Doubly loaded cycloSaligenyl-pronucleotides - 5,5′-bis- (cycloSaligenyl-2′,3′-dideoxy-2′,3′-didehydrothymidine monophosphates)

Article abstract of DOI:10.1021/jm900164g

Recently, we reported on 3,3′-bis-(cycloSaligenyl-2′,3′- dideoxy-2′,3′-didehydrothymidine monophosphates) (3,3′-bis-(cycloSal-d4TMPs) 4) as the first pronucleotides with a mask-to-drug ratio of 1:2 that is still a novelty in the field of pronucleotides. H

Full text of DOI:10.1021/jm900164g

3464
J. Med. Chem. 2009, 52, 3464–3473
Doubly Loaded cycloSaligenyl-Pronucleotides – 5,5′-Bis-
(cycloSaligenyl-2′,3′-dideoxy-2′,3′-didehydrothymidine Monophosphates)
Nicolas Gisch,† 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 February 9, 2009
Recently, we reported on 3,3′-bis-(cycloSaligenyl-2′,3′-dideoxy-2′,3′-didehydrothymidine monophosphates)
(3,3′-bis-(cycloSal-d4TMPs) 4) as the first pronucleotides with a mask-to-drug ratio of 1:2 that is still a
novelty in the field of pronucleotides. Here, we report on a new set of compounds of these unique type of
cycloSaligenyl prodrugs 5 that bear a biaryl axis at the 5-position of the cycloSal residue. All compounds
5 showed pronounced in vitro activity against HIV-1 and HIV-2 in wild-type CEM cell cultures and better
retained their antiviral activities in thymidine kinase-deficient CEM cells than the compound 4 series.
Moreover, compound 5b is the first bis-(cycloSal-d4TMP) that even showed complete retention of antiviral
activity in TK-deficient CEM cells. The complex hydrolysis behavior of 5 was investigated, and the proposed
hydrolysis mechanism was proven by means of 31P NMR spectroscopy and HPLC analysis.
Introduction
diastereomers (RP and SP configuration).3 Therefore, bis-(cycloSal-
d4TMPs) as 4 were formed as mixtures of three diastereomers
(RP,RP; RP,SP; SP,SP) in a theoretical ratio of 1:2:1. However,
this ratio was not observed. For example, 4a was isolated in a
diastereomeric ratio of 1:2:2.4 In conclusion, there may be a
steric interaction between the two cycloSal-d4TMP units in 3,3′-
bis-(cycloSal-d4TMPs) 4 that led to some extent to stereodif-
ferentiation.
Here, we report on the synthesis, properties, and antiviral
evaluation of bis-(cycloSal-d4TMPs) linked via the two 5-posi-
tions of the cycloSal-residue. These 5,5′-bis-(cycloSal-d4TMPs)
5 (Figure 1) were expected to have two sterically independent
cycloSal-d4TMP units. Moreover, a detailed investigation of
the complex hydrolysis pathway of 5a was carried out.
The action of antiviral or antitumor active nucleoside
analogues is highly dependent on the efficient stepwise conver-
sion into the bioactive 5′-triphosphates via mono- and diphos-
phate formation mediated by cellular kinases. In the case of
the anti-HIV nucleoside 2′,3′-dideoxy-2′,3′-didehydrothymidine
(d4T 1, Figure 1), the first phosphorylation to its nucleotide
(d4TMP 2, Figure 1) by the salvage pathway enzyme thymidine
kinase (TK) is the anabolism-limiting step.1 To overcome this
bottleneck, pronucleotide systems for the selective intracellular
delivery of nucleotides have been developed.2 Among these
approaches, the cycloSala concept has been established as the
only system that releases nucleoside monophosphates by a pure
chemical hydrolysis mechanism.3 Moreover, cycloSal-pronucle-
otides (general structure: 3, Figure 1) are unique because they
display a mask-to-drug ratio of 1:1 while all other pronucleotide
systems have a higher ratio (2:1 to 4:1).2
Results and Discussion
Chemistry. For the synthesis of 5,5′-bis-(cycloSal-d4TMPs)
5 one of the synthetic routes developed for the synthesis of the
3,3′-counterpart 4a also proved to be suitable after a few
modifications.4 Starting from 4-bromosalicylisopropylidene ac-
etals 6a-c,5 a halogen-lithium exchange was performed, and
the resulting intermediate was used for an oxidative homocou-
pling with iron(III) acetylacetonate to yield the respective bis-
(isopropylidene)-3,3′-bis(hydroxymethyl)biphenyl-4,4′-di-
ols 7a-c in moderate to good yields (37-75%). For the
acidic deprotection of acetals 7 our previously reported
ultrafast method with hydrochloric acid5a was used to yield
biphenyl tetrols 8a-c. These reactions are summarized in
Scheme 1.
Target molecules 5a-c were synthesized using the method
via saligenyl chlorophosphites (9a-c, Scheme 2).6 These
phosphitylating agents were synthesized in yields of about
54-55% with moderate purities (purity grade, 59-68%;
impurity: pyridine hydrochloride) from tetrols 8. Subsequent
treatment of crude chlorophosphites 9a-c with d4T (1) and
oxidation with tert-butyl hydroperoxide (t-BuOOH) led to the
desired 5,5′-bis-(cycloSal-d4TMPs) 5a-c (28-37%). Neverthe-
less, the yields obtained here are 4- to 5-fold higher compared
to their 3,3′-bis-(cycloSal)-counterparts.
To improve the number of equivalents of released nucleotide
per masking unit (mask-to-drug ratio of 1:2), we recently
developed “dimeric” cycloSal pronucleotides 4 (Figure 1) using
the antiviral active nucleoside analogue d4T (1) for initial
studies.4 The 3,3′-bis-(cycloSal-d4TMPs) (4) showed good anti-
HIV activity in wild-type CEM cells and were significantly more
active than 1 in TK-deficient CEM cells (Table 2) although the
antiviral activity was not fully retained.
In the synthesis of compounds 4 a major problem was the
formation of a side product in considerable amounts. This
compound was formed during the formation of the bis-saligenyl
chlorophosphites starting from the corresponding tetrols. As a
consequence, the following reaction of the crude chlorophos-
phites with d4T (1) led to compounds 4 in only poor yields
(8% (4a), 11% (4b)).
Because of the nonstereoselective synthesis, the cycloSal-
pronucleotides 3 were always obtained as mixtures of two
* To whom correspondence should be addressed. Phone: +49-40-42838-
4324. Fax: +49-40-42838-5592. E-mail: chris.meier@chemie.uni-hamburg.de.
†
University of Hamburg.
Katholieke Universiteit Leuven.
‡
a Abbreviations: cycloSal, cycloSaligenyl; DTBS, di-tert-butylsilyl;
Fe(acac)3, iron(III) acetylacetonate.
10.1021/jm900164g CCC: $40.75
2009 American Chemical Society
Published on Web 05/13/2009

cycloSaligenyl-Pronucleotides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3465
Figure 1. Structures of d4T 1, d4TMP 2, “monomeric” cycloSal-d4TMP 3, and bis-(cycloSal-d4TMPs) 4 and 5.
Scheme 1. Synthesis of Biphenyl tetrols 8 Starting from Acetals 6a
a Reagents and conditions: Method A: (i) n-BuLi, THF, -80 °C, 1 h; (ii) Fe(acac)3, THF, -80 °C to room temp, 36-42 h. Method B: conc HCl,
CH3CN/H2O/THF), heat, 30 s or 3 min.
For 5c, Oxone (2 KHSO5 ·KHSO4 ·K2SO4) was tested as an
alternative oxidation reagent.7 This attempt resulted in a
comparable yield of 5c of 30%. For all bis-pronucleotides 5,
final purification was achieved by preparative HPLC.
and are summarized in Table 1. For comparison, the corresponding
data of triesters 4a,b are given as well.
Hydrolysis studies at physiological pH 7.3 revealed that 5,5′-
bis-(cycloSal-d4TMPs) 5a-c are less stable against chemical
hydrolysis than their 3,3′-counterparts 4a,b. This has been
expected from the earlier reported data of phenyl- or methyl-
substituted cycloSal-d4TMPs (for X (in 3, Figure 1) ) 3-Ph,
In the case of 5c, all three diastereomers could be separated
and were isolated in the theoretically expected ratio of 1:2:1.
The first (5c-fast) and the slowest eluting diastereomer (5c-slow)
showed only one signal in the 31P NMR spectra (-8.83 ppm
(fast) and -8.59 ppm (slow)), while for 5c-middle two signals
were observed in the spectra. This points to the fact that 5c-
middle is the (RP,SP)-isomer. Moreover, both diastereomers of
5c with identically configurated phosphorus atoms also showed
identical signals in the 1H NMR for both cycloSal-d4TMP units.
1H NMR spectra of 5c-middle appears like the formal “sum”
of the spectra of 5c-fast and 5c-slow. Diastereomers of 5a could
only be partially separated; the first eluting peak contained two
diastereomers, while the slow-eluting compound was identical
to 5a-slow. NMR spectra of these diastereomers led to the same
conclusions as made for 5c. The separation of the diastereomers
of 5b was not possible.
t1/2 ) 5.1 h; for X ) 5-Ph, t1/2 ) 3.1 h; for X ) 3-Me, t1/2
)
15 h; for X ) 5-Me, t1/2 ) 5.1 h).8
Additional alkyl substituents in the aromatic rings of 5 led
to a marked increase of the hydrolysis half-life with the tert-
butyl group having a significantly stronger influence than the
methyl group. For the unsubstituted compounds 4a and 5a a
remarkable influence of the stereochemistry at the phosphorus
centers was observed.
Interestingly, the unsubstituted compound 5a showed a
decrease in hydrolysis stability in RPMI/FCS (10%). While the
half-lives of 5a-mix and 5a-fast dropped 2-fold, the half-life of
5a-slow decreased from 3.3 to 1.0 h. This can be clearly
attributed to the more basic pH 7.6 as can be seen from the
values determined in PBS (pH 7.6). In contrast, the half-life of
the methyl-substituted 5b was identical in all three media
(t1/2 ) 2.2-2.3 h). The half-lives of 5c in PBS (pH 7.6) were
only slightly shorter than in PBS (pH 7.3) but are significantly
increased in RPMI/FCS (10%). For all samples of 5c (X) t-Bu)
Hydrolysis Studies. Compounds 5a-c were studied for their
hydrolytic stability at pH 7.3 (physiological) and pH 7.6 in aqueous
25 mM phosphate buffer (PBS) and in the incubation medium for
the antiviral tests (RPMI/heat-inactivated FCS (10%), pH 7.6). All
given half-lives refer to the disappearance of starting pronucleotides

3466 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Scheme 2. Synthesis of 5,5′-Bis-(cycloSal-d4TMPs) 5a
Gisch et al.
The only stable intermediate in this hydrolysis cascade is
compound 13. In order to use it as reference, a route for its
preparation was developed. Analogous studies have already been
made for the hydrolysis of 4a. However, there the corresponding
hydrolysis intermediate was neither detectable in the 31P NMR
spectra nor detectable in HPLC analysis.4
Synthesis of 13 was achieved starting from 4-bromosalicyl
alcohol 14.5a Compound 14 was protected using the di-tert-
butylsilyl protecting group (DTBS) to give 15 (81% yield).9
Then 15 was reacted with 4-bromosalicyl alcohol isopropylidene
acetal 6a (1:1 mixture) using the conditions of the oxidative
coupling described above. This reaction resulted in heterocou-
pling product 16 in 31% yield as well as in both homocoupling
products 7a (31% yield) and 17 (46% yield). The last two
compounds can be deprotected in excellent yields (7a, 89%;
17, 88%) to tetrol 8a and then can be used as starting material
in further syntheses, for example, for compound 5a (Scheme
2). These reactions are depicted in Scheme 4.
The DTBS group of 16 was removed selectively with
triethylamine trihydrofluoride (NEt3 ·3HF) to give salicyl alcohol
18 (96% yield). Subsequently, isopropylidene-protected pro-
nucleotide 19 (5-(isopropylidenesaligen-5′-yl)-cycloSal-d4TMP,
45% yield) was prepared via the chlorophosphite. Finally, acidic
deprotection of 19 led to reference compound 13 (85% yield).
These steps are also summarized in Scheme 4.
a Reagents and conditions. Method C: (i) PCl3, Et2O(THF), -40 °C, 10
min; (ii) Et2O, pyridine, -40 °C, 3 h; (iii) room temp, 1 h. Method D: (i)
d4T (1), DIPEA, CH3CN, -20 °C to room temp, 1-1.5 h; (ii) t-BuOOH,
-20 °C to room temp, 1 h. Method E: (i) d4T (1), DIPEA, CH3CN, -20
°C to room temp, 1 h; (ii) Oxone in H2O, -10 °C to room temp, 10 min.
The hydrolysis stability of 13 was analyzed as described
above for bis-(cycloSal-d4TMPs) 5 (Table 1). At the physi-
ological pH, reference compound 13 was found to be slighly
more stable (t1/2 ) 2.2 h; Table 1) in comparison to 5a-mix (t1/2
) 1.7 h). Interestingly, for compound 13 no influence of the
more basic pH was found here (t1/2(PBS, pH 7.6) ) 2.0 h; t1/
2(RPMI/FCS) ) 2.1 h). Again, as with triester 5, compound 13
hydrolyzed selectively to d4TMP 2. The formation of 13 during
the hydrolysis of bis-(cycloSal-d4TMP) 5a-mix was clearly
proven by HPLC analysis. In Figure 2 the HPL-chromatograms
of the hydrolysis of 5a-mix at the beginning (top) and after an
incubation time of 1 h at 37 °C as well as the reference
chromatogram of 13 (bottom) are shown. Obviously, after 1 h
a significant amount of 13 was formed. With the used HPLC
method (method I; Supporting Information) polar compounds
like d4TMP 2 (with two negative charges) are eluted together
with the injection peak from the RP column. A signal for the
possible bis(benzyl phosphate diester) 10 would appear here,
too. But because of a possible overlapping with the signals of
2 and DMSO, it cannot be clearly determined whether this
intermediate is formed or not. The intermediates 11 and 12 with
only one negative charge should elute later compared to 10.
Signals a-c in Figure 2 are increasing at the beginning but are
disappearing during the course of hydrolysis (data not shown).
So, these signals should result from such intermediates. Signal
a could be attributed to triol 12 (Scheme 3) because this
intermediate was present in the hydrolysis of 13, too (see C in
Figure S1 in Supporting Information). Accordingly, signals b
and c should be attributable to the two formed diastereomers
(RP and SP) of intermediate 11. The small signal d corresponds
to the formed tetrol 8a (see A in Figure S1 in Supporting
Information).
Table 1. Hydrolysis Data of 5a-c and 13 Compared to 4a,b
t1/2 (h)a
PBSb
triester
pH 7.3
pH 7.6
RPMI/FCSc
5a (mix)
5a (fast)
5a (slow)
5b (mix)
5c (mix)
5c (fast)
5c (middle)
5c (slow)
13 (mix)
4a (mix)
4a (fast)
4a (slow)
4b (mix)
5,5′-(H)
5,5′-(H)
5,5′-(H)
5,5′-(Me)
5,5′-(t-Bu)
5,5′-(t-Bu)
5,5′-(t-Bu)
5,5′-(t-Bu)
5-Sald
3,3′-(H)
3,3′-(H)
3,3′-(H)
3,3′-(Me)
1.7
1.0
3.3
2.2
4.7
4.3
4.7
5.3
2.2
8.2
3.2
8.5
12
0.5
0.4
0.7
2.3
4.1
3.9
4.1
4.4
2.0
nde
nde
nde
nde
0.8
0.6
1.0
2.3
8.3
7.9
8.0
9.1
2.1
nde
nde
nde
nde
a Hydrolysis half-lives. b 25 mM phosphate buffer. c RPMI/10% heat-
inactivated fetal calf serum (FCS), pH 7.6. d 5-(Saligen-5′-yl)-cycloSal-d4TMP.
e nd: not determined.
the half-life in cell culture medium is approximately doubled
compared to the half-life in PBS (pH 7.6). It is important to
mention that the final products in all hydrolyses were exclusively
d4TMP and the respective tetrol 8.
Hydrolysis Pathway. To get further insight in the hydrolysis
behavior of such 5,5′-bis-(cycloSal-d4TMPs) 5, the chemical
hydrolysis pathway of 5a was studied in detail. The hydrolysis
of 5a may proceed via different pathways. Triester 5a can be
hydrolyzed, either leading to bis(benzyl phosphate diester) 10
or leading to benzyl phosphate ester 11. From the labile
intermediate 10, 2 equiv of d4TMP 2 can be released either
simultaneously (direct release of 2 and 8a) or consecutively
(release of 2 and 8a via benzyl phosphate ester 12). If the
hydrolysis takes place via 11, 5-(saligen-5′-yl)-cycloSal-d4TMP
13 has to be formed. Then 2 and 8a would be released via 12.
All these theoretically possible hydrolytic routes are shown in
Scheme 3.
Because a simultaneous hydrolysis pathway could not be ruled
out completely by this method, further analysis using 31P NMR
spectroscopy (NMR hydrolysis in imidazole/HCl buffer, pH 7.3,
37 °C) was performed. In an initial experiment reference
compound 13 was hydrolyzed (Figure 3a). During hydrolysis
the formation of the benzyl phosphate diester 12 (Figure 3b)
and d4TMP 2 (Figure 3c) was observed. At the end, d4TMP 2

cycloSaligenyl-Pronucleotides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3467
Table 2. Antiviral Activity and Cytotoxicity of Pronucleotides 5a-c, 13, and 4a,b and Tetrols 8 Compared to d4T 1
EC50 (µM)a
d
CEM/0c
CEM/TK-
CEM/0c
compound
HIV-1
HIV-2
HIV-2
CC50 (µM)b
5a (mix)
5a (fast)
5a (slow)
13
5b (mix)
5c (mix)
5c (fast)
5c (middle)
5c (slow)
8a
5,5′-(H)
5,5′-(H)
0.51 ( 0.21
0.46 ( 0.27
0.47 ( 0.20
0.73 ( 0.042
0.40 ( 0.0
0.88 ( 0.18
0.81 ( 0.25
0.49 ( 0.45
0.86 ( 0.31
99 ( 4.9
0.62 ( 0.51
1.2 ( 0.0
7.5 ( 2.5
4.4 ( 2.1
3.3 ( 1.8
3.7 ( 1.1
0.85 ( 0.61
2.9 ( 1.2
2.8 ( 1.7
3.2 ( 0.64
2.6 ( 0.85
18 ( 0.71
>50
6.9 ( 2.7
29 ( 3.8
2.33 ( 0.58
3.33 ( 2.31
2.37 ( 1.48
1.60 ( 0.57
115 ( 58
107 ( 39
78 ( 2.1
82 ( 1.4
64 ( 7.1
20 ( 0.71
18 ( 1.4
19 ( 0.0
20 ( 1.4
>250
96 ( 0.71
20 ( 0.71
>250
27.8 ( 11.0
107 ( 13
90.7 ( 16.1
67.1 ( 7.1
5,5′-(H)
1.2 ( 0.21
0.91 ( 0.28
0.83 ( 0.24
1.3 ( 0.071
1.6 ( 0.95
1.0 ( 0.085
2.0 ( 0.0
117 ( 41
>50
7.9 ( 3.0
0.79 ( 0.19
0.27 ( 0.11
0.30 ( 0.09
0.22 ( 0.16
0.41 ( 0.19
5-Sale
5,5′-(Me)
5,5′-(t-Bu)
5,5′-(t-Bu)
5,5′-(t-Bu)
5,5′-(t-Bu)
8b
8c
20 ( 2.8
7.9 ( 3.0
d4T 1
0.56 ( 0.20
0.19 ( 0.05
0.25 ( 0.14
0.21 ( 0.17
0.37 ( 0.25
4a (mix)f
4a (fast)f
4a (slow)f
4b (mix)f
3,3′-(H)
3,3′-(H)
3,3′-(H)
3,3′-(Me)
a Antiviral activity in T-lymphocytes: 50% effective concentration (shown values are the mean of two to three independent experiments). b Cytostatic
activity: 50% cytostatic concentration. c Wild-type CEM cells. d Thymidine kinase-deficient CEM cells. e 5-(saligen-5′-yl)-cycloSal-d4TMP. f Data taken
from ref 4.
Scheme 3. Theoretical Pathways of Chemical Hydrolysis of 5,5′-Bis-(cycloSal-d4TMP) 5a
was found to be the sole phosphorus containing hydrolysis
product (Figure 3d).
spectrum was almost identical to the spectrum d in Figure 3. In
conclusion, for bis-(cycloSal-d4TMP) 5a a consecutive hydroly-
sis pathway leading to a selective release of 2 equiv of d4TMP
2 as the exclusive phosphorus containing product can be
concluded.
Antiviral Evaluation and Cytotoxicity. CycloSal-triesters
5 and 13 and tetrols 8 were evaluated for their in vitro anti-
HIV activity and cytostatic activity (Table 2). For comparison,
the values of d4T 1 and 3,3′-bis-(cycloSal-d4TMPs) 4 are given
as well.
In a second experiment 5a-mix was hydrolyzed under
the same conditions (Figure 4). As in the HPLC analysis the
formation of 13 was detected (Figure 4ii). Furthermore, the
formation of two different benzyl phosphate diesters 11 and 12
was observed. The later appearing one (Figure 4iii) is the known
intermediate 12 that was also formed in the hydrolysis of 13
(for a detailed comparable assignment, see Figure S2 in
Supporting Information). Consequently, the benzyl phosphate
diester in Figure 4ii should be intermediate 11 (Scheme 3).
Almost at the end of the hydrolysis (Figure 4iv) only signals
for compounds 12 and 2 were present. Finally, the 31P NMR
All pronucleotides 4, 5, and 13 showed comparable antiviral
potency against HIV-1 and HIV-2 in wild-type CEM/0 cells to
d4T 1 within the experimental error. Almost all compounds

3468 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Gisch et al.
Scheme 4. Synthesis of 4,7-O-di-tert-Butylsilyl-4′,7′-O-isopropylidene-3,3′-bis(hydroxymethyl)biphenyl-4,4′-diol 16 and
5-(Saligen-5′-yl)-cycloSal-d4TMP 13a
a Reagents and conditions. Method F: (t-Bu)2Si(OTf)2, pyridine, CH2Cl2, room temp, 2.5 h. Method G: (i) 6a, n-BuLi, THF, -80 °C, 1 h; (ii) Fe(acac)3,
THF,-80 °C to room temp, 60 h. Method H: Et3N·3HF, THF, room temp, 20 min. Method I: Et3N·3HF, THF, room temp, 10 min. Method K: (i) PCl3,
Et2O, -20 °C, 10 min; (ii) Et2O, pyridine, -20 °C, 3 h; (iii) room temp, 1 h. Method L: (i) d4T 1, DIPEA, CH3CN, -20 °C to room temp, 1 h; (ii) Oxone
in H2O, -10 °C to room temp, 10 min. Method M: conc HCl, CH3CN/H2O/THF, heat, 15 s.
showed a loss in antiviral potency in the HIV-2-infected CEM/
TK-deficient cells. Interestingly, the decrease of anti-HIV-2
activity for 5,5′-bis-(cycloSal-d4TMPs) 5 in CEM/TK- cell
cultures is significantly less than in the case of the 3,3′-
counterparts 4. Most striking is the full retention of anti-HIV-2
activity in the CEM/TK- cell line for 5b-mix. This is the first
example showing that this kind of “high-loaded” pronucleotides
can act as successful nucleotide delivery systems. For all
separated diastereomers (4a, 5a, 5c) no dependence of the
antiviral activity to the configuration of the phosphorus atom
was observed.
The cytostatic activity of prodrugs 5 was found to be higher
than for d4T 1 and increased with the electron-donating effect
of the alkyl substituents. Taking the cytostatic effects of the
tetrols 8 into account, the toxicity of 5c-mix (CC50 ) 20 µM
for the diastereomeric mixture) can definitely be attributed to
the toxicity of tetrol 8c (CC50 ) 20 µM) that is released upon
hydrolysis. However, tetrol 8b that is hydrolytically released
from pronucleotide 5b was less cytostatic. Consequently,
cycloSal-pronucleotide 5b showed clearly the most selective
antiviral and modest antiproliferative properties and deserves
further studies.
a mask-to-drug ratio of 1:2 has been successfully developed.
These compounds were analyzed with regard to their hydrolytic
and antiviral behavior. Although compounds 5 were chemically
less stable compared to their 3,3′-counterparts 4, they showed
anti-HIV-1 and HIV-2 activity in wild-type CEM cells. More-
over, the 5,5′-bis-(cycloSal-d4TMPs) 5 were only slightly less
active in CEM/TK- than in CEM/0 cells. In particular, 5,5′-
bis(3-methyl-cycloSal-d4TMP) 5b has to be highlighted because
it is the first member of this class that completely retained its
anti-HIV-2 activity in CEM/TK- cells. Obviously, this com-
pound is able to penetrate cell membranes and release d4TMP
inside cells, which makes the compound independent of the
presence of thymidine kinase. The complex hydrolysis pathway
of these compounds to release 2 equiv of d4TMP 2 was studied
in detail. For these studies, mono-d4TMP-loaded intermediate
13 was synthesized as a reference compound. The formation
of compound 13 during the hydrolysis of 5a was definitely
proven by HPLC analysis and 31P NMR hydrolysis studies,
which demonstrated a consecutive hydrolysis pathway of 5a.
Experimental Section
NMR spectra were recorded with a Bruker AMX 400, Bruker
AV 400, or a Bruker DRX 500 Fourier transform spectrometer.
Conclusion
1
All H and 13C NMR chemical shifts (δ) are quoted in parts per
An efficient synthetic route to 5,5′-bis-(cycloSal-d4TMPs) 5
as new members of the class of cycloSal-pronucleotides having
million (ppm) downfield from tetramethylsilane and calibrated on
solvent signals. The 31P NMR chemical shifts (proton decoupled)

cycloSaligenyl-Pronucleotides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3469
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.
Et2O was dried over sodium/benzophenone and distilled under
nitrogen. The purity of the new compounds 5 was checked by means
of HPLC using two different gradients. Purity was in all cases >96%
(details are given in the Supporting Information). THF was dried
over potassium/benzophenone and distilled under nitrogen. Pyridine,
CH2Cl2, and CH3CN were destilled from calcium hydride under
nitrogen. N,N-Diisopropylethylamine was destilled from sodium
prior to use. The solvent for HPLC (CH3CN) was obtained from
Acros (HPLC grade).
General Procedure A: Homocoupling of 4-Bromosalicyl
Alcohol Isopropylidene Acetals. A solution of the 4-bromosalicyl
alcohol isopropylidene acetal (1.0 equiv) in dry THF was cooled
to -80 °C under nitrogen, and a 1.6 M solution of n-butyllithium
in hexane (1.0 equiv) was added. The reaction mixture was stirred
at this temperature for 1 h and then added to a solution of iron(III)
acetylacetonate (1.1 equiv) in dry THF (-80 °C). After 5 min the
cooling was removed. The reaction mixture was allowed to warm
up to room temperature and then stirred for the time given below.
The reaction mixture was diluted with 2 N HCl, and the phases
were separated with ethyl acetate. The aqueous layer was extracted
with EtOAc twice. Then the combined organic layers were washed
with 2 N HCl (3×), water (2×), and brine (2×) and dried with
sodium sulfate. The solvent was removed in vaccuo, and the
resulting crude products were purified by column chromatography
(CH2Cl2).
General Procedure B: Deprotection of Isopropylidene Ac-
etals. Concentrated HCl was added to a solution of the respective
isopropylidene acetal in CH3CN/H2O and THF. The reaction
mixture was heated to reflux with a heat gun for the given time.
This procedure was repeated until the deprotection has been
completed (monitoring by TLC, CH2Cl2). The reaction mixture was
diluted with water, and the phases were separated with ethyl acetate.
The aqueous layer was extracted three times with EtOAc. The
combined organic layers were dried with sodium sulfate, and the
solvent was removed under reduced pressure. The crude products
were purified by column chromatography (CH2Cl2/CH3OH, 9:1).
General Procedure C: Preparation of cycloSal-d4T-mono-
phosphates. Variant A. Synthesis of the Saligenyl Chlorophos-
phite. A solution of the respective salicyl alcohol in dry Et2O (and
dry THF) was cooled to -40 °C in an inert atmosphere. After
dropwise addition of freshly distilled PCl3 and stirring at -40 °C
for 10 min, a solution of dry pyridine in dry diethyl ether was added
at the same temperature within 3 h. After completion the reaction
mixture was allowed to warm up to room temperature and stirred
for 1 h. It was kept at -20 °C overnight to precipitate the formed
pyridinium chloride almost completely. Filtration under nitrogen
and concentration of the filtrate under reduced pressure yielded the
saligenyl chlorophosphite as a crude product which was directly
used for the synthesis of the cycloSal-d4T-monophosphate without
further purification. The grade of purity was determined by
integration of 1H NMR signals (two protons of C5H5N·HCl vs two
aromatic protons of the saligenyl chlorophosphite).
Synthesis of the cycloSal Nucleoside Monophosphate. Under
nitrogen, d4T (1, 1.2 equiv per 1.0 equiv of saligenyl chlorophos-
phite unit) was dissolved in dry CH3CN and cooled to -20 °C.
Then DIPEA (1.9 equiv per 1.0 equiv of saligenyl chlorophosphite
unit) and the respective saligenyl chlorophosphite in dry CH3CN
were added. The reaction mixture was stirred at room temperature
for the given time. Subsequently, tert-butyl hydroperoxide (5.5 M
in n-nonane, 3.6 equiv per 1.0 equiv of saligenyl chlorophosphite
unit) was added at -20 °C. The solution was stirred at room
temperature until the oxidation was completed and then poured into
water. The phases were separated with EtOAc, the aqueous layer
was extracted with EtOAc three times, and the combined organic
layers were dried with sodium sulfate and concentrated under
reduced pressure. The resulting residues were purified by flash
chromatography (CH2Cl2/CH3OH, 9:1) and preparative RP-HPLC.
The isolated products were lyophilized from CH3CN/H2O (1:1).
Figure 2. HPLC analytical proof of the formation of 13 during the
hydrolysis of 5a-mix.
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], and 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-methoxybenzal-
dehyde, 9 mL of EtOH, 0.5 mL of concentrated sulfuric acid, and
0.1 mL of glacial acetic acid). All preparative TLC 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
chromatography, Merck silica gel 60, 230-400 mesh, was used.
UV spectra were recorded on a Varian Cary 1E UV-visible
spectrophotometer, and absorption maximum wavelengths λmax are
given in nm. UV absorptions of cycloSal nucleotides were
determined from their HPLC data (diode array detector). 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

3470 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Gisch et al.
Figure 3. 31P NMR hydrolysis study of 13 (diastereomeric mixture).
Figure 4. 31P NMR hydrolysis study of 5a-mix.
Variant B. Variant B was performed as described for variant
A, but the preparation of the saligenyl chlorophosphite was
performed at -20 °C and addition of the mixture of dry pyridine
and dry Et2O was carried out in only 1.5 h.
General Procedure D: Cleavage of DTBS Protecting Group.
The corresponding DTBS-protected salicyl alcohol derivative was
dissolved in THF, and NEt3 · 3HF was added. The solution was
stirred at room temperature for the given time, and afterwards the
phases were separated with ethyl acetate and water. The aqueous
layer was extracted with EtOAc once, the combined organic layers
were washed with a 5% sodium bicarbonate solution, and the
solvent was removed in vacuo. The products were purified by flash
chromatography (CH2Cl2/CH3OH, 9:1 (8a) or CH2Cl2/CH3OH
gradient 0-7%, 18).

cycloSaligenyl-Pronucleotides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3471
Bis-(isopropylidene)-3,3′-bis(hydroxymethyl)biphenyl-4,4′-
diol (7a). Corresponding to Method A (Scheme 1). General
procedure A was carried out with 1.1 g (4.5 mmol) of
4-bromosalicyl alcohol isopropylidene acetal (6a), dissolved in
9.5 mL of dry THF, 2.8 mL (1.6 M solution in hexane, 4.5 mmol)
of n-BuLi, and 1.78 g (5.03 mmol) of iron(III) acetylacetonate
in 4 mL of dry THF. Reaction time: 42 h. Yield: 558 mg (1.71
H-6), 7.22-7.19 (m, 2H, aryl-H-2), 5.84 (t, J ) 5.2 Hz, 2H, benzyl-
OH), 4.69 (d, J ) 4.8 Hz, 4H, benzyl-H), 1.40 (s, 18H, t-Bu-CH3)
ppm.
5,5′-Bis-(cycloSal-d4TMP) (5a). Synthesis of Saligenyl Chlo-
rophosphite 9a. (Corresponding to Method C (Scheme 2)).
General Procedure C (variant A) was carried out with 105 mg (0.426
mmol) of 3,3′-bis(hydroxymethyl)biphenyl-4,4′-diol (8a) dissolved
in 22 mL of dry Et2O/dry THF (1:1), 0.10 mL (1.1 mmol) of PCl3,
and 0.17 mL (2.1 mmol) of dry pyridine in 0.85 mL of dry Et2O.
Yield: 130 mg (55%; purity, 68%).
Synthesis of cycloSal Nucleoside Monophosphate 5a (Corre-
sponding to Method D (Scheme 2)). The synthesis involved 130
mg of crude saligenyl chlorophosphite 9a, dissolved in 4 mL of
dry CH3CN, 125 mg (0.557 mmol) of d4T (1), dissolved in 13 mL
of dry CH3CN, 0.15 mL (0.86 mmol) DIPEA, and 0.30 mL (5.5 M
in n-nonane, 1.7 mmol) of tert-butyl hydroperoxide. Reaction time:
1.5 h. Oxidation time: 1 h. Preparative RP-HPLC: CH3CN/H2O
(1:2). Yield: 51 mg (65 µmol, 28%) of a diastereomeric mixture
as a colorless foam. 1H NMR (400 MHz, DMSO-d6): δ )
11.36-11.32 (m, 6H, 6 × NH), 7.67-7.59 (m, 6H, 6 × aryl-H-4),
7.59-7.54 (m, 6H, 6 × aryl-H-6), 7.26-7.17 (m, 12H, 6 × aryl-
H-3, 6 × thymine-H-6), 6.84-6.77 (m, 6H, 6 × 1′-H), 6.46-6.36
(m, 6H, 6 × 3′-H), 6.06-6.00 (m, 6H, 6 × 2′-H), 5.61-5.41 (m,
12H, 12 × benzyl-H), 4.98-4.95 (m, 6H, 6 × 4′-H), 4.40-4.26
(m, 12H, 12 × 5′-H), 1.69 (s, 9H, 3 × thymine-CH3), 1.63 (s, 9H,
3 × thymine-CH3) ppm. 31P NMR (162 MHz, DMSO-d6): δ )
-9.48, -9.52 ppm. The diastereomers were partially separated by
preparative RP-HPLC (CH3CN/H2O 2:5).
1
mmol, 75%) as a colorless solid. H NMR (400 MHz, CDCl3):
δ ) 7.38 (dd, J ) 8.5, 2.4 Hz, 2H, aryl-H-6), 7.31 (d, J ) 2.4
Hz, 2H, aryl-H-2), 6.83 (d, J ) 8.5 Hz, 2H, aryl-H-5), 4.86 (s,
4H, benzyl-H), 1.48 (s, 12H, acetal-CH3) ppm.
Bis-(isopropylidene)-3,3′-bis(hydroxymethyl)-5,5′-dimethyl-
biphenyl-4,4′-diol (7b). Corresponding to Method A (Scheme
1). General procedure A was carried out with 451 mg (1.75
mmol) of 4-bromo-6-methylsalicyl alcohol isopropylidene acetal
(6b), dissolved in 4 mL of dry THF, 1.1 mL (1.6 M solution in
hexane, 1.8 mmol) of n-BuLi, and 686 mg (1.94 mmol) of
iron(III) acetylacetonate in 1.5 mL of dry THF. Reaction time:
36 h. Yield: 120 mg (0.339 mmol, 37%) as a colorless solid. 1H
NMR (400 MHz, CDCl3): δ ) 7.20-7.18 (m, 2H, aryl-H-6),
6.97-6.94 (m, 2H, aryl-H-2), 4.88 (s, 4H, benzyl-H), 2.22 (s,
6H, aryl-CH3), 1.57 (s, 12H, acetal-CH3) ppm.
Bis-(isopropylidene)-3,3′-bis(hydroxymethyl)-5,5′-di-tert-bu-
tylbiphenyl-4,4′-diol (7c). Corresponding to Method A (Scheme
1). General procedure A was carried out with 1.1 g (3.7 mmol) of
4-bromo-6-tert-butylsalicyl alcohol isopropylideneacetal (6c), dis-
solved in 9.5 mL of dry THF, 2.3 mL (1.6 M solution in hexane,
3.7 mmol) of n-BuLi, and 1.44 g (4.07 mmol) of iron(III)
acetylacetonate in 4 mL of dry THF. Reaction time: 36 h. Yield:
593 mg (1.23 mmol, 67%) as a colorless solid. 1H NMR (400 MHz,
CDCl3): δ ) 7.29 (d, J ) 2.2 Hz, 2H, aryl-H-6), 6.97-6.94 (m,
2H, aryl-H-2), 4.92 (s, 4H, benzyl-H), 1.59 (s, 12H, acetal-CH3),
1.41 (s, 18H, t-Bu-CH3) ppm.
Analytical Data of 5a-fast (Two Diastereomers). 1H NMR (400
MHz, DMSO-d6): δ ) 11.32 (s, 4H, 4 × NH), 7.64-7.59 (m, 4H,
4 × aryl-H-4), 7.59-7.57 (m, 4H, 4 × aryl-H-6), 7.26-7.17 (m,
8H, 4 × aryl-H-3, 4 × thymine-H-6), 6.84-6.77 (m, 4H, 4 × 1′-
H), 6.43 (ddd, J ) 6.0, 1.8, 1.5 Hz, 3H, 3 × 3′-H), 6.37 (ddd, J )
6.0, 1.8, 1.8 Hz, 1H, 1 × 3′-H), 6.06-6.00 (m, 4H, 4 × 2′-H),
5.61-5.41 (m, 8H, 8 × benzyl-H), 4.99-4.94 (m, 4H, 4 × 4′-H),
4.40-4.25 (m, 8H, 8 × 5′-H), 1.69 (d, J ) 0.7 Hz, 9H, 3 ×
thymine-CH3), 1.63 (d, J ) 1.3 Hz, 3H, 1 × thymine-CH3) ppm.
31P NMR (162 MHz, DMSO-d6): δ ) -9.48, -9.52 ppm.
Analytical Data of 5a-slow (One Diastereomer). 1H NMR (400
MHz, DMSO-d6): δ ) 11.33 (s, 2H, 2 × NH), 7.64-7.59 (m, 2H,
2 × aryl-H-4), 7.59-7.54 (m, 2H, 2 × aryl-H-6), 7.22-7.17 (m,
4H, 2 × aryl-H-3, 2 × thymine-H-6), 6.80-6.77 (m, 2H, 2 × 1′-
H), 6.38 (ddd, J ) 6.0, 1.8, 1.8 Hz, 2H, 2 × 3′-H), 6.04-6.00 (m,
2H, 2 × 2′-H), 5.61-5.50 (m, 2H, 2 × benzyl-H), 5.50-5.41 (m,
2H, 2 × benzyl-H), 4.98-4.95 (m, 2H, 2 × 4′-H), 4.41-4.25 (m,
4H, 4 × 5′-H), 1.63 (d, J ) 1.3 Hz, 6H, 2 × thymine-CH3) ppm.
31P NMR (162 MHz, DMSO-d6): δ ) -9.52 ppm.
3,3′-Bis(hydroxymethyl)biphenyl-4,4′-diol (8a). Method I
(Corresponding to Method B (Scheme 1). General procedure B
was carried out with 969 mg (2.97 mmol) of bis-(isopropylidene)-
3,3′-bis(hydroxymethyl)biphenyl-4,4′-diol (7a), dissolved in 90 mL
of CH3CN/H2O (7:3) and 25 mL of THF, and 20 drops of
concentrated HCl. Reaction time: 30 s. Yield: 651 mg (2.64 mmol,
1
89%) as a colorless solid. H NMR (400 MHz, DMSO-d6): δ )
9.32 (s, 2H, phenol-OH), 7.51 (d, J ) 2.3 Hz, 2H, aryl-H-2), 7.24
(dd, J ) 8.3, 2.5 Hz, 2H, aryl-H-6), 6.80 (d, J ) 8.3 Hz, 2H, aryl-
H-5), 5.01 (t, J ) 5.7 Hz, 2H, benzyl-OH), 4.52 (d, J ) 5.7 Hz,
4H, benzyl-H) ppm.
Method II (Corresponding to Method H (Scheme 4). General
procedure D was carried out with 610 mg (1.15 mmol) of bis-di-
tert-butylsilyl-3,3′-bis(hydroxymethyl)biphenyl-4,4′-diol (17) dis-
solved in 50 mL of THF, and 1.1 mL (6.5 mmol) of NEt3 ·3HF.
Reaction time: 20 min. Yield: 250 mg (1.01 mmol, 88%) as a
slightly yellow solid. The analytical data were identical with those
reported above.
3,3′-Bis(hydroxymethyl)-5,5′-dimethylbiphenyl-4,4′-diol (8b).
Corresponding to Method B (Scheme 1). General procedure B
was carried out with 162 mg (0.457 mmol) of bis-(isopropylidene)-
3,3′-bis(hydroxymethyl)-5,5′-dimethyl-biphenyl-4,4′-diol (7b), dis-
solved in 20 mL of CH3CN/H2O (7:3) and 5 mL of THF, and 5
drops of concentrated HCl. Reaction time: 30 s. Yield: 115 mg
(0.419 mmol, 92%) as a colorless solid. 1H NMR (400 MHz,
DMSO-d6): δ ) 8.38 (s, 2H, phenol-OH), 7.30 (d, J ) 2.3 Hz,
2H, aryl-H-2), 7.20 (d, J ) 2.0 Hz, 2H, aryl-H-6), 5.30 (t, J ) 5.5
Hz, 2H, benzyl-OH), 4.60 (d, J ) 5.3 Hz, 4H, benzyl-H), 2.21 (s,
6H, aryl-CH3) ppm.
5,5′-Bis(3-methyl-cycloSal-d4TMP) (5b). Synthesis of Salig-
enyl Chlorophosphite 9b (Corresponding to Method C (Scheme
2)). General procedure C (variant A) was carried out with 90 mg
(0.33 mmol) of 3,3′-bis(hydroxymethyl)-5,5′-dimethylbiphenyl-4,4′-
diol (8b) dissolved in 18 mL of dry Et2O/dry THF (1:1), 70 µL
(0.79 mmol) of PCl3, and 0.14 mL (1.7 mmol) of dry pyridine in
0.70 mL of dry Et2O. Yield: 125 mg (54%; purity: 59%).
Synthesis of cycloSal Nucleoside Monophosphate 5b (Corre-
sponding to Method D (Scheme 2)). The synthesis involved 122
mg of crude saligenyl chlorophosphite 9b, dissolved in 4 mL of
dry CH3CN (+0.8 mL dry THF), 96 mg (0.43 mmol) of d4T (1),
dissolved in 9 mL of dry CH3CN, 0.14 mL (0.82 mmol) of DIPEA,
and 0.23 mL (5.5 M in n-nonane, 1.3 mmol) of tert-butyl
hydroperoxide. Reaction time: 1 h. Oxidation time: 1 h. Preparative
RP-HPLC: CH3CN/H2O (2:3). Yield: 43 mg (53 µmol, 30%) of a
1
diastereomeric mixture as a colorless foam. H NMR (400 MHz,
3,3′-Bis(hydroxymethyl)-5,5′-di-tert-butylbiphenyl-4,4′-diol (8c).
Corresponding to Method B (Scheme 1). General procedure B
was carried out with 206 mg (0.407 mmol) of bis-(isopropylidene)-
3,3′-bis(hydroxymethyl)-5,5′-di-tert-butylbiphenyl-4,4′-diol (7c),
dissolved in 50 mL of CH3CN/H2O (5:1) and 5 mL of THF, and
3.5 mL of concentrated HCl. Reaction time: 3 min. Yield: 120 mg
(0.335 mmol, 71%) as a colorless solid. 1H NMR (400 MHz,
DMSO-d6): δ ) 8.67 (s, 2H, phenol-OH), 7.25-7.22 (m, 2H, aryl-
DMSO-d6): δ ) 11.33 (s, 6H, 6 × NH), 7.56-7.52 (m, 6H, 6 ×
aryl-H-4), 7.41-7.37 (m, 6H, 6 × aryl-H-6), 7.23-7.18 (m, 6H, 6
× thymine-H-6), 6.83-6.79 (m, 6H, 6 × 1′-H), 6.43 (ddd, J )
6.0, 1.8, 1.8 Hz, 3H, 3 × 3′-H), 6.38 (ddd, J ) 6.0, 1.8, 1.5 Hz,
3H, 3 × 3′-H), 6.06-6.00 (m, 6H, 6 × 2′-H), 5.56-5.47 (m, 6H,
6 × benzyl-H), 5.45-5.36 (m, 6H, 6 × benzyl-H), 5.00-4.95 (m,
6H, 6 × 4′-H), 4.37-4.23 (m, 12H, 12 × 5′-H), 2.28 (s, 9H, 3 ×
aryl-CH3), 2.25 (s, 9H, 3 × aryl-CH3), 1.66 (d, J ) 0.8 Hz, 9H, 3

3472 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Gisch et al.
× thymine-CH3), 1.63 (d, J ) 1.0 Hz, 9H, 3 × thymine-CH3) ppm.
31P NMR (162 MHz, DMSO-d6): δ ) -8.75, -8.88 ppm.
Di-tert-butylsilyl-4-bromosalicyl Alcohol (15). Correspond-
ing to Method F (Scheme 4). Under nitrogen, an amount of 3.06 g
(15.1 mmol) of 4-bromosalicyl alcohol (14) was dissolved in 100
mL of dry CH2Cl2. After the addition of 5.3 mL (66 mmol) of dry
pyridine and 6.4 mL (20 mmol) of di-tert-butylsilyl bis(trifluo-
romethanesulfonate), the solution was stirred at room temperature
for 2.5 h. At 0 °C the reaction mixture was diluted with water and
the phases were separated. The aqueous layer was extracted with
CH2Cl2 twice. Then the combined organic layers were dried with
sodium sulfate and concentrated under reduced pressure. The crude
product was purified by flash column chromatography (petroleum
ether 50-70/CH2Cl2, 5:1). Yield: 4.15 g (12.1 mmol, 81%) as a
5,5′-Bis(3-tert-butyl-cycloSal-d4TMP) (5c). Synthesis of
Saligenyl Chlorophosphite 9c (Corresponding to Method C
(Scheme 2)). General procedure C (variant A) was carried out with
170 mg (0.474 mmol) of 3,3′-bis(hydroxymethyl)-5,5′-di-tert-
butylbiphenyl-4,4′-diol (8c) dissolved in 36 mL of dry Et2O, 0.10
mL (1.1 mmol) of PCl3, and 0.19 mL (2.4 mmol) of dry pyridine
in 0.95 mL of dry Et2O. Yield: 206 mg (55%; purity: 62%).
Synthesis of cycloSal Nucleoside Monophosphate 5c. Method
I (Corresponding to Method E (Scheme 2)). The synthesis
involved 96 mg of crude saligenyl chlorophosphite 9c, dissolved
in 4 mL of dry CH3CN, 70 mg (0.31 mmol) of d4T (1), dissolved
in 7 mL of dry CH3CN, and 84 µL (49 µmol) of DIPEA. Deviating
from the general procedure C for the oxidation reaction, Oxone
was used. Therefore, the reaction mixture was cooled to -20 °C
and a suspension of 0.32 g (0.52 mmol) of Oxone in 1.5 mL of
cold water was added. The cooling was removed immediately, and
the reaction mixture was stirred for 10 min. Subsequently, the
mixture was extracted with EtOAc twice. The combined organic
layers were dried with sodium sulfate and concentrated under
reduced pressure. Preparative RP-HPLC: CH3CN/H2O (4:5). Yield:
33 mg (37 µmol, 30%) of a diastereomeric mixture as a colorless
1
colorless solid. H NMR (400 MHz, CDCl3): δ ) 7.26 (dd, J )
8.6, 2.5 Hz, 1H, aryl-H-5), 7.07 (d, J ) 2.5 Hz, 1H, aryl-H-3),
6.80 (d, J ) 8.6 Hz, 1H, aryl-H-6), 4.95 (s, 2H, benzyl-H), 1.03
(s, 18H, Si-t-Bu-CH3) ppm.
4,7-O-Di-tert-butylsilyl-4′,7′-O-isopropylidene-3,3′-bis(hy-
droxymethyl)biphenyl-4,4′-diol (16). Corresponding to Method
G (Scheme 4). This reaction was carried out analogously to the
homocoupling of 4-bromosalicyl alcohol isopropylidene acetals 6
(general procedure A). An equimolar mixture of 4-bromosalicyl
alcohol isopropylidene acetal (1.28 g, 5.27 mmol, 6a) and di-tert-
butylsilyl-4-bromosalicyl alcohol (1.81 g, 5.27 mmol, 15), dissolved
in 25 mL of dry THF, 6.6 mL (1.6 M solution in hexane, 11 mmol)
of n-BuLi, and 4.15 g (11.8 mmol) of Fe(acac)3 in 10 mL of dry
THF, was used. Reaction time: 60 h. Yield: 701 mg (1.64 mmol,
31%) as well as 17 (642 mg, 1.22 mmol, 46%) and 7a (267 mg,
0.818 mmol, 31%) as colorless solids. 1H NMR (400 MHz, CDCl3):
δ ) 7.38-7.30 (m, 2H, aryl-H-6, aryl-H-6′), 7.15-7.12 (m, 1H,
aryl-H-2′), 7.09 (d, J ) 2.2 Hz, 1H, aryl-H-2), 6.96 (d, J ) 8.2
Hz, 1H, aryl-H-5), 6.86 (d, J ) 8.5 Hz, 1H, aryl-H-5′), 5.05 (s,
2H, benzyl-H), 4.89 (s, 2H, benzyl-H′), 1.57 (s, 6H, acetal-CH3),
1.06 (s, 18H, Si-t-Bu-CH3) ppm.
1
foam. H NMR (400 MHz, DMSO-d6): δ ) 11.34 (s, 6H, 6 ×
NH), 7.50-7.44 (m, 12H, 6 × aryl-H-4, 6 × aryl-H-6), 7.24 (d, J
) 1.3 Hz, 3H, 3 × thymine-H-6), 7.21 (d, J ) 1.0 Hz, 3H, 3 ×
thymine-H-6), 6.85-6.79 (m, 6H, 6 × 1′-H), 6.46-6.39 (m, 6H, 6
× 3′-H), 6.07-6.01 (m, 6H, 6 × 2′-H), 5.56-5.38 (m, 12H, 12 ×
benzyl-H), 5.01-4.96 (m, 6H, 6 × 4′-H), 4.41-4.28 (m, 12H, 12
× 5′-H), 1.66-1.59 (m, 18H, 6 × thymine-CH3), 1.39 (s, 27H, 3
× t-Bu-CH3), 1.36 (s, 27H, 3 × t-Bu-CH3) ppm. 31P NMR (162
MHz, DMSO-d6): δ ) -8.59, -8.84 ppm.
Analytical Data of Bis-di-tert-butylsilyl-3,3′-bis(hydroxy-
Method II (Corresponding to Method D (Scheme 2). The
synthesis involved 110 mg of crude saligenyl chlorophosphite 9c,
dissolved in 4 mL of dry CH3CN, 70 mg (0.31 mmol) of d4T (1),
dissolved in 7 mL of dry CH3CN, 84 µL (49 µmol) of DIPEA, and
0.17 mL (5.5 M in n-nonane, 0.94 mmol) of tert-butyl hydroper-
oxide. Reaction time: 1 h. Oxidation time: 1 h. Preparative RP-
HPLC: CH3CN/H2O (2:3). Yield: 9 mg of 5c-fast 25 mg of 5c-
middle and 12 mg of 5c-slow (in total, 46 mg, 51 µmol, 37%) as
colorless foams.
1
methyl)-biphenyl-4,4′-diol (17). H NMR (400 MHz, CDCl3): δ
) 7.34 (dd, J ) 8.2, 2.2 Hz, 2H, aryl-H-6), 7.10 (d, J ) 2.2 Hz,
2H, aryl-H-2), 6.95 (d, J ) 8.2 Hz, 2H, aryl-H-5), 5.05 (s, 4H,
benzyl-H), 1.06 (s, 36H, Si-t-Bu-CH3) ppm.
4′,7′-O-Isopropylidene-3,3′-bis(hydroxymethyl)biphenyl-4,4′-
diol (18). Corresponding to Method I (Scheme 4). General
procedure D was carried out with 0.35 g (0.82 mmol) of 4,7-O-
di-tert-butylsilyl-4′,7′-O-isopropylidene-3,3′-bis(hydroxymethyl)bi-
phenyl-4,4′-diol (16) dissolved in 20 mL of THF and 0.38 mL (2.3
mmol) of NEt3 ·3HF. Reaction time: 10 min. Yield: 226 mg (0.789
mmol, 96%) as a slightly yellow solid. 1H NMR (400 MHz, DMSO-
d6): δ ) 9.41 (s, 1H, phenol-OH), 7.52 (d, J ) 2.3 Hz, 1H, aryl-
H-2′), 7.35 (dd, J ) 8.7, 2.3 Hz, 1H, aryl-H6), 7.27 (dd, J ) 8.1,
2.3 Hz, 1H, aryl-H6′), 7.26 (d, J ) 2.3 Hz, 1H, aryl-H-2), 6.82 (d,
J ) 8.4 Hz, 1H, aryl-H-5′), 6.81 (d, J ) 8.1 Hz, 1H, aryl-H-5),
5.00 (t, J ) 5.6 Hz, 1H, benzyl-OH), 4.87 (s, 2H, benzyl-H′), 4.52
(d, J ) 5.6 Hz, 2H, benzyl-H), 1.48 (s, 6H, acetal-CH3) ppm.
5-(Isopropylidenesaligen-5′-yl)-cycloSal-d4TMP (19). Syn-
thesis of Saligenyl Chlorophosphite (Corresponding to Method
K (Scheme 4). General procedure C (variant B) was carried out
with 188 mg (0.657 mmol) of 4′,7′-O-isopropylidene-3,3′-bis(hy-
droxymethyl)biphenyl-4,4′-diol (18) dissolved in 10 mL of dry Et2O,
69 µL (79 µmol) PCl3 and 0.10 mL (1.3 mmol) of dry pyridine in
0.5 mL of dry Et2O. Yield: 192 mg.
Synthesis of cycloSal Nucleoside Monophosphate 19 (Corre-
sponding to Method L (Scheme 4). The synthesis involved 129
mg of crude saligenyl chlorophosphite, dissolved in 3 mL of dry
CH3CN, 101 mg (0.446 mmol) of d4T (1), dissolved in 14 mL of
dry CH3CN, and 0.13 mL (0.75 mmol) of DIPEA. Reaction time:
1 h. Deviating from the general procedure C for the oxidation
reaction, Oxone was used. Therefore, the reaction mixture was
cooled to -10 °C and a suspension of 1.1 g (1.8 mmol) of Oxone
in 5 mL of cold water was added. The cooling was removed
immediately, and the reaction mixture was stirred for 10 min.
Subsequently, the mixture was extracted with EtOAc twice. The
combined organic layers were washed with water (2×), dried with
sodium sulfate, and concentrated under reduced pressure. Yield:
Analytical Data of 5c-fast. 1H NMR (400 MHz, DMSO-d6): δ
) 11.36 (s, 2H, 2 × NH), 7.50-7.47 (s, 4H, 2 × aryl-H-4, 2 ×
aryl-H-6), 7.24 (d, J ) 1.3 Hz, 2H, 2 × thymine-H-6), 6.84-6.80
(m, 2H, 2 × 1′-H), 6.43 (ddd, J ) 6.0, 1.8, 1.5 Hz, 2H, 2 × 3′-H),
6.05-6.01 (m, 2H, 2 × 2′-H), 5.55-5.39 (m, 4H, 4 × benzyl-H),
5.03-4.95 (m, 2H, 2 × 4′-H), 4.40-4.28 (m, 4H, 4 × 5′-H), 1.61
(d, J ) 1.0 Hz, 6H, 2 × thymine-CH3), 1.36 (s, 18H, 2 × t-Bu-
CH3) ppm. 31P NMR (162 MHz, DMSO-d6): δ ) -8.83 ppm.
1
Analytical Data of 5c-middle. H NMR (400 MHz, DMSO-
d6): δ ) 11.37-11.32 (s, 2H, 2 × NH), 7.50-7.44 (m, 4H, 2 ×
aryl-H-4, 2 × aryl-H-6), 7.24 (d, J ) 1.3 Hz, 1H, 1 × thymine-
H-6), 7.21 (d, J ) 1.3 Hz, 1H, 1 × thymine-H-6), 6.84-6.80 (m,
2H, 2 × 1′-H), 6.43 (ddd, J ) 5.8, 1.8, 1.5 Hz, 1H, 1 × 3′-H),
6.42 (ddd, J ) 5.8, 1.8, 1.5 Hz, 1H, 1 × 3′-H), 6.07-6.02 (m, 2H,
2 × 2′-H), 5.55-5.39 (m, 4H, 4 × benzyl-H), 5.01-4.96 (m, 2H,
2 × 4′-H), 4.40-4.28 (m, 4H, 4 × 5′-H), 1.61 (d, J ) 1.0 Hz, 3H,
1 × thymine-CH3), 1.60 (d, J ) 1.0 Hz, 3H, 1 × thymine-CH3),
1.39 (s, 9H, 1 × t-Bu-CH3), 1.36 (s, 9H, 1 × t-Bu-CH3) ppm. 31P
NMR (162 MHz, DMSO-d6): δ ) -8.59, -8.84 ppm.
Analytical Data of 5c-slow. 1H NMR (400 MHz, DMSO-d6): δ
) 11.33 (s, 2H, 2 × NH), 7.49-7.46 (m, 2H, 2 × aryl-H-4),
7.46-7.44 (m, 2H, 2 × aryl-H-6), 7.21 (d, J ) 1.3 Hz, 2H, 2 ×
thymine-H-6), 6.83-6.79 (m, 2H, 2 × 1′-H), 6.42 (ddd, J ) 5.8,
1.8, 1.5 Hz, 2H, 2 × 3′-H), 6.07-6.03 (m, 2H, 2 × 2′-H),
5.55-5.38 (m, 4H, 4 × benzyl-H), 5.01-4.96 (m, 2H, 2 × 4′-H),
4.41-4.29 (m, 4H, 4 × 5′-H), 1.60 (d, J ) 1.0 Hz, 6H, 2 ×
thymine-CH3), 1.39 (s, 18H, 2 × t-Bu-CH3) ppm. 31P NMR (162
MHz, DMSO-d6): δ ) -8.59 ppm.

cycloSaligenyl-Pronucleotides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3473
93 mg (0.17 mmol, 45%) of a diastereomeric mixture as a colorless
foam. H NMR (400 MHz, DMSO-d6): δ ) 11.36 (s, 1H, 1 ×
Acknowledgment. We are grateful to the Deutsche Fors-
chungsgemeinschaft (DFG), the University of Hamburg, the
“Geconcerteerde Onderzoeksacties (GOA No. 05/19), 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.
1
NH), 11.35 (s, 1H, 1 × NH), 7.62-7.55 (m, 2H, 2 × aryl-H-4),
7.54-7.52 (m, 2H, 2 × aryl-H-6), 7.46-7.42 (m, 2H, 2 × aryl-
H-4′), 7.38-7.35 (m, 2H, 2 × aryl-H-6′), 7.22 (d, J ) 1.3 Hz, 1H,
1 × thymine-H-6), 7.19 (d, J ) 1.3 Hz, 1H, 1 × thymine-H-6),
7.18 (d, J ) 8.5 Hz, 1H, 1 × aryl-H-3), 7.15 (d, J ) 8.5 Hz, 1H,
1 × aryl-H-3), 6.87 (d, J ) 8.5 Hz, 2H, 2 × aryl-H-3′), 6.83-6.77
(m, 2H, 2 × 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, 1.8 Hz, 1H, 1 × 3′-H), 6.05-5.99 (m, 2H,
2 × 2′-H), 5.56 (dd, J ) 14.6, 5.3 Hz, 1H, 1 × benzyl-H), 5.52
(dd, J ) 14.6, 5.3 Hz, 1H, 1 × benzyl-H), 5.44 (dd, J ) 14.6, 10.5
Hz, 2H, 2 × benzyl-H), 4.99-4.95 (m, 2H, 2 × 4′-H), 4.88 (s,
4H, 4 × benzyl-H′), 4.42-4.25 (m, 4H, 4 × 5′-H), 1.69 (d, J )
1.0 Hz, 3H, 1 × thymine-CH3), 1.63 (d, J ) 1.0 Hz, 3H, 1 ×
thymine-CH3), 1.49 (s, 12H, 4 × acetal-CH3) ppm. 31P NMR (162
MHz, DMSO-d6): δ ) -9.37 ppm.
Supporting Information Available: 13C NMR and UV spec-
troscopic data, mass spectrometric data, Rf values, melting points,
analytical HPLC data of new compounds; methods for HPLC
analysis; general nomenclature of 3,3′-bis(hydroxymethyl)-5,5′-
biphenyl-4,4′-diol derivatives and 5-(saligen-5′-yl)-cycloSaligenyl
derivatives; two figures. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
5-(Saligen-5′-yl)-cycloSal-d4TMP (13). Corresponding to
Method M (Scheme 4). Fifteen drops of concentrated hydrochloric
acid were added to a solution of 35 mg (63 µmol) of 5-(isopropyl-
idenesaligen-5′-yl)-cycloSal-d4TMP (19), dissolved in 20 mL of
CH3CN/H2O (7:3). The reaction mixture was heated to reflux with
a heat gun for 15 s. The reaction mixture was diluted with water,
and the phases were separated with EtOAc. The aqueous layer was
extracted three times with EtOAc. The combined organic layers
were washed with water (2×) and dried with sodium sulfate, and
the solvent was removed under reduced pressure. The residue was
lyophilized from CH3CN/H2O (1:1). Yield: 28 mg (53 µmol, 85%)
(1) (a) Balzarini, J. Metabolism and Mechanism of Antiretroviral Action
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1
of a diastereomeric mixture (ratio 1.0:1.0) as a colorless foam. H
NMR (400 MHz, DMSO-d6): δ ) 11.37 (s, 2H, 2 × NH), 9.58 (s,
2H, 2 × phenol-OH), 7.60-7.47 (m, 6H, 2 × aryl-H-4, 2 × aryl-
H-6, 2 × aryl-H-6′), 7.33 (dd, J ) 8.3, 1.8 Hz, 2H, 2 × aryl-H-4′),
7.22 (d, J ) 1.3 Hz, 1H, 1 × thymine-H-6), 7.19 (d, J ) 1.3 Hz,
1H, 1 × thymine-H-6), 7.17 (d, J ) 8.5 Hz, 1H, 1 × aryl-H-3),
7.14 (d, J ) 8.5 Hz, 1H, 1 × aryl-H-3), 6.84 (d, J ) 8.3 Hz, 2H,
2 × aryl-H-3′), 6.83-6.77 (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), 6.05-5.99 (m, 2H, 2 × 2′-H), 5.58 (dd, J ) 14.6, 6.0 Hz,
1H, 1 × benzyl-H), 5.53 (dd, J ) 14.6, 6.0 Hz, 1H, 1 × benzyl-
H), 5.45 (dd, J ) 14.6, 10.5 Hz, 2H, 2 × benzyl-H), 5.04 (t, J )
5.5 Hz, 2H, 2 × benzyl-OH), 4.99-4.94 (m, 2H, 2 × 4′-H), 4.52
(d, J ) 5.5 Hz, 4H, 4 × benzyl-H′), 4.38-4.26 (m, 4H, 4 × 5′-H),
1.69 (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
) -9.24, -9.29 ppm.
Hydrolysis Studies of cycloSal Phosphate Triesters. Hydrolysis
studies of cycloSal nucleotides (phosphate buffer, pH 7.3) by HPLC
analysis (method I, Supporting Information) have been described
before.8,10 Studies in RPMI/FCS(10%) were carried out as described
in ref 5b.
Antiretroviral Evaluation. The method of antiviral evaluation
has been described in ref 5a 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 5a-mix and 13
were carried out as described before.8
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Bis-cycloSal-d4T-monophosphates: Drugs That Deliver Two Mol-
ecules of Bioactive Nucleotides. J. Med. Chem. 2007, 50, 1335–1346.
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d4T-monophosphates: The Third Generation of cycloSal-Pronucle-
otides. J. Med. Chem. 2007, 50, 1658–1667. (b) Gisch, N.; Perten-
breiter, F.; Balzarini, J.; Meier, C. 5-(1-Acetoxyvinyl)-cycloSaligenyl-
2′,3′-dideoxy-2′,3′-didehydrothymidine Monophosphates, a Second
Type of New, Enzymatically Activated cycloSaligenyl Pronucleotides.
J. Med. Chem. 2008, 51, 8115–8123.
(6) Meier, C.; De Clercq, E.; Balzarini, J. Nucleotide Delivery from
cycloSaligenyl-3′-azido-3′-deoxythymidine Monophosphates (cycloSal-
AZTMP). Eur. J. Org. Chem. 1998, 837–846.
(7) Wendicke, S.; Warnecke, S.; Meier, C. Efficient Synthesis of Nucleo-
side Diphosphate Glycopyranoses. Angew. Chem. 2008, 120, 1365-
1376; Angew. Chem., Int. Ed. 2008, 47, 1500-1502.
(8) Ducho, C.; Balzarini, J.; Naesens, L.; De Clercq, E.; Meier, C. Aryl-
Substituted and Benzo-Annulated cycloSal-Derivatives of 2′,3′-Dideoxy-
2′,3′-didehydrothymidine Monophosphate. Correlation of Structure,
Hydrolysis Properties and Anti-HIV Activity. AntiViral Chem. Chemoth-
er. 2002, 13, 129–141.
(9) Kita, Y.; Takeda, Y.; Iio, K.; Yokogawa, K.; Takahashi, K.; Akai, S.
An Efficient Preparation of peri-Hydroxy Dihydroquinone Derivatives
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Hydroxy Aromatic Sulfoxides. Tetrahedron Lett. 1996, 37, 7545–7548.
(10) Pearson, A. J.; Zhang, P.; Lee, K. Application of Arene-Ruthenium
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JM900164G