Second-generation cycloSal-d4TMP pronucleotides bearing esterase-cleavable sites - The "trapping" concept

Article abstract of DOI:10.1002/ejoc.200500490

An extension of the cycloSal-pronucleotide approach is presented. Attachment of an enzyme-cleavable ester/acylal group to the cycloSal-d4TMP triesters should allow these compounds to be trapped intracellularly after cleavage. The ester/acylal groups were introduced in the 3- or 5-position of the cycloSal ring system, and surprising differences were observed in hydrolysis studies in CEM cell extracts with respect to the ester/acylal moiety. While acetyl and levulinyl esters were readily cleaved, alkyl esters of cycloSal-d4TMP acids proved to be resistant to enzymatic cleavage. In contrast, AM-, POM- and POC-acylals were rapidly cleaved in the extracts, leading to cycloSal-d4TMP acids. The antiviral activity of the compounds against HIV is also presented. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejoc.200500490

FULL PAPER
DOI: 10.1002/ejoc.200500490
Second-Generation cycloSal-d4TMP Pronucleotides Bearing Esterase-
Cleavable Sites − The “Trapping” Concept
Chris Meier,*[a] Christian Ducho,[a] Henning Jessen,[a] Dalibor Vukadinovic´-Tenter,[a] and
Jan Balzarini[b]
Antiviral agents / Bioorganic chemistry / Nucleosides / Phosphates / Pronucleotides
Keywords:
An extension of the cycloSal-pronucleotide approach is pre- esters were readily cleaved, alkyl esters of cycloSal-d4TMP
sented. Attachment of an enzyme-cleavable ester/acylal
group to the cycloSal-d4TMP triesters should allow these
compounds to be trapped intracellularly after cleavage. The
ester/acylal groups were introduced in the 3- or 5-position
of the cycloSal ring system, and surprising differences were
acids proved to be resistant to enzymatic cleavage. In con-
trast, AM-, POM- and POC-acylals were rapidly cleaved in
the extracts, leading to cycloSal-d4TMP acids. The antiviral
activity of the compounds against HIV is also presented.
observed in hydrolysis studies in CEM cell extracts with re- (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
spect to the ester/acylal moiety. While acetyl and levulinyl
Germany, 2006)
Introduction
the pronucleotide, which leads to high concentrations of the
nucleotide. Thus, in order to trap the lipophilic cycloSal-
triester, the attachment of a (carboxy)esterase-cleavable es-
ter site in the cycloSal aromatic ring should lead to interest-
ing new properties. However, ester groups are electron-with-
drawing substituents and would lead to a considerable re-
duction of the chemical stability of the triesters. To avoid
this, an alkyl spacer/isolator has to be introduced. An enzy-
matic cleavage reaction releases a highly polar (carboxylic
acid) or at least a more polar cycloSal derivative (alcohol;
Y-prodrug, Figure 1, step c).[12] The nucleotide is finally re-
leased from the polar Y-prodrug by a chemical mechanism
(identical to step a).
This concept is based on the higher intracellular concen-
trations of esterases compared to the extracellular medium.
This use of an “esterase strategy” is well known in prodrug
development. Two drugs that are based on this strategy
have recently been approved by the FDA as antivirals:
bis{pivaloxymethyl (POM)}-PMEA (2002, Adefovir dipi-
voxyl, Hepsera®) and bis{isopropyloxycarbonylmethoxy
(POC)}-PMPA (2001, Tenofovir disoproxil fumarate, Vi-
read®).[13]
The antiviral action of nucleoside analogues like 2Ј,3Ј-
dideoxy-2Ј,3Ј-didehydrothymidine (d4T, 1) depends on their
conversion into the ultimately bioactive triphosphates via
the mono- and diphosphates (nucleotides) by cellular kin-
ases. However, the first phosphorylation step to the mono-
phosphates catalysed by the salvage-pathway enzyme thymi-
dine kinase (TK) is often the metabolism-limiting step.[1,2]
The use of nucleotides as therapeutic agents is impossible
due to their high polarity and their catabolism in the blood
by unspecific nucleotidases. In contrast, lipophilic nucleo-
tide-releasing systems (pronucleotides) could circumvent
these limitations (TK bypass).[3] We have developed
cycloSal-pronucleotides as a new class of chemically cleav-
able nucleotide prodrugs of biologically active nucleoside
analogues.[4] These lipophilic precursors
2 (cycloSal-
d4TMP; Scheme 1) deliver d4TMP inside cells and achieve
an efficient thymidine-kinase bypass.[5] cycloSal-Phosphate
triesters 2 deliver the nucleotides by a chemically triggered
cascade reaction (Figure 1, step a). This approach has been
applied successfully to various nucleoside analogues.[6–11]
Due to the lipophilic character of cycloSal-triesters like 2
and the chemically triggered delivery mechanism, a concen-
tration equilibrium is formed through the membrane (X-
prodrug, Figure 1, step b). For effective antiviral efficiency
it is necessary to have high intracellular concentrations of
Two types of ester-bearing cycloSal-triesters were envis-
aged, namely cyclosal-d4TMP acid esters 3 and 4, which
release the cycloSal-d4TMP acids 5a,b, or cycloSal-d4TMP
alcohol esters 6, which liberate the cycloSal-d4TMP alcohol
7 after enzymatic cleavage (Scheme 1). In both cases, a more
polar cycloSal derivative than the precursor is formed that
should lead to intracellular accumulation (Y-prodrug, Fig-
ure 1; “lock-in” or “trapping” concept).
[a] Institute of Organic Chemistry, University of Hamburg,
Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
Fax: +49-40-42838-2495
E-mail: chris.meier@chemie.uni-hamburg.de
[b] Rega Institute for Medical Research, K. U. Leuven,
Minderbroedersstraat 10, 3000 Leuven, Belgium
Here, the synthesis, properties and the antiviral evalu-
ation of these new potentially enzyme-cleavable cycloSal-
d4TMP triesters are reported.
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FULL PAPER
group of ethyl acetate or ethyl levulinate in the presence of
SiO2·NaHSO4 as catalyst in 95% and 82% yield, respec-
tively.[16] The pivaloyl ester (9f) of 12 was synthesised from
an activated pivaloyl donor by the “twisted amide” method
reported previously (80% yield).[17]
Phenols 9a–f were then transformed into the salicyl
alcohols 8a–f by treatment with paraformaldehyde and
phenylboronic acid in the presence of 0.5 equiv. of propi-
onic acid (50–85% yield).[4] Diols 8 were then treated with
PCl3 to yield cyclic chlorophosphanes 13, which were used
subsequently for the phosphitylation with d4T 1.[18] The in-
termediately formed phosphite triesters were oxidized in a
one-pot reaction with tert-butyl hydroperoxide (tBuOOH)
to give triesters 3 and 6 (n = 2) in 30–60% yield (not op-
timised).[4] Interestingly, the yields here were found to be
lower than reported previously for some unknown reasons.
However, it was noticed that the yields depend markedly on
the quality of the tBuOOH. Finally, cycloSal-d4TMP acid
5a was prepared by cleavage of the tert-butyl ester in 3c
with TFA (87% yield), and cycloSal-d4TMP alcohol 7 was
obtained after delevulinylation of 6c with hydrazine hydrate
(25% yield).
Beside cycloSal triesters carrying a C2 linker between the
cycloSal moiety and the ester group, a C3 linker was intro-
duced in order to prove the efficiency of isolating the ester
group from the aromatic ring (cycloSal-d4TMP ester 3d, n
= 3). However, the starting material needed for this synthe-
sis is not commercially available. Thus, a Suzuki cross-coup-
ling procedure was used for the synthesis of the required
salicyl alcohol 13. The required precursors for the Suzuki
coupling are isopropylidene acetal 14 and trialkylborane
15.[19] The former compound was prepared starting from 2-
bromophenol (16) by selective ortho-hydroxymethylation to
yield 17 and subsequent acetalisation. The latter compound
15 is the hydroboration product of methyl butenoate 18 and
9-BBN. Trialkylborane 15 was immediately coupled with
the acetal 14 to give the 3-alkylated acetal 19 in 83% yield.
Cleavage of the acetal with catalytic amounts of HCl re-
sulted in the formation of salicyl alcohol 13 (70% yield),
which was converted into the cycloSal triester 3d by the
standard procedure (Scheme 3). The advantage of this Su-
zuki cross-coupling protocol is that it can be used as a gene-
ral procedure for the synthesis of cycloSal nucleotides with
variable linker lengths.
Scheme 1. Target cycloSal pronucleotides 3–7 of d4T 1.
Results and Discussion
Chemistry
In order to be able to use our previous protocol[4] for the
In addition to the compounds carrying the function-
synthesis of triesters 3–7, the required salicyl alcohols 8 alised alkyl residue in the 3-position, cycloSal-d4TMP esters
were prepared from the corresponding phenols 9. All syn- 4a–c with the alkyl group attached at the 5-position of the
thetic steps are depicted in Schemes 2, 3 and 4. While the cycloSal-moiety were prepared starting from 5-propionyl-
methyl ester 9a was prepared by transesterification of dihy- cycloSal-d4TMP acid (5b). Carboxylic acid 5b was prepared
drocoumarin (10) with methanol in the presence of H2SO4 starting from 3-(4-hydroxyphenyl)propionic acid (20) by the
in 96% yield, the benzyl ester 9b was synthesised by alky- same synthetic route as described for 5a (Scheme 4).
lation of 3-(2-hydroxyphenyl)propionic acid (11) with ben- cycloSal-d4TMP acid 5b was used in an alkylation reaction
zyl bromide in the presence of DBU in toluene (97% with bromomethyl acetate in the presence of DIPEA in
yield).[14] The tert-butyl ester 9c was obtained by esterifica- CH3CN at 10 °C for 3 h to yield acetoxymethyl (AM) acylal
tion of carboxylic acid 11 in the presence of DMF/dineo- 4a in 65% yield.[20] A general problem of such alkylation
pentylacetal in toluene (85% yield).[15] The acetyl (9d) and reactions is the concomitant alkylation of the nucleobase.
levulinyl esters (9e) of 2-(2-hydroxyphenyl)ethanol (12) were To avoid this, the reaction temperature was kept as low as
prepared by a transesterification of the acetyl or levulinyl possible. Although the yield is only 65%, no base-alkylated
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Second-Generation cycloSal-d4TMP Pronucleotides Bearing Esterase-Cleavable Sites
FULL PAPER
Figure 1. Trapping concept for the cycloSal-pronucleotides and the chemically induced cleavage of the triesters.
Scheme 2. Synthesis of the corresponding salicyl alcohols and the target triesters 3, 5, 6 and 7. Method A: methanol, H2SO4, reflux, 5–
8 h; method B: benzyl bromide, DBU, toluene, reflux, 7 h; method C: (CH3)2NCH(OCH2tBu)2, tert-butyl alcohol, toluene, reflux, 5 h;
method D: ethyl acetate or ethyl levulinate, n-hexane, SiO2·NaHSO4, 67 °C, 6–18 h; method E: 3-pivaloyl-1,3-thiazolidine-2-thione, tolu-
ene, 65 °C, 48 h; method F: i) phenylboronic acid, propionic acid (cat.), p-formaldehyde, toluene, reflux, 6–8 h; ii) H2O2, THF, 0 °C,
30 min; method G: PCl3, pyridine, Et2O, 0–21 °C, 1 h; method H: i) d4T 1, CH3CN, DIPEA, 0–20 °C; ii) tBuOOH, CH3CN, room temp.,
30 min; method I: NH2NH2·H2O, pyridine/HOAc (3:2), pyridine, 0 °C, 10 min; method J: TFA (10 equiv.), CH2Cl2, room temp., 1 h.
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The isopropyloxycarbonylmethoxy (POC) derivative 4c was
prepared with the corresponding chloromethyl alkylation
reagent. This reagent was easily synthesised from chlo-
romethyl formate and 2-propanol. However, chloromethyl
isopropyl carbonate is even less reactive than chloromethyl
pivalate. Thus, at ambient temperature nearly no reaction
took place even after 16 d. However, heating of the reaction
mixture in CH3CN to 50 °C for 7 d gave the target triester
4c in 47% yield (Scheme 4).
1
All triesters 3–7 were characterised by means of H, 13C
and 31P NMR spectroscopy as well as high-resolution mass
spectrometry. Experimental details are given here and in a
previous report.[12]
Chemical Stability Studies
Scheme 3. Synthesis of the ester-functionalised cycloSal-d4TMP
triester 3d with a C3 linker. Method A: i) phenylboronic acid, propi-
onic acid, (CH2O)n, toluene, reflux, 18 h; ii) H2O2, THF, 0 °C,
45 min; method B: 2,2-dimethoxypropane, pTsOH, acetone,
Na2SO4; method C: 9-BBN, THF, 0 °C Ǟ 55 °C, 16 h; method D:
K2CO3, [Pd(dppf)Cl2], DMF, 55 °C, 4 d; method E: cat. HCl,
CH3CN /H2O, 5 min; method F: i) PCl3, pyridine, Et2O, –20 °C to
room temp., 2 h; ii) 1, DIPEA, CH3CN, –20 °C to room temp., 1 h;
iii) tBuOOH, CH3CN, –20 °C to room temp., 1 h.
Triesters 3–7 were studied for their stability in aqueous
25 mm phosphate buffer (pH = 7.3). The half-lives are sum-
marised in Table 1. As expected, the hydrolysis of the cy-
cloSal triester was observed in these studies without ad-
ditional hydrolysis of the carboxylic acid esters. Thus, the
half-lives given in Table 1 refer to the cleavage of the tri-
esters and formation of d4TMP; cycloSal-d4TMP triesters
3a–c and 5a,b have half-lives of between 7.3 and 13.5 h.
product was isolated from the above reaction. In contrast From these values, and comparison with the half-lives of
to bromomethyl acetate, bromomethyl pivalate is not com- two cycloSal triesters studied before [unsubstituted triester
mercially available. Therefore, attempts were made to con- 2a (t1/2 = 4.4 h) and 3-methyl-cycloSal-d4TMP 2b (t1/2
=
vert the less reactive chloromethyl pivalate into the iodome- 17.5 h), Table 1],[6] it was concluded that the ethylene spacer
thyl pivalate by a Finkelstein reaction. Chloromethyl pival- length is not entirely sufficient to separate the electron-with-
ate was dissolved in acetone in the presence of NaI. After drawing ester moiety from the aromatic ring. However, tri-
3 h, NaCl had formed and iodomethyl pivalate was used ester 3d, which has a propylene linker, is more stable (t1/2
=
immediately for the alkylation. In a second attempt, the al- 17.6 h), and its stability is similar to that of 3-methyl-cy-
kylation reagent was formed in the presence of the acid 5b, cloSal-ester 2b.
DIPEA and NaI in CH3CN. However, in both cases a pre-
The chemical stability of cycloSal-d4TMP acids 5 was
cipitation of the sodium salt of acid 5b was observed. Con- surprisingly 1.8- to 3.0-fold higher than those of esters 3
sequently, an overalkylation of the remaining acid in solu- and 4. This may be due to the overall negative charge of the
tion was performed leading, again, to complex and insepa- deprotonated acid 5 at pH = 7.3, which avoids an efficient
rable mixtures. Finally, pivaloxymethyl (POM) triester 4b nucleophilic attack at the phosphorus atom. In contrast,
was prepared from the less reactive chloromethyl pivalate cycloSal-d4TMP alcohol 7 shows a similar chemical
in CH3CN at ambient temperature by leaving the reaction stability to the esters. In all cases the products were
to proceed for 9 d. Triester 4b was isolated in 46% yield. d4TMP and the corresponding diol. This was proven by
Scheme 4. Synthesis of enzyme-cleavable 5-functionalised cycloSal-d4TMP triesters 4. Method A: (CH3)2NCH(OCH2tBu)2, tert-butyl
alcohol, toluene, reflux, 5 h; method B: i) phenylboronic acid, propionic acid (cat.), p-formaldehyde, toluene, reflux, 6–8 h; ii) H2O2, THF,
0 °C, 30 min; method C: PCl3, pyridine, Et2O, 0–21 °C, 1 h; method D: i) d4T 1, CH3CN, DIPEA, 0–20 °C; ii) tBuOOH, CH3CN, room
temp., 30 min; method F: TFA (10 equiv.), CH2Cl2, room temp., 1 h; method G: bromomethyl acetate, DIPEA, CH3CN, 10 °C, 3 h;
method H: chloromethyl acetate, DIPEA, CH3CN, room temp., 9 d; method I: chloromethyl isopropyl carbonate, DIPEA, CH3CN,
50 °C, 7 d.
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Second-Generation cycloSal-d4TMP Pronucleotides Bearing Esterase-Cleavable Sites
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Table 1. Lipophilicity, hydrolysis data and antiviral activity of the triesters 3–7 compared to the prototypes 2 and d4T 1.
[a]
X or Y
n
logPcalcd.
t1/2 [h][b]
EC50 [μm][c]
CC50 [μm][d]
PBS[e]
CE[f]
CEM/O[g]
CEM/TK–[h]
HIV-2
HIV-1
HIV-2
3a
3b
3c
3d
4a
4b
4c
5a
5b
6a
6b
6c
7
COOMe
COOBn
COOtBu
COOMe
AM-Pr
POM-Pr
POC-Pr
COOH
COOH
OAc
OPiv
OLev
OH
H
2
2
2
3
–
–
–
2
2
2
2
2
2
–
–
–
–
0.46
2.19
1.65
0.79
2.18
4.04
3.56
–3.96
–3.96
0.42
1.65
0.37
-0.53
0.28
0.77
0.77
–0.48
7.3
9.1
13.5
17.6
4.3
5.6
3.9
22.9
12.5
13.6
13.1
12.5
12.6
4.4
7.2
6.0
9.0
0.09 0.05
0.15 0.09
0.33 0.11
0.35 0.09
0.20 0.11
0.23 0.04
0.26 0.20
0.19 0.08
0.14 0.06
0.16 0.09
0.16 0.09
0.13 0.04
0.24 0.01
0.10 0.02
0.073 0.05
0.46 0.24
0.23 0.04
0.25 0.15
1.00 0.28
0.50 0.14
0.47 0.12
0.53 0.39
0.33 0.11
0.60 0.00
1.4 0.60
0.40 0.42
2.89 1.96
1.14 0.96
1.35 0.92
25 19.1
0.7 0.08
1.73 1.97
20.0 0.00
50 30
0.15 0.0
0.40 0.96
0.15 0.1
0.49 0.3
0.90 0.28
0.55 0.21
1.25 0.90
15.0 7.1
57
44
43
85
81
24
74
100
76
40
55
58
96
31
29
38
60
6.0
0.25
0.38
0.9
20.4
12.4
1.9
6.6
2.0
15.0
4.0
14.5
6.0
0.8 0.08
0.33 0.24
0.70 0.42
0.33 0.11
0.33 0.11
0.13 0.04
0.10 0.02
0.46 0.48
0.24 0.02
2a
2b
2c
1
3-Me
5-Me
–
16
6.2
n.a.
n.a.
[a] Calculated partition coefficients (logP) calculated using the logP function implemented in ChemDraw 6.0. [b] Hydrolysis half-lives.
[c] Antiviral activity in T-lymphocytes: 50% effective concentration, values shown are means of two to three independent experiments.
[d] Cytostatic activity: 50% cytostatic concentration. [e] 25 mm phosphate buffer, pH = 7.3. [f] CEM cell extracts, pH = 6.9. [g] Wild-type
CEM cells. [h] Thymidine kinase-deficient CEM cells.
HPLC co-elution experiments or 31P NMR spectroscopy ture, the stability of triesters 4a–c was determined in citrate/
(step c, Figure 1).
HCl buffer at pH = 2.0. However, compounds 4 were not
As expected, the chemical stability half-lives of the 5- cleaved either at the acylal function or at the phosphate
modified cycloSal-d4TMP triesters 4 dropped to 4–5.3 h. ester moiety within 30 min.
This reduced stability was expected because 5-methyl-
cycloSal-d4TMP 2c was also found to be less stable than 3-
methyl-cycloSal-d4TMP 2b (Table 1). Again, only d4TMP
was released from triesters 4, whereas the AM, POM or
POC moieties proved to be stable under these conditions
Cell Extract Studies
(Figure 2). Due to the potentially acid-labile acylal struc-
Hydrolysis studies in T-lymphocyte cell extracts (CE;
Table 1) proved the cleavage of the ester groups in the case
of triesters 6a (OAc ester; t1/2 = 1.9 h vs. 13.6 h in the chem-
ical hydrolysis) and 6c (OLev ester; t1/2 = 2.0 h vs. 12.5 h).
Thus, triester alcohol 7 was formed (HPLC co-elution ex-
periments). The Piv triester 6b is more stable due to its
branched alkyl group. Thus, an about 9-fold more polar
product [logP = –0.53 (7) vs. 0.42 (6a) to 0.37 (6c)] is deliv-
ered compared to the precursors. However, although a more
polar compound (7) is liberated, it cannot be excluded that
the alcohol group is still not polar enough to achieve the
desired intracellular trapping.
Surprisingly, triesters 3, which were used to release the
much more polar carboxylate group (cycloSal-d4TMP acid
5a, logP = –3.96), were found to be inert to enzymatic cleav-
age. Although the half-lives measured in the cell extracts
were lower than those found in the chemical hydrolysis
studies, no formation of the free acid 5a was observed by
means of HPLC. This resistance to the cell extract enzymes
was unexpected because carboxyl groups in drugs are often
blocked bioreversibly by esterification.[20] Moreover, in pre-
vious test experiments methylpropionyl salicyl alcohol (8a)
was incubated with pig liver esterase (PLE).[21] In those ex-
periments, the methyl ester was cleaved extremely rapidly.
Figure 2. Chemical hydrolysis of the AM-cycloSal-d4TMP triester
4a followed by 31P NMR spectroscopy {solvent: [D6]DMSO/50 mm
imidazole·HCl buffer (pH = 7.3), 1:1 (v/v); spectra were recorded
once a week; H3PO4 was used as external reference}.
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In contrast to the unsuccessful delivery of cycloSal tri- enough to cross the cell membrane. Consequently, a passive
ester 5a from triesters 3, the AM, POM and POC acylals 4 efflux from the cells cannot be excluded. Nevertheless, en-
were efficiently cleaved in cell extracts with formation of zyme-degradable cycloSal-triester alcohol esters 6a and 6c
cycloSal triester 5b. Thus, the carboxylate group can be lib- showed full retention of antiviral activity in the CEM/TK–
erated (Figure 3).
cells. However, enzyme-stable triester 3a also retained its
antiviral activity entirely (Table 1).
A striking difference was observed for cycloSal-d4TMP
acylals 4. In contrast to triesters 4b,c, the AM acylal 4a lost
its activity against HIV (Table 1). This compound shows
the same antiviral activity profile as cycloSal-d4TMP acid
5b. Taking the results of the hydrolysis in RPMI/FCS cul-
ture medium into account, the AM acylal is already signifi-
cantly cleaved in this medium to the acid, which is unable
to pass the membrane. In contrast, triesters 4b and 4c were
found to be stable in the RPMI/FCS medium and therefore
led to full retention of antiviral activity in the mutant CEM/
TK– cells.
Figure 3. Cell extract incubation of AM-cycloSal-d4TMP triester
4a followed by HPLC (HPLC method: HPLC analysis method II
was performed as reported in ref.[7]).
Conclusions
For AM acylals 4a the half-life drops 17-fold (t1/2
=
The results disclosed here prove that the delivery of
highly polar cycloSal-d4TMP acids such as 5 is possible in
principal. However, it seems that the delivery of cycloSal-
d4TMP alcohols 7 may not lead to sufficient “trapping”,
although the compounds proved to be entirely independent
of the presence of thymidine kinase in the cells. Assuming
an efficient intracellular trapping of the cycloSal-d4TMP
acids, one should expect a considerably higher intracellular
concentration of these compounds. The reason why the bio-
activity of the precursors is only comparable to that of the
cycloSal-d4TMP alcohol 7 remains unclear and will be
studied further in our laboratories. Maybe this is due to the
metabolism profile of the nucleoside d4T itself or to the
higher chemical stability of the intermediate cycloSal-
d4TMP acid 5, which prevents the fast build-up of higher
d4TMP concentrations in the cells during the incubation
period. Further experiments will be conducted in order to
obtain more labile ester sites and enzyme-cleavable precur-
sors of cycloSal-d4TMP acid 5.
0.25 h) with respect to the chemical stability. Subsequently,
triester 5b delivered d4TMP. The POM acylal 4b is slightly
more stable than the AM acylal, but a 15-fold reduction of
the half-life was still observed. The most stable compound
was the POC-acylal 4c, which only shows a fourfold re-
duction in stability, although the acylal cleavage is still the
predominant reaction. In addition, cycloSal triesters 4 were
also studied for their stability in human serum. The stability
in serum was found to be identical to that found in the
chemical hydrolysis studies (4a: t1/2 = 3.9 h; 4b: 5.2 h; 4c:
5.1 h). Finally, these compounds were also incubated with
RPMI culture medium containing 10% fetal calf serum.
Surprisingly, AM-acylal 4a was cleaved significantly in this
medium. In contrast, the POM and POC acylals proved to
be resistant to the calf serum enzymes (4a: t1/2 = 2.4 h; 4b:
4.2 h; 4c: 3.7 h).
Antiviral Evaluation
Finally, triesters 3–7 were tested for their anti-HIV ac-
tivity in wild-type CEM and mutant thymidine-kinase-de-
ficient CEM/TK– cells (Table 1). D4T (1) was used as the
reference compound as it is active against HIV-1 and HIV-
2 in wild-type CEM/O but inactive in TK-deficient CEM/
TK– cells. The same activity has been found for cycloSal-
Experimental Section
General Remarks: NMR spectra were recorded with Bruker AMX
400 and Bruker DRX 500 Fourier-transform spectrometers. All 1H
and 13C NMR chemical shifts (δ) are quoted in ppm and calibrated
with respect to solvent signals. The 31P NMR chemical shifts (pro-
d4TMP acids 5, which can be attributed to a lack of mem- ton-decoupled) are quoted in ppm using H3PO4 as the external
reference. The spectra were recorded at room temperature. EI mass
spectra were measured with a VG Analytical VG/70-250S spec-
trometer (double focussing), ESI mass spectra with a Finnigan
ThermoQuest MAT 95 XL spectrometer and FAB high-resolution
(HR) mass spectra with a VG Analytical 70-250S spectrometer
using an MCA method and poly(ethylene glycol) as support.
Merck precoated 60 F254 plates with a 0.2 mm layer of silica gel
were used for thin layer chromatography (TLC). All preparative
TLCs were performed with a chromatotron® (Harrison Research,
Model 7924T) using glass plates coated with 1- or 2-mm layers of
brane penetration due to the charged carboxylate. The re-
maining activity in the wild-type CEM cells should be due
to an extracellular delivery of d4TMP, dephosphorylation
to d4T 1 and subsequent cellular uptake of d4T, which is
then rephosphorylated and consequently shows activity in
CEM/O cells (but not in CEM/TK– cells due to lack of
TK). As assumed, cycloSal-d4TMP triester 7, which con-
tains the hydroxyethyl group, proved to be antivirally active
in the wild-type CEM cell culture but also in the mutant
TK-deficient cells. Thus, the compound is still lipophilic Merck 60 PF254 silica gel containing a fluorescent indicator. For
202 Eur. J. Org. Chem. 2006, 197–206
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Second-Generation cycloSal-d4TMP Pronucleotides Bearing Esterase-Cleavable Sites
FULL PAPER
3
3
column chromatography, Merck silica gel 60 (230–400) mesh was
used. Analytical HPLC was performed with a Merck–Hitachi
HPLC system (D-7000) equipped with a LiChroCART 125-3 col-
umn containing reversed-phase silica gel Lichrospher 100 RP 18
(5 μm; Merck, Darmstadt, Germany). The lyophilized products 2–
7 did not give useful microanalytical data, most probably due to
incomplete combustion of the compounds or varying amounts of
water, but were found to be pure by rigorous HPLC analysis (gradi-
ent of 5–100% CH3CN in water within 25 min., flow
0.5 mLmin–1). All reactions were carried out under dry nitrogen,
except for the synthesis of 13, 14 and 17. Solvents used in the inert-
gas syntheses were commercially available dry solvents stored under
argon and over molecular sieves (Fluka). N,N-Dimethylformamide
was additionally degassed and stored under nitrogen and over mol-
ecular sieves. Diethyl ether was dried with sodium/benzophenone
and distilled under nitrogen. Potassium carbonate (water-free,
Merck) was dried and stored under nitrogen.
5-H), 7.11 (d, JH,H = 7.4 Hz, 1 H, 6-H), 7.46 (dd, JH,H = 7.9,
4JH,H = 0.8 Hz, 1 H, 4-H) ppm. 13C NMR (101 MHz, [D6]DMSO):
δ = 24.75 (acetal-CH3), 60.09 (benzyl-C), 100.81 (acetal-C), 110.02
(C-3), 121.52 (C-5), 121.81 (C-1), 124.74 (C-6), 131.40 (C-4), 147.60
(C-2) ppm. MS (EI): m/z (%) = calcd. 242 [M]; found 242 (25), 184
(100), 156 (6), 105 (45), 77 (26), 51 (16).
3-[3-(Methoxycarbonyl)propyl]salicyl Alcohol Isopropylidene Acetal
(19): 9-BBN (22 mL, 11 mmol; 0.5 m in THF) was added dropwise
to methyl 3-butenoate (1.00 g, 10.0 mmol) at 0 °C. After removing
the cooling bath, the solution was stirred at 55 °C overnight. After-
wards, acetal 14 (1.86 g, 8.00 mmol), potassium carbonate (2.49 g,
16.0 mmol), [Pd(dppf)Cl2] (198 mg, 0.27 mmol) and 50 mL of
DMF were added and the mixture stirred at 55 °C for 4 d. The
reaction was monitored by TLC (petroleum ether 50–70/ethyl ace-
tate, 4:1). The solution was allowed to cool to room temp. and then
poured into 1 m HOAc/NaOAc buffer (pH = 5) and stirred for
10 min. The aqueous layer was extracted twice with Et2O, the com-
bined organic layers were dried with sodium sulfate and the sol-
vents removed in vacuo. The brown crude was purified by column
chromatography on silica gel (petroleum ether 50–70/ethyl acetate,
4:1). Yield: 1.25 g (4.70 mmol, 59%) of a colourless oil. Rf = 0.36
(petroleum ether 50–70/ethyl acetate, 4:1). 1H NMR (400 MHz,
3-Bromosalicyl Alcohol (17): A solution of 2-bromophenol (16;
50.0 mL, 81.5 g, 0.471 mol), phenylboronic acid (68.9 g,
0.565 mol), paraformaldehyde (28.3 g, 0.942 mol) and propionic
acid (17.6 mL, 17.5 g, 0.236 mol) in 1 L of toluene was heated un-
der reflux in a Dean–Stark apparatus. Every 3 h, the same amount
of paraformaldehyde was added. This procedure was continued un-
til a total amount of paraformaldehyde (170 g, 5.66 mol) and a
reaction period of 18 h were reached. The reaction was monitored
by TLC (CH2Cl2/MeOH, 9:1). The solvent was removed under re-
duced pressure and the resulting residue was dissolved in CH2Cl2
and water. The aqueous phase was then extracted twice with
CH2Cl2. The combined organic phases were washed with water,
dried with sodium sulfate and concentrated under reduced pressure
to yield the dioxaborine as a crude product (98.2 g, dark red oil)
which was dissolved in 340 mL of THF. After cooling to 0 °C,
340 mL of 30% hydrogen peroxide was added and the reaction was
continued at that temperature for 45 min. After completion of the
oxidation, the mixture was diluted with water and extracted five
times with diethyl ether. The combined organic phases were washed
with 39% sodium bisulfite solution and brine, dried with sodium
sulfate and concentrated under reduced pressure. The product was
purified by column chromatography on silica gel [CH2Cl2/MeOH
gradient (0–2%)]. Yield: 50.5 g (0.248 mol, 53% over 2 steps) of a
yellow-brown oil which crystallised at –20 °C after several days. Rf
3
[D6]DMSO): δ = 1.76 (quint, JH,H = 7.4 Hz, 2 H, 9-H), 1.43 (s, 6
3
3
H, acetal-CH3), 2.24 (t, JH,H = 7.4 Hz, 2 H, 10-H), 2.49 (t, JH,H
= 7.4 Hz, 2 H, 8-H), 3.55 (s, 3 H, 12-H), 4.77 (s, 2 H, 7-H), 6.73
(dd, 3JH,H = 7.2 Hz, 1 H, aryl-5-H), 6.87 (dd, 3JH,H = 7.2 Hz, 1 H,
aryl-6-H), 6.97 (dd, 3JH,H = 7.2 Hz, 1 H, aryl-4-H) ppm. 13C NMR
(101 MHz, [D6]DMSO): δ = 24.56 (acetal-CH3), 24.63 (C-9), 28.05
(C-8), 32.67 (C-10), 51.18 (C-12), 60.14 (C-7), 99.08 (acetal-C),
119.22 (C-1), 119.78 (C-5), 122.81 (C-6), 125.37 (aryl-C-4), 127.90
(aryl-C-1), 128.21 (aryl-C-4), 128.65 (aryl-C-3), 148.91 (aryl-C-2),
172.18 (C-11) ppm. MS (ESI+): m/z = calcd. 287.13 [M + Na+];
found 287.23.
3-[3-(Methoxycarbonyl)propyl]salicyl Alcohol (13): Two drops of
concd. hydrochloric acid were added to a solution of 19 (940 mg,
3.60 mmol) in 30 mL of CH3CN/H2O (7:1), and the reaction mix-
ture was heated with a heat gun until it was boiling. The heating
was continued until the deprotection was complete (monitoring by
TLC, CH2Cl2/MeOH, 19:1, 0.5–5 min) and then the mixture was
diluted with CH2Cl2 and water. The aqueous layer was extracted
three times with CH2Cl2 and the combined organic layers were
washed subsequently with satd. ammonium hydrogencarbonate
solution and water, dried with sodium sulfate and the solvent was
removed under reduced pressure. The residue was purified by col-
umn chromatography on silica gel (CH2Cl2/MeOH, 14:1). Yield:
524 mg (2.30 mmol, 65%) of a colourless oil. Rf = 0.53 (CH2Cl2/
1
= 0.13 (CH2Cl2). H NMR (400 MHz, [D6]DMSO): δ = 4.58 (s, 2
3
3
H, benzyl-H), 5.38 (s, 1 H, OH), 6.80 (dd, JH,H = 7.9, JH,H
=
3
7.6 Hz, 1 H, 5-H), 7.25–7.32 (m, 1 H, 6-H), 7.40 (dd, JH,H = 7.9,
4JH,H = 1.2 Hz, 1 H, 4-H), 9.13 (s, 1 H, OH) ppm. 13C NMR
(101 MHz, [D6]DMSO): δ = 59.48 (benzyl-C), 110.76 (C-3), 121.04
(C-5), 126.89 (C-6), 131.02 (C-4), 131.40 (C-1), 150.90 (C-2) ppm.
MS (EI): m/z (%) = calcd. 202 [M]; found 202 (25), 184 (100), 156
(14), 105 (83), 94 (17), 77 (47), 63 (12), 51 (20).
1
MeOH, 19:1). H NMR (400 MHz, [D6]DMSO): δ = 1.77 (quint,
3JH,H = 7.5 Hz, 2 H, 9-H), 2.30 (t, 3JH,H = 7.5 Hz, 2 H, 10-H), 2.56
3
3
(t, JH,H = 7.5 Hz, 2 H, 8-H), 3.58 (s, 3 H, 13-H), 4.57 (d, JH,H
=
3-Bromosalicyl Alcohol Isopropylidene Acetal (14): A mixture of 3-
bromosalicyl alcohol (17; 45.4 g, 0.224 mol), 2,2-dimethoxypro-
pane (139 mL, 117 g, 1.12 mol), para-toluenesulfonic acid monohy-
drate (4.26 g, 22.4 mmol) and 89.6 g of anhydrous sodium sulfate
in 900 mL of acetone was heated at 40 °C for 3 d. The solvent was
removed under reduced pressure and the residue was dissolved in
ethyl acetate and water. The aqueous phase was extracted twice
with ethyl acetate. The combined organic phases were washed twice
with 1 m sodium hydroxide and water, dried with sodium sulfate
and concentrated under reduced pressure. The product was purified
by column chromatography on silica gel (CH2Cl2). Yield: 54.0 g
3
5.3 Hz, 2 H, benzyl-H), 5.37 (t, JH,H = 5.3 Hz, 1 H, benzyl-OH),
3
3
6.74 (dd, JH,H = 7.5 Hz, 1 H, aryl-5-H), 6.95 (dd, JH,H = 7.5,
4JH,H = 1.5 Hz, 1 H, aryl-6-H) 7.06 (dd, JH,H = 7.5, JH,H
=
3
4
1.5 Hz, 1 H, aryl-4-H), 8.42 (s, 1 H, phenol-OH) ppm. 13C NMR
(100 MHz, [D6]DMSO): δ = 24.85 (C-9), 28.82 (C-8), 32.97 (C-10),
51.21 (C-12), 60.04 (benzyl-C), 119.10 (aryl-C-5), 125.34 (aryl-C-
4), 127.92 (aryl-C-3), 128.07 (aryl-C-1), 128.36 (aryl-C-6), 152.39
(aryl-C-2), 173.29 (C-11) ppm. HRMS (FAB): m/z = calcd.
224.1049 [M]; found 224.1054.
3-[3-(Methoxycarbonyl)propyl]-cycloSal-d4T-monophosphate
(3-
(0.222 mol, 99%) of a brown oil. Rf = 0.64 (CH2Cl2/MeOH, 30:1). MeBu-cycloSal-d4TMP) (3d): A solution of the salicyl alcohol de-
1H NMR (400 MHz, [D6]DMSO): δ = 1.52 (s, 6 H, acetal-CH3), rivative 13 (451 mg, 2.00 mmol) in dry Et2O was cooled to –20 °C.
3
4.86 (s, 2 H, benzyl-H), 6.87 (dd, 3JH,H = 7.9, JH,H = 7.4 Hz, 1 H,
After addition of freshly distilled phosphorus(iii) chloride (330 mg,
Eur. J. Org. Chem. 2006, 197–206
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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203

C. Meier, C. Ducho, H. Jessen, D. Vukadinovic´-Tenter, J. Balzarini
FULL PAPER
2.40 mmol) and stirring at –20 °C for 10 min, a solution of dry
pyridine (364 mg, 4.60 mmol) in dry diethyl ether was added at the
same temperature over a period of 3 h. After completion of the
addition, the reaction mixture was allowed to warm to room tem-
perature and stirred for 2 h. It was kept at –20 °C overnight for
a complete precipitation of pyridinium chloride. Filtration under
nitrogen and concentrated of the filtrate under reduced pressure
afforded the phosphitylating agent (saligenyl chlorophosphite) as a
crude product to be directly used for the synthesis of the cycloSal
1.69 (s, 3 H, thymine-CH3), 2.05 (s, 3 H, acetyl-CH3), 2.06 (s, 3 H,
3
acetyl-CH3), 2.70 (t, JH,H = 7.3 Hz, 4 H, 2×9-H), 2.84 (t, 3JH,H
=
7.3 Hz, 4 H, 2×8-H), 4.24–4.35 (m, 4 H, 2×5Ј-H), 4.94–5.00 (m,
2 H, 2×4Ј-H), 5.34–5.58 (m, 4 H, 2×benzyl-H), 5.66 (s, 2 H, 11-
H), 5.67 (s, 2 H, 1×11-H), 6.00–6.04 (m, 2 H, 2×2Ј-H), 6.36 (ddd,
3
4
3JH,H = 6.0, JH,H = 1.6, JH,H = 1.6 Hz, 1 H, 3Ј-H), 6.42 (ddd,
3JH,H = 6.0, 3JH,H = 1.6, 4JH,H = 1.6 Hz, 1 H, 3Ј-H), 6.78 (dd, 3JH,H
= 2.6, JH,H = 1.6 Hz, 1 H, 1Ј-H), 6.80 (dd, JH,H = 2.6, JH,H
1.6 Hz, 1 H, 1Ј-H), 7.02 (d, JH,H = 8.5 Hz, 1 H, aryl-3-H), 7.05
4
3
4
=
3
3
phosphate triester 3d without further purification. The general syn- (d, JH,H = 8.5 Hz, 1 H, aryl 3-H), 7.15–7.26 (m, 63 H, 2×aryl 4-
thesis of cycloSal-d4T-monophosphates has been published be-
fore.[3,5,13] Diisopropylethylamine (DIPEA; 194 mg, 1.50 mmol)
was added to a solution of d4T 1 (224 mg, 1.00 mmol) in dry
CH3CN. The resulting solution was cooled to –20 °C and the chlo-
rophosphite (350 mg, 1.20 mmol) was added. The solution was al-
lowed to warm to room temperature, and stirring was continued
for 3 h. Subsequently, tert-butyl hydroperoxide (6 m in n-decane;
H, 2×aryl 6-H, 2×thymine 6-H), 11.34 (s, 2 H, 2×NH) ppm. 13C
NMR (101 MHz, [D6]DMSO): δ = 12.00 (thymine-CH3), 12.09
(thymine-CH3), 20.64 (2 × acetyl-CH3), 29.16 (2 × C-8), 34.59
2
2
(2×C-9), 68.38 (d, JC,P = 6.1 Hz, 2×benzyl-C), 68.52 (d, JC,P
=
3
6.1 Hz, 2×C-5Ј), 79.15 (2×C-11), 84.28 (d, JC,P = 8.0 Hz, 2×C-
4Ј), 89.35 (2×C-1Ј), 109.82 (2×thymine-C-5), 118.14 (d, JC,P
3
=
8.0 Hz, 2×aryl-C-3), 125.94 (aryl-C-6), 126.00 (aryl-C-6), 127.46
0.50 mL, 3.00 mmol) was added at –20 °C. After warming to room (2×C-2Ј), 127.97 (2×aryl-C-1), 129.78 (aryl-C-4), 129.96 (aryl-C-
temperature and stirring for 1 h, the solvent was removed under
reduced pressure. The resulting residue was purified by preparative
TLC [Chromatotron®; 1. ethyl acetate/MeOH (0.1% HOAc), 9:1;
2. CH2Cl2/MeOH gradient (0–5%)]. Lyophilisation yielded 277 mg
4), 132.96 (C-3Ј), 133.03 (C-3Ј), 135.82 (thymine-C-6), 135.86 (thy-
mine-C-6), 136.65 (2×aryl-C-5), 148.08 (d, 2JC,P = 7.1 Hz, 2×aryl-
C-2), 150.85 (thymine-C-2), 150.88 (thymine-C-2), 163.88 (thymine-
C-4), 163.90 (thymine-C-4), 169.44 (2 ×C-12), 171.22 (2 ×C-10)
(0.56 mmol, 56%) of a diastereomeric mixture (ratio 1.0:1.0) as a ppm. 31P NMR (202 MHz, [D6]DMSO): δ = –8.00, –8.07 ppm.
colourless foam. Rf = 0.47 (CH2Cl2/MeOH, 9:1). 1H NMR
(500 MHz, [D6]DMSO): δ = 1.59 (d, 4JH,H = 1.0 Hz, 3 H, thymine-
HRMS (ESI+): m/z = calcd. 559.1094 [M + Na+]; found 559.1113.
HPLC: tR = 14.0 min (method I); tR = 11.9 min (method II). UV/
Vis (water/CH3CN): λmax = 265 nm.
4
CH3), 1.62 (d, JH,H = 1.0 Hz, 3 H, thymine-CH3), 1.75–1.83 (m,
3
3
4 H, 2×9-H), 2.30 (t, JH,H = 7.4 Hz, 2 H, 10-H), 2.32 (t, JH,H
=
5-[2-(Pivaloyloxymethoxycarbonyl)ethyl]-cycloSal-d4T-monophos-
phate [5-(POM-Pr)-cycloSal-d4TMP] (4b): Under nitrogen,
1.53 mL of a solution of DIPEA in dry CH3CN (1:100; 94.7 μmol
of DIPEA) and 1.37 mL of a solution of chloromethyl pivalate in
dry CH3CN (1:100; 94.7 μmol of chloromethyl pivalate) were
added dropwise to a solution of 5-(2-carboxyethyl)-cycloSal-
d4TMP (5b; 40 mg, 86 μmol) in 10 mL of dry CH3CN at room
temperature. The reaction mixture was stirred at room temperature
for 9 d and the reaction monitored by TLC (CH2Cl2/MeOH, 9:1).
The solvent was removed in vacuo and the residue was purified
by preparative TLC [Chromatotron®; CH2Cl2/MeOH gradient (0–
3%)]. The isolated product was lyophilized. Yield: 23 mg (39 mmol,
45 %) of a colourless solid as a 1.0:0.8 mixture of two dia-
7.4 Hz, 2 H, 10-H), 2.54–2.66 (m, 4 H, 2×8-H), 3.57 (s, 3 H, 12-
H), 3.58 (s, 3 H, 12-H), 4.24–4.34 (m, 4 H, 2×5Ј-H), 4.97–4.93 (m,
3
2 H, 2×4Ј-H), 5.34 (dd, 2JH,H = 16.9, JH,P = 6.9 Hz, 1 H, benzyl-
2
3
H), 5.38 (dd, JH,H = 13.2, JH,P = 6.3 Hz, 1 H, benzyl-H), 5.44
2
3
2
(dd, JH,H = 16.4, JH,P = 5.7 Hz, 1 H, benzyl-H), 5.48 (dd, JH,H
= 14.5, JH,P = 6.3 Hz, 1 H, benzyl-H), 5.99–6.05 (m, 2 H, 2×2Ј-
3
H), 6.35–6.38 (m, 1 H, 3Ј-H), 6.78–6.82 (m, 2 H, 2×1Ј-H), 7.10–
7.28 (m, 8 H, 2×aryl 4-H 2×aryl 5-H, 2×aryl 6-H, 2×thymine 6-
H), 11.32 (s, 1 H, NH), 11.34 (s, 1 H, NH) ppm. 13C NMR
(101 MHz, [D6]DMSO): δ = 11.75 (thymine-CH3), 11.85 (thymine-
CH3), 24.56 (C-10), 24.59 (C-10), 27.89 (2×C-8), 32.64 (C-9), 32.66
(C-9), 68.19 (d, 2JC,P = 3.2 Hz, benzyl-C), 68.26 (d, 2JC,P = 3.5 Hz,
2
benzyl-C), 68.51 (d, 2J = 4.0 Hz, C-5Ј), 68.61 (d, JC,P = 4.5 Hz,
1
stereomers. Rf = 0.43 (CH2Cl2/MeOH, 9:1). H NMR (500 MHz,
C-5Ј), 84.10 (C-4Ј), 84.18 (C-4Ј), 89.11 (C-1Ј), 89.19 (C-1Ј), 109.64
(thymine-C-5), 109.69 (thymine-C-5), 124.18 (2×C-aryl), 127.38
(2×C-2Ј), 130.30 (C-aryl), 130.35 (C-aryl), 132.74 (C-3Ј), 132.81
(C-3Ј), 135.62 (C-aryl), 135.68 (C-aryl), 150.70 (2×C-2), 163.71
(2×thymine-C-4), 172.97 (2×C-11) ppm. 31P NMR (162 MHz,
[D6]DMSO): δ = –8.37, –8.51 ppm. HRMS (FAB): m/z = calcd.
493.1376 [M + H]; found 493.1377. HPLC: tR = 14.5 min (method
I); tR = 11.1, 11.2 min (method II). UV/Vis (water/CH3CN): λmax
= 267 nm.
[D6]DMSO): δ = 1.11 (s, 9 H, tBu), 1.12 (s, 9 H, tBu), 1.63 (s, 3 H,
3
thymine-CH3), 1.70 (s, 3 H, thymine-CH3), 2.71 (t, JH,H = 7.6 Hz,
3
4 H, 2×9-H), 2.84 (t, JH,H = 7.6 Hz, 4 H, 2×8-H), 4.25–4.37 (m,
4 H, 2×5Ј-H), 4.96–5.00 (m, 2 H, 2×4Ј-H), 5.33–5.48 (m, 4 H,
2×benzyl-H), 5.69 (s, 2 H, 11-H), 5.70 (s, 2 H, 11-H), 6.02–6.05
3
3
4
(m, 2 H, 2×2Ј-H), 6.36 (ddd, JH,H = 5.7, JH,H = 2.5, JH,H
=
=
=
3
3
4
2.5 Hz, 1 H, 3Ј-H), 6.42 (ddd, JH,H = 5.7, JH,H = 2.5, JH,H
3
2.5 Hz, 1 H, 3Ј-H), 6.79–6.82 (m, 2 H, 2×1Ј-H), 7.02 (d, JH,H
8.2 Hz, 1 H, aryl 3-H), 7.05 (d, JH,H = 8.2 Hz, 1 H, aryl 3-H),
3
5-[2-(Acetoxymethoxycarbonyl)ethyl]-cycloSal-d4T-monophosphate 7.16–7.25 (m, 6 H, 2 × aryl 4-H, 2 × aryl 6-H, 2 × thymine 6-H),
[(5-AM-Pr)-cycloSal-d4TMP] (4a): Under nitrogen, 2.7 mL of a
solution of DIPEA in dry CH3CN (1:100; 0.17 mmol of DIPEA)
and 1.7 mL of a solution of bromomethyl acetate in dry CH3CN
(1:100; 0.17 mmol of bromomethyl acetate) were added dropwise
to a solution of 5-(2-carboxyethyl)-cycloSal-d4TMP (5b; 70 mg,
0.15 mmol) in 22 mL of dry CH3CN at 10 °C. The reaction mixture
was stirred at 10 °C for 3 h and the reaction monitored by TLC
(CH2Cl2/MeOH, 9:1). The solvent was removed in vacuo and the
residue was purified by preparative TLC [Chromatotron®; CH2Cl2/
11.35 (s, 2 H, 2×NH) ppm. 13C NMR (101 MHz, [D6]DMSO): δ
= 12.02 (thymine-CH3), 12.10 (thymine-CH3), 26.61 (2×tBu-CH3),
29.16 (2×C-8), 34.52 (2×C-9), 38.33 (2×tBu-C), 68.41–68.52 (m,
3
2×C-5Ј, 2×benzyl-C), 79.48 (2×C-11), 84.29 (d, JC,P = 8.1 Hz,
C-4Ј), 84.53 (C-4Ј), 89.35 (2 × C-1Ј), 109.84 (2 × thymine-C-5),
3
118.06 (d, JC,P = 3.1 Hz, 2×aryl-C-3), 125.94 (aryl-C-6), 126.00
(aryl-C-6), 127.48 (C-2Ј), 127.51 (C-2Ј), 128.43 (2 × aryl-C-1),
129.77 (aryl-C-4), 129.86 (aryl-C-4), 132.95 (C-3Ј), 133.02 (C-3Ј),
135.86 (2×thymine-C-6), 136.64 (2×aryl-C-5), 147.41 (2×aryl-C-
MeOH gradient (0–3 %)]. The isolated product was lyophilized. 2), 150.67 (thymine-C-2), 150.85 (thymine-C-2), 163.89 (thymine-
Yield: 53 mg (99 μmol, 65%) of a colourless solid as a mixture of
two diastereomers in a 1:1 ratio. Rf = 0.36 (CH2Cl2/MeOH, 9:1).
C-4), 163.91 (thymine-C-4), 167.17 (C-12), 167.32 (C-12), 171.19
(C-10), 171.24 (C-10) ppm. 31P NMR (202 MHz, [D6]DMSO): δ
1H NMR (500 MHz, [D6]DMSO): δ = 1.62 (s, 3 H, thymine-CH3), = –8.01, –8.08 ppm. HRMS (ESI+): m/z = calcd. 601.1533 [M +
204
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Second-Generation cycloSal-d4TMP Pronucleotides Bearing Esterase-Cleavable Sites
FULL PAPER
Na+]; found 601.1559. HPLC: tR = 17.6 min (method I); tR
14.8 min (method II). UV/Vis (water/CH3CN): λmax = 267 nm.
=
acid buffer (0.55 mm, pH = 3.8) with an acetonitrile gradient from
0 to 70%, flow 0.6 mLmin–1]. Two separate samples for each time
point and test compound were prepared and analysed. Determi-
nation of t1/2 was performed analogously to that for the chemical
hydrolysis studies except that the absolute integral values were used
instead of standardised IU. Identification of the hydrolysis prod-
ucts was based on the retention times of the reference compounds
and co-elution experiments under identical analytical conditions.
5-[2-(Isopropyloxycarbonyloxymethoxycarbonyl)ethyl]-cycloSal-
d4T-monophosphate [5-(POC-Pr)-cycloSal-d4TMP] (4c): Under ni-
trogen, 1.47 mL of a solution of DIPEA in dry CH3CN (1:50;
0.17 mmol of DIPEA) and 0.76 mL of a POC chloride solution
in dry CH3CN (33 mg/mL, 0.17 mmol POC chloride) were added
dropwise to a solution of 5-(2-carboxyethyl)-cycloSal-d4TMP (5b;
70 mg, 0.15 mmol) in 18 mL of dry CH3CN at room temperature.
The reaction mixture was stirred at 50 °C for 7 d and the reaction
monitored by TLC (CH2Cl2/MeOH, 9:1). The solvent was removed
in vacuo and the residue was purified by preparative TLC [Chro-
matotron®; CH2Cl2/MeOH gradient (0–5%)]. The isolated product
was lyophilized. Yield: 41 mg (71 μmol, 47%) of a colourless solid
as a 0.5:1.0 mixture of two diastereomers. Rf = 0.53 (CH2Cl2/
Chemical Hydrolysis in Citrate Buffer: 10 μL of a 5 mm solution of
the prodrug in DMSO was mixed with 20 μL of DMSO and 30 μL
of citrate/HCl buffer (pH = 2.0) and incubated at 37 °C for 30 min.
An additional reference sample using water instead of buffer was
also prepared. Both samples were analysed by RP-HPLC as de-
scribed previously.[7]
31P NMR Hydrolysis Studies of the cycloSal Phosphate Triesters:
Approximately 7 μmol of the cycloSal triesters was dissolved in
500 μL of deuterated DMSO and 500 μL of a 50 mm imidazole/
hydrochloric acid buffer (pH = 7.3). The resulting kinetic solutions
were transferred into an NMR tube and investigated by 31P NMR
spectroscopy (proton-decoupled, 202 MHz, 256 scans each sam-
ple). All samples were stored at room temperature. Furthermore,
some proton-coupled 31P NMR spectra (202 MHz, 512 scans each
sample) were recorded for the identification of the hydrolysis prod-
ucts.[22]
1
3
MeOH, 9:1). H NMR (500 MHz, [D6]DMSO): δ = 1.25 (d, JH,H
= 6.2 Hz, 12 H, 2×iPr-CH3), 1.63 (d, 4JH,H = 0.6 Hz, 3 H, thymine-
CH3), 1.70 (s, 3 H, thymine-CH3), 2.73 (t, JH,H = 7.4 Hz, 4 H,
2×9-H), 2.85 (t, JH,H = 7.4 Hz, 4 H, 2×8-H), 4.25–4.36 (m, 4 H,
3
3
3
2×5Ј-H), 4.82 (sept, JH,H = 6.2 Hz, 2 H, 2×iPr-CH), 4.95–4.99
(m, 2 H, 2×4Ј-H), 5.35–5.39 (m, 2 H, 2×benzyl-H), 5.43 (dd, 2JH,H
3
2
= 15.6, JH,P = 5.8 Hz, 1 H, benzyl-H), 5.47 (dd, JH,H = 15.6,
3JH,P = 5.9 Hz, 1 H, benzyl-H), 5.69 (s, 4 H, 2×11-H), 6.01–6.05
(m, 2 H, 2×2Ј-H), 6.35–6.38 (m, 1 H, 3Ј-H), 6.42–6.45 (m, 1 H,
3Ј-H), 6.79–6.81 (m, 1 H, 1Ј-H), 6.81–6.83 (m, 1 H, 1Ј-H), 7.03 (d,
3JH,H = 8.4 Hz, 1 H, aryl-3-H), 7.05 (d, JH,H = 8.4 Hz, 1 H, aryl-
3
3-H), 7.16–7.27 (m, 6 H, 2×aryl 4-H, 2×aryl 6-H, 2×thymine 6- Acknowledgments
H), 11.35 (s, 1 H, 1×NH), 11.36 (s, 1 H, 1×NH) ppm. 13C NMR
(101 MHz, [D6]DMSO): δ = 12.00 (thymine-CH3), 12.09 (thymine-
CH3), 21.49 (2×iPr-CH3), 29.12 (2×C-8), 34.57 (2×C-9), 68.44 (d,
Financial support by the Deutsche Forschungsgemeinschaft, Ger-
many, and the René Descartes Prize 2001 of the European Com-
mission is gratefully acknowledged.
2
2JC,P = 7.1 Hz, 2×benzyl-C), 68.55 (d, JC,P = 3.8 Hz, 2×C-5Ј),
72.78 (2×iPr-CH), 81.86 (2×C-11), 84.27 (d, 3JC,P = 8.1 Hz, 2×C-
4Ј), 89.35 (2×C-1Ј), 109.81 (thymine-C-5), 109.83 (thymine-C-5),
[1] J. Balzarini, M. Baba, R. Pauwels, P. Herdewijn, Biochem.
Pharmacol. 1988, 37, 2847–2856.
[2] J. Balzarini, E. De Clercq, Biochem. Pharmacol. 1994, 49, 751–
772.
3
118.16 (d, JC,P = 2.0 Hz, 2×aryl-C-3), 125.92 (aryl-C-6), 125.99
3
(aryl-C-6), 127.46 (C-2Ј), 127.49 (C-2Ј), 129.73 (d, JC,P = 8.1 Hz,
2×aryl-C-1), 129.80 (aryl-C-4), 129.85 (aryl-C-4), 133.02 (C-3Ј),
133.04 (C-3Ј), 135.80 (thymine-C-6), 135.85 (thymine-C-6), 136.59
[3] a) C. Meier, Synlett 1998, 233–242; b) C. R. Wagner, V. V. Iyer,
E. J. McIntee, Med. Res. Rev. 2000, 20, 417–451.
[4] a) C. Meier, Mini Rev. Med. Chem. 2002, 2, 219–234; b) C.
Meier, J. Renze, C. Ducho, J. Balzarini, Curr. Top. Med. Chem.
2002, 2, 1111–1121; c) C. Meier, Advances in Antiviral Drug
Design (Ed.: E. De Clercq), Elsevier, Amsterdam, 2004, vol. 4,
p. 147–213.
[5] a) J. Balzarini, S. Aquaro, T. Knispel, C. Rampazzo, V. Bianchi,
C.-P. Perno, E. De Clercq, C. Meier, Mol. Pharmacol. 2000, 58,
928–935; b) J. Balzarini, L. Naesens, S. Aquaro, T. Knispel, C.-
F. Perno, E. De Clercq, C. Meier, Mol. Pharmacol. 1999, 56,
1354–1361.
2
(aryl-C-5), 136.62 (aryl-C-5), 148.06 (d, JC,P = 3.4 Hz, 2×aryl-C-
2), 150.93 (2×thymine-C-2), 152.87 (2×C-12), 163.86 (thymine-C-
4), 163.89 (thymine-C-4), 171.19 (2 × C-10) ppm. 31P NMR
(202 MHz, [D6]DMSO): δ = –8.01, –8.09 ppm. HRMS (FAB): m/z
= calcd. 581.1536 [M + H+]; found 581.1530. HPLC: tR = 14.1 min
(method I); tR = 13.3 min (method II). UV/Vis (water/CH3CN):
λmax = 265 nm.
Antiretroviral Evaluation: The experimental setup for the antiviral
testing has been described previously.[6]
[6] C. Meier, M. Lorey, E. De Clercq, J. Balzarini, J. Med. Chem.
1998, 41, 1417–1427.
[7] C. Meier, C. A. Lomp, A. A. Meerbach, A. P. Wutzler, J. Med.
Chem. 2002, 45, 5157–5172.
[8] J. Balzarini, F. Haller-Meier, E. De Clercq, C. Meier, Antiviral
Chem. Chemother. 2002, 12, 301–306.
[9] C. Meier, L. Habel, F. Haller-Meier, A. Lomp, M. Herderich,
R. Klöcking, A. Meerbach, P. Wutzler, Antiviral Chem. Chem-
other. 1998, 9, 389–402.
[10] C. Meier, T. Knispel, E. De Clercq, J. Balzarini, J. Med. Chem.
1999, 42, 1604–1614.
[11] C. Meier, T. Knispel, V. E. Marquez, E. De Clercq, J. Balzarini,
J. Med. Chem. 1999, 42, 1615–1624.
[12] C. Meier, M. Ruppel, D. Vukadinovic, J. Balzarini, Nucleosides
Nucleotides 2004, 23, 89–115.
[13] M. N. Arimilli, C. U. Kim, J. Dougherty, A. Mulato, R. Oliyai,
J. P. Shaw, K. C. Cundy, N. Bischofberger, Antiviral Chem.
Chemother. 1997, 8, 557–564.
Hydrolysis Studies of the cycloSal Phosphate Triesters: Hydrolysis
studies of cycloSal nucleotides (phosphate buffer, pH = 7.3) by
HPLC analysis (method I) have been described before.[14,22] Studies
in cell extracts were performed as reported in ref.[7] Thus, 100 μL
of human CEM/O cell extracts was mixed with 20 μL of a 70 mm
aqueous magnesium chloride solution. The reaction was started by
addition of 20 μL of a 1.5 mm solution of the cycloSal phosphate
triester in DMSO. Four separate samples of the above-mentioned
mixtures were incubated for 0, 2, 4 and 8 h, respectively. This was
done to avoid contamination of the biological media by taking mul-
tiple samples from only one solution. All solutions were incubated
at 37 °C. To stop the reaction and precipitation of protein, 300 μL
of acidified methanol (1 mL glacial acetic acid in 20 mL methanol)
was added and the cap was kept at 0 °C for 5 min. After centrifuga-
tion (13000 rpm, 10 min) and filtration of the supernatant, the sam-
ple was subjected to HPLC analysis [0–50 min TBAH/phosphoric
Eur. J. Org. Chem. 2006, 197–206
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
205

C. Meier, C. Ducho, H. Jessen, D. Vukadinovic´-Tenter, J. Balzarini
FULL PAPER
[14] A. J. Pearson, P. Zhang, K. Lee, J. Org. Chem. 1996, 61, 6581– [20] W. von Daehne, E. Frederiksen, E. Gundersen, F. Lund, P.
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fredsen, J. Med. Chem. 1970, 13, 607–612.
[21] C. Meier, M. Ruppel, D. Vukadinovic, J. Balzarini, Mini Rev.
Med. Chem. 2004, 4, 383–394.
[22] C. Ducho, J. Balzarini, L. Naesens, E. De Clercq, C. Meier,
Antiviral Chem. Chemother. 2002, 13, 129–141.
Received: July 4, 2005
[15] H. Brechbühler, H. Büchi, E. Hatz, J. Schreiber, A. Esch-
enmoser, Helv. Chim. Acta 1965, 48, 1746–1771.
[16] G. W. Breton, J. Org. Chem. 1997, 62, 8952–8954.
[17] S. Yamada, T. Sugaki, K. Matsuzaki, J. Org. Chem. 1996, 61,
5932–5938.
[18] C. Meier, E. De Clercq, J. Balzarini, Eur. J. Org. Chem. 1998,
837–846.
Published Online: November 3, 2005
[19] J. C. Boehm, J. G. Gleason, I. Pendrak, H. M. Sarau, D. B.
Schmidt, J. J. Foley, W. D. Kingsbury, J. Med. Chem. 1993, 36,
3333–3340.
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Eur. J. Org. Chem. 2006, 197–206