Anti-HIV-Active Nucleoside Triphosphate Prodrugs

Article abstract of DOI:10.1021/acs.jmedchem.0c00271

We disclose a study on nucleoside triphosphate (NTP) analogues in which the γ-phosphate is covalently modified by two different biodegradable masking units and d4T as nucleoside analogue that enable the delivery of d4TTP with high selectivity in phosphate buffer (pH 7.3) and by enzyme-triggered reactions in human CD4+ T-lymphocyte CEM cell extracts. This allows the bypass of all steps normally needed in the intracellular phosphorylation. These TriPPPro-nucleotides comprising an acyloxybenzyl (AB; ester) or an alkoxycarbonyloxybenzyl (ACB; carbonate) in combination with an ACB moiety are described as NTP delivery systems. The introduction of these two different groups led to the selective formation of γ-(ACB)-d4TTPs by chemical hydrolysis and in particular by cell extract enzymes. γ-(AB)-d4TTPs are faster cleaved than γ-(ACB)-d4TTPs. In antiviral assays, the compounds are highly active against HIV-1 and HIV-2 in wild-type CEM/O cells and more importantly in thymidine kinase-deficient CD4+ T-cells (CEM/TK-).

Full text of DOI:10.1021/acs.jmedchem.0c00271

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Article
Anti-HIV-active Nucleoside Triphosphate Prodrugs
Xiao Jia, Dominique Schols, and Chris Meier
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00271 • Publication Date (Web): 18 May 2020
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Anti-HIV-active Nucleoside Triphosphate Prodrugs
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1*
Xiao Jia, 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 We disclose a study on nucleoside triphosphate (NTP) analogues in which the -
phosphate is covalently modified by F8butwo different biodegradable masking units and d4T as
nucleoside analogue that enable the delivery of d4TTP with high selectivity in phosphate buffer
+
(
pH 7.3) and by enzyme-triggered reactions in human CD4 T-lymphocyte CEM cell extracts.
This allows the bypass of all steps normally needed in the intracellular phosphorylation. These
TriPPPro-nucleotides comprising an acyloxybenzyl- (AB; ester) or an
alkoxycarbonyloxybenzyl- (ACB; carbonate) in combination with an ACB-moiety are described
as NTP delivery systems. The introduction of these two different groups led to the selective
formation of -(ACB)-d4TTPs by chemical hydrolysis and in particular by cell extract enzymes.

-(AB)-d4TTPs are faster cleaved than -(ACB)-d4TTPs. In antiviral assays, the compounds are
highly active against HIV-1 and HIV-2 in wild-type CEM/O cells and more importantly in
+
-
thymidine kinase-deficient CD4 T-cells (CEM/TK ).
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Introduction
For many decades, nucleoside analogues have been applied in antiviral and antitumoral
chemotherapy and they still comprise the frontline of drugs used to combat infections caused by
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HIV, herpes virus, hepatitis B and C virus. The antiviral efficacy of many nucleoside analogues
such as 3’-deoxy-2’,3’-dehydrothymidine (d4T, 1; Scheme 1) or 3’-deoxy-3’-azidothymidine
(AZT) are strongly dependent on their intracellular activation by host cell kinases to give, via the
monophosphate (2, NMP) and the diphosphate (3, NDP), ultimately the bioactive nucleoside
3
,4
analogue triphosphate derivatives (4, NTP). The targets of effective nucleoside analogue drugs
are the virus-encoded DNA- or RNA polymerases, such as the HIV reverse transcriptase (RT)5,6
7
or the HCV-encoded RNA-dependent RNA-polymerase NS5B, which are the key enzymes in
the replication cycle of HIV and HCV, respectively. Up to now, many nucleoside analogues have
8
been approved as HIV-RT inhibitors (NRTIs) and they are used as the backbone of the
9
combined antiretroviral therapy (cART). However, cellular kinases often catalyse these
biotransformation insufficiently, resulting in low or no biological activity of the given
compound.3,10,11 Moreover, the clinical efficacy of nucleoside analogues is hampered by
limitations such as poor biological half-lives due to catabolic elimination from the body,
mutations of nucleoside transporters, variable bioavailability after oral administration or
development of drug resistance.12-14 These hurdles can be overcome by using lipophilic prodrugs
of the phosphorylated parent nucleosides, which are able to bypass the rate-limiting, kinase-
catalyzed conversion steps, such as tenofovir and sofosbuvir.15,16 In the past, this task has been
successfully achieved for the intracellular delivery of NMP using prodrug strategies17-19 such as
the cycloSal-,20-22 SATE-,23,24 bisPOM- or phosphoramidate nucleotides.
25
26,27
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extracellular
intracellular
O
NH
HO
d4T
N
O
O
TK
d4T 1
O
nucleoside
monophosphate
prodrug 5
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mask P
O
d4T
O
O
O
O
OH d4TMP 2
TMP-K
mask
O
P
O
P
nucleoside
diphosphate
prodrug - DiPPro
concept 6
mask P P
O
d4T
d4T
O
O
O
d4T d4TDP
3
O
O
NDP-K
mask
fast chemical or
enzymatical cleavage
nucleoside
triphosphate
prodrug - TriPPPro
concept 7
O
O
O
P
mask P P P
O
d4TTP 4
O
P
O
P
O
O
d4T
O
O
O
cell membrane
mask
Scheme 1: Metabolism of nucleoside analogues such as d4T 1 and the corresponding nucleotide
prodrugs.
In the case of the anti-HIV drug d4T 1 within the stepwise biotransformation, the first
phosphorylation step catalyzed by the host cell enzyme thymidine kinase (TK) has been
identified as the metabolism-limiting step because of the rather modest affinity of d4T 1 to TK as
an alternative substrate and because TK activity is S-phase dependent.4,28 In addition to the
cycloSal-nucleoside monophosphate prodrug approach, we have reported on a successful
approach to deliver NDPs inside cells using lipophilic, but still partially charged NDP derivatives
(
DiPPro-approach 6; Scheme 1).29-33 However, although both approaches deliver phosphorylated
forms of nucleosides, the released nucleotides still need further phosphorylation into their
triphosphate forms by cellular kinases in order to interact with the viral polymerases. As a
consequence, the development of NTP prodrugs (TriPPPro-concept 7; Scheme 1) is still highly
interesting and desirable because this would bypass all steps of intracellular phosphorylation and
would in principle maximize the intracellular concentration of the ultimately bioactive NTP
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although the formidable challenges are not to be underestimated. A triphosphate has (a) four
negative charges that require masking, has (b) an inherent lability within the triphosphate unit
due to two reactive phosphate anhydride linkages and (c) the chemical synthesis of a prodrug
might be difficult. Earlier, very few reports on potential triphosphate prodrugs have been
reported,37,38 which may be due to their low chemical stability, complicated synthesis, poor
deliverability and their high sensitivity for enzymatic dephosphorylation. Recently, we disclosed
the first delivery system of NTPs through a prodrug technology (TriPPPro-approach 7).40-42 It
was proven for d4T 1 and meanwhile also for other nucleoside analogues that such compounds
deliver successfully the corresponding triphosphate inside cells by an uptake study using a
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1
-
fluorescent nucleoside analogue and by the observed significant anti-HIV activity in CEM/TK
cell cultures whereas the parent 1 was virtually inactive in these cells due to the lack of
phosphorylation. The membrane permeability was achieved by esterification of the -phosphate
group with two covalently attached but bioreversible 4-acceptor-substituted benzyl esters
(
acyloxybenzyl; AB groups), which led also to a marked increase in enzymatic stability of the
triphosphate unit. The enzyme-driven cleavage of both masks by an initial cleavage of the
phenolic acyl ester and a subsequent spontaneous cleavage of the remaining part of the mask led
to the formation of d4TTP 4 from TriPPPro-prodrugs 7 (Scheme 2). Further studies showed that
the -dimasked TriPPPro-prodrugs were not substrates for polymerases such as HIV-RT or
DNA-pol .
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Delivery mechanism of d4TTP from TriPPPro-compounds 7
O
O
R
O
N
esterase
esterase
O
P
O
P
O
P
O
P
O
P
O
P
NH
O
P
O
P
O
P
O
O
O
O
d4T
Cat O
O
O
O
d4T
Cat O
Cat
O
O
O
or
O
O
O
O
O
O
O
O
O
O
1
1
1
1
1
1
1
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chemically
induced
O
Cat
Cat
Cat
Cat
Cat
Cat
O
R
O
R
O
O
TriPPPro-compounds 7
Intermediate 7b
d4TTP 4
Scheme 2: TriPPPro-nucleoside triphosphate prodrugs 7 and the delivery pathway.
Interestingly, in the case of non-symmetric DiPPro-compounds, a highly selective conversion of
the DiPPro-compounds into nucleoside analogue diphosphates was finally achieved in CEM cell
3
2
extracts. We have shown before that the stability of such compounds could be adjusted over a
3
0
wide range. Moreover, the symmetric TriPPPro-compounds bearing long alkyl chains such as a
40
8
Z-C17 residue (t1/2 = 27 h) proved to be unstable because of altered solubility behavior or
micelle formation. In addition to the ester functional group in the AB masking moiety also first
examples of carbonate linked compounds were studied. The chemical stability of these carbonate
TriPPPro-compounds and intermediates were found to be higher than the corresponding ester
4
0
TriPPPro-compounds and intermediates, respectively. These observations guided us here to
conduct a study on a series of triphosphate derivatives 8,13 bearing two different biodegradable
1
3
2
masking units (R or R not identical to R ). One of the bioreversible groups is an
acyloxybenzyl- (AB; ester) or an alkoxycarbonyloxybenzyl- (ACB; carbonate) moiety while the
second group is always an ACB-moiety. It was expected that such a combination would be more
rapidly cleaved by chemical or particularly by enzymatic means to form the ACB-carbonate-
intermediate 20 (Scheme 3) and thereby would help to avoid the side reaction that is responsible
for the formation of the unwanted NMP or NDP. The introduced ACB-moiety comprise a long,
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lipophilic aliphatic chain, which not only added high lipophilicity to the molecule but also should
slowly cleaved to form the triphosphate. It was expected that with these non-symmetric
compounds a selective conversion of the TriPPPro-compounds 8,13 into nucleoside
triphosphates such as d4TTP 4 can be achieved.
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Delivery mechanism of d4TTP from TriPPPro-compounds 8,13
O
O
1
st cleavage
R3
R1
O
O
O
Low stability
Low lipophilicity
O
O
P
O
P
O
P
esterase
O
O
O
O
d4T
esterase
Cat
O
O
O
P
O
O
P
O
O
O
O
P
O
P
O
O
d4T
O
P
O
O
P
O
d4T
or
or
Cat Cat
O
O
O
O
O
O
chemically
induced
chemically
induced
Cat Cat
Cat Cat
O
O
R
2
R = R or R3
O
O
O
O
O
R2
R2
high lipophilicity
nd cleavage
O
intermediates 20
O
high lipophilicity
2
esterase
TriPPPro-compounds 8
TriPPPro-compounds 13
d4TTP 4
Scheme 3: The prodrug of d4TTP 8,13 and the -alkoxycarbonyloxybenzyl-modified-d4TTP
derivatives 20.
Here we present the synthesis and characterization of these new TriPPPro-compounds 8,13 as
well as their hydrolysis behavior in different media and their antiviral activity.
RESULTS AND DISCUSSION.
Synthesis of -acyloxybenzyl (AB)-alkoxycarbonyloxybenzyl (ACB)-d4TTPs 8 and -
(ACB;ACB)-d4TTPs 13
For the synthesis of -acyloxybenzyl (AB)-alkoxycarbonyloxybenzyl (ACB)-d4TTPs 8, the H-
phosphonate route was used as reported previously by us for the preparation of symmetric
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TriPPPro-derivatives (Scheme 4).41 In the first step, d4TMP 2 was prepared according to a
known procedure.44 Mixed H-phosphonates 11 were prepared from diphenyl
hydrogenphosphonate (DPP), 4-acyloxybenzyl alcohols 9 and 4-alkoxycarbonyloxybenzyl
alcohols 10. Next, compounds 11 were converted into the corresponding chlorophosphate using
N-chlorosuccinimide (NCS)45 followed by a phosphorylation with tetra-n-butylammonium
phosphate to yield pyrophosphates 12 in almost quantitative yield. The final coupling reaction
was accomplished using modified literature methods46,47 by a stepwise activation of 12 with
trifluoroacetic acid anhydride (TFAA) and N-methylimidazole, followed by addition of d4TMP 2
1
1
1
1
1
1
1
1
1
1
2
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2
2
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9
0
+
+
to give -(AB;ACB)-d4TTPs 8 (n-Bu N form). The different -(AB;ACB)-d4TTPs 8 (NH
4
4
form) were isolated as white solids after reversed-phase (rp) column chromatography, a Dowex
+
5
0WX8 (NH ) ion exchange and freeze-drying. The advantage of the H-phosphonate route used
4
here is that d4TMP was easier to prepare than d4TDP, which was the limiting step in the overall
yield of the phosphoramidite method. A second advantage of this route is that after the formation
of the P-O-P-linkage, no oxidation is needed in contrast to the phosphoramidite chemistry. The
conversion of the parent nucleoside d4T 1 to the target TriPPPro-compounds 8 was improved
and the overall yields varied between 23%-78%. For comparison, three -(ACB;ACB)-d4TTPs
1
3 were synthesized as well using the same approach (Scheme 4). These compounds have two
alkoxycarbonyloxybenzyl (ACB)-moieties but bearing different lipophilic alkyloxy residues.
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O
O
O
R1
R1
R1
O
O
O
OH
O
O
N
O
R1
O
P
O
P
O
P
NH
O
O
O
P
O
O
O
O
i
O
P
H
ii
O
P
O
OH
iv
O
O
O
O
O
O
O
O
O
O
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0
O
R2
HO
nBu N
NH4 NH4
4
10r-z
9a-j
O
O
O
O
O
O
O
O
O
R2
12
R2
R2
-(AB,ACB)-d4TTPs 8
11
1
R : a=CH , b=C H , c=C H (n-propyl), d=C H7
3
2
5
3
7
3
O
O
(isopropyl), e=C H9 (n-butyl), f=C H9 (isobutyl),
4 4
g=C H , h=C H , i=C H17, j=C11H23
6
13
8
17
9
NH
O
P
NH
2
HO
iii
O
O
R : r=C6H13, s=C9H19, t=C10H21,
u=C11H23, v=C12H25, w=C14H29
x=C15H31, y=C16H33, z=C18H37
N
O
N
O
nBu N
4
O
,
O
O
nBu N
4
3
d4T 1
d4TMP 2
R : k=C2H5, l=C4H9
O
O
O
R3
R3
R3
O
O
O
O
O
O
OH
O
O
N
R3
O
O
O
O
O
P
O
P
O
P
O
P
NH
O
P
O
P
O
OH
iv
O
O
O
O
i
H
ii
O
O
O
O
O
O
O
O
O
NH4 NH4
O
nBu N
4
R2
HO
1
0v,z
10k,l
O
O
O
O
O
O
O
O
R2
O
R2
R2
14
15
-(ACB,ACB)-d4TTPs 13
8ay: 50%; 8by: 51%; 8ey: 71%; 8fy: 73%; 8gy: 50%; 8hy: 46%; 8iy: 23%; 8bs: 51%; 8bt: 37%; 8bu: 69%; 8bv: 43%; 8bw: 66%; 8cv: 52%;
dv: 44%; 8ev: 59%; 8ew: 78%; 8ex: 74%; 8ez: 39%; 8is: 78%; 8jr: 54%; 13ss: 70%; 13kv: 59%; 13lv: 55%; 13lz: 52%
8
Scheme 4. Reagents and conditions: i) DPP, pyridine, 0 °C-rt, 12 h; ii) a. NCS, CH CN, rt, 2 h,
3
b) N(Bu) (H PO ), CH CN, rt, 1 h; iii) d4T 1, POCl , pyridine, H O, CH CN, 0 °C-rt, 5h; iv) a.
4
2
4
3
3
2
3
TFAA, Et N, CH CN, 0 °C, 10 min, b. 1-methylimidazole, Et N, CH CN, 0 °C-rt, 10 min, c.
3
3
3
3
d4TMP 2, rt, 2-5 h.
Synthesis of -alkoxycarbonyloxybenzyl (ACB)-d4TTPs 20 and -(ACB; -cyanoethyl)-
d4TTPs 16
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
-Alkoxycarbonyloxybenzyl-d4TTPs 20 were synthesized using the H-phosphonate route as
well. However, the -cyanoethyl group was introduced as protection group for the -phosphate
group. In the first step, DPP was successively reacted with 3-hydroxypropionitrile and 4-
alkoxycarbonyloxybenzyl alcohols 10 to form H-phosphonates 18. Compounds 18 were
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
4
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
converted into the pyrophosphates 19 as above. After the coupling reaction of compounds 19 and
+
d4TMP 3 to give the -protected triphosphates 16 (n-Bu N form), the crude reaction product
4
was deprotected during the ion-exchange to yield the mixture of -(ACB; -cyanoethyl)-d4TTPs
+
+
+
1
6 (NH form) and -ACB-d4TTPs 20 (NH form). Finally, -(ACB)-d4TTPs 20 (NH form)
4
4
4
were obtained as white solids after rp column chromatography and freeze-drying (Scheme 5).
O
NC
NC
NC
NC
O
P
O
P
O
P
NH
O
P
O
P
O
P
O
O
O
O
OH
N
O
O
H
O
O
OH
iv
i
ii
+
O
O
O
O
O
O
O
OH
NH4 NH4
nBu4N
16v,y
O
O
O
O
O
O
O
O
O
R2
O
O
O
R2
R2
+
O
N
R2
O
P
O
P
O
P
NH
1
0v,y
18
19
NH4 O
O
O
O
O
O
O
O
O
O
N
O
N
NH4 NH4
NH
O
NH
HO
iii
O
P
O
O
O
O
O
nBu4N
O
O
O
20v,y
O
nBu4N
R2
d4T 1
d4TMP 2
6v: 52%[b]; R = C12H ; 20v: 23%[a]
2
1
1
25
6y: 63%[b]; R = C16H ; 20y: 10%[a]
2
33
Scheme 5. Reagents and conditions: i) DPP, pyridine, 0 °C-rt, 12 h; ii) a. NCS, CH CN, rt, 2 h,
3
b) N(Bu) (H PO ), CH CN, rt, 1 h; iii) d4T 1, POCl , pyridine, H O, CH CN, 0 °C-rt, 5h; iv) a.
4
2
4
3
3
2
3
TFAA, Et N, CH CN, 0 °C, 10 min, b. 1-methylimidazole, Et N, CH CN, 0 °C-rt, 10 min, c.
3
3
3
3
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1
1
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1
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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
4
5
5
5
5
5
5
5
5
5
5
6
d4TMP 2, rt, 5 h. [a] yields are calculated for the conversion from 18 to 20. [b] yields are
calculated for the conversion from 18 to 16.
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
The TriPPPro-d4TTP prodrugs 8,13,16 and the -ACB-d4TTPs 20 were incubated in PBS (25
+
mM, pH 7.3), or were exposed to human CD4 T-lymphocyte cell extracts and to pig liver
esterase (PLE) to study their stability and to identify the formed hydrolysis products. The
hydrolysis mixtures were analyzed by means of analytical RP18-HPLC. The calculated half-lives
of prodrugs 8 (Table 1, t ) reflect either the removal of the bioreversible AB-group or the ACB-
1
/2
group to yield the corresponding intermediates 7b and 20, respectively. Possible hydrolysis
pathways and products are summarized in Scheme 6.
OH
O
O
17
O
N
O
P
O
P
O
P
NH
O
O
O
O
O
O
P
O
P
O
P
NH
HO
N
O
O
O
O
O
O
O
O
O
O
H2O
a1
O
O
O
O
O
Nu
a1
O
d1
R1
O
d4TMP
2
0
O
d1
R2
O
N
c
O
O
O
R2
O
O
P
O
P
O
P
NH
NH
O
Nu
O
P
O
P
O
O
O
O
O
O
O
b
O
c
P
O
O
N
O
O
O
O
O
O
O
O
O
d2
Nu
Nu
O
Nu
b
R1
d4TTP
O
OH
O
R1
d4TDP
d2
a2
O
N
O
a2
Nu
O
17
HO
O
O
P
O
P
O
P
NH
O
O
P
O
O
NH
R2
O
O
O
O
O
O
O
O
P
O
P
O
O
O
O
N
O
O
H2O
O
O
O
O

-(AB; ACB)-d4TTPs 8
7b
Scheme 6. Hydrolysis and delivery mechanism of -(AB;ACB)-NTP prodrugs 8.
a. Chemical stability in aqueous phosphate buffer (PBS, pH 7.3).
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In PBS, the stability of TriPPPro-d4TTP prodrugs 8bs–8bw (C2-AB; ACB: C9-C14, Table 1)
increased with increasing alkyl chain lengths. However, chemical stabilities of compounds 8ew–
8ez,fy (C4-AB; ACB: C14-C18) were in the same range as TriPPPro-d4TTP prodrugs 8cv–8dv
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
4
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
(
C3-AB; C12-ACB) (t = 69-77h, Table 1). The half-lives of intermediates 20 were found to be
1/2
significantly higher than those of the prodrugs 8, supposingly caused by repulsive interaction
between the increase in negative charges of the intermediate and the approaching nucleophile.
40
Interestingly, as compared to previously studied -(C17-AB)-d4TTP 7b (t = 583 h), the half-
1
/2
lives for -(C16-ACB)-d4TTP 20y (t > 1600 h) was found to be significantly higher by almost
1
/2
4
0
a factor of 3. In intermediates -(C4-AB)-d4TTP 7be (t1/2 = 270 h) and -(C12-ACB)-d4TTP
2
0v (t = 625 h), the short alkanoyl ester moieties were more rapidly hydrolyzed as compared to
1
/2
the long alkyl carbonate bearing moieties to form d4TTP 4. Moreover, in contrast to compounds
8bv (C2-AB; C12-ACB) and 8ev (n-C4-AB; C12-ACB), the half-lives of compounds 13kv (C2-
ACB; C12-ACB) and 13lv (n-C4-ACB; C12-ACB) with two different alkoxycarbonyloxybenzyl
groups, which were included for comparative reasons, increased due to altered chemical stability
of the two carbonate residues.
Table 1: Half-lives of TriPPPro-d4TTPs 8,13,16 and -ACB-d4TTPs 20 in PBS, PH 7.3.
PBS
PBS
1
3
R2
Comp.
R1
R2
Comp.
R or R
pH=7.3
t [h]
pH=7.3
t [h]
1/2
1
/2
8
ay
by
8ey
fy
8gy
hy
CH3
C H
81
8ev
8ew
8ex
8ez
8is
C H
C H
12 25
87
69
74
74
81
95
16 33
4
9
9
9
9
8
C H
C H
16 33
83
74
77
89
90
C H
4
C H
14 29
2
5
n-C H
C H
16 33
C H
4
C H
15 31
4
9
8
iso-C H
C H
16 33
C H
4
C H
18 37
4
9
C H
C H
16 33
C H
C H
6
13
17
9
19
9
19
8
C H
8
C H
16 33
8jr
C H
11 23
C H
6
13
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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
4
5
5
5
5
5
5
5
5
5
5
6
8
iy
8bs
bt
8bu
bv
bw
C H
C H
85
25
13ss
13kv
13lv
13lz
16v
C H
C H
19
101
84
9
19
16 33
9
19
9
C H
2
C H
C H
2
C H
12 25
5
5
5
5
5
9
19
5
9
9
8
C H
2
C H
10 21
34
C H
4
C H
12 25
107
111
30
C H
2
C H
11 23
52
C H
4
C H
18 37
8
C H
2
C H
12 25
66
C H CN
C H
12 25
2
4
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
8
C H
2
C H
14 29
74
16y
C H CN
C H
16 33
56
2
4
8
cv
n-C H
C H
12 25
75
20v
C H
12 25
625
>1600
3
7
8
dv
iso-C H
C H
12 25
70
20y
C H
16 33
3
7
d4TTP
>500
The hydrolysis experiments of -modified-NTP 8,13,16 and 20 were conducted in aqueous 25
mM phosphate buffer (PBS, pH=7.3). The hydrolysis products were detected by analytical rp18
HPLC.
The cleavage of the AB group or the ACB group was initiated by an ester or a carbonate
hydrolysis and thus proceed similar to the previously published cleavage pathway for TriPPPro-
NTPs 7. However, it can not be excluded that the initial cleavage under chemical conditions
4
0
proceeded at least in part also by a nucleophilic attack at the benzyl position. Nevertheless, in
both cases identical products would be the result.
As an example, the hydrolysis of compound 8by (C2-AB; C16-ACB) is shown in Figure 1. The
chemical hydrolysis was followed over a period of 50 days. Clearly, the starting material
disappeared and the expected -(C16-ACB)-d4TTP 20y was formed. The hydrolysis of
TriPPPro-d4TTP prodrugs 8ay-8iy (AB: C1-C9; C16-ACB) in PBS released intermediates -
(
C16-ACB)-d4TTP 20y and -(AB: C1-C9)-d4TTP 7b, respectively, indicating that both
masking groups of TriPPPro-d4TTP prodrugs 8 were involved in the chemical hydrolysis
1
2
(
pathways a and a , Scheme 6). While -(C2-AB;C16-ACB)-d4TTPs 8by is hydrolyzed mainly
to -(C16-ACB)-d4TTP 20y with some concomitant cleavage to d4TTP occurring as well.
Before complete consumption of the starting material, an increase of d4TTP 4 and -(C16-ACB)-
d4TTP 20y concentrations were observed and a very small amount of -(C2-AB)-d4TTP was
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detected. Because of the very long hydrolysis time periods, also a side-reaction occurred as
proven by the appearance of the nucleobase thymine by the cleavage of the glycosidic bond as
reported before.33,40 However, in the case of 13lv (C4-ACB; C12-ACB), the formation of
intermediate -(C4-ACB)-d4TTP proceeded with nearly the same rate to -(C12-ACB)-d4TTP
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
4
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
2
0v (Figure 4, A). In the case of the prodrug 8jr (C11-AB; C6-ACB), the -(C6-ACB)-d4TTP
0r/-(C11-AB)-d4TTP 7bj ratio was found to be 3:1 (Figure 2, A). The half-lives for -(C6-
2
ACB)-d4TTP 20r (t1/2 = 544h) was found to be higher than -(C11-AB)-d4TTP 7bj (t1/2
=
4
60h),40 which supports that the chemical stability of the carbonate intermediates -(ACB)-
d4TTP 20 were found to be higher than the corresponding ester intermediates -(AB)-d4TTP 7b
(Figure 2, A). Also some d4TDP was formed in this hydrolysis. However, after full conversion
of the starting TriPPPro-compound 8jr, no further increase of the d4TDP concentration was
detected which led to the conclusion that d4TDP was formed directly from the starting
compound. The moment a charge appears at the -phosphate group due to the cleavage of the
first biodegradable masking moiety, the cleavage of the anhydride bond between the - and the
-phosphate is prevented. Additionally, no d4TMP could be detected in these hydrolysis studies.
In addition to the two types of bioreversible triphosphate prodrugs, we studied the two -
cyanoethyl-ACB-d4TTP derivatives 16v,16y as well. Both compounds were hydrolysed
surprisingly fast and faster than most of the ACB-prodrugs. We concluded that these compounds
seem to be cleaved preferentially at the -cyanoethyl-moiety first and in the second step the
ACB-bioreversible groups is hydrolysed. Mechanistically this may happen either by the known
-elimination or a (less probably) nucleophilic attack at the first carbon atom resulting in a
substitution reaction leading in both cases to compounds 20v and 20y.
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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
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
Figure 1: HPLC profile for TriPPPro-d4TTP prodrug 8by after incubation in PBS (pH 7.3).
(
(
A): -(AB-C11;ACB-C6)-d4TTP 8jr in PBS (B): -(AB-C11;ACB-C6)-d4TTP 8jr in
pH 7.3). CEM/0 cell extracts.
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1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
4
4
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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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8
9
0
1
2
3
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8
9
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0
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8
9
0
(
D): -(AB-C2;ACB-C16)-d4TTP 8by in
(
C): -(AB-C2;ACB-C16)-d4TTP 8by in PBS
CEM/0 cell extracts.
(pH 7.3).
Figure 2: Hydrolysis study of 8jr in PBS (pH 7.3) and in CEM/0 cell extracts.
b. Hydrolysis study using Esterase.
1
TriPPPro-d4TTP prodrugs 8 bearing as R a C H or a C H residue in the AB-group in
2
5
4
9
combination with long alkyl chains in the carbonate masking group such as the OC H or the
1
2
25
OC H residue were incubated with PLE in phosphate buffer (pH 7.3) to investigate the impact
1
6
33
of the chain length on the enzymatic cleavage by this esterase and to study the chemoselectivity.
The hydrolysis of TriPPPro-compound 8by (C2-AB;C16-ACB) in PLE is shown in Figure 3,
which was in agreement with the results obtained from the studies of compounds 8by in PBS. As
can be seen in Figure 3, there is a highly selective cleavage of one biodegradable moiety which
led to the formation of intermediate 20y. Intermediate 20y first accumulated and later is cleaved
as well and finally formed d4TTP. Almost no d4TDP and no d4TMP was detected. Furthermore,
TriPPPro-d4TTP prodrug 13kv (C2-ACB; C12-ACB) was also included to study whether the
attached two different carbonate functional group have an effect on the hydrolysis pathway or on
the compound stability. As compared to the chemical hydrolyses, all compounds were rapidly
hydrolyzed and delivered the nucleoside triphosphates d4TTP much faster than in PBS,
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demonstrating a significant contribution of the enzymatic cleavage (Table 2). The half-lives
determined for compounds 8bv (C2-AB; C12-ACB), 8by (C2-AB; C16-ACB) as well as 8ev
(C4-AB; C12-ABC) and 8ey (C4-AB; C16-ACB) increased with increasing of the alkyl
0
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9
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8
9
0
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8
9
0
2
carbonate chain lengths (R ). In contrast to the above study with the OC H -bearing TriPPPro-
1
6
33
1
compounds 8by,8ey, here the slightly longer aliphatic chain (R =C H ) in the AB moiety was
4
9
1
cleaved faster than the short chain acyl group (R =C H ). The cleavage of the acyloxybenzyl
2
5
masking unit in 8ev (C4-AB;C12-ABC) occurred readily under the experimental conditions (t1/2
0.17 h) leading selectively to the formation of -(C12-ACB)-d4TTP 20v. Moreover, for
=
prodrug 8hy (C8-AB;C16-ABC) containing a long alkyl chain, the half-life was found to be 13.8
h. Again, the TriPPPro-NTPs 8 proved to be more stable as compared to the studies of
TriPPPro-compounds 7 described before.40 Remarkably, in the case of the hydrolysis of
compound 13lv (C4-ACB;C12-ABC) as compared to the hydrolysis in PBS, both hydrolysis
intermediates bearing the long aliphatic chain were formed, although in markedly different
amounts as can be seen in Figure 4 (B). In chemical hydrolysis also both possible intermediates
were formed but in almost identical amounts (Figure 4; A).
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0
1
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1
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1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3: HPLC profile for TriPPPro-d4TTP prodrug 8by after incubation in PLE.
(
A): -(ACB-C4;ACB-C12)-d4TTP 13lv in (B): -(ACB-C4;ACB-C12)-d4TTP 13lv in
PBS. PLE.
Figure 4: Hydrolysis study of 13lv in PBS (PH 7.3) and PLE.
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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
4
4
4
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5
5
5
5
5
5
5
5
6
Table 2. Half-lives of TriPPPro-d4TTPs 8 and 13 in the presence of PLE
and CEM/0 cell extracts.
CEM/0
cell extracts
t [h]
CEM/0
Comp.
Comp.
PLE
cell extracts
PLE
t [h]
1/2
t [h]
1/2
t [h]
1/2
1
/2
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
1
.9
.7
.4
4.7
1.6
1.2
2.5
0.17
8
by
ey
8ev
8jr
n.d.a
3
6
8
13.8
0.44
0.94
0.51
0.28
8
8
hy
bv
13kv
13lv
n.d.a
n.d.a
an.d.: not determined.
c. Hydrolysis in cell extracts.
+
The hydrolysis of the prodrugs 8,13 was further investigated in human CD4 T-lymphocyte
CEM cell extracts. Again, the half-lives of the prodrugs correlated well with the chain length and
were found to be significantly lower than the half-lives in PBS. Here, half-lives as low as 0.94 h
(
13kv) to 6.4 h (8hy) were determined which clearly points as in the PLE studies to an enzymatic
cleavage (Table 2). The prodrug 8by (C2-AB; C16-ACB) comprising a short AB-group in the
prodrug moiety was found to be more readily cleaved to form intermediate -(C16-ACB)-d4TTP
2
0y shown in Figure 5. In addition, the amount of d4TDP increased most probably because of
the presence of dephosphorylating enzymes such as phosphatases/kinases, which is in
accordance to our previous results of the DiPPro- or the TriPPPro-compounds 7. There, it was
almost impossible to detect significant concentrations of d4TTP due to its fast dephosphorylation
(
t = 38 min) to form first d4TDP 3 and ultimately d4TMP 2. Interestingly, in the case of the
1/2
hydrolysis of TriPPPro-prodrug 8jr (C11-AB; C6-ABC) as compared to the hydrolysis in PBS,

-(C6-ACB)-d4TTP 20r was also detected as the main monomasked intermediate product
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(
Figure 2, B). After 8 h incubation, the ratio of -(C6-ACB)-d4TTP and -(C11-AB)-d4TTP was
1
0:1. This means that an almost selective cleavage process took place in cell extracts with the
AB-moiety being cleaved first. This was in sharp contrast to the studies performed with
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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3
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3
3
3
3
4
4
4
4
4
4
4
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
TriPPPro-compounds 7.
Figure 5: HPLC profile of compound 8by after incubation in CEM/0 cell extracts at different
times.
d. Antiviral evaluation.
TriPPPro-d4TTP prodrugs 8,13,16 and -alkoxycarbonyloxybenzyl-d4TTPs 20 were evaluated
for their activity to inhibit the HIV replication in HIV-1- and HIV-2-infected wild-type CEM/0
-
cell cultures and in HIV-2-infected mutant thymidine kinase-deficient CEM/TK cell cultures.
Table 3 summarizes the antiviral and cytostatic data of the TriPPPro-d4TTP prodrugs 8,13,16, -
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2
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3
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5
5
6
ACB-d4TTPs 20 and the parent nucleoside analogues d4T 1 as reference compound. As can be
seen, most of the TriPPPro-d4TTP prodrugs showed virtually similar or even slightly better
activities against HIV-1 and HIV-2 than the parent nucleoside d4T 1 in wild-type CEM/O cells.
In addition and more importantly, all TriPPPro-d4TTP prodrugs 8,13 were also highly potent in
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
-
CEM/TK cell cultures whereas d4T 1 lacked any relevant anti-HIV activity in this thymidine
kinase-deficient cell model (EC : 31.05 µM). However, as compared to TriPPPro-d4TTP
50
prodrug 8by (C2-AB; C16-ACB), no increased antiviral activity of prodrug 8iy (C9-AB; C16-
ACB) was observed, although the compound is more lipophilic but maybe also too low in
solubility. D4TTP derivatives 8bs-8by or 8ev-8ez bearing aliphatic carbonate functions in the
ACB-units proved to be antivirally active against HIV-1 and HIV-2 in the same concentration
range as compared to the parent compound d4T 1 in wild-type CEM/0 cell cultures. The three
TriPPPro-compounds comprising either two OC9 chains (13ss), C9-AB and C9-ACB alkyl
group (8is) or a mixture of a short C6-ACB and a long C11-AB alkyl group (8jr) were also
active in TK-deficient CEM cells in contrast to the parent d4T and d4TTP. Interestingly,
TriPPPro-d4TTP prodrugs 8is (C9-AB;C9-ACB) and 8jr (C11-AB;C6-ACB) also showed
marked activity in TK-deficient cell cultures due to sufficient lipophilicity of the compounds
combined with a relatively slow cleavage of the bioreversible moieties which led to the
formation of different long-chain -modified-d4TTPs. The -cyanoethyl-comprising compounds
16 and even more interestingly the monomasked -ACB-d4TTPs 20 were found to be in the
-
same range antivirally active in CEM/TK cell cultures although the latter compound has an
additional charge as compared to all the other prodrugs. Consequently, it seems that even one
long aliphatic chain in the ACB-units provides enough lipophilicity to enable a cellular uptake of
the aliphatic TriPPPro-d4TTP prodrugs. It should also be noticed that none of the TriPPPro-
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d4TTP prodrugs 8,13,16 and -ACB-d4TTPs 20 were endowed with a marked cytotoxicity
compared to the parent d4T 1. Compounds 16v and 20v (CC50b [µM] >100) exhibited even lower
toxicity than d4T 1. However, it should be mentioned that it can not be excluded that the
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
4
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
-
antiviral activity observed in the infected CEM/TK deficient cells is at least in part due to the
formation of d4TMP and d4TDP. As seen in the hydrolysis studies particularly in the cell
extracts, the d4TTP is quickly dephosphorylated to give d4TDP and d4TMP. So, during the
assay the delivered triphosphate as well as the rephosphorylated d4TMP and d4TDP are most
probably responsible for the observed antiviral effect.
Table 3. Antiviral activity and cytotoxicity of TriPPPro-d4TTPs 8,13,16 and -ACB-d4TTPs 20
in comparison with the parent nucleoside d4T 1.
cellular
toxicity
HIV-1 (HE)
HIV-2 (ROD)
CEM/TK-
HIV-2 (ROD)
EC50a [µM]
0.11 ± 0.0071
0.12 ± 0.048
0.24 ± 0.0071
1.76±0.13
selectivity
index (SI)
Comp.
EC50a [µM]
0.027 ± 0.0092
0.032 ± 0.017
0.16 ± 0.085
0.073±0.028
0.061±0.027
0.040 ± 0.029
0.055±0.006
0.11±0.021
0.30±0.18
EC50a [µM]
0.0048 ± 0.0065
0.014 ± 0.015
0.078 ± 0.044
0.040±0.011
0.13±0.072
0.017 ± 0.015
0.025±0.007
0.09±0.011
0.07±0.003
0.09±0.03
CC50b [µM]
34 ± 9.3
21 ± 17
16 ± 1.1
84±23
33±21
27 ± 4.9
58±22
37±2
8
by
8ey
iy
8bs
bv
ev
ez
is
8jr
1259
656
100
1150
541
675
1054
336
137
100
112
74
8
8
0.64±0.12
8
0.073 ± 0.036
0.56±0.13
8
8
0.15±0.02
0.21±0.02
41±9
1
3ss
0.34±0.24
0.23±0.11
34±2
13kv
0.57±0.33
0.24±0.17
1.75±0.25
64±16
54±13
>100
1
3lv
0.73±0.53
0.17±0.014
0.23±0.021
0.30±0.05
1.12±0.21
1
6v
6y
0v
0.47±0.29
2.48±0.46
>212
100
>303
1
0.53±0.26
3.26±0.28
53±22
>100
2
0.33±0.13
0.25±0.06
1.98±1.67
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2
0y
0.50±0.29
0.43±0.23
0.29±0.06
1.46±1.34
31.05±5.25
61±36
>50
122
d4T
0.31±0.13
>116
+
[
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
50%; values are the mean ±SD of n=2-3 independent experiments.
+
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
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The cleavage product in all cases is 4-hydroxybenzyl alcohol which is known to be mainly and
quickly oxidized to 4-hydroxybenzoic acid which is a non-toxic compound. Previously, this
compound has been added to the antiviral assays and CC50 values >100 M were observed.
However, from the report, when applying the water-soluble tetrazolium (WST-1) assay to
eEND2 cells incubated with a range of concentrations of 4-hydroxybenzyl alcohol, a loss in cell
viability was found with a LD 50 of ~50 mM. In line with these findings, cytotoxicity of 4-
hydroxybenzyl alcohol progressively increased between 1 and 100 mM, as indicated by an
enhanced lactate dehydrogenase (LDH) release from damaged eEND2 cells. However, these are
concentrations that are about a 1000-fold higher than the concentration that we generate with our
compounds.48
CONCLUSIONS
In summary, TriPPPro-NTPs 8 and 13 of the nucleoside analogue d4T 1 bearing two different
biodegradable masking units attached to the -phosphate group of the corresponding nucleoside
triphosphate are disclosed here. The TriPPPro-d4TTP prodrugs 8,13 were synthesized using the
H-phosphonate approach described previously with modest to high yields (up to 78%). Most of
the enzyme-cleavable TriPPPro-d4TTPs 8,13 were as active as or even more active against HIV-
1 and HIV-2 in wild-type CEM/0 cell cultures. Moreover, also high activities were obtained
depending on the lipophilicity of the TriPPPro-d4TTP prodrugs 8,13 against HIV-2 in mutant
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-
CEM/ TK cell cultures. Interestingly, as compared to the symmetric ester bearing AB groups
TriPPPro-NTPs 7 with two identical acyloxybenzyl masks reported previously,40,41 we have
proven that the AB-prodrug group was more readily cleaved to give -(C H2n+1-ACB)-nucleoside
n
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
4
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
triphosphates 20 by chemical hydrolysis (slow process) and in particular by cell extract enzymes
(
fast process). The extent of differentiation was dependent on the medium. It is lower in PBS but
very pronounced in PLE.
-
The -alkoxycarbonyloxybenzyl (ACB)-d4TTPs 20 were also highly potent in CEM/TK cell
cultures. This confirmed that these compounds were taken-up by the cells and delivered
intracellularly a phosphorylated form of d4T, most likely d4T triphosphate or it acted as such.
Thus, obviously the modification at the -phosphate group by one lipophilic, biodegradable
moiety and the 4-ACB-group gave the molecules sufficient lipophilicity to cross the biological
barriers. We disclosed the non-symmetric TriPPPro-concept in which the -phosphate of NTPs
is bioreversibly modified to deliver d4TTP with high selectivity by an enzyme-triggered
mechanism which enabled the bypass of all steps of the intracellular phosphorylation. This
concept is warrant to be applied to nucleoside analogues that show severe limitations in their
activation to give the corresponding nucleoside triphosphates, e.g. nucleoside analogs such as
AZT or FTC. We are convinced that the TriPPPro-strategy offers high potential in antiviral and
antitumoral chemotherapies. Highly active TriPPPro-prodrugs may be used in the future as
commercial drugs. However, many development steps still have to be achieved, e.g. toxicity
assay, PK studies, testing phases and the development of reaction routes that allow production of
industrial size quantities of the compounds.
EXPERIMENTAL SECTION
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6
General: All experiments involving water-sensitive compounds were carried out under anhydride
conditions and nitrogen atmosphere. Solvents and Reagents: Acetonitrile, pyridine and THF were
purchased from Acros Organics (Extra Dry over molecular sieves) and dried with activated
molecular sieves. Triethylamine (Et N) was refluxed over CaH for three days and distilled under
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
2
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8
9
0
1
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3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
nitrogen. Trifluoroacetic anhydride (TFAA) was dried over phosphorus pentoxide for one hour
and distilled under nitrogen. 1-Methylimidazole was dried over sodium and distilled under
nitrogen. All further reagents commercially available were used as received. Thin layer
chromatography (TLC): For thin layer chromatography Macherey-Nagel pre-coated TLC sheets
®
Alugram Xtra SIL G/UV254 were used. Column chromatography: Normal phase column
chromatography were performed with Macherey-Nagel silica gel 60 M (0.04-0.063 mm).
Automatic RP-18 chromatography: For reversed phase chromatography an Interchim Puriflash
®
4
30 in combination with Chromabond Flash RS40 C ec was used. High Performance Liquid
1
8
Chromatography (HPLC): HPLC was required for analytical studies and monitoring reactions. A
VWR-Hitachi LaChromElite HPLC system (L-2130, L-2200, L-2455), EzChromElite software
and equipped with a Nucleodur 100-5 C ec or Nucleodur 100-5 C ec (Macherey-Nagel) was
1
8
8
available. Acetonitrile for HPLC was obtained from VWR (HPLC grade) and ultrapure water
was produced by a Sartorius Aurium® pro (Sartopore 0.2 µm, UV). 2 mM tetra-n-
Butylammonium acetate solution (TBAA, pH 6.3) or 10 mM triethylammonium acetate (TEAA,
pH 6.2) were used for buffering. Method: Nucleodur 100-5 C18ec; 0-20 min: TBAA
buffer/acetonitrile gradient (5-80%); 20-30 min: buffer/acetonitrile (80%); 30-33 min:
buffer/acetonitrile (80-5%); 33-38 min: buffer/acetonitrile (5%); flow: 1 mL/min. Compound
purity: All final compounds were isolated analytically pure, ≥95% purity by HPLC and NMR
spectroscopy.
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Nuclear Magnetic Resonance (NMR): NMR spectra were recorded at room temperature in
automation mode with a Varian Gemini 2000BB, Bruker Fourier 300, Bruker AMX 400, Bruker
1
13
DRX 500 or Bruker AVIII 600. All H- and C-NMR chemical shifts (δ) were quoted in parts
per million (ppm) downfield from tetramethylsilane (TMS) and calibrated on solvent signal. The
31P-NMR chemical shifts (proton decoupled) are also quoted in ppm using phosphoric acid as the
external standard. Mass Spectrometry (MS): HRMS (ESI) mass spectra were acquired with a VG
Analytical Finnigan ThermoQuest MAT 95 XL or an Agilent 6224 EIS-TOF spectrometer.
MALDI measurements (matrix: 9-aminoacridine [9-AA] or 2,5-dihydroxybenzoic acid [DHB])
were performed with a Bruker UltrafleXtreme spectrometer. Infrared spectroscopy (IR): IR
spectra were recorded on a Bruker Alpha P FT-IR at room temperature in the range of 400-4000
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
4
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
-1
cm .
Syntheses and characterization
The syntheses and characterization of 4-(hydroxymethyl)phenylalkanoates 9 were described
previously.29,32 The synthesis of (n-Bu N) ▪d4TMP 2 was performed using the Sowa-Ouichi
4
2
procedure starting from d4T 1.44
General Procedure 1: Preparation of 4-(hydroxymethyl)phenylalkylcarbonate 10.
a) The reactions carried out under nitrogen (N ) atmosphere under dry conditions. A mixture of
2
triphosgene (1.0 equiv.), K CO (2.0 equiv.) and DMF (0.72 equiv.) as a catalyst in toluene
2
3
stirred for 30 min and cooled to 0 °C. A solution of alkyl alcohol C H2n+1OH in toluene was
n
added dropwise to the mixture (n>10, added dropwise to the mixture at room temperature in case
of solidification). The mixture was warmed to room temperature and stirred for 12 h. The solvent
was removed in vaccum and the residue was purified using column chromatography (petroleum
ether/ethyl acetate 97:3 v/v) to give alkyl chloroformates. b) 4-Hydroxybenzyl alcohol 17 and
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trimethylamine (TEA) in DCM or THF were cooled to 0 °C. The corresponding alkyl
chloroformate in DCM or THF was added dropwise to the mixture and stirred overnight. The
solvent was removed in vaccum and the residue was washed once with saturated sodium
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bicarbonate solution and twice with water. The organic layer was dried with MgSO and the
4
solvent was removed in vaccum. The crude material was purified using column chromatography
to give compound 4-(hydroxymethyl)phenylalkylcarbonate 10.
General Procedure 2: Preparation of H-phosphonate 11
General Procedure b1: Under dry conditions, diphenyl H-phosphonate (DPP, 1.0 equiv.) was
dissolved pyridine and cooled to 0 °C. 4-(Hydroxymethyl)phenylalkanoate 9 (1.05 equiv.) was
added and stirred at 0 °C for 1h and then stirred at room temperature (rt) for 1h. Following, 4-
(hydroxymethyl)phenylalkyl carbonate 10 (1.0 equiv.) was added and the mixture was stirred for
1
2 h. Then the solvent was removed in vacuum. The residue was purified by flash column
chromatography (silica) with EtOAc/petroleum ether/0.5% acetic acid as eluent.
General Procedure b2: Under dry conditions, DPP (1.0 equiv.) was dissolved in pyridine and
cooled to 0 °C. 4-(Hydroxymethyl)phenylalkylcarbonate 10 (1.0 equiv.) was added and stirred at
0
°C for 1 h and then stirred at room temperature (rt) for 1 h. Following, 4-
(hydroxymethyl)phenylalkanoate 9 (1.05 equiv.) was added and the mixture was stirred for 12 h.
Then the solvent was removed in vacuum. The residue was purified by column chromatography
with EtOAc/petroleum ether/0.5% acetic acid as eluent.
General Procedure 3: Preparation of -(AB;ACB)-d4TTPs 8 and -(ACB;ACB)-d4TTPs 13
The reactions were performed in a nitrogen (N ) atmosphere and dry conditions. a) H-
2
phosphonate (1.0 equiv.) was dissolved in 3 mL CH CN and N-chlorosuccinimide (NCS, 2.0
3
equiv.) was added. After stirring for 2 h at room temperature, tetrabutylammonium phosphate
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solution (0.4 M in acetonitrile) (3.0 equiv.) was added quickly. The mixture was stirred for 1 h
and the solvent was removed in vacuum. The residue was extracted with CH Cl /H O. The
2
2
2
organic phase was dried over sodium sulfate and the solvent was removed by evaporation to
afford pyrophosphate in almost quantitative yield. b) The corresponding pyrophosphate was
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dissolved in 3 mL CH CN and cooled down to 0 °C. A mixture of trifluoroacetic anhydride
3
(
TFAA, 5.0 equiv.) and Et N (8.0 equiv.) in 3 mL CH CN was cooled to 0 °C and added to the
3 3
mixture. After stirring for 10 min, all volatile components were removed in vacuum. The residue
was subsequently dissolved in 3 mL CH CN at 0 °C. 1-Methylimidazole (3.0 equiv.) and Et N
3
3
(TEA, 5.0 equiv.) was added. The mixture was warmed to room temperature and stirred for 10
min. The resulting activated imidazolidate formed and d4TMP (0.6-0.85 equiv.) in 4 mL CH CN
3
was added. The reaction was stirred at rt for 2-5 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
were obtained as white solids.
Synthesis of 4-(hydroxymethyl)phenylalkylcarbonate 10.
Butyl (4-(hydroxymethyl)phenyl) carbonate 10l
According to general procedure 1 with 4.47 g 4-hydroxybenzyl alcohol 17 (36 mmol, 1.0 equiv.)
and 5.0 mL triethylamine (36 mmol, 1.0 equiv.) in 40 mL THF at 0 °C and dropwise addition of
4
.6 mL butyl chloroformate (36 mmol, 1.0 equiv.) in 10 mL THF. Reaction time was 12 h at
room temperature. Column chromatography (petroleum ether/ethyl acetate 7:3 v/v). Yield: 5.83 g
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(
26 mmol, 72%) colourless oil. H-NMR (400 MHz, CDCl ):  [ppm] = 7.36-7.32 (m, 2H, H-c),
3
3
7
.16-7.12 (m, 2H, H-d), 4.63 (s, 2H, H-a), 4.24 (t, J = 6.7 Hz, 2H, H-g), 2.05 (s, 1H, OH),
HH
3
13
1.76-1.68 (m, 2H, H-h), 1.50-1.40 (m, 2H, H-i), 0.97 (t, J = 7.3 Hz, 3H, H-j). C-NMR (101
HH
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3
4
5
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7
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9
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2
3
4
5
6
7
8
9
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MHz, CDCl ):  [ppm] = 153.7 (C-f), 150.3 (C-e), 138.7 (C-b), 127.9 (C-c), 121.0 (C-d), 68.6
3
+
(
C-g), 64.8 (C-a), 30.4 (C-h), 18.8 (C-i), 13.5 (C-j). HRMS (ESI , m/z): calculated for C H O ,
12 16
4
+
-1
[
M+Na] 247.0941; found 247.0901. IR: ν [cm ] = 3380, 2961, 2934, 2874, 1756, 1607, 1508,
460, 1391, 1247, 1207, 1118, 1103, 960, 924, 868, 820, 778, 740, 602, 507, 435.
1
Hexyl (4-(hydroxymethyl)phenyl) carbonate 10r
According to general procedure 1 with 1.48 g triphosgene (5.0 mmol, 1.0 equiv.), 1.38 g K CO
2
3
(
10.0 mmol, 2.0 equiv.), 0.29 mL DMF (3.6 mmol, 0.72 equiv.) in 10 mL toluene at 0 °C and
dropwise addition of 1.02 g 1-Hexanol (10.0 mmol, 2.0 equiv) in 10 mL toluene. Yield: 1.24 g
8.3 mmol, 83%) colorless oil. b) 1.13 g 4-Hydroxybenzyl alcohol 17 (9.1 mmol, 1.1 equiv.) and
.15 mL TEA (8.3 mmol 1.0 equiv.) in 10 mL DCM were cooled to 0 °C followed by a dropwise
(
1
addition of hexyl chloroformate (8.3 mmol, 1.0 equiv.) in 10 mL DCM. Column chromatography
1
(
petroleum ether/ethyl acetate 8:2 v/v). Yield: 1.60 g (6.4 mmol, 77%) white solid. H-NMR
(
400 MHz, CDCl ):  [ppm] = 7.34-7.29 (m, 2H, H-c), 7.15-7.10 (m, 2H, H-d), 4.59 (s, 2H, H-a),
3
3
3
4.23 (t, J = 6.7 Hz, 2H, H-g), 2.55 (s, 1H, OH), 1.73 (quint, J = 6.8 Hz, 2H, H-h), 1.46-1.37
HH
HH
3
13
(
m, 2H, H-i), 1.36-1.20 (m, 4H, H-j, H-k), 0.90 (t, J = 6.7 Hz, 3H, H-l). C-NMR (101 MHz,
HH
CDCl ):  [ppm] = 153.8 (C-f), 150.3 (C-e), 138.6 (C-b), 127.9 (C-c), 121.0 (C-d), 69.0 (C-g),
3
+
64.3 (C-a), 28.4 (C-h), 25.2 (C-i), 31.2, 22.4 (C-j, C-k), 13.9 (C-l). HRMS (ESI , m/z):
+
-1
calculated for C H O , [M+Na] 275.1254; found 275.1234. IR: ν [cm ] = 3387, 2957, 2929,
1
4
20
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871, 2157, 1757, 1607, 1508, 1466, 1392, 1247, 1209, 1046, 1014, 920, 848, 779, 603, 504,
04.
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4
-(Hydroxymethyl)phenyl nonyl carbonate 10s
According to general procedure 1 with 2.97 g triphosgene (10.0 mmol, 1.0 equiv), 2.76 g K CO
2
3
(
20.0 mmol, 2.0 equiv), 0.58 mL DMF (7.2 mmol, 0.72 equiv) in 20 mL toluene at 0 °C and
dropwise addition of 3.5 mL 1-nonanol (20.0 mmol, 2.0 equiv) in 20 mL toluene. Yield: 1.10 g
5.3 mmol, 27%) colorless oil. b) 0.73 g 4-Hydroxybenzyl alcohol 17 (5.9 mmol, 1.1 equiv.) and
.74 mL TEA (5.3 mmol 1.0 equiv.) in 10 mL DCM were cooled to 0 °C followed by a dropwise
(
0
addition of nonyl chloroformate (5.3 mmol, 1.0 equiv.) in 10 mL DCM. Column chromatography
1
(petroleum ether/ethyl acetate 8:2 v/v). Yield: 1.10 g (10.5 mmol, 70%) white solid. H-NMR
(500 MHz, CDCl ):  [ppm] = 7.31-7.27 (m, 2H, H-c), 7.13-7.08 (m, 2H, H-d), 4.54 (s, 2H, H-a),
3
3
3
4.21 (t, J = 6.7 Hz, 2H, H-g), 2.85(s, 1H, OH), 1.72 (quint, J = 7.4 Hz, 2H, H-h), 1.45-1.37
HH
HH
3
13
(m, 2H, H-i), 1.36-1.20 (m, 10H, H-j, H-k, H-l, H-m, H-n), 0.88 (t, J = 6.90 Hz, 3H, H-o). C-
HH
NMR (126 MHz, CDCl ):  [ppm] = 153.8 (C-f), 150.3 (C-e), 138.7 (C-b), 127.9 (C-c), 121.0
3
(C-d), 69.0 (C-g), 64.3 (C-a), 28.5 (C-h), 25.6 (C-i), 31.7, 29.3, 29.1, 22.6 (C-j, C-k, C-l, C-m, C-
+
+
n), 14.0 (C-o). HRMS (ESI , m/z): calculated for C H O , [M+Na] 317.1723; found 317.1701.
1
7
26
4
-1
IR: ν [cm ] = 3311, 2954, 2922, 2855, 2778, 1750, 1508, 1469, 1456, 1420, 1239, 1211, 1036,
1012, 955, 820, 782, 746, 722, 519, 496, 463.
Decyl (4-(hydroxymethyl)phenyl) carbonate 10t
According to general procedure 1 with 4.45 g triphosgene (15.0 mmol, 1.0 equiv.), 4.15 g
K CO (30.0 mmol, 2.0 equiv.), 0.87 mL DMF (10.8 mmol, 0.72 equiv.) in 45 mL toluene at 0
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