Bis-cycloSal-d4T-monophosphates: Drugs that deliver two molecules of bioactive nucleotides

Article abstract of DOI:10.1021/jm0611713

Bis-cycloSal-d4T-monophosphates have been synthesized as potentially anti-HIV active "dimeric" prodrugs of 2′,3′-dideoxy- 2′,3′-didehydrothymidine monophosphate (d4TMP). These pronucleotides display a mask-drug ratio of 1:2, a novelty in the field of pron

Full text of DOI:10.1021/jm0611713

J. Med. Chem. 2007, 50, 1335-1346
1335
Bis-cycloSal-d4T-monophosphates: Drugs That Deliver Two Molecules of Bioactive Nucleotides
Christian Ducho,† Ulf Go¨rbig,† So¨nke Jessel,† Nicolas Gisch,† Jan Balzarini,‡ and Chris Meier*,†
Department of Chemistry, Organic Chemistry, Faculty of Science, UniVersity of Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg,
Germany, and Rega Institute for Medical Research, K. U. LeuVen, Minderbroedersstraat 10, B-3000 LeuVen, Belgium
ReceiVed October 10, 2006
Bis-cycloSal-d4T-monophosphates have been synthesized as potentially anti-HIV active “dimeric” prodrugs
of 2′,3′-dideoxy-2′,3′-didehydrothymidine monophosphate (d4TMP). These pronucleotides display a mask-
drug ratio of 1:2, a novelty in the field of pronucleotides. Both bis-cycloSal-d4TMP 6 and bis-5-methyl-
cycloSal-d4TMP 7 showed increased hydrolytic stability as compared to their “monomeric” counterparts
and a completely selective hydrolytic release of d4TMP. The hydrolysis pathway was investigated Via 31P
NMR spectroscopy. Moreover, due to the steric bulkiness, compound 6 already displayed strongly reduced
inhibitor potency toward human butyrylcholinesterase (BChE), while compound 7 turned out to be devoid
of any inhibitory activity against BChE. Partial separation of the diastereomeric mixture of 6 revealed strong
dependence of the pronucleotides’ properties on the stereochemistry at the phosphorus centers. Both 6 and
7 showed good activity against HIV-1 and HIV-2 in wild-type CEM cells in Vitro. These compounds were
significantly more potent than the parent nucleoside d4T 1 in HIV-2-infected TK-deficient CEM cells,
indicating an efficient TK-bypass.
Introduction
ribo-ddAMP while the parent nucleoside is entirely inactive.8
A further example of such a dramatic effect is the application
of the cycloSal technology to (E)-5-(2-bromovinyl)-2′-deoxy-
uridine (BVDU): while BVDU itself is inactive against the
Epstein-Barr virus (EBV), vaccinia virus, and cowpox virus,
several cycloSal derivatives of BVDU showed significant
potency against these viruses.11
A major problem in the application of nucleoside analogues
as antivirally active agents is their requirement for bioactivation
and conversion to the nucleoside triphosphates, the biologically
active species. In the case of the anti-HIV active 3′-deoxy-2′,3′-
didehydrothymidine (d4T) 1 (Figure 1), thymidine kinase (TK)-
mediated phosphorylation yields d4T monophosphate (d4TMP)
and is followed by two additional enzymatic steps to lead to
d4T diphosphate (d4TDP) and finally d4T triphosphate (d4TTP).1
D4TTP is antivirally active as it inhibits the reverse transcriptase
(RT) of HIV. The first phosphorylation step yielding d4TMP
represents the metabolic bottleneck of this bioactivation cascade.
Often, nucleoside analogues have a poor affinity for the first
phosphorylation reaction.2 Hence, several attempts have been
made to bypass the first phosphorylation step by application of
lipophilic prodrugs of nucleoside monophosphates (NMPs).3
These pronucleotides are designed to allow cellular uptake and
intracellular release of NMPs. Among these approaches, the
cycloSaligenyl (cycloSal) concept was established as a nucle-
otide delivery system.4 The intracellular cleavage of cycloSal
pronucleotides (cycloSal-d4TMP 2, Figure 1) is based on an
entirely pH-driven chemical hydrolysis mechanism, which has
been reported extensively and is briefly summarized in Figure
1.5 It should be pointed out that cycloSal pronucleotides display
a mask-drug ratio of 1:1 while all other pronucleotide ap-
proaches lead to the delivery of a relatively higher number of
masking groups (mask-drug ratio usually 2-4:1).3
As cycloSal pronucleotides represent reactive organophos-
phates, which may be potential cholinesterase inhibitors,
investigations concerning such an unwanted effect had to be
carried out.12 Cholinesterase inhibition might potentially lead
to serious side effects in a possible therapeutic application of
cycloSal compounds. However, in a recent study we have clearly
proven that cycloSal pronucleotides do not inhibit the physi-
ologically important acetylcholinesterase (AChE, EC 3.1.1.7,
neither that from calf serum nor from beef erythrocytes nor from
Electrophorus electricus nor human AChE).12,13 In contrast, a
structure-activity relationship was found for the inhibition of
human butyrylcholinesterase (BChE, EC 3.1.1.8). BChE is a
serum enzyme, much less specific than AChE. Its physiological
role is yet unclear. It is known though that BChE can be used
for the treatment of succinylcholine apnea in humans,14 and it
is known to protect rodents from the toxic effects of cocaine.15
However, we also could show that substitution patterns of
increasing steric demand in the cycloSal moiety could prevent
the cycloSal pronucleotides from displaying the unwanted BChE
inhibition or at least significantly reduce their inhibitory potency,
possibly due to a steric repulsion in the active site of the
enzyme.12,16 Hence, there was an interest in the development
of cycloSal derivatives with increased “bulkiness”.
Besides d4T 1,5,6 the cycloSal pronucleotide approach led to
remarkable improvement of the antiviral activity with several
other nucleoside analogues, including 2′,3′-dideoxyadenosine
(ddA),7 2′,3′-dideoxy-2′,3′-didehydroadenosine (d4A),7 2′-fluoro-
2′,3′-dideoxyadenosine (2′-F-ddA),8 and the carbocyclic nucleo-
sides carbovir (CBV), abacavir,9 and acyclovir (ACV).10
Impressive is the significant anti-HIV activity of cycloSal-F-
We already described the synthesis and properties of 3-phen-
yl-cycloSal-d4TMP 5 (Figure 2).17 While this derivative showed
a satisfying hydrolytic profile as well as high antiviral potency,
it was a surprisingly strong BChE inhibitor.12 Nevertheless, its
otherwise beneficial properties led us to the design of so-called
bis-cycloSal-d4TMP 6 and bis-5-methyl-cycloSal-d4TMP 7,
“dimeric” forms of the prototype cycloSal-d4TMP 8 and
5-methyl-cycloSal-d4TMP 9 (Figure 2). Compound 6 derives
from 5 by formal attachment of a second nucleotidyl moiety to
* To whom correspondence should be addressed. Tel.: +49-40-
42838-4324; Fax:
+49-40-42838-2495. E-mail:
chris.meier@
chemie.uni-hamburg.de.
† University of Hamburg.
‡ Rega Institute for Medical Research.
10.1021/jm0611713 CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/01/2007

1336 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Ducho et al.
Figure 1. The chemical formula of d4T 1 and the hydrolysis pathway of cycloSal pronucleotides with cycloSal-d4TMP 2 shown as the example.
Figure 2. Structures of “monomeric” cycloSal-d4TMPs 5, 8, 9 and novel “dimeric” target structures 6, 7.
the second aromatic ring of the biphenyl system of 5. The design
of 6, 7 was based on three rationales: first, they display a formal
mask-drug ratio of 1:2, an unprecedented novelty in the field
of pronucleotides. Second, their massively increased steric
bulkiness should lead to a complete loss or at least strong
reduction of BChE inhibitor activity. Third, the additional methyl
groups in 7 as compared to 6 should lead to a higher hydrolytic
stability which might be advantageous with respect to nucleotide
delivery in the intact cells and thus the prodrug’s antiviral
activity.5
homo-coupled using iron(III) acetylacetonate leading to 14 in
84% yield (Scheme 1).
A similar reaction had been observed before by Katz and
Cram as an unwanted side reaction in an attempted cross-
coupling.20 However, acidic deprotection of 14 employing ion-
exchange resin (Dowex), a method otherwise identified to be
useful for cleavage of salicyl alcohol isopropylidene acetals in
our laboratories, turned out to be difficult. Multiple amounts of
methylated byproduct 15 (24-60% isolated yields) were formed
depending on the reaction conditions. Several attempts to avoid
the formation of compound 15 by changing and optimizing the
conditions (reaction time, amount of Dowex and methanol)
failed. The best isolated yield for tetrol 10 upon Dowex
treatment of 14 on a larger scale was 28%. Although this
drawback seems to make the second route for the preparation
of 10 unfavorable as compared to the first one, avoidance of
the use of the highly toxic MOM-chloride and easier purification
have to be mentioned as two key advantages of the second
approach.
In this study, we describe the synthesis of 6, 7 and investiga-
tions concerning their hydrolytic properties, hydrolysis path-
ways, BChE inhibitor potencies, and anti-HIV activities.
Results and Discussion
Chemistry. Generally, for the synthesis of cycloSal pro-
nucleotides, an approach using phosphorus(III) chemistry has
been established. The required cyclic saligenyl chlorophosphites
can be obtained from appropriately substituted salicyl alcohol
derivatives.4
For the synthesis of bis-5-methyl-cycloSal-d4TMP 7, alcohol
16 was needed (Scheme 1). Thus, methylated biphenyl diol 17
was synthesized starting from 2-bromo-4-methylanisole 18
according to procedures reported before21 and employing an
iron-cross coupling reaction as described above (52% yield over
two steps). Subsequent hydroxymethylation of 17 using form-
aldehyde under basic conditions directly led to the isolation of
16, though in moderate yield (48%). An application of this rather
simple hydroxymethylation method for the preparation of 10
had been impossible as 2,2′-biphenyldiol has both an unsub-
stituted ortho- and para-position relative to the phenolic hydroxyl
group which can react with formaldehyde, probably resulting
in tedious product mixtures.
Accordingly, for the synthesis of bis-cycloSal-d4TMP 6 the
hydroxymethylated biphenyl diol (“dimeric” salicyl alcohol) 10
was prepared first. The synthesis of 10 could be achieved Via
two different routes. For the first route, dialdehyde 11 has been
synthesized according to a previously published procedure18 with
few modifications regarding purification: commercially avail-
able biphenyl 2,2′-diol was protected as a methoxymethyl
(MOM) ether (66% yield) and subsequently bis-formylated using
DMF as a formylating agent after ortho-lithiation. However,
chromatographic purification of the protected dialdehyde turned
out to be extremely difficult. Hence, a crude mixture of this
material was deprotected to yield dialdehyde 11 (48% over two
steps). Final reduction of 11 using LiAlH4 led to isolation of
the desired alcohol 10 in 94% yield (30% over four steps,
Scheme 1).
For the conversion of alcohols 10, 16 into the corresponding
phosphitylating agents 19, 20, tetrols 10, 16 were treated with
phosphorus(III) chloride under basic conditions. However,
besides formation of the desired six-membered rings, formation
of seven-membered rings leading to unwanted byproducts also
seemed to be possible (shown for 10 in Scheme 2). 31P NMR
investigation of the crude product from the reaction of 10 with
The second route for the preparation of 10 was based on
3-bromosalicyl alcohol isopropylidene acetal 12 which can be
obtained from 2-bromophenol 13 in three steps (overall yield
52%).19 Acetal 12 was lithiated and subsequently oxidatively

Bis-cycloSal-d4T-monophosphates
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1337
Scheme 1. Synthesis of Biphenyl Tetrols 10,16a
a Reagents and conditions: (a) (i) NaH, THF, 0 °C to r.t., 1 h; (ii) MOM-Cl, 0 °C to r.t., 16 h; (b) (i) n-BuLi, Et2O, r.t., 2 h; (ii) DMF, 0 °C to r.t., 5 h;
(c) 6 M HCl, CH2Cl2, EtOH, reflux, 16 h; (d) (i) LiAlH4, THF, r.t. to reflux, 3 h; (ii) H+/H2O; (e) see ref 19; (f) (i) n-BuLi, THF, -80 °C, 1 h; (ii) Fe(acac)3,
THF, -80 °C to r.t., 17 h; (g) Dowex 50X8, CH2Cl2/MeOH 1:1, r.t., 65 h; (h) see ref 21; (i) (i) 37% HCHO/H2O, NaOH, 40 °C, 4 d; (ii) H+/H2O;
MOM ) methoxymethyl.
Scheme 2. Possible Reaction Products for the Reaction of 10
with PCl3
to be distilled in Vacuo, a way to prepare saligenyl phosphor-
amidites from crude chlorophosphites had to be elaborated. In
a model reaction, crude 3-phenyl saligenyl chlorophosphite 2117
was treated with diisopropylamine (DIPA). However, instead
of saligenyl N,N-diisopropylaminophosphoramidite 22, the air-
stable ring contraction product 23 was isolated. Formation of
phosphonamidate 23 probably was the result from an Arbuzov-
Michaelis rearrangement of 22 catalyzed by traces of acidic
pyridinium chloride present in the crude mixture of 21. Traces
of pyridinium chloride are often present because the preparation
of chlorophosphites like 21 is achieved using pyridine as base.23
In contrast, it was demonstrated that saligenyl phosphoramidites
can be prepared from crude chlorophosphites if these had been
prepared in the absence of pyridine, e.g., if triethylamine was
used instead of pyridine. Triethylammonium chloride as a
potential impurity of 24 probably is too weak as an acid to
catalyze an Arbuzov-Michaelis rearrangement of 25 similar
to that of 22. The crude phosphoramidite 25 can subsequently
be used for pronucleotide synthesis in satisfying yields as can
be seen in the case of cycloSal-d4TMP 8 (Scheme 3).
Application of this synthetic methodology led to the suc-
cessful preparation of N,N-diisopropylaminophosphoramidite 22
from chlorophosphite 19. Treatment of d4T 1 with 22 and
subsequent oxidation afforded target compound 6. However,
the yield (5%) was found to be similar to that obtained on the
chlorophosphite route. In order to circumvent these purification
problems, N,N-diethylaminophosphoramidite 26 was synthesized
and applied on nucleotide synthesis instead. Unfortunately, this
approach did not lead to a considerable improvement in the yield
of 6 (6%) either. Thus, the synthesis of the second target
compound 7 was performed using chlorophosphite chemistry
Via phosphitylating agent 20 (prepared analogously to 19) finally
leading to the isolation of 7 in 11% yield (Scheme 3).
PCl3 using the standard conditions (pyridine at -20 °C)4 indeed
indicated significant formation of seven-membered ring byprod-
ucts (integral ratio 70% of product 19 to 30% of byproducts).
Further reduction of the reaction temperature helped to decrease
the amount of byproducts formed significantly (-40 °C: (87:
13); -60 °C: (88:12)), so that the synthesis of 19 was finally
carried out at -40 °C instead of -20 °C.
Subsequent treatment of d4T 1 with crude chlorophosphite
19 (most saligenyl chlorophosphites are too sensitive for
standard purification procedures)4 led to target pronucleotide
6, but only after tedious chromatography and in poor yield (8%,
Scheme 3).
Consequently, ways to improve the yield of nucleotide
synthesis were investigated. The synthesis of cycloSal com-
pounds Via phosphoramidite chemistry was described before,22
but preparation of the required phosphoramidite reagents from
the saligenyl chlorophosphites was performed starting from
purified chlorophosphites which were obtained in the case of
low molecular weight saligenyl chlorophosphites by vacuum
Kugelrohr distillation. As compound 19 was not volatile enough
CycloSal-pronucleotides were always obtained as mixtures
of two diastereomers (RP and SP configuration).4 In the case of
bis-cycloSal-d4TMPs 6, 7, two stereogenic centers are formed
in the course of preparation. Hence, they should be obtained as
mixtures of three stereoisomers (RP/RP, RP/SP, and SP/SP con-

1338 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Ducho et al.
Scheme 3. Synthesis of Pronucleotides 6, 7 from Alcohols 10, 16 and Model Reactionsa
a Reagents and conditions: (a) (i) PCl3, Et2O, -40 °C, 10 min; (ii) pyridine or NEt3 (if subsequent conversion to a phosphoramidite was envisaged),
-40 °C, 2 h; (iii) r.t., 2 h; (b) (alkyl)2NH, Et2O, r.t., 3 h, no purification; (c) (i) d4T 1, DIPEA, CH3CN, -20 °C to r.t., 1 h; (ii) t-BuOOH, -20 °C to r.t.,
1 h; (d) like c, but 6 h reaction period in i; (e) (i) d4T 1, pyridine‚HCl, CH3CN, 0 °C, 3 h; (ii) t-BuOOH, 0 °C to r.t.,1 h; (f) (i-Pr)2NH, Et2O, r.t., 20 h; (g)
(i) PCl3, Et2O, -20 °C, 10 min; (ii) NEt3, -20 °C, 2.5 h; (iii) r.t., 2 h; (h) like b with alkyl ) i-Pr; DIPEA ) diisopropylethylamine (Hu¨nig’s base).
Table 1. Hydrolytic Stabilities of Bis-cycloSal-d4TMPs 6, 7 Compared
to “Monomeric” Reference Compounds
on the hydrolytic stability of 6 was significant: 6 slow (t1/2
)
8.5 h) possessed a half-life slightly higher than that of the
diastereomeric mixture. However, 6 fast (t1/2 ) 3.2 h) proved
to be even more labile than the “monomeric” reference 8
(diastereomeric mixture).
cycloSal-d4TMP
t
1/2 [h]a
6 (mix, X ) H)
6 fast (X ) H)
6 slow (X ) H)
7 (mix, X ) Me)
5 (mix, X ) 3-Ph)
8 (mix, X ) H)
9 (mix, X ) 5-Me)
8.2
3.2
8.5
12
5.1
4.4
6.2
Hydrolysis Pathway. It was of major importance to inves-
tigate whether the chemical hydrolysis of 6, 7 led to d4TMP
formation. Therefore, the pathway of hydrolysis of 6 was studied
in detail. The prodrug 6 might either undergo simultaneous
cleavage of both phosphate triester moieties according to the
mechanism proven for cycloSal pronucleotides (Figure 1)
leading to the bis-(benzyl phosphate diester) 27, which then
yields 2 equiv of d4TMP and alcohol 10 as the byproduct, or
might be hydrolyzed consecutively, implying that product 29
is formed Via the labile intermediate 28 prior to the liberation
of the second d4TMP molecule. These theoretically possible
hydrolytic routes are depicted in Scheme 4.
The only intermediate in this hydrolysis pattern not being
intrinsically instable is pronucleotide 29 (which will be named
3-Sal-cycloSal-d4TMP). Thus, an independent synthesis of 29
was necessary in order to be used as reference for hydrolysis
studies on 6. Its individual preparation was achieved starting
from alcohol 10 (Scheme 5).
After several unsuccessful attempts to selectively protect only
one of the salicyl alcohol moieties of tetrol 10 as an isopropy-
lidene acetal, one of the benzyl alcohol functions in 10 was
blocked as tert-butyldiphenyl silyl (TBDPS) ether. The best
results for this reaction were achieved using 0.75 equiv of
TBDPS chloride and DMAP as an activator, allowing the
isolation of monosilyl ether 30 in 53% yield with concomitant
formation of the unwanted byproduct 31 in 11% yield.
Subsequent protection of 30 as isopropylidene acetal (32) was
achieved using 2,2-dimethoxypropane under acidic conditions
in 60% yield. However, after desilylation of 32 to give the salicyl
alcohol derivative (89% yield), isopropylidene protected pro-
nucleotide 33 (3-Isopr-Sal-cycloSal-d4TMP) could be prepared
a 25 mM phosphate buffer, pH 7.3, 37 °C; t1/2 values refer to disappear-
ance of starting pronucleotides.
1
figuration, respectively) in a ratio of 1:2:1. According to H
and 31P NMR spectroscopy, 6 indeed was obtained as a mixture
of three diastereomers, but in a ratio of 1:2:2. The minor
stereoisomer (6 fast) could be separated by preparative RP-
HPLC from the remaining mixture of stereoisomers (6 slow),
which proved to be inseparable. Interestingly, 7 was isolated
as a mixture of only two diastereomers (ratio 0.7:1.0). Most
probably, the expected third stereoisomer was lost from the
product mixture during chromatographic purification. However,
the diastereomeric mixture of 7 turned out to be inseparable
Via RP-HPLC just like 6 slow. It should be pointed out that
neither 6 nor 7 formed atropisomers due to potentially hindered
rotation around the biphenyl axis.
Hydrolytic Stabilities. The hydrolytic stabilities of 6,7 in
phosphate buffer (pH 7.3, 37 °C) were investigated (release of
d4TMP was measured by HPLC analysis) and compared to
several reference compounds (Table 1). Both 6 (diastereomeric
mixture, t1/2 ) 8.2 h) and 7 (t1/2 ) 12 h) turned out to be 2-fold
more stable than their “monomeric” counterparts, cycloSal-
d4TMP 8 (t1/2 ) 4.4 h) and 5-methyl-cycloSal-d4TMP 9 (t1/2
) 6.2 h). This was clearly attributed to the presence of the
second nucleotidyl moiety, as a comparison of the stabilities of
6 and 3-phenyl-cycloSal-d4TMP 5 (t1/2 ) 5.1 h) has shown.
The influence of the stereochemistry at the phosphorus centers

Bis-cycloSal-d4T-monophosphates
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1339
Scheme 4. Potential Pathways for the Hydrolysis of Bis-cycloSal-d4TMP 6
Table 2. Hydrolytic Stabilities of the cycloSal-d4TMPs 29, 33
Scheme 5. Synthesis of 3-Sal-cycloSal-d4TMP 29, a Potential
Intermediate in the Hydrolysis of 6a
cycloSal-d4TMP
t1/2 [h]a
29 (mix)
29 (RP)
29 (SP)
33 (mix)
33 (RP)
33 (SP)
1.3
1.4
1.1
16
21
14
a 25 mM phosphate buffer, pH 7.3, 37 °C; t1/2 values refer to disappear-
ance of starting pronucleotides.
reaction before complete conversion of the acetal in order to
prevent extensive side reactions. As attempts to crystallize either
29 or 33 in order to obtain an X-ray structure failed, assignment
of the stereochemistry at the phosphorus centers was performed
using CD spectroscopy and comparison of the spectra to those
of a cycloSal reference compound with known stereochemical
configuration.4c
The pronucleotides 29, 33 were investigated for their
hydrolytic stability as described above (Table 2). While 29
turned out to be remarkably labile (t1/2 ) 1.3 h) as compared to
both the 3-phenyl derivative 5 and “dimer” 6 (t1/2 ) 5.1 h and
t1/2 ) 8.2 h, Table 1), its protected precursor 33 displayed a
significant stability (t1/2 ) 16 h) in comparison to the reference
compounds, pointing to a major influence of the polarity or the
steric effects of the phenyl ring substituents on the hydrolytic
stabilities. Lipophilic and sterically demanding substituents may
prevent a nucleophilic attack on the phosphate triester, as the
most lipophilic derivative 33 showed the longest hydrolysis half-
life of the pronucleotides studied. In both cases (29 and 33),
the RP-configured diastereomer turned out to be more stable
than the SP isomer, but while this effect was rather small for 29
(∆t1/2 ∼ 0.3 h), it was more pronounced for 33 (∆t1/2 ∼ 7 h).
Following the preparation of the potential hydrolytic inter-
mediate 29, the hydrolysis of 6 (mixture of three diastereomers)
was investigated using 31P NMR spectroscopy (imidazole/HCl
buffer, pH 7.3, r.t.). The changes in conditions compared to
the stability studies in phosphate buffer allowed the monitoring
of the hydrolytic reaction over several days. In an initial
experiment, the mixture of stereoisomers of 6 was incubated
and only the final hydrolysis products were of interest. D4TMP
was detected as the sole hydrolysis product of 6 (Figure 3).
a Reagents and conditions: (a) (i) 0.75 equiv of TBDPS-Cl, NEt3, DMAP,
THF, 0 °C to r.t., 2 h; (ii) CH3OH; (b) 2,2-dimethoxypropane, p-TsOH,
Na2SO4, acetone, 40 °C, 3 d; (c) NH4F, CH3OH, r.t., 61 h; (d) (i) PCl3,
Et2O, -40 °C; (ii) pyridine/Et2O, -40 °C, 1 h; (iii) r.t., 1 h; (e) (i) d4T 1,
DIPEA, CH3CN, -20 °C to r.t., 1 h; (ii) t-BuOOH, -20 °C to r.t., 1 h; (f)
Dowex 50X8, CH2Cl2/MeOH 1:1, r.t., 8 d; TBDPS ) tert-butyldiphenylsilyl;
DMAP ) 4-dimethylaminopyridine; DIPEA ) diisopropylethylamine
(Hu¨nig’s base).
in 60% yield using the chlorophosphite method. The diastere-
omeric mixture of 33 (RP/SP) was separated using preparative
RP-HPLC, and the diastereomerically pure material was then
deprotected to afford two diastereomers of 29 separately. Mild
cleavage of the isopropylidene acetal was achieved by treatment
of 33 with Dowex ion-exchange resin in moderate yields of
54% and 46%, respectively, due to the necessity to stop the

1340 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Ducho et al.
Figure 3. 31P NMR hydrolysis study of pronucleotide 6 (diastereomeric mixture).
Table 3. BChE Inhibitory Potency and Anti-HIV Activity of the Novel cycloSal-d4TMPs
antiviral activity EC50 [µM]b
CEM/0
CC50 [µM]c
CEM/TK-
HIV-2
CEM/0
compound
IC50 (BChE) [µM]a
HIV-1
HIV-2
6 (mix, X ) H)
6 fast (X ) H)
6 slow (X ) H)
7 (X ) Me)
29 (mix)
29 (RP)
29 (SP)
33 (mix)
33 (RP)
40
14
>50
>50
2.3
>50
0.79
4.7
0.19 ( 0.05
0.25 ( 0.14
0.21 ( 0.17
0.37 ( 0.25
n.d.d
0.27 ( 0.11
0.30 ( 0.09
0.22 ( 0.16
0.41 ( 0.19
n.d.
2.33 ( 0.58
3.33 ( 2.31
2.37 ( 1.48
1.60 ( 0.57
n.d.
0.70 ( 0.05
1.50 ( 0.70
1.07 ( 0.83
0.55 ( 0.21
3.0 ( 1.4
0.28 ( 0.18
0.30 ( 0.25
1.25 ( 0.90
n.d.
27.8 ( 11.0
107 ( 13
90.7 ( 16.1
67.1 ( 7.1
n.d.
0.27 ( 0.05
0.34 ( 0.10
0.35 ( 0.09
0.36 ( 0.07
0.50 ( 0.14
0.13 ( 0.05
0.15 ( 0.07
0.46 ( 0.24
>150
0.50 ( 0.05
0.40 ( 0.00
0.40 ( 0.00
0.40 ( 0.00
0.40 ( 0.00
0.27 ( 0.13
0.13 ( 0.06
0.46 ( 0.48
>150
69.4 ( 11.7
78.7 ( 6.2
65.0 ( 6.7
55.3 ( 23.0
89.3 ( 11.7
22.0 ( 5.0
26.5 ( 22.4
37.9 ( 20.0
>150
>50
33 (SP)
3.6
5 (X ) 3-Ph)
8 (X ) H)
9 (X ) 5-Me)
10
16
d4T 1
0.35
0.77
4.6
>50
>50
-
>150
0.25 ( 0.00
>150
0.19 ( 0.10
n.d.
51.0 ( 10.4
>150
105 ( 78
a Inhibitor activity: 50% inhibitory concentration against human BChE (human serum). b Antiviral activity: 50% effective concentration. c Cytostatic
activity: 50% cytostatic concentration. d n.d. ) not determined.
A more detailed investigation on the hydrolysis of partially
separated diastereomers 6 fast/slow followed with more 31P
NMR spectra recorded at shorter time intervals. However, for
both hydrolyses the formation of 29 (RP/SP) could not be
observed. If consecutive hydrolysis takes place, then 29 is
hydrolyzed significantly faster than it is formed, making its
detection impossible. Hence, a consecutive hydrolysis pathway
cannot be ruled out. Analogously proven by 31P NMR spec-
troscopy, the hydrolysis of the methyl-substituted derivative 7
(mixture of two diastereomers) led also exclusively to d4TMP
as the sole hydrolysis product.
Biological Activity
BChE Inhibitor Activity. All synthesized pronucleotides as
well as the tetrols 10, 16 were tested for their inhibitory potency
toward human BChE as described before12 using human serum
as a BChE source. Their inhibitor activities were compared with
several reference compounds (Table 3).
The results for the bis-cycloSal-d4TMPs 6, 7 met the
expectations, as they showed significantly reduced BChE
inhibitory activity. With IC50 values of 40 µM for 6 (diaster-
eomeric mixture) and out of the range of physiological
importance (>50 µM) for 7, they turned out to inhibit human

Bis-cycloSal-d4T-monophosphates
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1341
BChE more than 50-fold (6) and more than 10-fold (7) weaker
than their “monomeric” counterparts cycloSal-d4TMP 8
corresponding SP isomer for both 29 and 33. However, differ-
ences in hydrolytic stabilities alone cannot be responsible for
the observed differences in antiviral activities in the CEM/TK-
cells. For example, 29 (RP) was hydrolytically much more labile
than 33 (SP) (t1/2 ) 1.4 h vs t1/2 ) 14 h), but it possessed a
better anti-HIV-2 activity in CEM/TK- cells, whereas the anti-
HIV-2 activities of 6 fast and 6 slow in this cell line turned out
to be comparable.
The cytotoxicity of most of the cycloSal derivatives tested
seemed to be slightly higher than that of d4T 1, but a specific
trend with respect to certain masking structures or a correlation
to the antiviral activities could not be elucidated. Finally, as
expected the tetrols 10, 16 showed no antiviral activity against
HIV and were found to be non-cytotoxic. This is in complete
agreement with a lot of other salicyl alcohols that we have
synthesized and tested before.
(IC50 ) 0.77 µM)12 and 5-methyl-cycloSal-d4TMP 9 (IC50
)
4.6 µM).12 Furthermore, a comparison of the IC50 value of the
isomeric mixture of 6 to those of several other compounds
reveals the crucial role of the second nucleotidyl moiety to
successfully overcome BChE inhibition. While 3-phenyl-cy-
cloSal-d4TMP 5 (IC50 ) 0.35 µM)12 inhibited the enzyme even
stronger than prototype 8, 3-Sal derivative 28 (mix, IC50 ) 2.3
µM) and its synthetic precursor 33 (mix, IC50 ) 4.7 µM)
displayed a ∼10-fold weaker inhibitory potency. However, this
effect becomes remarkably pronounced only due to the simple
attachment of a second nucleotidyl residue as in 6, leading to a
further 9- to 17-fold decrease of inhibitory activity. The further
reduction in inhibitory activity upon the attachment of two
additional methyl substituents (7) is in accordance to previous
results proving disubstituted “monomeric” cycloSal nucleotides
to be rather weak BChE inhibitors.12
Conclusion
The essential influence of the stereochemistry at the phos-
phorus center on the BChE inhibitor potency of the cycloSal
pronucleotides has been described before12 and could be
confirmed within this study. The RP diastereomers of the
monomeric cycloSal derivatives 29, 33 showed no BChE
inhibitory activity at all, and the effect solely occurred from
the SP isomer. Similarly, the single diastereomer 6 fast displayed
weak BChE inhibition (IC50 ) 14 µM) while the remaining two
diastereomers 6 slow turned out to be noninhibitory (IC50 > 50
µM). Thus, it can be assumed that 6 fast should be the (SP,SP)
diastereomer though this needs further confirmation in future
investigations. As expected the tetrols 10, 16 showed no
inhibitory activity against BChE.
In summary, the first synthesis of bis-cycloSal-d4TMPs 6, 7
as “dimeric” pronucleotides bearing a mask-drug ratio of 1:2
was accomplished. The novel derivatives proved to be hydro-
lytically more stable than their “monomeric” counterparts and
exclusively hydrolyzed to d4TMP. They showed significantly
reduced inhibitory potency toward human BChE, making this
bis-cycloSal-nucleotide approach attractive for further applica-
tions. The anti-HIV-2 activities of 6, 7 in TK-deficient human
CEM cells were significantly higher than that of the parent
nucleoside d4T 1, providing a rather efficient TK-bypass
capacity of these prodrugs. Additional work to further improve
the mask structure is envisaged and is currently in progress.
Experimental Section
Antiviral Activity and Cytotoxicity. The synthesized pro-
nucleotides and tetrols 10, 16 were evaluated for their anti-
HIV activity and cytotoxicity in Vitro (Table 3). All cycloSal
derivatives investigated showed activities against HIV-1 and
HIV-2 in wild-type CEM cells (CEM/0) comparable to those
of the parent nucleoside 1. The efficiency to bypass TK-
mediated phosphorylation of d4T as a consequence of intra-
cellular d4TMP delivery is reflected by the prodrugs’ retention
of marked anti-HIV-2 activities in TK-deficient CEM cells
(CEM/TK-). Several pronucleotides (29 (RP), 33 (RP) and
references 5, 8) displayed virtually full retention of their anti-
HIV-2 activities in the TK-deficient cell line, indicating a highly
successful TK-bypass. Although still showing activities about
25-fold more pronounced than d4T, bis-cycloSal-d4TMPs 6, 7
slightly lost some antiviral activity in the TK- cells as compared
to the wild-type cells. It cannot be ruled out that the cellular
uptake of this new type of pronucleotide was partially hindered
due to its markedly increased molecular size. Furthermore, it
seems remarkable that 33 (diastereomeric mixture) displayed a
slight loss of activity in CEM/TK- cells as well since its
significant hydrolytic stability should allow an efficient cell
penetration prior to its cleavage. Interestingly, the stereochem-
istry at the phosphorus atoms also had an influence on the
antiviral activities. For both 29 and 33, the RP diastereomer led
to a successful TK-bypass while the SP diastereomer lost some
activity in the TK- cells. It would be tempting to speculate that
the RP diastereomer could be better taken up by the intact CEM
cells than the SP diastereomer, suggesting a degree of preference
or selectivity of uptake of diastereomeric compounds. However,
this finding is also important since the more active RP isomer
did not show any BChE inhibitor potency, while the less
active SP isomer did. Moreover, it should be pointed out
that the RP isomer was hydrolytically more stable than the
Chemistry. All air-sensitive reactions were carried out under
an atmosphere of dry nitrogen. Solvents used in the inert gas
syntheses were commercially available dry solvents stored under
argon and over molecular sieves (Fluka). Diethyl ether and THF
were dried over sodium/benzophenone and distilled under nitrogen.
Merck precoated 60 F254 plates with a 0.2 mm layer of silica gel
were used for thin layer chromatography (TLC). All preparative
TLCs were performed on a Chromatotron (Harrison Research, Model
7924T) using glass plates coated with 1 mm or 2 mm layers of
Merck 60 PF254 silica gel containing a fluorescent indicator. For
column chromatography, Merck silica gel 60, 230-400 mesh was
used. NMR spectra were recorded using Bruker AMX 400 and
Bruker DRX 500 Fourier transform spectrometers. All 1H and 13
C
NMR chemical shifts (δ) are quoted in ppm 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. EI mass spectra were
measured on a VG Analytical VG/70-250S spectrometer (double
focusing). FAB low and high resolution (HR) mass spectra were
recorded on a VG Analytical 70-250S spectrometer using an MCA
method and poly(ethylene glycol) as support. ESI mass spectra were
obtained on a Finnigan ThermoQuest MAT 95 XL spectrometer.
IR spectra were measured on a Thermo Nicolet AVATAR 370 FT-
IR spectrometer, and wavenumbers ν are quoted in cm-1. 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). 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

1342 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Ducho et al.
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. The appropri-
ate fractions were pooled and lyophilized. The lyophilized cycloSal
compounds did not give useful microanalytical data most probably
due to incomplete combustion of the compounds or varying amounts
of water but were found to be pure by rigorous HPLC analysis.
General Procedure A (Deprotection of Salicyl Alcohol Iso-
propylidene Acetals). The salicyl alcohol isopropylidene acetal
was dissolved in DCM/MeOH 1:1 and vigorously stirred with
Dowex 50X8 (H+ form, previously washed with DCM and MeOH)
at room temperature for the indicated time. The reaction was
monitored Via TLC or HPLC. After filtration, the filtrate was
evaporated under reduced pressure, and the crude product was
purified by chromatography.
General Procedure B (Synthesis of Saligenyl Chlorophos-
phites). Under a nitrogen atmosphere, freshly distilled PCl3 was
added to a solution of the salicyl alcohol derivative in Et2O at the
indicated temperature. After ca. 10 min, a solution of pyridine or
NEt3 in Et2O was added dropwise at the same temperature over
2-3 h. The reaction mixture was warmed to room temperature and
further stirred at room temperature for 1-2 h. Pyridinium or
triethylammonium chloride was completely precipitated by storing
the mixture at -20 °C overnight. After filtration under nitrogen,
the solvent was removed in Vacuo. The resulting crude products
were used in pronucleotide synthesis without further purification.
General Procedure C (Synthesis of Saligenyl Phosphoramid-
ites). Under a nitrogen atmosphere, a solution of dialkylamine in
Et2O was added dropwise to a solution of crude saligenyl chloro-
phosphite in Et2O at room temperature. The mixture was stirred
for 3 h at room temperature. After filtration under nitrogen, the
solvent was removed in Vacuo. The resulting crude products were
used in pronucleotide synthesis without further purification.
General Procedure D (Synthesis of cycloSal Nucleotides Using
Chlorophosphites). Under a nitrogen atmosphere, DIPEA and the
crude saligenyl chlorophosphite, dissolved in MeCN, were added
to a solution of d4T 1 in MeCN at -20 °C. The reaction mixture
was warmed to room temperature and stirred for 1 h. The reaction
was monitored Via TLC (DCM/MeOH 9:1). After cooling to
-20 °C, a solution of tert-butyl hydroperoxide in n-decane was
added. The solution was warmed to room temperature again and
further stirred for 1 h (TLC monitoring). The solvent was removed
in Vacuo, and the resulting crude products were purified by
preparative TLC (Chromatotron, 1. EtOAc/MeOH 9:1, 2. DCM/
MeOH gradient, MeOH contained 0.1% HOAc). The oily products
were lyophilized to yield colorless foams.
General Procedure E (Synthesis of cycloSal Nucleotides Using
Phosphoramidites). Under a nitrogen atmosphere, crude saligenyl
phosphoramidite, dissolved in MeCN, was added to a solution of
d4T 1 and pyridinium chloride in MeCN at 0 °C. The reaction
mixture was stirred 3 h at 0 °C. The reaction was monitored Via
TLC (DCM/MeOH 9:1). Subsequently, a solution of tert-butyl
hydroperoxide in n-decane was added at 0 °C. The solution was
warmed to room temperature and further stirred for 1 h (TLC
monitoring). The solvent was removed in Vacuo, and the resulting
crude products were purified as described in general procedure D.
Bis-cycloSal-d4TMP (6). Method I: General Procedure D with
d4T 1 (300 mg, 1.34 mmol), crude chlorophosphite 19 (938 mg),
DIPEA (0.47 mL, 0.35 g, 2.7 mmol), 5.5 M solution of tert-butyl
hydroperoxide in n-decane (0.86 mL, 4.8 mmol), and MeCN (30
mL). The product 6 (42 mg, 8%) was obtained as a colorless foam
and mixture of three diastereomers (1.1:2.0:1.9): 1H NMR (500
MHz, DMSO-d6) δ 11.34 (s, 6H), 7.42-7.36 (m, 6H), 7.36-7.27
(m, 6H), 7.27-7.23 (m, 6H), 7.17 (s, 2H), 7.16 (s, 2H), 7.12 (s,
2H), 6.81-6.76 (m, 6H), 6.32-6.27 (m, 6H), 6.03-5.92 (m, 6H),
5.64-5.40 (m, 12H), 4.99-4.88 (m, 6H), 4.29-4.18 (m, 12H), 1.64
(s, 6H), 1.60 (s, 12H); 13C NMR (101 MHz, DMSO-d6) δ 164.1,
151.1, 147.2, 136.0, 135.9, 133.1, 131.3, 131.1, 127.7, 126.8, 122.4,
110.0, 89.8, 84.4, 69.0, 68.3, 12.2, 12.1; 31P NMR (202 MHz,
DMSO-d6) δ -7.75, -8.21, -8.47; MS (FAB) m/z calcd 783.1
(M + H+), found 783.3 (M + H+); HR-MS (ESI+) m/z calcd
805.1228 (M + Na+), found 805.1290 (M + Na+); UV (HPLC)
λmax 265; TLC Rf value 0.40 (DCM/MeOH 9:1).
Method II: General Procedure E with d4T 1 (379 mg, 1.69
mmol), crude phosphoramidite 22 (1.10 g, dissolved in 25 mL
MeCN), pyridinium chloride (781 mg, 6.76 mmol), 5.5 M solution
of tert-butyl hydroperoxide in n-decane (0.90 mL, 4.9 mmol), and
MeCN (20 mL). Preparative TLC on the Chromatotron did not
provide the pure product, so final purification was achieved using
preparative HPLC (MeCN/H2O 24:76, H2O contained 0.5% HOAc).
This also allowed partial separation of the 3 diastereomers to furnish
6 fast (6 mg, 1 diastereomer) and 6 slow (28 mg, 2 diastereomers,
1.0:1.0) as colorless foams, providing an overall yield of 34 mg
(5%). Analytical data of 6 fast: 1H NMR (500 MHz, DMSO-d6) δ
7.41-7.36 (m, 2H), 7.36-7.32 (m, 2H), 7.32-7.27 (m, 2H), 7.16
(s, 2H), 6.83-6.78 (m, 2H), 6.35-6.29 (m, 2H), 6.04-5.98 (m,
2H), 5.57 (dd, J ) 14.5, 15.1 Hz, 2H), 5.46 (dd, J ) 12.0, 14.5
Hz, 2H), 4.98-4.88 (m, 2H), 4.29-4.18 (m, 4H), 1.61 (s, 6H);
13C NMR (101 MHz, DMSO-d6) δ 164.1, 151.0, 147.2, 136.0,
135.9, 133.3, 133.0, 131.8, 131.1, 127.7, 126.8, 122.3, 110.1, 89.6,
84.3, 68.9, 68.3, 12.1; 31P NMR (202 MHz, DMSO-d6) δ -7.76;
Preparative HPLC tR ) 25.4 min (MeCN/H2O 24:76, H2O contained
0.5% HOAc). Analytical data of 6 slow: 1H NMR (500 MHz,
DMSO-d6) δ 11.33 (s, 4H), 7.41 (dd, J ) 1.9, 6.9 Hz, 4H), 7.37-
7.28 (m, 4H), 7.28-7.24 (m, 4H), 7.18 (s, 2H), 7.12 (s, 2H), 6.82-
6.76 (m, 4H), 6.32-6.27 (m, 4H), 6.05-5.99 (m, 2H), 5.96-5.90
(m, 2H), 5.61 (dd, J ) 14.5, 15.1 Hz, 2H), 5.59 (dd, J ) 14.5,
15.1 Hz, 2H), 5.50 (dd, J ) 13.1, 14.5 Hz, 2H), 5.46 (dd, J )
12.6, 14.5 Hz, 2H), 4.99-4.88 (m, 4H), 4.29-4.18 (m, 8H), 1.64
(s, 6H), 1.61 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ 164.1,
151.0, 147.3, 136.7, 135.9, 133.0, 131.8, 131.6, 127.6, 126.8, 126.8,
122.1, 110.3, 89.5, 89.3, 84.4, 69.0, 68.7, 12.2, 12.1; 31P NMR
(202 MHz, DMSO-d6) δ -8.22, -8.46; Preparative HPLC tR
27.6 min (MeCN/H2O 24:76, H2O contained 0.5% HOAc).
)
Method III: General Procedure E with d4T 1 (539 mg, 2.40
mmol), crude phosphoramidite 26 (1.40 g, dissolved in 35 mL
MeCN), pyridinium chloride (1.11 g, 9.61 mmol), 5-6 M solution
of tert-butyl hydroperoxide in n-decane (1.41 mL, g 7.05 mmol),
and MeCN (30 mL). The product 6 (53 mg, 6%) was obtained as
a colorless foam and mixture of three diastereomers (1.0:2.5:2.5).
Partial separation of diastereomers was carried out as described
above to yield 6 fast (4 mg, 1 diastereomer) and 6 slow (24 mg, 2
diastereomers, 1.0:1.0) as colorless foams. The anaytical data were
identical with those reported above.
Bis-5-methyl-cycloSal-d4TMP (7). General Procedure D with
d4T 1 (779 mg, 3.47 mmol), crude chlorophosphite 20 (1.18 mg,
dissolved in 30 mL MeCN), DIPEA (1.22 mL, 903 mg, 7.00 mmol),
5-6 M solution of tert-butyl hydroperoxide in n-decane (2.34 mL,
g 11.7 mmol), and MeCN (80 mL). The time for the phosphityl-
ation reaction was increased to 6 h, and addition of the chloro-
phosphite 20 was carried out portionwise. The product 7 (160 mg,
11%) was obtained as a colorless foam and mixture of two
diastereomers (0.7:1.0): 1H NMR (500 MHz, DMSO-d6) δ 11.34
(s, 4H), 7.19-7.08 (m, 12H), 6.81-6.77 (m, 4H), 6.31-6.28 (m,
4H), 6.03-6.00 (m, 2H), 5.94-5.91 (m, 2H), 5.56 (dd, J ) 14.5,
14.5 Hz, 2H), 5.53 (dd, J ) 14.1, 14.1 Hz, 2H), 5.45 (dd, J )
14.5, 14.5 Hz, 2H), 5.40 (dd, J ) 14.1, 14.1 Hz, 2H), 4.92-4.88
(m, 4H), 4.26-4.16 (m, 8H), 2.33 (s, 6H), 2.30 (s, 6H), 1.65 (s,
6H), 1.62 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ 163.9, 150.9,
150.8, 150.5 (d, J ) 7.1 Hz), 150.4 (d, J ) 7.1 Hz), 135.9, 133.7,
133.7, 132.9, 132.8, 131.8, 127.4, 126.7, 125.7, 125.4, 125.4, 109.8,
89.3, 84.2 (d, J ) 7.8 Hz), 68.7 (d, J ) 6.1 Hz), 68.6 (d, J ) 8.8
Hz), 20.2, 12.0, 11.9; 31P NMR (202 MHz, DMSO-d6) δ -8.13,
-8.26; MS (FAB-HR) m/z calcd 833.1601 (M + Na+), found
833.1594 (M + Na+); UV (HPLC) λmax 267; TLC Rf value 0.34
(DCM/MeOH 9:1).
cycloSal-d4TMP (8). General Procedure E with d4T 1 (84 mg,
0.37 mmol), crude phosphoramidite 25 (241 mg, dissolved in 5
mL MeCN), pyridinium chloride (179 mg, 1.51 mmol), 5.5 M
solution of tert-butyl hydroperoxide in n-decane (0.23 mL, 1.3
mmol), and MeCN (10 mL). The product 8 (77 mg, 54%) was

Bis-cycloSal-d4T-monophosphates
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1343
obtained as a colorless foam and mixture of two diastereomers.
The anaytical data were identical with those reported previously.6
3,3′-Bis(hydroxymethyl)biphenyl 2,2′-Diol (10). Method I:
Under a nitrogen atmosphere, a solution of aldehyde 11 (6.03 g,
25.1 mmol) in THF (100 mL) was added dropwise over 40 min to
a suspension of LiAlH4 (1.58 g, 41.6 mmol) in THF (160 mL) at
room temperature. The reaction mixture was stirred at room
temperature for 2 h and heated under reflux for 1 h. The reaction
was quenched by addition of 2 N HCl at 0 °C up to pH 4. After
addition of water, the phases were separated and the aqueous phase
extracted with EtOAc (5×). The combined organics were dried over
anhydrous Na2SO4, and the solvent evaporated under reduced
pressure. The resulting crude product was purified by filtration
through a thin pad of silica (EtOAc) to yield 10 (5.77 g, 94%) as
a yellow-brown solid: 1H NMR (400 MHz, DMSO-d6) δ 7.26 (dd,
J ) 1.8, 7.4 Hz, 2H), 7.05 (dd, J ) 1.8, 7.6 Hz, 2H), 6.89 (dd,
J ) 7.4, 7.6 Hz, 2H), 4.62 (s, 4H); 13C NMR (101 MHz, DMSO-
d6) δ 153.1, 130.2, 129.6, 126.5, 124.2, 120.1, 60.1; MS (EI) m/z
calcd 246 (M), found 246 (M, 1%), 214 (35), 200 (66), 181 (58),
152 (50), 139 (22), 128 (45), 115 (49), 102 (11), 91 (28), 77 (39),
63 (26), 51 (32), 39 (100); IR (KBr) ν 3384, 2927, 1589, 1446,
1325, 1228, 1202, 1013, 768, 744; UV (MeOH) λmax 284, 243,
225; TLC Rf value 0.45 (DCM/MeOH 9:1), 0.09 (DCM/MeOH
30:1).
Method II: General Procedure A with acetal 14 (9.78 g, 30.0
mmol), Dowex 50X8 (117 g), DCM/MeOH 1:1 (400 mL) and a
reaction period of 65 h. The reaction was monitored Via TLC
(DCM/MeOH 30:1). Purification was carried out by column
chromatography (DCM/MeOH gradient (3-10%)) to yield 10 (2.08
g, 28%) as a yellow-brown solid. The anaytical data were identical
with those reported above. Analytical data of byproduct 3-(meth-
oxymethyl)-3′-(hydroxymethyl)biphenyl 2,2′-diol 15 (isolated from
various test reactions as a yellowish solid): 1H NMR (400 MHz,
DMSO-d6) δ 7.31 (dd, J ) 1.7, 7.5 Hz, 1H), 7.27 (dd, J ) 1.6, 7.5
Hz, 1H), 7.12 (dd, J ) 1.7, 7.5 Hz, 1H), 7.09 (dd, J ) 1.6, 7.5 Hz,
1H), 6.94 (dd, J ) 7.5, 7.5 Hz, 2H), 4.67 (s, 2H), 4.52 (s, 2H),
3.37 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 151.8, 151.5, 130.9,
130.1, 129.4, 128.0, 127.0, 126.8, 126.5, 126.1, 119.8, 119.8, 69.8,
59.9, 57.9; MS (EI) m/z calcd 260 (M), found 260 (M, 25%), 228
(100), 210 (78), 197 (52), 181 (58), 153 (39), 141 (12), 128 (16),
115 (20), 105 (9), 91 (7), 76 (12), 61 (7), 40 (28); IR (KBr) ν
3411, 3215, 2995, 2920, 2869, 1590, 1462, 1443, 1385, 1230, 1071,
1016, 943, 840, 776, 750; UV (MeOH) λmax 282, 243, 225; TLC
Rf value 0.38 (DCM/MeOH 30:1).
The resulting crude product was purified by repeated column
chromatography (petroleum ether/DCM gradient (25-100%)) to
yield 14 (26.2 g, 84%) as a brown oil: 1H NMR (400 MHz, DMSO-
d6) δ 7.05 (dd, J ) 1.8, 7.5 Hz, 2H), 7.02 (dd, J ) 1.5, 7.4 Hz,
2H), 6.93 (dd, J ) 7.4, 7.5 Hz, 2H), 4.87 (s, 4H), 1.43 (s, 12H);
13C NMR (101 MHz, DMSO-d6) δ 148.4, 130.1, 126.3, 124.4,
119.7, 99.4, 60.4, 24.9; MS (EI) m/z calcd 326 (M), found 326
(M, 25%), 268 (29), 269 (6), 211 (32), 210 (100), 183 (10), 182
(61), 181 (28), 154 (6), 153 (25), 152 (17), 115 (5), 91 (8), 43 (6);
UV (MeOH) λmax 282, 226; TLC Rf value 0.12 (petroleum ether/
DCM 3:1).
3,3′-Bis(hydroxymethyl)-5,5′-dimethylbiphenyl 2,2′-Diol (16).
5,5′-Dimethylbiphenyl 2,2′-diol 17 (3.00 g, 13.8 mmol, obtained
from 2-bromo-4-methylanisole 18 in two steps with 52% overall
yield21) and sodium hydroxide (18.0 g, 450 mmol) were dissolved
in 37% aqeous formaldehyde solution (200 mL) at 0 °C and stirred
at 40 °C for 4 d. The reaction was monitored Via TLC (DCM/
MeOH 9:1) and quenched by addition of 10% HCl up to pH 4.
The aqueous solution was extracted with EtOAc (5×). The
combined organics were dried over anhydrous Na2SO4 and the
solvent evaporated under reduced pressure. The resulting crude
product was purified by filtration through a thin pad of silica (DCM/
MeOH 9:1) and column chromatography (DCM/MeOH 19:1) to
yield 16 (1.86 g, 48%) as a colorless solid: 1H NMR (400 MHz,
DMSO-d6) δ 7.10 (d, J ) 1.9 Hz, 2H), 6.88 (d, J ) 1.9 Hz, 2H),
4.61 (s, 4H), 2.27 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ 149.0,
130.3, 129.4, 128.2, 127.2, 126.6, 59.7, 20.5; MS (EI) m/z calcd
274 (M), found 274 (M, 23%), 256 (100), 238 (60), 225 (93), 210
(70), 195 (48), 182 (14), 165 (39), 152 (23), 141 (14), 128 (20),
115 (24), 104 (17), 91 (8), 77 (20), 65 (6), 40 (100); IR (KBr) ν
3277, 2917, 1472, 1355, 1220, 1198, 1045, 862, 641, 606; UV
(MeOH) λmax 291, 225; TLC Rf value 0.45 (DCM/MeOH 9:1).
Bis-saligenylchlorophosphite (19). Method I: General Proce-
dure B with alcohol 10 (1.03 g, 4.11 mmol), PCl3 (0.84 mL, 1.3 g,
9.6 mmol), pyridine (1.50 mL, 1.47 g, 18.6 mmol, dissolved in 7.5
mL Et2O), Et2O (100 mL), and a reaction temperature of -40 °C.
The crude product 19 (938 mg) was obtained as a yellow oil and
mixture of two diastereomers: 1H NMR (500 MHz, CDCl3) δ 7.16
(d, J ) 7.6 Hz, 4H), 7.08 (dd, J ) 7.6, 7.6 Hz, 4H), 6.86 (d, J )
7.6 Hz, 4H), 5.60-4.85 (m, 8H); 13C NMR (101 MHz, CDCl3) δ
143.7, 131.1, 127.7, 125.6, 123.5, 121.7, 121.5, 61.1; 31P NMR
(202 MHz, CDCl3) δ 140.40.
Method II: General Procedure B with alcohol 10 (990 mg, 4.00
mmol), PCl3 (0.85 mL, 1.3 g, 9.6 mmol), NEt3 (2.55 mL, 1.85 g,
18.3 mmol), Et2O (50 mL), and a reaction temperature of -40 °C.
The crude product 19 (1.01 mg) was obtained as a yellow oil and
mixture of two diastereomers. The anaytical data were identical
with those reported above.
Bis-5-methyl-saligenylchlorophosphite (20). General Procedure
B with alcohol 16 (1.72 g, 6.27 mmol), PCl3 (1.32 mL, 2.07 g,
15.1 mmol), pyridine (2.33 mL, 2.28 g, 28.8 mmol, dissolved in
12 mL of Et2O), Et2O (155 mL), and a reaction temperature of
-40 °C. The crude product 20 (1.54 g) was obtained as a colorless
solid and mixture of two diastereomers. The compound was
extremely poorly soluble in CDCl3 and was therefore used for
pronucleotide synthesis without further characterization.
3,3′-Bis-formylbiphenyl 2,2′-Diol (11). The title compound was
obtained according to the previously published procedure18 with
the following modifications: After the MOM protection, purifica-
tion was performed using column chromatography (dichlo-
romethane) furnishing a yellow oil, yield 66%. Attempts to fully
purify the formylated MOM-protected compound (column chro-
matography, dichloromethane/methanol gradient) failed, so purifica-
tion had to be carried out after MOM deprotection employing
column chromatography (dichloromethane) and preparative TLC
(Chromatotron, petroleum ether/dichloromethane gradient) to obtain
a yellowish solid, yield 48% over two steps. The anaytical data
were identical with those reported.18
Bis(isopropylidene)-3,3′-bis(hydroxymethyl)biphenyl 2,2′-Diol
(14). Under a nitrogen atmosphere, a 1.6 M solution of n-
butyllithium in n-hexane (120 mL, 0.192 mol) was added to a
solution of 3-bromosalicyl alcohol isopropylidene acetal 12 (46.5
g, 0.191 mol, obtained from 2-bromophenol 13 in three steps with
52% overall yield19) in THF (400 mL) at -80 °C and stirred for 1
h at the same temperature. The resulting mixture was added to a
solution of iron(III) acetylacetonate (74.2 g, 0.210 mol) in THF
(160 mL) at -80 °C. The reaction mixture was warmed to room
temperature and stirred for 17 h at room temperature. The solvent
was evaporated under reduced pressure and the residue dissolved
in EtOAc and 2 N HCl. After separation of phases, the aqueous
phase was extracted with EtOAc (2×). The combined organics were
washed with 2 N HCl (4×), water (2×) and brine (2×), and dried
over Na2SO4. The solvent was evaporated under reduced pressure.
Bis-saligenyl-N,N-diisopropylaminophosphoramidite (22). Gen-
eral Procedure C with crude chlorophosphite 19 (1.01 g, prepared
using NEt3 according to method II), diisopropylamine (1.80 mL,
1.30 g, 12.8 mmol, dissolved in 4 mL of Et2O), and Et2O (20 mL).
The crude product 22 (1.14 g) was obtained as a colorless oil and
mixture of two diastereomers (0.7:1.0): 1H NMR (500 MHz,
CDCl3) δ 7.37 (dd, J ) 1.9, 7.3 Hz, 2H), 7.32 (dd, J ) 2.0, 7.2
Hz, 2H), 6.97-6.91 (m, 8H), 5.18 (dd, J ) 5.4, 13.4 Hz, 2H),
5.15 (dd, J ) 5.3, 13.6 Hz, 2H), 4.93 (dd, J ) 3.5, 13.4 Hz, 2H),
4.90 (dd, J ) 3.9, 13.6 Hz, 2H), 3.63-3.53 (m, 8H), 1.19 (d, J )
6.9 Hz, 24H), 1.08 (d, J ) 6.4 Hz, 12H), 1.07 (d, J ) 6.7 Hz,
12H); 13C NMR (101 MHz, CDCl3) δ 151.1, 150.8, 131.8, 131.4,
128.3, 128.0, 124.8, 124.7, 120.4, 120.3, 65.1, 64.8 (d, J ) 4.1
Hz), 44.6, 44.5, 25.1, 24.7; 31P NMR (202 MHz, CDCl3) δ 137.12,
136.28.

1344 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Ducho et al.
N,N-Diisopropyl-7-phenyl-2,3-dihydro-1,2-benzoxaphosphol-
2-amine-2-oxide (23). General Procedure C with crude chloro-
phosphite 2117 (1.13 g, prepared using pyridine according to method
I), diisopropylamine (1.20 mL, 866 mg, 8.56 mmol, dissolved in 5
mL of Et2O), Et2O (20 mL), and a reaction period of 20 h. The
crude product was purified by preparative TLC (Chromatotron,
DCM). Instead of the desired product 22, rearrangement product
23 (1.26 g, 3.83 mmol) was obtained as a colorless crystalline
solid: 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J ) 7.6 Hz, 2H),
7.42 (dd, J ) 7.6, 7.6 Hz, 2H), 7.35-7.33 (m, 2H), 7.21 (d, J )
7.6 Hz, 1H), 7.08 (dd, J ) 7.6 Hz, 1H), 3.40-3.30 (m, 2H), 3.22
(dd, J ) 18.3, 18.3 Hz, 1H), 3.01 (dd, J ) 11.4, 18.3 Hz, 1H),
1.29 (d, J ) 6.9 Hz, 12H); 13C NMR (101 MHz, CDCl3) δ 144.7,
129.2, 128.8, 128.2, 127.4, 126.1, 125.9, 122.7, 46.5 (d, J ) 5.6
Hz), 28.0 (d, J ) 111.9 Hz), 22.9, 22.3, 22.3; 31P NMR (202 MHz,
CDCl3) δ 47.78; MS (EI) m/z calcd 329 (M), found 329 (M, 26%),
314 (54), 286 (14), 272 (100), 229 (19), 183 (15), 165 (9), 43 (7);
IR (KBr) ν 2968, 2933, 1468, 1451, 1429, 1412, 1391, 1369, 1260,
1218, 1201, 1186, 1153, 1127, 1031, 1010, 888, 854, 778, 762,
703, 565; UV (MeOH) λmax 282, 246; TLC Rf value 0.73 (DCM/
MeOH 9:1).
Saligenylchlorophosphite (24). General Procedure B with salicyl
alcohol (1.01 g, 8.10 mmol), PCl3 (0.85 mL, 1.3 g, 9.5 mmol),
NEt3 (2.60 mL, 1.89 g, 18.7 mmol), Et2O (30 mL), and a reaction
temperature of -20 °C. The crude product 24 (1.30 g) was obtained
as a yellow oil. The anaytical data were identical with those reported
previously.24
Saligenyl-N,N-diisopropylaminophosphoramidite (25). Gen-
eral Procedure C with crude chlorophosphite 24 (757 mg, prepared
using NEt3 as described above), diisopropylamine (1.20 mL, 866
mg, 8.56 mmol, dissolved in 4 mL of Et2O), and Et2O (9 mL). The
crude product 25 (1.04 g) was obtained as a yellow oil: 1H NMR
(500 MHz, CDCl3) δ 7.11-7.06 (m, 1H), 6.89-6.79 (m, 3H), 5.05
(dd, J ) 4.1, 14.5 Hz, 1H), 4.78 (dd, J ) 4.2, 14.5 Hz, 1H), 3.60-
3.50 (m, 2H), 1.19-1.13 (m, 12H); 13C NMR (101 MHz, CDCl3)
δ 153.2 (d, J ) 4.2 Hz), 129.2, 128.5, 125.2, 121.0, 119.0, 64.2
(d, J ) 4.7 Hz), 44.2, 44.1, 24.7 (d, J ) 7.6 Hz), 24.5 (d, J ) 8.2
Hz); 31P NMR (202 MHz, CDCl3) δ 137.04.
Bis-saligenyl-N,N-diethylaminophosphoramidite (26). General
Procedure C with crude chlorophosphite 19 (1.37 g, prepared using
NEt3 according to method II), diethylpropylamine (1.77 mL, 1.25
g, 17.1 mmol, dissolved in 5 mL of Et2O), and Et2O (27 mL). The
crude product 26 (1.46 g) was obtained as a colorless oil and
mixture of 2 diastereomers (1.0:0.7): 1H NMR (500 MHz, CDCl3)
δ 7.33-7.30 (m, 4H), 7.02-6.97 (m, 8H), 5.11-5.04 (m, 4H),
4.97-4.89 (m, 4H), 3.11-3.04 (m, 16H), 1.01 (t, J ) 6.9 Hz, 12H),
1.01 (t, J ) 6.9 Hz, 12H); 13C NMR (101 MHz, CDCl3) δ 149.9,
149.8 (d, J ) 6.1 Hz), 131.3, 131.2, 127.8, 127.7, 125.6, 125.5,
124.6 (d, J ) 6.1 Hz), 120.5, 120.4, 64.0, 63.7, 38.0, 37.8, 14.8,
14.8; 31P NMR (202 MHz, CDCl3) δ 131.03, 130.55.
(RP)-3-Sal-cycloSal-d4TMP (29 (RP)). General Procedure A with
pronucleotide 33 (RP) (166 mg, 0.299 mmol), Dowex 50X8 (8.3
g), DCM/MeOH 1:1 (14 mL), and a reaction period of 8 d. The
reaction was monitored Via analytical HPLC (see Supporting
Information). Purification was carried out by preparative HPLC
(MeCN/H2O 2:5) to yield 29 (RP) (84 mg, 54%) as a colorless
foam: 1H NMR (500 MHz, DMSO-d6) δ 11.35 (s, 1H), 8.61 (s,
1H), 7.31-7.24 (m, 4H), 7.18 (q, J ) 1.1 Hz, 1H), 7.02 (dd, J )
1.5, 7.5 Hz, 1H), 6.89 (dd, J ) 7.5, 7.5 Hz, 1H), 6.82-6.80 (m,
1H), 6.31 (ddd, J ) 1.6, 1.6, 6.0 Hz, 1H), 5.97-5.95 (m, 1H),
5.55 (dd, J ) 14.3, 14.3 Hz, 1H), 5.51 (dd, J ) 5.4, 5.4 Hz, 1H),
5.45 (dd, J ) 14.3, 14.3 Hz, 1H), 4.96-4.93 (m, 1H), 4.65 (d, J )
5.4 Hz, 2H), 4.28 (ddd, J ) 2.5, 6.4, 11.2 Hz, 1H), 4.22 (ddd, J )
4.1, 6.9, 11.2 Hz, 1H), 1.60 (s, 3H); 13C NMR (101 MHz, DMSO-
d6) δ 163.9, 152.2, 150.9, 148.4, 135.8, 132.9, 132.0, 129.6, 128.7,
127.6, 127.5, 125.4, 124.1, 123.7, 121.8, 121.7, 119.2, 109.9, 89.2,
84.3 (d, J ) 8.1 Hz), 68.5 (d, J ) 7.1 Hz), 68.5 (d, J ) 7.0 Hz),
60.2, 11.9; 31P NMR (202 MHz, DMSO-d6) δ -7.88; MS (FAB)
calcd 515.1 (M + H+), found 515.2 (M + H+); HR-MS (ESI+)
m/z calcd 537.1039 (M + Na+), found 537.1048 (M + Na+); UV
(HPLC) λmax 269; TLC Rf value 0.38 (DCM/MeOH 9:1); Prepara-
tive HPLC tR ) 13.8 min (MeCN/H2O 2:5).
(SP)-3-Sal-cycloSal-d4TMP (29 (SP)). General Procedure A with
pronucleotide 33 (SP) (181 mg, 0.326 mmol), Dowex 50X8 (9.1
g), DCM/MeOH 1:1 (15 mL), and a reaction period of 8 d. The
reaction was monitored Via analytical HPLC (see Supporting
Information). Purification was carried out by preparative HPLC
(MeCN/H2O 2:5) to yield 29 (SP) (76 mg, 46%) as a colorless
foam: 1H NMR (500 MHz, DMSO-d6) δ 11.34 (s, 1H), 8.60 (s,
1H), 7.32-7.23 (m, 4H), 7.21 (q, J ) 0.9 Hz, 1H), 7.03 (d, J )
7.5 Hz, 1H), 6.91 (dd, J ) 7.5, 7.5 Hz, 1H), 6.83-6.81 (m, 1H),
6.31-6.28 (m, 1H), 6.02-6.00 (m, 1H), 5.53 (dd, J ) 14.4, 14.4
Hz, 1H), 5.49 (dd, J ) 5.7, 5.7 Hz, 1H), 5.44 (dd, J ) 14.4, 14.4
Hz, 1H), 4.94-4.91 (m, 1H), 4.65 (d, J ) 5.7 Hz, 2H), 4.28-4.20
(m, 2H), 1.65 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 163.9,
152.1, 150.9, 147.5 (d, J ) 7.1 Hz), 135.9, 133.0, 132.0, 129.6,
128.9, 127.6, 127.5, 125.4, 124.1, 123.7, 121.8, 121.7, 119.3, 109.9,
89.3, 84.3 (d, J ) 7.6 Hz), 68.6 (d, J ) 6.3 Hz), 68.3 (d, J ) 6.5
Hz), 60.1, 12.0; 31P NMR (202 MHz, DMSO-d6) δ -7.98; MS
(FAB) calcd 515.1 (M + H+), found 515.2 (M + H+); UV (HPLC)
λ
max 269; TLC Rf value 0.38 (DCM/MeOH 9:1); Preparative HPLC
tR ) 14.1 min (MeCN/H2O 2:5).
3-(O-(tert-Butyldiphenylsilyl)hydroxymethyl)-3′-(hydroxy-
methyl)biphenyl 2,2′-Diol (30). Under a nitrogen atmosphere, NEt3
(2.41 mL, 1.75 g, 17.3 mmol), a catalytic amount of DMAP, and
TBDPS chloride (2.25 mL, 2.38 g, 8.65 mmol) were added to a
solution of alcohol 10 (2.84 g, 11.5 mmol) in THF (50 mL) at
0 °C. The reaction mixture was warmed to room temperature and
stirred at room temperature for 2 h. The reaction was monitored
Via TLC (DCM/MeOH 30:1) and quenched by addition of MeOH
(10 mL) and stirring at room temperature for 5 min. The solvent
was evaporated under reduced pressure, and the residue was
dissolved in EtOAc and water. After separation of the phases, the
aqueous solution was extracted with EtOAc (3×). The combined
organics were washed with water (1×) and dried over anhydrous
Na2SO4, and the solvent evaporated under reduced pressure. The
resulting crude product was purified by column chromatography
(DCM/MeOH gradient (0-10%)) to yield 30 (2.98 g, 53%) as a
yellow oil: 1H NMR (400 MHz, DMSO-d6) δ 7.72-7.69 (m, 4H),
7.54-7.45 (m, 7H), 7.28 (dd, J ) 1.7, 7.6 Hz, 1H), 7.10 (dd, J )
1.8, 7.6 Hz, 1H), 7.07 (dd, J ) 1.5, 7.6 Hz, 1H), 7.03 (dd, J ) 7.4,
7.6 Hz, 1H), 6.93 (dd, J ) 7.6, 7.6 Hz, 1H), 4.85 (s, 2H), 4.63 (s,
2H), 1.09 (s, 9H); 13C NMR (101 MHz, DMSO-d6) δ 151.5, 150.5,
135.2, 133.1, 130.2, 130.1, 130.1, 129.4, 128.9, 128.2, 126.9, 126.7,
126.4, 120.1, 119.9, 119.8, 61.5, 59.9, 26.9, 19.2; UV (MeOH) λmax
284, 224; TLC Rf value 0.57 (DCM/MeOH 30:1). Byproduct 3,3′-
bis-(O-(tert-butyldiphenylsilyl)-hydroxymethyl)biphenyl 2,2′-diol 31
(904 mg, 11%) could also be isolated as a yellowish oil: 1H NMR
(400 MHz, DMSO-d6) δ 7.71-7.68 (m, 8H), 7.56-7.52 (m, 2H),
7.52-7.44 (m, 12H), 7.10 (dd, J ) 1.6, 7.5 Hz, 2H), 7.02 (dd, J )
7.5, 7.5 Hz, 2H), 4.83 (s, 4H), 1.08 (s, 18H); 13C NMR (101 MHz,
DMSO-d6) δ 150.7, 135.2, 133.1, 130.2, 130.1, 129.4, 128.2, 127.7,
125.5, 120.0, 61.5, 26.9, 19.1; UV (MeOH) λmax 282, 244, 224;
TLC Rf value 0.77 (DCM/MeOH 30:1).
3-(O-(tert-Butyldiphenylsilyl)-hydroxymethyl)-3′-(hydroxy-
methyl)biphenyl 2,2′-Diol 2′,3′-O-Isopropylideneacetal (31). A
mixture of salicyl alcohol 30 (2.96 g, 6.11 mmol), 2,2-dimethoxy-
propane (3.9 mL, 3.3 g, 32 mmol), p-TsOH (120 mg, 0.631 mmol),
and anhydrous Na2SO4 (2.5 g) in acetone (120 mL) was stirred at
40 °C for 3 d. The solvent was evaporated under reduced pressure,
and the residue was dissolved in EtOAc and water. After separation
of the phases, the aqueous solution was extracted with EtOAc (2×).
The combined organics were washed with water (1×) and dried
over anhydrous Na2SO4, and the solvent evaporated under reduced
pressure. The resulting crude product was purified by column
chromatography (petroleum ether/DCM 1:1) to yield 32 (1.92 g,
60%) as a colorless oil: 1H NMR (400 MHz, DMSO-d6) δ 7.98
(s, 1H), 7.71-7.68 (m, 4H), 7.52-7.44 (m, 7H), 7.09-7.02 (m,
3H), 6.97 (dd, J ) 7.4, 7.6 Hz, 1H), 6.95 (dd, J ) 7.4, 7.6 Hz,
1H), 4.87 (s, 2H), 4.83 (s, 2H), 1.42 (s, 6H), 1.08 (s, 9H); 13C
NMR (101 MHz, DMSO-d6) δ 150.9, 148.5, 135.2, 133.1, 130.3,

Bis-cycloSal-d4T-monophosphates
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1345
130.2, 130.1, 128.5, 128.2, 126.6, 125.7, 124.4, 120.1, 120.0, 119.5,
99.6, 61.7, 60.4, 26.9, 24.8, 19.1; MS (ESI+) m/z calcd 547.2 (M
+ Na+), found 547.4 (M + Na+); UV (MeOH) λmax 284, 244, 224;
TLC Rf value 0.83 (DCM/MeOH 30:1).
124.2, 124.2, 121.7, 121.6, 120.0, 119.9, 109.9, 99.8, 89.3, 84.2
(d, J ) 7.7 Hz), 68.5 (d, J ) 7.0 Hz), 68.3 (d, J ) 7.3 Hz), 60.3,
24.8, 24.3, 11.8; 31P NMR (202 MHz, DMSO-d6) δ -7.62;
Preparative HPLC tR ) 38.2 min (MeCN/H2O 2:5). Analytical data
of 33 (SP): 1H NMR (500 MHz, DMSO-d6) δ 11.34 (s, 1H), 7.31-
7.24 (m, 3H), 7.17-7.08 (m, 3H), 6.99 (dd, J ) 7.5, 7.5 Hz, 1H),
6.81-6.79 (m, 1H), 6.26-6.24 (m, 1H), 6.02-5.99 (m, 1H), 5.54
(d, J ) 14.2, 14.2 Hz, 1H), 5.45 (dd, J ) 14.2, 14.2 Hz, 1H), 4.93-
4.86 (m, 3H), 4.21 (dd, J ) 4.3, 7.1 Hz, 2H), 1.58 (s, 3H), 1.47 (s,
3H), 1.42 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 163.9, 150.8,
148.4, 147.2 (d, J ) 7.1 Hz), 135.8, 132.8, 131.9, 129.8, 127.5,
125.7, 125.3, 124.2, 124.1, 121.6, 121.5, 120.1, 120.0, 109.9, 99.8,
89.3, 84.2 (d, J ) 7.2 Hz), 68.6 (d, J ) 6.2 Hz), 68.2 (d, J ) 6.1
Hz), 60.3, 25.1, 24.0, 11.8; 31P NMR (202 MHz, DMSO-d6) δ
-7.72; Preparative HPLC tR ) 41.1 min (MeCN/H2O 2:5).
3-Isopr-Sal-cycloSal-d4TMP (33). To a solution of silylated
alcohol 32 (1.89 g, 3.60 mmol) in MeOH (60 mL) was added NH4F
(1.34 g, 36.2 mmol) at room temperature. The reaction mixture
was stirred at room temperature for 61 h and the reaction monitored
Via TLC (DCM/MeOH 30:1). The solvent was evaporated under
reduced pressure, and the residue was dissolved in EtOAc and water.
After separation of the phases, the aqueous solution was extracted
with EtOAc (3 ×). The combined organics were washed with water
(1×) and dried over anhydrous Na2SO4, and the solvent evaporated
under reduced pressure. The resulting crude product was purified
by preparative TLC (Chromatotron, petroleum ether/DCM gradient
(0-100%)) to yield the deprotected salicyl alcohol derivative (915
mg, 89%) as a yellow oil: 1H NMR (400 MHz, DMSO-d6) δ 8.19
(s, 1H), 7.24-7.22 (m, 1H), 7.10-7.08 (m, 1H), 7.07-7.04 (m,
1H), 7.01 (dd, J ) 1.8, 7.4 Hz, 1H), 6.95 (dd, J ) 7.3, 7.6 Hz,
1H), 6.86 (dd, J ) 7.4, 7.6 Hz, 1H), 5.41 (s, 1H), 4.88 (s, 2H),
4.63 (s, 2H), 1.44 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ 152.0,
148.4, 130.3, 130.0, 128.9, 126.9, 126.7, 125.4, 124.1, 119.9, 119.9,
119.0, 99.5, 60.4, 60.1, 24.8; MS (EI) m/z calcd 286 (M), found
286 (M, 4%), 228 (100), 210 (54), 198 (40), 182 (55), 153 (28),
139 (6), 128 (9), 115 (14), 91 (8), 77 (6), 43 (20); UV (MeOH)
λmax 283, 242, 225; TLC Rf value 0.51 (DCM/MeOH 30:1).
Chlorophosphite synthesis was carried out using General Pro-
cedure B with the deprotected salicyl alcohol derivative (896 mg,
3.13 mmol), PCl3 (0.32 mL, 0.50 g, 3.6 mmol), pyridine (0.56 mL,
0.55 g, 7.0 mmol, dissolved in 2.5 mL of Et2O), Et2O (30 mL),
and a reaction temperature of -40 °C. The crude chlorophosphite
(696 mg) was obtained as a yellow solid: 1H NMR (500 MHz,
CDCl3) δ 7.28-7.25 (m, 1H), 7.16-7.11 (m, 2H), 7.02-6.95 (m,
3H), 5.54 (d, J ) 13.2 Hz, 1H), 5.12-5.04 (m, 1H), 4.96-4.89
(m, 2H), 1.55 (s, 3H), 1.54 (s, 3H); 13C NMR (101 MHz, CDCl3)
δ 148.8, 143.8 (d, J ) 8.1 Hz), 131.5, 130.1, 129.2 (d, J ) 2.0
Hz), 125.4, 125.0 (d, J ) 2.0 Hz), 124.5, 123.3, 121.3 (d, J ) 13.2
Hz), 119.7, 119.5, 99.9, 61.4, 61.0, 25.3, 24.2; 31P NMR (202 MHz,
CDCl3) δ 142.42.
Pronucleotide synthesis was carried out using General Procedure
D with d4T 1 (354 mg, 1.58 mmol), crude chlorophosphite (665
mg), DIPEA (0.41 mL, 0.30 g, 2.3 mmol), 5-6 M solution of tert-
butyl hydroperoxide in n-decane (0.95 mL, g4.8 mmol), and MeCN
(36 mL). The product 33 (525 mg, 60%) was obtained as a colorless
foam and mixture of two diastereomers (1.0:0.9): 1H NMR (500
MHz, DMSO-d6) δ 11.34 (s, 2H), 7.31-7.24 (m, 6H), 7.17-7.06
(m, 6H), 6.99 (dd, J ) 7.5, 7.5 Hz, 1H), 6.97 (dd, J ) 7.5, 7.5 Hz,
1H), 6.81-6.79 (m, 2H), 6.32-6.30 (m, 1H), 6.26-6.24 (m, 1H),
6.02-5.99 (m, 1H), 5.95-5.92 (m, 1H), 5.57-5.51 (m, 2H), 5.47-
5.41 (m, 2H), 4.94-4.86 (m, 6H), 4.30-4.17 (m, 4H), 1.58 (s,
3H), 1.52 (s, 3H), 1.46 (s, 3H), 1.45 (s, 3H), 1.42 (s, 3H), 1.40 (s,
3H); 13C NMR (101 MHz, DMSO-d6) δ 163.9, 163.9, 150.9, 148.4,
148.4, 147.3 (d, J ) 2.0 Hz), 147.2, 135.9, 135.7, 132.9, 132.9,
131.9, 131.9, 129.8, 127.6, 127.5, 125.7, 125.7, 125.3, 124.2-124.1
(m), 121.7, 121.6, 121.5, 120.1, 120.0, 119.9, 110.0, 109.9, 99.8,
89.3, 89.3, 84.3, 68.6 (d, J ) 6.1 Hz), 68.3 (d, J ) 5.1 Hz), 68.2
(d, J ) 6.1 Hz), 60.3, 25.1, 24.8, 24.3, 24.0, 11.9, 11.8; 31P NMR
(202 MHz, DMSO-d6) δ -7.62, -7.71; MS (FAB) m/z calcd 555.2
(M + H+), found 555.2 (M + H+); HR-MS (ESI+) m/z calcd
577.1352 (M + Na+), found 577.1359 (M + Na+); UV (HPLC)
λmax 267, 248; TLC Rf value 0.52 (DCM/MeOH 9:1). Preparative
HPLC (MeCN/H2O 2:5) allowed separation of the two diastereo-
mers to furnish 33 (RP) (192 mg) and 33 (SP) (206 mg) as colorless
foams. Analytical data of 33 (RP): 1H NMR (500 MHz, DMSO-
d6) δ 11.35 (s, 1H), 7.30-7.24 (m, 3H), 7.13-7.06 (m, 3H), 6.97
(dd, J ) 7.5, 7.5 Hz, 1H), 6.81-6.79 (m, 1H), 6.32-6.30 (m, 1H),
5.95-5.92 (m, 1H), 5.54 (dd, J ) 14.3, 17.9 Hz, 1H), 5.43 (dd,
J ) 10.6, 14.3 Hz, 1H), 4.96-4.92 (m, 1H), 4.88 (s, 2H), 4.30-
4.25 (m, 1H), 4.19 (ddd, J ) 4.9, 6.1, 11.0 Hz, 1H), 1.52 (s, 3H),
1.45 (s, 3H), 1.40 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 163.8,
150.9, 148.4, 147.2, 135.7, 132.9, 131.9, 129.8, 127.5, 125.7, 125.3,
Hydrolysis Studies. Hydrolytic stabilities were determined and
31P NMR hydrolysis experiments carried out as described previ-
ously.17
BChE Assay. The procedure for the BChE assay using human
serum has been described before.12
Antiviral Activity and Cytotoxicity. Antiviral activities and
cytotoxicities were determined as published previously.6
Acknowledgment. We thank the Deutsche Forschungsge-
meinschaft, Germany, and the “Geconcerteerde Onderzoeksac-
ties (GOA nr. 019/05) for financial support. C. D. is grateful to
a Ph. D. fellowship from the Fonds der Chemischen Industrie/
BMBF, Germany. The authors also thank Mrs. Ann Absillis
for excellent technical assistance.
Supporting Information Available: Analytical HPLC data of
new cycloSal compounds. This material is available free of charge
Via the Internet at http://pubs.acs.org.
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