated and the aqueous extracted with
ethyl acetate (2 × 20 mL). The pooled org. layers were washed with
brine, dried over sodium sulfate, and the solvent was removed in
vacuo. The residue was purified by column chromatography on silica
(PE/ethyl acetate 5:1 → 1:1).
General Procedure 2: Syntheses of (AB)-Phosphordiamidites
(12). The protocol was adapted to a previously described protocol.39
(4-Hydroxymethyl)phenylalkanoate (1.0 equiv) and dry NEt3 (1.4
equiv) dissolved in Et2O (1 mL/1 mmol) were added to
bis(diisopropylamino)chlorophosphine (1.1 equiv) dissolved in Et2O
(5 mL/1 mmol) at 0 °C and stirred for 16−18 h at rt. The precipitant
was filtered off, washed with n-hexane, and the solvent of the filtrate
was removed under reduced pressure. The residue was purified by
column chromatography on silica (petrol ether + 5−10% NEt3).
General Procedure 3: Syntheses of Nonsymmetrical (AB,kb)-
Phosphoramidites (11). The protocol was adapted to a literature-
known protocol.30 O-(4-Alkanoyloxybenzyl)-N,N,N′,N′-tetraisopro-
pylphosphordiamidite (1.5 equiv) and (4-(hydroxymethyl)phenyl)-
alkyl ketone (1.0 equiv) were co-evaporated with CH3CN and
dissolved in CH3CN/CH2Cl2. Afterward, 4,5-dicyanoimidazol (0.25
M in CH3CN, 1.0 equiv) was added at 0 °C in portions or dropwise,
and the suspension was stirred 1−2 h at rt. After filtration and
removal of the solvent under reduced pressure, the residue was
purified by column chromatography on silica (petrol ether + 10%
NEt3).
General Procedure 4: Syntheses of Nonsymmetric (AB,kb)-NTP
Prodrugs (9). (n-Bu4N)2·Nucl-DP49 (1.0 equiv) was co-evaporated
with CH3CN, dried in addition to (AB,kb)-phosphoramidite 11
(1.0−2.0 equiv) 1−2 h in vacuo, and dissolved in CH3CN (1−2 mL).
To this solution was added a solution of 4,5-dicyanoimidazole (0.25
M in CH3CN, 1.2−1.8 equiv) at rt and stirred for 20−120 min. t-
BuOOH (5.5 M in n-decane, 1.2−2.0 equiv) was subsequently added
dropwise at −20 to −30 °C and the solution was stirred for 30 min at
rt. The solvent was removed via cold distillation. The residue was
purified by using automated Flash-RP-CC (H2O/CH3CN 90:10 →
30:70) and transformed into the corresponding ammonium form by
the Dowex 50WX8 ion-exchange resin. In most cases, a further
purification step was necessary by using automated Flash-RP-CC
(H2O/CH3CN).
Nuclear Magnetic Resonance (NMR). NMR spectra were
recorded at room temperature in automation mode with a Varian
Gemini 2000BB, Bruker Fourier 300, Bruker AMX 400, Bruker DRX
1
500, or Bruker AVIII 600. All H- and 13C NMR chemical shifts (δ)
are quoted in parts per million (ppm) downfield from tetramethylsi-
lane and calibrated on a solvent signal. The 31P NMR chemical shifts
(proton decoupled) are also quoted in ppm using phosphoric acid as
the external standard. Mass Spectrometry (MS): HRMS (ESI) mass
spectra were acquired with a VG Analytical Finnigan ThermoQuest
MAT 95 XL or a Agilent 6224 EIS-TOF spectrometer. MALDI
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General Procedure 5: Syntheses of Nonsymmetric (Fm,kb)-
phosphoramidites (17). The protocol was adapted to an earlier
reported procedure.43
(FmO)P(N(i-Pr2))2 (1.2 equiv) and (4-(hydroxymethyl)phenyl)-
alkylketone 13 (1.0 equiv) were dissolved in THF (5 mL). A solution
of 4,5-dicyanoimidazolel (0.25 M in CH3CN, 4.00 mL, 1.0 mmol, 1.0
equiv) was added dropwise at 0 °C and the suspension was stirred for
1 h at 0 °C. After removal of the solvent in vacuo, the residue was
purified by column chromatography on silica (n-hexane/ethylacetate
9:1 + 1% NEt3).
2H, 2-H), 4.79 (s, 2H, 1-CH2O), 2.94 (t, J = 7.4 Hz, 2H, 6-H), 1.73
(p, J = 7.4 Hz, 2H, 7-H), 1.44−1.22 (m, 12H, 8-H, 9-H, 10-H, 11-H,
12-H, 13-H), 0.95 (s, 9H, OTBDMS), 0.93−0.82 (m, 3H, 14-H),
0.11 (s, 6H, OTBDMS). 13C NMR (75 MHz, CDCl3): δ [ppm] 200.5
(C-5), 146.9 (C-1), 136.0 (C-4), 128.3 (C-3), 126.0 (C-2), 64.7 (1-
CH2O), 38.8 (C-6), 32.0 (C-12), 29.6 (C-8−C-11), 29.6, 29.6, 29.4,
26.1 (OTBDMS), 24.6 (C-7), 22.8 (C-13), 18.5 (OTBDMS), 14.3
(C-14), −5.1 (OTBDMS). IR (film): ν
̃
[cm−1] 2955, 2928, 2855,
1686, 1610, 1463, 1256, 1094, 839, 778. HRMS (ESI+): m/z calcd,
377.2870 [M + H]+; found, 377.2881 [M + H]+.
Preparation of the Ketones (13). Syntheses of N-Methoxy-N-
methylalkylamides (14). N-Methoxy-N-methyldecanoylamide
(14a). N,O-Dimethylhydroxylamine hydrochloride (879 mg, 9.20
mmol, 1.0 equiv) was dissolved in CH2Cl2 (24 mL) and cooled to 0
°C. Dry pyridine (1.65 mL, 20.3 mmol, 2.2 equiv) and
decanoylchloride (1.93 mL, 9.20 mmol, 1.0 equiv.) were added and
the solution was stirred for 3 h at 0 °C. Then, water and CH2Cl2 were
added, the phases were separated, and the aqueous phase extracted
with CH2Cl2. The pooled organic layers were washed with brine,
dried over sodium sulfate, and the solvent was removed in vacuo. The
crude product was purified by column chromatography on silica (PE/
ethylacetate 2:1). Yield: 1.91 g (8.88 mmol, 96%; colorless oil). TLC:
(4-((tert-Butyldimethylsilyl)oxymethyl)phenyl)heptadecylketone.
According to general procedure 1, using bis(4-((tert-
butyldimethylsilyl)oxymethyl)phenyl)-magnesium·LiCl 15 (0.4 M in
THF/dioxane 9:1, 4.59 mmol, 1.2 equiv) and N-methoxy-N-
methyloctadecanylamide 14b (1.25 g, 3.82 mmol, 1.0 equiv.) in
THF (8 mL). Yield: 1.12 g (2.48 mmol, 65%; colorless oil). TLC: Rf
= 0.3 (PE/DCM 3:1, VSS: orange). 1H NMR (400 MHz, CDCl3): δ
[ppm] 7.93 (d, J = 8.3 Hz, 2H, 3-H), 7.40 (d, J = 8.7 Hz, 2H, 2-H),
4.79 (s, 2H, 1-CH2O), 2.94 (t, J = 7.4 Hz, 2H, 6-H), 1.73 (p, J = 7.3
Hz, 2H, 7-H), 1.43−1.21 (m, 28H, 8-H−21-H), 0.95 (s, 9H,
OTBDMS), 0.91−0.85 (m, 3H, 22-H), 0.11 (s, 3H, OTBDMS). 13C
NMR (101 MHz, CDCl3): δ [ppm] 200.5 (C-5), 146.9 (C-1), 136.0
(C-4), 128.3 (C-3), 126.0 (C-2), 64.7 (1-CH2O), 38.8 (C-6), 32.1
(C-20), 29.9, 29.8, 29.8, 29.8, 29.7, 29.6, 29.6, 29.5 (C-8−C-19), 26.1
(OTBDMS), 24.6 (C-7), 22.8 (C-21), 18.5 (OTBDMS), 14.3 (C-
22), −5.1 (OTBDMS).
1
Rf = 0.4 (PE/ethylacetate 1:1, iodine). H NMR (300 MHz, CDCl3-
d1): δ [ppm] 3.67 (s, 3H, OMe), 3.17 (s, 3H, N-Me), 2.40 (t, J = 7.6
Hz, 2H, 2-H), 1.74−1.50 (m, 2H, 3-H), 1.41−1.17 (m, 12H, 4-H−9-
H), 0.97−0.79 (m, 3H, 10-H). 13C NMR (75 MHz, CD3OD-d1): δ
[ppm] 174.8 (C-1, HMBC), 61.3 (OMe), 32.1, 32.0 (C-2, NMe),
29.6, 29.6, 29.4 (C-4, C-5, C-6, C-7, C-8), 24.8 (C-3), 22.8 (C-9),
(4-(Hydroxymethyl)phenyl)nonylketone (13a). To a solution of
silylethers (434 mg, 1.15 mmol, 1.0 equiv) in THF (10 mL) was
added TBAF (1 M in THF, 1.7 mL, 1.73 mmol, 1.5 equiv) at 0 °C
and stirred at rt for 45 min. The solvent was removed in vacuo
subsequently. The residue was purified by column chromatography on
silica (PE/ethylacetate 2:1). Yield: 297 mg (1.13 mmol, 98%) as a
14.2 (C-10). IR (film): ν
̃
[cm−1] 2923, 2853, 1667, 1462, 1381, 1176,
1001, 495. HRMS (ESI+): m/z calcd, 216.1958 [M + H]+; found,
216.1964 [M + H]+.
N-Methoxy-N-methyloctadecanoylamide (14b). N,O-Dimethyl-
hydroxylamine hydrochloride (595 mg, 6.10 mmol, 1.0 equiv) was
dissolved in CH2Cl2 (30 mL) and cooled to 0 °C. Dry pyridine (1.04
mL, 12.8 mmol, 2.1 equiv) and decanoylchloride (2.10 mL, 6.30
mmol, 1.0 equiv) were added and the solution was stirred for 2.5 h at
0 °C. Afterward, water and CH2Cl2 were added, the phases were
separated, and aqueous phase extracted with CH2Cl2 (20 mL). The
pooled org. layers were washed with brine, dried over sodium sulfate,
and the solvent was removed in vacuo. The crude product was
purified by column chromatography on silica (petrol ether/ethyl-
acetate 2:1 → 1:1). Yield: 1.92 g (5.85 mmol, 96%) as a colorless
1
colorless solid. TLC: Rf = 0.5 (PE/ethylacetate 1:1, VSS). H NMR
(600 MHz, CDCl3): δ [ppm] 7.95 (d, J = 8.3 Hz, 2H, 3-H), 7.45 (d, J
= 8.2 Hz, 2H, 2-H), 4.77 (s, 2H, 1-CH2O), 2.96 (mc, 2H, 6-H), 1.83
(s, OH), 1.73 (p, J = 7.5 Hz, 2 H, 7-H), 1.44−1.20 (m, 12H, 8-H, 9-
H, 10-H, 11-H, 12-H, 13-H), 0.88 (t, J = 7.0 Hz, 3H, 14-H). 13C
NMR (151 MHz, CDCl3): δ [ppm] 200.5 (C-5), 146.0 (C-1), 136.5
(C-4), 128.5 (C-3), 126.8 (C-2), 64.8 (1-CH2O), 38.8 (C-6), 32.0
(C-12), 29.6, 29.6, 29.5, 29.4 (C-8−C-11), 24.6 (C-7), 22.8 (C-13),
14.2 (C-14). HRMS (ESI+): m/z calcd, 263.2006 [M + H]+; found,
263.1974 [M + H]+.
1
solid. TLC: Rf = 0.5 (PE/ethylacetate 2:1, iodine). H NMR (300
(4-(Hydroxymethyl)phenyl)heptadecylketone (13b). For the
procedure, see ketone 15a. The TBDMS-protected precursor (1.91
g, 2.44 mmol, 1.0 equiv) was used in THF (19 mL) and TBAF (1 M
in THF, 3.65 mL, 3.65 mmol, 1.5 equiv). Yield: 870 mg (2.32 mmol,
95%) as a colorless solid. TLC: Rf = 0.4 (PE/ethyl acetate 2:1, VSS:
MHz, CDCl3): δ [ppm] 3.67 (s, 3H, OMe), 3.17 (s, 3H, N-Me), 2.40
(t, J = 7.6 Hz, 2H, 2-H), 1.62 (p, J = 7.3 Hz, 2H, 3-H), 1.41−1.17 (m,
28H, 4-H−17-H), 0.97−0.82 (m, 3H, 18-H). 13C NMR (75 MHz,
CDCl3): δ [ppm] 174.9 (C-1, HMBC), 61.3 (OMe), 32.1 (C-2,
NMe), 29.8, 29.8, 29.8, 29.7, 29.6, 29.6, 29.5 (C-4−C-16), 24.8 (C-
1
orange). H NMR (400 MHz, CDCl3): δ [ppm] 7.95 (d, J = 8.3 Hz,
3), 22.8 (C-17), 14.3 (C-18). IR (film): ν
̃
[cm−1] = 2921, 2852, 1670,
2H, 3-H), 7.45 (d, J = 8.1 Hz, 2H, 2-H), 4.77 (s, 2H, 1-CH2O), 2.95
(mc, 2H, 6-H), 1.73 (m, 2H, 7-H), 1.49−1.17 (m, 28H, 8-H−21-H),
0.91−0.85 (m, 3H, 22-H). 13C NMR (101 MHz, CDCl3): δ [ppm]
200.4 (C-5), 146.0 (C-1), 136.5 (C-4), 128.5 (C-3), 126.8 (C-2),
64.8 (1-CH2O), 38.8 (C-6), 32.1 (C-12), 29.8, 29.8, 29.8, 29.8, 29.7,
29.6, 29.5, 29.5 (C-8−C-19), 24.6 (C-7), 22.8 (C-21), 14.3 (C-22).
HRMS (ESI+): m/z calcd, 375.3258 [M + H]+; found, 375.3237 [M +
H]+.
1463, 1412, 1381, 1176, 1381, 999, 721. HRMS (ESI+): m/z calcd,
328.3210 [M + H]+; found, 328.3217 [M + H]+.
Synthesis of the Grignard Reagent. Bis(4-((tert-
butyldimethylsilyl)oxymethyl)phenyl)magnesium·LiCl (15). Prepa-
ration of the reagent iPrMgCl·LiCl: a solution of LiCl (0.5 M in THF,
11.6 mL, 5.81 mmol, 1.0 equiv) was mixed with a solution of iPrMgCl
(2 M in THF, 2.9 mL, 5.81 mmol, 1.0 equiv) at rt. The concentration
was determined to 0.4 M by calculation. Br-/Mg-exchange: OTBS-4-
bromobenzyl alcohol (1.20 mL, 1.38 g, 4.59 mmol, 1.0 equiv) was
added to a solution of iPrMgCl·LiCl (0.4 M in THF, 12.0 mL, 4.8
mmol, 1.0 equiv) and 1,4-dioxane (10 vol %, 1.2 mL) and stirred 16−
30 h at rt until consumption of bromide. The gained suspension was
used within 24 h.
Syntheses of (AB)-Phosphordiamidites (12). O-(4-Pentanoy-
loxybenzyl)-N,N,N,N′-tetraisopropylphosphordiamidite (12x). Ac-
cording to general procedure 2, using bis(N,N-diisopropylamino)-
chlorophosphine (1.59 g, 5.97 mmol, 1.1 equiv), dry NEt3 (1.10 mL,
769 mg, 7.60 mmol, 1.4 equiv), and (4-hydroxymethyl)-
phenylpentanoate (1.13 g, 5.43 mmol, 1.0 equiv). Yield: 1.88 g
(4.28 mmol, 79%) as a colorless oil. The compound was
contaminated with 3% (31P NMR) of the corresponding phosphor-
Synthesis of the ketones. (4-((tert-Butyldimethylsilyl)-
oxymethyl)phenyl)nonanylketone. According to general procedure
1, using N-methoxy-N-methyldecanylamide 14a (358 mg, 1.66 mmol,
1.0 equiv.) in THF (5 mL) and bis(4-((tert-butyldimethylsilyl)-
oxymethyl)phenyl)-magnesium·LiCl 15 (0.91 M in THF/dioxane 9:1,
1.83 mmol, 1.1 equiv). Yield: 437 mg (1.16 mmol, 70%; colorless oil).
1
amidate. TLC: Rf = 0.70 (PE/NEt3 9:1 v/v). H NMR (400 MHz,
3
3
CDCl3): δ [ppm] 7.36 (d, JHH = 8.5 Hz, 2H, 2-H), 7.03 (d, JHH
=
3
8.5 Hz, 2H, 3-H), 4.63 (d, JHP = 7.3 Hz, 2H, 1-CH2O), 3.63−3.50
(mc, 4H, 1′a-H), 2.55 (t, 3JHH = 7.5 Hz, 2H, 6-H), 1.74 (qi, 3JHH = 7.5
1
3
TLC: Rf = 0.5 (PE/ethyl acetate 3:1, VSS). H NMR (300 MHz,
Hz, 2H, 7-H), 1.50−1.40 (mc, 2H, 8-H), 2 × 1.18 (2× d, JHH = 6.7
CDCl3): δ [ppm] 7.93 (d, J = 8.3 Hz, 2H, 3-H), 7.40 (d, J = 8.5 Hz,
Hz, 3JHH = 6.8 Hz, 24H, 2′a-H, 2′b-H), 0.97 (t, 3JHH = 7.4 Hz, 3H, 9-
J
https://dx.doi.org/10.1021/acs.jmedchem.0c01293
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
H). 13C NMR (101 MHz, CDCl3): δ [ppm] 172.5 (C-5), 149.7 (C-
3JCP = 7.3 Hz, C-1′), 136.3 (C-4), 128.3 (C-2), 128.2 (C-2′), 126.9
2 2
3
4), 138.2 (d, JCP = 10.3 Hz, C-1), 127.9 (C-2), 121.3 (C-3), 65.9,
(C-3), 121.5 (C-3′), 65.1 (d, JCP = 18.5 Hz, 1-CH2O), 65.0 (d, JCP
= 18.3 Hz, 1′-CH2O), 43.3 (d, 2JCP = 12.4 Hz, 2× C-1″a), 38.8 (C-6),
34.3 (C-6′), 32.0 (C-12), 29.6, 29.6, 29.6, 29.4 (C-8, C-9, C-10, C-
11), 27.2 (C-7′), 24.9 (C-7), 24.8, 24.8, 24.6 (2× C-2″a, 2× C-2″b),
22.8 (C-13), 22.4 (C-8′), 14.3 (C-14), 13.9 (C-9′). 31P NMR (243
MHz, CDCl3): δ [ppm] 148.4. HRMS (ESI+, m/z): calcd, 600.3813
[M + H]+; found, 600.3808 [M + H]+.
65.7 (1-CH2O), 44.6 (d, 2JCP = 12.5 Hz, 4× C-1′a), 34.3 (C-6), 27.2
(C-7), 24.8 (d, 3JCP = 8.0 Hz, 4× C-2′a), 24.0 (d, 3JCP = 5.8 Hz, 4× C-
2′b), 22.4 (C-8), 13.9 (C-9). 31P NMR (162 MHz, CDCl3): δ [ppm]
123.5. IR (Film): ν
̃
[cm−1] 2963, 2930, 2869, 1760, 1507, 1361, 1183,
1016, 951, 748, 526. HRMS (ESI+, m/z): calcd, 457.3190 [M +
H3O]+; found, 457.3205 [M + H3O]+.
O-(4-Heptanoyloxybenzyl)-N,N,N′,N′-tetraisopropylphosphor-
diamidite (12y). According to general procedure 2, using bis(N,N-
diisopropylamino)chlorophosphine (1.33 g, 5.00 mmol, 1.1 equiv),
dry NEt3 (894 μL, 6.37 mmol, 1.4 equiv), and (4-hydroxymethyl)-
phenylheptanoate (1.08 g, 4.55 mmol, 1.0 equiv). Yield: 788 mg (1.69
mmol, 37%; colorless oil). The compound was contaminated with 2%
(31P NMR) of the corresponding phosphoramidate. TLC: Rf = 0.70
O′-(4′-Heptanoyloxybenzyl)-O-(4-octadecylcarbonylbenzyl)-
N,N-diisopropylphosphor Amidite (11by). According to general
procedure 3, using O-(4-heptanoyloxybenzyl)-N,N,N′,N′-tetraisopro-
pylphosphordiamidite 12y (366 mg, 784 μmol, 1.5 equiv) and (4-
(hydroxymethyl)phenyl)heptadecylketone 13b (196 mg, 452 μmol,
1.0 equiv) dissolved in CH3CN/CH2Cl2/THF (5/8/5 mL) and 4,5-
dicyanoimidazole (0.25 M in CH3CN, 2.10 mL, 523 μmol, 1.0 equiv).
Yield: 345 mg (466 μmol, 89%; colorless solid). The compound was
contaminated with 2% (31P NMR) of the corresponding phosphor-
1
(n-hexane/NEt3 9:1 v/v). H NMR (600 MHz, CDCl3): δ [ppm]
3
3
7.36 (d, JHH = 8.4 Hz, 2H, 2-H), 7.02 (d, JHH = 8.5 Hz, 2H, 3-H),
3
1
4.62 (d, JHP = 7.2 Hz, 2H, 1-CH2O), 3.63−3.50 (mc, 4H, 1′a-H),
amidate. TLC: Rf = 0.8 (PE/NEt3 9:1, VSS: orange). H NMR (400
3
3
2.54 (t, JHH = 7.5 Hz, 2H, 6-H), 1.74 (qi, JHH = 7.5 Hz, 2H, 7-H),
1.50−1.40 (mc, 2H, 8-H), 1.36−1.30 (m, 4H, 9-H, 10-H), 2 × 1.18
MHz, CDCl3): δ [ppm] 7.92 (d, J = 8.3 Hz, 2H, 3-H), 7.42 (d, J = 8.1
Hz, 2H, 2-H), 7.35 (d, J = 8.5 Hz, 2H, 2′-H), 7.03 (d, J = 8.5 Hz, 2H,
3’-H), 4.90−4.61 (m, 4H, 1-CH2O, 1’-CH2O), 3.70 (mc, 2H, 1″a-H),
2.94 (t, J = 7.4 Hz, 2H, 6-H), 2.55 (t, J = 7.5 Hz, 2H, 6′-H), 1.81−
1.67 (m, 4H, 7-H, 7′-H), 1.47−1.17 (m, 46H, 8′-H, 9′-H, 10′-H, 8-
H−21-H, 2″a-H, 2″b-H), 0.94−0.85 (m, 6H, 11′-H, 22-H). 13C NMR
(101 MHz, CDCl3): δ [ppm] 200.5 (C-5), 172.5 (C-5′), 150.1 (C-
3
3
(2× d, JHH = 6.7 Hz, JHH = 6.9 Hz, 24H, 2′a-H, 2′b-H), 0.93−0.88
(m, 3H, 11-H). 13C NMR (151 MHz, CDCl3): δ [ppm] 172.5 (C-5),
3
149.7 (C-4), 138.2 (d, JCP = 10.3 Hz, C-1), 127.9 (C-2), 121.3 (C-
3), 65.8 (d, 2JCP = 23.0 Hz, 1-CH2O), 44.6 (d, 2JCP = 12.5 Hz, 4× C-
1′a), 34.6 (C-6), 31.6 (C-9), 28.9 (C-8), 25.1 (C-7), 24.8 (d, 4× C-
2′a), 24.0 (d, 3JCP = 5.8 Hz, 4× C-2′b), 22.4 (C-10), 13.9 (C-11). 31
P
3
4′), 144.8 (d, JCP = 7.5 Hz, C-1), 136.3 (C-1′), 136.3 (C-4), 128.3
NMR (243 MHz, CDCl3): δ [ppm] 123.5. HRMS (ESI+, m/z): calcd,
2
(C-2), 128.2 (C-2′), 126.9 (C-3), 121.5 (C-3′), 65.1 (d, JCP = 18.5
467.3397 [M + H]+; found, 467.3406 [M + H]+.
Hz, 1-CH2O), 65.0 (d, 2JCP = 18.2 Hz, 1′-CH2O), 43.3 (d, 2JCP = 12.5
Hz, 2× C-1″a), 38.8 (C-6), 34.6 (C-6′), 32.1 (C-20), 31.6 (C-10′),
29.9, 29.8, 29.8, 29.7, 29.7, 29.6, 29.5, 28.9 (C-8′, C-8, C-9, C-10, C-
11, C-12, C-13, C-14, C-15, C-16, C-17, C-18, C-19), 25.1, 24.9, 24.8,
24.8, 24.8, 24.6 (C-7, C-7′, 2× C-2″a, 2× C-2″b), 22.8 (C-21), 22.6
(C-10′), 14.3 (C-11′), 14.2 (C-22). 31P NMR (162 MHz, CDCl3): δ
[ppm] 148.4. HRMS (ESI+, m/z): calcd, 740.5378 [M + H]+; found,
740.5362 [M + H]+.
O-(4-Octadecanoyloxybenzyl)-N,N,N′,N′-tetraisopropylphos-
phordiamidite (12z). According to general procedure 2, using
bis(N,N-diisopropylamino)chlorophosphine (1.65 g, 6.19 mmol, 1.0
equiv) dissolved in Et2O (40 mL) and dry NEt3 (1.10 mL, 792 mg,
7.84 mmol, 1.3 equiv) and (4-hydroxymethyl)phenyloctadecanoate
(2.59 g, 6.63 mmol, 1.1 equiv) dissolved in CH2Cl2 (12 mL). The
reaction mixture was stirred for 1 h at 0 °C. Yield: 3.48 g (5.60 mmol,
91%; colorless solid). The compound was contaminated with 7% (31P
NMR) of (C17)2-phosphoramidite. The side product is removable by
CC on silica (n-hexane/NEt3 95:5) with a loss of material. TLC: Rf =
O′-(4′-Octadecanoyloxybenzyl)-O-(4-decanoylbenzyl)-N,N-diiso-
propylphosphoramidite (11az). According to general procedure 3,
using O-(4-octadecanoyloxybenzyl)-N,N,N′,N′-tetraisopropylphos-
phordiamidite 12z (700 mg, 1.16 mmol, 1.5 equiv) and (4-
(hydroxymethyl)phenyl)nonylketone 14a (199 mg, 758 μmol, 1.0
equiv) dissolved in CH3CN/CH2Cl2 (7/5 mL) and 4,5-dicyanoimi-
dazole (0.25 M in CH3CN, 3.00 mL, 758 μmol, 1.0 equiv). Yield: 599
mg (766 μmol, quant.; colorless solid). The compound was
contaminated with 13% (31P NMR) of the corresponding (AB-C17/
AB-C17)-phosphoramidite. TLC: Rf = 0.8 (PE/NEt3 9:1, VSS:
1
0.80 (n-hexane/NEt3 9:1). H NMR (400 MHz, CDCl3): δ [ppm]
7.37 (d, J = 8.3 Hz, 2H, 2-H), 7.03 (d, J = 8.5 Hz, 2H, 3-H), 4.63 (d,
3JHP = 7.3 Hz, 2H, 1-CH2O), 3.64−3.50 (mc, 4H, 1′a-H), 2.54 (t, 3JHH
= 7.5 Hz, 2H, 6-H), 1.75 (qi, J = 7.5 Hz, 2H, 7-H), 1.42−1.24 (m,
28H, 8−21-H), 2 × 1.18 (2× d, J = 6.8 Hz, J = 6.8 Hz, 24H, 2′a-H,
2′b-H), 0.91−0.86 (m, 3H, 22-H). 13C NMR (101 MHz, CDCl3): δ
3
[ppm] 172.6 (C-5), 149.7 (C-4), 138.2 (d, JCP = 10.2 Hz, C-1),
1
127.9 (C-2), 121.3 (C-3), 65.8 (d, 2JCP = 23.7 Hz, 1-CH2O), 44.6 (d,
2JCP = 12.4 Hz, 4× C-1′a), 34.6 (C-6), 32.1 (C-20), 29.9, 29.8, 29.8,
29.8, 29.6, 29.5, 29.4, 29.3, (C-8, C-9, C-10, C-11, C-12, C-13, C-14,
C-15, C-16, C-17), 25.1 (C-7), 24.8 (d, 3JCP = 7.9 Hz, 4× C-2′a), 24.0
orange). H NMR (400 MHz, CDCl3): δ [ppm] 7.92 (d, J = 8.3
Hz, 2H, 3-H), 7.42 (d, J = 8.2 Hz, 2H, 2-H), 7.35 (d, J = 8.5 Hz, 2H,
2′-H), 7.03 (d, J = 8.5 Hz, 2H, 3′-H), 4.86−4.62 (m, 4H, 1-CH2O, 1’-
CH2O), 3.70 (mc, 2H, 1″a-H), 2.94 (t, J = 7.4 Hz, 2H, 6-H), 2.54 (t, J
= 7.5 Hz, 2H, 6′-H), 1.82−1.66 (m, 4H, 7-H, 7′-H), 1.46−1.15 (m,
52H, 8-H, 9-H, 10-H, 11-H, 12-H, 13-H, 8′-H−21′-H, 2″a-H, 2″b-
H), 0.88 (t, J = 6.7 Hz, 6H, 22’-H, 14-H). 13C NMR (101 MHz,
CDCl3): δ [ppm] 200.5 (C-5), 172.5 (C-5′), 150.1 (C-4′), 144.9 (d,
3JCP = 7.2 Hz, C-1), 136.3 (C-4), 128.3 (C-2), 128.2 (C-2′), 126.9
(C-3), 121.5 (C-3′), 65.2, 65.1, 65.0, 64.9 (1-CH2O, 1′-CH2O), 43.4,
43.3 (2× C-1″a), 38.8 (C-6), 34.6 (C-6′), 32.1 (C-20′), 32.0 (C-12),
29.9, 29.8, 29.8, 29.8, 29.7, 29.6, 29.6, 29.5, 29.4, 29.4, 29.3 (C-8−C-
11, C-8′−C-19′), 25.1, 24.9, 24.8, 24.8, 24.8, 24.6 (C-7, C-7′, 2× C-
2″a, 2× C-2″b), 22.8 (C-13), 22.8 (C-21′), 14.3 (C-22′), 14.3 (C-
14). 31P NMR (162 MHz, CDCl3): δ [ppm] 148.4. HRMS (ESI+, m/
z): calcd, 782.5847 [M + H]+; found, 782.5829 [M + H]+.
O′-(4′-Octadecanoyloxybenzyl)-O-(4-octadecanoylbenzyl)-N,N-
diisopropylphosphoramidite (11bz). According to general procedure
3, using O-(4-octadecanoyloxybenzyl)-N,N,N′,N′-tetraisopropylphos-
phordiamidite 12z (357 mg, 575 μmol, 1.5 equiv) and (4-
(hydroxymethyl)phenyl)heptadecanylketone 13b (142 mg, 379
μmol, 1.0 equiv′) in CH3CN/THF (8/5 mL) and 4,5-dicyanoimi-
dazole (0.25 M in CH3CN, 1.52 mL, 380 μmol, 1.0 equiv). Yield: 350
mg (391 μmol, quant.; colorless solid). TLC: Rf = 0.7 (petrol ether/
NEt3 9:1, VSS: orange). 1H NMR (400 MHz, CDCl3): δ [ppm] 7.92
3
(d, JCP = 5.7 Hz, 4× C-2′b), 22.9 (C-21), 14.3 (C-22). 31P NMR
(162 MHz, CDCl3): δ [ppm] 123.6. HRMS (ESI+, m/z): calcd,
639.5046 [M + H3O]+; found, 639.5240 [M + H3O]+.
Syntheses of Nonsymmetric (AB,kb)-Phosphoramidites
(11). O′-(4′-Pentanoyloxybenzyl)-O-(4-decanoylbenzyl)-N,N-diiso-
propylphosphoramidite (11ax). According to general procedure 3,
using O-(4-pentanoyloxybenzyl)-N,N,N′,N′-tetraisopropylphosphor-
diamidite 12x (193 mg, 450 μmol, 1.5 equiv), (4-(hydroxymethyl)-
phenyl)nonanylketone 13a (79.0 mg, 300 μmol, 1.0 equiv), and 4,5-
dicyanoimidazole (0.25 M in CH3CN, 1.20 mL, 300 μmol, 1.0 equiv).
Yield: 100 mg (167 μmol, 37%; colorless oil). The compound was
contaminated with 2% (31P NMR) of the corresponding phosphor-
1
amidate. TLC: Rf = 0.6 (PE/NEt3 9:1, VSS). H NMR (300 MHz,
CDCl3): δ [ppm] = 7.92 (d, J = 8.3 Hz, 2H, 3-H), 7.42 (d, J = 8.7 Hz,
2H, 2-H), 7.35 (d, J = 8.5 Hz, 2H, 2′-H), 7.03 (d, J = 8.6 Hz, 2H, 3′-
H), 4.86−4.63 (m, 4H, 1-CH2O, 1′-CH2O), 3.78−3.62 (mc, 2H, 1″a-
H), 2.99−2.88 (m, 2H, 6-H), 2.59−2.53 (m, 2H, 6′-H), 1.81−1.65
(m, 4H, 7-H, 7′-H), 1.54−1.16 (m, 26H, 8′-H, 8-H, 9-H, 10-H, 11-H,
12-H, 13-H, 2″a-H, 2″b-H), 0.97 (t, J = 7.3 Hz, 3H, 9′-H), 0.92−0.84
(m, 3H, 14-H). 13C NMR (151 MHz, CDCl3): δ [ppm] 200.5 (C-5),
3
172.5 (C-5′), 150.1 (C-4′), 144.9 (d, JCP = 7.2 Hz, C-1), 137.0 (d,
K
https://dx.doi.org/10.1021/acs.jmedchem.0c01293
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(d, J = 8.1 Hz, 2H, 3-H), 7.42 (d, J = 8.2 Hz, 2H, 2-H), 7.35 (d, J =
8.3 Hz, 2H, 2′-H), 7.03 (d, J = 8.4 Hz, 2H, 3′-H), 4.86−4.64 (m, 4H,
1-CH2O, 1′-CH2O), 3.70 (mc, 2H, 1″a-H), 2.94 (t, J = 7.4 Hz, 2H, 6-
H), 2.54 (t, J = 7.5 Hz, 2H, 6′-H), 1.82−1.66 (m, 4H, 7-H, 7’-H),
1.48−1.12 (m, 68H, 8-H−21-H, 8′-H−21′-H, 2″a-H, 2″b-H), 0.88 (t,
J = 6.7 Hz, 6H, 22′-H, 22-H). 13C NMR (101 MHz, CDCl3): δ
Nonsymmetric γ-(AB,kb)-NTP Prodrugs (9). (AB-C4H9,kb-
C9H19)-d4TTP (9ax). According to general procedure 4 using (n-
Bu4N)2·d4TDP 4 (61.0 mg, 74.0 μmol, 1.0 equiv), (AB-C4/ab-C9)-
phosphoramidite 11ax (90.0 mg, 149 μmol, 1.8 equiv), 4,5-
dicyanoimidazole (0.25 M in CH3CN, 533 μL, 134 μmol, 1.8
equiv), and t-BuOOH (5.5 M in n-decane, 27.0 μL, 150 μmol, 2.0
equiv). Yield: after freeze drying 35.5 mg (38.0 μmol, 51%) were
obtained as colorless cotton. RP18-HPLC: tR [min] (λmax) = 15.3
3
[ppm] 200.5 (C-5), 172.5 (C-5′), 150.1 (C-4′), 144.9 (d, JCP = 7.3
3
Hz, C-1), 137.0 (d, JCP = 7.3 Hz, C-1′), 136.3 (C-4), 128.3 (C-2),
1
(249 nm), Method A. H NMR (600 MHz, MeOH-d4): δ [ppm] =
128.2 (C-2′), 126.9 (C-3), 121.5 (C-3′), 65.2, 65.1, 65.0, 64.9 (1-
CH2O, 1′-CH2O), 43.4, 43.3 (2× C-1″a), 38.8 (C-6), 34.6 (C-6′),
32.1 (C-20, C-20′), 29.9, 29.8, 29.8, 29.8, 29.7, 29.7, 29.6, 29.6, 29.5,
29.4, 29.3 (C-8−C-19, C-8′−C-19′), 25.1, 24.9, 24.8, 24.8, 24.8, 24.6
(C-7, C-7′, 2× C-2″a, 2× C-2″b), 22.9 (C-21, C-21′), 14.3 (C-22′),
14.3 (C-22). 31P NMR (162 MHz, CDCl3): δ [ppm] 148.4. HRMS
(ESI+, m/z): calcd, 894.7099 [M + H]+; found, 894.6557 [M + H]+.
Syntheses of Nonsymmetric (Fm,kb)-Phosphoramidites
(17). O′-1-(9H-Fluoren-9-yl)methoxy-O-(4-decanoylbenzyl)-N,N-
diisopropylphosphoramidite (17a). According to general procedure
5, using (FmO)P(Ni-Pr2)2 (515 mg, 1.21 mmol, 1.2 equiv) and (4-
(hydroxymethyl)phenyl)nonanylketone 13a (263 mg, 1.0 mmol, 1.0
equiv) in THF (5 mL) and 4,5-dicyanoimidazole (0.25 M in CH3CN,
4.00 mL, 1.00 mmol, 1.0 equiv). Yield: 633 mg (935 mmol, 93%;
colorless oil). The final compound was contaminated with 5% (31P
NMR) phosphoramidite (FmO)2PN(i-Pr)2 and with 23% dibenzo-
7.93 (dd, J = 8.4, 1.8 Hz, 2H, 3″-H), 7.68 (mc, 1H, 6-H), 7.45 (dd, J =
8.4, 2.8 Hz, 2H, 2″-H), 7.40 (dd, J = 8.6, 3.2 Hz, 2H, 2‴-H), 7.03 (dd,
J = 8.7, 2.3 Hz, 2H, 3‴-H), 6.92 (mc, 1H, 1′-H), 6.49 (mc, 1H, 3′-H),
5.80 (mc, 1H, 2′-H), 5.22 (d, 3JHP = 8.1 Hz, 2H, 1″-CH2O), 5.20 (d,
3JHP = 8.4 Hz, 2H, 1‴-CH2O), 4.94 (mc, 1H, 4′-H), 4.29 (m, 1H, 5′-
Ha), 4.20 (m, 1H, 5′-Hb), 3.00 (mc, 2H, 6″-H), 2.58 (mc, 2H, 6‴-H),
1.91−1.85 (m, 3H, 5-CH3), 1.76−1.66 (m, 4H, 7″-H, 7‴-H), 1.50−
1.42 (m, 2H, 8‴-H), 1.42−1.26 (m, 12H, 8″-H−13″-H), 0.99 (t, J =
7.4 Hz, 3H, 9‴-H), 0.93−0.87 (m, 3H, 14″-H). 13C NMR (151 MHz,
MeOH-d4): δ [ppm] 202.5 (C-5), 173.7 (C-5‴), 166.5 (C-4), 152.8
(C-2), 152.4 (C-4‴), 141.3 (C-1″*, HMBC), 138.7 (C-6), 138.1 (C-
4″*), 135.8 (C-3′), 135.0 (d, 3JCP = 7.4 Hz, C-1‴*), 130.5 (d, 4JCP
=
4
5.4 Hz, C-2‴), 129.4 (C-3″), 128.9 (d, JCP = 6.0 Hz, C-2″), 127.1
(C-2′), 122.8 (d, 4JCP = 2.3 Hz, C-3‴), 112.1 (C-5), 90.8 (C-1′), 87.3
3
(d, JCP = 9.1 Hz, C-4′), 70.5 (m, 1″-CH2O), 70.1 (m, 1‴-CH2O),
1
67.9 (d, 2JCP = 5.8 Hz, C-5′), 39.6 (C-6″), 34.8 (C-6‴), 33.1 (C-12″),
30.7, 30.6, 30.4, 30.4 (C-8″−11″), 28.1 (C-7‴), 25.6 (C-7″), 23.7 (C-
13″*), 23.3 (C-8‴*), 14.4 (C-14″), 14.1 (C-9‴), 12.5 (5-CH3). 31P
fulvene. TLC: Rf = 0.7 (PE/ethylacetate 2:1 + 1% NEt3, VSS)· H
NMR (600 MHz, CDCl3): δ [ppm] 7.93 (d, J = 8.3 Hz, 2H, 3-H),
7.78−7.68 (m, 2H, Fm), 7.66 (d, J = 7.5 Hz, 1H, Fm), 7.61 (d, J = 7.6
Hz, 1H, Fm), 7.42 (d, J = 8.1 Hz, 2H), 7.40−7.36 (m, 2H, Fm),
7.34−7.25 (m, 2H, Fm), 4.75 (dd, J = 13.5, 8.1 Hz, 1H, 1-CH2,aO),
4.69 (dd, J = 13.6, 8.5 Hz, 1H, 1-CH2,bO), 4.21 (t, J = 7.1 Hz, 1H,
Fm-2′-H), 4.09−4.03 (m, 1H, Fm-1′-Ha), 3.89−3.83 (m, 1H, Fm-1′-
Hb), 3.69 (m, 2H, 1″a-H), 2.94 (t, J = 7.4 Hz, 2H, 6-H), 1.73 (p, J =
7.4 Hz, 2H, 7-H), 1.42−1.23 (m, 12H, 8-H−13-H), 1.20 (d, J = 6.8
Hz, 6H, 2″a-H), 1.17 (d, J = 6.8 Hz, 6H, 2″b-H), 0.91−0.87 (m, 3H,
14-H). 13C NMR (151 MHz, CDCl3): δ [ppm] 200.5 (C-5), 145.0
(C-1), 145.0 (Fm), 144.7 (Fm), 141.5 (Fm), 141.4 (Fm), 136.2 (C-
4), 128.9, 128.3 (C-2), 127.6 (Fm), 127.2 (Fm), 127.0 (Fm), 127.0
(Fm), 126.9 (C-3), 125.5 (Fm), 125.3 (Fm), 120.0 (Fm), 119.9
2
NMR (162 MHz, MeOH-d4): δ [ppm] −11.84 (d, JPP = 20.2 Hz,
Pγ), −13.26 (d, 2JPP = 17.3 Hz, Pα), −23.82 (dd, 2JPP = 22.2, 17.4 Hz,
Pβ). HRMS (ESI−): m/z calcd, 448.1231 [M − H]−; found, 448.1235
[M − 2H]2−.
(AB-C6H13,kb-C17H35)-d4TTP (9by). According to general proce-
dure 4 using (n-Bu4N)2·d4TDP (100 μmol, 1.0 equiv), (AB-
C6H13,ab-C17H35)-phosphoramidite 11by (127 mg, 172 μmol, 1.7
equiv), 4,5-dicyanoimidazole (0.25 M in CH3CN, 680 μL, 170 μmol,
1.70 equiv) and t-BuOOH (5.5 M in n-decane, 31.0 μL, 170 μmol, 1.7
equiv). Yield: after freeze-drying 56.0 mg (52.0 μmol, 52%) were
1
obtained as colorless cotton in a purity of 96% (via H NMR, 31P
2
2
NMR). RP18-HPLC: tR [min] (λmax) = 20.3 (249 nm), Method B. 1H
NMR (600 MHz, MeOH-d4): δ [ppm] 7.92 (dd, J = 8.4, 1.7 Hz, 2H,
3″-H), 7.67 (mc, 1H, 6-H), 7.44 (dd, J = 8.4, 2.8 Hz, 2H, 2″-H), 7.39
(dd, J = 8.6, 3.1 Hz, 2H, 2‴-H), 7.03 (dd, J = 8.6, 2.4 Hz, 2H, 3‴-H),
6.96−6.89 (mc, 1H, 1′-H), 6.51−6.45 (mc, 1H, 3′-H), 5.83−5.79 (mc,
1H, 2′-H), 5.21 (d, 3JHP = 8.2 Hz, 2H, 1″-CH2O), 5.18 (d, 3JHP = 8.5
Hz, 2H, 1‴-CH2O), 4.96−4.92 (mc, 1H, 4′-H), 4.29 (ddd, J = 11.7,
6.8, 3.3 Hz, 1H, 5′-Ha), 4.24−4.16 (m, 1H, 5′-Hb), 3.00 (mc, 2H, 6″-
H), 2.58 (mc, 2H, 6‴-H), 1.88 (s, 3H, 5-CH3), 1.77−1.65 (m, 4H, 7″-
H, 7‴-H), 1.48−1.23 (m, 34H, 8″-H−21″-H, 8‴-H−10‴-H), 0.96−
0.92 (m, 3H, 11″-H), 0.90 (t, J = 7.0 Hz, 3H, 22″-H).13C NMR (151
MHz, MeOH-d4): δ [ppm] 202.4 (C-5), 173.7 (C-5‴), 166.5 (C-4),
152.8 (C-2), 152.4 (C-4‴), 142.6 (C-1″, HMBC), 138.7 (C-6), 138.1
(C-4″), 135.8 (C-3′), 134.9 (C-1‴, HMBC), 130.5 (d, 4JCP = 5.4 Hz,
(Fm), 66.2 (d, JCP = 18.2 Hz, Fm-1′), 64.8 (d, JCP = 17.3 Hz, 1-
CH2O), 49.4 (d, 3JCP = 7.7 Hz, Fm-2′), 43.3 (d, 2JCP = 12.3 Hz, 2× C-
1″a), 38.8 (C-6), 32.0 (C-12), 29.7, 29.6, 29.6, 29.4 (C-8, C-9, C-10,
C-11), 24.9 (C-7), 24.8, 24.8, 24.7, 24.6 (2× C-2″a, 2× C-2″b), 22.8
(C-13), 14.3 (C-14). 31P NMR (162 MHz, CDCl3): δ [ppm] 147.7.
O′-1-(9H-Fluoren-9-yl)methoxy-O-(4-octadecanoylbenzyl)-N,N-
diisopropylphosphoramidite (17b). According to general procedure
5, using (FmO)P(Ni-Pr2)2 (522 mg, 1.22 mmol, 1.8 equiv) and (4-
(hydroxymethyl)phenyl)heptadecanylketone 13b (250 mg, 667 μmol,
1.0 equiv) in CH2Cl2/THF (8/10 mL). 4,5-Dicyanoimidazole (0.25
M in CH3CN, 3.50 mL, 875 μmol, 1.3 equiv). Yield: 546 mg (780
μmol, quant.; colorless solid). The compound was contaminated with
22% (31P NMR) phosphoramidite (FmO)2PN(i-Pr)2. TLC: Rf = 0.8
(PE/ethylacetate 2:1 + 1% NEt3, VSS). 1H NMR (400 MHz, CDCl3):
δ [ppm] 7.93 (d, J = 8.3 Hz, 2H, 3-H), 7.79−7.75 (m, 2H, Fm),
7.69−7.56 (m, 2H, Fm), 7.47−7.34 (m, h H, 2-H, Fm), 7.33−7.24
(m, 2H, Fm), 4.76 (dd, J = 13.6, 8.2 Hz, 1H, 1-CH2,aO), 4.69 (dd, J =
13.6, 8.5 Hz, 1H, 1-CH2,bO), 4.25−4.15 (m, 1H, Fm-2′-H), 4.11−
3.96 (m, 1H, Fm-1′-Ha), 3.90−3.77 (m, 1H, Fm-1′-Hb), 3.75−3.61
(mc, 2H, 1″a-H), 2.94 (t, J = 7.4 Hz, 2H, 6-H), 1.73 (p, J = 7.4 Hz,
2H, 7-H), 1.43−1.17 (m, 40H, 8-H−21-H, 2″a-H, 2″b-H), 0.98−0.83
(m, 3H, 22-H). 13C NMR (151 MHz, CDCl3): δ [ppm] 200.4 (C-5),
145.1, 145.0, 144.8 (2× Fm, C-1), 141.5 (Fm), 141.4 (Fm), 136.2 (C-
4), 128.3 (C-2), 127.6 (Fm), 127.5 (Fm), 127.0 (Fm), 127.0 (Fm),
126.9 (C-3), 125.5 (Fm), 125.3 (Fm), 120.0 (Fm), 119.9 (Fm), 66.2
(d, 2JCP = 17.1 Hz, Fm-1′), 64.8 (d, 2JCP = 18.2 Hz, 1-CH2O), 49.3 (d,
3JCP = 7.7 Hz, Fm-2′), 43.3 (d, 2JCP = 12.7 Hz, 2× C-1″a), 38.8 (C-6),
4
C-2‴), 129.4 (C-3″), 128.9 (d, JCP = 6.3 Hz, C-2″), 127.1 (C-2′),
122.8 (d, 4JCP = 2.5 Hz, C-3‴), 112.1 (C-5), 90.8 (C-1′), 87.2 (d, 3JCP
= 8.9 Hz, C-4′), 70.5 (m, 1″-CH2O), 70.1 (m, 1‴-CH2O), 67.9 (d,
2JCP = 5.8 Hz, C-5′), 39.6 (C-6″), 35.0 (C-6‴), 33.1 (C-20″), 32.7
(C-10″), 30.8, 30.8, 30.8, 30.7, 30.7, 30.5, 30.4, 29.9 (C-8‴, C-9‴, C-
8″−19″), 25.9 (C-7‴), 25.6 (C-7″), 23.7 (C-21″*), 23.6 (C-10‴*),
14.4 (C-22″), 14.4 (C-11‴), 12.5 (5-CH3).31P NMR (243 MHz,
MeOH-d4): δ [ppm] −11.83 (d, 2JPP = 20.0 Hz, Pγ), −13.19 (d, 2JPP
=
2
17.0 Hz, Pα), −23.81 (t, JPP = 18.5 Hz, Pβ). HRMS (ESI−): m/z =
calcd, 1037.4095 [M − H]−; found, 1037.4102 [M − H]−.
(AB-C17H35,kb-C9H19)-d4TTP (9az). According to general procedure
4 using (n-Bu4N)2·d4TDP (100 μmol, 1.0 equiv), phosphoramidite
11az (132 mg, 169 μmol, 1.7 equiv), 4,5-dicyanoimidazole (0.25 M in
CH3CN, 680 μL, 170 μmol, 1.7 equiv), and t-BuOOH (5.5 M in n-
decane, 31.0 μL, 170 μmol, 1.7 equiv). Yield: 51 mg (46 μmol, 46%;
colorless cotton). RP18-HPLC: tR [min] (λmax) = 21.4 (249 nm),
32.1 (C-20), 31.7, 29.8 (m), 29.7, 29.7, 29.6, 29.5 (C-8, C-9, C-10, C-
11, C-12, C-13, C-14, C-15, C-16, C-17, C-18, C-19), 24.9, 24.8, 24.8,
24.7, 24.6 (C-7, 2× C-2″a, 2× C-2″b), 22.9, 22.8 (C-21), 14.3 (C-
22). 31P NMR (162 MHz, CDCl3): δ [ppm] 147.7. HRMS (ESI+, m/
z): calcd, 700.4855 [M + H]+; found, 700.4853 [M + H]+.
1
Method B. H NMR (600 MHz, MeOH-d4): δ [ppm] 7.92 (dd, J =
8.4, 1.7 Hz, 2H, 3″-H), 7.67 (mc, 1H, 6-H), 7.43 (dd, J = 8.3, 3.1 Hz,
L
https://dx.doi.org/10.1021/acs.jmedchem.0c01293
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
2H, 2″-H), 7.39 (dd, J = 8.5, 3.1 Hz, 2H, 2‴-H), 7.02 (dd, J = 8.6, 2.5
Hz, 2H, 3‴-H), 6.94−6.91 (mc, 1H, 1′-H), 6.50−6.45 (mc, 1H, 3′-H),
Hz, Pβ). HRMS (ESI+): m/z calcd, 1122.4735 [M − H]−; found,
1122.4751 [M − H]−.
3
5.83−5.75 (mc, 1H, 2′-H), 5.21 (d, JHP = 8.2 Hz, 2H, 1″-CH2O),
γ-Ketobenzyl-NTPs (10). (Kb-C9H19)-d4TTP 10a (d4T). D4TTP
prodrug 9ax (5 mg, 5.4 μmol, 1.0 equiv) dissolved in water (200 μL)
and PLE (3 mg/mL in 50 mM PB, 0.3 → 0.8 mL, 41 U) were
incubated for 11 h at 37 °C in a thermomixer. The solvent was
removed by freeze-drying. The residue was purified by using
automated Flash-RP-CC (H2O/CH3CN 90:10 → 30:70). Yield:
after freeze-drying, 2.2 mg (2.9 μmol, 53%) were obtained as a
colorless solid. RP18-HPLC: tR [min] (λmax) = 13.4 (253 nm),
method A. 1H NMR (600 MHz, MeOH-d4): δ [ppm] 7.94 (d, J = 8.3
Hz, 2H, 3″-H), 7.71 (mc, 1H, 6-H), 7.58 (d, J = 8.2 Hz, 2H, 2″-H),
6.93 (mc, 1H, 1′-H), 6.54 (mc, 1H, 3′-H), 5.81 (mc, 1H, 2′-H), 5.14
(d, 3JHP = 6.0 Hz, 2H, 1″-CH2O), 4.97 (mc, 1H, 4′-H), 4.32−4.26 (m,
1H, 5′-Ha), 4.19−4.15 (m, 1H, 5′-Hb), 3.00 (t, J = 7.4 Hz, 2H, 6″-H),
1.95−1.87 (m, 3H, 5-CH3), 1.69 (p, J = 7.4 Hz, 2H, 7″-H), 1.44−1.23
(m, 12H, 8″-H−13″-H), 0.90 (t, J = 7.0 Hz, 3H, 14″-H). 13C NMR
(151 MHz, MeOH-d4): δ [ppm] 202.8 (C-5″, HMBC), 166.7 (C-4,
HMBC), 145.8 (C-1″, HMBC), 138.8 (C-4″), 135.6 (C-3′, HSQC),
129.2 (C-3″), 128.3 (C-2″), 126.4 (C-2′, HSQC), 112.2 (C-5,
HMBC), 90.3 (C-1′, HSQC), 87.0 (C-4′, HSQC), 67.5 (1″-CH2O,
HSQC), 67.3 (C-5′, HSQC), 39.5 (C-6″), 33.0 (C-12″), 30.6, 30.6,
30.4, 30.4 (C-8″−11″), 25.7 (C-7″), 23.7 (C-13″*), 14.4 (C-14″),
12.5 (5-CH3). 31P NMR (243 MHz, MeOH-d4): δ [ppm] −10.03 (d,
3
5.18 (d, JHP = 8.7 Hz, 2H, 1‴-CH2O), 4.96−4.93 (mc, 1H, 4′-H),
4.29 (ddd, J = 11.6, 6.9, 3.2 Hz, 1H, 5′-Ha), 4.20 (ddd, J = 11.6, 5.5,
3.1 Hz, 1H, 5′-Hb), 3.00 (mc, 2H, 6″-H), 2.57 (mc, 2H, 6‴-H), 1.88
(s, 3H, 5-CH3), 1.77−1.66 (m, 4H, 7″-H, 7‴-H), 1.48−1.25 (m, 40H,
8″-H−13″-H, 8‴-H−21‴-H), 0.90 (mc, 6H, 14″-H, 22‴-H). 13C
NMR (151 MHz, MeOH-d4): δ [ppm] 202.4 (C-5), 173.7 (C-5‴),
166.5 (C-4), 152.8 (C-2), 152.4 (C-4‴), 142.6 (C-1″, HMBC), 138.7
(C-6), 138.0 (C-4″), 135.8 (C-3′), 134.9 (C-1‴, HMBC), 130.5 (d,
4
4JCP = 5.4 Hz, C-2‴), 129.4 (C-3″), 128.9 (d, JCP = 6.3 Hz, C-2″),
5
127.2 (C-2′), 122.8 (d, JCP = 2.5 Hz, C-3‴), 112.1 (C-5), 90.8 (C-
3
1′), 87.2 (d, JCP = 8.9 Hz, C-4′), 70.5 (m, 1″-CH2O), 70.1 (m, 1‴-
CH2O), 67.9 (d, 2JCP = 6.1 Hz, C-5′), 39.6 (C-6″), 35.0 (C-6‴), 33.1
(C-12″, C-20‴), 30.8, 30.8, 30.8, 30.7, 30.7, 30.7, 30.6, 30.5, 30.5,
30.4, 30.2 (C-8‴−C-19‴, C-8″−11″), 26.0 (C-7‴), 25.6 (C-7″), 23.8
(C-21‴*), 23.7 (C-13″*), 14.5 (C-22″), 14.5 (C-14‴), 12.5 (5-CH3).
31P NMR (243 MHz, MeOH-d4): δ [ppm] −11.82 (d, 2JPP = 20.1 Hz,
2
Pγ), −13.17 (mc, Pα), −23.80 (t, JPP = 18.5 Hz, Pβ). HRMS (ESI−):
m/z calcd, 1079.4564 [M − H]−; found, 1079.4610 [M − H]−.
(AB-C17H35,kb-C17H35)-d4TTP (9bz). According to general proce-
dure 4 using (n-Bu4N)2·d4TDP (88 mg, 100 μmol, 1.0 equiv),
phosphoramidite 11bz (136 mg, 153 μmol, 1.5 equiv), 4,5-
dicyanoimidazole (0.25 M in CH3CN, 612 μL, 153 μmol, 1.5
equiv), and t-BuOOH (5.5 M in n-decane, 28.0 μL, 150 μmol, 1.5
equiv). Yield: 15 mg (12.4 μmol) of the NH4 form and 32 mg (19.8
μmol) of the n-Bu4N form as a colorless solid. Overall yield: 63%.
2
2JPP = 16.7 Hz, Pγ), −10.25 (d, JPP = 16.6 Hz, Pα), −20.43 (m, Pβ).
HRMS (ESI−): m/z calcd, 707.1614 [M − H]−; found, 707.1554 [M
− H]−.
(Kb-C17H35)-d4TTP (10b). According to general procedure 6, using
(n-Bu4N)2·d4TDP 4 (95.0 μmol, 1.0 equiv), phosphoramidite 19b
(100 mg, 143 μmol, 1.5 equiv), 4,5-dicyanoimidazol (0.25 M in
CH3CN, 570 μL, 1.5 equiv), and t-BuOOH (5.5 M in n-decane, 26.0
μL, 143 μmol, 1.5 equiv). Yield: after freeze-drying, 22.1 mg (25.9
μmol, 27%) were obtained as a colorless solid. RP18-HPLC: tR [min]
(λmax) = 16.4 (253 nm), Method B. 1H NMR (600 MHz, MeOH-d4):
δ [ppm] 7.95 (d, J = 8.3 Hz, 2H, 3″-H), 7.69 (mc, 1H, 6-H), 7.57 (d, J
= 8.2 Hz, 2H, 2″-H), 6.93 (mc, 1H, 1′-H), 6.53 (mc, 1H, 3′-H), 5.82
(mc, 1H, 2′-H), 5.14 (d, 3JHP = 6.4 Hz, 2H, 1″-CH2O), 4.97 (mc, 1H,
4′-H), 4.30 (ddd, J = 11.7, 6.8, 3.3 Hz, 1H, 5′-Ha), 4.18 (ddd, J =
11.6, 5.4, 3.1 Hz, 1H, 5′-Hb), 3.01 (t, J = 7.3 Hz, 2H, 6″-H), 1.90 (s,
3H, 5-CH3), 1.69 (p, J = 7.3 Hz, 2H, 7″-H), 1.43−1.22 (m, 28H, 8″-
H−21″-H), 0.90 (t, J = 7.0 Hz, 3H, 22″-H). 13C NMR (151 MHz,
MeOH-d4): δ [ppm] 202.7 (C-5″), 166.6 (C-4), 152.8 (C-2), 145.5
(C-1″, HMBC), 138.7 (C-6), 137.4 (C-4″), 136.0 (C-3′), 129.2 (C-
3″), 128.3 (C-2″), 127.0 (C-2′), 112.0 (C-5), 90.9 (C-1′), 87.3 (d,
1
RP18-HPLC: tR [min] (λmax) = 25.4 (265 nm), method B. H NMR
(600 MHz, THF-d8): δ [ppm] 10.12−9.95 (m, 1H, NH), 8.04 (mc,
1H, 6-H), 7.93 (d, J = 8.1 Hz, 2H, 3″-H), 7.68−7.49 (m, 4H, 2″-H,
2‴-H), 7.03 (d, J = 8.1 Hz, 2H, 3‴-H), 6.98−6.93 (mc, 1H, 1′-H),
6.74−6.62 (mc, 1H, 3′-H), 5.63−5.55 (m, 1H, 2′-H), 5.47−5.23 (m,
1″-CH2O, 1‴-CH2O) 4.84−4.79 (mc, 1H, 4′-H), 4.56−4.31 (m, 1H,
5′-Ha), 4.17−3.99 (m, 1H, 5′-Hb), 3.54−3.38 (m, 16H, n-Bu4N), 2.98
(t, J = 7.3 Hz, 2H, 6″-H), 2.56 (t, J = 7.5 Hz, 2H, 6‴-H), 2.03−1.93
(m, 3H, 5-CH3), 1.83−1.64 (m, 16H, n-Bu4N), 1.52−1.29 (m, 72H,
8″-H−21″-H, 8‴-H−21‴-H n-Bu4N), 0.98 (t, J = 7.3 Hz, 24H),
0.95−0.90 (m, 6H, 22″-H, 22‴-H). 13C NMR (151 MHz, THF-d8): δ
[ppm] 199.3 (C-5), 172.1 (C-5‴), 152.1 (C-2), 151.7 (C-4‴), 138.5
4
(C-6), 137.6 (C-4″), 130.2 (d, JCP = 4.8 Hz, C-2‴), 128.7 (C-3″),
128.6 (d, 4JCP = 5.0 Hz, C-2″), 125.9 (C-2′), 122.2 (C-3‴), 111.3 (C-
5), 90.1 (C-1′), 88.1 (d, 3JCP = 7.9 Hz, C-4′), 59.2 (n-Bu4N), 39.2 (C-
6″), 34.9 (C-6‴), 33.1 (C-20″, C-20‴), 30.8, 30.8, 30.7, 30.5, 30.5,
30.3 (C-8‴−C-19‴, C-8″−19″), 25.3 (C-7″*), 25.0 (C-7‴*), 23.7
(C-21″*, C-21‴*, n-Bu4N), 20.8 (n-Bu4N), 14.6, 14.4 (n-Bu4N, C-
22″, C-22‴), 12.7 (5-CH3). 31P NMR (162 MHz, THF-d8): δ [ppm]
−11.90 (d, 2JPP = 20.8 Hz, Pγ), −12.97 (d, 2JPP = 19.8 Hz, Pα), −21.85
(m, Pβ). HRMS (ESI−): m/z calcd, 1191.5816 [M − H]−; found,
1191.5860 [M − H]−.
3JCP = 9.3 Hz, C-4′), 68.1 (d, 2JCP = 5.5 Hz, 1″-CH2O), 67.7 (d, 2JCP
=
5.9 Hz, C-5′), 39.5 (C-6″), 33.1 (C-20″), 30.8, 30.8, 30.8, 30.7, 30.7,
30.7, 30.5, 30.4 (C-8″−13″), 25.7 (C-7″), 23.7 (C-21″), 14.4 (C-
22″), 12.5 (5-CH3). 31P NMR (243 MHz, MeOH-d4): δ [ppm] =
−11.20 (d, 2JPP = 19.5 Hz, Pγ), −11.52 (d, 2JPP = 19.4 Hz, Pα), −22.53
2
(t, JPP = 19.5 Hz, Pβ). HRMS (ESI−): m/z calcd, 819.2788 [M −
(AB-C17H35,kb-C9H19)-ATP (9ay). According to general procedure 4
using ADP·(n-Bu4N)2 (71 mg, 73.0 μmol, 1.0 equiv) in DMF (1 mL)
and phosphoramidite 11az (100 mg, 109 μmol, 1.5 equiv.) in CH2Cl2
(2 mL), BTT (0.3 M in CH3CN, 487 μL, 146 μmol, 2.0 equiv), and t-
BuOOH (5.5 M in n-decane, 27.0 μL, 146 μmol, 2.0 equiv) at 0 °C.
The residue was a several times purified by auto Flash RP-CC (H2O/
CH3CN), transformed via ion-exchange into the triethylammonium
form and purified by auto Flash RP-CC (H2O/CH3CN). Yield: after
freeze-drying, 31 mg (24.5 μmol, 34%) were obtained as colorless
strong hygroscopic cotton. RP18-HPLC: tR [min] (λmax) = 20.0 (249
H]−; found, 819.2786 [M − H]−.
(Kb-C9H19)-TTP (10c). According to general procedure 6, using (n-
Bu4N)2·dTDP (46 μmol, 1.0 equiv), phosphoramidite 17a (39 mg, 60
μmol, 1.3 equiv), 4,5-dicyanoimidazol (0.25 M in CH3CN, 0.20 mL,
51 μmol, 1.1 equiv), and t-BuOOH (5.5 M in n-decane, 11.0 μL, 60
μmol, 1.3 equiv). Yield: after freeze-drying, 23.1 mg (30.3 μmol, 66%)
were obtained as a colorless solid. RP18-HPLC: tR [min] (λmax) =
13.7 (253 nm), Method A. 1H NMR (600 MHz, MeOH-d4): δ [ppm]
7.94 (d, J = 8.3 Hz, 2H, 3″-H), 7.82 (s, 1H, 6-H), 7.57 (d, J = 8.1 Hz,
3
2H, 2″-H), 6.29 (mc, 1H, 1′-H), 5.14 (d, JHP = 6.4 Hz, 2H, 1″-
1
nm), method B. H NMR (400 MHz, MeOH-d4/CDCl3 2:1 v/v): δ
CH2O), 4.64−4.58 (m, 1H, 3′-H), 4.31−4.25 (m, 1H, 5′-Ha), 4.23−
4.15 (m, 1H, 5′-Hb), 4.00 (mc, 1H, 4′-H), 3.00 (t, J = 7.3 Hz, 2H, 6″-
H), 2.25 (ddd, J = 13.5, 7.4, 6.1 Hz, 1H, 2′-Ha), 2.19 (ddd, J = 13.5,
6.2, 3.6 Hz, 1H, 2′-Hb), 1.92 (d, J = 1.2 Hz, 3H, 5-CH3), 1.69 (p, J =
7.3 Hz, 2H, 7″-H), 1.42−1.25 (m, 12H, 8″-H−14″-H), 0.90 (t, J =
7.0 Hz, 3H, 14″-H). 13C NMR (151 MHz, MeOH-d4): δ [ppm] 202.7
(C-5), 166.5 (C-4), 152.4 (C-2), 145.5 (C-1″), 138.2 (C-6), 137.4
(C-4″), 129.2 (C-3″), 128.2 (C-2″), 111.9 (C-5), 87.4 (C-4′), 86.0
[ppm] 8.67 (s, 1H, 8-H), 8.16 (s, 1H, 2-H), 7.88−7.78 (m, 2H, 3″-
H), 7.41−7.24 (m, 4H, 2″-H, 2‴-H), 7.01−6.88 (m, 3H, 3‴-H), 6.01
(mc, 1H, 1′-H), 5.20−5.09 (m, 4H, 1″-CH2O, 1‴-CH2O), 4.50−4.42
(m, 2H, 2′-H, 3′-H), 4.42−4.34 (m, 1H, 4′-H), 4.32−4.23 (m, 2H,
5′-H), 3.01−2.85 (mc, 2H, 6″-H), 2.53 (t, J = 7.4 Hz, 2H, 6‴-H),
1.79−1.59 (m, 4H, 7″-H, 7‴-H), 1.48−1.17 (m, 40H, 8″-H−13″-H,
8‴-H−21‴-H), 0.85 (t, J = 6.8 Hz, 6H, 14″-H, 22‴-H). 31P NMR
2
2
(162 MHz, MeOH-d4/CDCl3 2:1 v/v): δ [ppm] −11.27 (d, JPP
=
(C-1′), 72.0 (C-3′), 68.1, (d, JCP = 5.3 Hz, 1″-CH2O), 66.6 (C-5′),
2
2
17.2 Hz, Pγ), −12.86 (d, JPP = 15.7 Hz, Pα), −22.65 (t, JPP = 16.6
40.7 (C-2′), 39.5 (C-6″), 33.1 (C-12″), 30.7, 30.6, 30.4, 30.4 (C-8″−
M
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11″), 25.7 (C-7″), 23.7 (C-13″), 14.4 (C-14″).31P NMR (243 MHz,
addition of 100 μL of dioxane. The resulting suspension was kept on
ice for 5 min followed by centrifugation for 5 min (13,000 rpm). The
supernatants were filtered (Chromafil RC-20/15 MS, 0.2 μm) and
stored in liquid nitrogen. (c) For 9by, the reactions were stopped by
the addition of 100 μL of THF (HPLC grade). The resulting
suspension was kept on ice for 5 min followed by centrifugation for 5
min (13,000 rpm). The supernatants were filtered (Chromafil AO-
20/3, 0.2 μm) and stored in liquid nitrogen. (d) For 9bz, the
reactions were stopped by the addition of 100 μL THF (HPLC
grade). The resulting suspension was kept on ice for 5 min, followed
by defrosting, ultrasonication for 10 min, and by centrifugation for 5
min (13,000 rpm). The supernatant was directly injected to HPLC.
The samples were defrosted and 30−50 μL were subjected to HPLC
analysis. The calculation of t1/2 was performed analogously to that for
the chemical hydrolysis studies.
MeOH-d4): δ [ppm] −11.12 (d, 2JPP = 19.0 Hz, Pγ), −11.40 (d, 2JPP
=
2
19.2 Hz, Pα), −22.31 (t, JPP = 19.1 Hz, Pβ). HRMS (ESI−): m/z
calcd, 726.1642 [M − H]−; found, 725.1645 [M − H]−.
(Kb-C9H19)-dATP (10d). According to general procedure 6, using
(n-Bu4N)2-dADP (50.0 μmol, 1.0 equiv) in DMF (1 mL),
phosphoramidite 17a (49.0 mg, 75.0 μmol, 1.5 equiv), 4,5-
dicyanoimidazole (0.25 M in CH3CN, 300 μL, 75.0 μmol, 1.5
equiv), and t-BuOOH (5.5 M in n-decane, 14.0 μL, 75.0 μmol, 1.5
equiv). Yield: after freeze-drying, 14.5 mg (18.9 μmol, 38%) were
obtained as colorless cotton. RP18-HPLC: tR [min] (λmax) = 13.3
(253 nm), Method A. 1H NMR (600 MHz, MeOH-d4): δ [ppm] 8.75
(s, 1H, 8-H), 8.17 (s, 1H, 2-H), 7.83 (d, J = 8.3 Hz, 2H, 3″-H), 7.50
(d, J = 8.1 Hz, 2H, 2″-H), 6.39 (t, J = 6.3 Hz, 1H, 1′-H), 5.13 (d, J =
6.4 Hz, 2H, 1″-CH2O), 4.76−4.66 (mc, 1H, 3′-H), 4.42−4.34 (m,
1H, 5′-Ha), 4.31−4.26 (m, 1H, 5′-Hb), 4.18−4.14 (m, 1H, 4′-H),
2.93 (t, J = 7.2 Hz, 1H, 6″-H), 2.64−2.57 (m, 1H, 2′-Ha), 2.49−2.42
(m, 1H, 2′-Hb), 1.65 (p, J = 7.2 Hz, 2H, 7″-H), 1.40−1.24 (m, 12H,
8″-H−13″), 0.89 (t, J = 6.9 Hz, 3H, 14″-H). 13C NMR (151 MHz,
MeOH-d4): δ [ppm] 202.4 (C-5″), 152.8 (C-6, HMBC), 149.1 (C-
Preparation of Cell Extracts. Human CD4+ T-lymphocyte CEM
cells were grown in RPMI-1640-based cell culture medium to a final
density of ∼3 × 106 cells/mL. Then, cells were centrifuged for 10 min
at 1250 rpm at 4 °C, washed twice with cold PB, and the pellet was re-
suspended at 108 cells/mL and sonicated (Hielscher Ultrasound
Techn., 100% amplitude, 3·times for 10 s) to destroy cell integrity.
The resulting cell suspension was then centrifuged at 10,000 rpm to
remove cell debris, and the supernatant was divided in aliquots before
being frozen at −80 °C and used.
Anti-HIV Activity Assay. Inhibition of HIV-1(IIIB)- and HIV-
2(ROD)-induced cytopathicity in wild-type CEM/0 and TK-deficient
CEM/TK− cell cultures was measured in microtiter 96-well plates
containing ∼3 × 105 CEM cells/mL infected with 100 CCID50 of
HIV per milliliter and containing appropriate dilutions of the test
compounds. After 4−5 days of incubation at 37 °C 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%.
3
4), 145.2 (d, JCP = 8.9 Hz, C-1″), 143.0 (C-8), 137.2 (C-4″), 129.1
(C-3″), 128.2 (C-2″), 119.2 (C-5), 88.0 (d, 3JCP = 9.0 Hz, 4′-H), 85.6
(C-1′), 71.9 (3′-H), 68.0 (d, 2JCP = 5.1 Hz, 1″-CH2O), 66.4 (d, 2JCP
=
5.9 Hz, C-5′), 42.1 (C-2′), 39.5 (C-6″), 33.1 (C-12″), 30.7, 30.6,
30.4, 30.4 (C-8″−11″), 25.6 (C-7″), 23.7 (C-13″), 14.4 (C-14″). 31P
2
NMR (162 MHz, MeOH-d4): δ [ppm] −10.91 (d, JPP = 17.8 Hz,
2
2
Pγ), −11.19 (d, JPP = 17.8 Hz, Pα), −21.75 (t, JPP = 17.8 Hz, Pβ).
HRMS (ESI−): m/z calcd, 734.1757 [M − H]−; found, 734.1794 [M
− H]−.
Chemical Hydrolysis of Prodrugs of γ-Ketobenzyl-(9)-d4TTP
as Well as the γ-Ketobenzyl-d4TTPs (10). Stock solutions (50
mM in DMSO) of compounds 9, 10 were prepared. After dilution of
11 μL of the stock solution with 100 μL of Milli-Q water and 189 μL
of DMSO to 1.9 mM hydrolysis solutions, the reaction was started by
the addition of 300 μL of phosphate buffer saline (PB, 50 mM, pH
7.3). The solution was incubated at 37 C in a thermomixer. An initial
aliquot (25 μL) was taken directly and analyzed by analytical HPLC
at 265−266 nm. Further, aliquots were taken for monitoring the
kinetic hydrolysis. The exponential decay curves (pseudo-first order)
based on absolute integral values were calculated with commercially
available software (OriginPro 9.0G) and yielded the half-lives t1/2 of
the prodrugs via one determination.
Enzymatic Hydrolysis of Compounds (9) with PLE. The
appropriate 50 mM DMSO stock solution (20 μL) were diluted to 6.0
mM by the addition of 83.3 μL of DMSO as well as 83.3 μL of
Millipore water. Furthermore, 140 μL of the 6.0 mM solution was
diluted with 105 μL of DMSO and 700 μL of 50 mM phosphate
buffer. The reaction was started by the addition of 52.5 μL of PLE in
PB buffer (3 mg/mL) and the mixture was incubated at 37 °C in a
thermomixer. At different times, aliquots (125 μL) were taken and the
sample was directly frozen in liquid nitrogen. The solution was
defrosted followed by ultrasonication, centrifugation, filtration, and
stored. Samples of 50−80 μL were subjected to HPLC analysis. The
calculation of t1/2 was performed analogously to that for the chemical
hydrolysis studies.
Enzyme-Catalyzed Hydrolysis of Prodrugs of γ-Ketobenzyl-
Modified d4TTPs (9) and γ-Modified NTPs 10 in CEM Cell
Extracts. The appropriate 50 mM DMSO stock solution (18 μL) was
diluted to a 6.0 mM hydrolysis solution by the addition of 132.0 μL
DMSO. Different samples (7−10), including 10 μL of water and 10
μL of hydrolysis solution, were prepared in 2 mL Eppendorf vials. The
reaction was started by the addition of 50 μL of the human CEM cell
extract and the mixture was incubated at 37 °C for different time
periods. The work-up depended on the particular compound because
of solubility problems using CH3OH for longer chain lengths: (a) For
9ax, 10a, and 10c, the reactions were stopped by the addition of 150
μL of MeOH. The resulting suspension was kept on ice for 5 min
followed by centrifugation for 5 min (13,000 rpm). The supernatants
were filtered (Chromafil RC-20/15 MS, 0.2 μm) and stored in liquid
nitrogen. (b) For 9az and 10b, the reactions were stopped by the
Primer-Extension Reactions. HIV-RT and human polymerase β
and γ were obtained from Roboklon and human DNA polymerase α
was obtained from Chimerx. The primer and template for the
radiolabeled experiment was purchased from Life Technologies. The
fluorescently labeled primer was purchased from Microsynth. The size
of the gels was 450 mm × 200 mm × 0.4 mm, adjusted to the
electrophoresis chamber.
Primer-sequence for the 32P-labeled primer extension experiment:
3′-T TGG ATA GGA GGA AGT CCT GGT TGC-5′
Primer-sequence for the Cy3-fluorescent primer extension experi-
ment:
5′-Cy3-CGTTG GTCCT GAAGG AGGAT AGGTT-3′
Template-sequence for both experiments:
5′-ACA GAA ACC TAT CCT CCT TCA GGA CCA ACG-3′
The primer extension assays were performed under the following
conditions:
(A) For the 32P-labeled primer extension experiment: the primer
was labeled using [γ32P]-ATP according to standard techniques. After
5 min incubation at 95 °C in 20 mM Tris-HCl (pH 7.6) and 50 mM
NaCl, the hybridization/annealing of the primer to the template
strand was achieved by a cooling phase from 95 to −20 °C over 3 h.
The spots were visualized by phosphorimaging.
(B) For the Cy3-labeled primer extension experiment: after 5 min
incubation at 95 °C in 20 mM Tris-HCl (pH 7.6) and 50 mM NaCl,
the hybridization/annealing of the primer to the template strand was
achieved by a cooling phase from 95 to −20 °C over 2 h. The results
were visualized by fluorescence imaging.
(1a) For the HIV-RT assay (radiolabeled): the final assay solution
(20 μL) consists of 50 mM Tris-HCl (pH 8.6 at 22 °C), 10 mM
MgCl2, 40 mM KCl, dNTPs 66 μM, HIV-RT 0.2 U, and duplex 0.32
μM in a reaction volume of 20 μL, incubated 37 °C for 15 min. The
reaction was stopped by heating up to 80 °C for 3 min. The assay
mixture was loaded on a denaturating PAGE (15%).
(1b) For the HIV-RT assay (fluorescent): the final assay solution
(10 μL) consists of 50 mM Tris-HCl (pH 8.6 at 22 °C), 10 mM
MgCl2, 40 mM KCl, dNTPs 250 μM, HIV-RT 6 U, and duplex 0.20
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μM in a reaction volume of 10 μL, incubated 37 °C for 30 min and 80
°C for 3 min. The assay mixture was loaded on a denaturating PAGE
(15%; 50 mA, 45 W for 3 h).
(2a) For the human DNA pol β assay (radiolabeled): the final assay
solution (10 μL) consists of 50 mM Tris-HCl (pH 8.7), 10 mM
MgCl2, 100 mM KCl, 1.0 mM dithiothreitol, 0.4 mg/mL of bovine
serum albumin, 15% glycerol, dNTPs 66 μM, DNA polymerase β 6 U,
and duplex 0.32 μM in a reaction volume of 10 μL, incubated 37 °C
for 60 min and 80 °C for 3 min. The assay mixture was loaded on a
denaturating PAGE (15%; 50 mA, 45 W for 3 h).
(2b) For the human DNA pol β assay (fluorescent): the final assay
solution (10 μL) consists of 50 mM Tris-HCl (pH 8.7), 10 mM
MgCl2, 100 mM KCl, 1.0 mM dithiothreitol, 0.01% (w/v) of bovine
serum albumin, 15% glycerol, dNTPs 250 μM, DNA polymerase β 7.5
U, and duplex 0.2 μM in a reaction volume of 10 μL, incubated 37 °C
for 60 min and 80 °C for 3 min. The assay mixture was loaded on a
denaturating PAGE (15%; 50 mA, 45 W for 3 h).
(3) For the human DNA pol α assay (fluorescent): the final assay
solution (25 μL) consists of 60 mM Tris-HCl (pH 8), 5 mM MgOAc,
1.0 mM dithiothreitol, 0.1 mM spermine, 0.01% (w/v) of bovine
serum albumin, dNTPs 250 μM, DNA polymerase α 2 U, and duplex
0.20 μM in a reaction volume of 25 μL, incubated 37 °C for 5 min
without dNTPs, then incubated 60 min at 37 °C and 80 °C for 3 min.
The assay mixture was loaded on a denaturating PAGE (15%; 50 mA,
45W for 3 h).
Jan Balzarini − Laboratory of Virology and Chemotherapy,
Department of Microbiology and Immunology and
Transplantation, Rega Institute for Medical Research, KU
Leuven, B-3000 Leuven, Belgium
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01293
Author Contributions
C.M. headed the project; T.N. performed the chemical
synthesis; T.D.d.O. and S.W. contributed with the biochemical
assays, D.S. and J.B. carried out the antiviral testing of the
synthesized compounds. All the authors were involved in the
preparation of the manuscript.
Funding
The work conducted by C.M. was supported by the Deutsche
Forschungsgemeinschaft (DFG; Me1161/15-1) and that of
D.S. and J.B. has been supported by the KU Leuven (GOA 15/
19 TBD).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(4) For the human DNA pol γ assay (fluorescent): the final assay
solution (25 μL) consists of 60 mM Tris-HCl (pH 8), 5 mM MgOAc,
1.0 mM dithiothreitol, 0.1 mM spermine, 0.01% (w/v) of bovine
serum albumin, dNTPs 250 μM, DNA polymerase γ 4U, and duplex
0.20 μM in a reaction volume of 25 μL, incubated 37 °C for 5 min
without dNTPs, then incubated 60 min at 37 °C and 80 °C for 3 min.
The assay mixture was loaded on a denaturating PAGE (15%; 50 mA,
45 W for 3 h).
■
The authors are grateful to Lizette van Berckelaer, Ria van
Berwaer, Kristien Minner, Sandra Claes, and Evelyne Van
Kerckhove for excellent technical assistance.
DEDICATION
■
Dedicated to Prof. Dr. Dr. h.c. mult. Wittko Francke on the
occasion of his 80th birthday.
ASSOCIATED CONTENT
* Supporting Information
■
sı
ABBREVIATIONS
■
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01293.
d4T, 3′-deoxy-2′,3′-didehydrothymidine; NTP, nucleoside
triphosphate; NDP, nucleoside diphosphate; NMP, nucleoside
monophosphate
HPLC results and NMR-spectra of representative
compounds (PDF)
REFERENCES
Molecular formula strings (CSV)
■
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