γ-Non-Symmetrically Dimasked TriPPPro Prodrugs as Potential Antiviral Agents against HIV

Article abstract of DOI:10.1002/cmdc.202000712

Nucleoside analogue reverse transcriptase inhibitors (NRTI) and nucleoside analogue monophosphate prodrugs are used in combination antiretroviral therapy (cART). The design of antivirally active nucleoside triphosphate prodrugs is a recent and an important advancement in the field of nucleoside analogue drug development. Here, we report on TriPPPro-derivatives of nucleoside analogue triphosphates (NTPs) that comprised two different acyloxybenzyl-masks at the γ-phosphate of the NTP aiming to achieve the metabolic bypass. Thus, γ-non-symmetrically dimasked TriPPPro-compounds (γ-(AB,ab)-d4TTPs) were synthesized and they proved to be active against HIV-1 and HIV-2 in cultures of infected wild-type human CD4⁺ T-lymphocyte (CEM/0) cells and more importantly also in thymidine kinase-deficient CD4⁺ T-cells (CEM/TK-). From hydrolysis studies both in phosphate buffer (PB, pH 7.3) and CEM cell extracts, there was surprisingly no differentiation in the cleavage of the two acyloxybenzyl prodrug-masks. However, if within one of the two acyloxybenzyl groups a short PEG-type methoxytriglycol group was introduced, the “standard” acyloxybenzyl-mask was cleaved with high preference.

Full text of DOI:10.1002/cmdc.202000712

Accepted Article
Title: gamma-Non-symmetrically-dimasked TriPPPro-prodrugs as
potential antivirals against HIV
Authors: Chenglong Zhao, Xiao Jia, Dominique Schols, Jan Balzarini,
and Chris Meier
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000712
Link to VoR: https://doi.org/10.1002/cmdc.202000712
01/2020

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
-Non-symmetrically-dimasked TriPPPro-prodrugs as potential
antivirals against HIV
Chenglong Zhao,[a] Xiao Jia,[a] Dominique Schols,[b] Jan Balzarini,[b] Chris Meier*[a]
[a]
Dr. C. L. Zhao,. Dr. X. Jia, Prof. Dr. C. Meier
Organic Chemistry, Department of Chemistry,
Faculty of Mathematics, Informatics and Natural Sciences, Universität Hamburg
Martin-Luther-King-Platz 6, D-20146 Hamburg (Germany)
E-mail: chris.meier@chemie.uni-hamburg.de
[b]
Prof. Dr. D. Schols, Prof. Dr. J. Balzarini
Laboratory of Virology and Chemotherapy
Department of Microbiology and Immunology and Transplantation
Rega Institute for Medical Research
KU Leuven, Herestraat 49, B-3000 Leuven (Belgium)
Supporting information for this article is given via a link at the end of the document.
Abstract: Nucleoside analogue reverse transcriptase inhibitors
(NRTI) and nucleoside analogue monophosphate prodrugs are used
in combination antiretroviral therapy (cART). The design of antivirally
active nucleoside triphosphate prodrugs is a recent and an important
advancement in the field of nucleoside analogue drug development.
Here, we report on TriPPPro-derivatives of nucleoside analogue
triphosphates (NTPs) that comprised two different acyloxybenzyl-
masks at the -phosphate of the NTP aiming to achieve the
metabolic bypass. Thus, -non-symmetrically dimasked TriPPPro-
compounds (-(AB,ab)-d4TTPs) were synthesized and they proved
to be active against HIV-1 and HIV-2 in cultures of infected wild-type
human CD4+ T-lymphocyte (CEM/0) cells and more importantly also
in thymidine kinase-deficient CD4+ T-cells (CEM/TK-). From
hydrolysis studies both in phosphate buffer (PB, pH 7.3) and CEM
cell extracts, there was surprisingly no differentiation in the cleavage
of the two acyloxybenzyl prodrug-masks. However, if within one of
the two acyloxybenzyl groups a short PEG-type methoxytriglycol
group was introduced, the “standard” acyloxybenzyl-mask was
cleaved with high preference.
transformation into the triphosphates often occurs insufficiently
due to the substrate specificity of the involved kinases. Often the
first metabolic step, the monophosphorylation, is rate limiting but
examples for bottlenecks in the following second and third
phosphorylation steps are known as well. Further limitations
such as poor biological half-lives due to catabolic elimination,
mutations of nucleoside transporters, variable bioavailability
after oral administration or selection of drug resistance have
been observed for nucleoside analogues.9 To overcome some of
these hurdles, nucleoside monophosphate (NMP) prodrugs have
been explored in the past and resulted in orally administrable
forms of some antiviral NMPs and others are currently under
continuing development.10-13 Amongs others, two examples of
efficient NMP-prodrugs are the nucleoside phosphoramidates
and cycloSal-nucleoside phosphate triesters.14-19
Introduction
In the past a number of nucleoside analogues was discovered
and applied in antitumor and antiviral chemotherapy. These
compounds still play an important role to combat viral infections
in the clinic, such as HIV, hepatitis B and C, influenza or most
recently SARS-CoV-2 infections.1,2 Generally, the targets of
these nucleoside analogues are the viral DNA- or RNA
polymerases which are involved in the virus replication, such as
HIV’s reverse transcriptase (HIV-RT).3,4 Till now, several
nucleoside analogues have been approved as HIV-RT inhibitors
(NRTIs)6 and they are nowadays part of the highly effective
combination antiretroviral therapy (cART). However, the antiviral
activity of nucleoside analogues such as 3´-deoxy-2´,3´-
didehydrothymidine 1 (d4T), is strongly dependent on an in-vivo
phosphorylation into the nucleoside triphosphate forms (NTPs)
mainly by host cell kinases. D4TTP 1t is formed via a stepwise
phosphorylation from the nucleoside analogue into the
nucleoside mono- (1m, d4TMP), the diphosphate (1d, d4TDP)
and finally to d4TTP (1t) (Scheme 1).7,8 However, the stepwise
Scheme 1: Metabolism of nucleoside analogues such as d4T 1 and the
corresponding nucleotide prodrugs.
The aim of the above-mentioned nucleotide prodrugs was the
delivery of NMPs into cells, to bypass the first phosphorylation
step and finally improve antiviral activity. Next, an approach
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
towards nucleoside diphosphate prodrugs was developed by
attaching two lipophilic, bioreversible masks (R1=R2 or R1 ≠ R2)
at the -phosphate,20-23 such as DiPPro-d4TDPs 6 (Scheme 1).23
Moreover, we also reported on a first delivery system of NTPs.
These first generation TriPPPro-compounds 2 comprised two
identical acyloxybenzyl-masking moieties at the -phosphate
moiety to achieve membrane permeability (Scheme 1).24-28 It
was shown that these TriPPPro-d4TTPs 2 with long-chain
acyloxybenzyl masks (AB-mask) exhibited also higher antiviral
activity and longer half-life in phosphate buffer (PB) and CEM
cell extracts than those containing two short acyloxybenzyl-
masks. Such TriPPPro-compounds not only proved to be
antivirally active against HIV-1 and HIV-2 in wild-type CEM/0
cells, but they even retained high anti-HIV activity in HIV-2-
infected mutant CEM/TK--cell cultures whereas the parent
nucleoside d4T 1 was virtually inactive due to the lack of
phosphorylation. The stepwise enzyme-driven cleavage of the
two AB-masks of the TriPPPro-compounds 2 was achieved by
an ester bond cleavage within the AB-moiety forming an
intermediate of type 4 and a subsequent spontaneous cleavage
of the remaining part of the mask leading to d4TTP 1t and two
equivalents of 4-hydroxybenzyl alcohol (Scheme 2).26 The
cellular uptake of these compounds was proven by using a
fluorescent nucleoside analogue.27 However, although this
approach worked satisfactory and NTPs were formed in
chemical and PLE-catalyzed studies predominately, we also
observed some concomitant formation of the corresponding
NDPs and also (very) small amounts of the NMPs. Particularly in
cell extracts, very small amounts of d4TTP and large amounts of
d4TDP were detected due to the fast dephosphorylation of
d4TTP by cellular phosphorylases or kinases.26 During our
development of the previously described DiPPro-compounds 6,
we observed the exclusive formation of NDPs when we
introduced two different acyloxybenzyl masking groups.23 Thus,
a combination of a short-chain acyloxybenzyl moiety and a long-
chain, lipophilic acyloxybenzyl moiety led for DiPPro-compounds
6 to a selective first fast cleavage of the short-chain residue and
formation of the intermediate comprising the long lipophilic AB-
moiety. Moreover, we have shown that some concomitant
cleavage of the pyrophosphate linkage of the DiPPro-
compounds to yield NMPs only happened at the level of the
doubly-masked
starting
DiPPro-compound.
From
the
intermediate no NMP formation was detected and only the
second bioreversible group was cleaved leading to NDPs.20-23
Recently, we also reported on bis-alkoxycarbonyloxybenzyl
(ACB)-TriPPPro-compounds as well as on non-symmetric-
TriPPPro compounds which are active against HIV-2 in mutant
CEM/TK--cell cultures. The prodrugs comprised two different
ACB-moieties or a combination of an ACB and an AB mask at -
phosphate. For the latter compounds it has proven that the
acyloxybenzyl-mask was faster cleaved to give almost
selectively the -ACB-NTP-intermediates in chemical hydrolysis
studies and also in cell extracts.29
In this report, we transferred the promising results from the
DiPPro-compounds to the TriPPPro-approach and studied a
series of non-symmetrically esterified TriPPPro-compounds 3
(Scheme 1). We disclose here the synthesis and the properties
of such non-symmetrically modified TriPPPro-derivatives of the
nucleoside d4T
1
comprising an ab-moiety with an
alkyl/polyether chain and a mask with different alkyl AB-moiety.
The aim was to achieve a highly selective delivery of d4TTP, as
in the case of the DiPPro-counterparts. It was expected that the
short mask should be cleaved fast in cells forming the
monomasked intermediate which subsequently would be
converted into the target d4TTP (Scheme 2).
In addition to acyl groups bearing simple alkyl chains we also
studied PEG-units linked by an ester group as the acyl-moiety
(ab-PEG mask) in order to increase the hydrophilicity due to the
low solubility of some TriPPPro-compounds 2 comprising two
long lipophilic ab-groups in aqueous media.26
Scheme 2: Proposed hydrolysis mechanism of non-symmetric TriPPPro-compounds 3.
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
Results and Discussion
conversions of d4TMP 1m to give the TriPPPro-compounds 3
varied between 28%-64% (Scheme 3).
Synthesis
Compounds 3bh, 3eh and 3ei are TriPPPro-derivatives with one
PEG-comprising ab-moiety. The hydrophilic PEG-bearing benzyl
alcohols 7h and 7i were synthesized starting from 2-(2-(2-
D4TMP 1m was prepared applying the method described by
Sowa and Ouchi.30 For the synthesis of -(AB,ab)-d4TTPs 3, the
H-phosphonate route was used as disclosed previously by
us.27,29 Briefly, diphenyl hydrogen phosphonate (DPP) was
selectively reacted with two different acyloxybenzyl alcohols 7
which led to H-phosphonates 8 in moderate yields ranging from
44% to 52%. Next, H-phosphonates 8 were converted into the
corresponding phosphorochloridates by an oxidative chlorination
using N-chlorosuccinimide (NCS) which were then reacted with
tetra-n-butylammonium phosphate to yield pyrophosphates 9.
The conversion was almost quantitative and after extraction in
CH2Cl2/water, the crude products were immediately used for the
next step without further purification. Compounds 9 were
stepwise activated with trifluoroacetic acid anhydride (TFAA)
and N-methylimidazol and coupled with d4TMP 1m to yield -
(AB,ab)-d4TTPs 3 (Bu4N+ form). After a reverse-phase column
methoxyethoxy)ethoxy)ethan-1-ol
10
(MEEE).
Succinic
anhydride 11h and glutaric anhydride 11i were used to form a
diester linker between the PEG moiety and 4-hydroxybenzyl
alcohol (Scheme 4). MEEE-succinate 7h (MEEES) and MEEE-
glutarate 7i (MEEEG) are hygroscopic and should be carefully
handled when exposed to air.
chromatography of the crude products, ion-exchange using
+
Dowex 50WX8 (NH4 ),
a second reverse-phase column
Scheme 4: Reagents and conditions: i) DMAP, CH2Cl2, rt; ii) oxalyl chloride,
chromatography and freeze-drying, -(AB,ab)-d4TTPs 3 in their
NH4 -form were isolated. The total yields obtained in these
cat. DMF, CH2Cl2, 0 °C-rt; iii) 4-hydroxybenzyl alcohol, Et3N, CH2Cl2, 0 °C-rt.
+
Scheme 3: Reagents and conditions: i) diphenyl hydrogen phosphonate (DPP), pyridine, 0 °C-38 °C, 3.3 h; ii) a. NCS, CH3CN, rt, 2 h, b) (H2PO4)Bu4N, CH3CN, rt,
1 h; iii) d4T 1, POCl3, pyridine, H2O, CH3CN, 0 °C-rt, 5 h; iv) a. TFAA, Et3N, CH3CN, 0 °C, 10 min, b. 1-methylimidazol, Et3N, CH3CN, 0 °C-rt, 10 min, c. d4TMP
1m, rt, 3-5 h, RP-chromatography, Dowex 50WX8 (NH4+ form) ion exchange, RP-chromatography.
lymphocyte cell extracts or pig liver esterase (PLE) in PB, pH 7.3.
Stability Studies
To study the hydrolytic stability of the prodrugs and the products
distribution, TriPPPro-compounds
phosphate buffer (PB, 25 mM, pH 7.3), human CD4 T-
Samples of the hydrolysis mixtures at different time points were
analyzed by analytical RP18-HPLC. The calculated half-lives
(t1/2) of compounds 3 reflect the removal of the first bioreversible
AB- or ab-group to yield the corresponding monomasked
3
were incubated in
+
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
intermediates 5. The half-lives (t1/2) of prodrugs 3 in phosphate
buffer (PB, 25 mM, pH 7.3) and human CD4 T-lymphocyte cell
extracts are summarized in Table 1.
PEG-bearing masking group unchanged, the stability increased
by a 3-fold. For prodrugs 3eh and 3ei, in which the linker
structure changed from succinic diester (3eh) to glutaric diester
(3ei), the t1/2 of the prodrug increased by 1.3-fold. The
distribution of the hydrolysis products was identical to the results
of the non-PEG-containing TriPPPro-compounds 3 showing
almost no selectivity. Both intermediates -(AB-C14)-d4TTP 5e
+
Table 1. Hydrolysis of TriPPPro-compounds 3 in different media.
and -(ab-MEEES)-d4TTP 5h were formed to
a highest
PB pH=7.3
t1/2 [h]
CEM/0 cell
extracts
t1/2 [h]
concentration after 200 h incubation and decreased when the
hydrolysis proceeded. TriPPPro-compounds 3eh was totally
consumed after incubation of 500 h, and at that time point the
ratio of 5e:5h:1t:1d:1m was 3:5:15:8:1. During the hydrolysis,
almost no d4TMP 1m was formed.
Prodrug
R1
R2
3ae
3be
3de
3ce
3bf
3bg
3bh
3eh
3ei
C2H5
n-C4H9
n-C6H13
iso-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C14H29
n-C14H29
/
n-C14H29
n-C14H29
n-C14H29
n-C14H29
n-C15H31
n-C17H35
MEEES
MEEES
MEEEG
/
59
45
61
64
49
50
22
65
86
0.8
2.5
2.3
3.8
3.1
3.3
0.8
1.1
2.4
n.a.[a]
0.6
Hydrolysis in CEM cell extracts
The stability of TriPPPro-prodrugs 3 was further investigated in
+
human CD4 T-lymphocyte CEM cell extracts. Here, an
enzymatic cleavage reaction took place, because the half-lives
of the prodrugs 3 in CEM/0 cell extracts ranging from 0.8 h to
3.8 h were significantly lower than the half-lives in PB buffer.
Compounds 3ae and 3bh, comprising a propanoyl-ester and
pentanoyl-ester moiety, respectively, were found to be the most
labile compounds. This is in accordance to our previous results
of the DiPPro-compounds23 or the TriPPPro-compounds 2.26 In
all those cases the predominate formation of d4TDP 1d was
observed. Mono-masked Intermediates 5 and d4TTP 1t were
only observed in very low concentration. This result was in
accordance to our previous studies.26 In CEM/0 cell extracts, the
half-life of d4TDP 1d was 59 h and that of d4TTP 1t was only 38
min,26 which means that the observed low levels of d4TTP 1t in
these hydrolysis studies were the result of a fast enzymatic
dephosphorylation of d4TTP 1t. It can not fully exclude that the
d4TDP found in these studies is a result of a direct cleavage of
the anhydride bond between the - and -phosphate. However, it
seems to be unlikely because here in the cell extracts a
considerable amount of esterases is present which are
responsible for the ester cleavage within the masking group.
This pathway might be more relevant in the phosphate buffer
hydrolysis studies due to the lack of the esterase. But even in
those studies the main product, e.g. for compound 3bf was still
d4TTP 1t. And in contrast to the phosphate buffer, pH 7.3, the
cell extracts contain phosphatases. No thymine was detected
during the incubation period within 10 h for all prodrugs. It was
not possible to calculate the exact peak area for d4TMP 1m
using this HPLC method because of the overlapped peaks
between d4TMP 1m and cell extracts.
D4T
D4TTP31
>40
>40
/
/
The hydrolysis experiments of TriPPPro-compounds 3 were performed in
aqueous 25 mM phosphate buffer (PB, pH=7.3) and in CEM/0 cell extracts.
The hydrolysis products were detected by analytical RP18 HPLC. [a] n.a.: not
available.
Chemical stability in phosphate buffer (PB, pH 7.3)
In PB, the chemical stability of the TriPPPro-compounds 3ae-
3bg bearing two different acyloxybenzyl masking groups
hydrolyzed with half-lives between 45 h and 64 h without
showing a clear trend following the cleavage mechanism
depicted in Scheme 2. A stability difference was observed for
TriPPPro-derivatives 3be (45 h) and 3ce (64 h) bearing a n-butyl
moiety (3be) and a branched i-butyl group (3ce), respectively.
Generally, d4TTP 1t and d4TDP 1d were detected as main
hydrolysis products in addition to d4TMP 1m which was
detected as well although in very low concentrations (<4%).
After complete consumption of the TriPPPro-prodrugs 3, an
increase in the d4TTP 1t concentration was formed in the case
of TriPPPro-compounds 3 in PB (pH 7.3). As an example, when
TriPPPro-compounds 3bf was totally consumed after incubation
of 480 h, the ratio of 1t:1d:1m was 14:11:1. Both intermediates
-(ab-C15)-d4TTP 5f and-(AB-C4)-d4TTP 5b were formed in
almost identical concentrations and thus, the hydrolysis
proceeded without marked selectivity, which was in agreement
with the results obtained from the studies of -(ACB-C4,ACB-
C12)-d4TTPs in PB, but in contrast with results as -(AB-
C2,ACB-C16)-d4TTPs.29 Compared to the half-lives in PB of
symmetric -(AB-C4,AB-C4)-d4TTP (t1/2 = 22 h) and -(AB-
C9,AB-C9)-d4TTP (t1/2 = 44 h), the half-life of all non-symmetric
TriPPPro-compounds studied here showed half-lives close to
that of -(AB-C9,AB-C9)-d4TTP, e.g. -(AB-C4,AB-C14)-d4TTP
3be; t1/2 = 45 h, although most of them comprise also one short
acyloxybenzyl moieties.26 As reported previously for the
TriPPPro-compounds 2, the half-lives of the mono-masked
intermediates 5 were also much higher than those of the
TriPPPro-prodrugs 3. A side reaction occurred and thymine was
formed due to the cleavage of the glycosidic bond.
In addition, both intermediates 5f and 5b were observed in low
concentration. Thus, as above and in contrast to our previous
work on the DiPPro-compounds 623 and -(AB,ACB)-d4TTPs29
no selective cleavage of the different masks was observed in the
hydrolysis in CEM cell extracts. In our previous work, the half-
lives of symmetric -(AB-C4,AB-C4)-d4TTP and -(AB-C9,AB-
C9)-d4TTP in CEM/0 cell extracts were t1/2 = 0.43 h and t1/2 = 2.8
h, respectively. Thus, similar to the results in PB, the determined
half-life of non-symmetric -(AB-C4,AB-C14)-d4TTP 3be (t1/2
=
2.5 h) was almost identical to that of symmetric -(AB-C9,AB-
C9)-d4TTP but comprising a short AB-group.26 This points to a
compensation of the lability of short chain comprising TriPPPro-
derivatives if a lipophilic alkyl masking group is also present in
the molecule.
Interestingly, for the PEG-containing MEEES- and MEEEG-
TriPPPro-derivatives 3bh (t1/2= 22 h), 3eh (t1/2= 65 h) and 3ei
(t1/2= 86h) a broad range of stability was determined. Just by
changing the alkyl residue in one of the acyloxybenzyl-groups
from n-butyl- (3bh) to a n-C14-fragment (3eh) and keeping the
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
The situation was different in the case of the PEG-containing
derivatives 3bh, 3eh, and 3ei. After 3h incubation in the CEM/0
cell extracts, low concentrations d4TTP 1t (3%) and more
importantly only small amounts of the intermediate -(ab-C14)-
d4TTP 5e (9%) were detected. Here, the predominant product
was intermediate -(ab-MEEES)-d4TTP 5h (52%). Intermediate
PEG-d4TTP 5h was detected and found to be much more stable
than -(AB-C14)-d4TTPs 5e (Figure 2, B). D4TDP 1d increased
constantly to an amount of 15% after 3h incubation and 32%
after 9h incubation. Thus, by introducing the hydrophilic PEG-
Hydrolysis in Pig Liver Esterase (PLE)
TriPPPro- d4TTP 3bf was treated with pig liver esterase (PLE)
to prove a possible selectivity in the removal of the masking
groups (Figure 1, C). The determined half-life here was 0.75 h.
The cleavage of the masking units in 3bf occurred much faster
than that in PB (t1/2 = 49 h) and also faster as compared to the
study in CEM/0 cell extracts (t1/2 = 3.1 h). Again, both 5f and 5b
were formed in identical concentrations. Both, d4TTP 1t and
d4TDP 1d were observed in low levels during PLE hydrolysis.
The half-lives for intermediates 5f, 5b were also found to be
higher than that for -(AB-C4,ab-C15)-d4TTP 3bf, probably due
to the additional negative charge at the -phosphate.
bearing
acyloxybenzyl-mask,
the
“normal”
lipophilic
acyloxybenzyl-mask was cleaved preferably in these hydrolysis
studies, which is in agreement with the results obtained from the
studies as reported before.29
(A) Hydrolysis in PBS
3x107
-(AB-C4,ab-C15)-d4TTP 3bf
-(ab-C15)-d4TTP 5f
-(AB-C4)-d4TTP 5b
d4TTP 1t
d4TDP 1d
d4TMP 1m
thymine
2x107
1x107
0
0
100
200
300
400
500
Time (h)
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
(B) Hydrolysis in CEM cell extracts
-(AB-C4,ab-C15)-d4TTP 3bf
3x107
2x107
1x107
0
-(ab-C15)-d4TTP 5f
-(AB-C4)-d4TTP 5b
d4TTP 1t
d4TDP 1d
0
1
2
3
4
5
6
7
8
9
Time (h)
4x107
3x107
2x107
1x107
0
(C) Hydrolysis with PLE
-(AB-C4,ab-C15)-d4TTP 3bf
-(ab-C15)-d4TTP 5f
-(AB-C4)-d4TTP 5b
d4TTP 1t
d4TDP 1d
0
1
2
3
4
5
6
7
8
Time (h)
Figure 1: Hydrolysis of -(AB-C4,ab-C15)-d4TTP 3bf in PB (A), CEM cell extracts (B) and pig liver esterase (PLE; C).
4x107
(A) Hydrolysis in PBS
-(AB-C14,ab-MEEES)-d4TTP 3eh
-(AB-C14)-d4TTP 5e
-(ab-MEEES)-d4TTP 5h
3x107
d4TTP 1t
d4TDP 1d
d4TMP 1m
2x107
thymine
1x107
0
0
100
200
300
400
500
Time (h)
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
4x107
3x107
2x107
1x107
0
(B) Hydrolysis in CEM cell extracts
-(AB-C14,ab-MEEES)-d4TTP 3eh
-(AB-C14)-d4TTP 5e
-(ab-MEEES)-d4TTP 5h
d4TTP 1t
d4TDP 1d
0
1
2
3
4
5
6
7
8
9
10
Time (h)
Figure 2: Hydrolysis of -(ab-C14,ab-MEEES)-d4TTP 3eh in PB (A) and in CEM cell extracts (B).
membrane passage of the non-symmetrically-masked TriPPPro-
compounds 3, one of the two masking groups should comprise a
long lipophilic alkyl chain which then ensures that the prodrug is
active against HIV, which in agreement with the results as -
(AB,ACB)-d4TTPs.29
Interestingly, the antiviral activity and the chemical or biological
stability of prodrug 3ce are 1.5-fold higher than that of prodrug
3be. Due to this favorable hydrolysis data, the antiviral activity
observed in the wild-type CEM/0 cell cultures was nearly
retained in the case of -(AB-C4,ab-C14)-d4TTP 3ce (EC50: 0.39
µM) in mutant thymidine kinase-deficient CEM cell cultures
(CEM/TK-). TriPPPro-compound 3ce is the most potent
compound of all the listed derivatives (>128-fold more active in
the TK-def. cell assay than the parent d4T).
Anti-HIV activities in CEM/0 and CEM/TK- cells
All TriPPPro-compounds 3 were evaluated for their antiviral
activity. For this purpose, HIV-1- or HIV-2-infected wild-type
CEM/0 as well as mutant thymidine kinase-deficient CEM cell
cultures (CEM/TK-) were treated with the TriPPPro-d4TTPs 3.
Table 2. Anti-HIV activity and Cytotoxicity of TriPPPro-compounds 3.
EC50 [µM][a]
CEM/0
CC50 [µM][b]
CEM/0
Prodrug
CEM/TK-
HIV-1
HIV-1
HIV-2
3ae
3be
3de
3ce
3bf
3bg
3bh
3eh
3ei
0.24 ± 0.17
0.14 ± 0.11
0.15 ± 0.087
0.17 ± 0.085
0.15 ± 0.12
0.12 ± 0.05
0.34 ± 0.23
0.26 ± 0.14
0.31 ± 0.16
0.33 ± 0.13
1.05 ± 0.35
0.39 ± 0.22
0.32 ± 0.26
0.29 ± 0.24
0.23 ± 0.18
0.27 ± 0.25
0.10 ± 0.03
0.76 ± 0.58
0.43 ± 0.30
0.65 ± 0.59
0.97 ± 0.50
0.40 ± 0.26
1.1 ± 0.82
0.69 ± 0.52
0.74 ± 0.46
0.39 ± 0.21
1.1 ± 0.81
0.54 ± 0.41
16 ± 4.0
1.3 ± 0.64
2.0 ± 1.1
>50
20 ± 0
18 ± 0
22 ± 3
24 ± 3
24 ± 15
33 ± 7
23 ± 9
28 ± 3
34 ± 14
>50
Conclusion
In summary, a series of TriPPPro-compounds 3 bearing two
different AB-masks attached to the -phosphate was synthesized
by using H-phosphonate route. Hydrolysis in CEM cell extracts
and pig liver esterase proved the delivery mechanism that the
hydrolysis is mainly triggered by enzymes. However, compared
to -(AB,ACB)-d4TTPs,29 hydrolysis studies showed that no
obvious selective cleavage occurred between short acetyl ester
and long acetyl ester. Antiviral evaluation showed that most of
the compounds exhibited higher activity against HIV-1 and HIV-2
in cultures of infected wild-type human CD4+ T-lymphocyte
(CEM/0) cells and more importantly in thymidine kinase-deficient
CD4+ T-cells (CEM/TK–). Interestingly, if one AB-mask was
modified with methoxytriglycol group with a diester linker, the
other AB-mask was cleaved predominately. However, these
compounds with hydrophilic groups are less potent than the
TriPPPro-compounds with two alkylacyl AB-masks.
d4T 1
d4TTP31
>100
>100
[a] Antiviral activity of the compounds in CD4+ T-lymphocyte CEM cell
cultures: 50% effective concentration; values are the mean ±SD of n=2-3
independent experiments. [b] Cytotoxicity: 50% cytostatic concentration or
compound concentration required to inhibit CD4+ T-cell (CEM) proliferation by
50%; values are the mean ±SD of n=2-3 independent experiments.
In wild-type CEM/0 cell cultures, all -(AB,ab)-d4TTPs 3 showed
higher activities against HIV-2 and similar or slightly better
activities against HIV-1 as compared to the parent nucleoside
d4T 1 or d4TTP 1t (Table 2). More importantly, except
compound 3bh (C4/PEG) all prodrugs 3 were highly potent in
thymidine kinase-deficient CEM/TK- cells whereas d4T 1 and
d4TTP 1t lacked any relevant anti-HIV activity in this assay
(EC50: >50 µM for d4T 1 and >100 µM for d4TTP 1t). Correlated
with a substantial increase of the lipophilicity of the mask,
TriPPPro-compound 3eh was 12-fold more active than 3bh
against HIV-2 in CEM/TK- cells. This point to sufficient
lipophilicity of compound 3eh combined with a relatively slow
cleavage of the bioreversible AB-moiety which led to the
formation of -(ab-MEEES)-d4TTP 5h (Figure 2, B). To enable
Experimental Section
1.1 Chemical and Instruments. General: Without further noticed, all
manipulations were carried out under an atmosphere of nitrogen using
standard Schlenk techniques and all solvents were dried by using
standard procedures. Triethylamine (Et3N) were dried by being heated
under reflux over calcium hydride for 5 days and followed by distillation.
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
Trifluoroacetic anhydride (TFAA) was dried over phosphorus pentoxide
for one hour and distilled under nitrogen. Anhydrous CH3CN (CH3CN),
tetrahydrofuran (THF), dichloromethane (CH2Cl2),) and pyridine were
obtained by the MBraun solvent purification system (MB SPS-800). Other
dry solvents were purchased from Acros Organics (extra dry over
molecular sieves). For eluent of column chromatography, technique
grade ethyl acetate (EA), petroleum ether (PE) 50-70, CH2Cl2, and
CH3OH were distilled before use. Tetra-n-butylammonium phosphate
monobasic solution (0.4 M in CH3CN) was purchased from Acros
Organics directly. Column chromatography: Normal phase column
chromatography were performed with Macherey-Nagel silica gel 60 M
(0.04-0.063 mm). For automatic reversed-phase chromatography an
Interchim Puriflash 430 in combination with Chromabond® Flash C18ec
was used. Analytical thin-layer chromatography (TLC): For thin layer
chromatography Macherey-Nagel pre-coated TLC sheets Alugram® Xtra
SIL G/UV254 were used. Phosphomolybdic acid (PMA) stain was used
and prepared by dissolving 10 g of phosphomolybdic acid in 100 mL of
ethanol. Potassium permanganate stain was used and prepared by 1.5 g
of KMnO4, 10 g K2CO3, and 1.25 mL 10% NaOH in 200 mL water. High
General Procedure B: Preparation of non-symmetric (AB,ab)-H-
phosphonates 8. Under dry conditions, diphenyl phosphonate (1.2
equiv.) was dissolved in 3 mL pyridine and cooled to 0 °C. Compounds
7e-h (1.0 equiv.) were added and stirred at 0 °C for 30 min and then
heated up to 38 °C. Following, compounds 7a-e (1.4 equiv.) were added
and the mixture was stirred for 3 h. Then the solvent was removed in
vacuo. The crude products were purified by flash column
chromatography (silica) with EtOAc/petroleum ether/0.5% acetic acid as
eluent.
General Procedure C: Preparation of non-symmetric TriPPPro-
compounds 3. The reactions were performed under dry conditions. a) H-
Phosphonates 8 (1.0 eq.) were dissolved in 6 mL CH3CN and N-
chlorosuccinimide (2.0 eq.) was added. After stirring for 2 h at rt, tetra-n-
butylammonium phosphate monobasic solution (0.4 M in CH3CN) (3.0
eq.) was added dropwise. The mixture was stirred for 1 h and the solvent
was removed in vacuum. The residue was extracted with CH2Cl2/H2O.
The organic phase was dried over sodium sulfate and the solvent was
removed by evaporation to afford corresponding pyrophosphates in
nearly quantitative yield. B) The corresponding pyrophosphates were
dissolved in CH3CN and cooled down to 0 °C. A mixture of trifluoroacetic
anhydride (TFAA, 5.0 eq.) and trimethylamine (Et3N, 8.0 eq.) in 3 mL
CH3CN was cooled to 0 °C and added to the mixture. After stirring for 10
min, all volatile components were removed in vacuum. The residue was
once again co-evaporated with 3 mL CH3CN and subsequently dissolved
in 3 mL CH3CN at 0 °C. 1-methylimidazole (3.0 eq.) and trimethylamine
(Et3N, 8.0 eq.) was added. The suspension was warmed up to rt and
stirred for 10 min. The resulting activated imidazolidate formed and the
corresponding d4TMP 1m (0.7-1.0 eq.) in 3 mL CH3CN was added. The
reaction was stirred at room temperature for 2-3 h and dried in vacuum.
The crude product was purified by automatic RP18 flash chromatography,
and then followed by ion-exchange to the ammonium form with Dowex
50WX8 cation-exchange resin and a second RP18 chromatography
purification step. Product-containing fractions were collected, and the
organic solvent evaporated. The remaining aqueous solutions were
freeze-dried, and the desired product obtained as a white solid.
performance liquid chromatography (HPLC):
A
VWR-Hitachi
LaChromElite HPLC system (L-2130, L-2200, L-2455) with an
EzChromElite software and a Nucleodur 100-5 C18ec column (Macherey-
Nagel) was used. HPLC grade CH3CN was obtained from VWR and
ultrapure water (Mili-Q water) from a Sartorius Aurium® pro apparatus
(Sartopore 0.2 μm, UV). 2 mM tetra-n-butylammonium acetate solution
(TBAA, pH 6.3) was used as buffer. Method: Nucleodur 100-5 C18ec; 0-
20 min: TBAA buffer/CH3CN gradient (5-80%); 20-30 min: buffer/CH3CN
(80%); 30-33 min: buffer/CH3CN (80-5%); 33-38 min: buffer/CH3CN (5%);
flow: 1 mL/min. TBAA buffer (2 mM): 4.05 mL tetra-n-butylammonium
hydroxide in water (ca. 40%) was diluted with 3000 mL ultrapure water.
Then glacial acetic acid was added to adjust the buffer to pH 6.3. Nuclear
Magnetic Resonance spectroscopy (NMR): NMR spectra were recorded
at room temperature in automation mode on either of a Varian Gemini
2000BB, Bruker AMX 400, Bruker DRX 500 or Bruker AVIII 600
spectrometer. Chloroform-d1 (CDCl3), methanol-d4 (MeOD), dimethyl
sulfoxide-d6 (DMSO) and deuterium oxide (D2O) were purchased form
1
Euriso-Top. All H, 31P, and 13C NMR chemical shifts are quoted in parts
per million (ppm). Reference peaks for chloroform-d in 1H NMR and 13C
4-(Hydroxymethyl)phenyl propionate 7a: According to General Procedure
A, under atmosphere, 3.41 g 4-hydroxybenzylalcohol (27.5 mmol, 1.1
equiv.) and 3.47 mL triethylamine (25 mmol, 1.0 equiv.) was dissolved in
15 mL CH2Cl2 at 0 °C. 2.17 mL propionyl chloride (25 mmol, 1.0 equiv.)
in 25 mL CH2Cl2 was added dropwise. Reaction time was 2.5 h at rt.
Column chromatography (SiO2, PE/EE 6:4 v/v) Yield: 60%, 2.70 g, as
colorless oil. 1H NMR (400 MHz, CDCl3):  [ppm] = 7.36 (d, 3J = 8.0 Hz,
NMR spectra were set at 7.26 ppm and 77.0 ppm, respectively. For
1
methanol-d4 reference peaks in H NMR and 13C NMR spectra were set
at 3.31 ppm and 49.0 ppm respectively. All final compounds were
isolated analytically pure, ≥95% purity by HPLC and NMR spectroscopy.
Mass Spectrometry (MS): HRMS (ESI) mass spectra were recorded on
an Agilent 6224 ESI-TOF spectrometer. MALDI measurements (matrix:
9-aminoacridine [9AA] or 2,5-dihydroxybenzoic acid [DHB]) were
3
2H), 7.06 (d, J = 8.0 Hz, 2H), 4.66 (s), 2.59 (q, J = 7.5 Hz, 2H), 1.91 (s,
performed with
a
Bruker UltrafelXtreme spectrometer. Infrared
1H), 1.26 (t, JHH = 7.5 Hz, 3H). 13C-NMR (101 MHz, CDCl3):  [ppm] =
173.0, 150.1, 138.4, 128.0, 121.6, 64.7, 27.7, 9.0. HRMS (ESI-TOF) m/z:
calculated for C10H12NaO3 [M+Na]+ 203.0679, found 203.0618. IR: ν
[cm−1] = 3405, 2982, 2941, 2883, 1752, 1606, 1509, 1473, 1456, 1413,
1383, 1351, 1201, 1170, 1138, 1073, 1025, 999, 946, 891, 830, 804, 761,
727, 577, 559, 515, 423, 409.
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. was used. Ultrapure water:
Arium®pro ultrapure water system was used. pH meter: pH value was
tested with ProLab 300 from Schott Co. Thermomixer: Eppendorf
Thermomixer 5436 and CellMedia Thermoschüttler basic were used.
Ultrasonic cleaning device: Sonorex RK512H from Bandelin Co. was
used. Centrifuges: Heraeus Biofuge Pico (13000 u/min) and Biofuge
Primo R (8000 u/min) was used.
4-(Hydroxymethyl)phenyl pentanoate 7b: According to General
Procedure A, under atmosphere, 1.22 g 4-hydroxybenzylalcohol (9.79
mmol, 1.2 equiv.) and 1.36 mL triethylamine (9.79 mmol, 1.2 equiv.) was
dissolved in 20 mL CH2Cl2 at rt. 1 mL pentanoyl chloride (8.16 mmol, 1.0
equiv.) in 30 mL CH2Cl2 was added dropwise. Reaction time was 4 h at rt.
Column chromatography (SiO2, PE/EE 7:3 v/v). Yield: 78%, 1.33 g, as
colorless oil. 1H NMR (400 MHz, CDCl3):  [ppm] = 7.36-7.27 (d, J = 8.1
Hz, 2H), 7.08-6.97 (d, J = 8.4 Hz, 2H), 4.57 (s, 2H), 2.54 (t, J = 7.5 Hz,
2H), 2.75-2.45 (br, s, 1H), 1.73 (tt, J = 7.5, 7.5 Hz, 2H), 1.44 (tq, J = 8.0,
7.2 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H). 13C-NMR (101 MHz, CDCl3):  [ppm]
= 172.4, 149.9, 138.4, 127.9, 121.5, 64.4, 34.0, 26.9, 22.1, 13.6. HRMS
(ESI-TOF) m/z: calculated for C12H16NaO3 [M+Na]+ 231.0992, found
231.0972. IR: ν [cm−1] = 3393, 2958, 2932, 2872, 1754, 1606, 1506,
1464, 1417, 1365, 1345, 1310, 1198, 1163, 1143, 1101, 1046, 1014, 940,
919, 848, 811, 753, 733, 643, 561, 505, 458, 405, 389.
1.2 General Synthesis Procedures.
General
Procedure
A:
Preparation
of
4-
(hydroxymethyl)phenylalkanoates 7. 4-Hydroxybenzyl alcohol and
triethylamine in CH2Cl2 were cooled down to 0 °C or at room temperature.
The corresponding acyl chloride in CH2Cl2 was added dropwise and the
mixture was stirred at room temperature (rt). The precipitate was filtered,
and the solvent was removed in vacuum. The residue was diluted with
CH2Cl2 and washed once with water. The organic layer was dried with
Na2SO4 and the solvent was removed. The crude was purified by SiO2
column chromatography to give compounds 7.
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
4-(Hydroxymethyl)phenyl 3-methylbutanoate 7c: According to General
Procedure A, under atmosphere, 3.0 g 4-hydroxybenzylalcohol (24.0
mmol, 1.0 equiv.) and 3.67 mL triethylamine (26.4 mmol, 1.1 equiv.) was
dissolved in 15 mL CH2Cl2 at 0 °C. 3.21 mL 3-methylbutanoyl chloride
(25 mmol, 1.0 equiv.) in 25 mL CH2Cl2 was added dropwise. The mixture
was stirred for 2.5 h at rt. Column chromatography (SiO2, PE/EE 7:3 v/v).
Yield: 64%, 3.21 g, as colorless oil. 1H NMR (400 MHz, CDCl3):  [ppm] =
7.40-7.27 (m, 2H), 7.10-7.01 (m, 2H), 4.66 (s, 2H), 2.43 (d, J = 7.2 Hz,
2H), 2.34-2.18 (m, 1H), 2.09-1.77 (m, 1H), 1.06 (d, J = 6.6 Hz, 6H). 13C-
NMR (101 MHz, CDCl3):  [ppm] = 172.4, 149.9, 138.4, 127.9, 121.5,
64.4, 34.0, 26.9, 22.1, 13.6. HRMS (ESI-TOF) m/z: calculated for
C12H16NaO3 [M+Na]+ 231.0992, found 231.0989. IR: ν [cm−1] = 3350,
2959, 2932, 2873, 1753, 1606, 1466, 1417, 1388, 1369, 1292, 1245,
1197, 1152, 1099, 1042, 1014, 964, 914, 890, 850, 832, 811, 628, 562,
500.
added in 100 mL CH2Cl2. 5.57 mL octadecanoyl chloride (16.5 mmol, 1.0
equiv.) in 150 mL CH2Cl2 was added dropwise and the mixture was
stirred at rt overnight. The precipitate was filtered, and the solvent was
removed in vacuum. The residue was diluted with CH2Cl2 and washed
once with water. The organic layer was dried with Na2SO4 and the
solvent was removed. Column chromatography (SiO2, petrol
ether/ethylacetate 6:4 v/v). Yield: 78%, 5.0 g, as white solid. 1H NMR
(500 MHz, CDCl3):  [ppm] = 7.37 (d, J = 8.4 Hz, 2H), 7.07 (dt, J = 8.4 Hz,
2H), 4.68 (s, 2H), 2.55 (t, J = 7.5 Hz, 2H), 1.74 (p, J = 7.5 Hz, 2H), 1.64
(br, s, 1H), 1.49-1.12 (m, 28H), 0.88 (t, J = 6.8 Hz, 3H). 13C-NMR (126
MHz, CDCl3):  [ppm] = 172.38, 150.16, 138.32, 128.03, 121.69, 64.79,
34.39, 31.92, 29.69, 29.67, 29.65, 29.64, 29.59, 29.45, 29.35, 29.25,
29.10, 24.94, 22.68, 14.11. HRMS (ESI-TOF) m/z: calculated for
C25H46NO3 [M+NH4]+ 408.3472, found 408.3478. IR: ν [cm−1] = 3318,
2955, 2914, 2847, 1748, 1605, 1509, 1471, 1463, 1410, 1387, 1331,
1312, 1292, 1272, 1252, 1232, 1218, 1166, 1150, 1101, 1034, 1014, 950,
924, 846, 817, 760, 727, 719, 580, 511.
4-(Hydroxymethyl)phenyl heptanoate 7d: According to General
Procedure A, under atmosphere, 3.0 g 4-hydroxybenzylalcohol (24.0
mmol, 1.0 equiv.) and 3.67 mL triethylamine (26.4 mmol, 1.1 equiv.) was
dissolved in 20 mL CH2Cl2 at 0 °C. 4.1 mL n-heptanoyl chloride (26.4
mmol, 1.1 equiv.) in 30 mL CH2Cl2 was added dropwise. The mixture was
stirred for 2.5 h at rt. Column chromatography (SiO2, PE/EE 7:3 v/v).
Yield: 58%, 3.31 g, as white solid. 1H NMR (400 MHz, CDCl3):  [ppm] =
7.45-7.32 (m, 2H), 7.13-7.02 (m, 2H), 4.66 (s, 2H), 2.55 (t, J = 7.5 Hz,
2H), 1.75 (quint, J = 7.0 Hz, 2H), 1.69 (br, s), 1.49-1.21 (m, 2H), 0.91 (t, J
= 7.0 Hz, 3H). 13C-NMR (101 MHz, CDCl3):  [ppm] = 172.4, 150.0, 138.4,
128.0, 121.6, 64.6, 34.3, 31.4, 28.7, 22.4, 24.8, 14.0. HRMS (ESI-TOF)
m/z: calculated for C14H20NaO3 [M+Na]+ 259.1305, found 259.1230. IR: ν
[cm−1] = 3380, 2956, 2928, 2860, 1754, 1510, 1459, 1417, 1379, 1195,
1164, 1140, 1015, 848, 811, 503.
12-Oxo-2,5,8,11-tetraoxapentadecan-15-oic acid 12h: Under atmosphere,
10 g 2-(2-(2-Methoxyethoxy)ethoxy)ethan-1-ol 10 (60.9 mmol, 1.0 equiv.)
and 7.3 g succinic anhydride 11h (73.1 mmol, 1.2 equiv.) was added in
40 mL CH2Cl2. Then 1.49 g DMAP (12.2 mmol, 0.2 equiv.) was added
and the mixture was stirred at rt overnight. The reaction mixture was then
quenched with 4 mL water, diluted with 20 mL CH2Cl2 and extract with
(3×10 mL) 10% NaHSO4 and (1×10 mL) brine. The organic phase was
dried with MgSO4 and the solvent was removed in vacuum. The product
was used directly without further purification. Yield: 72%, 11.6 g, as
colorless liquid. 1H NMR (400 MHz, DMSO-d6):  [ppm] = 4.16-4.07 (m,
2H), 3.65-3.56 (m, 2H), 3.56-3.48 (m, 6H), 3.48-3.38 (m, 2H) 3.24 (s, 3H),
2.57 -2.42 (m, 4H). 13C-NMR (101 MHz, DMSO-d6):  [ppm] = 173.24,
172.07, 71.23, 69.76, 69.68, 69.56, 68.20, 63.33, 58.00, 28.60. HRMS
(ESI-TOF): calculated for C11H20NaO7 [M+Na]+ 287.1101, found
287.1072. IR: ν [cm−1] = 2877, 1730, 1452, 1383, 1349, 1244, 1199,
1160, 1097, 1027, 988, 942, 849, 623, 564, 405.
4-(Hydroxymethyl)phenyl pentadecanoate 7e: According to General
Procedure A, under atmosphere, 1.22 g 4-hydroxybenzylalcohol (9.9
mmol, 1.2 equiv.) and 1.36 mL triethylamine (9.9 mmol, 1.2 equiv.) was
dissolved in 20 mL CH2Cl2 at room temperature. 2.14 g n-pentadecanoyl
chloride (8.25 mmol, 1.0 equiv.) in 30 mL CH2Cl2 was added dropwise.
The mixture was stirred for overnight at rt. Column chromatography (SiO2,
PE/EE 7:3 v/v). Yield: 63%, 1.82 g, as white solid. 1H NMR (400 MHz,
4-(Hydroxymethyl)phenyl
(2-(2-(2-methoxyethoxy)ethoxy)ethyl)
succsinate 7h: 5.0 g 12-oxo-2,5,8,11-tetraoxapentadecan-15-oic acid
12h (18.9 mmol, 1.0 equiv.) was dissolved in 100 mL CH2Cl2 and cooled
to 0 °C. 0.7 mL oxalyl chloride (22.7 mmol, 1.2 equiv.) was added to the
flask and 3 drops of DMF was then added. Afterwards, the mixture was
warm up to rt and stirred until no more gas generated (around 2-4 h).
Then oxalyl chloride was evaporated to afford target acyl chloride as
colorless liquid. The yield was calculated as quantitative. The crude
product 13h was used directly without further purification. According to
General Procedure A, under atmosphere, 2.25 g 4-hydroxybenzylalcohol
(18.1 mmol, 1.2 equiv.) and 2.52 mL triethylamine (18.1 mmol, 1.2
3
CDCl3):  [ppm] = 7.35 (dt, J = 8.8, 2.6 Hz, 2H), 7.06 (dt, JHH = 8.4, 2.4
Hz, 2H), 4.65 (s, 2H), 2.55 (t, J = 7.6 Hz, 2H), 1.75 (p, J = 7.5 Hz, 2H),
1.46-1.19 (m, 22H), 0.89 (t, J = 6.8 Hz, 3H). 13C-NMR (101 MHz, CDCl3):
 [ppm] = 172.4, 150.1, 138.4, 128.0, 121.6, 64.7, 34.4, 31.9, 29.66,
29.65, 29.57, 29.43, 29.33, 29.23, 29.08, 22.7, 24.9, 14.1. HRMS (ESI-
TOF) m/z: calculated for C22H40NO3 [M+NH4]+ 366.3003, found 366.3000.
IR: ν [cm−1] = 3321, 2955, 2914, 2847, 1748, 1605, 1509, 1463, 1411,
1386, 1318, 1294, 1270, 1246, 1218, 1166, 1150, 1094, 1037, 1014, 950,
925, 846, 817, 760, 745, 719, 580, 515.
equiv.) was added in 20 mL CH2Cl2. 4.27
g
2-(2-(2-
methoxyethoxy)ethoxy)ethyl 4-chloro-4-oxobutanoate 13h (15.1 mmol,
1.0 equiv.) in 30 mL CH2Cl2 was added dropwise and the mixture was
stirred at room temperature overnight. The precipitate was filtered and
the solvent was removed in vacuum. The residue was diluted with CH2Cl2
and washed once more with water. The organic layer was dried with
Na2SO4 and the solvent was removed. Column chromatography (SiO2,
petrol ether/ethylacetate 2:8 v/v). Yield: 50%, 3.0 g, as colorless oil. 1H
NMR (400 MHz, CDCl3)  [ppm] = 7.36 (d, J = 8.5 Hz, 2H), 7.07 (d, J =
8.5 Hz, 2H), 4.66 (s, 2H), 4.32-4.22 (m, 2H), 3.74-3.67 (m, 2H), 3.67-3.58
(m, 6H), 3.56-3.50 (m, 2H) 3.37 (s, 3H), 2.87 (t, J = 6.8 Hz, 2H), 2.76 (t, J
= 6.8 Hz, 2H), 1.97 (s, 1H). 13C-NMR (101 MHz, CDCl3):  [ppm] =
172.07, 170.89, 149.97, 138.58, 128.00, 121.54, 71.88, 70.53, 70.50,
69.02, 64.66, 63.96, 58.98, 29.26, 29.04. HRMS (ESI-TOF): calculated
for C18H26NaO8 [M+Na]+ 393.1520, found 393.1514. IR: ν [cm−1] = 3437,
2875, 1856, 1732, 1607, 1507, 1453, 1411, 1350, 1244, 1196, 1164,
1131, 1099, 1015, 997, 944, 888, 847, 811, 550, 506.
4-(Hydroxymethyl)phenyl hexadecanoate 7f: According to General
Procedure A, under atmosphere,1.22 g 4-hydroxybenzylalcohol (9.8
mmol, 1.2 equiv.) and 1.36 mL triethylamine (9.8 mmol, 1.2 equiv.) was
dissolved in 20 mL CH2Cl2 at rt. 2.48 mL n-hexadecanoyl chloride (8.16
mmol, 1.0 equiv.) in 30 mL CH2Cl2 was added dropwise. The mixture was
stirred for overnight at rt. Column chromatography (SiO2, PE/EE 7:3 v/v).
1
Yield: 80%, 2.36 g, as white solid. H NMR (400 MHz, CDCl3):  [ppm] =
7.37 (d, J = 8.5 Hz, 2H), 7.06 (dt, J = 8.4, 2.4 Hz, 2H), 4.68 (s, 2H), 2.55
(t, J = 7.5 Hz, 2H), 1.75 (p, J = 7.5 Hz, 2H), 1.48-1.18 (m, 24H), 0.88 (t, J
= 7.0 Hz, 3H). 13C-NMR (101 MHz, CDCl3):  [ppm] = 172.4, 150.1, 138.3,
128.0, 121.7, 64.8, 34.4, 31.9, 29.68, 29,67, 29.64, 29.59, 29.45, 29.35,
29.25,29.10, 22.68, 24.9, 14.1. HRMS (ESI-TOF) m/z: calculated for
C23H38NaO3 [M+Na]+ 385.2719, found 385.2782. IR: ν [cm−1] = 3322,
2955, 2914, 2847, 1748, 1606, 1509, 1471, 1463, 1410, 1387, 1348,
1330, 1308, 1286, 1263, 1241, 1218, 1166, 1150, 1097, 1039, 1014, 950,
925, 846, 817, 779, 960, 739, 727, 719, 581, 515.
12-Oxo-2,5,8,11-tetraoxahexadecan-16-oic acid 12i: Under atmosphere,
10 g 2-(2-(2-Methoxyethoxy)ethoxy)ethan-1-ol 10 (60.9 mmol, 1.0 equiv.)
and 8.4 g glutaric anhydride 11i (73.1 mmol, 1.2 equiv.) was added in 50
mL CH2Cl2. Then 1.49 g DMAP (12.2 mmol, 0.2 equiv.) was added and
4-(Hydroxymethyl)phenyl octadecanoate 7g: According to General
Procedure A, under atmosphere, 2.46 g 4-hydroxybenzylalcohol (19.8
mmol, 1.2 equiv.) and 2.75 mL triethylamine (19.8 mmol, 1.2 equiv.) was
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
the mixture was stirred at rt overnight. The reaction mixture was then
quenched with 4 mL water, diluted with 20 mL CH2Cl2 and extract with
(3×10 mL) 10% NaHSO4 and (1×10 mL) brine. The organic phase was
dried with MgSO4 and the solvent was removed in vacuum. The product
was used directly without further purification. Yield: 59%, 9.8 g, as
colorless liquid. 1H NMR (400 MHz, CDCl3)  [ppm] = 4.31-4.20 (m, 2H),
3.75-3.61 (m, 8H), 3.60-3.52 (m, 2H), 3.38 (s, 3H), 2.44 (td, J = 7.1, 3.5
Hz, 4H), 1.98 (quint, J = 7.2 Hz, 2H). 13C-NMR (101 MHz, CDCl3): 
[ppm] = 172.9, 71.9, 70.61, 70.49, 70.46, 69.10, 63.57, 58.95, 33.10,
32.74, 19.88. HRMS (ESI-TOF): calculated for C12H22NaO7 [M+Na]+
301.1258, found 301.1263. IR: ν [cm−1] = 2880, 1731, 1451, 1385, 1344,
1240, 1189, 1155, 1098, 1024, 978, 951, 832, 611, 542, 410.
pentadecanoate 7e (1.0 mmol, 1.0 equiv.) was added and followed with
0.29 g 4-(hydroxymethyl)phenyl pentanoate 7b (1.4 mmol, 1.4 equiv.).
The mixture was stirred for 3 h at rt. Column chromatography (SiO2,
petrol ether/ethylacetate/CH3COOH 7:3:0.005 v/v/v). Yield: 48%, 0.29 g,
as white solid. 1H NMR (400 MHz, CDCl3):  [ppm] = 7.44-7.31 (m, 4H,
1
H-2´´), 7.17-7.01 (m, 4H, H-3´´), 6.94 (d, JPH = 708 Hz, 1H, P-H), 5.16-
4.93 (m, 4H, Ph-CH2), 2.56 (t, 3JHH = 7.5 Hz, 2H, H-q), 2.55 (t, 3JHH = 7.5
Hz, 2H, H-b), 1.85-1.65 (m, 4H, H-c, H-r), 1.53-1.17 (m, 24H, H-d, H-e,
H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-n, H-s), 0.97 (t, 3JHH = 7.3 Hz, 3H,
3
H-t), 0.88 (t, JHH = 6.7 Hz, 3H, H-o). 13C-NMR (101 MHz, CDCl3): 
[ppm] = 172.08 (C-p),172.06 (C-a), 151.0 (2×C-4´´), 132.98, 132.92
(2×C-1´´), 129.2 (4×C-2´´), 121.90 (4×C-3´´), 66.69, 66.63 (2×Ph-CH2),
34.4 (C-b), 34.1 (C-q), 31.9, 29.66, 29,65, 29.62, 29.57, 29.43, 29.33,
29.23, 29.08 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m), 26.9 (C-r),
24.9 (C-c), 22.7 (C-n), 22.2 (C-s), 14.1 (C-o), 13.7 (C-t). 31P-NMR (162
MHz, CDCl3):  [ppm] = 7.70. HRMS (ESI-TOF) m/z: calculated for
C34H55NO7P [M+NH4]+ 620.3711, found 620.3709. IR: ν [cm−1] = 2956,
2915, 2848, 1748, 1608, 1510, 1465, 1413, 1383, 1350, 1317, 1295,
1268, 1250, 1219, 1167, 1150, 1105, 1061, 1009, 996, 925, 897, 878,
834, 769, 720, 692, 580, 559, 538, 515, 456, 420, 408.
4-(Hydroxymethyl)phenyl (2-(2-(2-methoxyethoxy)ethoxy)ethyl) glutarate
7i: 4.3 g 12-oxo-2,5,8,11-tetraoxahexadecan-16-oic acid 12i (15.6 mmol,
1.0 equiv.) was dissolved in 50 mL CH2Cl2 and cooled to 0 °C. 1.58 mL
oxalyl chloride (18.7 mmol, 1.2 equiv.) was added to the flask and 3
drops of DMF was then added. Afterwards, the mixture was warm up to rt
and stirred until no more gas generated (around 2-4 h). Then oxalyl
chloride was evaporated to afford 4.3 g target acyl chloride as colorless
liquid. The yield was calculated as 77%. The crude product 13i was used
directly without further purification. According to General Procedure A,
under atmosphere, 2.32 g 4-hydroxybenzylalcohol (18.7 mmol, 1.2
equiv.) and 2.59 mL triethylamine (18.7 mmol, 1.2 equiv.) was added in
20 mL CH2Cl2. 4.3 g 2-(2-(2-methoxyethoxy)ethoxy)ethyl 5-chloro-5-
oxopentanoate 13i (15.6 mmol, 1.0 equiv.) in 30 mL CH2Cl2 was added
dropwise and the mixture was stirred at rt overnight. The precipitate was
filtered, and the solvent was removed in vacuum. The residue was
diluted with CH2Cl2 and washed once more with water. The organic layer
was dried with Na2SO4 and the solvent was removed. Column
chromatography (SiO2, petrol ether/ethylacetate 2:8 v/v). Yield: 50%, 3.0
g, as colorless oil. 1H NMR (400 MHz, CDCl3)  [ppm] = 7.37 (d, J = 8.5
Hz, 2H), 7.07 (d, J = 9.2 Hz, 2H), 4.68 (d, J = 4.3 Hz, 2H), 4.32-4.20 (m,
2H), 3.75-3.67 (m, 2H), 3.67-3.60 (m, 6H), 3.57-3.51 (m, 2H) 3.37 (s, 3H),
2.64 (t, J = 7.4 Hz, 2H,), 2.49 (t, J = 7.3 Hz, 2H), 2.07 (quint, J = 7.3 Hz,
2H), 1.80 (s, 1H). 13C-NMR (101 MHz, CDCl3):  [ppm] = 172.8, 171.5,
150.0, 138.5, 128.0, 121.6, 71.93, 70.60, 70.57, 69.11, 64.73, 63.63,
59.0, 33.30, 33.06, 20.03. HRMS (ESI-TOF) m/z: calculated for
C19H28NaO8 [M+Na]+ 407.1676, found 407.1703. IR: ν [cm−1] = 3439,
2874, 1731, 1601, 1507, 1452, 1417, 1381, 1352, 1195, 1163, 1127,
1016, 944, 849, 562, 507, 385.
(AB-C6H13,ab-C14H29)-H-phosphonate 8de: According to General
Procedure B, 0.23 mL diphenyl phosphonate (1.2 mmol, 1.2 equiv.) was
added to 5 mL pyridine at 0 °C. Then 0.35 g 4-(hydroxymethyl)phenyl
pentadecanoate 7d (1.0 mmol, 1.0 equiv.) was added and followed with
0.33 g 4-(hydroxymethyl)phenyl heptanoate 7e (1.4 mmol, 1.4 equiv.).
The mixture was stirred for 3 h at rt. Column chromatography (SiO2,
petrol ether/ethylacetate/CH3COOH 7:3:0.005 v/v/v). Yield: 52%, 0.33 g,
as white solid. 1H NMR (400 MHz, CDCl3):  [ppm] = 7.51-7.32 (m, 4H,
1
H-2´´), 7.18-6.97 (m, 4H, H-3´´), 6.93 (d, JPH = 708 Hz, 1H, P-H), 5.20-
4.85 (m, 4H, Ph-CH2), 2.55 (m, 4H, H-q, H-b), 1.75 (m, 4H, H-c, H-r),
1.55-1.18 (m, 28H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-n, H-
s, H-t, H-u), 0.940.84 (m, 6H, H-v, H-o). 13C-NMR (101 MHz, CDCl3): 
[ppm] = 172.05 (C-p, C-a), 151.0 (2×C-4´´), 132.97, 132.91 (2×C-1´´),
129.2 (4×C-2´´), 121.90 ( 4×C-3´´), 66.67, 66.61 (2×Ph-CH2), 34.3 (C-b,
C-q), 31.88, 31.38, 29.64, 29,63, 29.60, 29.56, 29.42, 29.31, 29.21,
29.07, 28.72 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m, C-s, C-t),
24.87,24,82 (C-r, C-c), 22.6 (C-n), 22.4 (C-u), 14.1 (C-o), 14.0 (C-v). 31P-
NMR (162 MHz, CDCl3):  [ppm]
= 7.70. HRMS (ESI-TOF) m/z:
calculated for C36H59NO7P [M+NH4]+ 648.4024, found 648.4037. IR: ν
[cm−1] = 2956, 2915, 2848, 1748, 1608, 1510, 1465, 1411, 1384, 1293,
1270, 1250, 1236, 1218, 1167, 1149, 1116, 1061, 996, 925, 878, 834,
769, 739, 721, 581, 539, 515, 453, 427.
(AB-C2H5,ab-C14H29)-H-phosphonate 8ae: According to General
Procedure B, 0.23 mL diphenyl phosphonate (1.2 mmol, 1.2 equiv.) was
added to 5 mL pyridine at 0 °C. Then 0.35 g 4-(hydroxymethyl)phenyl
pentadecanoate 7e (1.0 mmol, 1.0 equiv.) was added and followed with
0.25 g 4-(hydroxymethyl)phenyl propionate 7a (1.4 mmol, 1.4 equiv.).
The mixture was stirred for 3 h at rt. Column chromatography (SiO2,
petrol ether/ethylacetate/CH3COOH 7:3:0.005 v/v/v). Yield: 50%, 0.29 g,
as white solid. 1H NMR (400 MHz, CDCl3):  [ppm] = 7.39-7.30 (m, 4H,
(AB-iso-C4H9,ab-C14H29)-H-phosphonate 8ce: According to General
Procedure B, 0.23 mL diphenyl phosphonate (1.2 mmol, 1.2 equiv.) was
added to 5 mL pyridine at 0 °C. Then 0.35 g 4-(hydroxymethyl)phenyl
pentadecanoate 7e (1.0 mmol, 1.0 equiv.) was added and followed with
0.29 g 4-(hydroxymethyl)phenyl 3-methylbutanoate 7c (1.4 mmol, 1.4
equiv.). The mixture was stirred for 3 h at rt. Column chromatography
(SiO2, petrol ether/ethylacetate/CH3COOH 7:3:0.005 v/v/v). Yield: 48%,
1
H-2´´), 7.17-7.02 (m, 4H, H-3´´), 6.94 (d, JPH = 708 Hz, 1H, P-H), 5.13-
4.93 (m, 4H, Ph-CH2), 2.59 (q, 3JHH = 8.0 Hz, 2H, H-q), 2.55 (t, 3JHH = 6.3
Hz, 2H, H-b), 1.75 (p, J = 7.5 Hz, 2H, H-c), 1.47-1.25 (m, 25H, H-d, H-e,
H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-n, H-r), 0.88 (t, 3JHH = 6.7 Hz, 3H,
H-o). 13C-NMR (101 MHz, CDCl3):  [ppm] = 172.7 (C-p),172.1 (C-a),
151.0 (2×C-4´´), 132.98, 132.92 (2×C-1´´), 129.2 (4×C-2´´), 121.90,
0.28 g, as white solid. H NMR (400 MHz, CDCl3):  [ppm] = 7.33-7.23 (m,
1
1
4H, H-2´´), 7.08-6.95 (m, 4H, H-3´´), 6.87 (d, JPH = 708 Hz, 1H, P-H),
5.16-4.89 (m, 4H, Ph-CH2), 2.48 (t, 3JHH = 7.5 Hz, 2H, H-b), 2.36 (d, 3JHH
3
3
= 7.2 Hz, 2H, H-q), 2.17 (tq, JHH = 6.8 Hz, 1H, H-r), 1.67 (p, JHH = 7.5
Hz, 2H, H-c), 1.40-1.11 (m, 22H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l,
H-m, H-n), 0.99 (d, 3JHH = 6.7 Hz, 6H, H-t), 0.81 (t, 3JHH = 6.8 Hz, 3H, H-
o). 13C-NMR (101 MHz, CDCl3):  [ppm] = 172.08 (C-a), 171.31 (C-p),
150.99, 150.94 (2×C-4´´), 133.01, 132.95 (2×C-1´´), 129.22, 129.21
(4×C-2´´), 121.94, 121.92 (4×C-3´´), 66.70, 66.65 (2×Ph-CH2), 43.30 (C-
q), 34.36 (C-b), 31.90, 29.66, 29.65, 29.62, 29.58, 29.44, 29.33, 29.23,
29.09 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m), 25.83 (C-r), 24.89
(C-c), 22.66 (C-n), 22.37 (C-s, C-t), 14.09 (C-o). 31P-NMR (162 MHz,
3
121.87 (4×C-3´´), 66.65 (d, JCP = 6.1 Hz, Ph-CH2), 34.4 (C-b), 31.9,
29.65, 29,64, 29.61, 29.57, 29.43, 29.32, 29.22, 29.08 (C-d, C-e, C-f, C-g,
C-h, C-i, C-j, C-k, C-l, C-m), 27.7 (C-q), 24.9 (C-c), 22.7 (C-n), 14.1 (C-o),
9.0 (C-r). 31P-NMR (162 MHz, CDCl3):  [ppm] = 7.71. HRMS (ESI-TOF)
m/z: calculated for C32H47NaO7P [M+Na]+ 597.2952, found 597.2952. IR:
ν [cm−1] = 2956, 2915, 2848, 1754, 1607, 1510, 1463, 1412, 1386, 1359,
1318, 1295, 1269, 1249, 1219, 1167, 1149, 1061, 996, 925, 896, 876,
827, 806, 769, 720, 806, 769, 720, 581, 538, 515, 455, 422, 410.
CDCl3):
 [ppm] = 7.70. HRMS (ESI-TOF) m/z: calculated for
C34H51NaO7P [M+Na]+ 625.3265, found 625.3262. IR: ν [cm−1] = 2956,
2915, 2848, 1748, 1608, 1510, 1464, 1413, 1385, 1317, 1295, 1250,
1218, 1166, 1150, 1106, 1060, 996, 924, 877, 832, 768, 719, 693, 580,
558, 538, 515, 456, 423.
(AB-C4H9,ab-C14H29)-H-phosphonate 8be: According to General
Procedure B, 0.23 mL diphenyl phosphonate (1.2 mmol, 1.2 equiv.) was
added to 5 mL pyridine at 0 °C. Then 0.35 g 4-(hydroxymethyl)phenyl
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
(AB-C4H9,ab-C15H31)-H-phosphonate 8bf: According to General
Procedure B, 0.23 mL diphenyl phosphonate (1.2 mmol, 1.2 equiv.) was
added to 5 mL pyridine at 0 °C. Then 0.36 g 4-(hydroxymethyl)phenyl
hexadecanoate 7f (1.0 mmol, 1.0 equiv.) was added and followed with
0.29 g 4-(hydroxymethyl)phenyl pentanoate 7b (1.4 mmol, 1.4 equiv.).
The mixture was stirred for 3 h at rt. Column chromatography (SiO2,
petrol ether/ethylacetate/CH3COOH 7:3:0.005 v/v/v). Yield: 44%, 0.27 g,
as white solid. 1H NMR (600 MHz, CDCl3):  [ppm] = 7.39-7.34 (m, 4H,
[cm−1] = 3435, 2874, 1756, 1733, 1607, 1508, 1456, 1417, 1350, 1248,
1198, 1165, 1131, 1101, 1029, 1016, 947, 888, 849, 811, 553, 504, 429.
(AB-C14H29,ab-MEEES)-H-phosphonate 8eh: According to General
Procedure B, 0.23 mL diphenyl phosphonate (1.2 mmol, 1.2 equiv.) was
added to 5 mL pyridine at 0 °C. Then 0.35 g 4-(hydroxymethyl)phenyl
pentadecanoate 7e (1.0 mmol, 1.0 equiv.) was added and followed with
0.52
g 4-(hydroxymethyl)phenyl (2-(2-(2-methoxyethoxy)ethoxy)ethyl)
1
H-2´´), 7.10-7.04 (m, 4H, H-3´´), 6.93 (d, JPH = 708 Hz, 1H, P-H), 5.12-
4.95 (m, 4H, Ph-CH2), 2.56 (t, 3JHH = 7.6 Hz, 2H, H-q), 2.55 (t, 3JHH = 7.5
succinate 7h (1.4 mmol, 1.4 equiv.). The mixture was stirred for 3 h at rt.
Column chromatography (SiO2, petrol ether/ethylacetate/ CH3COOH
2:8:0.005 v/v/v). Yield: 52%, 0.39 g, as white solid. 1H NMR (600 MHz,
CDCl3):  [ppm] = 7.47-7.29 (m, 4H, H-2´´), 7.15-7.00 (m, 4H, H-3´´), 6.92
Hz, 2H, H-b), 1.74 (m, 4H, H-c, H-r), 1.54-1.19 (m, 26H, H-d, H-e, H-f, H-
3
g, H-h, H-i, H-j, H-k, H-l, H-m, H-n, H-o, H-s), 0.97 (t, JHH = 7.4 Hz, 3H,
3
H-t), 0.88 (t, JHH = 7.0 Hz, 3H, H-u). 13C-NMR (151 MHz, CDCl3): 
1
3
(d, JPH = 708 Hz, 1H, P-H), 5.11-4.93 (m, 4H, Ph-CH2), 4.26 (t, JHH
=
[ppm] = 172.09 (C-p),172.08 (C-a), 151.0 (2×C-4´´), 132.97, 132.93
(2×C-1´´), 129.2 (4×C-2´´), 121.90 (4×C-3´´), 66.68, 66.64 (2×Ph-CH2),
34.4 (C-b), 34.1 (C-q), 31.9, 29.66, 29,65, 29.63, 29.62, 29.58, 29.44,
29.33, 29.23, 29.09 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m, C-n),
26.9 (C-r), 24.9 (C-c), 22.7 (C-o), 22.2 (C-s), 14.1 (C-u), 13.7 (C-t). 31P-
5.7 Hz, 2H, H-t), 3.76-3.67 (m, 2H, H-u), 3.68-3.57 (m, 6H, H-v, H-w, H-x),
3.57-3.49 (m, 2H, H-y), 3.36 (s, 3H, H-z), 2.87 (t, 3JHH = 6.4 Hz, 2H, H-q),
3
3
2.76 (t, JHH = 6.7 Hz, 2H, H-r), 2.54 (t, JHH = 7.5 Hz, 2H, H-b), 1.73
(quint, 3JHH = 7.5 Hz, 2H, H-c), 1.43-1.36 (m, 2H, H-d), 1.35-1.16 (m, 20H,
H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-n), 0.87 (t, 3JHH = 7.0 Hz, 3H,
H-o). 13C-NMR (151 MHz, CDCl3):  [ppm] = 172.07, 171.98 (C-p, C-s),
NMR (243 MHz, CDCl3):  [ppm]
= 7.70. HRMS (ESI-TOF) m/z:
calculated for C35H57NO7P [M+NH4]+ 634.3867, found 634.3861. IR: ν
[cm−1] = 2956, 2915, 2848, 1748, 1608, 1510, 1465, 1413, 1383, 1348,
1250, 1240, 1219, 1167, 1150, 1105, 1061, 1008, 996, 925, 897, 879,
833, 769, 720, 580, 538, 514, 451, 421, 403.
3
170.66 (C-a), 150.95, 150.85 (2×C-4´´), 133.14 (d, JCP = 6.1 Hz, C-1´´),
3
132.96 (d, JCP = 6.2 Hz, C-1´´), 129.21 (2×C-2´´), 121.88 (d, JCP = 8.5
Hz, C-3´´), 71.91 (C-y), 70.55, 70.54 (C-v, C-w, C-x), 69.03 (C-u), 66.70,
66.66, 66.60 (Ph-CH2), 63.98 (C-t), 58.99 (C-z), 34.40 (C-b), 31.90, 29.64,
29.64, 29.62, 29.52, 29.44, 29.35, 29.27, 29.11 (C-d, C-e, C-f, C-g, C-h,
C-i, C-j, C-k, C-l, C-m, C-q), 28.99 (C-r), 24.91 (C-c), 22.68 (C-n), 14.15
(C-o). 31P-NMR (243 MHz, CDCl3):  [ppm] = 7.69. HRMS (ESI-TOF)
m/z: calculated for C40H65NO12P [M+NH4]+ 782.4239, found 782.4233. IR:
ν [cm−1] = 2955, 2915, 2848, 1747, 1607, 1510, 1464, 1423, 1412, 1387,
1340, 1317, 1296, 1270, 1250, 1222, 1200, 1166, 1138, 1062, 1009, 995,
925, 835, 770, 727, 720, 657, 580, 554, 538, 516, 455, 441, 421.
(AB-C4H9,ab-C17H35)-H-phosphonate 8bg: According to General
Procedure B, 0.23 mL diphenyl phosphonate (1.2 mmol, 1.2 equiv.) was
added to 5 mL pyridine at 0 °C. Then 0.39 g 4-(hydroxymethyl)phenyl
octadecanoate 7g (1.0 mmol, 1.0 equiv.) was added and followed with
0.29 g 4-(hydroxymethyl)phenyl pentanoate 7b (1.4 mmol, 1.4 equiv.).
The mixture was stirred for 3 h at rt. Column chromatography (SiO2,
petrol ether/ethylacetate/CH3COOH 8:2:0.005 v/v/v). Yield: 43%, 0.28 g,
as white solid. 1H NMR (600 MHz, CDCl3):  [ppm] = 7.39-7.33 (m, 4H,
(AB-C14H29,ab-MEEEG)-H-phosphonate 8ei: According to General
Procedure B, 0.23 mL diphenyl phosphonate (1.2 mmol, 1.2 equiv.) was
added to 5 mL pyridine at 0 °C. Then 0.35 g 4-(hydroxymethyl)phenyl
pentadecanoate 7e (1.0 mmol, 1.0 equiv.) was added and followed with
1
H-2´´), 7.11-7.05 (m, 4H, H-3´´), 6.93 (d, JPH = 708 Hz, 1H, P-H), 5.16-
4.91 (m, 4H, Ph-CH2), 2.56 (t, 3JHH = 7.5 Hz, 2H, H-q), 2.55 (t, 3JHH = 7.5
Hz, 2H, H-b), 1.74 (m, 4H, H-c, H-r), 1.49-1.20 (m, 30H, H-d, H-e, H-f, H-
g, H-h, H-i, H-j, H-k, H-l, H-m, H-n, H-o, H-u, H-v, H-s), 0.97 (t, 3JHH = 7.3
0.54
g 4-(hydroxymethyl)phenyl (2-(2-(2-methoxyethoxy)ethoxy)ethyl)
3
Hz, 3H, H-t), 0.88 (t, JHH = 7.0 Hz, 3H, H-w). 13C-NMR (151 MHz,
glutarate 7i (1.4 mmol, 1.4 equiv.). The mixture was stirred for 3 h at rt.
Column chromatography (SiO2, petrol ether/ethylacetate/CH3COOH
2:8:0.005 v/v/v). Yield: 46%, 0.36 g, as white solid. 1H NMR (400 MHz,
CDCl3):  [ppm] = 7.48-7.30 (m, 4H, H-2´´), 7.17-7.00 (m, 4H, H-3´´), 6.92
CDCl3):  [ppm] = 172.08 (C-p),172.06 (C-a), 151.0 (2×C-4´´), 132.95,
132.91 (2×C-1´´), 129.2 (4×C-2´´), 121.90 (4×C-3´´), 66.67, 66.63 (2×Ph-
CH2), 34.3 (C-b), 34.1 (C-q), 31.9, 29.66, 29,64, 29.62, 29.61, 29.57,
29.43, 29.33, 29.22, 29.08 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m,
C-n, C-o, C-u), 26.9 (C-r), 24.9 (C-c), 22.7 (C-v), 22.2 (C-s), 14.1 (C-w),
13.7 (C-t). 31P-NMR (243 MHz, CDCl3):  [ppm] = 7.71. HRMS (ESI-TOF)
m/z: calculated for C37H61NO7P [M+NH4]+ 662.4180, found 662.4200. IR:
ν [cm−1] = 2956, 2915, 2848, 1748, 1608, 1510, 1465, 1383, 1251, 1235,
1220, 1167, 1150, 1104, 1062, 1010, 997, 925, 878, 834, 769, 720, 510.
1
3
(d, JPH = 712 Hz, 1H, P-H), 5.15-4.90 (m, 4H, Ph-CH2), 4.25 (t, JHH
=
4.9 Hz, 2H, H-u), 3.75-3.67 (m, 2H, H-v), 3.67-3.60 (m, 6H, H-w, H-x, H-
y), 3.57-3.51 (m, 2H, H-z), 3.36 (s, 3H, -OCH3), 2.64 (t, J = 7.3 Hz, 2H,
3
H-q), 2.54 (t, JHH = 7.5 Hz, 2H, H-b), 2.49 (t, J = 7.2 Hz, 2H, H-s), 2.06
3
(quint, 3JHH = 7.0 Hz, 2H, H-r), 1.74 (quint, JHH = 7.4 Hz, 2H, H-c), 1.46-
1.19 (m, 22H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-n), 0.87 (t,
3JHH = 6.5 Hz, 3H, H-o). 13C-NMR (151 MHz, CDCl3):  [ppm] = 172.7 (C-
t), 172.1 (C-a), 171.2 (C-p), 151.0, 150.8 (2×C-4´´), 133.07 (d, 4JCP = 6.0
(AB-C4H9,ab-MEEES)-H-phosphonate 8bh: According to General
Procedure B, 0.23 mL diphenyl phosphonate (1.2 mmol, 1.2 equiv.) was
added to 5 mL pyridine at 0 °C. Then 0.28 g 4-(hydroxymethyl)phenyl
pentanoate 7b (1.0 mmol, 1.0 equiv.) was added and followed with 0.52
3
Hz, C-1´´), 133.92 (d, JCP = 6.0 Hz, C-1´´), 129.2 (2×C-2´´), 121.92,
121.86 (2×C-3´´), 71.9 (C-z), 70.60, 70.54 (C-w, C-x, C-y), 69.1 (C-v),
66.69, 66.66 (Ph-CH2), 64.7 (Ph´´-CH2), 63.6 (C-u), 59.0 (-OCH3), 34.4
(C-b), 33.3 (C-q), 33.0 (C-s), 31.89, 29.65, 29.64, 29.61, 29.56, 29.43,
29.32, 29.22, 29.07 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m), 24.9
(C-c), 22.7 (C-n), 20.0 (C-r), 14.1 (C-o). 31P-NMR (162 MHz, CDCl3): 
g
4-(hydroxymethyl)phenyl
(2-(2-(2-methoxyethoxy)ethoxy)ethyl)
succinate 7h (1.4 mmol, 1.4 equiv.). The mixture was stirred for 3 h at rt.
Column chromatography (SiO2, petrol ether/ethylacetate/CH3COOH
2:8:0.005 v/v/v). Yield: 52%, 0.33 g, as white solid. 1H NMR (400 MHz,
CDCl3):  [ppm] = 7.40-7.32 (m, 4H, H-2´´), 7.09 (m, 4H, H-3´´), 6.93 (d,
[ppm]
= 7.71. HRMS (ESI-TOF) m/z: calculated for C41H67NO12P
[M+NH4]+ 796.4395, found 796.4416. IR: ν [cm−1] = 2915, 2849, 1756,
1747, 1607, 1510, 1464, 1412, 1390, 1270, 1250, 1223, 1196, 1167,
1136, 1063, 1021, 1011, 997, 925, 877, 835, 770, 726, 582, 540, 516,
455.
3
1JPH = 708 Hz, 1H, P-H), 5.13-4.93 (m, 4H, Ph-CH2), 4.27 (t, JHH = 4.8
Hz, 2H, H-t), 3.74-3.68 (m, 2H, H-u), 3.68-3.60 (m, 6H, H-v, H-w, H-x),
3.57-3.51 (m, 2H, H-y), 3.37 (s, 3H, H-z), 2.88 (t, 3JHH = 6.9 Hz, 2H, H-q),
3
3
2.77 (t, JHH = 6.9 Hz, 2H, H-r), 2.56 (t, JHH = 7.6 Hz, 2H, H-b), 1.73
(quint, 3JHH = 7.5 Hz, 2H, H-c), 1.44 (tq, 3JHH = 7.6, 7.5 Hz, 2H, H-d), 0.97
(t, 3JHH = 7.3 Hz, 3H, H-e). 13C-NMR (101 MHz, CDCl3):  [ppm] = 172.06,
171.99 (C-p, C-s), 170.67 (C-a), 150.97, 150.81 (2×C-4´´), 133.13 (d,
-(AB-C2H5,ab-C14H29)-d4TTP (ammonium salt) 3ae: According to
General Procedure C, the reactions were performed under dry conditions
using 100 mg H-phosphonate 8ae (0.174 mmol, 1.0 equiv.) and 137 mg
d4TMP 2nBu4N+ salt (0.174 mmol, 1.0 equiv.). Yield: 64%, 111 mg, as
3
3JCP = 6.1 Hz, C-1´´), 132.93 (d, JCP = 6.2 Hz, C-1´´), 129.20 (2×C-2´´),
white solid. 1H-NMR (600 MHz, MeOD):  [ppm] = 7.69-7.62 (m, 1H, Hhet
-
121.86 (d, JCP = 8.5 Hz, C-3´´), 71.90 (C-z), 70.56, 70.54 (C-v, C-w, C-x),
69.02 (C-u), 66.70, 66.64, 66.58 (Ph-CH2), 63.98 (C-t), 58.99 (C-z), 34.05
(C-b), 29.25 (C-q), 28.99 (C-r), 26.92 (C-c), 22.20 (C-d), 13.67 (C-e) 31P-
6), 7.40 (dt, 3JHH = 8.6, 2.5 Hz, 4H, H-2´´), 7.08-7.03 (m, 4H, H-3´´), 6.92
(m, 1H, H-1´), 6.45 (dt, 3JHH = 5.9, 4JHH = 1.8 Hz, 1H, H-3´), 5.80 (dt, 3JHH
4
3
NMR (162 MHz, CDCl3):  [ppm]
= 7.71. HRMS (ESI-TOF) m/z:
= 5.8 Hz, JHH = 1.8 Hz, 1H, H-2´), 5.15 (d, JHP = 8.1Hz, 4H, Ph-CH2),
calculated for C30H45NO12P [M+NH4]+ 642.2674, found 642.2687. IR: ν
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
3
4.96-4.91 (m, 1H, H-4´), 4.30-4.15 (m, 2H, H-5´), 2.60 (q, JHH = 7.8 Hz,
IR: ν [cm−1] = 3184, 3045, 2955, 2922, 2852, 1754, 1690, 1509, 1464,
1380, 1249, 1220, 1168, 1128, 1113, 1084, 1007, 910, 838, 784, 768,
722, 696, 643, 577, 494, 421, 400.
3
2H, H-q), 2.57 (t, JHH = 7.2 Hz, 2H, H-b), 1.89 (s, 3H, Hhet-7), 1.76-1.69
(m, 2H, H-c), 1.47-1.25 (m, 22H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l,
H-m, H-n), 1.23 (t, 3JHH = 7.6 Hz, 3H, H-r), 0.90 (t, 3JHH = 6.9 Hz, 3H, H-o).
13C-NMR (151 MHz, MeOD):  [ppm] = 174.5 (C-p), 173.8 (C-a), 166.5.3
(Chet-4), 152.8 (Chet-2), 152.4 (2×C-4´´), 138.6 (Chet-6), 135.7 (C-3´),
134.9 (d, 3JCP = 7.6 Hz, 2×C-1´´), 130.5 (d, 4JCP = 2.9 Hz, 4×C-2´´), 127.2
(C-2´), 122.89, 122.86, 122.82 (4×C-3´´), 112.1 (Chet-5), 90.8 (C-1´), 87.2
-(AB-iso-C4H9,ab-C14H29)-d4TTP (ammonium salt) 3ce: According to
General Procedure C, the reactions were performed under dry conditions
using 90.8 mg H-phosphonate 8ce (0.151 mmol, 1.0 equiv.) and 106 mg
d4TMP 2nBu4N+ salt (0.151 mmol, 1.0 equiv.). Yield: 39%, 60 mg, as
white solid. 1H-NMR (600 MHz, MeOD):  [ppm] = 7.68 (d, 4JHH = 1.2 Hz,
1H, Hhet-6), 7.47-7.35 (m, 4H, H-2´´), 7.09-7.04 (m, 4H, H-3´´), 6.94 (ddd,
(d, 3JCP = 9.1 Hz, C-4´), 70.39 (d, 3JCP = 5.7 Hz, Ph-CH2), 67.9 (d, 2JCP
=
5.8 Hz, C-5´), 35.0 (C-b), 33.0 (C-q), 30.78, 30.77, 30.75, 30.71, 30.60,
30.46, 30.40, 30.16, 28.4 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m),
26.0 (C-c), 23.7 (C-n), 14.4 (C-o), 12.5 (Chet-7), 9.3 (C-r). 31P-NMR (243
MHz, MeOD):  [ppm] = -11.81 (d, 2JPP = 19.6 Hz, P-), -13.23 (d, 2JPP
17.2 Hz, P-), -23.74 (br, s, P-). HRMS (ESI-TOF) m/z: calculated for
C42H58N2O17P3 [M-H]- 955.2954, found 955.2911. IR: ν [cm−1] = 2922,
2852, 1757, 1689, 1509, 1460, 1422, 1248, 1218, 1168, 1128, 1079,
1008, 903, 837, 806, 784, 768, 721, 697, 645, 577, 488, 426, 401.
4
4
3JHH = 3.5 Hz, JHH = 1.6, 1.3 Hz, 1H, H-1´), 6.48 (dt, 3JHH = 6.0 Hz, JHH
= 1.7 Hz, 1H, H-3´), 5.81 (ddd, 3JHH = 6.1 Hz, 3JHH = 2.4 Hz, 4JHH = 1.4 Hz,
1H, H-2´), 5.17 (d, JHP = 8.1 Hz, 4H, Ph-CH2), 4.98-4.93 (m, 1H, H-4´),
4.29 (ddd, JHH = 11.6 Hz, JHH = 6.8 Hz, 4JHH = 3.3 Hz, 1H, H-5´), 4.21
(ddd, JHH = 11.6 Hz, JHH = 5.4 Hz, JHH = 3.1 Hz, 1H, H-5´), 2.59 (td,
3JHH = 7.4 Hz, 4JHH = 1.3 Hz 2H, H-b), 2.47 (dd, 2JHH = 7.2 Hz, 4JHH = 1.3
Hz, 2H, H-q), 2.28-2.17 (m, 1H, H-r), 1.91 (d, JHH = 1.2 Hz, 3H, Hhet-7),
3
=
2
3
2
3
4
4
1.80-1.69 (m, 2H, H-c), 1.51-1.22 (m, 22H, H-d, H-e, H-f, H-g, H-h, H-i,
3
4
-(AB-C4H9,ab-C14H29)-d4TTP (ammonium salt) 3be: According to
General Procedure C, the reactions were performed under dry conditions
using 276 mg H-phosphonate 8be (0.458 mmol, 1.0 equiv.) and 360 mg
d4TMP 2nBu4N+ salt (0.458 mmol, 1.0 equiv.). Yield: 28%, 131 mg, as
white solid. 1H-NMR (400 MHz, MeOD):  [ppm] = 7.68 (d, 4JHH = 1.2 Hz,
1H, Hhet-6), 7.55-7.25 (m, 4H, H-2´´), 7.12-6.99 (m, 4H, H-3´´), 6.92 (dt,
3JHH = 3.5 Hz, 4JHH = 1.6 Hz, 1H, H-1´), 6.46 (dt, 3JHH = 6.0, 4JHH = 1.8 Hz,
H-j, H-k, H-l, H-m, H-n), 1.08 (dd, JHH = 6.7 Hz, JHH = 0.8 Hz, 6H, H-t,
H-s), 0.92 (t, JHH = 7.0 Hz, 3H, H-o). 13C-NMR (151 MHz, MeOD): 
3
[ppm] = 173.8 (C-p), 173.0 (C-a), 166.5 (Chet-4), 152.8 (Chet-2), 152.4,
152.3 (2×C-4´´), 138.7 (Chet-6), 135.8 (C-3´), 135.8 (2×C-1´´), 130.5 (d,
4JCP = 4.5 Hz, 4×C-2´´), 127.2 (C-2´), 122.9 (4×C-3´´), 112.1 (Chet-5),
90.9 (C-1´), 87.2 (d, 3JCP = 9.6 Hz, C-4´), 70.4 (2×Ph-CH2), 67.88 (d, 2JCP
= 5.6 Hz, C-5´), 44.0 (C-q), 35.0 (C-b), 33.1, 30.79, 30.78, 30.76, 30.72,
30.61, 30.48, 30.41, 30.17 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-
m), 27.0 (C-r), 26.0 (C-c), 23.7 (C-n), 22.7 (C-s, C-t), 14.4 (C-o), 12.5
(Chet-7) 31P-NMR (243 MHz, MeOD):  [ppm] = -11.80 (d, J = 19.6 Hz, P-
), -13.21 (d, J = 17.3 Hz, P-), -23.72 (t, J = 19.3 Hz, P-). HRMS (ESI-
TOF) m/z: calculated for C44H62N2O17P3 [M-H]- 983.3267, found 983.3160.
IR: ν [cm−1] = 3046, 2957, 2923, 2853, 1756, 1690, 1509, 1464, 1369,
1247, 1218, 1202, 1167, 1128, 1112, 1083, 1009, 909, 837, 784, 769,
722, 696, 644, 573, 490, 425.
3
1H, H-3´), 5.83-5.76 (m, 1H, H-2´), 5.16 (d, JHP = 8.0 Hz, 4H, Ph-CH2),
3
4.99-4.93 (m, 1H, H-4´), 4.36-4.16 (m, 2H, H-5´), 2.58 (t, JHH = 7.2 Hz,
2H, H-q), 2.57 (t, 3JHH = 7.2 Hz, 2H, H-b), 1.90 (d, 4JHH = 1.2 Hz, 3H, Hhet
-
7), 1.84-1.66 (m, 4H, H-c, H-r), 1.57-1.23 (m, 24H, H-d, H-e, H-f, H-g, H-
h, H-i, H-j, H-k, H-l, H-m, H-n, H-s), 0.99 (t, 3JHH = 7.4 Hz, 3H, H-t), 0.90 (t,
3JHH = 6.8 Hz, 3H, H-o). 13C-NMR (101 MHz, MeOD):  [ppm] = 174.6 (C-
p, C-a), 167.3 (Chet-4), 153.6 (Chet-2), 153.2 (2×C-4´´), 139.5 (Chet-6),
3
4
136.6 (C-3´), 135.8 (d, JCP = 7.5 Hz, 2×C-1´´), 131.4 (d, JCP = 2.9 Hz,
4×C-2´´), 128,0 (C-2´), 123.73, 123.72 (4×C-3´´), 112.9 (Chet-5), 91.7 (C-
1´), 88.1 (d, 3JCP = 9.1 Hz, C-4´), 71.3 (d, JCP = 6.1 Hz, Ph-CH2), 71.2 (d,
JCP = 5.3 Hz, Ph-CH2), 68.9 (d, 2JCP = 5.8 Hz, C-5´), 35.9 (C-b), 35.6 (C-
q), 33.9, 31.64, 31.63, 31.61, 31.57, 31.46, 31.32, 31.26, 31.03 (C-d, C-e,
C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m), 28.9 (C-r), 26.9 (C-c), 24.6 (C-n),
24.1 (C-s), 15.3 (C-o), 15.0 (C-t), 12.5 (Chet-7). 31P-NMR (81 MHz,
MeOD):  [ppm] = -11.81 (br, s, P-), -13.24 (d, J = 17.2 Hz, P-), -23.77
(br, s, P-). HRMS (ESI-TOF) m/z: calculated for C44H62N2O17P3 [M-H]-
983.3267, found 983.3229. IR: ν [cm−1] = 3183, 3042, 2923, 2853, 1756,
1689, 1509, 1462, 1380, 1248, 1218, 1202, 1167, 1128, 1112, 1082,
1008, 908, 837, 784, 723, 697, 644, 488, 421, 401.
-(AB-C4H9,ab-C15H31)-d4TTP (ammonium salt) 3bf: According to
General Procedure C, the reactions were performed under dry conditions
using 100 mg H-phosphonate 8bf (0.162 mmol, 1.0 equiv.) and 128 mg
d4TMP 2nBu4N+ salt (0.162 mmol, 1.0 equiv.). Yield: 47%, 78 mg, as
white solid. 1H-NMR (600 MHz, MeOD):  [ppm] = 7.68 (d, 4JHH = 1.2 Hz,,
1H, Hhet-6), 7.44-7.38 (m, 4H, H-2´´), 7.14-7.01 (m, 4H, H-3´´), 6.94 (dt,
3JHH = 3.5, 4JHH = 1.6 Hz, 1H, H-1´), 6.48 (dt, 3JHH = 6.1 Hz, 4JHH = 1.7 Hz,
1H, H-3´), 5.81 (ddd, JHH = 6.1, JHH = 2.4 Hz, JHH = 1.3 Hz, 1H, H-2´),
5.17 (d, JHP = 8.4 Hz, 4H, Ph-CH2), 4.99-4.92 (m, 1H, H-4´), 4.32-4.16
(m, 2H, H-5´), 2.63-2.56 (m, 4H, H-b, H-q), 1.91 (d, JHH = 1.2 Hz, 3H,
3
3
4
3
4
Hhet-7), 1.79-1.69 (m, 4H, H-c, H-r), 1.52-1.23 (m, 26H, H-d, H-e, H-f, H-g,
-(AB-C6H13,ab-C14H29)-d4TTP (ammonium salt) 3de: According to
General Procedure C, the reactions were performed under dry conditions
using 100 mg H-phosphonate 8de (0.159 mmol, 1.0 equiv.) and 125 mg
d4TMP 2nBu4N+ salt (0.159 mmol, 1.0 equiv.). Yield: 52%, 87 mg, as
white solid. 1H-NMR (600 MHz, MeOD):  [ppm] = 7.70 -7.62 (m, 1H,
H-h, H-i, H-j, H-k, H-l, H-m, H-n, H-o, H-s), 1.01 (t, 3JHH = 7.4 Hz, 3H, H-t),
3
0.92 (t, JHH = 7.0 Hz, 3H, H-u). 13C-NMR (151 MHz, MeOD):  [ppm] =
173.78 (C-p, C-a), 166.52 (Chet-4), 152.76 (Chet-2), 152.35 (2×C-4´´),
138.66 (Chet-6), 135.76 (C-3´), 134.93 (d, 3JCP = 7.4 Hz, 2×C-1´´), 130.48
(d, 4JCP = 2.9 Hz, 4×C-2´´), 127.16 (C-2´), 122.87 (d, 3JCP = 2.1 Hz, 4×C-
3
3
Hhet-6), 7.44-7.37 (m, 4H, H-2´´), 7.12-7.01 (m, 4H, H-3´´), 6.94 (dt, JHH
3´´), 112.06 (Chet-5), 90.84 (C-1´), 87.20 (d, JCP = 9.1 Hz, C-4´), 70.41,
= 3.5 Hz, 4JHH = 1.7 Hz, 1H, H-1´), 6.47 (dt, 3JHH = 5.9 Hz, 4JHH = 1.8 Hz,
70.38, 70.36 (2×Ph-CH2), 67.88 (d, 2JCP = 5.6 Hz, C-5´), 35.03, 34.76 (C-
q, C-b), 33.07, 30.78, 30.77, 30.75, 30.71, 30.60, 30.46, 30.40, 30.17(C-
d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m, C-n), 28.07 (C-r), 25.96 (C-c),
23.73 (C-o), 23.25 (C-s), 14.44 (C-u), 14.10 (C-t), 12.48 (Chet-7). 31P-
NMR (243 MHz, MeOD):  [ppm] = -11.73 (br, s, P-), -13.18 (d, J = 17.2
Hz, P-), -23.68 (br, s, P-). HRMS (ESI-TOF) m/z: calculated for
C45H64N2O17P3 [M-H]- 997.3423, found 997.3389. IR: ν [cm−1] = 3191,
3045, 2956, 2921, 2852, 1755, 1689, 1509, 1464, 1380, 1249, 1219,
1168, 1128, 1082, 1008, 908, 838, 784, 769, 721, 696, 644, 492, 422,
401.
3
1H, H-3´), 5.84-5.79 (m, 1H, H-2´), 5.17 (d, JHP = 8.2 Hz, 4H, Ph-CH2),
3
4.99-4.93 (m, 1H, H-4´), 4.33-4.17 (m, 2H, H-5´), 2.59 (t, JHH = 7.4 Hz,
4
4H, H-q, H-b), 1.91 (d, JHH = 1.3 Hz, 3H, Hhet-7), 1.82-1.69 (m, 4H, H-c,
H-r), 1.52-1.23 (m, 28H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m,
H-n, H-s, H-t, H-u), 0.95 (t, 3JHH = 7.2 Hz, 3H, H-v), 0.92 (t, 3JHH = 7.0 Hz,
3H, H-o). 13C-NMR (151 MHz, MeOD):  [ppm] = 173.76 (C-a, C-p),
166.5 (Chet-4), 152.8 (Chet-2), 152.4 (2×C-4´´), 138.6 (Chet-6), 135.7 (C-3´),
3
4
134.9 (d, JCP = 7.6 Hz, 2×C-1´´), 130.49 (d, JCP = 4.5 Hz, 4×C-2´´),
127.2 (C-2´), 122.88, 122.87 (4×C-3´´), 112.1 (Chet-5), 90.9 (C-1´), 87.12
3
(d, JCP = 9.1 Hz, C-4´), 70.42 (d, JCP = 3.8 Hz, Ph-CH2), 70.39 (d, JCP
=
2
4.4 Hz, Ph-CH2), 67.96 (d, JCP = 5.3 Hz, C-5´), 35.0 (C-b, C-q), 33.07,
32.66, 30.79, 30.77, 30.75, 30.72, 30.60, 30.47, 30.41, 30.17, 29.8 (C-d,
C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m, C-s, C-t), 25.96 (C-r), 25.93 (C-
c), 23.73 (C-n), 23.58 (C-u), 14.44 (C-o), 14.39 (C-v), 12.5 (Chet-7). 31P-
NMR (243 MHz, MeOD):  [ppm] = -11.84 (d, 2JPP = 18.2 Hz, P-), -13.18
(d, 2JPP = 16.8 Hz, P-), -23.82 (t, 2JPP = 16.3 Hz, P-). HRMS (ESI-TOF)
m/z: calculated for C46H66N2O17P3 [M-H]- 1011.3580, found .1011.3472.
--(AB-C4H9,ab-C17H35)-d4TTP (ammonium salt) 3bg: According to
General Procedure C, the reactions were performed under dry conditions
using 97 mg H-phosphonate 8bg (0.150 mmol, 1.0 equiv.) and 118 mg
d4TMP 2nBu4N+ salt (0.150 mmol, 1.0 equiv.). Yield: 40%, 64 mg, as
white solid. 1H-NMR (600 MHz, MeOD):  [ppm] = 7.71-7.66 (m, 1H, Hhet
6), 7.44-7.38 (m, 4H, H-2´´), 7.09-7.04 (m, 4H, H-3´´), 6.94 (dt, 3JHH = 3.5
-
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
4
3
4
Hz, JHH = 1.6 Hz, 1H, H-1´), 6.48 (dt, JHH = 6.0 Hz, JHH = 1.8 Hz, 1H,
67.84 (d, 2JCP = 5.5 Hz, C-5´), 65.03 (C-t), 59.09 (C-z), 35.03 (C-b), 33.07,
30.79, 30.78, 30.76, 30.72, 30.61, 30.47, 30.42, 30.18, 30.12, 29.92 (C-d,
C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m, C-q, C-r), 25.97 (C-c), 23.74
(C-n), 14.44 (C-o), 12.49 (Chet-7). 31P-NMR (243 MHz, MeOD):  [ppm] =
-11.71 (d, J = 19.6 Hz, P-), -13.16 (d, J = 17.1 Hz, P-), -23.60 (t, J =
17.1 Hz, P-). HRMS (ESI-TOF) m/z: calculated for C50H72N2O22P3 [M-H]-
1145.3795, found 1145.3786. IR: ν [cm−1] = 3184, 2923, 2853,1756,
1738, 1689, 1509, 1456, 1367, 1248, 1219, 1200, 1167, 1128, 1084,
1009, 906, 838, 807, 784, 722, 645, 486, 423, 399.
3
3
4
H-3´), 5.81 (ddd, JHH = 6.1 Hz, JHH = 2.4 Hz, JHH = 1.3 Hz, 1H, H-2´),
5.17 (d, JHP = 8.2 Hz, 4H, Ph-CH2), 4.98-4.93 (m, 1H, H-4´), 4.33-4.15
(m, 2H, H-5´), 2.63-2.56 (m, 4H, H-b, H-q), 1.91 (d, JHH = 1.2 Hz, 3H,
Hhet-7), 1.79-1.69 (m, 4H, H-c, H-r), 1.52-1.23 (m, 30H, H-d, H-e, H-f, H-g,
H-h, H-i, H-j, H-k, H-l, H-m, H-n, H-o, H-u, H-v, H-s), 1.01 (t, JHH = 7.4
Hz, 3H, H-t), 0.92 (t, JHH = 7.0 Hz, 3H, H-w). 13C-NMR (151 MHz,
MeOD):  [ppm] = 173.77 (C-p, C-a), 166.53 (Chet-4), 152.76 (Chet-2),
152.35 (2×C-4´´), 138.67 (Chet-6), 135.76 (C-3´), 134.93 (d, 3JCP = 7.5 Hz,
2×C-1´´), 130.48 (d, 4JCP = 2.9 Hz, 4×C-2´´), 127.16 (C-2´), 122.88 (4×C-
3
4
3
3
3
3´´), 112.05 (Chet-5), 90.83 (C-1´), 87.20 (d, JCP = 9.1 Hz, C-4´), 70.38
(2×Ph-CH2), 67.87 (d, JCP = 5.6 Hz, C-5´), 35.03, 34.76 (C-q, C-b),
-(AB-C14H29,ab-MEEEG)-d4TTP (ammonium salt) 3ei: According to
General Procedure C, the reactions were performed under dry conditions
using 117 mg H-phosphonate 8ei (0.150 mmol, 1.0 equiv.) and 118 mg
d4TMP 2nBu4N+ salt (0.150 mmol, 1.0 equiv.). Yield: 35%, 62 mg, as
colorless solid. 1H-NMR (600 MHz, MeOD):  [ppm] = 7.69 (d, 4JHH = 1.4
Hz, 1H, Hhet-6), 7.47-7.38 (m, 4H, H-2´´), 7.13-7.03 (m, 4H, H-3´´), 6.94
2
33.07, 30.79, 30.77, 30.75, 30.72, 30.61, 30.47, 30.42, 30.18 (C-d, C-e,
C-f, C-g, C-h, C-i, C-j, C-k, C-l, C-m, C-n, C-o, C-u), 28.07 (C-r), 25.96
(C-c), 23.74 (C-v), 23.26 (C-s), 14.45 (C-w), 14.11 (C-t), 12.49 (Chet-7).
31P-NMR (243 MHz, MeOD):  [ppm] = -11.77 (d, J = 19.6 Hz, P-), -
13.20 (d, J = 17.3 Hz, P-), -23.70 (t, J = 18.1 Hz, P-). HRMS (ESI-TOF)
m/z: calculated for C47H68N2O17P3 [M-H]- 1025.3736, found 1025.3703.
IR: ν [cm−1] = 3040, 2920, 2851, 1755, 1690, 1509, 1465, 1380, 1249,
1219, 1168, 1128, 1082, 1007, 910, 838, 784, 768, 721, 644, 491, 420.
(dt, 3JHH = 3.5 Hz, 4JHH = 1.6 Hz, 1H, H-1´), 6.48 (dt, 3JHH = 6.0 Hz, 4JHH
=
1.7 Hz, 1H, H-3´), 5.81 (ddd, 3JHH = 5.9 Hz, 3JHH = 2.4 Hz, 4JHH = 1.9 Hz,
3
1H, H-2´), 5.17 (d, JHP = 8.1 Hz, 4H, Ph-CH2), 4.98-4.93 (m, 1H, H-4´),
4.32-4.17 (m, 4H, H-5´, H-u), 3.74-3.70 (m, 2H, H-v), 3.67-3.61 (m, 6H,
H-w, H-x, H-y), 3.56-3.52 (m, 2H, H-z), 3.36 (s, 3H, -OCH3), 2.69 (td, 3JHH
4
3
4
-(AB-C4H9,ab-MEEES)-d4TTP (ammonium salt) 3bh: According to
General Procedure C, the reactions were performed under dry conditions
using 144 mg H-phosphonate 8bh (0.231 mmol, 1.0 equiv.) and 187 mg
d4TMP 2nBu4N+ salt (0.231 mmol, 1.0 equiv.). Yield: 45%, 101 mg, as
colorless solid. 1H-NMR (600 MHz, MeOD):  [ppm] = 7.69 (d, 4JHH = 1.3
Hz, 1H, Hhet-6), 7.46-7.39 (m, 4H, H-2´´), 7.12-7.04 (m, 4H, H-3´´), 6.94
= 7.4 Hz, JHH = 1.3 Hz, 2H, H-q), 2.60 (td, JHH = 7.4 Hz, JHH = 1.3 Hz,
3
3
2H, H-b), 2.52 (t, JHH = 7.2 Hz, 2H, H-s), 2.04 (quint, JHH = 7.2 Hz, 2H,
H-r), 1.91 (d, 4JHH = 1.3 Hz, 3H, Hhet-7), 1.75 (quint, 3JHH = 7.4 Hz, 2H, H-
c), 1.49-1.25 (m, 22H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-
n), 0.92 (t, JHH = 7.0 Hz, 3H, H-o). 13C-NMR (151 MHz, MeOD):  [ppm]
3
= 174.6 (C-t), 173.8 (C-a), 173.1 (C-p), 166.5 (Chet-4), 152.8 (Chet-2),
(dt, 3JHH = 3.5 Hz, 4JHH = 1.7 Hz, 1H, H-1´), 6.48 (dt, 3JHH = 6.0 Hz, 4JHH
=
152.36, 152.27 (C-4´´), 138.7 (Chet-6), 135.80 (C-3´), 135.03 (d, JCP
=
4
3
1.8 Hz, 1H, H-3´), 5.83-5.76 (m, 1H, H-2´), 5.17 (dd, JHP = 8.1, 4.8 Hz,
4H, Ph-CH2), 4.98-4.93 (m, 1H, H-4´), 4.32-4.17 (m, 4H, H-5´, H-t), 3.74-
3.51 (m, 10H, H-u, H-v, H-w, H-x, H-y), 3.26 (s, 3H, H-z), 2.93-2.87 (m,
2H, H-q), 2.78 (t, 3JHH = 6.6 Hz, 2H, H-r) 2.61 (td, 3JHH = 7.4, 1.3 Hz, 2H,
H-b), 1.91 (d, JHH = 1.2 Hz, 3H, Hhet-7), 1.78-1.70 (m, 2H, H-c), 1.52-
1.44 (m, 2H, H-d), 1.01 (t, JHH = 7.4 Hz, 3H, H-e). 13C-NMR (151 MHz,
MeOD):  [ppm] = 173.9,173.8 (C-p, C-s), 172.6 (C-a), 166.53 (Chet-4),
152.8 (Chet-2), 152.4, 152.3 (2×C-4´´), 138.7 (Chet-6), 135.8 (C-3´), 135.8
(2×C-1´´), 130.50,130.46 (4×C-2´´), 127.1 (C-2´), 122.88, 122.86, 122.84,
122.83 (4×C-3´´), 112.1 (Chet-5), 90.8 (C-1´), 87.2 (d, 3JCP = 9.5 Hz, C-4´),
72.9, 71,54, 71.50, 71,3, 70.07 (C-u, C-v, C-w, C-x, C-y), 70.38, 70.35,
7.6 Hz, C-1´´), 134.96 (d, 3JCP = 7.7 Hz, C-1´´), 130.50, 130.47 (4×C-2´´),
127.2 (C-2´), 122.89, 122.87 (4×C-3´´), 112.1 (Chet-5), 90.8 (C-1´), 87.23
3
(d, JCP = 9.0 Hz, C-4´), 73.0 (C-z), 71.53, 71.39 (C-w, C-x, C-y), 70.39,
2
70.36, 70.33 (2×Ph-CH2), 70.12 (C-v), 67.86 (d, JCP = 6.0 Hz, C-5´),
4
64.7 (C-u), 59.1 (-OCH3), 35.03 (C-b), 33.98, 33.91 (C-q, C-s), 33.08,
30.80, 30.79, 30.76, 30.73, 30.62, 30.48, 30.42, 30.18 (C-d, C-e, C-f, C-g,
C-h, C-i, C-j, C-k, C-l, C-m), 25.97 (C-c), 23.74 (C-n), 21.19 (C-r), 14.45
(C-o), 12.5 (Chet-7). 31P-NMR (243 MHz, MeOD):  [ppm] -11.80 (d, J =
19.9 Hz, P-), -13.23 (d, J = 17.2 Hz, P-), -23.75 (t, J = 18.6 Hz, P-
).HRMS (ESI-TOF) m/z: calculated for C51H74N2O22P3 [M-H]- 1159.3952,
found 1159.3885.IR: ν [cm−1] = 3184, 2922, 2853, 1755, 1736, 1689,
1509, 1455, 1247, 1219, 1200, 1167, 1127, 1083, 1009, 905, 838, 784,
722, 643, 489, 420, 399.
3
2
70.35, 70.32 (2×Ph-CH2), 67.85 (d, JCP = 6.0 Hz, C-5´), 65.02 (C-t),
59.08 (C-z), 34.8 (C-b), 30.1, 29.9 (C-q, C-r), 28.1 (H-c), 23.3 (C-d), 14.1
(C-e), 12.5 (Chet-7) 31P-NMR (243 MHz, MeOD):  [ppm] = -11.77 (d, J =
19.8 Hz, P-), -13.24 (d, J = 17.0 Hz, P-), -23.71 (t, J = 19.0 Hz, P-).
HRMS (ESI-TOF) m/z: calculated for C40H52N2O22P3 [M-H]- 1005.2230,
found 1005.2268. IR: ν [cm−1] = 3187, 2932, 2874, 1755, 1736, 1688,
1509, 1453, 1422, 1247, 1218, 1200, 1167, 1127, 1082, 1007, 903, 837,
806, 783, 731, 696, 644, 480, 422, 401.
Preparation of phosphate buffer (PB, pH 7.3):
5.47 g disodium hydrogen and phosphate 1.55 g Potassium dihydrogen
phosphate were dissolved in 1L ultrapure water. Then titrated with diluted
phosphoric acid to pH 7.3. All prodrugs were incubated in this buffer to
study their chemical stability.
-(ab-C14H29,ab-MEEES)-d4TTP (ammonium salt) 3eh: According to
General Procedure C, the reactions were performed under dry conditions
using 100 mg H-phosphonate 8eh (0.162 mmol, 1.0 equiv.) and 127 mg
d4TMP 2nBu4N+ salt (0.162 mmol, 1.0 equiv.). Yield: 39%, 75 mg, as
1.3 Hydrolysis Studies.
Chemical hydrolysis in PB
colorless solid. 1H-NMR (600 MHz, MeOD):  [ppm] = 7.69 (m, 1H, Hhet
6), 7.46-7.38 (m, 4H, H-2´´), 7.13-7.03 (m, 4H, H-3´´), 6.94 (dt, 3JHH = 3.5
-
4
3
4
Stock solutions (50 mM in DMSO) of TriPPPro-NTPs were prepared.
After dilution of 11 µL stock solution with 189 µL ultrapure water and 100
µL DMSO to 1.83 mM hydrolysis solutions the reaction was started by
addition of 300 µL phosphate buffer (PB, 50 mM, pH 7.3). The solution
was incubated with 800 rpm and at 37 °C in a thermomixer. An initial
aliquot (25 µL) was taken directly and analyzed by analytical HPLC with
UV detector. For compound containing d4T, λ = 265 nm. Further aliquots
were taken for monitoring the kinetic hydrolysis.
Hz, JHH = 1.6 Hz, 1H, H-1´), 6.49 (dt, JHH = 5.6 Hz, JHH = 1.7 Hz, 1H,
3
3
4
H-3´), 5.81 (ddd, JHH = 5.6 Hz, JHH = 1.9 Hz, JHH = 1.8 Hz, 1H, H-2´),
5.17 (d, JHP = 7.3 Hz, 4H, Ph-CH2), 4.98-4.93 (m, 1H, H-4´), 4.33-4.17
3
(m, 4H, H-5´, H-t), 3.75-3.69 (m, 2H, H-u), 3.67-3.59 (m, 6H, H-v, H-w, H-
3
x), 3.56-3.51 (m, 2H, H-y), 3.36 (m, 3H, H-z), 2.91 (t, JHH = 6.6 Hz, 2H,
3
3
H-q), 2.78 (t, JHH = 6.3 Hz, 2H, H-r), 2.60 (t, JHH = 7.5 Hz, 2H, H-b),
4
3
1.91 (d, JHH = 2.9 Hz, 3H, Hhet-7), 1.75 (quint, JHH = 7.5 Hz, 2H, H-c),
1.49-1.24 (m, 22H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-n),
3
0.92 (t, JHH = 5.7 Hz, 3H, H-o). 13C-NMR (151 MHz, MeOD):  [ppm] =
173.88, 173.79, 172.59 (C-p, C-s, C-a), 166.53 (Chet-4), 152.77 (Chet-2),
Enzyme-catalyzed hydrolysis in CEM cell extracts
4
152.36, 152.29 (C-4´´), 138.70 (Chet-6), 135.83 (C-3´), 135.10 (d, JCP
=
7.4 Hz, C-1´´), 134.97 (d, 3JCP = 7.7 Hz, C-1´´), 130.50, 130.47 (4×C-2´´),
10 µL 50 mM DMSO stock solution of TriPPPro-d4TTPs was diluted to
6.0 mM hydrolysis solution by addition of 73.3 µL DMSO. 7 different
samples including 10 µL water and 10 µL hydrolysis solution were
127.12 (C-2´), 122.88, 122.86, 122.84, 122.82 (4×C-3´´), 112.08 (Chet-5),
3
90.83 (C-1´), 87.25 (d, JCP = 8.9 Hz, C-4´), 72.95 (C-y), 71.55, 71.51,
71.38 (C-w, C-x, C-y), 70.39, 70.36, 70.33 (2×Ph-CH2), 70.08 (C-u),
13
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
prepared. The reaction was started by addition of 50 µL human CEM cell
extract and the mixture incubated with 800 rpm at 37 °C for different time
periods of hydrolysis. The reactions were stopped by addition of 150 µL
MeOH. The solution was kept on ice for 5 min followed by centrifugation
for 5 min (13000 rpm). The supernatants were filtered (Chromafil® RC-
20/15 MS, 0.2 µm) and stored in liquid nitrogen. When testing, the
samples were defrosted and injection volume with 80 µL was used for
HPLC analysis. The calculation of t1/2 was performed analogously to that
for the chemical hydrolysis studies.
[7] J. Balzarini, P. Herdewijn, E. De Clercq, J. Biol. Chem. 1989, 264, 6127-
6133.
[8] A. R. Van Rompay, M. Johansson, A. Karlsson, Pharmacol. Therapeut.
2000, 87, 189-198.
[9] E. L. Asahchop, M. A. Wainberg, R. D. Sloan, C. L. Tremblay, Antimicrob.
Agents Chemother. 2012, 56, 5000-5008.
[10] C. Meier, Synlett 1998, 233-242.
[11] C. R. Wagner, V. V. Iyer, E. J. McIntee, Med. Res. Rev. 2000, 20, 417-
451.
[12] S. J. Hecker, M. D. Erion, J. Med. Chem. 2008, 51, 2328-2345.
[13] U. Pradere, E. C. Garnier-Amblard, S. J. Coats, F. Amblard, R. F.
Schinazi, Chem. Rev. 2014, 114, 9154-9218.
Enzyme-catalyzed hydrolysis in pig liver esterase (PLE)
[14] D. Cahard, C. McGuigan, J. Balzarini, Mini-Rev. Med. Chem. 2004, 4,
371-381.
10 µL 50 mM DMSO stock solution of TriPPPro-d4TTPs was diluted to
6.0 mM hydrolysis solution by addition of 31.7 mL DMSO and 41.7 mL
ultrapure water. Then 83.3 mL of the 6.0 mM solution was diluted with
125 µL DMSO and 833 µl 50 mM PB (pH 7.3). The reaction was started
by addition of 62.5 mL of PLE in PB (3 mg/mL) and the mixture was
incubated with 800 rpm at 37 °C in a thermomixer. At different times,
aliquots (100 mL) were taken and the reaction was stopped by addition to
106 mL MeOH. The mixture was kept for 5min on ice followed by
centrifugation for 5min (13000 rpm). The mixture was filtered (Chromafil
RC-20/15 MS, 0.2 mm) and stored in liquid nitrogen. When testing, the
samples were defrosted and injection volume with 80 µL was used for
HPLC analysis.
[15] F. J. Li, H. Maag, T. Alfredson, J. Pharm. Sci. 2008, 97, 1109-1134.
[16] Y. Zhang, Y. Gao, X. Wen, H. Ma, Asian J. Pharm. Sci. 2014, 9, 65-74.
[17] C. Meier, Eur. J. Org. Chem. 2006, 5, 1081-1102.
[18] I. S. Krylov, B. A. Kashemirov, J. M. Hilfinger, C. E. McKenna, Mol. Pharm.
2013, 10, 445-458.
[19] C. Meier, J. Balzarini, Antiviral Res. 2006, 71, 282-292.
[20] H. J. Jessen, T. Schulz, J. Balzarini, C. Meier, Angew. Chem., Int. Ed.
2008, 47, 8719-8722.
[21] T. Schulz, J. Balzarini, C. Meier, ChemMedChem 2014, 9, 762-775.
[22] L. Weinschenk, T. Gollnest, D. Schols, J. Balzarini, C. Meier,
Chemmedchem 2015, 10, 891-900.
[23] L. Weinschenk, D. Schols, J. Balzarini, C. Meier, J. Med. Chem. 2015, 58,
6114-6130.
Preparation of the cell extracts:
[24] X. L. Tan, C. K. Chu, F. D. Boudinot, Adv. Drug. Deliver. Rev. 1999, 39,
117-151.
+
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 1,250 rpm at 4 C, washed twice with cold PB,
and the pellet was resuspended at 108 cells/mL and sonicated (Hielscher
Ultrasound Techn., 100% amplitude, 3·times for 10 sec) to destroy cell
integrity. The resulting cell suspension was then centrifuged at 10000
rpm to remove cell debris, and the supernatant divided in aliquots before
being frozen at -80 °C and used.
[25] M. J. Camarasa, ChemMedChem 2018, 13, 1885-1889.
[26] T. Gollnest, T. D. de Oliveira, D. Schols, J. Balzarini, C. Meier, Nat.
Commun. 2015, 6, 8716.
[27] T. Gollnest, T. D. de Oliveira, A. Rath, I. Hauber, D. Schols, J. Balzarini, C.
Meier, Angew. Chem., Int. Ed. 2016, 55, 5255-5258.
[28] C. Meier, Antiviral Chem. Chemother. 2017, 25, 69-82.
[29] X. Jia, D. Schols, C. Meier, J. Med. Chem. 2020, 63, 6003-6027.
[30] T. Sowa, S. Quchi, Bull. Chem. Soc. Jpn. 1975, 48, 2084−2090.
[31] T. Gollnest, Ph.D. thesis, University of Hamburg, 2015.
1.4 Antiviral Assay against HIV.
Inhibition of HIV-1(IIIB)- and HIV-2(ROD)-induced cytopathicity in wild-
type CEM/0 and thymidine kinase-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%.
Acknowledgements
C.M. is grateful to the Deutsche Forschungsgemeinschaft for
financial support (ME 1161/15-1). C.Z. and X.J. are grateful for
CSC fellowships from the Chinese Ministry of Education.
Keywords: antivirals• nucleoside analogues• nucleoside
triphosphates prodrugs bioreversible protection nucleotides NTP
[1] L. P. Jordheim, D. Durantel, F. Zoulim, C. Dumontet, Nat. Rev. Drug Discov.
2013, 12, 447-464.
[2] J. Deval, Drugs 2009, 69, 151-166.
[3] T. Cihlar, A. S. Ray, Antivir Res. 2010, 85, 39-58.
[4] Y. El Safadi, V. Vivet-Boudou, R. Marquet, Appl. Microbiol. Biotechnol.
2007, 75, 723-737.
[5] J. R. Burton, G. T. Everson, Clin. Liver Dis. 2009, 13, 453-465.
[6] E. De Clercq, J. Clin. Virol. 2004, 30, 115-133.
14
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000712
ChemMedChem
FULL PAPER
Entry for the Table of Contents
Lipophilic nucleoside triphosphate prodrugs bearing two different bioreversible masking groups attached to the -position of the triphosphate moiety
were studied. The aim to use two different masking groups was to enhance the selectivity of their removal during the cleavage process in order to
deliver nucleoside triphosphates intracellularly. The compounds proved to be active against HIV even in cells lacking a phosphorylating enzyme.
15
This article is protected by copyright. All rights reserved.