Interaction of cycloSal-pronucleotides with cholinesterases from different origins. A structure-activity relationship

Article abstract of DOI:10.1021/jm031032a

A large number of cycloSal-nucleotide triesters 1-49 have been studied concerning their ability to inhibit cholinesterases of different origins as well as to inhibit HIV replication in cell culture. It was shown that none of the triesters showed inhibitor

Full text of DOI:10.1021/jm031032a

J . Med. Chem. 2004, 47, 2839-2852
2839
In ter a ction of cycloSa l-P r on u cleotid es w ith Ch olin ester a ses fr om Differ en t
Or igin s. A Str u ctu r e-Activity Rela tion sh ip †
Chris Meier,*,‡ Christian Ducho,‡ Ulf Go¨rbig,‡ Robert Esnouf,§ and J an Balzarini|
Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany,
Structural Biology Division, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, United Kingdom,
and Rega Institute for Medical Research, K. U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received September 12, 2003
A large number of cycloSal-nucleotide triesters 1-49 have been studied concerning their ability
to inhibit cholinesterases of different origins as well as to inhibit HIV replication in cell culture.
It was shown that none of the triesters showed inhibitory effects against human acetyl-
cholinesterase (AChE; isolated enzyme) as well as against AChE from beef erythrocytes and
calf serum. In contrast, inhibition of butyrylcholinesterase (BChE) has been observed for some
triesters in human and mouse serum. cycloSal pronucleotides showed strong competitive
inhibition with respect to the substrate acetylcholine chloride (Ki/Km: ∼2 × 10-5) and acted by
time-dependent irreversible inhibition of the human serum BChE. Detailed studies demon-
strated that the inhibitory effect against BChE is dependent on the nucleoside analogue, the
substitution pattern of the cycloSal-moiety, and particularly on the stereochemistry at the
phosphorus atom. Structural requirements to avoid the inhibition of BChE by cycloSal-
nucleotide triesters have been elucidated in the reported study.
In tr od u ction
However, as cycloSal-phosphate triesters belong to the
class of hydrolytically labile phosphate esters, they may
interfere with cholinesterases. If an interaction with
acetylcholinesterase (AChE; EC 3.1.1.7) is involved,
serious side effects may be expected due to the impor-
tant physiological role of this enzyme.12 Acetylcholinest-
erase inactivates acetylcholine and thus terminates its
action at the junctions of various cholinergic nerve
endings with their effector organs or postsynaptic sites.
If acetylcholine is not destroyed, it can again activate
the cholinergic receptor resulting in continuous stimula-
tion of the cholinergic systems throughout the central
and peripheral nervous systems, permanent contraction
of skeletal muscle, and eventual death.13 Anti-cholinest-
erase (anti-AChE) agents that inhibit AChE have been
extensively used as agricultural insecticides and nerve
gases, e.g. tabun, sarin, and VX,14 and are also used as
therapeutic agents.15 The organophosphates represent
the most characteristic type of anti-AChE agents. The
mode of action of these agents is their irreversible
inactivation of AChE by phosphorylating a serine
residue in the active site of the enzyme. The phos-
phorylated adduct is hydrolytically labile but becomes
extremely resistant to further hydrolysis after “aging”,
resulting in virtually permanent inactivation of the
enzyme (suicide inhibition mechanism). The general
formula of these AChE inhibitors is depicted in Figure
1A. The residue R and the leaving group X can be of a
large variety of substituents including alkyl, alkoxy,
aryloxy, amido, mercapto, halo, cyano, thiocyano, and
phenoxy. Diisopropylfluorophosphate (DFP) and para-
thion (metabolized to paraoxon) are probably the most
extensively studied compounds among this group of
anti-AChE organophosphates. Moreover, it was previ-
ously reported that several S-alkyl- and S-benzyl-
saligenin cyclic phosphorothioate derivatives (Figure
1B) act as AChE inhibitors.16 The structure-activity
cycloSal-phosphate triesters represent a particular
class of pronucleotides.1 These lipophilic derivatives of
various antiviral and antitumor active nucleoside ana-
logues have been developed as intracellular delivery
forms of the corresponding nucleotide analogues.2 By
releasing the nucleotide, the metabolic bottleneck of the
conversion of the nucleoside into the nucleotide by
cellular or viral enzymes can be bypassed (chemical
trojan horse concept). The cycloSal-approach relies on
a chemically driven hydrolysis reaction. The initial
chemical hydrolysis step intrinsically activates the
remaining masking group which is then spontaneously
cleaved to yield the nucleotide (tripartite prodrug).
Often, nucleoside analogues, e.g. 3′-deoxy-2′,3′-didehydro-
thymidine (d4T),4 have a poor affinity for the first
activation (phosphorylation) step, and this may mark-
edly impair their biological (i.e. antiviral, antitumor)
efficacy.5 The approach has been applied successfully
to a number of different nucleoside analogues, e.g. d4T,6
2′,3′-dideoxyadenosine (ddA),7 2′,3′-dideoxy-2′,3′-di-
dehydroadenosine (d4A),7 fluorinated 2′,3′-dideoxy-
adenosine (2′-F-ddA),8 carbocyclic nucleosides (CBV and
abacavir),9 and acyclovir (ACV).10 Recently, we have
reported that by attaching the cycloSal-masking group
on (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdU), the
resulting pronucleotide showed considerable anti-
Epstein-Barr-virus (EBV) activity, while the parent
nucleoside BVdU was entirely inactive.11
* To whom correspondence should be addressed. Tel: +49-40-
42838-4324; Fax:
+49-40-42838-2495; e-mail:
chris.meier@
chemie.uni-hamburg.de.
† Dedicated to Professor J oachim W. Engels on the occasion of his
60th birthday.
‡ University of Hamburg.
§ Wellcome Trust Centre for Human Genetics.
| Rega Institute for Medical Research.
10.1021/jm031032a CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/23/2004

2840 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Meier et al.
cycloSal-d4TMP 22, 3-phenyl-cycloSal-BVdUMP 48, and
5-methoxy-cycloSal-d4AMP 49 have been separated into
their diastereomers by preparative HPLC using isocratic
acetonitrile/water eluents. The metabolic fates and
antiviral activities of most of these test compounds have
been reported previously.6-11,20
Deter m in a tion of th e IC50 Va lu es of th e Tr iester s
1-49 a ga in st Ch olin ester a ses fr om Differ en t Or i-
gin s. Studies were done using human, mouse, and calf
serum. The first two sera contain butyrylcholinesterase
(BChE). The latter serum is known to contain acetyl-
cholinesterase (AChE) but no BChE. In addition, iso-
lated enzymes were used: human AChE, human BChE,
beef erythrocyte AChE, and AChE from electric eel
(electrophorus electricus). The assay is a modified assay
of Rappaport measuring the hydrolysis of acetylcholine
catalyzed by these enzymes to yield acetic acid at pH
7.8.21 As indicator, m-nitrophenol is used. The progres-
sion of the reaction was therefore followed photo-
metrically (absorption at λ ) 420 nm) due to the
decreasing amounts of m-nitrophenolate.
F igu r e 1. Structures of known acetylcholinesterase (AChE)
inhibitors.
relationship for enzyme inhibition of these compounds
revealed three major effects: a lipophilic effect, an
electronic effect, and a steric effect. More recently, a
study of substituted [1.3.2]-benzodioxaphosphorin-2-
oxide derivatives containing alkyl, alkoxy, or aryloxy
groups at the phosphorus atom (Figure 1C) showed that
several of these compounds displayed inhibitory effects
on neuropathy targeted esterase in vitro.17 In those
studies, all compounds were tested as enantiomeric
mixtures.
In addition to AChE, a closely related cholinesterase
is known which is present in various vertebrate tissues
(e.g. liver, brain, lung) and predominately in human
serum: the unspecific serum butyrylcholine esterase
(BChE; EC 3.1.1.8). The physiological role of this
enzyme is still unknown. However, plasma BChE is of
pharmacological and toxicological importance because
it hydrolyzes ester-containing drugs such as succinyl-
choline and cocaine. Therefore, purified BChE has been
used for treatment of succinylcholine apnea in humans18
and it is known to protect rodents from the toxic effects
of cocaine.19 Both AChE and BChE belong to the group
of serine hydrolases employing a catalytic triad and so
the catalytic site and mechanism of both enzymes
should be comparable.
Effect of P r ein cu ba tion of Hu m a n Ser u m ACh E
w ith 3-Me-cycloSa l-d d AMP 14 on En zym e Activity.
In a first set of experiments, human serum BChE was
preincubated with 0.05 µM 3-Me-cycloSal-ddAMP 14 for
different time periods (varying from 0 to 30 min). The
reaction was allowed to proceed upon addition of the
substrate, after which the conversion of the substrate
was recorded at 5 and 15 min. Whereas no preincuba-
tion of the enzyme with the inhibitor resulted in 25 to
40% inhibition, preincubation resulted in a striking
time-dependent inactivation. A preincubation time as
short as 1 min resulted in 65 to 70% inhibition, and a
preincubation time of 3 to 4 min afforded complete
inhibition of BChE (Figure 3). cycloSal-phosphate tri-
esters are chemically hydrolyzable in a pH-dependent
manner3. Thus, to avoid any interference between
chemical (half-lives at pH 7.3: 1-75 h) and enzyme-
catalyzed hydrolysis (100% inhibition within 3-4 min)
and to ascertain proper IC50 measurements within the
linear time-scale of the reaction, IC50 values were
determined after no longer than 5 min incubation.
In this study, we have evaluated a large number of
cycloSal-nucleotide derivatives for their inhibitory ac-
tivities against acetyl- and butyrylcholinesterases from
different origins. Structural features to avoid interaction
with butyrylcholinesterase have been elucidated.
In a second set of experiments, different (human
serum) BChE concentrations were exposed to fixed
3-Me-cycloSal-ddAMP concentrations, after which the
remaining enzyme activity was recorded. Whereas 0.015
µM inhibitor completely inactivated 25 µL of (human
serum) enzyme, the BChE reaction resumed and pro-
ceeded linearly and proportionally with control (in the
absence of inhibitor) at higher serum concentrations
(data not shown). The presence of 0.025 µM inhibitor
inactivated 50 µL of (human serum) enzyme, but at
higher enzyme concentrations, reaction again proceeded
linearly and proportionally with control. The enzyme
activity values in the presence of 0.02 µM inhibitor were
between those recorded for 0.015 and 0.025 µM inhibitor
(data not shown). These data point to a stoichiometric
irreversible inhibition of BChE by the inhibitor.
Resu lts a n d Discu ssion
Ch em istr y. The cycloSal-triesters 1-49 used in this
study have been prepared as previously reported6a,7,8,11b
except triester 25 (Figure 2).
Starting from 3-fluorophenol, both tert-butyl groups
were introduced by an acid-catalyzed Friedel-Crafts
alkylation using isobutene to yield 2,4-bis-tert-butyl-5-
fluorophenol in 70% yield. Hydroxymethylation of this
material was performed using a solution of formalde-
hyde in water (71% yield of the salicyl alcohol deriva-
tive). Conversion into the cyclic chlorophosphite and
preparation of the corresponding cycloSal-d4TMP tri-
ester 25 was carried out as described before (59%
yield).6a,7,8,11b The triesters have been characterized by
means of 1H, 13C, and 31P NMR spectroscopy, by
analytical reversed-phase high-performance liquid chro-
matography (RP-HPLC), and by mass spectrometry.
Most of the studied cycloSal-phosphate triesters were
used as diastereomeric mixtures. However, 3-methyl-
cycloSal-d4TMP 4, 3-methyl-cycloSal-ganciclovir mono-
phosphate (3-Me-cycloSal-GCVMP) 10, 3-tert-butyl-
Kin etic P r op er ties of th e In h ibition of 3-Me-
cycloSa l-d d AMP 14 a ga in st Hu m a n Ser u m BCh E.
Two concentrations of 3-Me-cycloSal-ddAMP 14 (0.05
and 0.1 µM) were combined with six different concen-
trations of the substrate acetylcholine chloride in a
human serum BChE-catalyzed reaction, and the data

SAR of cycloSal-Pronucleotides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2841
F igu r e 2. Structural formulas of the studied cycloSal-phosphate triesters.
µM (measured after 5 and 15 min incubation, respec-
tively), whereas the Km values ranged between 862 and
977 µM which results in a Ki/Km ratio of ∼2 × 10-5
(Figure 4). These results prove that the reaction of
3-methyl-cycloSal-ddAMP 14 took place in the active
site and again point to the known suicide mechanism.
Va r ia tion of th e Nu cleosid e P a r t. First, a variety
of 3-methyl-cycloSal-nucleotides 1-18 were evaluated
for their inhibitory effects on human BChE present in
human serum. 3-Methyl-substituted cycloSal derivatives
have been selected because these methyl-substituted
triesters showed optimal antiviral activities so far. First,
the use of the competitive BChE-selective inhibitor
ethopropazine proved that the enzyme activity and the
inhibitory effects of the compounds discussed below are
solely a result of the inhibition of BChE. As expected,
there was no AChE activity in human serum. The enzy-
matic activity observed in the serum could be sup-
pressed by ethopropazine in a concentration-dependent
F igu r e 3. Effect of preincubation of human serum AChE with
3-Me-cycloSal-ddAMP 14 on enzyme activity.
were plotted as a Lineweaver-Burk diagram. The
inhibition of the enzyme proved competitive with respect
to the substrate. The Ki values were 0.017 and 0.027

2842 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Meier et al.
Ta ble 1. Inhibitory Activity and Bioactivity of Diastereomeric
Mixtures of 3-Methyl-cycloSal-nucleotides: Variations in the
Nucleoside Part
a
a
IC50 (µM) IC50 (µM) biol. activity
triester nucleoside
AChE
BChE
(µM) [ref]
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
dT
AZT
ddT
d4T
3‘-O-Lev-dT
5-FdU
BVdU
dG
ACV
GCV
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
36
40
1.0
n.a.b
0.006c [19]
4.3c [22]
0.087c [5]
n.a.b
1.2
>50
>50
>50
>50
>50
>50
>50
2.5
28
0.91
1.2
5.0
1.1
0.04d [21]
4.1e [10]
n.a.b
9
9
0.15f [9]
3.3f [22]
17.7f [23]
0.47c [8]
n.a.b
10
11
12
13
14
15
16
17
18
10
11
12
13
14
15
16
17
18
PCV
CBV
dA
ddA
0.047c [6]
0.065c [6]
3.67c [7]
11.7c [7]
0.70c [8]
d4A
F-ara-ddA
F-ribo-ddA
ABC
1.3
a
IC50: 50% inhibitory concentration for the acetylcholine cleav-
age by the AChE (Electrophorus electricus) and human BChE.b Not
available. c Anti-HIV-1 activity (EC50): 50% effective concentration
d
in human lymphocytic CEM cells. Antitumor activity (IC50): 50%
inhibitory concentration in L1210 cells. e Anti-EBV activity (EC50
)
50% effective concentration blocking EBV-DNA synthesis. f Anti-
HSV-1 activity (EC50): 50% effective concentration in Vero cells.
triester derivative ABC 18, proved to be entirely non-
inhibitory toward BChE irrespective of having a cyclic
or an acyclic “glycon” residue. 3-Methyl-cycloSal-ACVMP
9 proved also noninhibitory against BChE. This com-
pound has no 3′-substituent and a very flexible back-
bone, which should be able to adopt the structural
requirement in the active site of the enzyme. In contrast,
carbovir triester 12 bearing a guanine moiety but a d4-
sugar showed an IC50 of 2.5 µM. This result clearly
points to the predominant importance of the presence
of a lipophilic “glycon” in the nucleoside leading to high
BChE inhibitory potency.
F igu r e 4. Lineweaver-Burk plots for the inhibitory activity
of 3-Me-cycloSal-ddAMP 14 against human serum AChE (a)
after 5 min and (b) after 15 min incubation.
manner. At 46 µM ethopropazine, no cleavage of acetyl-
choline was observed. Moreover, such experiments were
also carried out with isolated human butyrylcholinest-
erase (Sigma). No difference was observed in the experi-
ments using human serum or isolated human BChE.
Most of the 3-methyl-cycloSal-phosphate triesters were
tested as diastereomeric mixtures. The results are
summarized in Table 1.
None of the tested cycloSal-triesters was found to be
inhibitory to human AChE and electric eel AChE (only
data from electrophorus electricus AChE are shown in
Table 1). In human serum, clear differences can be
observed within the series of pyrimidine nucleotide
prodrugs. Nucleotide prodrugs bearing nucleosides with
a 3′-substituent like the azido or the hydroxy group
showed very weak if any inhibitory potency to BChE
(entries 1, 2, 6, 7). It seems that even the nature of the
heterocycle has an influence on the inhibitory effect
against the enzyme. While the thymine-bearing nucleo-
tide triester 1 showed weak inhibition (IC50 ) 36 µM),
modified uracil heterocycles bearing nucleotide triesters
such as 5-fluorouracil or (E)-5-(2-bromovinyl)uracil were
devoid of any inhibitory potency (entries 6, 7).
As the pyrimidine nucleotide prodrugs, purine nucleo-
tide-bearing derivatives showed a broad range of IC50
values. The most inhibiting derivatives of all compounds
tested are 3-methyl-cycloSal-triesters bearing a 2′,3′-
dideoxy (d2) or a 2′,3′-dideoxy 2′,3′-didehydro (d4)-sugar
residue (triesters 3, 4, 14, 15, 17, 18). IC50 values varied
between 0.9 µM and 1.3 µM. However, the 2′-ara-fluoro
counterpart 16 (entry 16) proved to be 5-fold less
inhibitory as compared to the ddA triester 14 (entry 14).
Interestingly, all guanine bearing triesters 8-11, except
the carbovir triester 12 and the modified guanine
BCh E In h ibition a s a F u n ction of th e Su bstitu -
tion P a tter n in th e cycloSa l Moiety. Next, the effect
of the different substituents in different positions of the
cycloSal-group was studied. We have chosen d4T as the
nucleoside analogue because 3-methyl-cycloSal-d4TMP
4 belongs to the most potent BChE-inhibiting triesters
(Table 1, entry 4). Moreover, so far we have the largest
number of different cycloSal-derivatives prepared with
d4T available. The results are summarized in Table 2.
The nature of the substituent had a pronounced effect
on the prodrug’s inhibition of the enzyme. As compared
to 3-methyl triester 4, the unsubstituted cycloSal-
d4TMP 19 was even more inhibitory to BChE (0.77 µM).
This increased potency has been expected from previous
results obtained by Casida et al.16 However, the inhibi-
tory effect decreases significantly by introducing bulky
alkyl substituents. For instance, the 3-tert-butyl group
resulted in a 3.5-fold decreased inhibitory activity
compared to the 3-methyl-substituted prodrug 4 (Table
2). Modification of the 5-position of the cycloSal-group
(triesters 20, 23, 27) showed rather identical inhibitory
activity as compared to corresponding modifications at
the 3-position (triesters 4, 22, 26). However, the in-
hibitory potency was most efficiently decreased by
concomitant introduction of substituents in the 3- and
the 5-position. 3,5-Dimethyl-cycloSal-d4TMP 21 and 3,5-

SAR of cycloSal-Pronucleotides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2843
Ta ble 2. Inhibitory Activity and Anti-HIV-1 Activity of
Diastereomeric Mixtures of cycloSal-D4TMP Phosphate
Triesters: Variations in the cycloSal Moiety
at least some resemblance exists in the overall struc-
tural features of BChE and AChE. In contrast to BChE,
crystallographic structures of AChE have been resolved
for different species including Drosophila,26 Torpedo
californica,27 mouse28 and man.29 Nevertheless, due to
the observation that cycloSal-triesters are not inhibitors
of AChE, this enzyme has not been used as an appropri-
ate BChE model for molecular modeling studies. How-
ever, from the observed potential for variation in the
substitution pattern on the BChE inhibition it can be
concluded that the cycloSal-moiety as such does not
entirely fill the active site pocket.
a
a
IC50 (µM) IC50 (µM) EC50
triester
substituent
AChE
BChE
(µM)b
1
2
3
4
5
6
7
8
19
4
H
3-Me
5-Me
3,5-Me
3-tBu
5-tBu
3,5-tBu
3,5-tBu,6-F
3-sBu
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.77
1.2
4.6
46
4.2
5.6
>50
48
2.0
3.3
2.7
8.8
3.4
4.4
2.8
22
0.28
0.10
0.18
0.10
0.18
0.14
1.1
0.16
0.08
1.9
0.16
0.24
0.33
0.18
0.15
0.08
0.19
0.14
0.13
0.40
0.14
0.41
0.09
0.09
0.19
0.19
0.14
0.19
0.16
0.19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5-sBu
From the studies described above, structural require-
ments can be derived in order to make the binding of
the triester weaker. It was shown in our experiments
using isolated BChE and BChE in human serum that
substituents particularly in the 3- and 5-position of the
cycloSal-moiety should have a pronounced effect on the
binding. So, we included cycloSal-d4TMP triesters 36
and 37 (Table 2, entries 19, 20) that bear larger
substituents than the tert-butyl group.30 A phenyl
residue in the 3- or 5-position is a rigid substituent
which spans a distance of about 5 Å. Surprisingly,
triester 36 was found to be one of the most inhibitory
triesters among the whole d4TMP series (0.35 µM)! We
conclude that due to the presence of a number of
aromatic amino acids in the active site of BChE, the
phenyl ring may accommodate a lipophilic aromatic
pocket stabilized by π-π-interactions. However, as
expected, introduction of the phenyl ring in the 5-posi-
tion in triester 37 led to a 6-fold reduction of the
inhibitory potency as compared to the unsubstituted
cycloSal-ring in triester 19. The same trend was found
for the ring anullated benzo[a]-, benzo[b]-, and benzo-
[c]-cycloSal-d4TMPs 38-40.30 The more the additional
aromatic ring is shifted away from the 3-position, the
weaker the inhibitory activity (entries 21-23). Obvi-
ously, the highly lipophilic nature of the active site of
BChE can also be deduced from the values found for
propionic acid-modified cycloSal-d4TMP triesters 34 and
35. The acid group is deprotonated under the reaction
conditions used in the assay and the presence of the
highly polar carboxylate is the reason for these com-
pounds being devoid of any inhibitory activity against
BChE (entries 17, 18). Interestingly, substitutions at the
7-position by methyl (42), butyl (43), or ethoxycar-
bonylmethyl (ECM; 44) led to a reduction of the binding
properties.31 Moreover, large substituents such as the
chlorinated 7-methyl-groups also considerably decrease
the binding affinity (triesters 45-47, entries 28-30).31
3-MeOOC Et
5-MeOOC Et
3-tBuOOC Et
5-tBuOOC Et
3-BnOOC Et
5-BnOOC Et
3-HOOC Et
5-HOOC Et
3-Ph
>50
>50
0.35
4.4
5-Ph
benzo[a]
benzo[b]
benzo[c]
6-Cl
6-Cl, 7-Me
6-Cl, 7-Bu
6-Cl, 7-ECM
7-CH2Cl
7-CHCl2
7-CCl3
0.25
0.41
3.9
0.57
1.8
13
>50
3.9
26
27
a
Sp and Rp represent the configuration of the diastereomers.
Rp, Sp represents an equal mixture of both diastereomers that
b
could not be separated. IC50: 50% inhibitory concentration for
the acetylcholine cleavage by cholinesterase.
F igu r e 5. Graphical representation of the anti-AChE and
anti-BChE potency and the antiviral activity of alkyl-substi-
tuted cycloSal-d4TMP triesters.
Finally, it should be added that for most of the studied
cases, the described compounds bearing different sub-
stitution patterns in the cycloSal-part are still anti-
virally active and retained the antiviral potency in
thymidine-kinase (TK)-deficient cell lines and thus
achieved the TK-bypass. This can be demonstrated for
example with 3,5-bis-tert-butyl-6-fluoro-cycloSal-3′-
deoxy-2′,3′-didehydrothymidine monophosphate 25. This
compound showed a chemical half-life of 6.2 h, only
weak if any inhibitory activity against BChE (IC50 48
µM; Table 2), no AChE inhibition (IC50 >50 µM), strong
anti-HIV-1 (EC50 0.13 µM, Table 2) and anti-HIV-2
activity (EC50 0.33 µM) and full retention of activity in
HIV-2-infected TK-deficient CEM cells (EC50 0.60 µM).32
di-tert-butyl-cycloSal-d4TMP 24 proved to be virtually
noninhibitory to BChE (entries 4 and 7). The varying
inhibitory effects of triesters 20-24 due to the substitu-
tion pattern on the cycloSal part of the masking
molecule has no impact on the interaction with AChE
(all compounds were found to be entirely inactive) and
also not on the observed anti-HIV-1 activity (all com-
pounds showed virtually identical HIV-1 activity). This
has been summarized in Figure 5.
Unfortunately, until today there is no crystal struc-
ture available from butyrylcholinesterase.25 However,
from the amino acid sequence it can be concluded that

2844 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Meier et al.
Ta ble 3. Dependence of the Inhibitory Activity against AChE
(Electrophorus electricus) and Human BChE on the
Stereochemistry of the cycloSal-nucleotides
S-enantiomer (Ki ) 17.5 nM). In contrast, among the
cycloSal-nucleotide prodrugs, the difference in IC50
between the two diastereomers was found to be up to
>200-fold.
b
b
IC50
(µM)
configa AChE BChE
IC50
antiviral
act.
(µM)
(µM)
Moreover, an interesting inverse correlation between
the inhibitory potency and the antiviral activity was
observed. As a rule, the most antivirally active dia-
stereomer corresponded to the least BChE-inhibitory
diastereomer. Thus, the Rp diastereomer of 3-Me-
cycloSal-d4TMP 4, while not being inhibitory to human
BChE at 50 µM, was 5-fold more anti-HIV active (0.087
µM) than the Sp diastereomer.6a In contrast, the Sp
diastereomer was markedly more inhibitory against
BChE (0.24 µM). Also, 5-OMe-d4AMP 49 (Rp) was 11-
fold more inhibitory to HIV than 5-OMe-d4AMP (Sp)
(EC50: 0.043 and 0.50 µM, respectively).7 Furthermore,
the Rp diastereomer was not inhibitory against human
BChE at 50 µM whereas the Sp diastereomer was
inhibitory at 1.8 µM (Table 3). In deciding which
cycloSal anti-HIV prodrug(s) should be chosen as clinical
candidate(s) the optimal phosphorus configuration is Rp,
but it would be advantageous to choose cycloSal sub-
stituents with reduced anti-BChE activity of the Sp
configuration to avoid the need of separating the dia-
stereomers.
St u d ies w it h ACh E fr om Differ en t Sou r ces.
Finally, selected cycloSal-phosphate triesters were stud-
ied against AChE and BChE from different origins. As
summarized in Table 4, cycloSal-phosphate triesters
were found to show 10-30-fold lower affinities for the
murine serum BChE enzyme as compared to the human
serum BChE and lack of measurable inhibitory effect
against the calf and erythrocyte AChE.
Thus, the results obtained with beef erythrocyte
AChE were in full agreement with the results found
with AChE from electric eel and man. In the case of
BChE, the results clearly prove that even functionally
similar enzymes may behave differently with respect
to the source of the species they were derived from. This
may be related to subtle differences in the structure of
the enzymes.
triester
1
2
3
4
5
6
7
8
9
3-Me-cycloSal-d4TMP 4
3-Me-cycloSal-d4TMP 4
3-tBu-cycloSal-d4TMP 22
3-tBu-cycloSal-d4TMP 22
3-Me-cycloSal-FdUMP 6
3-Me-cycloSal-FdUMP 6
3-Ph-cycloSal-BVdUMP 48
3-Ph-cycloSal-BVdUMP 48
5-OMe-cycloSal-d4AMP 49
Rp
Sp
Rp
Sp
Rp
Sp
Rp
Sp
Rp
Sp
Rp
Sp
>50 >50
0.08c
>50
0.24 0.42c
>50 >50
0.13c
0.6c
0.040d
0.044d
-
>50
2.6
>50 >50
>50 >50
>50 >50
>50
3.7
-
>50 >50
0.043c
0.5c
3.29e
-
10 5-OMe-cycloSal-d4AMP 49
11 3-Me-cycloSal-GCVMP 10
12 3-Me-cycloSal-GCVMP 10
>50
1.8
>50 >50
>50
23
a
IC50: 50% inhibitory concentration for the acetylcholine cleav-
b
age by cholinesterase. Anti-HIV-1 activity (EC50): 50% effective
concentration in CEM cells
Due to the fact that the active site of the enzyme is
chiral, a difference in the inhibition may be observed
with respect to the stereochemistry at the phosphorus
atom of the cycloSal-triesters.
Dep en d en ce of t h e In h ib it or y P ot en cy on t h e
P -Ster eoch em istr y. Some of the prepared cycloSal-
NMP triesters have been separated into their diastereo-
isomers by semipreparative HPLC. Compounds were
found to be stereochemically purely separated as judged
by 1H NMR, 31P NMR, and HPLC. These separated
diastereomers then were subjected to the assays with
AChE from electric eel and BChE from human serum.
Again, no inhibitory activity has been found against
AChE for both diastereoisomers. Much more interesting
was the behavior of the triesters against human BChE
(Table 3). As expected, a marked difference in the
inhibition was observed. In all cases, the Sp diastereo-
isomer was found to be inhibitory toward the enzyme
while the Rp isomers were entirely noninhibiting, e.g.
the Rp diastereomer of the d4TMP derivatives 4,22 were
devoid of appreciable inhibitory activity while their Sp
derivatives showed pronounced inhibitory activity. This
behavior was independent of the nature of the nucleo-
side (Table 3). Thus, human BChE is able to markedly
discriminate between the Sp and Rp diastereomers of
the cycloSal-nucleotide prodrugs. To some extent, such
stereoselectivity has also been reported for T. californica
AChE against E-2020 ((R,S)-1-benzyl-4-[(5,6-dimethoxy-
1-indanon)-2-yl)methylpiperidine (donepezil hydro-
chloride; Aricept:33 the enzyme showed a 5-fold higher
affinity for the R-enantiomer (Ki ) 3.3 nM) than for the
Con clu sion
In summary, we can conclude from the presented data
that cycloSal-phosphate triesters of various nucleosides
and nucleoside analogues are noninhibitory to human
AChE. Some of the compounds show significant inhibi-
tory activity against human serum BChE and to a minor
extent against mouse serum BChE. However, several
structural factors clearly determine the inhibitory po-
tency of the compounds:
Ta ble 4. Inhibitory Activities of cycloSal NMP Derivatives against Cholinesterases from Different Species
b
IC50 (µM)
human serum
BChE
mouse serum
BChE
calf serum
AChE
beef erythrocytes
AChE
cycloSal-triester
configurationa
3-Me-cycloSal-ddAMP
3-Me-cycloSal-d4AMP
3-Me-cycloSal-F-ara-ddAMP
3-Me-cycloSal-d4TMP
3-Me-cycloSal-d4TMP
3-Me-cycloSal-AZTMP
14
15
16
4
4
2
(Rp, Sp)
(Rp, Sp)
(Rp, Sp)
(Sp)
(Rp)
(Rp, Sp)
0.91
1.2
5.0
2.35
10.5
>50
g5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.24
>50
>50
a
b
Stereochemistry at the phosphorus atom of the cycloSal-phosphate triester. IC50: 50% inhibitory concentration for the acetylcholine
cleavage by the AChE (Electrophorus electricus) and human BChE. c Anti-HIV-1 activity (EC50): 50% effective concentration in wild-type
CEM cell cultures. Cytostatic activity (IC50): 50% inhibitory concentration against L1210 cells. e Anti-HSV-1 activity (EC50): 50% effective
d
concentration in Vero cell cultures.

SAR of cycloSal-Pronucleotides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2845
multinuclear NMR spectroscopy, and mass spectroscopy.
Synthesis of some of the cycloSal-nucleoside monophosphates
has been described before.6a,7,8,11b
(i) polar or bulky substituents in the 3′-position of the
glycon ring reduce or destroy the inhibitory effect;
(ii) polar or bulky substituents in the 3-, 5-, and/or
7-position of the cycloSal-moiety reduce the inhibitory
potency;
(3) the inhibitory effect depends on the stereo-
chemistry at the phosphorus atom. While the Rp stereo-
isomers were devoid of any enzyme inhibition, only the
Sp diastereomers showed an inhibitory effect. Impor-
tantly, the Rp diastereomer was found to be antivirally
more active as compared to the Sp isomer.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of t h e
cycloSa l-n u cleosid e Mon op h osp h a tes. (A) The appropri-
ate salicyl alcohol derivatives needed for the preparation of
the cycloSal phosphotriesters were obtained by established
methods2a,20,31 except of the synthesis of 51. They were
converted into the chlorophosphites (phosphitylating agents)
by the procedure published before.1,20 The highly moisture
sensitive chlorophosphites were used in the subsequent syn-
thetic procedure without further purification.
To a solution of the nucleoside analogue (0.40 mmol) in 10
mL of dry acetonitrile was added diisopropylethylamine (0.80
mmol, DIPEA). The resulting solution was cooled to -20 °C,
and the appropriate chlorophosphite (0.80 mmol if calculated
as hypothetically pure product) was added. The solution was
allowed to warm to room temperature, and stirring was
continued for 1 h (TLC monitoring). Afterward, tert-butyl
hydroperoxide (1.4 mmol, solution in n-decane) was added at
-20 °C. After warming up to room temperature and stirring
for 1 h again (TLC monitoring), the solvent was removed under
reduced pressure. The resulting residue was purified by
preparative TLC (Chromatotron, 1. ethyl acetate/methanol 9:1,
2. Dichloromethane/methanol gradient). Subsequent lyophiliza-
tion yielded the products as colorless foams.
Moreover, a few characteristics of the enzyme can be
concluded. The active site seems to be highly lipophilic
and is able to accommodate the cycloSal-triesters.
However, there should be significant structural differ-
ences between the two enzymes because cycloSal-
triesters are inhibitory to human and murine BChE but
not to human and bovine AChE. Work in order to
eliminate the inhibition of BChE by structural changes
of the cycloSal-moiety seem to be possible with the help
of the presented data and work along this is under
current investigation in our laboratories.
(B) Analogously to procedure A, but with the use of dry
DMF/THF 2:1 as solvent and 0.60 mmol chlorophosphite.
(C) Analogously to procedure A, but with the use of dry
DMF/THF 1:5 as solvent and 0.60 mmol chlorophosphite.
Addition of the chlorophosphite was carried out at -40 °C, and
the solution was allowed to warm to 0 °C to complete the
phosphitylation reaction.
(D) Analogously to procedure A, but with the use of dry
DMF/THF 1:10 as solvent. The phosphitylation reaction was
carried out at -70 °C for 15 min.
Exp er im en ta l Section
All experiments involving water-sensitive compounds were
conducted under scrupulously dry conditions (argon or nitrogen
atmosphere). Solvents: Anhydrous methylene chloride (CH2Cl2),
anhydrous tetrahydrofuran (THF), and anhydrous acetonitrile
(CH3CN) were obtained in a Sure/Seal bottle from Fluka and
stored over 4 Å molecular sieves; ethyl acetate, methylene
chloride, and methanol employed in chromatography were
distilled prior to use. Diisopropyethylamine (DIPEA) was
distilled from sodium prior to use. Evaporation of solvents was
carried out on a rotary evaporator under reduced pressure or
using a high-vacuum pump. Chromatography: Chromatotron
(Harrison Research 7924), silica gel 60Pf (Merck, “gipshaltig”),
UV detection at 254 nm; for column chromatography, Merck
silica gel 60, 230-400 mesh was used. TLC: analytical thin-
layer chromatography was performed on Merck precoated
aluminum plates 60 F254 with a 0.2 mm layer of silica gel
containing a fluorescence indicator; sugar-containing com-
pounds were visualized with the sugar spray reagent (0.5 mL
of 4-methoxybenzaldehyde, 9 mL of ethanol, 0.5 mL of con-
centrated sulfuric acid, and 0.1 mL of glacial acetic acid) by
heating with a fan or on a hot plate. NMR spectra were
recorded using (1H NMR) Bruker AC 250 at 250 MHz, Bruker
WM 400 at 400 MHz, Bruker AMX 400 at 400 MHz or Bruker
DMX 500 at 500 MHz; (13C NMR) Bruker WM 400 at 101 MHz,
Bruker AMX 400 at 101 MHz or Bruker DMX 500 at 126 MHz
(calibration was done in both cases with the solvent); (31P
NMR) Bruker AMX 400 at 162 MHz or Bruker DMX 500 at
202 MHz (H3PO4 as external standard); (19F NMR) Bruker WH
270 at 254 MHz or Bruker DMX 500 at 471 MHz (CFCl3 as
external standard). All 1H and 13C NMR chemical shifts (δ)
are quoted in parts per million (ppm) downfield from tetra-
methylsilane. 31P NMR chemical shifts are quoted in ppm
using H3PO4 as external reference. 19F NMR chemical shifts
are quoted in ppm using CFCl3 as external reference. The
spectra were recorded at room temperature, and all 13C and
31P NMR were recorded in proton-decoupled mode. UV/vis
extinction was measured with a Varian Cary 1E UV/Vis
spectrometer. Mass spectra were obtained with a Finnigan
electrospray MAT 95 Trap XL (ESI) or a VG Analytical VG/
70-250 F spectrometer (FAB, matrix was m-nitrobenzyl
alcohol). The test compounds were isolated as mixtures of
diastereomers arising from the mixed stereochemistry at the
phosphate center. In some cases, separation of diastereomers
was performed using semipreparative or preparative RP-HPLC
(Merck, acetonitrile/water eluents). The resulting lyophilized
triesters were found to be pure by HPLC analysis, high-field
(E) The appropriate DMTr-protected nucleoside analogues
have been converted into the corresponding cycloSal-nucleoside
monophosphates according to procedure A. Afterward, depro-
tection was carried out using benzenesulfonic acid or tri-
fluoroacetic acid in dichloromethane/methanol 7:3 at room
temperature. After evaporation of the solvent under reduced
pressure and purification by preparative TLC (Chromatotron,
dichloromethane/methanol 17:3), the product was lyophilized.
3-Meth yl-cycloSa l-th ym id in e m on op h osp h a te 1: pro-
1
cedure B; yield 31%; H NMR (500 MHz, DMSO-d6) δ 11.31
(s, 2H, NH); 7.45 (s, 1H, H6-thymine); 7.43 (s, 1H, H6-
thymine); 7.29-7.24 (m, 2H, H4-aryl); 7.12-7.08 (m, 4H, H5-
aryl, H6-aryl); 6.19 (dd, 1H, H1′); 6.18 (dd, 1H, H1′); 5.50 (dd,
1H, H-benzyl); 5.48 (dd, 1H, H-benzyl); 5.45 (s, 2H, OH); 5.44
(dd, 1H, H-benzyl); 5.41 (dd, 1H, H-benzyl); 4.38-4.21 (m, 6H,
H4′, H5′); 3.93-3.91 (m, 2H, H3′); 2.23 (s, 3H, CH3-aryl); 2.22
(s, 3H, CH3-aryl); 2.18-2.08 (m, 4H, H2′); 1.75 (s, 3H, CH3-
thymine); 1.72 (s, 3H, CH3-thymine); 13C NMR (101 MHz,
DMSO-d6) δ 163.8 (C4-thymine); 163.8 (C4-thymine); 150.5
(C2-thymine); 148.1 (d, C2-aryl); 148.1 (d, C2-aryl); 136.0 (C6-
thymine); 135.9 (C6-thymine); 131.1 (C4-aryl); 127.0 (C3-aryl);
127.0 (d, C3-aryl); 124.1 (d, C6-aryl); 123.8 (C5-aryl); 121.2
(d, C1-aryl); 121.1 (d, C1-aryl); 110.0 (C5-thymine); 110.0 (C5-
thymine); 84.3 (d, C4′); 84.3 (d, C4′); 84.2 (C1′); 84.1 (C1′); 70.1
(C3′); 70.0 (C3′); 68.6 (d, C5′); 68.5 (d, C5′); 68.0 (d, C-benzyl);
67.9 (C-benzyl); 38.8 (C2′); 38.6 (C2′); 15.0 (CH3-aryl); 15.0
(CH3-aryl); 12.2 (CH3-thymine); 12.2 (CH3-thymine); 31P NMR
(202 MHz, DMSO-d6) δ -7.72; -7.75; Rf value 0.47 (CH2Cl2/
MeOH, 9:1); MS (FAB, m/z) calcd 425.1114 (M + H+), found
425.1121 (M + H+).
3-Meth yl-cycloSa l-3′-d eoxyth ym id in e m on op h osp h a te
1
3: procedure A; yield 21%; H NMR (500 MHz, DMSO-d6) δ
11.28 (s, 2H, NH); 7.45 (s, 1H, H6-thymine); 7.44 (s, 1H, H6-
thymine), 7.28-7.25 (m, 2H, H4-aryl); 7.12-7.08 (m, 4H, H5-
aryl, H6-aryl); 6.02 (dd, 1H, H1′); 6.00 (dd, 1H, H1′); 5.51 (dd,
1H, H-benzyl); 5.47 (dd, 1H, H-benzyl); 5.43 (dd, 1H, H-benzyl);
5.40 (dd, 1H, H-benzyl); 4.40-4.23 (m, 4H, H5′); 4.22-4.16 (m,
2H, H4′); 2.30-2.23 (m, 2H, H2′); 2.23 (s, 3H, CH3-aryl); 2.22

2846 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Meier et al.
(s, 3H, CH3-aryl); 2.06-1.90 (m, 4H, H2′, H3′); 1.89-1.82 (m,
2H, H3′); 1.75 (s, 3H, CH3-thymine); 1.72 (s, 3H, CH3-thymine);
13C NMR (101 MHz, DMSO-d6) δ 163.9 (C4-thymine); 150.5
(C2-thymine); 150.5 (C2-thymine); 148.1 (d, C2-aryl); 148.1 (d,
C2-aryl); 135.9 (C6-thymine); 135.9 (C6-thymine); 131.1 (C4-
aryl); 131.0 (C4-aryl); 127.0 (d, C3-aryl); 124.1 (C6-aryl); 123.7
(C5-aryl); 121.2 (d, C1-aryl); 109.6 (C5-thymine); 85.1 (C1′);
85.0 (C1′); 78.0 (C4′); 77.9 (C4′); 69.2 (d, C5′); 69.1 (d, C5′);
68.6 (d, C-benzyl); 68.4 (d, C-benzyl); 30.5 (C2′); 30.4 (C2′); 25.4
(C3′); 25.3 (C3′); 15.0 (CH3-aryl); 15.0 (CH3-aryl); 12.2 (CH3-
thymine); 12.2 (CH3-thymine); 31P NMR (202 MHz, DMSO-
d6) δ -7.65; -7.73; Rf value 0.42 (CH2Cl2/MeOH, 9:1); MS
(FAB, m/z) calcd 409.1165 (M + H+), found 409.1159 (M + H+).
3-Meth yl-cycloSa l-3′-O-levu lin yl-th ym id in e m on op h os-
p h a te 5: procedure A; yield 33%; 1H NMR (500 MHz, DMSO-
d6) δ 11.37 (s, 1H, NH); 11.37 (s, 1H, NH); 7.52 (s, 1H, H6-
thymine); 7.49 (s, 1H, H6-thymine); 7.29-7.25 (m, 2H, H4-
aryl); 7.13-7.09 (m, 4H, H5-aryl, H6-aryl); 6.17 (dd, 2H, H1′);
5.54-5.40 (m, 4H, H-benzyl); 5.18 (ddd, 1H, H3′); 5.16 (ddd,
1H, H3′); 4.42-4.31 (m, 4H, H5′); 4.16-4.12 (m, 2H, H4′); 2.75
(t, 2H, H3-Lev); 2.75 (t, 2H, H3-Lev); 2.51-2.49 (m, 4H, H2-
Lev); 2.40-2.25 (m, 4H, H2′); 2.23 (s, 3H, CH3-aryl); 2.21 (s,
3H, CH3-aryl); 2.13 (s, 6H, H5-Lev); 1.76 (s, 3H, CH3-thymine);
1.74 (s, 3H; CH3-thymine); 13C NMR (101 MHz, DMSO-d6) δ
207.0 (C4-Lev); 172.1 (C1-Lev); 163.8 (C4-thymine); 163.8 (C4-
thymine); 150.5 (C2-thymine); 148.1 (d, C2-aryl); 148.1 (d, C2-
aryl); 135.9 (C6-thymine); 135.7 (C6-thymine); 131.1 (C4-aryl);
127.1 (d, C3-aryl); 127.0 (C3-aryl); 124.1 (d, C6-aryl); 123.7
(C5-aryl); 121.1 (d, C1-aryl); 110.2 (C5-thymine); 110.1 (C5-
thymine); 84.3 (C1′); 84.2 (C1′); 81.7 (d, C4′); 81.6 (d, C4′); 73.7
(C3′); 73.6 (C3′); 68.7 (d, C-benzyl); 68.5 (d, C-benzyl); 67.8 (d,
C5′); 67.7 (d, C5′); 37.6 (C3-Lev); 35.7 (C2′); 35.7 (C2′); 29.6
(C5-Lev); 27.8 (C2-Lev); 15.0 (CH3-aryl); 14.9 (CH3-aryl); 12.2
(CH3-thymine); 12.1 (CH3-thymine); 31P NMR (202 MHz,
DMSO-d6) δ -7.87; -7.90; Rf value 0.44 (CH2Cl2/MeOH, 9:1);
MS (FAB, m/z) calcd 523.1482 (M + H+), found 523.1492 (M
+ H+).
3-Meth yl-cycloSa l-5-flu or o-2′-d eoxyu r id in e m on op h os-
p h a te 6: procedure D; yield 20%; 1H NMR (400 MHz, DMSO-
d6) δ 11.80 (s, 2H, NH); 7.85 (d, 2H, H6-thymine); 7.24 (m,
2H, H4-aryl); 7.08 (m, 4H, H5-aryl, H6-aryl); 6.12 (dd, 2H, H1′);
5.48 (dd, 2H, H-benzyl); 5.40 (m, 4H, H-benzyl, OH); 4.35 (ddd,
2H, H5′); 4.25 (ddd, 2H, H5′); 4.17 (m, 2H, H3′); 3.89 (m, 2H,
H4′); 2.20 (s, 6H, CH3-aryl); 2.11 (ddd, 4H, H2′); 13C NMR (63
MHz, DMSO-d6) δ 157.0 (C4-thymine); 149.0 (C2-thymine);
147.9 (C2-aryl); 140.1 (C5-thymine); 130.9 (C4-aryl); 126.9 (C3-
aryl); 124.9 (C6-thymine); 123.9 (C5-aryl); 123.5 (C6-aryl);
121.0 (C1-aryl); 84.5 (C1′); 84.3 (C4′); 69.6 (C3′); 68.5 (C5′);
67.6 (C-benzyl); 38.5 (C2′); 14.8 (CH3-aryl); 31P NMR (162 MHz,
DMSO-d6) δ -7.87; -7.94; 19F NMR (254 MHz, DMSO-d6) δ
-182.2; Rf value 0.33 (CH2Cl2/MeOH, 9:1); MS (ESI-, m/z)
calcd 428.1 (M), found 427.4 (M).
3-Meth yl-cycloSa l-2′-d eoxygu a n osin e m on op h osp h a te
8: procedure C; yield 4%; 1H NMR (400 MHz, DMSO-d6) δ
10.60 (s, 2H, NH); 7.78 (s, 2H, H8-guanine); 7.26-7.07 (m, 6H,
H4-aryl, H5-aryl, H6-aryl); 6.45 (s, 4H, NH2); 6.13 (dd, 1H,
H1′); 6.13 (dd, 1H, H1′); 5.50-5.33 (m, 6H, H-benzyl, OH);
4.42-4.20 (m, 6H, H3′, H5′); 4.01-3.96 (m, 2H, H4′); 2.62-
2.55 (m, 2H, H2′); 2.30-2.25 (m, 2H, H2′); 2.17 (s, 6H, CH3-
aryl); 13C NMR (101 MHz, DMSO-d6) δ 156.9 (C6-guanine);
153.8 (C2-guanine); 151.0 (C4-guanine); 148.0 (d, C2-aryl);
135.3 (C8-guanine); 135.3 (C8-guanine); 131.0 (C4-aryl); 127.1
(d, C3-aryl); 127.0 (d, C3-aryl); 124.1 (C6-aryl); 123.7 (C5-aryl);
123.7 (C5-aryl); 121.1 (d, C1-aryl); 121.0 (d, C1-aryl); 117.0
(C5-guanine); 116.9 (C5-guanine); 84.8 (C1′); 84.7 (C1′); 82.8
(C4′); 82.7 (C4′); 70.4 (C3′); 68.6 (d, C5′); 68.5 (d, C5′); 68.2 (d,
C-benzyl); 68.0 (d, C-benzyl); 40.0 (C2′); 15.1 (CH3-aryl); 15.0
(CH3-aryl); 31P NMR (202 MHz, DMSO-d6) δ -7.48; -7.98; Rf
value 0.20 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd 450.1179
(M + H+), found 450.1165 (M + H+).
6.45 (s, 8H, NH2); 5.40 (dd, 4H, H-benzyl); 5.37 (s, 8H, H1‘);
5.28 (dd, 4H, H-benzyl); 4.82 (t, 4H, OH); 4.17 (ddd, 4H, H5‘);
4.05 (ddd, 4H, H5‘); 3.75 (ddd, 4H, H4‘); 3.34 (dd, 4H, H4‘);
3.28 (dd, 4H, H3‘); 2.19 (s, 12H, CH3-aryl); 13C NMR (101 MHz,
DMSO-d6) δ 156.9 (C6-guanine); 154.0 (C2-guanine); 151.4 (C4-
guanine); 148.1 (C2-aryl); 137.7 (C8-guanine); 131.0 (C4-aryl);
127.1 (C3-aryl); 124.1 (C5-aryl); 123.6 (C6-aryl); 116.6 (C5-
guanine); 77.4 (C3‘); 71.2 (C1‘); 68.4 (C-benzyl); 67.2 (C5‘); 59.7
(C4‘); 15.1 (CH3-aryl); 31P NMR (162 MHz, DMSO-d6) δ -7.89;
Rf value 0.18 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd 438.1
(M + H+), found 438.4 (M + H+).
3-Meth yl-cycloSa l-p en ciclovir m on op h osp h a te 11: pro-
cedure E; yield 43% (four diastereomers); 1H NMR (400 MHz,
DMSO-d6) δ 10.53 (s, 4H, NH); 7.61 (s, 2H, H8-guanine); 7.59
(s, 2H, H8-guanine); 7.21 (dd, 4H, H5-aryl); 7.07-7.02 (m, 8H,
H4-aryl, H6-aryl); 6.39 (s, 8H, NH2); 5.44 (dd, 4H, H-benzyl);
5.36 (dd, 4H, H-benzyl); 4.65 (t, 2H, OH); 4.63 (t, 2H, OH);
4.18-3.85 (m, 16H, H1′, H4‘); 3.40-3.30 (m, 8H, H5‘); 2.19 (s,
6H, CH3-aryl); 2.17 (s, 6H, CH3-aryl); 1.72-1.60 (m, 12H, H2‘,
H3‘); 13C NMR (101 MHz, DMSO-d6) δ 156.9 (C6-guanine);
153.6 (C2-guanine); 151.2 (C4-guanine); 148.1 (C1-aryl); 148.0
(C1-aryl); 137.3 (C8-guanine); 131.0 (C4-aryl); 127.0 (C3-aryl);
127.0 (C3-aryl); 124.0 (C5-aryl); 123.6 (C6-aryl); 116.7 (C5-
guanine); 68.6 (C-benzyl); 68.5 (C-benzyl); 68.1 (C4‘); 59.9 (C5‘);
59.8 (C5‘); 41.2 (C1‘); 38.9 (C3‘); 38.8 (C3‘); 28.0 (C2‘); 28.0 (C2‘);
15.1 (CH3-aryl); 15.0 (CH3-aryl); 31P NMR (162 MHz, DMSO-
d6) δ -9.09; Rf value 0.31 (CH2Cl2/MeOH, 9:1); MS (ESI+, m/z)
calcd 436.1 (M + H+), found 436.3 (M + H+).
3-Meth yl-cycloSa l-2′-d eoxya d en osin e m on op h osp h a te
1
13: procedure C; yield 14%; H NMR (500 MHz, DMSO-d6) δ
8.27 (s, 1H, H2-adenine); 8.25 (s, 1H, H2-adenine); 8.12 (s, 1H,
H8-adenine); 8.11 (s, 1H, H8-adenine); 7.27-7.21 (m, 6H, H4-
aryl, NH2); 7.10-7.03 (m, 4H, H5-aryl, H6-aryl); 6.36 (dd, 2H,
H1′); 5.52 (s, 2H, OH); 5.48-5.31 (m, 4H, H-benzyl); 4.49-
4.45 (m, 2H, H3′); 4.38 (ddd, 1H, H5′); 4.36 (ddd, 1H, H5′);
4.31-4.22 (m, 2H, H5′); 4.04-4.00 (m, 2H, H4′); 2.85-2.77 (m,
2H, H2′); 2.32 (ddd, 2H, H2′); 2.20 (s, 3H, CH3-aryl); 2.14 (s,
3H, CH3-aryl); 13C NMR (101 MHz, DMSO-d6) δ 156.2 (C6-
adenine); 152.7 (C2-adenine); 149.2 (C4-adenine); 148.1 (C2-
aryl); 139.6 (C8-adenine); 139.5 (C8-adenine); 131.0 (C4-aryl);
127.0 (d, C3-aryl); 124.0 (C6-aryl); 123.7 (C5-aryl); 123.6 (C5-
aryl); 121.1 (d, C1-aryl); 119.4 (C5-adenine); 119.4 (C5-
adenine); 84.9 (C1′); 84.8 (C1′); 83.6 (d, C4′); 70.4 (C3′); 68.5
(d, C5′); 68.5 (d, C5′); 68.0 (d, C-benzyl); 67.9 (d, C-benzyl);
38.6 (C2′); 38.4 (C2′); 15.0 (CH3-aryl); 14.9 (CH3-aryl); 31P NMR
(202 MHz, DMSO-d6) δ -7.93; -8.09; Rf value 0.23 (CH2Cl2/
MeOH, 9:1); MS (FAB, m/z) calcd 434.1229 (M + H+), found
434.1232 (M + H+).
3-ter t-Bu t yl-cycloSa l-3′-d eoxy-2′,3′-d id eh yd r ot h ym i-
1
d in e m on op h osp h a te 22: procedure A; yield 91%; H NMR
(400 MHz, DMSO-d6) δ 11.37 (s, 1H, NH); 11.35 (s, 1H, NH);
7-37-7.35 (m, 2H, H4-aryl); 7.25 (d, 1H, H6-thymine); 7.22
(d, 1H, H6-thymine); 7.21-7.13 (m, 4H, H5-aryl, H6-aryl); 6.83
(ddd, 1H, H1′); 6.83 (ddd, 1H, H1′); 6.44 (ddd, 1H, H3′); 6.42
(ddd, 1H, H3′); 6.07-6.03 (m, 2H, H2′); 5.48-5.35 (m, 4H,
H-benzyl); 5.01-4.96 (m, 2H, H4′); 4.35-4.31 (m, 4H, H5′);
1.61 (d, 3H, CH3-thymine); 1.60 (d, 3H, CH3-thymine); 1.36
(s, 9H, CH3-tBu); 1.33 (s, 9H, CH3-tBu); 13C NMR (101 MHz,
DMSO-d6) δ 164.0 (C4-thymine); 163.9 (C4-thymine); 150.9
(C2-thymine); 150.9 (C2-thymine); 148.9 (d, C2-aryl); 148.9 (d,
C2-aryl); 138.6 (d, C3-aryl); 138.5 (d, C3-aryl); 136.0 (C6-
thymine); 135.9 (C6-thymine); 133.1 (C3′); 133.0 (C3′); 127.5
(C4-aryl); 127.5 (C4-aryl); 127.5 (C2′); 127.5 (C2′); 124.7 (C6-
aryl); 124.4 (C5-aryl); 123.3 (d, C1-aryl); 123.2 (d, C1-aryl);
109.9 (C5-thymine); 109.8 (C5-thymine); 89.4 (C1′); 89.3 (C1′);
84.4 (d, C4′); 84.3 (d, C4′); 69.1 (d, C5′); 69.0 (d, C5′); 68.4 (d,
C-benzyl); 68.4 (d, C-benzyl); 34.5 (C-tBu); 34.5 (C-tBu); 29.8
(CH3-tBu); 29.7 (CH3-tBu); 12.0 (CH3-thymine); 12.0 (CH3-
thymine); 31P NMR (202 MHz, DMSO-d6) δ -7.23; -7.49; Rf
value 0.36 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd 471.1 (M
+ Na+), 487.1 (M + K+), found 471.4 (M + Na+), 487.3 (M +
K+).
3-Meth yl-cycloSa l-ga n ciclovir m on op h osp h a te 10: pro-
1
cedure E; yield 35% (four diasteromers); H NMR (500 MHz,
DMSO-d6) δ 10.61 (s, 4H, NH); 7.72 (s, 4H, H8-guanine); 7.24-
7.20 (m, 4H, H5-aryl); 7.09-7.02 (m, 8H, H4-aryl, H6-aryl);
5-ter t-Bu t yl-cycloSa l-3′-d eoxy-2′,3′-d id eh yd r ot h ym i-
d in e m on op h osp h a te 23: procedure A; yield 34%; H NMR
1

SAR of cycloSal-Pronucleotides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2847
(400 MHz, DMSO-d6) δ 11.37 (s, 1H, NH); 11.36 (s, 1H, NH);
7.41 (d, 1H, H4-aryl); 7.38 (d, 1H, H4-aryl); 7.32 (s, 2H, H6-
aryl); 7.22 (d, 1H, H6-thymine); 7.18 (d, 1H, H6-thymine); 7.07
(d, 1H, H3-aryl); 7.04 (d, 1H, H3-aryl); 6.82 (ddd, 1H, H1′);
6.81 (ddd, 1H, H1′); 6.44 (ddd, 1H, H3′); 6.38 (ddd, 1H, H3′);
6.03 (ddd, 1H, H2′); 6.02 (ddd, 1H, H2′); 5.52 (dd, 1H,
H-benzyl); 5.48 (dd, 1H, H-benzyl); 5.41 (d, 1H, H-benzyl); 5.38
(d, 1H, H-benzyl); 5.00-4.95 (m, 2H, H4′); 4.36-4.26 (m, 4H,
H5′); 1.69 (d, 3H, CH3-thymine); 1.61 (CH3-thymine); 1.28 (s,
9H, CH3-tBu); 1.27 (s, 9H, CH3-tBu); 13C NMR (101 MHz,
DMSO-d6) δ 163.9 (C4-thymine); 163.9 (C4-thymine); 150.9
(C2-thymine); 150.9 (C2-thymine); 147.5 (d, C2-aryl); 147.5 (d,
C2-aryl); 147.2 (C5-aryl); 135.9 (C6-thymine); 135.8 (C6-
thymine); 133.1 (C3′); 133.0 (C3′); 127.5 (C2′); 127.5 (C2′); 126.9
(C4-aryl); 126.8 (C4-aryl); 123.1 (C6-aryl); 123.0 (C6-aryl);
120.7 (d, C1-aryl); 120.6 (d, C1-aryl); 117.8 (d, C3-aryl); 117.7
(d, C3-aryl); 109.8 (C5-thymine); 89.3 (C1′); 84.3 (d, C4′); 84.3
(d, C4′); 68.8 (d, C5′); 68.7 (d, C5′); 68.5 (d, C-benzyl); 68.4 (d,
C-benzyl); 34.4 (C-tBu); 31.2 (CH3-tBu); 12.1 (CH3-thymine);
12.0 (CH3-thymine); 31P NMR (202 MHz, DMSO-d6) δ -7.84;
-7.86; Rf value 0.36 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd
471.1 (M + Na+), 487.1 (M + K+), found 471.4 (M + Na+), 487.3
(M + K+).
3,5-Bis-ter t-Bu t yl-cycloSa l-3′-d eoxy-2′,3′-d id eh yd r o-
th ym id in e m on op h osp h a te 24: procedure A; yield 50%; 1H
NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H, NH); 11.35 (s, 1H,
NH); 7.32 (s, 2H, H4-aryl); 7.23-7.21 (m, 4H, H6-aryl, H6-
thymine); 6.83 (ddd, 2H, H1′); 6.44 (ddd, 1H, H3′); 6.42 (ddd,
1H, H3′); 6.07-6.02 (m, 2H, H2′); 5.47-5.33 (m, 4H, H-benzyl);
5.01-4.96 (m, 2H, H4′); 4.41-4.29 (m, 4H, H5′); 1.58 (d, 3H,
CH3-thymine); 1.57 (d, 3H, CH3-thymine); 1.36 (s, 9H, CH3-
tBu); 1.33 (s, 9H, CH3-tBu); 1.28 (s, 18H, CH3-tBu); 13C NMR
(101 MHz, DMSO-d6) δ 163.9 (C4-thymine); 163.9 (C4-thy-
mine); 150.9 (C2-thymine); 150.9 (C2-thymine); 146.7 (C2-aryl);
146.6 (C2-aryl); 146.4 (C5-aryl); 137.7 (d, C3-aryl); 135.9 (C6-
thymine); 135.8 (C6-thymine); 133.1 (C3′); 133.0 (C3′); 127.5
(C2′); 127.5 (C2′); 123.9 (C6-aryl); 122.5 (d, C1-aryl); 121.5 (C4-
aryl); 109.9 (C5-thymine); 109.8 (C5-thymine); 89.3 (C1′); 89.2
(C1′); 84.4 (d, C4′); 84.3 (d, C4′); 68.9 (d, C5′); 68.9 (d, C5′);
68.7 (d, C-benzyl); 68.6 (d, C-benzyl); 34.6 (C-tBu); 34.6 (C-
tBu); 34.5 (C-tBu); 31.3 (CH3-tBu); 29.8 (CH3-tBu); 29.7 (CH3-
tBu); 11.9 (CH3-thymine); 11.9 (CH3-thymine); 31P NMR (202
MHz, DMSO-d6) δ -6.93; -7.23; Rf value 0.50 (CH2Cl2/MeOH,
9:1); MS (FAB, m/z) calcd 505.2104 (M + H+), found 505.2116
(M + H+).
3,5-Bis-ter t-Bu t yl-6-flu or o-cycloSa l-3′-d eoxy-2′,3′-d i-
d eh yd r oth ym id in e m on op h osp h a te 25: procedure A; yield
59%; 1H NMR (500 MHz, DMSO-d6) δ 11.37 (s, 1H, NH); 11.36
(s, 1H, NH); 7.26 (d, 2H, H4-aryl); 7.23 (d, 1H, H6-thymine);
7.22 (s, 1H, H6-thymine); 6.85-6.81 (m, 2H, H1′); 6.45-6.42
(m, 2H, H3′); 6.08-6.03 (m, 2H, H2′); 5.56 (dd, 1H, H-benzyl);
5.53 (dd, 1H, H-benzyl); 5.47 (dd, 1H, H-benzyl); 5.41 (dd, 1H,
H-benzyl); 5.02-4.98 (m, 2H, H4′); 4.37-4.35 (m, 4H, H5′);
1.61 (s, 6H, CH3-thymine); 1.35 (s, 9H, CH3-tBu); 1.34 (s, 18H,
CH3-tBu); 1.32 (s, 9H, CH3-tBu); 13C NMR (101 MHz, DMSO-
d6) δ 164.1 (C4-thymine); 164.1 (C4-thymine); 155.1 (d, C6-
aryl); 151.1 (C2-thymine); 147.3 (C2-aryl); 136.1 (C6-thymine);
133.7 (C5-aryl); 133.2 (C3′); 133.1 (C3′); 131.7 (C2′); 131.6 (C2′);
127.7 (d, C3-aryl); 125.2 (d, C4-aryl); 111.5 (dd, C1-aryl); 111.3
(dd, C1-aryl); 110.1 (C5-thymine); 110.0 (C5-thymine); 89.7
(C1′); 89.5 (C1′); 84.5 (d, C4′); 84.4 (d, C4′); 69.5 (d, C5′); 69.3
(d, C5′); 63.9 (C-benzyl); 34.7 (C-tBu); 34.6 (C-tBu); 34.3 (d,
C-tBu); 30.0 (d, CH3-tBu); 29.9 (CH3-tBu); 29.8 (CH3-tBu); 12.1
(CH3-thymine); 31P NMR (202 MHz, DMSO-d6) δ -8.71; -9.07;
19F NMR (471 MHz, DMSO-d6): δ -117.37 (d); -117.50 (d);
Rf value 0.50 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd
523.2009 (M + H+), found 523.2092 (M + H+).
4.91 (m, 4H, H4‘); 4.35-4.21 (m, 8H, H5‘); 3.00-2.88 (m, 4H,
CH-sBu); 1.61 (d, 6H, CH3-thymine); 1.60 (d, 6H, CH3-
thymine); 1.58-1.50 (m, 8H, CH2-sBu); 1.15 (d, 6H, CH-CH3-
sBu); 1.12 (d, 6H, CH-CH3-sBu); 0.77-0.71 (m, 12 H, CH2-
CH3-sBu); 13C NMR (101 MHz, DMSO-d6) δ 163.9 (C4-
thymine); 163.9 (C4-thymine); 150.9 (C2-thymine); 150.9 (C2-
thymine); 147.8 (C2-aryl); 147.8 (C2-aryl); 136.0 (C3-aryl);
135.9 (C3-aryl); 135.9 (C3-aryl); 133.1 (C3′); 133.0 (C3′); 133.0
(C3‘); 127.7 (C4-aryl); 127.6 (C4-aryl); 127.6 (C2′); 127.5 (C2‘);
124.5 (C6-aryl); 123.9 (C5-aryl); 122.1 (C1-aryl); 122.0 (C1-
aryl); 121.9 (C1-aryl); 109.9 (C5-thymine); 109.8 (C5-thymine);
89.4 (C1′); 89.2 (C1‘); 84.4 (C4′); 84.3 (C4′); 84.3 (C4′); 84.3 (C4‘);
68.9 (d, C5′); 68.9 (d, C5′); 68.7 (d, C5′); 68.6 (C5‘); 68.5 (C-
benzyl); 68.4 (C-benzyl); 68.4 (C-benzyl); 68.4 (C-benzyl); 33.4
(CH-sBu); 33.3 (CH-sBu); 33.2 (CH-sBu); 33.1 (CH-sBu); 29.4
(CH2-sBu); 29.4 (CH2-sBu); 29.1 (CH2-sBu); 28.9 (CH2-sBu);
20.7 (CH-CH3-sBu); 20.6 (CH-CH3-sBu); 20.3 (CH-CH3-sBu);
12.1 (CH2-CH3-sBu); 12.1 (CH2-CH3-sBu); 12.0 (CH3-thymine);
31P NMR (202 MHz, DMSO-d6) δ -6.80; -6.89; -7.21; -7.24;
Rf value 0.36 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd 471.1
(M + Na+), 487.1 (M + K+), found 471.4 (M + Na+), 487.3 (M
+ K+).
5-sek -Bu t yl-cycloSa l-3′-d eoxy-2′,3′-d id eh yd r ot h ym i-
d in e m on op h osp h a te 27: procedure A; yield 27% (four
diastereomers); 1H NMR (500 MHz, DMSO-d6) δ 11.34 (s, 4H,
NH); 7.22-7.19 (m, 4H, H4-aryl); 7.17 (s, 4H, H6-thymine);
7.12 (s, 4H, H6-aryl); 7.04 (dd, 4H, H3-aryl); 6.82-6.79 (m,
4H, H1‘); 6.43 (ddd, 2H, H3‘); 6.36 (ddd, 2H, H3‘); 6.02 (ddd,
2H, H2‘); 6.00 (ddd, 2H, H2‘); 5.51-5.35 (m, 8H, H-benzyl);
4.98-4.95 (m, 4H, H4‘); 4.35-4.26 (m, 8H, H5‘, H5‘); 2.62-
2.55 (m, 4H, CH-sBu); 1.67 (s, 6H, CH3-thymine); 1.60 (s, 6H,
CH3-thymine); 1.57-1.48 (m, 8H, CH2-sBu); 1.17 (d, 12H, CH-
CH3-sBu); 0.76 (t, 6H, CH2-CH3-sBu); 0.75 (t, 6H, CH2-CH3-
sBu); 13C NMR (126 MHz, DMSO-d6) δ 164.6 (C4-thymine);
164.6 (C4-thymine); 151.6 (C2-thymine); 151.6 (C2-thymine);
148.5 (d, C2-aryl); 148.4 (d, C2-aryl); 144.3 (C5-aryl); 136.6
(C6-thymine); 136.5 (C6-thymine); 133.7 (C3′); 133.7 (C3‘);
129.2 (C4-aryl); 129.2 (C4-aryl); 129.1 (C4-aryl); 128.2 (C2′);
128.2 (C2‘); 125.2 (C6-aryl); 125.2 (C6-aryl); 125.1 (C6-aryl);
125.1 (C6-aryl); 121.8 (d, C1-aryl); 121.7 (d, C1-aryl); 121.7
(d, C1-aryl); 118.7 (C3-aryl); 110.5 (C5-thymine); 110.5 (C5-
thymine); 90.0 (C1′); 90.0 (C1‘); 85.0 (C4′); 85.0 (C4‘); 69.3 (d,
C-benzyl); 69.3 (d, C-benzyl); 69.2 (d, C5′); 69.1 (d, C5‘); 41.0
(CH-sBu); 41.0 (CH-sBu); 31.4 (CH2-sBu); 31.3 (CH2-sBu); 31.3
(CH2-sBu); 31.3 (CH2-sBu); 22.5 (CH-CH3-sBu); 22.5 (CH-CH3-
sBu); 22.5 (CH-CH3-sBu); 22.5 (CH-CH3-sBu); 12.9 (CH2-CH3-
sBu); 12.8 (CH2-CH3-sBu); 12.8 (CH3-thymine); 12.7 (CH3-
thymine); 31P NMR (202 MHz, DMSO-d6) δ -7.77; -7.78;
-7.80; Rf value 0.36 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd
471.1 (M + Na+), 487.1 (M + K+), found 471.3 (M + Na+), 487.2
(M + K+).
3-(Me t h ylp r op ion yl)-cycloSa l-3′-d e oxy-2′,3′-d id e h y-
d r oth ym id in e m on op h osp h a te 28: procedure A; yield 32%;
1H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H, NH); 11.32 (s,
1H, NH); 7.26 (dd, 2H, H4-aryl); 7.20 (d, 1H, H6-thymine); 7.18
(d, 1H, H6-thymine); 7.16-7.08 (m, 4H, H5-aryl, H6-aryl); 6.79
(ddd, 1H, H1′); 6.79 (ddd, 1H, H1′); 6.40 (ddd, 1H, H3′); 6.36
(ddd, 1H, H3′); 6.02 (ddd, 1H, H2′); 6.00 (ddd, 1H, H2′); 5.48
(dd, 1H, H-benzyl); 5.44 (dd, 1H, H-benzyl); 5.38 (dd, 1H,
H-benzyl); 5.35 (dd, 1H, H-benzyl); 4.98-4.93 (m, 2H, H4′);
4.34-4.24 (m, 4H, H5′); 3.57 (s, 3H, OCH3); 3.56 (s, 3H, OCH3);
2.90-2.75 (m, 4H, CH2-propinoyl); 2.59 (t, 2H, CH2-propionyl);
2.57 (t, 2H, CH2-propionyl); 1.63 (d, 3H, CH3-thymine); 1.59
(d, 3H, CH3-thymine); 13C NMR (101 MHz, DMSO-d6) δ 172.5
(CO-propionyl); 163.9 (C4-thymine); 150.9 (C2-thymine); 150.8
(C2-thymine); 148.1 (d, C2-aryl); 147.9 (d, C2-aryl); 135.8 (C6-
thymine); 135.7 (C6-thymine); 133.0 (C3′); 132.9 (C3′); 130.3
(C4-aryl); 130.2 (C4-aryl); 129.6 (C1-aryl); 129.5 (C1-aryl);
127.6 (C2′); 127.5 (C2′); 124.5 (C6-aryl); 124.3 (C5-aryl); 121.7
(C3-aryl); 121.6 (C3-aryl); 109.9 (C5-thymine); 109.8 (C5-
thymine); 89.4 (C1′); 89.3 (C1′); 84.3 (C4′); 68.8 (d, C5′); 68.8
(d, C5′); 68.4 (d, C-benzyl); 51.5 (OCH3); 33.3 (CH2-propionyl);
33.2 (CH2-propionyl); 24.3 (CH2-propionyl); 24.2 (CH2-pro-
pionyl); 12.0 (CH3-thymine); 11.9 (CH3-thymine); 31P NMR (202
3-sek -Bu t yl-cycloSa l-3′-d eoxy-2′,3′-d id eh yd r ot h ym i-
d in e m on op h osp h a te 26: procedure A; yield 56% (four
diastereomers); 1H NMR (500 MHz, DMSO-d6) δ 11.34 (s, 2H,
NH); 11.31 (s, 2H, NH); 7.27 (d, 4H, H4-aryl); 7.22 (d, 2H, H6-
thymine); 7.18 (d, 2H, H6-thymine); 7.17-7.09 (m, 8H, H5-
aryl, H6-aryl); 6.82-6.77 (m, 4H, H1‘); 6.41-6.36 (m, 4H, H3‘);
6.03-6.00 (m, 4H, H2‘); 5.48-5.32 (m, 8H, H-benzyl); 4.96-

2848 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Meier et al.
MHz, DMSO-d6) δ -7.53; -7.60; Rf value 0.57 (CH2Cl2/MeOH,
9:1); MS (ESI+, m/z) calcd 479.1 (M + H+), found 478.9 (M +
H+).
value 0.49 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd 521.2 (M
+ H+), found 521.3 (M + H+).
3-(Be n zylp r op ion yl)-cycloSa l-3′-d e oxy-2′,3′-d id e h y-
d r oth ym id in e m on op h osp h a te 32: procedure A; yield 43%;
1H NMR (500 MHz, DMSO-d6) δ 11.33 (s, 1H, NH); 11.31 (s,
1H, NH); 7.36-7.28 (m, 10H, H-phenyl); 7.26-7.23 (m, 2H,
H4-aryl); 7.18 (d, 1H, H6-thymine); 7.17 (d, 1H, H6-thymine);
7.15-7.12 (m, 2H, H6-aryl); 7.09 (dd, 1H, H5-aryl); 7.08 (dd,
1H, H5-aryl); 6.79-6.77 (m, 2H, H1′); 6.37 (ddd, 1H, H3′); 6.32
(ddd, 1H, H3′); 5.99 (ddd, 1H, H2′); 5.97 (ddd, 1H, H2′); 5.47
(dd, 1H, H-benzyl); 5.44 (dd, 1H, H-benzyl); 5.37 (dd, 1H,
H-benzyl); 5.34 (dd, 1H, H-benzyl); 5.07 (s, 2H, OCH2); 5.06
(s, 2H, OCH2); 4.94-4.90 (m, 2H, H4′); 4.32-4.22 (m, 4H, H5′);
2.92-2.80 (m, 4H, CH2-propionyl); 2.68-2.62 (m, 4H, CH2-
propionyl); 1.64 (d, 3H, CH3-thymine); 1.58 (d, 3H, CH3-
thymine); 13C NMR (101 MHz, DMSO-d6) δ 172.4 (CO-
propionyl); 172.3 (CO-propionyl); 163.9 (C4-thymine); 150.8
(C2-thymine); 150.7 (C2-thymine); 148.0 (d, C2-aryl); 135.8
(C6-thymine); 135.7 (C6-thymine); 132.9 (C3′); 131.2 (C1-
phenyl); 130.3 (C4-aryl); 129.5 (C1-aryl); 128.6 (C3-phenyl, C5-
phenyl); 128.1 (C2-phenyl, C6-phenyl); 128.1 (C4-phenyl);
127.5 (C2′); 124.5 (C6-aryl); 124.3 (C5-aryl); 121.7 (C3-aryl);
109.9 (C5-thymine); 109.8 (C5-thymine); 89.4 (C1′); 89.3 (C1′);
84.3 (C4′); 84.2 (C4′); 68.7 (C5′); 68.4 (d, C-benzyl); 68.4 (d,
C-benzyl); 65.7 (OCH2); 33.4 (CH2-propionyl); 24.3 (CH2-
propionyl); 24.2 (CH2-propionyl); 12.0 (CH3-thymine); 11.9
(CH3-thymine); 31P NMR (202 MHz, DMSO-d6) δ -7.57; Rf
value 0.64 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd 555.1532
(M + H+), found 555.1532 (M + H+).
5-(Be n zylp r op ion yl)-cycloSa l-3′-d e oxy-2′,3′-d id e h y-
d r oth ym id in e m on op h osp h a te 33: procedure A; yield 59%;
1H NMR (500 MHz, DMSO-d6) δ 11.33 (s, 1H, NH); 11.32 (s,
1H, NH); 7.36-7.26 (m, 10H, H-phenyl); 7.23-7.18 (m, 2H,
H4-aryl); 7.18 (d, 1H, H6-thymine); 7.15 (d, 1H, H6-thymine);
7.11-7.09 (m, 2H, H6-aryl); 7.01 (d, 1H, H3-aryl); 6.99 (d, 1H,
H3-aryl); 6.79 (ddd, 1H, H1′); 6.77 (ddd, 1H, H1′); 6.40 (ddd,
1H, H3′); 6.34 (ddd, 1H, H3′); 6.01 (ddd, 1H, H2′); 5.99 (ddd,
1H, H2′); 5.41 (dd, 1H, H-benzyl); 5.38 (dd, 1H, H-benzyl); 5.33
(d, 1H, H-benzyl); 5.30 (d, 1H, H-benzyl); 5.06 (s, 4H, OCH2);
4.96-4.92 (m, 2H, H4′); 4.34-4.22 (m, 4H, H5′); 2.83 (t, 4H,
CH2-propionyl); 2.67 (t, 4H, CH2-propionyl); 1.66 (d, 3H, CH3-
thymine); 1.60 (d, 3H, CH3-thymine); 13C NMR (101 MHz,
DMSO-d6) δ 172.1 (CO-propionyl); 171.9 (CO-propionyl); 163.9
(C4-thymine); 150.9 (C2-thymine); 148.1 (C2-aryl); 136.9 (C1-
phenyl); 136.3 (C5-aryl); 135.9 (C6); 135.8 (C6); 133.0 (C3′);
129.9 (C4-aryl); 129.8 (C4-aryl); 128.6 (C3-phenyl, C5-phenyl);
128.5 (C1-aryl); 128.2 (C4-phenyl); 128.1 (C1-phenyl, C6-
phenyl); 127.5 (C2′); 126.0 (C6-aryl); 125.9 (C6-aryl); 118.2 (C3-
aryl); 118.1 (C3-aryl); 109.8 (C5-thymine); 89.3 (C1′); 84.3 (C4′);
84.2 (C4′); 68.5 (d, C5′); 68.4 (d, C5‘); 68.4 (d, C-benzyl); 68.3
(d, C-benzyl); 65.6 (OCH2); 35.0 (CH2-propionyl); 29.6 (CH2-
propionyl); 12.1 (CH3-thymine); 12.0 (CH3-thymine); 31P NMR
(202 MHz, DMSO-d6) δ -7.98; -8.05; Rf value 0.63 (CH2Cl2/
MeOH, 9:1); MS (FAB, m/z) calcd 555.2 (M + H+), found 555.2
(M + H+).
5-(Me t h ylp r op ion yl)-cycloSa l-3′-d e oxy-2′,3′-d id e h y-
d r oth ym id in e m on op h osp h a te 29: procedure A; yield 39%;
1H NMR (500 MHz, DMSO-d6) δ 11.33 (s, 1H, NH); 11.32 (s,
1H, NH); 7.23-7.18 (m, 2H, H4-aryl); 7.18 (d, 1H, H6-
thymine); 7.15 (d, 1H, H6-thymine); 7.13-7.11 (m, 2H, H6-
aryl); 7.03 (d, 1H, H3-aryl); 7.00 (d, 1H, H3-aryl); 6.79 (ddd,
1H, H1′); 6.78 (ddd, 1H, H1′); 6.40 (ddd, 1H, H3′); 6.34 (ddd,
1H, H3′); 6.01 (ddd, 1H, H2′); 5.99 (ddd, 1H, H2′); 5.45 (dd,
1H, H-benzyl); 5.41 (dd, 1H, H-benzyl); 5.35 (d, 1H, H-benzyl);
5.33 (d, 1H, H-benzyl); 4.96-4.91 (m, 2H, H4′); 4.34-4.22 (m,
4H, H5′); 3.57 (s, 6H, OCH3); 2.81 (t, 4H, CH2-propionyl); 2.60
(t, 4H, CH2-propionyl); 1.66 (d, 3H, CH3-thymine); 1.59 (d, 3H,
CH3-thymine); 13C NMR (101 MHz, DMSO-d6) δ 172.6 (CO-
propionyl); 163.9 (C4-thymine); 150.8 (C2-thymine); 148.0 (d,
C2-aryl); 137.0 (C5-aryl); 135.8 (C6-thymine); 133.0 (C3′); 132.9
(C3′); 129.8 (C4-aryl); 129.7 (C4-aryl); 129.0 (C1-aryl); 128.9
(C1-aryl); 127.5 (C2′); 127.4 (C2′); 125.9 (C6-aryl); 118.2 (C3-
aryl); 118.1 (C3-aryl); 109.8 (C5-thymine); 89.3 (C1′); 84.3 (C4′);
84.2 (C4′); 68.5 (d, C5‘); 68.5 (d, C5‘); 68.4 (d, C-benzyl); 51.5
(OCH3); 34.8 (CH2-propionyl); 29.5 (CH2-propionyl); 12.1 (CH3-
thymine); 12.0 (CH3-thymine); 31P NMR (202 MHz, DMSO-
d6) δ -7.98; -8.04; Rf value 0.52 (CH2Cl2/MeOH, 9:1); MS
(FAB, m/z) calcd 479.1 (M + H+), found 479.4 (M + H+).
3-(ter t-Bu tylp r op ion yl)-cycloSa l-3′-d eoxy-2′,3′-d id eh y-
d r oth ym id in e m on op h osp h a te 30: procedure A; yield 73%;
1H NMR (500 MHz, DMSO-d6) δ 11.32 (s, 1H, NH); 11.31 (s,
1H, NH); 7.27-7.24 (m, 2H, H4-aryl); 7.20 (d, 1H, H6-
thymine); 7.18 (d, 1H, H6-thymine); 7.15-7.08 (m, 4H, H5-
aryl, H6-aryl); 6.80-6.78 (m, 2H, H1′); 6.40 (ddd, 1H, H3′);
6.36 (ddd, 1H, H3′); 6.02 (ddd, 1H, H2′); 6.00 (ddd, 1H, H2′);
5.47 (dd, 1H, H-benzyl); 5.44 (dd, 1H, H-benzyl); 5.37 (dd, 1H,
H-benzyl); 5.35 (dd, 1H, H-benzyl); 4.97-4.93 (m, 2H, H4′);
4.34-4.25 (m, 4H, H5′); 2.87-2.73 (m, 4H, CH2-propionyl); 2.47
(t, 2H, CH2-propionyl); 2.46 (t, 2H, CH2-propionyl); 1.64 (d, 3H,
CH3-thymine); 1.57 (d, 3H, CH3-thymine); 1.35 (s, 9H, CH3-
tBu); 1.34 (s, 9H, CH3-tBu); 13C NMR (101 MHz, DMSO-d6) δ
171.4 (CO-propionyl); 171.3 (CO-propionyl); 163.9 (C4-thy-
mine); 150.8 (C2-thymine); 148.0 (d, C2-aryl); 148.0 (d, C2-
aryl); 135.8 (C6-thymine); 135.7 (C6-thymine); 133.0 (C3′);
132.9 (C3′); 130.3 (C4-aryl); 129.7 (C1-aryl); 129.6 (C1-aryl);
127.5 (C2′); 124.4 (C6-aryl); 124.2 (C5-aryl); 121.7 (C3-aryl);
121.6 (C3-aryl); 109.8 (C5-thymine); 89.3 (C1′); 84.3 (C4′); 80.0
(OC-tBu); 68.8 (d, C5‘); 68.7 (d, C5′); 68.4 (d, C-benzyl); 68.4
(d, C-benzyl); 34.6 (CH2-propionyl); 34.5 (CH2-propionyl); 27.9
(CH3-tBu); 24.4 (CH2-propionyl); 12.0 (CH3-thymine); 11.9
(CH3-thymine); 31P NMR (202 MHz, DMSO-d6) δ -7.50; -7.53;
Rf value 0.46 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd 521.2
(M + H+), found 521.2 (M + H+).
5-(ter t-Bu tylp r op ion yl)-cycloSa l-3′-d eoxy-2′,3′-d id eh y-
d r oth ym id in e m on op h osp h a te 31: procedure A; yield 44%;
1H NMR (500 MHz, DMSO-d6) δ 11.33 (s, 1H, NH); 11.32 (s,
1H, NH); 7.23-7.18 (m, 2H, H4-aryl); 7.18 (d, 1H, H6-
thymine); 7.15 (d, 1H, H6-thymine); 7.12 (d, 2H, H6-aryl); 7.03
(d, 1H, H3-aryl); 7.01 (d, 1H, H3-aryl); 6.79 (ddd, 1H, H1′);
6.77 (ddd, 1H, H1′); 6.40 (ddd, 1H, H3′); 6.34 (ddd, 1H, H3′);
6.00 (ddd, 1H, H2′); 5.99 (ddd, 1H, H2′); 5.44 (dd, 1H,
H-benzyl); 5.41 (dd, 1H, H-benzyl); 5.35 (dd, 1H, H-benzyl);
5.32 (dd, 1H, H-benzyl); 4.96-4.92 (m, 2H, H4′); 4.33-4.22 (m,
4H, H5′); 2.77 (dd, 4H, CH2-propionyl); 2.48 (dd, 4H, CH2-
propionyl); 1.67 (d, 3H, CH3-thymine); 1.60 (d, 3H, CH3-
thymine); 1.34 (s, 9H, CH3-tBu); 1.33 (s, 9H, CH3-tBu); 13C
NMR (101 MHz, DMSO-d6) δ 171.5 (CO-propionyl); 163.9 (C4-
thymine); 163.8 (C4-thymine); 150.8 (C2-thymine); 148.0 (C2-
aryl); 137.1 (C5-aryl); 135.8 (C6-thymine); 133.0 (C3′); 132.9
(C3′); 132.2 (C1-aryl); 129.8 (C4-aryl); 127.5 (C2′); 127.4 (C2′);
126.0 (C6-aryl); 125.9 (C6-aryl); 118.1 (C3-aryl); 118.0 (C3-
aryl); 109.8 (C5-thymine); 89.3 (C1′); 84.3 (C4′); 84.2 (C4′); 79.9
(OC-tBu); 68.5-68.3 (m, C5′, C-benzyl); 36.2 (CH2-propionyl);
29.8 (CH2-propionyl); 27.9 (CH3-tBu); 12.1 (CH3-thymine); 12.0
(CH3-thymine); 31P NMR (202 MHz, DMSO-d6) δ -8.02; Rf
3-(2-Ca r boxyet h yl)-cycloSa l-3′-d eoxy-2′,3′-d id eh yd r o-
th ym id in e Mon op h osp h a te 34. The title compound was
synthesized by treatment of ester 30 (15 mg) with trifluoro-
acetic acid (2.4 mL) in dichloromethane (15 mL) for 1 h at room
temperature. Afterward, the solvent was removed in vacuo and
the residue was purified by preparative TLC (Chromatotron,
dichloromethane/methanol gradient). Subsequent lyophiliza-
1
tion yielded the product (12 mg, 71%) as a colorless foam; H
NMR (500 MHz, DMSO-d6) δ 12.17 (s, 2H, COOH); 11.31 (s,
2H, NH); 7.27-7.25 (m, 2H, H4-aryl); 7.19 (d, 2H, H6-
thymine); 7.14-7.09 (m, 4H, H5-aryl, H6-aryl); 6.80-6.78 (m,
2H, H1′); 6.40 (ddd, 1H, H3′); 6.35 (ddd, 1H, H3′); 6.02 (ddd,
1H, H2′); 6.00 (ddd, 1H, H2′); 5.47 (dd, 1H, H-benzyl); 5.44
(dd, 1H, H-benzyl); 5.37 (dd, 1H, H-benzyl); 5.34 (dd, 1H,
H-benzyl); 4.96-4.92 (m, 2H, H4′); 4.34-4.24 (m, 4H, H5′);
2.85-2.75 (m, 4H, CH2-ethyl); 2.51 (t, 2H, CH2-ethyl); 2.50 (t,
2H, CH2-ethyl); 1.65 (d, 3H, CH3-thymine); 1.58 (d, 3H, CH3-
thymine); 13C NMR (101 MHz, DMSO-d6) δ 173.6 (COOH);
163.9 (C4-thymine); 150.9 (C2-thymine); 150.8 (C2-thymine);

SAR of cycloSal-Pronucleotides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2849
147.9 (d, C2-aryl); 147.9 (d, C2-aryl); 135.8 (C6-thymine); 135.7
(C6-thymine); 133.0 (C3′); 132.9 (C3′); 130.2 (C4-aryl); 130.0
(C1-aryl); 129.9 (C1-aryl); 127.5 (C2′); 124.4 (C6-aryl); 124.3
(C5-aryl); 121.7 (C3-aryl); 121.6 (C3-aryl); 109.9 (C5-thymine);
109.8 (C5-thymine); 89.4 (C1′); 89.3 (C1′); 84.3 (C4′); 68.7 (d,
C5‘); 68.7 (d, C5′); 68.4 (d, C-benzyl); 68.4 (d, C-benzyl); 33.5
(CH2-ethyl); 24.2 (CH2-ethyl); 12.0 (CH3-thymine); 11.9 (CH3-
thymine); 31P NMR (202 MHz, DMSO-d6) δ -7.51; -7.56; Rf
0.16 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd 464.1 (M), found
464.2 (M).
(CH3-benzyl); 12.1 (CH3-thymine); 31P NMR (202 MHz, DMSO-
d6) δ -9.18; -9.21; -9.50; -9.62; Rf value 0.36 (CH2Cl2/MeOH,
9:1); MS (FAB, m/z) calcd 441.062 (M + H+), found 441.062
(M + H+).
6-Ch lor o-7-b u t yl-cycloSa l-3′-d e oxy-2′,3′-d id e h yd r o-
th ym id in e m on op h osp h a te 43: procedure A; yield 83% (four
diastereomers); 1H NMR (500 MHz, DMSO-d6) δ 11.36 (s, 1H,
NH); 11.35 (s, 1H, NH); 11.34 (s, 1H, NH); 11.31 (s, 1H, NH);
7.43 (m, 4H, H4-aryl); 7.35 (m, 4H, H5-aryl); 7.22 (d, 1H, H6-
thymine); 7.21-7.15 (m, 4H, H3-aryl); 7.16 (d, 1H, H6-
thymine); 7.14 (d, 1H, H6-thymine); 7.13 (d, 1H, H6-thymine);
6.82 (m, 1H, H1′); 6.80 (m, 1H, H1′); 6.77 (m, 2H, H1′); 6.46
(ddd, 1H, H3′); 6.42 (ddd, 1H, H3′); 6.35 (ddd, 1H, H3′); 6.31
(ddd, 1H, H3′); 6.05 (m, 2H, H2′); 5.99 (m, 1H, H2′); 5.56 (m,
1H, H2′); 5.71-5.57 (m, 4H, H-benzyl); 5.00 (m, 2H, H4′); 4.87
(m, 2H, H4′); 4.47-4.40 (m, 2H, H5′); 4.37-4.29 (m, 2H, H5′);
4.23-4.14 (m, 4H, H5′); 2.03-1.91 (m, 4H, CH2-butyl); 1.89-
1.65 (m, 4H, CH2-butyl); 1.68 (d, 3H, CH3-thymine); 1.65 (d,
3H, CH3-thymine); 1.63 (d, 3H, CH3-thymine); 1.41 (d, 3H,
CH3-thymine); 1.55-1.21 (m, 16H, CH2-butyl, CH2-butyl); 0.88
(t, 3H, CH3-butyl); 0.87 (t, 3H, CH3-butyl); 0.85 (t, 3H, CH3-
butyl); 0.82 (t, 3H, CH3-butyl); 13C NMR (101 MHz, DMSO-
d6) δ 163.9 (C4-thymine); 163.8 (C4-thymine); 163.8 (C4-
thymine); 150.9 (C2-thymine); 150.8 (C2-thymine); 150.8 (C2-
thymine); 150.1 (d, C2-aryl); 150.1 (d, C2-aryl); 149.6 (d, C2-
aryl); 136.0 (C6-thymine); 135.9 (C6-thymine); 133.2 (C3′);
132.9 (C3′); 131.2 (C4-aryl); 131.2 (C4-aryl); 131.1 (C4-aryl);
130.3 (C6-aryl); 130.2 (C6-aryl); 127.5 (C2′); 127.5 (C2′); 126.0
(C5-aryl); 126.0 (C5-aryl); 120.8 (C1-aryl); 120.8 (C1-aryl);
120.7 (C1-aryl); 120.7 (C1-aryl); 118.3 (C3-aryl); 118.2 (C3-
aryl); 118.2 (C3-aryl); 118.1 (C3-aryl); 109.9 (C5-thymine);
109.7 (C5-thymine); 109.5 (C5-thymine); 89.3 (C1′); 89.2 (C1′);
89.2 (C1′); 84.4 (C4′); 84.3 (C4′); 84.2 (C4′); 84.2 (C4′); 80.1 (d,
C-benzyl); 80.0 (d, C-benzyl); 79.7 (d, C-benzyl); 69.5 (d, C5′);
68.9 (d, C5′); 68.8 (C5′); 68.7 (d, C5′); 34.7 (CH2-butyl); 34.0
(CH2-butyl); 33.7 (CH2-butyl); 27.1 (CH2-butyl); 26.9 (CH2-
butyl); 26.7 (CH2-butyl); 21.5 (CH2-butyl); 21.4 (CH2-butyl);
21.4 (CH2-butyl); 13.8 (CH3-butyl); 13.8 (CH3-butyl); 13.7 (CH3-
butyl); 12.1 (CH3-thymine); 12.0 (CH3-thymine); 12.0 (CH3-
thymine); 11.8 (CH3-thymine); 31P NMR: (202 MHz, DMSO-
d6) δ -8.47; -8.50; -9.59; Rf value 0.59 (CH2Cl2/MeOH, 9:1);
MS (FAB, m/z) calcd 483.109 (M + H+), found 483.107 (M +
H+).
5-(2-Ca r boxyet h yl)-cycloSa l-3′-d eoxy-2′,3′-d id eh yd r o-
th ym id in e Mon op h osp h a te 35. The title compound was
synthesized by treatment of ester 31 (14 mg) with trifluoro-
acetic acid (2.2 mL) in dichloromethane (15 mL) for 1 h at room
temperature. Afterward, the solvent was removed in vacuo and
the residue was purified by preparative TLC (Chromatotron,
dichloromethane/methanol gradient). Subsequent lyophiliza-
1
tion yielded the product (13 mg, 93%) as a colorless foam; H
NMR (500 MHz, DMSO-d6) δ 12.08 (s, 2H, COOH); 11.33 (s,
1H, NH); 11.32 (s, 1H, NH); 7.24-7.19 (m, 2H, H4-aryl); 7.18
(d, 1H, H6-thymine); 7.15 (d, 1H, H6-thymine); 7.13-7.11 (m,
2H, H6-aryl); 7.03 (d, 1H, H3-aryl); 7.01 (d, 1H, H3-aryl); 6.79
(ddd, 1H, H1′); 6.77 (ddd, 1H, H1′); 6.40 (ddd, 1H, H3′); 6.33
(ddd, 1H, H3′); 6.01 (ddd, 1H, H2′); 5.99 (ddd, 1H, H2′); 5.45
(dd, 1H, H-benzyl); 5.41 (dd, 1H, H-benzyl); 5.36 (d, 1H,
H-benzyl); 5.33 (dd, 1H, H-benzyl); 4.96-4.92 (m, 2H, H4′);
4.33-4.22 (m, 4H, H5′); 2.78 (t, 4H, CH2-ethyl); 2.51 (t, 4H,
CH2-ethyl); 1.67 (d, 3H, CH3-thymine); 1.61 (d, 3H, CH3-
thymine); 13C NMR (101 MHz, DMSO-d6) δ 173.7 (COOH);
163.9 (C4-thymine); 150.9 (C2-thymine); 150.8 (C2-thymine);
146.5 (d, C2-aryl); 146.2 (d, C2-aryl); 137.4 (C5-aryl); 135.8
(C6-thymine); 133.0 (C3′); 132.9 (C3′); 129.8 (C4-aryl); 129.7
(C4-aryl); 128.7 (C1-aryl); 127.5 (C2′); 127.4 (C2′); 125.9 (C6-
aryl); 125.8 (C6-aryl); 118.1 (C3-aryl); 109.8 (C5-thymine); 89.3
(C1′); 84.3 (C4′); 84.2 (C4′); 68.5 (d, C5‘); 68.5 (d, C5′); 68.4 (d,
C-benzyl); 35.1 (CH2-ethyl); 29.6 (CH2-ethyl); 12.1 (CH3-
thymine); 12.0 (CH3-thymine); 31P NMR (202 MHz, DMSO-
d6) δ -8.01; -8.06; Rf value 0.16 (CH2Cl2/MeOH, 9:1); MS
(FAB, m/z) calcd 464.1 (M), found 464.3 (M).
6-Ch lor o-cycloSa l-3′-d eoxy-2′,3′-d id eh yd r ot h ym id in e
1
m on op h osp h a te 41: procedure A; yield 51%; H NMR (400
MHz, DMSO-d6) δ 11.28 (s, 1H, NH); 11.26 (s, 1H, NH); 7.32
(m, 2H, H4-aryl); 7.24 (m, 2H, H5-aryl); 7.13-7.00 (m, 4H, H3-
aryl, H6-thymine); 6.71 (m, 1H, H1′); 6.68 (m, 1H, H1′); 6.33
(ddd, 1H, H3′); 6.27 (ddd, 1H, H3′); 5.93 (m, 2H, H2′); 5.39
(m, 4H, H-benzyl); 4.87 (m, 2H, H4′); 4.24 (m, 4H, H5′); 1.62
(d, 3H, CH3-thymine); 1.56 (d, 3H, CH3-thymine); 13C NMR
(126 MHz, DMSO-d6) δ 164.5 (C4-thymine); 164.1 (C4-thy-
mine); 151.8 (C2-thymine); 151.1 (C2-aryl); 136.1 (C6-thymine);
136.0 (C6-thymine); 134.0 (C6-aryl); 133.1 (C3′); 133.0 (C3′);
131.2 (C4-aryl); 131.1 (C4-aryl); 128.5 (C1-aryl); 127.7 (C2′);
125.7 (C5-aryl); 117.9 (C3-aryl); 108.1 (C5-thymine); 89.7 (C1′);
89.6 (C1′); 84.4 (C4′); 84.4 (C4′); 69.0 (C-benzyl); 67.1 (C5′);
67.0 (C5′); 12.3 (CH3-thymine); 12.2 (CH3-thymine); 31P NMR
(202 MHz, DMSO-d6) δ -9.74; -9.77; Rf value 0.68 (CH2Cl2/
MeOH, 9:1); MS (FAB, m/z) calcd 427.046 (M + H+), found
427.043 (M + H+).
6-Ch lor o-7-m et h yl-cycloSa l-3′-d eoxy-2′,3′-d id eh yd r o-
th ym id in e m on op h osp h a te 42: procedure A; yield 50% (four
diastereomers); 1H NMR (500 MHz, DMSO-d6) δ 11.30 (s, 4H,
NH); 7.38 (m, 8H, H4-aryl, H5-aryl); 7.23 (d, 1H, H6-thymine);
7.19 (dd, 1H, H3-aryl); 7.18 (d, 1H, H6-thymine); 7.18 (dd, 2H,
H3-aryl); 7.16 (dd, 1H, H3-aryl); 7.13 (d, 2H, H6-thymine); 6.79
(m, 4H, H1′); 6.44 (ddd, 2H, H3′); 6.35 (ddd, 1H, H3′); 6.33
(ddd, 1H, H3′); 6.05 (ddd, 1H, H2′); 6.03 (ddd, 1H, H2′); 5.98
(ddd, 1H, H2′); 5.96 (ddd, 1H, H2′); 5.83 (m, 4H, H-benzyl);
4.90 (m, 4H, H4′); 4.30 (m, 8H, H5′); 1.67 (d, 6H, CH3-thymine);
1.67 (d, 6H, CH3-benzyl); 1.65 (d, 3H, CH3-benzyl); 1.64 (d, 3H,
CH3-thymine); 1.62 (d, 3H, CH3-benzyl); 1.52 (d, 3H, CH3-
thymine); 13C NMR (101 MHz, DMSO-d6) δ 164.0 (C4-thy-
mine); 157.4 (C2-aryl); 151.0 (C2-thymine); 136.5 (C6-thymine);
133.9 (C4-aryl); 130.9 (C6-aryl); 129.0 (C3′); 127.2 (C1-aryl);
126.9 (C2′); 120.3 (C5-aryl); 115.8 (C3-aryl); 109.8 (C5-thy-
mine); 89.0 (C1′); 85.0 (C4′); 67.1 (C-benzyl); 65.9 (C5′); 22.3
6-Ch lor o-7-(eth oxyca r bon ylm eth yl)-cycloSa l-3′-d eoxy-
2′,3′-d id eh yd r oth ym id in e m on op h osp h a te 44: procedure
A; yield 49% (four diastereomers); 1H NMR (500 MHz, DMSO-
d6) δ 11.36 (s, 1H, NH); 11.35 (s, 1H, NH); 11.34 (s, 1H, NH);
11.31 (s, 1H, NH); 7.49-7.43 (m, 4H, H4-aryl); 7.40-7.36 (m,
4H, H5-aryl); 7.22 (d, 1H, H6-thymine); 7.21 (d, 1H, H6-
thymine); 7.19 (d, 1H, H6-thymine); 7.18 (d, 1H, H6-thymine);
7.17 (m, 2H, H3-aryl); 7.12 (m, 2H, H3-aryl); 6.82 (m, 2H, H1′);
6.76 (m, 2H, H1′); 6.44 (m, 2H, H3′); 6.33 (ddd, 1H, H3′); 6.26
(ddd, 1H, H3′); 6.08-5.94 (m, 8H, H-benzyl, H2′); 5.00 (m, 2H,
H4′); 4.87 (m, 2H, H4′); 4.43-4.29 (m, 4H, H5′); 4.21-4.09 (m,
4H, H5′); 4.18 (q, 2H, OCH2); 4.13 (q, 2H, OCH2); 3.98 (q, 2H,
OCH2); 3.96 (q, 2H, OCH2); 3.05-2.93 (m, 8H, CH2-ECM); 1.70
(d, 3H, CH3-thymine); 1.68 (d, 3H, CH3-thymine); 1.67 (d, 3H,
CH3-thymine); 1.53 (d, 3H, CH3-thymine); 1.20 (t, 3H, CH3-
ECM); 1.19 (t, 3H, CH3-ECM); 1.18 (t, 3H, CH3-ECM); 13C
NMR (126 MHz, DMSO-d6) δ 168.6 (CO-ECM); 168.6 (CO-
ECM); 168.4 (CO-ECM); 168.4 (CO-ECM); 163.9 (C4-thymine);
163.9 (C4-thymine); 163.9 (C4-thymine); 150.9 (C2-thymine);
150.9 (C2-thymine); 150.8 (C2-thymine); 150.2 (d, C2-aryl);
150.2 (d, C2-aryl); 149.6 (d, C2-aryl); 135.9 (C6-thymine); 135.8
(C6-thymine); 135.8 (C6-thymine); 133.0 (C3′); 132.9 (C3′);
132.8 (C3′); 132.8 (C3′); 131.7 (C4-aryl); 131.7 (C4-aryl); 131.7
(C4-aryl); 131.6 (C4-aryl); 130.4 (C6-aryl); 130.3 (C6-aryl);
130.3 (C6-aryl); 127.6 (C2′); 127.6 (C2′); 127.5 (C2′); 127.5 (C2′);
126.3 (C5-aryl); 126.2 (C5-aryl); 126.2 (C5-aryl); 122.0 (d, C1-
aryl); 121.4 (d, C1-aryl); 121.3 (d, C1-aryl); 118.4 (C3-aryl);
118.3 (C3-aryl); 118.2 (C3-aryl); 118.1 (C3-aryl); 109.9 (C5-
thymine); 109.8 (C5-thymine); 109.8 (C5-thymine); 89.5 (C1′);
89.3 (C1′); 89.3 (C1′); 89.2 (C1′); 84.2 (C4′); 84.1 (C4′); 84.1
(C4′); 76.5 (d, C-benzyl); 76.3 (d, C-benzyl); 76.2 (d, C-benzyl);

2850 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Meier et al.
76.2 (d, C-benzyl); 69.5 (d, C5′); 69.1 (d, C5′); 69.1 (d, C5′); 69.0
(d, C5′); 61.1 (OCH2); 61.0 (OCH2); 61.0 (OCH2); 61.0 (OCH2);
38.8 (CH2-ECM); 14.1 (CH3-ECM); 12.2 (CH3-thymine); 12.0
(CH3-thymine); 12.0 (CH3-thymine); 11.9 (CH3-thymine); 31P
NMR (202 MHz, DMSO-d6) δ -9.90; -9.95; -10.46; -10.49;
Rf value 0.48 (CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd
513.083 (M + H+), found 513.080 (M + H+).
(C2′); 124.9 (C5-aryl); 124.8 (C5-aryl); 119.8 (C3-aryl); 119.8
(C3-aryl); 109.9 (C5-thymine); 109.8 (C5-thymine); 99.4 (CCl3);
89.3 (C1′); 86.8 (C-benzyl); 86.8 (C-benzyl); 84.1 (C4′); 70.1
(C5′); 70.0 (C5′); 12.1 (CH3-thymine); 31P NMR (202 MHz,
DMSO-d6) δ -9.55; -11.06; -11.32; Rf value 0.46 (CH2Cl2/
MeOH, 9:1); MS (FAB, m/z) calcd 508.984 (M + H+), found
509.975 (M + H+).
3-P h en yl-cycloSa l-(E)-5-(2-b r om ovin yl)-2′-d eoxyu r i-
d in e m on op h osp h a te 48: procedure B; yield 57%; H NMR
7-(Mon och lor om eth yl)-cycloSa l-3′-d eoxy-2′,3′-d id eh y-
d r oth ym id in e m on op h osp h a te 45: procedure A; yield 11%
(four diastereomers); H NMR (500 MHz, DMSO-d6) δ 11.34
1
1
(500 MHz, DMSO-d6) δ 11.60 (s, 2H, NH); 7.77 (s, 1H, H6-
BVU); 7.76 (s, 1H, H6-BVU); 7.50-7.26 (m, 18H, H-aryl, H8-
BVU); 6.85 (d, 1H, H7-BVU); 6.82 (d, 1H, H7-BVU); 6.17 (dd,
1H, H1′); 6.14 (dd, 1H, H1′); 5.60-5.51 (m, 4H, H-benzyl); 5.47
(d, 2H, OH); 4.37-4.27 (m, 4H, H5′); 4.23-4.22 (m, 2H, H3′);
3.96-3.91 (m, 2H, H4′); 2.20-2.09 (m, 4H, H2′); 13C NMR (101
MHz, DMSO-d6) δ 161.7 (C4-BVU); 149.3 (C2-BVU); 149.3
(C2-BVU); 139.3 (C6-BVU); 135.6 (C2-aryl); 131.0 (C4-aryl);
131.0 (C1′-aryl); 130.9 (C1′-aryl); 129.9 (C7-BVU); 129.2 (C3′-
aryl, C5′-aryl); 128.6 (C2′-aryl, C6′-aryl); 128.5 (C2′-aryl, C6′-
aryl); 128.0 (C6-aryl); 127.9 (C6-aryl); 125.7 (C4′-aryl); 124.8
(C5-aryl); 124.8 (C5-aryl); 122.4 (C1-aryl); 121.5 (C3-aryl);
110.3 (C5-BVU); 110.2 (C5-BVU); 107.1 (C8-BVU); 107.1
(C8-BVU); 84.8 (C1′); 84.8 (C1′); 84.7 (C4′); 84.5 (d, C4′); 69.8
(C3′); 69.7 (C3′); 68.6 (d, C5′); 68.2 (d, C-benzyl); 40.0 (C2′);
31P NMR (202 MHz, DMSO-d6) δ -7.51; -7.54; Rf value 0.45
(CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd 577.0375 (M + H+),
found 577.0403 (M + H+).
2,4-Bis-ter t-b u t yl-5-flu or op h en ol. Isobutene gas was
bubbled through 3-fluorophenol (4.10 g) for 5 min at 40 °C.
Afterward, concentrated sulfuric acid (0.34 g) was added and
the inflow of isobutene was continued for 1 h. After the
addition of water, dichloromethane was added and the phases
were separated. The organic phase was neutralized with sat.
sodium bicarbonate solution and dried (Na2SO4). The solvent
was removed in vacuo, and the resulting residue was purified
by column chromatography (petroleum ether/CH2Cl2, 4:1)
yielding the title compound (5.73 g, 70%) as a colorless solid;
1H NMR (500 MHz, CDCl3) δ 7.19 (d, 1H, H5); 6.39 (d, 1H,
H2); 4.76 (s, 1H, OH); 1.42 (s, 9H, CH3-tBu); 1.37 (s, 9H, CH3-
tBu); 13C NMR (101 MHz, CDCl3) δ 159.9 (d, C3); 152.7 (d,
C1); 130.8 (d, C6); 128.1 (d, C4); 125.4 (d, C5); 104.9 (d, C2);
34.4 (C-tBu); 33.9 (d, C-tBu); 30.2 (d, CH3-tBu); 29.8 (CH3-tBu);
19F NMR (471 MHz, CDCl3) δ -113.34 (dd); Rf value 0.14
(petroleum ether/CH2Cl2, 4:1); MS (FAB, m/z) calcd 224.1576
(M), found 224.1559 (M).
3,5-Bis-ter t-bu tyl-6-flu or osa licyl Alcoh ol. 2,4-Bis-tert-
butyl-5-fluorophenol (5.55 g) and sodium hydroxide (1.18 g)
were dissolved in methanol (15 mL). To this solution was added
an aqueous formaldehyde solution (37%, 5.9 mL), and the
resulting mixture was stirred at room temperature for 3 d.
The reaction was monitored by TLC (CH2Cl2/MeOH, 9:1) and
finally quenched by the addition of water and concentrated
hydrochloric acid (resulting pH 4-5). The aqueous mixture
was extracted five times with dichloromethane. The combined
organic layers were dried (Na2SO4), and the solvent was
removed in vacuo. The resulting crude product was recrystal-
lized from petroleum ether to yield the title compound (4.49
g, 71%) as a colorless solid; 1H NMR (400 MHz, CDCl3) δ 7.92
(s, 1H, OH); 7.14 (d, 1H, H4); 5.02 (s, 2H, H-benzyl); 1.41 (s,
9H, CH3-tBu); 1.34 (d, 9H, CH3-tBu); 13C NMR (101 MHz,
CDCl3) δ 156.9 (d, C6); 154.7 (d, C2); 131.9 (d, C3); 126.9 (d,
C5); 124.6 (d, C4); 112.9 (d, C1); 58.1 (d, C-benzyl); 35.0 (C-
tBu); 34.3 (d, C-tBu); 30.5 (d, CH3-tBu); 30.0 (CH3-tBu); 19F
NMR (471 MHz, CDCl3) δ -119.74 (d); Rf value 0.74 (CH2Cl2/
MeOH, 9:1); MS (FAB, m/z) calcd 254.1682 (M), found 254.1681
(M).
(br, 4H, NH); 7.45-7.36 (m, 8H, H4-aryl, H6-aryl); 7.26-7.22
(m, 4H, H5-aryl); 7.22-7.20 (m, 2H, H3-aryl); 7.17-7.15 (m,
2H, H3-aryl); 7.14 (d, H6-thymine); 7.12 (d, H6-thymine); 7.11
(d, H6-thymine); 7.09 (d, H6-thymine); 6.82 (m, 1H, H1′); 6.80
(m, 1H, H1′); 6.77 (m, H, H1′); 6.75 (m, 1H, H1′); 6.42 (ddd,
1H, H3′); 6.40 (ddd, 1H, H3′); 6.36 (ddd, 1H, H3′); 6.26 (ddd,
1H, H3′); 6.05-5.89 (m, 8H, H-benzyl, H2′); 4.98-4.95 (m, 2H,
H4′); 4.92-4.88 (m, 2H, H4′); 4.36-4.17 (m, 16H, H5′, CH2Cl);
1.71 (d, 3H, CH3-thymine); 1.68 (d, 3H, CH3-thymine); 1.67
(d, 3H, CH3-thymine); 1.63 (d, 3H, CH3-thymine);13C NMR (126
MHz, DMSO-d6) δ 163.9 (C4-thymine); 150.9 (C2-thymine);
149.3 (C2-aryl); 135.9 (C6-thymine); 132.9 (C6-aryl); 130.8
(C3′); 128.9 (C4-aryl); 127.5 (C1-aryl); 127.4 (C1-aryl); 127.0
(C2′); 126.9 (C2′); 126.8 (C2′); 124.9 (C5-aryl); 124.9 (C5-aryl);
118.8 (C3-aryl); 118.7 (C3-aryl); 109.9 (C5-thymine); 89.2 (C1′);
88.0 (C1′); 86.4 (C4′); 84.2 (C4′); 80.9 (C-benzyl); 78.7 (C-
benzyl); 70.1 (C5′); 69.4 (C5′); 68.7 (C5′); 51.7 (CH2Cl); 51.3
(CH2Cl); 12.1 (CH3-thymine); 12.1 (CH3-thymine); 12.0 (CH3-
thymine); 31P NMR (202 MHz, DMSO-d6) δ -10.05; -10.08;
-10.51; -10.60; Rf value 0.49 (CH2Cl2/MeOH, 9:1); MS (FAB,
m/z) calcd 441.062 (M + H+), found 441.077 (M + H+).
7-(Dich lor om eth yl)-cycloSa l-3′-d eoxy-2′,3′-d id eh yd r o-
th ym id in e m on op h osp h a te 46: procedure A; yield 57% (four
diastereomers); 1H NMR (500 MHz, DMSO-d6) δ 11.35 (s, 1H,
NH); 11.34 (s, 2H, NH); 11.32 (s, 1H, NH); 7.51-7.40 (m, 8H,
H4-aryl, H6-aryl); 7.30-7.27 (m, 4H, H5-aryl); 7.26-7.23 (m,
4H, H3-aryl); 7.19 (d, H6-thymine); 7.18 (d, H6-thymine); 7.16
(d, H6-thymine); 7.15 (d, H6-thymine); 7.01 (m, 4H, CHCl2);
6.84 (m, 2H, H1′); 6.76-6.72 (m, 2H, H1′); 6.44 (m, 4H,
H-benzyl); 6.30 (m, 1H, H3′); 6.24 (m, 2H, H3′); 6.19 (m, 1H,
H3′); 6.07 (m, 1H, H2′); 6.04 (m, 2H, H2′); 5.96 (m, 1H, H2′);
5.01 (m, 3H, H4′); 4.88 (m, 1H, H4′); 4.43-4.15 (m, 8H, H5′);
1.74 (d, 3H, CH3-thymine); 1.73 (d, 3H, CH3-thymine); 1.67
(d, 3H, CH3-thymine); 1.66 (d, 3H, CH3-thymine); 13C NMR
(101 MHz, DMSO-d6) δ 163.9 (C4-thymine); 150.9 (C2-thy-
mine); 149.3 (C2-aryl); 136.0 (C6-thymine); 133.0 (C6-aryl);
131.4 (C3′); 130.5 (C1-aryl); 127.7 (C4-aryl); 127.6 (C4-aryl);
127.4 (C2′); 127.4 (C2′); 125.2 (C5-aryl); 125.1 (C5-aryl); 119.2
(C3-aryl); 119.1 (C3-aryl); 110.0 (C5-thymine); 109.9 (C5-
thymine); 89.4 (C1′); 89.3 (C1′); 84.2 (C4′); 84.1 (C4′); 81.8 (C-
benzyl); 81.8 (C-benzyl); 73.4 (CHCl2); 73.2 (CHCl2); 70.2 (C5′);
70.1 (C5′); 12.2 (CH3-thymine); 12.2 (CH3-thymine); 31P NMR
(202 MHz, DMSO-d6) δ -10.11; -10.16; -11.10; Rf value 0.42
(CH2Cl2/MeOH, 9:1); MS (FAB, m/z) calcd 475.023 (M + H+),
found 475.020 (M + H+).
7-(Tr ich lor om e t h yl)-cycloSa l-3′-d e oxy-2′,3′-d id e h y-
d r oth ym id in e m on op h osp h a te 47: procedure A; yield 57%
1
(four diastereomers); H NMR (400 MHz, DMSO-d6) δ 11.35
(s, 1H, NH); 11.34 (s, 2H, NH); 11.33 (s, 1H, NH); 7.69-7.63
(m, 4H, H6-aryl); 7.61-7.50 (m, 4H, H4-aryl); 7.35 (m, 4H, H5-
aryl); 7.30-7.25 (m, 3H, H3-aryl); 7.26 (d, 1H, H6-thymine);
7.24 (d, 1H, H6-thymine); 7.21 (dd, 1H, H3-aryl); 7.15 (d, 1H,
H6-thymine); 7.14 (d, 1H, H6-thymine); 6.85 (m, 2H, H1′); 6.81
(m, 1H, H1′); 6.74 (m, 1H, H1′); 6.58-6.48 (m, 4H, H-benzyl);
6.45 (m, 2H, H3′); 6.25 (ddd, 1H, H3′); 6.17 (ddd, 1H, H3′);
6.08 (ddd, 1H, H2′); 6.06 (ddd, 1H, H2′); 5.95 (m, 1H, H2′);
5.89 (m, 1H, H2′); 5.04 (m, 1H, H4′); 4.98 (m, 1H, H4′); 4.84
(m, 1H, H4′); 4.76 (m, 1H, H4′); 4.48 (m, 4H, H5′); 4.25-4.09
(m, 4H, H5′); 1.76 (d, 3H, CH3-thymine); 1.75 (d, 3H, CH3-
thymine); 1.69 (d, 3H, CH3-thymine); 1.68 (d, 3H, CH3-
thymine); 13C NMR (101 MHz, DMSO-d6) δ 163.9 (C4-
thymine); 150.9 (C2-thymine); 149.3 (C2-aryl); 136.1 (C6-
thymine); 132.7 (C6-aryl); 132.6 (C6-aryl); 131.3 (C3′); 130.4
(C1-aryl); 127.7 (C4-aryl); 127.6 (C4-aryl); 127.5 (C2′); 127.4
Ch olin ester a se Assa y. With BCh E (h u m a n ser u m ). A
modification of the Rappaport procedure was applied.21 Com-
mercially available solutions and reagents were used (Sigma,
kit 420-MC): sodium chloride solution (0.15 M in water),
m-nitrophenol (0.75 g/L in phosphate buffer, pH 7.8), acetyl-
choline chloride (preweighted solid, water was added for a
concentration of 150 g/L). All solutions were stored at 4 °C.
Human serum was freshly prepared from blood and stored at

SAR of cycloSal-Pronucleotides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2851
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E.; Balzarini, J . CycloSal-Pro-Nucleotides of 2′-fluoro-ara- and
2′-fluoro-ribo-2′, 3′-dideoxyadenosine as a strategy to bypass a
metabolic blockade. J . Med. Chem. 1999, 42, 1615-1624.
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activity of cyclosaligenyl prodrugs of acyclovir, carbovir and
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herpes simplex virus type 1 evaluation of cycloSal-nucleotides
of acyclic nucleoside analogues. Antiviral Chem. Chemother.
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-80 °C in small portions. For the enzyme assay, 40 µL of
sodium chloride solution, 370 µL of m-nitrophenol solution,
40 µL of acetylcholine chloride solution, 20 µL of a solution of
the appropriate cycloSal nucleotide in DMSO, and 430 µL of
water were transferred into a quarz cuvette. The enzymatic
reaction was started by the addition of 100 µL of human serum.
The cuvette was transferred into the UV/vis spectrometer
immediately, and the extinction (λ ) 420 nm, 25 °C) was
recorded at 0 and after 5 min. Different concentrations of the
cycloSal nucleotides in DMSO were used in order to achieve
final inhibitor concentrations of 0.1, 0.5, 1, 5, 10, 30, and 50
µM. For every inhibitor concentration, ∆E values were calcu-
lated and plotted. The IC50 values were determined from the
plot by interpolation in the pseudolinear region.
With BCh E (m ou se a n d ca lf ser u m ). Analogously to the
experiments with human serum, but using mouse or calf
serum instead.
With ACh E fr om Electr oph or u s electr icu s. As described
before, but using 100 µL of a 10 U/mL solution of purified
AChE from Electrophorus electricus (purchased from Sigma)
in water instead of human serum. The enzyme solution was
stored at 0 °C in order to avoid denaturation of the enzyme.
Only the experiments with 50 µM inhibitor concentration were
performed, as none of the tested cycloSal nucleotides exploited
any inhibitor activity.
With h u m a n ACh E. Analogously to the experiments with
AChE from E. electricus, but using purified human AChE
instead (purchased from Sigma).
With ACh E fr om beef er yth r ocytes: Analogously to the
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Ack n ow led gm en t . Financial support by the
Deutsche Forschungsgemeinschaft, Germany, the Fonds
der Chemischen Industrie, Germany, and the Rene´
Descartes Prize 2001 of the European Commission is
gratefully acknowledged. C.D. is grateful to a Ph.D.
fellowship from the Fonds der Chemischen Industrie/
BMBF, Germany.
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