5-(1-Acetoxyvinyl)-cyclosaligenyl-2′,3′-dideoxy-2′, 3′- didehydrothymidine monophosphates, a second type of new, enzymatically activated cyclosaligenyl pronucleotides

Article abstract of DOI:10.1021/jm801197f

In our attempt to further develop the cycloSal pronucleotide concept, we report on 5-(1-acetoxyvinyl)-cycloSal-d4TMPs as a new type of enzyme-activated pronucleotides. These compounds were converted into 5-acetyl-cycloSal-d4TMPs by (carboxy)esterase cleav

Full text of DOI:10.1021/jm801197f

J. Med. Chem. 2008, 51, 8115–8123
8115
5-(1-Acetoxyvinyl)-cycloSaligenyl-2′,3′-dideoxy-2′,3′- didehydrothymidine Monophosphates, a
Second Type of New, Enzymatically Activated cycloSaligenyl Pronucleotides
Nicolas Gisch,† Florian Pertenbreiter,† Jan Balzarini,§ and Chris Meier*,†
Organic Chemistry, Department of Chemistry, Faculty of Science, UniVersity of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany,
and Rega Institute for Medical Research, Katholieke UniVersiteit LeuVen, Minderbroedersstraat 10, 3000 LeuVen, Belgium
ReceiVed September 22, 2008
In our attempt to further develop the cycloSal pronucleotide concept, we report on 5-(1-acetoxyvinyl)-
cycloSal-d4TMPs as a new type of enzyme-activated pronucleotides. These compounds were converted
into 5-acetyl-cycloSal-d4TMPs by (carboxy)esterase cleavage inside the cells. The enzymatic reaction led
to the formation of a strong electron-withdrawing substituent that strongly accelerates the chemical hydrolysis
of the cycloSal nucleotide to give d4TMP. For some cycloSal-d4TMPs a separation into the diastereomers
was achieved. The absolute configuration was assigned by correlation of circular dichroism spectra with
similar compounds. Most of the compounds showed complete retention of antiviral activity in TK-deficient
CEM/TK- cells, which proves the TK-bypass potential of this approach. Interestingly, (SP)-isomers of cycloSal
phosphate triesters showed better antiviral activity in HIV-2-infected thymidine-kinase deficient CEM/TK-
cells than their (RP)-counterparts.
Scheme 1. Synthesis of 4-Acetylsalicyl Alcohols 8 and
5-Acetyl-cycloSal-d4TMPs 6a
Introduction
The cycloSala concept has been widely used for the delivery
of nucleoside monophosphates into cells.1,2 Because of the
lipophilic character of cycloSal phosphate triesters and their
chemically triggered delivery mechanism, a drug concentration
equilibrium is formed through the cell membrane. In order to
trap cycloSal triesters inside the cells, so-called “lock-in”
cycloSal pronucleotides were designed.3-5 These compounds
contained (carboxy)esterase-cleavable esters attached to the
aromatic ring. To avoid any influence on the chemical hydrolysis
properties, the ester moieties have been separated from the
aromatic ring by a C2 spacer. The CycloSal-d4TMP acid ester
and cycloSal-d4TMP alcohol ester,3 as well as AM- and POM-
acylals4 or amino acid ester functionalized cycloSal-d4TMPs,5
have been investigated. The common motif of these compounds
is that after enzymatic cleavage a highly polar carboxylate group
is formed. Hydrolysis studies in phosphate buffer (PBS, pH 7.3)
and in T-lymphocyte CEM cell extracts revealed that an
intracellular trapping is possible when highly polar, charged
cycloSal-d4TMP acids are released. The fast intracellular release
of these compounds is the primary aim of the “lock-in” concept.
D4TMP 1 is released from the cycloSal-d4TMP carboxylic acids
by chemical hydrolysis. However, the chemical hydrolysis of
the charged carboxylate bearing cycloSal phosphate triesters was
considerably slower than its lipophilic precursor. Therefore, a
conceptually different approach to enzymatic activation of
cycloSal pronucleotides has been introduced and was described
recently.6,7
a Reagents and conditions. Method A: (i) AlCl3, toluene, -45 °C, 2 h;
(ii) acetyl chloride, -45 °C, 2.5 h. Method B: formaldehyde solution (37%),
conc HCl, 50 °C, 4 h. Method C: formaldehyde solution (37%), conc HCl,
100 °C, 2.5 h. Method D: CaCO3, H2O, aq THF, room temp, 1 day. Method
E: CaCO3, H2O, aq THF, 50 °C, 6 h. Method F: (i) Et2O or THF, PCl3,
pyridine, -20 °C, 4 h; (ii) CH3CN, DIPEA, d4T 2, -20 °C to room temp,
2-3 h; (iii) CH3CN, t-BuOOH, -20 °C to room temp, 0.5-2 h.
withdrawing substituent by enzymatic cleavage with the con-
sequence that the so formed strong electron-withdrawing group
(an aldehyde function) in the cycloSal structure caused a
significant decrease in hydrolysis stability. Preferably, the enzy-
matic conversion does not occur in the extracellular environment
or is very slow. In contrast to earlier approaches,4 in this case
the enzymatic activation resulted in a rapid delivery of the
nucleotide (e.g., d4TMP 1, Figure 1). Thus, in contrat to our
earlier approach, the “lock-in” effect with these compounds
happens at the nucleotide level.
In this approach, prodrugs contained again a lipophilic,
functionalized substituent with electron-donating or only weak
electron-withdrawing properties attached to the aromatic ring
of the cycloSal mask. After passive transport into the cell the
functionalized substituent was converted into a strong electron
* To whom correspondence should be addressed. Phone: +49-40-42838-
4324. Fax: +49-40-42838-5592. E-mail: chris.meier@chemie.uni-ham-
burg.de.
†
University of Hamburg.
Katholieke Universiteit Leuven.
As the first type of this new generation of enzymatically
activated cycloSal prodrugs, different diacyloxymethyl-cycloSal-
d4TMPs were studied. 5-Diacetoxymethyl- (3a-c (5-di-AM),
§
a Abbreviations: acyl, acetoxyvinyl; CD, circular dichroism; di-AM,
diacetoxymethyl; di-iBOM, diisobutyroxymethyl; cycloSal, cycloSaligenyl.
10.1021/jm801197f CCC: $40.75
2008 American Chemical Society
Published on Web 12/03/2008

8116 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Gisch et al.
Figure 1. Structures of d4TMP 1, d4T 2, and cycloSal-d4TMPs 3-7.
Figure 2. Enzyme assisted hydrolysis cascade of 5-(1-acetoxyvinyl)-cycloSal-d4TMPs 7.
Figure 1),6 5-diisobutyroxymethyl-, and 3-diacetoxymethyl-
cycloSal-d4TMPs (3d,e (5-di-iBOM) and 4a,b (3-di-AM),
Figure 1) were synthesized. Indeed, these compounds showed
the expected hydrolytic behavior in several chemical and
biological media. Their enzymatic cleavage into the correspond-
ing formyl-substituted compounds (e.g., 5 (Figure 1) resulting
from the cleavage of 3c or 3e) was proven, and a general
relationship between the hydrolysis stability, the absolute
configuration at the phosphorus atom, and the antiviral activity
against HIV was found. By comparison of 5-di-AM-cycloSal-
d4TMPs 3a-c and 5-di-iBOM-cycloSal-d4TMPs 3d,e, respec-
tively, the 3-t-Bu-substituted compounds (3c,e) showed the
highest stability against chemical hydrolysis and the best
antiviral activity. Interestingly, the antiviral testing of the sepa-
rated diastereomers of compounds 3c,e and 5 revealed significant
differences. In all cases, the (SP)-diastereomer was more active
than the (RP)-isomer. The absolute configuration was assigned
on the basis of a single-crystal X-ray structure of the slow-
eluting isomer of 5, which showed (RP)-configuration. In CEM/0
cell extracts (RP)-5 was formed from 3c-slow and 3e-slow by
enzymatic cleavage. Thus, compounds 3c-slow and 3e-slow have
to be (RP)-isomers as well.7
Here, we report the synthesis, properties, and antiviral
evaluation of a second type of enzymatically activated cycloSal-
d4TMPs: 5-(1-acetoxyvinyl)-cycloSal-d4TMPs (7a-c, 5-(1-
acyl)-cycloSal-d4TMPs, Figure 1). These new compounds were
expected to be cleaved to 5-acetyl-cycloSal-d4TMPs 6a-c by
(carboxy)esterases. The proposed hydrolysis cascade is shown
in Figure 2.
Results and Discussion
Chemistry. In order to use our established method for the
preparation of cycloSal-NMPs starting from saligenyl chloro-
phosphites,2 4-acetyl- (8a-c) and 4-(1-acetoxyvinyl)salicyl
alcohols (9a-c) were synthesized. All synthetic steps are
summarized in Schemes 1 and 2. 4-Acetylsalicyl alcohols 8a-c
were synthesized starting from the respective 4-hydroxyac-

5-(1-AcetoxyVinyl)-cycloSal-d4TMPs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8117
Scheme 2. Synthesis of 5-(1-Acetoxyvinyl)-cycloSal-d4TMPs 7a
a Reagents and conditions. Method G: TBDPSCl, imidazole, abs pyridine, room temp, 4 days. Method H: isopropenyl acetate, p-TsOH, 110 °C, 18 or
90 h. Method I: THF, Et3N·3HF, room temp, 20-30 min. Method K: t-Bu2Si(OTf)2, abs CH2Cl2, abs pyridine, room temp, 2 h. Method L: (i) abs THF,
n-BuLi, -78 °C, 2 h; (ii) N,N-dimethylacetamide, -50 °C, 1 h. Method M: THF, TBAF, room temp, 1 h. Method N: (i) Et2O, PCl3, pyridine, -20 °C, 4 h;
(ii) CH3CN, DIPEA, d4T 2, -20 °C to room temp, 1.5 h; (iii) CH3CN, t-BuOOH, -20 °C to room temp, 0.5-1.5 h.
etophenones 10a-c. While 10a,b were commercially available,
10c was prepared from 2-tert-butylphenol 11 first. This was
achieved using the method described by Shimomura et al.8 The
4-hydroxyacetophenones 10a-c were converted into salicyl
alcohols 8a-c in two steps (chloromethylation and nucleophilic
substitution). The chloromethylation of 10a to 12a has been
described by Yanagi et al.,9 and the following nucleophilic
substitution reaction from 12a to 8a was performed by a
modified method from Buchanan et al.10 So 8a was synthesized
in a two-step procedure in 62% yield. Similar conditions were
used for the preparation of 8b,c in 59% (8b) and 29% yield
(8c), respectively. Because of an insufficient reactivity of the
starting material, the reactions leading to 8c had to be performed
at a higher temperature. However, this led to a significant
formation of a byproduct, which could be characterized as the
methylene protected derivative of 8c. This was formed in the
first step during the chloromethylation. 5-Acetyl-cycloSal-
d4TMPs 6a-c were finally synthesized from salicyl alcohols
8a-c using the corresponding chlorophosphites in moderate
yields (18-26%).
obviously steric reasons counteract this “double” protection.
Compounds 13a,b were subsequently converted into the cor-
responding 4-(1-acetoxyvinyl) derivatives 14a,b using isopro-
penyl acetate and p-toluenesulfonic acid in good yields (14a,
64%; 14b, 69%).11 Finally, deprotection with triethylamine
trihydrofluoride (NEt3 ·3HF) gave salicyl alcohols 9a (83%) and
9b (82%), respectively.
The preparation of 4-(1-acetoxyvinyl)-6-tert-butylsalicyl al-
cohol 9c was achieved starting from 4-bromo-6-tert-butylsalicyl
alcohol 15, which has already been used in the synthesis of
5-formyl-3-tert-butyl-cycloSal-d4TMP 5.6,12 The hydroxy func-
tions of salicyl alcohol 15 were protected as a cyclic di-tert-
butylsilyl ether to yield 16 in 90%.13 Then the acetyl function
was introduced via bromo/lithium exchange using N,N-dim-
ethylacetamide.14 Compound 17c was obtained in 63% yield.
As an alternative, compound 17c can also be deprotected using
TBAF to yield 4-acetyl-6-tert-butylsalicyl alcohol 8c in 95%.
On the other hand, conversion of 17c into enolic ester 18c (59%
yield) and deprotection (98% yield) finally led to 9c. 4-(1-
Acetoxyvinyl)-6-methylsalicyl alcohol 9b was synthesized using
the cyclic silyl protecting group as well. Protection of salicyl
alcohol 8b gave 17b (63% yield), and the enolic ester 18b was
then obtained in 69% yield. Deprotection with NEt3 ·3HF gave
9b in 85% yield. By this route, salicyl alcohol 9b was
synthesized in an overall yield of 37% from 8b. The overall
yield using TBDPS as the protecting group was only 19%.
Salicyl alcohols 9a-c were then converted into 5-(1-acetox-
yvinyl)-cycloSal-d4TMPs 7a-c in moderate yields (19-23%).
For the synthesis of 4-(1-acetoxyvinyl)salicyl alcohols (9a-c)
the hydroxyl groups of 4-acetylsalicyl alcohols 8a,b were
protected as TBDPS ethers first. Compound 8a was converted
into 13a in an excellent yield of 90%, while protection of salicyl
alcohol 8b gave 13b in only 33% yield. The reaction of 8c
resulted in the mono-TBDPS ether at the benzylhydroxyl group.
Even the use of the sterically less demanding TBDMS protecting
group again only yielded the monoprotected derivative. So

8118 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Gisch et al.
Figure 3. CD spectra of (SP)-5 (A) and (RP)-5 (B) compared to 6c-fast (C) and 6c-slow (D).
Characterization of all triesters was carried out by means of
1H, 13C, and 31P NMR spectroscopy as well as high-resolution
mass spectrometry (HR-MS).
d4TMPs 7 was examined in CEM/0 cell extracts (CE). All half-
lives are summarized in Table 1. For comparison, the corre-
sponding data of triesters 3c,e and 5 are given as well.
As for 5-formyl- (5) and 5-diacyloxymethyl-3-t-Bu-cycloSal-
d4TMPs 3c,e,7 a separation of the diastereomers of 5-acetyl-
(6c) and 5-(1-acetoxyvinyl)-3-t-Bu-cycloSal-d4TMP 7c was
achieved using preparative RP-HPLC. The slow-eluting dias-
tereomer of 6c crystallized under the same conditions as the
slow-eluting (RP)-5. Unfortunately, the obtained crystals were
not suitable for X-ray analysis. But because of the structural
similarity of compounds 5 and 6c, circular dichroism (CD) was
used for the attribution of the absolute configuration at the
phosphorus atom. The CD spectra of (SP)-5 and (RP)-5 were
compared with those of 6c-fast and 6c-slow (Figure 3). Because
of the similar spectra, it is obvious that the (SP)-configuration
can be attributed to 6c-fast and the (RP)-configuration to 6c-
slow. Analytical HPLC studies in CEM/0 cell extracts showed
the enzymatic cleavage of 7c-fast to (SP)-6c and of 7c-slow to
(RP)-6c, respectively (see SI). According to this, 7c-fast should
be in the (SP)-configuration while 7c-slow is the (RP)-isomer.
Hydrolysis Studies. Target compounds 6 and 7 have been
studied for their hydrolytic stability at physiological pH 7.3 in
aqueous 25 mM phosphate buffer (PBS) and in the incubation
medium for the antiviral test (RPMI/heat-inactivated FCS (10%),
pH 7.6). The given half-lives refer to the disappearance of the
triesters. The enzymatic cleavage of 5-(1-acetoxyvinyl)-cycloSal-
Chemical Hydrolysis. As expected from our work on
5-formyl- (as 5) and 5-di-AM-cycloSal-d4TMPs 3a-c,7 alkyl
groups in position 3 of the aromatic ring also led to a higher
hydrolysis stability in the case of the 5-acetyl triester (6a) or
5-(1-acetoxyvinyl) triester (7a). According to the studies of the
diastereomeric mixtures in phosphate buffer (PBS), pH 7.3, the
methyl group increases the stability of the acetyl compound 2.5-
fold (6b) and the tert-butyl group led to an 8-fold increase in
hydrolytic stability (6c). In the case of 5-(1-acetoxyvinyl)
compounds 7, the methyl group increases the hydrolysis half-
life 4.5-fold, while the tert-butyl group led to a 9-fold increase.
Similar changes were found for 5-di-AM compounds 3a-c
(t1/2 ) 1.2 h (3-H, 3a), 2.3 h (3-Me, 3b), 7.9 h (3-t-Bu, 3c))
and the 5-formyl-cycloSal-d4TMPs (t1/2 ) 0.18 h (3-H), 0.35 h
(3-Me), 0.95 h (3-t-Bu, 5)).7 Surprisingly, the separated dia-
stereomers of triester 6c showed no difference in the chemical
stability and the difference in half-lives of the fast-eluting 5-(1-
acetoxyvinyl)-cycloSal-d4TMP (SP)-7c (t1/2 ) 13.5 h) and the
slow-eluting (RP)-7c (t1/2 ) 11.3 h) was unusually small.
Hydrolysis studies of compounds 6 in a 1:1 mixture of
DMSO-d6 and imidazol/HCl buffer (pH 7.3) followed by 31P
NMR spectroscopy revealed that d4TMP 1 was the exclusive
phosphorus containing hydrolysis product. Analogous studies

5-(1-AcetoxyVinyl)-cycloSal-d4TMPs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8119
Table 1. Hydrolysis Data and Antiviral Activity of 6a-c and 7a-c Compared to 2, 3c,e, and 5
EC50 [µM]b
t1/2 [h]a
CEM/0g
substituent
5-acetyl
5-acetyl-3-Me
5-acetyl-3-t-Bu
5-acetyl-3-t-Bu
5-acetyl-3-t-Bu
5-(1-acyl)
5-(1-acyl)-3-Me
5-(1-acyl)-3-t-Bu
5-(1-acyl)-3-t-Bu
5-(1-acyl)-3-t-Bu
5-di-AM-3-t-Bu
5-di-AM-3-t-Bu
5-di-AM-3-t-Bu
5-di-iBOM-3-t-Bu
5-di-iBOM-3-t-Bu
5-di-iBOM-3-t-Bu
5-CHO-3-t-Bu
5-CHO-3-t-Bu
5-CHO-3-t-Bu
PBS,d pH 7.3
CEe
RPMI/FCSf
HIV-1
HIV-2
CEM/TK-,h HIV-2
CC50[µM]c
6a (mix)
6b (mix)
6c (mix)
6c (fast)
6c (slow)
7a (mix)
7b (mix)
7c (mix)
7c (fast)
7c (slow)
3c (mix)
3c (fast)
3c (slow)
3e (mix)
3e (fast)
3e (slow)
5 (mix)
0.3
0.75
2.3
2.3
2.3
1.4
6.1
12.3
13.5
11.3
7.9
7.9
8.3
3.5
3.3
3.8
1.0
1.3
1.0
nai
ndj
0.6
1.0
2.5
3.2
2.0
1.0
1.8
5.2
7.6
3.7
2.8
2.9
2.8
5.9
5.7
4.5
1.5
1.7
1.6
nai
1.3 ( 0.071
0.54 ( 0.19
0.45 ( 0.31
0.18 ( 0.0071
0.75 ( 0.049
0.65 ( 0.35
0.18 ( 0.071
0.47 ( 0.099
0.21 ( 0.035
0.47 ( 0.34
0.65 ( 0.35
0.54 ( 0.51
1.1 ( 1.3
0.53 ( 0.45
0.72 ( 0.68
0.89 ( 0.16
0.39 ( 0.40
0.16 ( 0.071
0.81 ( 0.042
0.85 ( 0.64
3.0 ( 3.0
2.2 ( 2.3
0.87 ( 0.12
0.51 ( 0.39
1.2 ( 1.1
0.80 ( 0.13
0.40 ( 0.23
0.88 ( 0.45
0.80 ( 0.57
1.1 ( 0.44
0.80 ( 0.13
0.75 ( 0.49
0.84 ( 0.085
2.0 ( 2.3
0.85 ( 0.64
1.9 ( 2.1
1.0 ( 0.22
0.69 ( 0.41
1.4 ( 0.81
1.1 ( 0.49
>50
148
96
49
56
64
111
18
49
ndj
36 ( 0.0
1.7 ( 0.42
0.55 ( 0.36
2.8 ( 1.1
>50
ndj
ndj
ndj
0.13
0.07
0.04
0.04
0.04
0.15
0.3
0.15
1.0
1.0
0.73 ( 0.042
1.7 ( 2.0
0.27 ( 0.12
1.7 ( 0.42
0.78 ( 0.0
0.29 ( 0.16
3.9 ( 2.7
2.4 ( 2.3
1.4 ( 0.91
3.6 ( 0.42
15 ( 6.4
2.6 ( 1.9
10 ( 0.0
69 ( 70
20
67
19 ( 0.0
17 ( 2.1
51 ( 21
20 ( 3.5
14 ( 1.4
40 ( 1.4
33 ( 7.8
27 ( 11
41 ( 1.4
220
0.8
ndj
5 (fast)
5 (slow)
2
ndj
ndj
nai
a Hydrolysis half-lives. b Antiviral activity in T-lymphocytes: 50% effective concentration (shown values are mean values of two to three independent
experiments). c Cytostatic activity: 50% cytostatic concentration. d 25 mM phosphate buffer. e CEM cell extracts (pH 6.9). f RPMI/10% heat-inactivated fetal
calf serum (FCS), pH 7.6. g Wild-type CEM cells. h Thymidine kinase-deficient CEM cells. i na: not applicable. j nd: not determined.
in a DMSO-d6/PBS mixture (pH 7.3) showed an identical picture
(except the appearance of a small amount of d4TDP resulting
from the hydrolysis of triesters 6 by phosphate).
FCS culture medium than 7b (t1/2 ) 5.2 h (7c) vs t1/2 ) 1.8 h
(7b)), the anti-HIV activity of these two compounds is similar.
So an effective trapping of the released acetyl compound seems
to be just as important as sufficient hydrolysis stability. In CEM/
TK- cells a complete loss of activity was only observed for
compounds showing very low hydrolysis stabilities (6a,b and
7a). According to the low chemical stabilities, these compounds
seem to hydrolyze almost completely in the extracellular
medium releasing d4TMP 1. D4TMP 1 is dephosphorylated to
d4T 2, which then acts in its usual way in CEM/0 cells. In
summary, the idea of our concept is most obvious when
compounds 6b and 7b are compared. Compound 6b is hardly
active in CEM/TK--deficient cells most probably because of
its low chemical stability (0.75 h), whereas derivative 7b, which
is the lipophilic precursor of 6b, is fully active in this cell line
(chemical stability, 6.1 h). All data presented show that 7b is
cleaved intracellularly after membrane passage into 6b that
rapidly releases d4TMP.
Enzymatic Hydrolysis. In contrast to chemical hydrolysis,
studies in T-lymphocyte CEM cell extracts showed an extremely
fast cleavage (t1/2 ) 0.04-0.13 h) of all 5-(1-acetoxyvinyl)-
cycloSal-d4TMPs 7 (t1/2 ) 1.4-13.5 h; up to a 300-fold
increase) to the corresponding 5-acetyl-cycloSal-d4TMP 6. Thus,
enolic esters 7 are suitable compounds for the concept of
enzymatic activation. The absolute configuration at the phos-
phorus atom had no influence on the enzymatic cleavage reaction
(compare compounds 7c). In contrast, the corresponding tests
for 5-diacyloxymethyl-cycloSal-d4TMPs 3c,e showed a slower
cleavage of the (SP)-isomers. The half-lives of triesters 7 in
RPMI-1640 culture medium containing 10% heat-inactivated
fetal calf serum (RPMI/FCS (10%)) were determined to be
t1/2 ) 1.0 h (7a), t1/2 ) 1.8 h (7b), and t1/2 ) 5.2 h (7c). These
values are lower than the stabilities found in the PBS studies.
A similar decrease of hydrolysis stability has previously been
found for the 5-di-AM-cycloSal-d4TMPs 3a-c, especially for
3c.7 The cause of the reduced stability in RPMI culture medium
seems to be the same in both series of compounds: on one hand
a partial cleavage of the enolic ester to the acetyl group has
been observed that may be a result of remaining esterase activity
(although the calf serum is heat-inactivated); on the other hand
the RPMI/FCS medium has a more basic pH value (7.6).
Interestingly, the separated diastereomers of 6c and 7c showed
the same result as the previously reported compounds 3c,e and
5:7 for all derivatives the fast-eluting (SP)-isomer is significantly
more antivirally active than its (RP)-counterpart.
Conclusion
A synthetic route to various 5-acetyl- (6) and 5-(1-acetox-
yvinyl)-cycloSal-d4TMPs 7 has been developed successfully.
These compounds were analyzed with regard to their hydrolytic
and antiviral behavior. It was shown that the enzymatic cleavage
of the acetoxyvinyl group to the acetyl group occurred rapidly
in CEM/0 cell extract in contrast to chemical hydrolysis.
Moreover, a selective release of d4TMP 1 from 5-acetyl triesters
6 was also proven in 31P NMR studies. All compounds proved
to be potent inhibitors of HIV-1 and HIV-2 replication in wild-
type CEM/0 cells, and at least 5-(1-acetoxyvinyl)-cycloSal-
d4TMPs 7b and 7c retained entirely their antiviral potency in
CEM/TK- cells. In addition, compounds 6c and 7c were
separated into their stereoisomers by preparative RP-HPLC and
the absolute configuration at the phosphorus was assigned. The
antiviral activity of the fast-eluting (SP)-diastereomers consis-
Antiviral Evaluation. The cycloSal triesters 6 and 7 were
evaluated for their in vitro anti-HIV activity (Table 1). All
compounds showed comparable or even higher antiviral potency
against HIV-1 and HIV-2 in wild-type CEM/0 cells than d4T
2. More importantly, 5-(1-acetoxyvinyl)-3-alkyl-cycloSal-
d4TMPs 7b and 7c-fast showed full retention of antiviral activity
in CEM/TK- cells. Surprisingly, 5-acetyl-cycloSal-d4TMP 6c
showed full retention of the activity as well, while 5-acetyl-3-
methyl-cycloSal derivative 6b entirely lost its antiviral potency.
This may be due to a higher lipophilicity of 6c compared to
6a,b in addition to a sufficient hydrolysis stability allowing the
transport into cells. Interestingly, although compound 7c is
significantly more stable against chemical hydrolysis in RPMI/

8120 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Gisch et al.
and the phases were separated with ethyl acetate. The aqueous
layer was extracted with ethyl acetate twice, and the combined
organic layers were dried with magnesium sulfate. The products
were purified by column chromatography (petroleum ether
50-70/ethyl acetate 1:1 (8b) or 4:1 (8c) or CH2Cl2/CH3OH 19:1
(8a).
tently proved to be superior to the antiviral activity of the (RP)-
diastereomers. In conclusion, 5-(1-acetoxyvinyl)-cycloSal-
d4TMPs 7 can be used as enzymatically activated prodrugs.
We are now focusing our attention to enolic esters in which
the ester moiety is stabilized against hydrolytic cleavage in the
RPMI/FCS (10%).
General Procedure B: Preparation of Bis-O-(tert-butyldiphe-
nylsilyl)-4-acetylsalicyl Alcohols (13). Under nitrogen, the respec-
tive 4-acetylsalicyl alcohol (8, 1.0 equiv) was dissolved in dry
pyridine. After the addition of imidazole (7.0 equiv) and tert-
butyldiphenylsilyl chloride (4.0 equiv), the solution was stirred
at room temperature until the reaction was completed. At 0 °C
the reaction mixture was diluted with water and the phases were
separated with ethyl acetate. The aqueous layer was extracted
with ethyl acetate three times. The combined organic layers were
dried with sodium sulfate and concentrated under reduced
pressure. The products were purified by column chromatography
(petroleum ether 50-70/CH2Cl2 2:1 (13b) or CH2Cl2 (13a)).
General Procedure C: Preparation of Di-tert-butylsilyl Pro-
tected Salicyl Alcohols (16, 17b). Under nitrogen, the respective
salicyl alcohol (8b or 15, 1.0 equiv) was dissolved in dry CH2Cl2.
After the addition of dry pyridine (4.4 equiv) and di-tert-butylsilyl
bis(trifluoromethanesulfonate) (1.3 equiv), the solution was stirred
at room temperature for 2 h. At 0 °C the reaction mixture was
diluted with water and the phases were separated. The aqueous layer
was extracted with CH2Cl2 twice, and the combined organic layers
were dried with sodium sulfate and concentrated under reduced
pressure. The products were purified by flash column chromatog-
raphy (petroleum ether 50-70/CH2Cl2 7:1 (16) or preparative TLC
[Chromatotron; petroleum ether 50-70/CH2Cl2 1:2 (17b)].
General Procedure D: Preparation of Silyl Protected 4-(1-
Acetoxyvinyl)salicyl Alcohols (14, 18). Under nitrogen, the cor-
responding silyl protected 4-acetylsalicyl alcohol (13 or 17, 1.0
equiv) was dissolved in isopropenyl acetate, and p-toluenesulfonic
acid (0.3 equiv) was added. The solution was stirred at 110 °C for
the given time. After the addition of water the phases were separated
with diethyl ether and the aqueous layer was extracted with diethyl
ether twice. The combined organic layers were dried with sodium
sulfate and concentrated under reduced pressure. The products were
purified by column chromatography (petroleum ether 50-70/CH2Cl2
1:1 (14a) or 1:2 (18b), petroleum ether 50-70/CH2Cl2 gradient
(25-50%, 14b) or CH2Cl2 (18c)).
General Procedure E: Cleavage of Silyl Protecting Groups.
The corresponding silyl protected 4-acetylsalicyl alcohol (17b, 1.0
equiv) or 4-(1-acetoxyvinyl)salicyl alcohol (14, 18, 1.0 equiv) was
dissolved in THF, and triethylamine trihydrofluoride (3.0 equiv)
was added. The solution was stirred at room temperature for the
given time, and afterward the phases were separated with diethyl
ether and water. The aqueous layer was extracted with diethyl ether
once, the combined organic layers were washed with a 5% sodium
bicarbonate solution, and the solvent was removed in vacuum. The
products were purified by preparative TLC [Chromatotron; petro-
leum ether 50-70/ethyl acetate 6:1 (8c), CH2Cl2 (9c) or CH2Cl2/
CH3OH gradient (0-2%, 9b (starting from 18b)) or (0-3%, 9a)
or (0-10%, 9b (starting from 14b))].
General Procedure F: Preparation of cycloSal-d4T Mono-
phosphates (6, 7). Method A. Under nitrogen, a solution of the
respective salicyl alcohol (8, 9) in dry diethyl ether was cooled to
-20 °C. After dropwise addition of freshly distilled phosphorus(III)
chloride and stirring at -20 °C for 10 min, a solution of dry pyridine
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 up to room temperature and stirred for 1.5 h. It
was kept at -20 °C overnight for the best possible precipitation of
pyridinium chloride. Filtration under nitrogen and concentration
of the filtrate under reduced pressure yielded the phosphitylating
agent (saligenyl chlorophosphite) as a crude product, which was
directly used for the synthesis of the cycloSal-d4T monophosphate
without further purification.
Experimental Section
NMR spectra were recorded with a Bruker AMX 400, Bruker
AV 400, or a Bruker DRX 500 Fourier transform spectrometer.
1
All H and 13C NMR chemical shifts (δ) are quoted in parts per
million (ppm) downfield from tetramethylsilane and calibrated
on solvent signals. The 31P NMR chemical shifts (proton
decoupled) are quoted in ppm using H3PO4 as the external
reference. The spectra were recorded at room temperature. Mass
spectra were obtained with a VG Analytical VG/70-250 F
spectrometer [FAB, (double focusing), matrix m-nitrobenzyl
alcohol]. ESI mass spectra were recorded with a VG Analytical
Finnigan ThermoQuest MAT 95 XL spectrometer. For thin layer
chromatography (TLC) VWR precoated 60 F254 plates with a
0.2 mm layer of silica gel (VWR no. 5554) were used; sugar-
containing compounds were visualized with sugar spray reagent
(0.5 mL of 4-methoxybenzaldehyde, 9 mL of EtOH, 0.5 mL of
concentrated sulfuric acid, and 0.1 mL of glacial acetic acid).
All preparative TLCs were performed on a chromatotron
(Harrison Research, model 7924T) using glass plates coated with
1, 2, or 4 mm layers of VWR 60 PF254 silica gel containing a
fluorescent indicator (VWR no. 7749). For column chromatog-
raphy, Merck silica gel 60, 230-400 mesh, was used. UV spectra
were recorded on a Varian Cary 1E UV-visible spectro-
photometer, and absorption maximum wavelengths λmax are given
in nm. UV absorptions of cycloSal nucleotides were determined
from their HPLC data (diode array detector). Circular dichroism
spectra were recorded on an AVIV CD instrument model 215.
Analytical HPLC was performed on a Merck-Hitachi HPLC
system (D-7000) equipped with a LiChroCART 125-3 column
containing reversed phase silica gel Lichrospher 100 RP 18 (5
µM; Merck, Darmstadt, Germany). Preparative HPLC was
carried out on an HPLC system consisting of a Merck-Hitachi
L-6250 Intelligent Pump, a Merck-Hitachi LaChrom UV detector
L-7400, and a Merck Hitachi D-2500A Chromato-Integrator
using a Merck Hibar RT 250-25 column containing reversed
phase silica gel Lichrospher 100 RP 18 (5 µM; Merck,
Darmstadt, Germany). The flow rate was 10 mL/min, and
detection was performed at a wavelength of 260 nm. Diethyl
ether was dried over sodium/benzophenone and distilled under
nitrogen. THF was dried over potassium/benzophenone and
distilled under nitrogen. Pyridine, CH2Cl2, and CH3CN were
distilled from calcium hydride under nitrogen. N,N-Diisopro-
pylethylamine and triethylamine were destilled from sodium prior
to use. Isopropenyl acetate was distilled under nitrogen prior to
use. The solvent for HPLC (CH3CN) was obtained from Acros
(HPLC grade).
General Procedure A: Preparation of 4-Acetylsalicyl Alco-
hols (8). The respective 4-hydroxyacetophenone (10) was
suspended in concentrated hydrochloric acid, and formaldehyde
solution (37%) was added. The reaction mixture was stirred at
the temperature mentioned below until the reaction was com-
pleted. In case of 4-hydroxyacetophenone (10a), the crude
chloromethylated product 12a was obtained by filtration as a
red solid. In case of compounds 10b,c ethyl acetate was added
to dissolve the generated violet precipitates, and the phases were
separated. The aqueous layer was extracted with ethyl acetate
twice, and the combined organic layers were dried with sodium
sulfate. The solvent was removed in vacuum to yield violet
crystals (12b,c). The crystals of 12a-c were dissolved in aqueous
THF, and additional water and calcium carbonate were added.
The reaction mixture was stirred at the temperature mentioned
below until the reaction was completed. Concentrated hydro-
chloric acid was added dropwise to adjust the pH to less than 7,
Under nitrogen, d4T (2, 1.0 equiv) was dissolved in dry CH3CN
and cooled to -20 °C. Then DIPEA (1.6 equiv) and the respective

5-(1-AcetoxyVinyl)-cycloSal-d4TMPs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8121
saligenyl chlorophosphite (2.0 equiv) in dry CH3CN were added.
The reaction mixture was stirred at room temperature until the
conversion of d4T 2 was completed. Subsequently, tert-butyl
hydroperoxide (5.5 M in n-nonane, 3.0 equiv) was added at -20
°C. The solution was stirred at room temperature until the oxidation
was completed and then poured into water or a suitable buffer
solution. The phases were separated with ethyl acetate, the aqueous
layer was extracted with ethyl acetate three times, and the combined
organic layers were dried with sodium sulfate and concentrated
under reduced pressure. The resulting residue was purified by
preparative TLC [Chromatotron; CH2Cl2/CH3OH gradient (0-5%)].
The isolated product was lyophilized from CH3CN/H2O 1:1.
Method B. Method B was performed as described for method
A, but a 3:1 mixture of dry diethyl ether and dry THF was used as
a solvent in the synthesis of the saligenyl chlorophosphite.
phite dissolved in 10 mL of dry CH3CN, 246 mg (1.10 mmol) of
d4T (2) dissolved in 25 mL of dry CH3CN, 0.30 mL (1.7 mmol)
of DIPEA, and 0.60 mL (5.5 M in n-nonane, 3.3 mmol) of tert-
butyl hydroperoxide. Reaction time: 3 h. Oxidation time: 2 h. Prior
to purification by preparative TLC, flash chromatography (ethyl
acetate/CH3OH 9:1) was performed. Yield: 97.3 mg (0.198 mmol,
18%) of a diastereomeric mixture (ratio 0.8:1) as a colorless foam.
1H NMR (500 MHz, DMSO-d6): δ ) 11.35 (s, 1H, 1 × NH), 11.32
(s, 1H, 1 × NH), 7.90-7.85 (m, 4H, 2 × H-4, 2 × H-6), 7.22 (d,
J ) 1.0 Hz, 1H, 1 × thymine-H-6), 7.19 (d, J ) 1.0 Hz, 1H, 1 ×
thymine-H-6), 6.86-6.78 (m, 2H, 2 × 1′-H), 6.46-6.39 (m, 2H, 2
× 3′-H), 6.07-6.01 (m, 2H, 2 × 2′-H), 5.59-5.39 (m, 4H, 4 ×
benzyl-H), 5.01-4.96 (m, 2H, 2 × 4′-H), 4.38-4.30 (m, 4H, 4 ×
5′-H), 2.56 (s, 6H, 2 × OCH3), 1.60 (d, J ) 1.0 Hz, 3H, 1 ×
thymine-CH3), 1.59 (d, J ) 1.0 Hz, 3H, 1 × thymine-CH3), 1.37
(s, 9H, 1 × t-Bu-CH3), 1.34 (s, 9H, 1 × t-Bu-CH3) ppm. 31P NMR
(162 MHz, DMSO-d6): δ ) -9.12, -9.42 ppm.
The diastereomers were separated by preparative RP-HPLC
(CH3CN/H2O 1:2) in which an amount of 45.0 mg of the
diastereomeric mixture of 6c was used to yield 21.2 mg of 6c-fast
and 17.4 mg of 6c-slow. Analytical data of 6c-fast are as follows.
1H NMR (500 MHz, DMSO-d6): δ ) 11.35 (s, 1H, NH), 7.88-7.85
(m, 2H, aryl-H-4, aryl-H-6), 7.22 (s, 1H, thymine-H-6), 6.83-6.79
(m, 1H, 1′-H), 6.46-6.40 (m, 1H, 3′-H), 6.06-6.01 (m, 1H, 2′-
H), 5.58-5.42 (m, 2H, benzyl-H), 5.01-4.96 (m, 1H, 4′-H),
4.38-4.30 (m, 2H, 5′-H), 2.56 (s, 3H, H-9), 1.60 (s, 3H, thymine-
CH3), 1.34 (s, 9H, t-Bu) ppm. 31P NMR (161 MHz, DMSO-d6): δ
) -9.42 ppm. Analytical data of 6c-slow are as follows. 1H NMR
(500 MHz, DMSO-d6): δ ) 11.32 (s, 1H, NH), 7.90-7.85 (m, 2H,
aryl-H-4, aryl-H-6), 7.19 (s, 1H, thymine-H-6), 6.83-6.78 (m, 1H,
1′-H), 6.43-6.39 (m, 1H, 3′-H), 6.07-6.02 (m, 1H, 2′-H),
5.59-5.39 (m, 2H, benzyl-H), 5.01-4.96 (m, 1H, 4′-H), 4.39-4.31
(m, 2H, 5′-H), 2.56 (s, 3H, COCH3), 1.59 (s, 3H, thymine-CH3),
1.37 (s, 9H, t-Bu-CH3) ppm. 31P NMR (161 MHz, DMSO-d6): δ
) -9.12 ppm.
5-(1-Acetoxyvinyl)-cycloSal-d4T Monophosphate (7a). General
procedure F (method A) was used, with 393 mg (1.89 mmol) of
4-(1-acetoxyvinyl)salicyl alcohol (9a) dissolved in 20 mL of dry
diethyl ether, 0.20 mL (2.3 mmol) of phosphorus(III) chloride, and
0.35 mL (4.4 mmol) of dry pyridine in 1.75 mL of dry diethyl
ether. Yield: 340 mg. Quantities for cycloSal-d4T monophosphate
synthesis were as follows: 335 mg of crude saligenyl chlorophos-
phite dissolved in 5 mL of dry CH3CN, 149 mg (0.665 mmol) of
d4T (2) dissolved in 15 mL of dry CH3CN, 0.17 mL (0.98 mmol)
of DIPEA, and 0.35 mL (5.5 M in n-nonane; 2.0 mmol) of tert-
butyl hydroperoxide. Reaction time: 1.5 h. Oxidation time: 30 min.
Buffer: phosphate (pH 7.3). Yield: 61.0 mg (0.128 mmol, 19%) of
a diastereomeric mixture (ratio 0.6:1) as a colorless foam. 1H NMR
(400 MHz, DMSO-d6): δ ) 11.34 (s, 2H, 2 × NH), 7.56-7.51
(m, 2H, 2 × aryl-H-4), 7.50-7.46 (m, 2H, aryl-H-6), 7.20 (d, J )
1.1 Hz, 1H, 1 × thymine-H-6), 7.18 (d, J ) 1.1 Hz, 1H, 1 ×
thymine-H-6), 7.17-7.11 (m, 2H, 2 × aryl-H-3), 6.84-6.76 (m,
2H, 2 × 1′-H), 6.42 (ddd, J ) 6.2, 1.7, 1.7 Hz, 1H, 1 × 3′-H),
6.36 (ddd, J ) 6.2, 1.8, 1.8 Hz, 1H, 1 × 3′-H), 6.05-5.95 (m, 2H,
1 × 2′-H), 5.65-5.64 (m, 2H, 2 × CHAHB), 5.58-5.35 (m, 4H, 4
× benzyl-H), 5.03 (d, J ) 2.3 Hz, 2H, 2 × CHAHB), 4.99-4.93
(m, 2H, 2 × 4′-H), 4.38-4.25 (m, 4H, 4 × 5′-H), 2.26 (s, 6H, 2 ×
OCOCH3), 1.68 (d, J ) 1.0 Hz, 3H, 1 × thymine-CH3), 1.63 (d, J
) 1.0 Hz, 3H, 1 × thymine-CH3) ppm. 31P NMR (162 MHz,
DMSO-d6): δ ) -9.46, -9.49 ppm.
5-(1-Acetoxyvinyl)-3-methyl-cycloSal-d4T Monophosphate
(7b). General procedure F (method A) was used, with 415 mg (1.87
mmol) of 4-(1-acetoxyvinyl)-6-methylsalicyl alcohol (9b), dissolved
in 20 mL of dry diethyl ether, 0.20 mL (2.3 mmol) of phospho-
rus(III) chloride, and 0.35 mL (4.3 mmol) of dry pyridine in 1.8
mL of dry diethyl ether. Yield: 435 mg. Quantities for cycloSal-
d4T monophosphate synthesis were as follows: 205 mg of crude
saligenyl chlorophosphite dissolved in 5 mL of dry CH3CN, 68
mg (0.30 mmol) of d4T (2) dissolved in 6 mL of dry CH3CN, 80
µL (0.45 mmol) of DIPEA, and 0.16 mL (5.5 M in n-nonane, 0.88
mmol) of tert-butyl hydroperoxide. Reaction time: 1.5 h. Oxidation
5-Acetyl-cycloSal-d4T Monophosphate (6a). General procedure
F (method B) was used, with 500 mg (3.01 mmol) of 4-acetylsalicyl
alcohol (8a) dissolved in 30 mL of dry diethyl ether and 10 mL of
dry THF, 0.25 mL (2.9 mmol) of phosphorus(III) chloride, and
0.44 mL (5.5 mmol) of dry pyridine in 2.5 mL of dry diethyl ether.
Yield: 616 mg. Quantities for cycloSal-d4T monophosphate syn-
thesis were as follows: 305 mg of crude saligenyl chlorophosphite
dissolved in 4 mL of dry CH3CN, 195 mg (0.870 mmol) of d4T
(2) dissolved in 9 mL of dry CH3CN, 0.22 mL (1.3 mmol) of
DIPEA, and 0.48 mL (5.5 M in n-nonane, 2.7 mmol) of tert-butyl
hydroperoxide. Reaction time: 3 h. Oxidation time: 1 h. Buffer: 1
M HOAc/NaOAc (pH 5). Yield: 83 mg (0.19 mmol, 22%) of a
1
diastereomeric mixture (ratio 0.4:1) as a colorless foam. H NMR
(400 MHz, DMSO-d6): δ ) 11.36-11.33 (m, 2H, 2 × NH),
7.97-7.91 (m, 4H, 2 × aryl-H-4, 2 × aryl-H-6), 7.26 (d, J ) 8.5
Hz, 1H, 1 × aryl-H-3), 7.22 (d, J ) 8.5 Hz, 1H, 1 × aryl-H-3),
7.17 (d, J ) 1.3 Hz, 1H, 1 × thymine-H-6), 7.16 (d, J ) 1.3 Hz,
1H, 1 × thymine-H-6), 6.81-6.78 (m, 1H, 1 × 1′-H), 6.78-6.75
(m, 1H, 1 × 1′-H), 6.43 (ddd, J ) 6.0, 1.8, 1.8 Hz, 1H, 1 × 3′-H),
6.37 (ddd, J ) 6.0, 1.8, 1.5 Hz, 1H, 1 × 3′-H), 6.04-6.00 (m, 2H,
2 × 2′-H), 5.62-5.53 (m, 2H, 2 × benzyl-H), 5.49-5.37 (m, 2H,
2 × benzyl-H), 4.98-4.94 (m, 2H, 2 × 4′-H), 4.37-4.24 (m, 4H,
4 × 5′-H), 2.55 (s, 6H, 2 × COCH3,), 1.67 (d, J ) 1.3 Hz, 3H, 1
× thymine-CH3), 1.62 (d, J ) 1.3 Hz, 3H, 1 × thymine-CH3) ppm.
31P NMR (162 MHz, DMSO-d6): δ ) -9.69, -9.89 ppm.
5-Acetyl-3-methyl-cycloSal-d4T Monophosphate (6b). General
procedure F (method B) was used, with 500 mg (2.77 mmol) of
4-acetyl-6-methylsalicyl alcohol (8b), dissolved in 30 mL of dry
diethyl ether and 10 mL of dry THF, 0.23 mL (2.6 mmol) of
phosphorus(III) chloride, and 0.40 mL (5.0 mmol) of dry pyridine
in 3 mL of dry diethyl ether. Yield: 610 mg. Quantities for cycloSal-
d4T monophosphate synthesis were as follows: 605 mg of crude
saligenyl chlorophosphite dissolved in 5 mL dry CH3CN, 283 mg
(1.26 mmol) of d4T (2) dissolved in 25 mL of dry CH3CN, 0.34
mL (2.0 mmol) of DIPEA, and 0.70 mL (5.5 M in n-nonane, 3.9
mmol) of tert-butyl hydroperoxide. Reaction time: 2 h. Oxidation
time: 30 min. Buffer: 1 M HOAc/NaOAc (pH 5). Yield: 143 mg
(0.319 mmol, 26%) of a diastereomeric mixture (ratio 0.5:1) as a
1
colorless foam. H NMR (400 MHz, DMSO-d6): δ ) 11.33 (s,
1H, 1 × NH), 11.31 (s, 1H, 1 × NH), 7.87 (s, 2H, 2 × aryl-H-4),
7.75 (s, 2H, 2 × aryl-H-6), 7.19 (d, J ) 1.3 Hz, 1H, 1 × thymine-
H-6), 7.15 (d, J ) 1.3 Hz, 1H, 1 × thymine-H-6), 6.81-6.77 (m,
2H, 2 × 1′-H), 6.43 (ddd, J ) 6.0, 1.8, 1.8 Hz, 1H, 1 × 3′-H),
6.38 (ddd, J ) 6.0, 1.8, 1.8 Hz, 1H, 1 × 3′-H), 6.05-6.00 (m, 2H,
2 × 2′-H), 5.61-5.49 (m, 2H, 2 × benzyl-H), 5.48-5.37 (m, 2H,
2 × benzyl-H), 5.00-4.92 (m, 2H, 2 × 4′-H), 4.36-4.23 (m, 4H,
4 × 5′-H), 2.54 (s, 6H, 2 × COCH3), 2.27 (s, 3H, 1 × CH3), 2.24
(s, 3H, 1 × CH3), 1.66-1.60 (m, 6H, 2 × thymine-CH3) ppm. 31
NMR (162 MHz, DMSO-d6): δ ) -8.87, -9.33 ppm.
P
5-Acetyl-3-tert-butyl-cycloSal-d4T Monophosphate (6c). Gen-
eral procedure F (method A) was used, with 500 mg (2.25 mmol)
of 4-acetyl-6-tert-butylsalicyl alcohol (8c), dissolved in 30 mL of
dry diethyl ether, 0.19 mL (2.2 mmol) of phosphorus(III) chloride,
and 0.32 mL (4.0 mmol) of dry pyridine in 3 mL of dry diethyl
ether. Yield: 629 mg. Quantities for cycloSal-d4T monophosphate
synthesis were as follows: 620 mg of crude saligenyl chlorophos-

8122 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Gisch et al.
time: 30 min. Prior to purification by preparative TLC, flash
chromatography (ethyl acetate/CH3OH 9:1) was performed. Pre-
parative TLC on the Chromatotron did not provide the pure product
(49 mg as a red foam), so final purification was achieved using
preparative RP-HPLC (CH3CN/H2O 1:1). Yield: 28.1 mg (57.3
µmol, 19%) of a diastereomeric mixture (ratio 0.8:1.0) as a colorless
extracts were performed as reported in ref 4 with different
incubation times but without using acetic acid to stop the reaction
for cycloSal nucleotides with acid sensitive substituents. Studies
in RPMI/FCS (10%) were carried out in the same way using culture
medium instead of cell extract.
Antiretroviral Evaluation. The method of antiviral evaluation
has been described in ref 6 and is based on the virus-induced
cytopathic effect (giant cell formation) in the CEM cell cultures.
31P NMR Hydrolysis Studies of cycloSal Phosphate Triesters.
31P NMR hydrolysis studies of cycloSal nucleotides 6 were carried
out as described before.16
1
foam. H NMR (400 MHz, DMSO-d6): δ ) 11.32 (s, 2H, 2 ×
NH), 7.47-7.43 (m, 2H, 2 × aryl-H-4), 7.30-7.27 (m, 2H, 2 ×
aryl-H-6), 7.21 (d, J ) 1.0 Hz, 1H, 1 × thymine-H-6), 7.20 (d, J
) 1.3 Hz, 1H, 1 × thymine-H-6), 6.83-6.79 (m, 2H, 2 × 1′-H),
6.41 (ddd, J ) 6.0, 1.8, 1.7 Hz, 1H, 1 × 3′-H), 6.36 (ddd, J ) 6.0,
1.8, 1.5 Hz, 1H, 1 × 3′-H), 6.06-5.99 (m, 2H, 2 × 2′-H), 5.62 (d,
J ) 2.3 Hz, 2H, 2 × CHAHB), 5.54-5.33 (m, 4H, 4 × benzyl-H),
5.01 (d, J ) 2.3 Hz, 2 × 1H, 2 × CHAHB), 4.98-4.92 (m, 2H, 2
× 4′-H), 4.34-4.22 (m, 4H, 4 × 5′-H), 2.26 (s, 6H, 2 × OCOCH3),
2.23 (s, 3H, 1 × CH3), 2.20 (s, 3H, 1 × CH3), 1.66 (d, J ) 1.0 Hz,
3H, 1 × thymine-CH3), 1.64 (d, J ) 1.0 Hz, 3H, 1 × thymine-
CH3) ppm. 31P NMR (161 MHz, DMSO-d6): δ ) -8.98, -9.02
ppm.
Acknowledgment. We are grateful to the Deutsche Fors-
chungsgemeinschaft (DFG), the University of Hamburg, the
“Geconcerteerde Onderzoeksacties” (GOA no. 05/019), and the
Fonds voor Wetenschappelijk Onderzoek (FWO)sVlaanderen
for financial support. The authors also thank Leen Ingels and
Ria Van Berwaer for excellent technical assistance.
5-(1-Acetoxyvinyl)-3-tert-butyl-cycloSal-d4T Monophosphate
(7c). General procedure F (method A) was used, with 500 mg (1.89
mmol) of 4-(1-acetoxyvinyl)-6-tert-butylsalicyl alcohol (9c), dis-
solved in 20 mL of dry diethyl ether, 0.20 mL (2.3 mmol) of
phosphorus(III) chloride, and 0.35 mL (4.3 mmol) of dry pyridine
in 1.8 mL of dry diethyl ether. Yield: 543 mg. Quantities for
cycloSal-d4T monophosphate synthesis were as follows: 534 mg
of crude saligenyl chlorophosphite dissolved in 8 mL of dry CH3CN,
145 mg (0.647 mmol) of d4T (2) dissolved in 14 mL of dry CH3CN,
0.17 mL (0.96 mmol) of DIPEA, and 0.34 mL (5.5 M in n-nonane,
1.9 mmol) of tert-butyl hydroperoxide. Reaction time: 1.5 h.
Oxidation time: 1.5 h. Prior to purification by preparative TLC,
flash chromatography (ethyl acetate/CH3OH 9:1) was performed.
Preparative TLC on the Chromatotron did not provide the pure
product (117 mg as a slightly fawn foam), so final purification was
achieved using preparative RP-HPLC. The diastereomeric mixture
was obtained by the use of CH3CN/H2O 1:1 as solvent for the
preparative RP-HPLC. The separated diastereomers were obtained
by the use of CH3CN/H2O 2:3. Yield: 79.2 mg (0.149 mmol, 23%);
39.6 mg of the diastereomeric mixture (ratio 0.8:1), as well as 17.7
mg of 7c-fast and 21.9 mg of 7c-slow as colorless foams. 1H NMR
(400 MHz, DMSO-d6): δ ) 11.34 (s, 2H, 2 × NH), 7.43-7.39
(m, 2H, 2 × aryl-H-4), 7.37-7.32 (m, 2H, 2 × aryl-H-6), 7.23 (d,
J ) 1.0 Hz, 1H, 1 × thymine-H-6), 7.20 (d, J ) 1.0 Hz, 1H, 1 ×
thymine-H-6), 6.84-6.79 (m, 2H, 2 × 1′-H), 6.44-6.38 (m, 2H, 2
× 3′-H), 6.06-6.00 (m, 2H, 2 × 2′-H), 5.67-5.63 (m, 2H, 2 ×
CHAHB), 5.50-5.35 (m, 4H, 4 × benzyl-H), 5.05-5.01 (m, 2H, 2
× CHAHB), 5.00-4.94 (m, 2H, 2 × 4′-H), 4.39-4.28 (m, 4H, 4 ×
5′-H), 2.26 (s, 6H, 2 × OCOCH3), 1.61 (s, 3H, 1 × thymine-CH3),
1.59 (s, 3H, 1 × thymine-CH3), 1.35 (s, 9H, 1 × t-Bu), 1.32 (s,
9H, 1 × t-Bu) ppm. 31P NMR (161 MHz, DMSO-d6): δ ) -8.80,
Supporting Information Available: Full characterization of
compounds 8a-c, 9a-c, 13a,b, 14a,b, 16, 17b,c, and 18b,c;
13C NMR and UV spectroscopic data, mass spectrometric data,
Rf values, melting points, analytical HPLC data of new com-
pounds; methods for HPLC analysis; general nomenclature of
salicyl alcohols and cycloSal-d4TMPs; HPLC chromatograms
of kinetic studies of 7c-fast and 7c-slow in CEM/0 cell extracts;
31P NMR hydrolysis of 6c in imidazole/HCl buffer (pH 7.3).
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
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1
-9.03 ppm. Analytical data of 7c-fast are as follows: H NMR
(400 MHz, DMSO-d6): δ ) 11.35 (s, 1H, NH), 7.43-7.39 (m, 1H,
aryl-H-4), 7.37-7.33 (m, 1H, aryl-H-6), 7.23 (d, J ) 1.3 Hz, 1H,
thymine-H-6), 6.84-6.80 (m, 1H, 1′-H), 6.44-6.39 (m, 1H, 3′-
H), 6.05-6.00 (m, 1H, 2′-H), 5.65 (d, J ) 2.3 Hz, 1H, CHAHB),
5.50-5.35 (m, 2H, 2 × benzyl-H), 5.03 (d, J ) 2.5 Hz, 1H,
CHAHB), 5.00-4.94 (m, 1H, 4′-H), 4.35-4.29 (m, 2H, 2 × 5′-H),
2.26 (s, 3H, OCOCH3), 1.61 (d, J ) 0.8 Hz, 3H, thymine-CH3),
1.32 (s, 9H, t-Bu) ppm. 31P NMR (161 MHz, DMSO-d6): δ ) -9.03
ppm. Analytical data of 7c-slow are as follows: 1H NMR (400 MHz,
DMSO-d6): δ ) 11.34 (s, 1H, NH), 7.44-7.38 (m, 1H, aryl-H-4),
7.37-7.31 (m, 1H, aryl-H-6), 7.20 (s, 1H, thymine-H-6), 6.84-6.77
(m, 1H, 1′-H), 6.44-6.36 (m, 1H, 3′-H), 6.07-5.99 (m, 1H, 2′-
H), 5.65 (d, J ) 1.5 Hz, 1H, CHAHB), 5.51-5.34 (m, 2H, 2 ×
benzyl-H), 5.03 (d, J ) 1.5 Hz, 1H, CHAHB), 5.00-4.93 (m, 1H,
4′-H), 4.39-4.28 (m, 2H, 2 × 5′-H), 2.26 (s, 3H, OCOCH3), 1.59
(s, 3H, thymine-CH3), 1.35 (s, 9H, t-Bu) ppm. 31P NMR (161 MHz,
DMSO-d6): δ ) -8.80 ppm.
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5-(1-AcetoxyVinyl)-cycloSal-d4TMPs
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