Membrane Permeable, Bioreversibly Modified Prodrugs of Nucleoside Diphosphate-γ-Phosphonates

Article abstract of DOI:10.1021/acs.jmedchem.0c01294

Nucleoside reverse transcriptase inhibitors (NRTIs) are widely used as antiviral and anticancer agents, although they require intracellular phosphorylation into their antivirally active form, the triphosphorylated nucleoside analogue metabolites. We report on the synthesis and characterization of a new class of nucleoside triphosphate analogues comprising a C-alkyl-phosphonate moiety replacing the γ-phosphate. These compounds were converted into bioreversibly modified lipophilic prodrugs at the γ-phosphonate by the attachment of an acyloxybenzyl (ester) or an alkoxycarbonyloxybenzyl (carbonate) group. Such compounds formed γ-C-(alkyl)-nucleoside triphosphate analogues with high selectivity because of an enzyme-triggered delivery mechanism. The latter compounds were very stable in CD4+ T-lymphocyte (CEM cell) extracts, and they were substrates for HIV-reverse transcriptase without being substrates for DNA-polymerases α, β, and γ. In antiviral assays, the excellent antiviral activity of the prodrugs that was found in CEM/0 cells was completely kept in CEM/TK- cells. The activity was improved by 3 logs as compared to the parent nucleoside d4T.

Full text of DOI:10.1021/acs.jmedchem.0c01294

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Article
Membrane permeable, bioreversibly modified
Prodrugs of Nucleoside Diphosphate-#-phosphonates
Xiao Jia, Stefan Weber, Dominique Schols, and Chris Meier
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01294 • Publication Date (Web): 29 Sep 2020
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Membrane permeable, bioreversibly modified
Prodrugs of Nucleoside Diphosphate--phosphonates
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Xiao Jia, Stefan Weber, Dominique Schols, Chris Meier
1Organic Chemistry, Department of Chemistry, Faculty of Mathematics, Informatics and Natural
Sciences, Universität Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany.
2Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology and
Transplantation, Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000
Leuven, Belgium.
ABSTRACT Nucleoside reverse transcriptase inhibitors (NRTIs) are widely used as antiviral
and anticancer agents although they require intracellular phosphorylation into their antivirally
active form, the triphosphorylated nucleoside analogue metabolites. We report on the synthesis
and characterisation of a new class of nucleoside triphosphate analogues comprising a C-alkyl-
phosphonate moiety replacing the -phosphate. These compounds were converted into
bioreversibly modified lipophilic prodrugs at the -phosphonate by the attachment of an
acyloxybenzyl- (AB; ester) or an alkoxycarbonyloxybenzyl- (ACB; carbonate) group. Such
compounds formed -C-(alkyl)-nucleoside triphosphate analogues with high selectivity due to an
enzyme-triggered delivery mechanism. The later compounds were very stable in CEM cell
extracts and they were substrates for HIV-RT without being substrates for DNA-polymerases ,

and . In antiviral assays, excellent antiviral activity of the prodrugs was found in CEM/0 cells
−
was completely kept in CEM/TK cells. The activity was improved by 3 logs as compared to the
parent nucleoside d4T.
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Introduction
Nucleoside analogues play an important role in combatting virus infections as can be seen in
their impressive potency against HIV, herpes virus, influenza, hepatitis B and hepatitis C and
since very recently SARS-CoV-2.1-5 Generally, they target the viral RNA- or DNA-polymerase
needed for virus replication. Today, several nucleoside analogues are approved as HIV reverse
transcriptase (RT)2,3 inhibitors (NRTIs) blocking RT's enzymatic function in the early phase of
the infection and prevent completion of the synthesis of the viral DNA, act as chain terminators
of DNA synthesis, thus preventing HIV from replicating. The efficacy of antiviral or antitumor
active nucleoside analogues is highly dependent on the efficient intracellular conversion by
cellular kinases to yield ultimately the bioactive nucleoside analogue triphosphate derivatives
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(
NTP) which block the function of the viral DNA polymerase. Former studies have shown that
cellular kinases often perform these transformations ineffectively, resulting in low or no antiviral
activity of the corresponding nucleoside analogue,8-10 and insufficient phosphorylation may led
to adverse effects.11,12 Within the stepwise biotransformation process, often the initial
phosphorylation step yielding the nucleoside monophosphate (NMP) performed by the salvage
pathway enzyme thymidine kinase (TK) was identified as limiting step, for example, for the
NRTI 3'-deoxy-2',3'-dehydrothymidine (d4T 1; Scheme 1) and the formation of its
monophosphate metabolite (d4TMP 2). Moreover, the clinical efficacy of such drugs is adversed
by further limitations such as poor biological half-lives, mutations of nucleoside transporters,
unsuitable bioavailability after oral administration, or drug resistant virus strain development.13-15
Prodrug strategies to overcome these problems resulted in orally administrable forms of a few
antiviral nucleoside monophosphates. Others are currently under ongoing development.10,16-25
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Two recent examples of a very successful nucleoside monophosphate prodrug are the anti-HCV
active drug sofosbuvir26-30 and Remdesivir.30-32
Recently, we reported on an technology to deliver nucleoside diphosphates (NDPs) to cells
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DiPPro-approach).33-37 However, although both approaches deliver the mono- or the
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diphosphorylated derivatives of the nucleosides, respectively, the delivered nucleotides need
further phosphorylation into the triphosphate forms in order to block the viral replication process.
As a consequence, the development of nucleoside triphosphate (NTP) prodrugs is still highly
desirable (TriPPPro-concept 5; Scheme 1) because all phosphorylation steps from the nucleoside
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analogue to the nucleoside triphosphate are bypassed. Thus, this approach should principle
optimise the intracellular amount of the antivirally active NTP.
Scheme 1: Cell uptake and metabolism of TriPPPro-d4TTP prodrugs.
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Despite these obvious advantages, almost no reports on attempts to design triphosphate prodrugs
have been disclosed.39,40 However, the significant challenges should not be overlooked : (a) a
triphosphate is highly negatively charged and thus requires extensive masking to produce a
lipophilic compound, (b) the intrinsic lability of the triphosphate moiety comprising reactive
phosphate anhydride linkages, (c) the requirement for the NTP to act selectively as a substrate
for viral polymerases and not for cellular polymerases, and (d) the risk of low stability of the
delivered NTP toward phosphatases/kinases leading to dephosphorylation and thus clearance of
the bioactive metabolite. Therefore, it was in the past common sense that the design of
nucleoside triphosphate prodrugs would be almost impossible.41
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We disclosed recently a unique pronucleotide approach based on partially masking the negative
charges and improving the lipophilicity of nucleoside triphosphates, namely, the TriPPPro-
approach 5.42-47 At first, TriPPPro-compounds 6 comprising two lipophilic and bioreversible
acyloxybenzyl- (AB) masking groups at the -phosphate and d4T 1 as a nucleoside analogue
42
were disclosed. The successful formation of a nucleoside triphosphate inside cells was proven
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in a celluptake study involving fluorescent nucleoside analogues. Recently, we also reported on
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non-symmetric TriPPPro-d4TTPs 7. In the case of TriPPPro-compounds 7, an enzymatic
break-down of the TriPPPro-derivatives to yield d4TTP was finally achieved in CEM cell
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extracts. TriPPPro-d4TTPs 6,7 retained their anti-HIV activity in CEM/TK cell assays whereas
the parent d4T 1 was inactive due to the lack of cytosolic monophosphorylating enzyme TK. The
TriPPPro-strategy was also used to convert so-far inactive nucleoside analogues into
biologically active metabolites.43,46 Both compounds 6,7 were rapidly hydrolysed in cell extracts.
However, it was very difficult to detect d4TTP 4 because of its relatively fast enzymatic
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dephosphorylation (t1/2 = 38 min) by phosphorylases/kinases to form d4TDP 3 first and
ultimately d4TMP 2.42,45
It should be added that we proved that the -double bioreversibly masked TriPPPro-compounds
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did not act as substrates for DNA polymerases, e.g HIV-RT or DNA pol . In contrast,
recently we have shown that NTP analogues such as -alkylketobenzyl-d4TTP and -(alkyl-
C18)-d4TTPs 9 that comprised one non-hydrolyzable lipophilic alkyl group were substrates for
HIV-RT and d4TMP 2 was incorporated in a primer extension assay into the primer strand while
these compounds were non-substrates for cellular DNA polymerases ,  and .47 More
importantly, prodrug versions of compounds 9 like -(AB;alkyl)-d4TTPs 8 showed even higher
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antiviral activity against HIV-2 in thymidine kinase-deficient cell assays (CEM/TK cells).
Further studies concerning the antiviral activity showed that also -(alkyl-C18)-d4TTP 9 was a
potent compound with EC values 3logs lower (EC : 0.05 µM) as compared to d4T 1 and in
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contrast to d4TTP 4, -(alkyl-C18)-d4TTP 9 proved to be very highly stable in cell extracts
towards dephosphorylation.47
Earlier studies showed that the carbonate TriPPPro-compounds 7 and intermediates were more
stable as compared to TriPPPro-compounds 6 and intermediates, respectively.42,45 The above
summarized results motivated us to develop a new series of -C-alkylphosphonate-d4TDP
prodrugs 10,11, bearing -C-n-alkyl chains of different length combined with an acyloxybenzyl-
(AB; ester) or an alkoxycarbonyloxybenzyl- (ACB; carbonate) moiety (Scheme 2). We expected
that such a design would lead to a rapid conversion of the TriPPPro-compounds 10,11 (Scheme
2
) into -C-(alkyl)-d4TTPs 23 and thereby should avoid a side reaction which we have observed
from prodrugs 6-8 that led to the formation of (although small amounts) of d4TMP 2 or d4TDP
3
. Moreover, the introduction of a P-C-bond in 10,11 instead of a P-O-bond in 6,7 will lead to a
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chemically and enzymatically stable compound because P-C-bond are not hydrolysed. This will
then also lead to metabolically very resistant nucleoside triphosphate analogues.
We report here on the synthesis of these -C-modified d4TTP prodrugs 10,11 as well as their
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hydrolysis products -C-(alkyl)-d4TTPs 23. The hydrolysis characteristics in phosphate buffer
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PB, pH 7.3), pig liver esterase (PLE), human CD4 T-lymphocyte CEM cell extracts and the
anti-HIV activity against HIV-1- and HIV-2 in infected wild-type CEM/0 cell cultures and in
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HIV-2-infected mutant thymidine kinase-deficient CEM/TK cell cultures will be described.
Primer extension assays were conducted studying the substrate properties of such -C-(alkyl)-
d4TTPs 23 for four different DNA-polymerases.
Scheme 2: -AB--C-alkyl-d4TTPs 10 and -ACB--C-alkyl-d4TTPs 11 and the cleavage
pathways leading to -C-alkyl-d4TTPs 23.
Results and Discussion
Synthesis of -acyloxybenzyl (AB)--C-alkyl-d4TTPs 10 and -alkyloxycarbonyloxybenzyl
(ACB)--C-alkyl-d4TTPs 11.
-AB--C-alkyl-d4TTPs 10 were synthesized using H-phosphinate chemistry. D4TMP 2 was
prepared from d4T 1 as described before.48 H-Phosphinates 15 were synthesized from H-
phosphinic acids 13, 4-dimethylaminopyridine (DMAP), 1-(3-dimethylaminopropyl)-3-
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ethylcarbodiimide (EDC) and 4-acyloxybenzyl alcohols 14 in yields between 48% and 85%.
Next, H-phosphinates 15 were reacted to give phosphonochloridates by an oxidative chlorination
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with N-chlorosuccinimide (NCS). Subsequent phosphorylation with tetra-n-butylammonium
phosphate yielded phosphonate-phosphates 16 in almost quantitative yields. Due to chemical
instability, compounds 16 were rapidly purified by extraction and immediately used in the next
step. The final coupling reaction was achieved by an activation of compounds 16 with
trifluoroacetic acid anhydride (TFAA) and N-methylimidazole,50,51 followed by the reaction with
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d4TMP 2 to give -AB--C-alkyl-d4TTPs 10 (n-Bu N form). -AB--C-alkyl-d4TTPs 10 (NH
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counterion) were obtained as colorless solids after reversed-phase (rp) column chromatography
+
(
18-68% yield), a Dowex 50WX8 (NH ) ion exchange and freeze-drying. A series of -ACB--
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C-alkyl-d4TTPs 11 was also prepared using the identical approach (Scheme 3). Using this H-
phosphinate pathway, the bond between the - and the -phosphate was formed without the need
of a subsequent oxidation after forming the P-O-P-linkage, which was in agreement with the H-
phosphonate synthesis sequence reported before.43 Thus, also oxidation sensitive nucleoside
analogues or masking units might be used in the future. Target compounds 10 and 11 were
isolated as diastereomeric mixtures. These diastereomeric mixtures were inseparable by
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chromatography and generally indistinguishable in the P-NMR spectra.
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Scheme 3. Reagents and conditions: i) EDC, DMAP, CH Cl , rt, 12 h; ii) a. NCS, CH CN, rt, 2
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h, b) N(Bu) (H PO ), CH CN, rt, 1 h; iii) d4T 1, POCl , pyridine, H O, CH CN, 0 °C-rt, 5 h; iv)
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a. TFAA, Et N, CH CN, 0 °C, 10 min, b. 1-methylimidazole, Et N, CH CN, 0 °C-rt, 10 min, c.
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d4TMP 2, rt, 2 h.
Synthesis of -(-cyanoethyl)--C-alkyl-d4TTPs 22 and -C-(alkyl)-d4TTPs 23.
As concluded from previous studies,45,47 a suitable access to -C-(alkyl)-d4TTPs 23 was
achieved starting from -(-cyanoethyl)--C-alkyl-d4TTPs 22 and treatment with tetra-n-
butylammonium phosphate. Thus, -(-cyanoethyl)--C-alkyl-d4TTPs 22 were synthesized by
the H-phosphinate route. The -cyanoethyl group was included here as protective group at the -
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phosphonate moiety. After final coupling of compounds 21 and d4TMP 2, leading to -(-
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cyanoethyl)--C-alkyl-d4TTPs 22 (n-Bu N form), the crude product was stirred in a mixture of
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0% nBu N OH (water solution) and CH CN at rt for 8h to obtain products 23 (n-Bu N form).
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After ion exchange (Dowex 50WX8 (NH )), followed by a rp-column chromatography and
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freeze-drying, the -C-(alkyl)-d4TTPs 23 (NH4+ form) were isolated as colorless solids. The
obtained yields for the conversions of d4TMP 2 to compounds 22 varied between 40% to 65%
and -C-(alkyl)-d4TTPs 23 were isolated in 15% to 42% yield.
Scheme 4. Reagents and conditions: i) EDC, DMAP, CH Cl , rt, 12 h; ii) a. NCS, CH CN, rt, 2
2
2
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h, b) N(Bu) (H PO ), CH CN, rt, 1 h; iii) d4T 1, POCl , pyridine, H O, CH CN, 0 °C-rt, 5 h; iv)
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a. TFAA, Et N, CH CN, 0 °C, 10 min, b. 1-methylimidazole, Et N, CH CN, 0 °C-rt, 10 min, c.
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+
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d4TMP 2, rt, 2 h. v) n-Bu N OH , 8h, Dowex 50WX8 (NH form) ion exchange. [a] yields are
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5
5
5
5
6
calculated for the conversion from 20 to 22. [b] yields are calculated for the conversion from 20
to 23.
Stability studies
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Previously we reported how nucleotides were released from different TriPPPro-compounds.42,45
In the case of compounds 6,7, the enzymatically-catalyzed cleavage of both bioreversible masks
led to d4TTP 4 formation which is subsequently dephosphorylated to yield d4TDP. However, in
the metabolism of compounds 8, a predominate formation of -(alkyl)-d4TTPs 9 companied by a
4
7
very small amount of d4TDP 3 was detected. As expected, no d4TTP formation was observed
in these studies using compounds 8. Prior to the antiviral evaluation of compounds 10,11,22,23,
we verified their chemical or enzyme-triggered nucleotide release. The determined half-lives of
prodrugs 10,11 (Table 1, t ) reflect the removal of the bioreversible AB-group or the ACB-
1
/2
group, respectively, to give -C-(alkyl)-d4TTPs 23. All mixtures were analyzed by means of
analytical RP18-HPLC.
a. Chemical stability in aqueous phosphate buffer (PB, pH 7.3)
TriPPPro-compounds 10,11,22,23 were incubated in aqueous phosphate buffer (PB, 25 mM,
pH 7.3) to study their stability and the product distribution. The stability of TriPPPro-d4TTP
prodrugs 10a-c (AB:C1-C11;alkyl-C12), 11a-c (ACB:C1-C11;alkyl-C12) increased with
increasing alkyl chain lengths (Table 1). As expected from previous work, the half-lives for the
carbonate compounds -ACB--C-alkyl-d4TTPs 11 were higher than the corresponding ester
derivatives -AB--C-alkyl-d4TTPs 10. Indeed, these compounds were more stable as compared
to the studies of TriPPPro-d4TTPs 6,7.42,45 Compared to previously studied -(AB-C4;alkyl-
4
7
C18)-d4TTP 8 (t = 237 h), the half-life for -(AB-C4)--C-(alkyl-C18)-d4TTP 10e was also
1
/2
significantly higher by almost a factor of 3 (t = 646 h, Table 1). Unexpectedly, the chemical
1/2
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stability of compounds 23a,c (alkyl:C12-C18) decreased with increasing alkyl chain lengths and
the half-life of -C-(alkyl-C18)-d4TTP 23c (t = 535 h) was found to be lower than those of the
1/2
prodrugs 10e (t = 646 h) or 11e (t = 1240 h), which was in contrast to the results as -(ACB)-
1
/2
1/2
1
1
1
1
1
1
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8
9
0
1
2
3
4
5
6
7
8
9
0
d4TTPs reported before.45
As an example, the hydrolysis of -(AB-C4)--C-(alkyl-C12)-d4TTP 10b is shown in Figure 1.
Clearly, as hydrolysis products starting from compound 10b a predominant formation of -C-
(alkyl-C12)-d4TTP 23a (85%) and also a small amount of d4TDP 3 (12%) was detected,
indicating an almost selective cleavage of the biodegradable AB group of compounds 10. In PB,
the cleavage of the AB group in compounds 10 or the ACB group in compounds 11 is initiated
by an ester or carbonate hydrolysis and thus proceed similar to the previously published cleavage
4
5
pathway for TriPPPro-d4TTPs 7. Due to the very long hydrolysis periods, side-reaction was
observed which has been reported previously by us.37,42,47 The nucleobase thymine was detected
which was the result of the cleavage of the glycosidic bond in d4T. Therefore, prior to complete
consumption of the starting material, an increase of thymine concentration was detected. In all
cases, -C-(alkyl-C12)-d4TTP 23a (t > 1000 h) was mainly formed (between 84% (11b) and
1
/2
9
6% (11a) and a very small amount of d4TMP 2 was detected (<4%) in chemical hydrolysis
studies (Figure S1-S3; Supporting Information). Additionally, after complete conversion of the
starting -(AB-C4)--C-(alkyl-C12)-d4TTP 10b, no increase of d4TDP 3 concentrations were
detected which leading to the conclusion that -(AB-C4)--C-(alkyl-C12)-d4TTP 10b were prone
to a breakage of the -phosphonate --phosphate bond. If an additional charge appears at the -
phosphonate group due to the cleavage of the biodegradable masking moiety, the hydrolysis of
the -phosphonate--phosphate anhydride bond is prevented. In the case of -(-cyanoethyl)--
C-alkyl-d4TTPs 22 in PB, a marked selective cleavage of -cyanoethyl moiety led to the
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formation of -(alkyl)-d4TTPs 23 by -elimination as can be seen in Figure S4-S5 (Supporting
Information). It should also be noted that d4TTP 4 was not detected using 10,11,22,23.
0
1
2
3
4
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8
9
0
1
2
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1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1: HPLC profile for -(AB-C4)--C-(alkyl-C12)-d4TTP 10b after incubation in PB (pH
7
.3).
b. Hydrolysis study using pig liver esterase
Next, the enzymatic stability of TriPPPro-compounds 10,11 was studied by incubation with pig
liver esterase (PLE) in phosphate buffer (pH 7.3) to confirm the -C-(alkyl)-d4TTPs (23)
formation. Compared to the chemical hydrolyses, TriPPPro-compounds 10,11 were quickly
hydrolyzed and delivered -C-alkyl-d4TTP derivatives 23 much faster than in PB, proving a
significant contribution of the enzymatic cleavage (Table 1). In PLE, the stability of TriPPPro-
compounds 10,11 was found to be between 0.009 h and 10.6 h. The enzymatic stability of
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TriPPPro-d4TTP prodrugs 10b,10d,10e (AB-C4;alkyl:C12,C14,C18), 11b,11d,11e (ACB-
C4;alkyl:C12,C14,C18), raised with increasing alkyl chain lengths (R2).
The AB masking group cleavage in 10b (AB-C4;alkyl-C12) occurred readily (t1/2 = 0.79 min)
leading selectively to -C-(alkyl-C12)-d4TTP 23a (Figure 2). For -(AB-C4)--C-(alkyl-C18)-
d4TTP 10e comprising a long, lipophilic alkyl chain, the half-life was found to be 2h, which was
in agreement with the results from the studies of TriPPPro-compounds 7 described before.45
However, the half-lives for -(ACB-C4)--C-(alkyl-C12)-d4TTP 11b (t = 0.53 min), -(ACB-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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8
9
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
/2
C4)--C-(alkyl-C18)-d4TTP 11e (t1/2 = 1.04 h) was found to be lower than the corresponding
compounds 10b (AB-C4;alkyl-C12) or 10e (AB-C4;alkyl-C18), respectively. Interestingly, the
stability of -(AB-C4)--C-(alkyl-C18)-d4TTP 10e (t = 2 h) was 15-fold higher as compared to
1
/2
the corresponding prodrug -(AB-C4;alkyl-C18)-d4TTP 8 (t1/2 = 8.1 min).47 Always, the
exclusive formation of -C-(alkyl)-d4TTP derivatives 23 and no d4TTP 4 (Figure S6-S10;
Supporting Information) was detected in contrast to the studies using the previously described
TriPPPro-compounds 6,742,45 and this is in agreement with the results reported before. More
importantly, the half-lives of -(-cyanoethyl)--C-(alkyl)-d4TTPs 22 (t > 80 h, Table 1) and -
47
1
/2
C-(alkyl)-d4TTP derivatives 23 (t1/2 > 80 h) showed that the delivery of d4TDP (Figure S11;
Supporting Information) was most probably due to a pure chemical cleavage of the -
phosphonate moiety, which proved our initial design of attaching an enzymaticallystable group
to the -phosphonate unit.
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1
1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 1: Half-lives of TriPPPro-d4TTP prodrugs 10,11,22 and -C-(alkyl)-d4TTPs 23 in different media.
PB
CEM/0
cell
PB
CEM/0 cell
extracts
Comp.
R1
R2
PLE
Comp.
R1
R2
PLE
pH=7.3
t [h]
extracts
pH=7.3
0
1
2
3
4
5
6
7
8
9
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8
9
0
1
2
3
4
5
6
7
8
t [h]
1/2
t [h]
1/2
t [h]
1/2
t [h]
1/2
t [h]
1/2
1
/2
1
0a
0b
10c
0d
0e
1a
1b
CH3
C H
103
215
919
509
646
123
345
0.043
0.81
>8
0.46
0.013
0.917
0.081
2.0
11c
11d
11e
22a
22c
23a
23c
C H
C H
12 25
1150
518
>10
6.4
10.6
0.032
1.04
>80
12
25
11 23
1
C H
C H
12 25
C H
4
C H
14 29
4
9
9
9
C H
C H
12 25
C H
4
C H
18 37
1240
148
>10
>15
>10
>30
>30
1
1
23
9
1
C H
4
C H
14 29
4.5
C H CN C H
2 4 12 25
1
C H
C H
18 37
6.7
C H CN C H
18 37
479
>150
>150
>80
4
9
2
4
1
CH3
C H
C H
0.65
4.3
0.7
C H
12 25
>3000
535
12
25
1
C H
12 25
0.009
C H
18 37
4
9
The hydrolysis experiments of -modified-d4TTPs 10,11,22 and 23 were conducted in aqueous 25 mM phosphate buffer (PB,
+
pH=7.3), pig liver esterase (PLE) and human CD4 T-lymphocyte cell extracts. The hydrolysis products were detected by analytical
rp18
HPLC.
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0
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8
9
0
Figure 2: HPLC profile for PLE hydrolysis of -(AB-C4)--C-(alkyl-C12)-d4TTP 10b.
c. Hydrolysis in cell extracts.
+
The incubation of TriPPPro-d4TTP prodrugs 10,11,22 with human CD4 T-lymphocyte CEM
cell extracts led to a significant acceleration of the formation of -C-(alkyl)-d4TTPs 23. The half-
lives of TriPPPro-d4TTPs 10,11 correlated with the alkyl chain length and were also
significantly lower than the half-lives in PB (up to 2300-fold; Table 1), which clearly indicates
the significant contribution of enzymatic cleavage as in the PLE studies.
The cleavage of the AB- or the ACB group is driven initially by an ester or a carbonate
4
5
hydrolysis and thus proceed similar as previously reported, showing a clear trend (Table 1).
The stability of TriPPPro-d4TTP prodrugs 10a-c (AB:C1,C4,C11;alkyl-C12), 11a-c
ACB:C1,C4,C11;alkyl-C12) increased with the raising lipophilicity of the alkyl chain (R1).
(
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While the half-lives determined for compounds 10b,10d,10e (AB-C4;alkyl:C12,C14,C18) as
well as 11b,11d,11e (ACB-C4;alkyl:C12,C14,C18) increased with the lipophilicity of the second
2
alkyl chain (R ). Moreover, the half-lives of compounds -ACB--C-alkyl-d4TTPs 11 were also
0
1
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3
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9
0
higher than the corresponding prodrugs -AB--C-alkyl-d4TTPs 10 (Table 1). As compared to -
(AB-C1)--C-(alkyl-C12)-d4TTP 10a (t = 0.043 h, Table 1), the half-life for -(ACB-C1)--C-
1
/2
(
alkyl-C18)-d4TTP 11a (t = 0.65 h, Table 1) was found to be remarkably higher by almost a
1/2
factor of 15. This is in accordance to our previously published results of TriPPPro-compounds
4
2
6. In all cases, before complete consumption of the starting compounds, an increase of -C-
(alkyl)-d4TTPs 23 concentrations and a very low concentration of d4TDP 3 (<3%) was
observed, but no d4TTP 4 was detected in these studies (Figure 3 and Figure S12-15; supporting
information). Additionally, in the case of -C-(alkyl)-d4TTPs 23 (t1/2 >30 h, Table 1), a very
small amount of d4TDP 3 was detected (Figure S16; supporting information), which supports the
hypothesis that -C-(alkyl)-d4TTPs 23 were subject to a bond breakage between the -
phosphonate and -phosphate in very small extend. Interestingly, the half-lives for -(AB-C4)--
C-(alkyl-C18)-d4TTP 10e (t = 6.7 h) and -(ACB-C4)--C-(alkyl-C18)-d4TTP 11e (t >10 h)
1
/2
1/2
4
7
were higher than the corresponding prodrug -(AB-C4; alkyl-C18)-d4TTP 8 (t = 4.8 h).
1
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0
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8
9
0
Figure 3: HPLC profiles of -(AB-C4)--C-(alkyl-C12)-d4TTP 10b; incubation in CEM/0 cell
extracts.
d. Antiviral evaluation
TriPPPro-d4TTP prodrugs 10,11,22 and -C-(alkyl)-d4TTPs 23 were evaluated for their ability
to inhibit HIV replication. Therefore, HIV-1- and HIV-2-infected wild-type CEM/0 as well as
−
HIV-2-infected mutant thymidine kinase-deficient CEM cell cultures (CEM/TK ) were treated
with these compounds. Table 2 summarizes the antiviral and cytostatic data of the TriPPPro-
d4TTP prodrugs 10,11,22, -C-(alkyl)-d4TTPs 23 and the parent nucleoside d4T 1 as reference
compound. As can be seen, the inhibition of the replication of HIV-1 and HIV-2 by prodrugs
10,11,22,23 was much higher (11e: EC : 0.0018 µM/HIV-1; EC : 0.026 µM/HIV-2), or at least
5
0
50
similar (11d: EC : 0.12 µM/HIV-1; EC : 0.22 µM/HIV-2), compared to their parent nucleoside
5
0
50
1
(EC : 0.43 µM/HIV-1; EC : 0.31 µM/HIV-2) in wild-type CEM/0 cells.
50 50
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For example, with compound 11e (EC : 0.0018 µM) the antiviral activity in this infected cell
5
0
line was improved by a 240-fold. In general, the antiviral activity determined for TriPPPro-
prodrugs 10a-c (AB:C1,C4,C11;alkyl-C12), 11a-c (ACB:C1,C4,C11;alkyl-C12) as well as
0
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0
1
0b,10d,10e (AB-C4;alkyl:C12,C14,C18), 11b,11d,11e (ACB-C4;alkyl:C12,C14,C18) was
1
2
dependent on the increasing lipophilicity due to the increasing alkyl chain lengths (R and R ).
However, prodrugs 10a,b (AB:C1,C4;alkyl-C12), 11a,b (ACB:C1,C4;alkyl-C12) showed a
marked loss of activity in thymidine-kinase-(TK)-deficient cell cultures, respectively. This points
to an insufficient lipophilicity of these compounds in addition to a fast cleavage of the
bioreversible AB- or ACB-moiety. This led to the fast formation of the more polar -C-(alkyl-
C12)-d4TTP 23a. -C-(alkyl-C12)-compound 23a lost its activity in the TK-deficient cell line. In
contrast, the antiviral activity observed in the wild-type CEM/0 cell cultures was almost fully
retained in the case of -(AB-C4)--C-(alkyl-C18)-d4TTP 10e (EC : 0.042 µM) and -(ACB-
5
0
−
C4)--C-(alkyl-C18)-d4TTP 11e (EC : 0.032 µM) in mutant TK-deficient CEM cells (TK ). -
5
0
(ACB-C4)--C-(alkyl-C18)-d4TTP 11e is the most active compounds of all the listed derivatives
1000-fold more active as d4T 1, which showed a 100-fold loss in activity in the TK-deficient
cell cultures; EC : 31 µM/HIV-2). The retention of the antiviral activity in TK-deficient cells
(
5
0
strongly supports a cell uptake of the TriPPPro-compounds 10,11. Although markedly less
active than the prodrugs 10e,11e (about a 38-fold and 49-fold, respectively), -C-(alkyl-C18)-
−
d4TTP 23c was also active against HIV-2 in CEM/TK cells (EC : 1.58 µM) indicating a cell
5
0
membrane passage of this compound. Compounds 22c (-cyanoethyl;alkyl-C18) and 23c (alkyl-
C18) bearing the C18 chain proved to be more active as compared to the compounds -C-(-
cyanoethyl;alkyl-C12)-d4TTP 22a, -C-(alkyl-C12)-d4TTP 23a, respectively. In contrast to -C-
(
alkyl-C12)-d4TTP 23a (EC : 41.1 µM), the antiviral activity of compound 10c (EC : 0.066
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µM) in cultures of infected wild-type CEM/0 cells was improved by a 623-fold, indicating the
advantage to combine the prodrug strategy with the stable -alkylphosphonate modification.
Ester prodrugs -AB--C-alkyl-d4TTPs 10 reported here had similar or even better activities
against HIV-1 and HIV-2 in cultures of infected wild-type CEM/0 cells and more importantly in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
TK-deficient CD4 T-cells as compared to the corresponding carbonate compounds -ACB--C-
alkyl-d4TTPs 11. For instance, -(AB-C11)--C-(alkyl-C12)-d4TTP 10c (EC : 0.0074 µM/HIV-
5
0
1
; EC : 0.091 µM/HIV-2) proved to be more active in cultures of infected wild-type CEM/0
50
cells than -(ACB-C11)--C-(alkyl-C12)-d4TTP 11c (EC : 0.04 µM/HIV-1; EC : 0.11
5
0
50
µM/HIV-2). Moreover, the antiviral activity of -(AB-C11)--C-(alkyl-C12)-d4TTP 10c was
found to be higher by almost a factor of 5 as compared to -(ACB-C11)--C-(alkyl-C12)-d4TTP
−
1
1c in CEM/TK cell cultures, probably due to the different stability of the prodrugs 10c (t > 8
1
/2
h),11c (t > 10 h) in CEM cell extracts.
1
/2
As compared to our previously reported -alkylphosphate compounds 8 (-(AB-C4;alkyl-C18)-
4
7
−
d4TTP; EC : 0.17 µM/HIV-2), the inhibitory effect in CEM/TK cells of -alkylphosphonates
5
0
such as -(AB-C4)--C-(alkyl-C18)-d4TTP 10e (EC : 0.042 µM/HIV-2) and -(ACB-C4)--C-
5
0
(
alkyl-C18)-d4TTP 11e (EC : 0.032 µM/HIV-2) disclosed here was improved by a 4-fold and 5-
50
fold, respectively. It should also be noticed that the cleavage of the AB masking unit or the ACB
masking unit in 10 and 11, respectively, occurred readily in the biological medium leading to the
formation of 4-hydroxybenzyl alcohol.52
Table 2: Antiviral activity and cytotoxicity of TriPPPro-d4TTPs 10,11,22 and -C-alkyl-d4TTPs
2
3 and d4T 1.
Comp.
HIV-1 (HE)
cellular toxicity
HIV-2 (ROD)
CEM/TK-
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2
2
2
2
2
2
2
2
2
2
3
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HIV-2 (ROD)
EC50a [µM]
0.023 ±0.025
0.065±0.064
0.0074±0.008
0.031±0.039
0.0074±0.0014
0.095±0.002
0.097±0.004
0.04±0.03
EC50a [µM]
0.021±0.07
0.025±0.021
0.091±0.008
0.16±0.021
0.021±0.006
0.35±0.01
EC50a [µM]
26.3±0.85
3.43±1.88
0.066±0.063
0.53±0.11
0.042±0.002
36.8±4
CC50b [µM]
>100±0
86±33
37±9
10a
0b
0c
0d
10e
1a
1b
1c
11d
1e
2a
2c
23a
3c
d4T
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
56±29
19±8
1
>100±0
38±2.5
39±6
1
0.20±0.2
9.9±4
1
0.11±0.1
0.32±0.7
0.12±0.2
0.22±0.3
1.76±2
32±6
1
0.0018±0.002
0.21±0.12
0.026±0.04
0.087±0.014
0.046±0.056
0.17±0.035
0.11±0.064
0.032±0.02
20.9±4.1
16±2
2
>100±0
38±2
2
0.095±0.032
0.15±0.039
0.046±0.021
0.43±0.23
0.14±0.02
41.1±0.71
1.58±0.75
31.05±5.25
>100±0
21±9
2
0.31±0.13
>50
+
[
a] Antiviral activity determined in CD4 T-lymphocytes: 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
5
0%; values are the mean ±SD of n=2-3 independent experiments.
Primer extension assays using -C-(alkyl)-d4TTPs 23 and cellular DNA polymerases and
HIV's reverse transcriptase (RT).
In antiviral assays, all compounds were highly active against HIV-1 and HIV-2 in cultures of
+
−
infected wild-type CEM/0 cells and also in thymidine kinase-deficient CD4 T-cells (CEM/TK ).
We have proven that no d4TTP was formed in all hydrolysis studies. From the results
summarized above, it appeared that the delivered -C-(alkyl)-d4TTPs 23 was responsible for the
inhibitory effect of these compounds, which is a marked difference to the formerly reported
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TriPPPro-compounds 6. Next, primer extension assays were done investigating -C-(alkyl)-
d4TTPs (23) substrate properties towards four DNA-polymerases: reverse transcriptase (RT)
which is an RNA-dependent-DNA-polymerase, DNA polymerase  DNA polymerase , and
1
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DNA polymerase , which is a mitochondrial polymerase and often made responsible for marked
toxicity effects caused by nucleoside analogue triphosphates.53,54
Figure 4 shows the results of a primer extension assay in which HIV-RT was used. In the case of
a missing RT no elongation happened (- lane) while full extension to the 30mer occurred (+ lane)
when all four canonical triphosphates were present (controls). -C-(alkyl-C18)-d4TTP 23c (lane
3
), -C-(alkyl-C12)-d4TTP 23a (lane 4), and d4TTP (lane 6), only show the n+1 band due to
d4TMP incorporation. Thus, -C-(alkyl)-d4TTPs 23 were accepted by HIV-RT as a substrate
although the elongation with -C-(alkyl-C18)-d4TTP 23c (lane 3) was not complete. As control
TTP (lane 5) was also used.
Figure 4. Primer extension assay using HIV's RT. lane 1 (+): dATP, dGTP, dCTP and TTP with
HIV-RT; lane 2 (-): dATP, dGTP, dCTP and TTP without HIV-RT; lane 3 (-C -d4TTP): -C-
1
8
(
alkyl-C18)-d4TTP 23c; lane 4 (-C -d4TTP): -C-(alkyl-C12)-d4TTP 23a; lane 5: TTP; lane 6:
1
2
d4TTP.
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Next, -C-(alkyl)-d4TTPs 23 were studied using human DNA polymerases ,  and  (Figure 5,
A-C). Figure 5 A shows the result using human DNA polymerase  and -C-(alkyl)-d4TTPs 23
compared to TTP. Obviously, with TTP the expected the canonical incorporation of TMP was
observed which led to the presence of the n + 1 band (26 nt, lane 6). However, when -C-(alkyl)-
d4TTPs 23 and d4TTP were used, no incorporation was detectable (Figure A; lanes 3-5). Thus,
0
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-C-(alkyl)-d4TTPs 23 were not substrates for DNA polymerase .
As is shown in Figure 5B and 5C, also no incorporation was detected in primer extension assays
using -C-(alkyl)-d4TTPs 23 in combination with DNA pol  (B) and DNA pol  (C).
Interestingly, in contrast to DNA polymerases  and , DNA polymerase  used d4TTP as a
good substrate which led to the n + 1 band in Figure 5B. As a conclusion DNA polymerase , 
and  did not recognize the -C-(alkyl)-d4TTPs 23 as a substrate. In contrast, these compounds
acted as substrates for HIV-RT.
Figure 5. Primer extension assay with human DNA polymerases  (A),  (B) and  (C).
A. For human DNA pol  assay: lane 1 (-): dATP, dGTP, dCTP and TTP without human Pol ;
lane 2 (+): dATP, dGTP, dCTP and TTP with human Pol ; lane 3 (-C18-d4TTP): -C-(alkyl-
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C18)-d4TTP 23c; lane 4 (-C12-d4TTP): -C-(alkyl-C12)-d4TTP 23a; lane 5 (d4TTP): d4TTP;
lane 6 (TTP): TTP.
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0
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0
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0
B. For human DNA pol  assay: lane 1 (-): dATP, dGTP, dCTP and TTP without human DNA
pol ; lane 2 (+): dATP, dGTP, dCTP and TTP with human DNA pol ; lane 3 (-C18-d4TTP):
-C-(alkyl-C18)-d4TTP 23c; lane 4 (-C12-d4TTP): -C-(alkyl-C12)-d4TTP 23a; lane 5
(d4TTP): d4TTP; lane 6 (TTP): TTP.
C. For human DNA pol  assay: lane 1 (-): dATP, dGTP, dCTP and TTP without human DNA
pol ; lane 2 (+): dATP, dGTP, dCTP and TTP with human DNA pol ; lane 3 (-C18-d4TTP): -
C-(alkyl-C18)-d4TTP 23c; lane 4 (-C12-d4TTP): -C-(alkyl-C12)-d4TTP 23a; lane 5 (TTP):
TTP; lane 6 (d4TTP): d4TTP.
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Summary and Conclusion
In summary, we disclosed the synthesis of a new class of nucleoside triphosphate prodrugs, -
AB--C-alkyl-d4TTPs 10 and -ACB--C-alkyl-d4TTPs 11, which differ from earlier reported
TriPPPro-derivatives 6,7 by bearing a non-cleavable -alkyl-phosphonate moiety instead of the
normal -phosphate group in addition to a biodegradable prodrug moiety at the -phosphonate
unit. As -C-alkyl-group three n-alkyl moieties were used in combination with the previously
used acyloxybenzyl- or alkoxycarbonyloxybenzyl-prodrug groups. TriPPPro-d4TTPs 10,11,22,
as well as -C-(alkyl)-d4TTPs 23, were synthesized by using the H-phosphinate route with
0
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0
satisfying yields (up to 68%). We have proven that the hydrolysis, stability, and antiviral activity
were significantly influenced by two different alkyl chain lengths of the prodrug moieties (R1
2
and R ). The prodrug group was cleaved to give -C-(alkyl)-d4TTPs 23 by chemical hydrolysis
or by enzymes present in cell extracts, no d4TTP 4 was detected in all hydrolysis studies using
prodrugs 10,11,22. Interestingly, -C-(alkyl)-d4TTPs 23 proved to be stable in PLE and cell
extracts. All these compounds showed at least similar, often even much better antiviral activity
against HIV-1 and HIV-2 than the parent nucleoside d4T 1 in wild-type CEM/0 cells. The strong
antiviral activities of TriPPPro-d4TTP prodrugs 10,11 (e,g. -(AB-C4)--C-(alkyl-C18)-d4TTP
10e and -(ACB-C4)--C-(alkyl-C18)-d4TTP 11e) in the wild-type CEM/0 cell cultures were
−
completely retained in mutant thymidine-deficient CEM cells (TK ), proving the successful
cellular uptake of these derivatives and an intracellular delivery of the nucleoside diphosphate--
alkylphosphonate. The half-lives of prodrugs -ACB--C-alkyl-d4TTPs 11 (t1/2 = 0.65-10 h,
Table 1) in cell extracts were found to be higher than those for -AB--C-alkyl-d4TTPs 10 (t =
1
/2
0.044-8 h, Table 1). However, the inhibition of the replication of HIV-1- and HIV-2 in infected
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wild-type CEM/0 as well as HIV-2-infected mutant thymidine kinase-deficient CEM cell
−
cultures (CEM/TK ) by prodrugs -ACB--C-alkyl-d4TTPs 11 was found to be lower or at its
best similar to the corresponding -AB--C-alkyl-d4TTPs 10. It was concluded that the antiviral
activity determined for TriPPPro-prodrugs 10,11 correlated well with the biological stability of
the prodrugs 10,11 in CEM cell extracts.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
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0
We have proven that -C-(alkyl-C18)-d4TTP 23c was also, although weakly potent against HIV-
−
2
in thymidine kinase-deficient cell cultures (CEM/TK , EC : 1.58 µM). However, this
5
0
compound was less active than its prodrug forms 10e,11e,22c (about a 38-fold, 49-fold, and 11-
fold, respectively). -C-(alkyl)-d4TTPs 23 were substrates for HIV-RT as shown in primer
extension assays while they proved to be non-substrates for the cellular DNA-polymerases , 
and . Thus, a high degree of differentiation between the viral polymerase and the cellular one
was discovered. We are currently working on the question if this increased selectivity towards
the viral enzyme is a general property of these -modified phosphonate compounds by using
different nucleoside analogues and further viral polymerases.
As a conclusion, the described prodrugs are able to efficiently enter cells and deliver -C-alkyl-
NTPs highly selectively by an enzyme-triggered reaction. This TriPPPro-approach offers high
potential to be used in antiviral and antitumoral chemotherapies: i) would bypass all
phosphorylation steps normally needed for the activation of a nucleoside analogue ii) the
delivered -C-alkyl-d4TTPs 23 proved to be highly stable in cell extracts against
dephosphorylation in contrast to natural NTPs and iii) -C-alkyl-d4TTPs 23 were substrates for a
viral polymerase (RT) but did not act as substrates for three cellular DNA-polymerases with
DNA pol  being the most important one. Moreover, we are convinced that this TriPPPro-
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strategy is not limited to HIV inhibition but may also be used for other viral targets. Using these
insights, -C-modified NTP-prodrugs of other nucleoside analogues will be explored next.
0
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Experimental Section
General: All experiments were carried out under anhydrous conditions and nitrogen atmosphere.
All anhydrous solvents were purchased from Acros Organics (Extra Dry over molecular sieves)
and obtained by the MBraun solvent purification system (MB SPS-800). CH CN for HPLC was
3
®
purchased from VWR (HPLC grade). Ultrapure water was produced by a Sartorius Aurium pro
(Sartopore 0.2 µm, UV). All other organic solvents were purchased technical grade and distilled
prior to use. Commercially available solvents and reagents were used as received without further
purification. General flash column chromatography was performed with silica gel 60 M (0.04-
0
.063 mm, Macherey-Nagel). For reversed phase automated flash chromatography, an Interchim
®
Puriflash 430 was used in combination with Chromabond Flash RS40 C ec. All High
1
8
Performance Liquid Chromatography (HPLC) measurements were carried out using a VWR-
Hitachi LaChromElite HPLC system (L-2130, L-2200, L-2455) and EzChromElite software.
TBAA buffer: 2 mM tetra-n-Butylammonium acetate solution (pH 6.3). Method: Nucleodur 100-
5
3
C18ec; 0-20 min: TBAA buffer/CH CN gradient (5-80%); 20-30 min: buffer/CH CN (80%);
3 3
0-33 min: buffer/CH CN (80-5%); 33-38 min: buffer/CH CN (5%); flow: 1 mL/min.
3
3
Compound purity: All final compounds were isolated analytically pure, ≥95% purity by HPLC
and NMR spectroscopy.
All NMR spectra were carried out using Bruker spectrometers: Bruker AMX 400, Bruker DRX
5
00 or Bruker AVIII 600. All chemical shifts (δ) were given in ppm and calibrated on solvent
signals. HRMS (ESI) mass spectra were performed with a VG Analytical Finnigan ThermoQuest
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MAT 95 XL or an Agilent 6224 EIS-TOF spectrometer. MALDI mass spectra were recorded on
an ultrafleXtreme MALDI-TOF-TOF mass spectrometer by Bruker Daltonik with 9-AA as
matrix.
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General procedures
General Procedure 1: Preparation of H-phosphinates 15.
The reactions carried out under nitrogen (N ) atmosphere under dry conditions. 1-(3-
2
Dimethylaminopropyl)-3-ethylcarbodiimide (EDC; 1.2 equiv.) was added to a solution of 4-
acyloxybenzyl alcohols 14 (2.0 equiv.), H-phosphinic acid 13 (1.0 equiv.) and 4-
dimethylaminopyridine (DMAP; 0.2 equiv.) in dry CH Cl . The mixture was stirred for 12 h at
2
2
room temperature (rt). The solvent was removed in vaccum and the residue was purified using
column chromatography to give compounds H-phosphinates 15.
General Procedure 2: Preparation of H-phosphinates 18.
The reactions carried out under nitrogen (N ) atmosphere under dry conditions. EDC (1.2 equiv.)
2
was added to a solution of and 4-alkoxycarbonyloxybenzyl alcohols 17 (2.0 equiv.), H-
phosphinic acid 13 (1.0 equiv.) and DMAP (0.2 equiv.) in dry CH Cl . The mixture was stirred
2
2
for 12 h at room temperature (rt). The solvent was removed in vaccum and the residue was
purified using column chromatography to obtain compounds H-phosphinates 18.
General Procedure 3: Preparation of -AB--C-alkyl-d4TTPs 10 and -ACB--C-alkyl-
d4TTPs 11.
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The reactions were performed in a nitrogen (N ) atmosphere and dry conditions. Firstly, H-
2
phosphinates 15,18 (0.3 mmol, 1.0 equiv.) were dissolved in 6 mL CH CN. N-chlorosuccinimide
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(NCS, 0.6 mmol, 2.0 equiv.) was added and the mixture was stirred for 2 h at room temperature.
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Subsequently, tetrabutylammonium phosphate solution (0.4 M in CH CN, 9 mmol, 3.0 equiv.)
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was added quickly and was stirred for 1 h at room temperature and the solvent was removed in
vacuum under reduced pressure. The residue was extracted with CH Cl /H O (three times), The
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organic phase was dried over sodium sulfate and the solvent was removed to afford
pyrophosphate. Then, pyrophosphate was dissolved in 4 mL CH CN at 0 °C. Then, a mixture of
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Et N (2.4 mmol, 8.0 equiv.) and trifluoroacetic anhydride (TFAA, 1.5 mmol, 5.0 equiv.) in 4 mL
3
CH CN was cooled to 0 °C and added to the mixture of pyrophosphate. All volatile components
3
were removed in vacuum under reduced pressure after 10 min. The residue was dissolved in 6
mL CH CN and cooled to 0 °C. 1-Methylimidazole (0.9 mmol, 3.0 equiv.) and Et N (TEA, 1.5
3
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mmol, 5.0 equiv.) were added at 0 °C. The mixture was warmed to room temperature and stirred
for 10 min. Finally, d4TMP (0.5-0.7 equiv.) dissolved in 6 mL CH CN was added to the mixture
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and stirred at room temperature for 2-5 h. All volatile components were removed in vacuum. The
crude product was purified by automatic RP18 flash chromatography, and 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 freeze-dried and the
products 10,11 were obtained as white solids.
Dodecyl-H-phosphinic acid 13a.
Under dry conditions, a solution of diethyl chlorophosphite (2.9 mL, 20 mmol) in dry Et 0 (40
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mL) was cooled to 0 °C. A 1.0 M solution of dodecylmagnesium bromide in Et O (20 mL, 20
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mmol) was added dropwise over 30 min and stirred at 0 °C. Following, the white suspension was
heated at 50 °C for 4 h, cooled, and filtered. The Et 0 was removed by distillation at atmospheric
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pressure and the residue distilled under reduced pressure to give the corresponding phosphonous
diester. 2) 2 mL concentrated HC1 was adeed to a solution of phosphonous diester and 20 mL
H O. After being stirred at room temperature for 1 h, the mixture was extracted with CH Cl .
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The organic phase was washed with saturated NaCl, dried over MgSO , and evaporated. The
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crude phosphonous monoester was taken up in 4.0 N NaOH (40 mL) and stirred at room
temperature for 1 h. The aqueous mixture was washed with Et 0, acidified with concentrated HCl
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pH 1), and extracted with CH Cl . The CH Cl was washed with saturated NaCl, dried over
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MgSO , and evaporated. Recrystallization of the crude product from CH Cl /Et O give pure
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phosphonous acid 13a (3.28 g, 70%) as a while solid. H-NMR (400 MHz, CDCl ):  [ppm] =
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1
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2.22 (m, 1H, OH), 7.05 (d, J = 540.1 Hz, 1H, P-H), 1.78-1.68 (m, 2H, H-a), 1.62-1.52 (m,
HH
2H, H-b), 1.41-1.34 (m, 2H, H-c), 1.31-1.24 (m, 16H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k),
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0
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.86 (t, J = 6.9 Hz, 3H, H-l). C NMR (101 MHz, CDCl ):  [ppm] = 31.9, 30.4, 30.3, 29.57,
HH
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9.53, 29.3, 29.1, 28.8, 22.6 (C-a, C-c, C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k), 20.6 (d, J = 2.7
CP
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+
H , C-b), 14.0 (C-l). P NMR (162 MHz, CDCl ):  [ppm] = 38.7. HRMS (ESI , m/z):
Z
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+
calculated for C H O P, [M+H] 235.1822; found, 235.2083.
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Tetradecyl-H-phosphinic acid 13b.
Under dry conditions, a solution of diethyl chlorophosphite (4.3 mL, 30 mmol) in dry THF (60
mL) was cooled to 0 °C. A 1.0 M solution of tetradecylmagnesium chloride solution in THF (30
mL, 30 mmol) was added dropwise over 30 min and stirred at 0 °C. Following, the mixture was
heated at 50 °C for 6 h, cooled, and the THF was removed under reduced pressure. 150 mL
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