cycloSal-2',3'-dideoxy-2',3'-didehydrothymidine monophosphate (cycloSal- d4TMP): Synthesis and antiviral evaluation of a new d4TMP delivery system

Article abstract of DOI:10.1021/jm970664s

The synthesis, hydrolysis, and antiviral evaluation of novel, lipophilic cycloSal-d4TMP derivatives 3a-h of the anti-HIV dideoxynucleoside 2',3'- dideoxy-2',3'-didehydrothymidine (d4T, 1) are reported. This pro-nucleotide concept has been designed to deliver d4TMP (2) by selective chemical hydrolysis. All compounds 3a-h were synthesized using phosphorus(III) chemistry in good yields and in somewhat lower yields using phosphorus(V) chemistry starting from substituted salicyl alcohols 6a-h. The phosphotriesters 3 were obtained without stereochemical preference with respect to the configuration at the phosphorus center as 1:1 diastereomeric mixtures. However, a few of the triesters 3 could be separated into the diastereomers by means of semipreparative HPLC. In a 1-octanol/phosphate buffer mixture, all compounds 3 exhibited 9-100-fold higher lipophilicity as judged from their Pa values as compared to d4T (1). Furthermore, in hydrolysis studies 3 decomposed under mild aqueous basic conditions releasing solely d4TMP (2) and the diols 6 following the designed tandem reaction sequence. A correlation of the electronic properties introduced by the substituents and the half-lives of triesters 3 was observed. Thus, by varying the substituent, the half-lives of 3 could be adjusted over a wide range of compounds still delivering d4TMP (2) selectively. Phosphotriesters 3 exhibited considerable activity against HIV-1 and HIV-2 in wild-type human T- lymphocyte (CEM/O) cells as well as mutant thymidine kinase-deficient (CEM/TK^-) cells. Surprisingly, we observed a 3-80-fold difference in antiviral activity between the two diastereomers. Our data clearly prove that the cycloSal-d4TMPs deliver exclusively the nucleotide d4TMP not only under simulated hydrolysis conditions but also under cellular conditions and thus fulfill the thymidine kinase-bypass premise. Therefore, the cycloSal- nucleotide concept is the first reported pro-nucleotide system that delivers the dideoxynucleotide by a pH-driven, chemically activated, tandem reaction without the requirement of an enzymatic contribution.

Full text of DOI:10.1021/jm970664s

J . Med. Chem. 1998, 41, 1417-1427
1417
cycloSa l-2′,3′-d id eoxy-2′,3′-d id eh yd r oth ym id in e Mon op h osp h a te
(cycloSa l-d 4TMP ): Syn th esis a n d An tivir a l Eva lu a tion of a New d 4TMP
Deliver y System
Chris Meier,*,† Martina Lorey,† Erik De Clercq,§ and J an Balzarini§
Institute of Organic Chemistry, J ulius-Maximilians-University of Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Germany, and
Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received October 1, 1997
The synthesis, hydrolysis, and antiviral evaluation of novel, lipophilic cycloSal-d4TMP
derivatives 3a -h of the anti-HIV dideoxynucleoside 2′,3′-dideoxy-2′,3′-didehydrothymidine (d4T,
1) are reported. This pro-nucleotide concept has been designed to deliver d4TMP (2) by selective
chemical hydrolysis. All compounds 3a -h were synthesized using phosphorus(III) chemistry
in good yields and in somewhat lower yields using phosphorus(V) chemistry starting from
substituted salicyl alcohols 6a -h . The phosphotriesters 3 were obtained without stereochemical
preference with respect to the configuration at the phosphorus center as 1:1 diastereomeric
mixtures. However, a few of the triesters 3 could be separated into the diastereomers by means
of semipreparative HPLC. In a 1-octanol/phosphate buffer mixture, all compounds 3 exhibited
9-100-fold higher lipophilicity as judged from their Pa values as compared to d4T (1).
Furthermore, in hydrolysis studies 3 decomposed under mild aqueous basic conditions releasing
solely d4TMP (2) and the diols 6 following the designed tandem reaction sequence. A correlation
of the electronic properties introduced by the substituents and the half-lives of triesters 3 was
observed. Thus, by varying the substituent, the half-lives of 3 could be adjusted over a wide
range of compounds still delivering d4TMP (2) selectively. Phosphotriesters 3 exhibited
considerable activity against HIV-1 and HIV-2 in wild-type human T-lymphocyte (CEM/O)
cells as well as mutant thymidine kinase-deficient (CEM/TK-) cells. Surprisingly, we observed
a 3-80-fold difference in antiviral activity between the two diastereomers. Our data clearly
prove that the cycloSal-d4TMPs deliver exclusively the nucleotide d4TMP not only under
simulated hydrolysis conditions but also under cellular conditions and thus fulfill the thymidine
kinase-bypass premise. Therefore, the cycloSal-nucleotide concept is the first reported pro-
nucleotide system that delivers the dideoxynucleotide by a pH-driven, chemically activated,
tandem reaction without the requirement of an enzymatic contribution.
In tr od u ction
three steps via the nucleoside monophosphate and the
nucleoside diphosphate.2a,4e,8 Although dideoxynucleo-
sides offer great promise for the inhibition of viral
replication, several shortcomings limit realization of
their full therapeutic efficacy. One problem is the
limited ability of some dideoxynucleosides to undergo
biotransformation catalyzed by host cell kinases into the
triphosphate due to structural differences as compared
to natural nucleosides.6 Furthermore, monocytes and
macrophages, which harbor high virus amounts, have
very low levels of nucleoside kinases. So, the presence
and activity of intracellular enzymes necessary for the
phosphorylation of nucleoside analogues are highly
dependent on the host species, the cell type, and the
stage in the cell cycle. Consequently, the rate and
extent to which the nucleoside analogues are converted
to their bioactive triphosphates may be at least as
important as the affinity of these compounds for the
target enzyme. As compared to the approved anti-HIV
drug AZT, d4T (1) shows comparable selective anti-HIV
activity in vitro.4e,9 Moreover, d4T (1) has been found
to be less toxic than AZT for bone marrow progenitor
cells and to be less inhibitory to mitochondria DNA
replication.10 In contrast to AZT, d4T (1) does not
interfere with the intracellular dTMP, dTDP, and dTTP
Due to the ultimately fatal nature of AIDS, an
intensive effort has been underway to develop new
therapies or to improve existing treatments. Among the
various structural classes of antiviral agents investi-
gated for the treatment of HIV infections, nucleoside
analogues are among the most effective.1 Most of these
nucleoside analogues are 2′,3′-dideoxynucleosides,2 e.g.,
3′-azido-2′,3′-dideoxythymidine (AZT, Zidovudine, Re-
trovir)3 and 2′,3′-dideoxy-2′,3′-didehydrothymidine (1)
(d4T, Stavudine, Zerit).4 The general mode of action of
these drugs is the inhibition of HIV reverse trans-
criptase5,6 and the incorporation into the growing DNA
chain which results in DNA chain termination.7 The
dideoxynucleosides are not active as such. After mem-
brane penetration, intracellular conversion of the nu-
cleoside analogues to their 5′-mono-, 5′-di-, and 5′-
triphosphates is a prerequisite for the expression of the
biological activity. So, formally dideoxynucleosides are
prodrugs of the ultimately bioactive triphosphates. This
biotransformation is established by host cell kinases in
* To whom correspondence should be addressed.
† J ulius-Maximilians-University of Wu¨rzburg.
§ Rega Institute for Medical Research.
S0022-2623(97)00664-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/03/1998

1418 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Meier et al.
Sch em e 2. Structures of d4T (1), d4TMP (2), and
Sch em e 1. Principle of a d4TMP
cycloSal-pro-nucleotide Approach
cycloSal-d4TMP Phosphotriesters 3
driven, selective chemical hydrolysis:19 the cyclosaligenyl-
2′,3′-dideoxy-2′,3′-didehydrothymidine monophosphates
3 (cycloSal-d4TMP).20 Before hydrolysis takes place,
the cycloSal-nucleotides 3 serve as lipophilic, neutral
prodrugs. This concept has been successfully introduced
with the anti-HIV nucleosides ddT21 as well as ddA.22
Although the general structure and the delivery mech-
anism were identical, the biological task of the com-
pounds was different: While the ddT phosphotriesters
were designed to follow the TK-bypass, the ddA ana-
logues were used for the adenosine deaminase (ADA)-
bypass.23
pools, and consequently, d4T does not cause the starva-
tion of the cellular dTTP.2a In comparison with AZTTP,
d4TTP has a longer half-life in CEM/O cells (3-3.5 h).8a
However, in contrast to AZT, d4T (1) is an example of
a dideoxynucleoside analogue with a rate-limiting step
in the first phosphorylation catalyzed by the host cell
enzyme thymidine kinase (TK) in CEM and MT-4
cells.8a The further conversion to the ultimate bioactive
drug d4TTP by thymidylate kinase and nucleoside
diphosphate kinase is not limiting. Consequently, the
direct application of the nucleotide 2′,3′-dideoxy-2′,3′-
didehydrothymidine monophosphate 2 (d4TMP) would
bypass the metabolization-limiting enzyme TK (TK-
bypass) and could thus improve the therapeutic poten-
tial of the drug (Scheme 1). But a well-known problem
in antiviral therapy is the bioavailability of this type of
phosphorylated derivative. This compound does not
easily penetrate cellular membranes due to its high
polarity. Furthermore, 2 is rapidly dephosphorylated
in the blood. Consequently, the delivery of d4TMP (2)
from neutral, lipophilic prodrugs12,13 is one effort to
achieve the TK-bypass (pro-nucleotide approach; Scheme
1).
Several prodrug strategies have been developed to
deliver nucleotides intracellularly. As a general motive,
uncharged nucleotide triesters are used as membrane-
permeable nucleotide precursors. The major differences
of these approaches are the delivery mechanisms to
d4TMP: While almost all approaches based on chemical
hydrolysis reported so far were unable to deliver the
nucleotide selectively and consequently serve only as
nucleoside depots,14 the newer concepts based on enzy-
matic activation processes reported by McGuigan (aryl-
oxyphosphoramidates)15 and Imbach (bis-SATE phos-
photriesters)16 demonstrated the successful intracellular
delivery of free nucleotides from highly lipophilic pre-
cursors.17
Here we report on the synthesis and the biological
evaluation of the cycloSal-2′,3′-dideoxy-2′,3′-didehy-
drothymidine monophosphates
3 (cycloSal-d4TMP;
Scheme 2) as potential neutral prodrugs of d4TMP (2)
of the approved antiviral drug d4T (1). Furthermore,
we report on their lipophilic properties (partition coef-
ficients, Pa values) and on hydrolysis studies in various
aqueous buffers at different pH values as well as on
hydrolysis studies under more biologically adapted
conditions using RPMI-1640 culture medium containing
10% heat-inactivated fetal calf serum (FCS) at 37 °C.
Resu lts a n d Discu ssion
Ch em istr y. d4T (1) was prepared from thymidine
following the procedure of Horwitz et al.24 or using the
methodology reported by Mansuri et al.9 Two ap-
proaches were used to yield the title compounds. The
first uses phosphorus(III) chemistry, whereas the second
involves phosphorus(V) chemistry. In the former meth-
odology, the title compounds 3a -h were synthesized as
outlined in Scheme 3a. Differently substituted salicyl
aldehydes 4 or salicylic acids 5 were used as starting
materials, which were reduced by standard procedures
to give the salicyl alcohols 6 in 75-90% yield.23a Diols
6 were reacted with phosphorus trichloride to yield the
cyclic saligenylchlorophosphanes 7a -h . These were
isolated by distillation under argon atmosphere in 50-
85% yield.23a,25 The cycloSal-d4TMPs 3a -h were ac-
cessible via 8a -h in a “one-pot reaction” by reacting d4T
(1) and 1.5 equiv of chlorophosphanes 7a -h at 0 °C in
the presence of dry, distilled diisopropylethylamine
(DIPEA) and subsequent in situ oxidation with tert-
butyl hydroperoxide (TBHP) at ambient temperature as
As part of our ongoing program to develop efficient
pro-nucleotide delivery systems,18 we were interested
in the design and the synthesis of a completely new pro-
nucleotide approach for d4TMP (2) based on a pH-

Synthesis, Evaluation of a New d4TMP Delivery System
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1419
Sch em e 3. Synthetic Pathways to the cycloSal-d4TMP Triesters 3a -h a
a
Reaction conditions: (a) NaBH4 for 4 (LiAlH4 for 5); (b) PCl3, pyridine, Et2O, -10 °C, 2 h; (c) d4T (1), DIPEA, CH3CN, 0 °C, 20 min;
(d) TBHP, CH3CN, rt, 30 min; (e) P(O)Cl3, DIPEA, THF, 0 °C, 12 h; (f) salicyl alcohol 6, DIPEA, THF, 0 °C-rt, 12 h.
1:1 diastereomeric mixtures. 3,5-Dimethylsalicylalde-
hyde (4h ) was prepared by orthoformylation of the
corresponding phenol using dichloromethyl methyl ether
in the presence of TiCl4.26
achieved for some derivatives. It was noted that the
3-substituted triesters were easier to separate than their
5-substituted counterparts. The configuration at the
phosphorus atom of the separated diastereomers was
assigned by comparison with the known stereochemistry
of a model compound using CD spectroscopy.27 It should
be mentioned that the Rp- or Sp-configuration, respec-
tively, correlated with the elution properties of the
diastereomers on a reversed-phase HPLC column. All
“slow”-eluting isomers showed Rp-configuration, whereas
all “fast”-eluting isomers are Sp-configurated at the
phosphorus center. However, in a few cases, the
separation of the diastereomers was still unsuccessful.
Thus, the diastereomeric mixtures as well as the “slow”
(Rp) and the “fast” (Sp) diastereomers of the phospho-
triesters 3 were isolated after the HPLC purification
followed by lyophilization.
An alternative synthetic approach toward the title
compounds 3 involved the less reactive phosphorus(V)
chemistry shown in Scheme 3b: The reaction of phos-
phorus oxychloride with d4T (1) yielded the 5′-O-d4T
phosphodichloridate 9 which was further reacted with-
out isolation with the salicyl alcohols 6 to give the
triesters 3. However, using this approach the yields
were markedly lower (∼37%) than obtained according
to the first described strategy (50-73% yield). Similar
to the phosphorus(III) approach, the phosphorus(V)
method gave no diastereoselectivity with respect to the
configuration at the phosphorus atom. After chromato-
graphic purification, the cycloSal-d4TMPs 3a -h were
1
characterized by means of H, 13C, and 31P NMR and
Deter m in a tion of th e P a r tition Coefficien ts (P a
Va lu es). The partition coefficients (Pa values) of the
cycloSal-d4TMPs 3a -h were determined in 1-octanol/
sodium phosphate buffer (pH 6.8). Triesters 3a -h were
partitioned between 1-octanol and phosphate buffer for
5 min, and after centrifugation the relative concentra-
tion in each solvent phase was determined by reverse-
phase HPLC analysis. This method for the determina-
tion of the partition coefficient is a very simple procedure
that gives a qualitative estimation of the lipophilicity
of the title compounds 3a -h .28 Like most nucleosides
and in contrast to AZT, which enters mammalian cells
by passive, nonfacilitated diffusion readily,29 d4T (1) is
taken up by the cells only to a limited extent by passive,
nonfacilitated diffusion. There seem to be also contri-
butions of active mechanisms such as nucleoside and
nucleobase carriers. Consequently, the Pa value of AZT
(1.0630) was used as a reference for the potential ability
of compounds 3 to diffuse passively through cellular
membranes. As can be seen in Table 1, all Pa values
UV spectroscopy as well as electrospray mass spectrom-
etry (ESI, negative mode). As expected, the phosphot-
riesters displayed two closely spaced signals in the 31P
NMR spectra (1:1 mixtures, -7.3 to -9.3 ppm), corre-
sponding to the presence of diastereomers, resulting
from mixed stereochemistry at the phosphorus center.
Similar diastereomeric splitting and phosphorus cou-
pling, where appropriate, were noted in the H-decoupled
13C NMR spectra. The purity of the cycloSal-d4TMPs
3a -h was checked by analytical HPLC analysis. In
almost all cases the two expected peaks of the diaster-
eomers overlapped completely, but in one case two peaks
with partial overlap were detected. For the biological
evaluation, small amounts of the cycloSal-d4TMP tri-
esters were additionally purified by semipreparative
HPLC using acetonitrile-water eluents in order to be
sure to eliminate any trace of the nucleoside d4T (1).
The second reason for using semipreparative HPLC was
the attempt to separate the diastereomers which was

1420 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Meier et al.
Ta ble 1. Hydrolysis in Different Aqueous Buffers and Pa
Values of cycloSal-d4TMPs 3a -h
prodrug concept is based on the above-mentioned dif-
ference in stability of the phenyl and benzyl phosphate
esters: This allows to discriminate between the different
phosphate ester bonds and results in a tripartate
prodrug system.36 The phenyl ester bond should be the
most labile one because the negative charge could be
delocalized in the aromatic ring leading to the 2-hy-
droxybenzyl phosphodiester 10. The alternative cleav-
age of the benzyl ester in 3 to yield the 2-(hydroxyme-
thyl)phenyl phosphodiester 11 is unfavorable (step c,
Scheme 4), because the phosphate residue in the ortho
position of the benzyl ester stabilizes this bond. Hence,
the hydrolysis should proceed as follows: In the initial
hydrolysis step, the phenyl ester is cleaved selectively
(step a, Scheme 4). As a consequence, the ortho sub-
stituent to the benzyl group has switched from an
acceptor group (phosphate) to a donor group (hydroxyl)
with the result of activation of the remaining masking
group caused by the “umpolung” of the 2-substituent.
This induces the spontaneous cleavage of diester 10 to
yield the nucleotide 2 and the diol 6 (tandem reaction;
step b, Scheme 4).
hydrolysis (t1/2, h)b
hydrolysis (t1/2, h)b
in CMe at 37 °C
in aqueous buffers
at 37 °C
without
FCSf
with
Pa
compd pH 6.9a pH 7.3c pH 8.9d
FCSe valueg
3a
3b
3c
3d
3e
3f
3g
3h
1
4.1
6.4
24.5
28.3
9.5
28.3
68.5
98.2
nai
0.15
0.7
4.5
7.2
1.4
8.0
0.06
0.3
1.1
1.07
0.4
1.3
ndh
2.5
7.2
7.2
3.2
0.12
1.0
3.9
4.3
2.0
8.4
9.1
1.5
7.6
1.9
2.3
1.4
8.9
5.4
5.1
10.2
1.5
16.0
17.5
nai
16.1
3.4
10.8
15.3
nai
nai
nai
0.15
a
b
pH 6.9: 30 mM TRIS buffer. Half-lives are given in h. c pH
d
7.3: 30 mM sodium phosphate buffer. pH 8.9: 30 mM sodium
borate buffer. e CM: RPMI-1640 culture medium, 37 °C. f FCS:
g
10% heat-inactivated fetal calf serum. Pa: partition coefficient.
Not determined. Not available.
h
i
Sch em e 4. Two Possible Hydrolysis Pathways of
cycloSal-d4TMP Phosphotriesters 3a -h
Thus, the major difference between our approach and
other concepts based on enzymatic activation15-17 is that
a second activation step (e.g., by enzymes) is not
required because we have an intrinsic activation by the
first reaction. The hydrolysis pathway has already been
verified by NMR spectroscopy.20 Furthermore, we have
already shown for the cycloSal-AZTMP derivatives that
the initial hydrolysis step can be controlled by substi-
tuents in the para position (position 5) of the phenolic
phosphate ester: the stronger the electron-withdrawing
activity of the substituent, the faster the degradation
reaction of triesters 3.23a
To prove the selective delivery of d4TMP (2), hydroly-
sis experiments of the cycloSal-d4TMPs 3a -h were
carried out in different aqueous buffers and RPMI-1640
culture medium with or without heat-inactivated fetal
calf serum (FCS). First, triesters 3 were hydrolyzed in
30 mM phosphate buffer, pH 7.3, at 37 °C as a model of
the physiological milieu. Furthermore, the pH depen-
dence of the hydrolysis was studied in borate buffer (30
mmol, pH 8.9) and TRIS buffer (30 mM, pH 6.9). The
degradation of 3a -h was followed by means of reversed-
phase HPLC analysis. The half-lives were determined
by quantifying the decreasing peaks of 3 vs time. The
half-lives are summarized in Table 1.
were found to be >1 and the log(Pa) was always positive.
The observed Pa values were 9-102-fold higher (Pa )
1.4-15.3) as compared to the value of d4T (1) and 1.3-
14-fold higher compared to that of AZT. It should be
added that the possibility of active transport has not
been investigated so far. Nevertheless, improved cel-
lular uptake by a passive way should be possible.
Kin et ic St u d ies. In contrast to other prodrug
concepts,15-17 the pro-nucleotide approach reported here
has been designed to selectively yield d4TMP (2) and
the two-component masking group after a controlled,
chemically induced tandem reaction involving a succes-
sive coupled cleavage of the phenyl and benzyl esters
of the cycloSal-phosphotriester. The dideoxynucleoside
is connected via an alkyl ester to the phosphate moiety
and this is the most stable bond. The difference in
hydrolytic stability of phenyl and benzyl phosphotri-
esters has already been reported: Whereas acceptor-
substituted bis-phenyl phosphotriesters are rapidly
hydrolyzed under alkaline conditions,31 a fast hydrolysis
of bis-benzyl phosphotriesters was observed only in the
case of donor-substituted derivatives.32 In general, both
resulting phosphodiesters are very stable against non-
enzymatic hydrolysis (step d, Scheme 4).33,34 Sometimes
even the enzymatic hydrolysis35 of phosphodiesters may
be difficult. Consequently, the critical point is the
second hydrolysis reaction after the first cleavage of the
triester to yield the diester. The rationale of our new
Using the mentioned buffers, cycloSal-d4TMPs 3a -h
were degraded following pseudo-first-order kinetics to
yield exclusively d4TMP (2) and the salicyl alcohols 6
in all studied media. At pH < 7, only the 3,5-dimethyl-
substituted derivative 3h yielded also in small amounts
the “wrong” phenyl-d4T-phosphodiester 11h , which is,
as expected, stable to further hydrolysis. As can be seen
in Table 1, the stability of the phosphotriesters 3a -h
increased considerably under mild acidic conditions (see
hydrolysis at pH 6.9 vs pH 7.3), whereas the half-lives
decreased markedly at more basic pH (see hydrolysis
at pH 8.9) and hence confirmed the anticipated pH
dependence of the hydrolysis. Furthermore, the tri-
esters were hydrolyzed in acetate buffer (30 mM, pH
4.6). As for the studies at pH 6.9, the half-lives were
largely increased with respect to the studies at pH 7.3
indicating a high increase in stability under acidic

Synthesis, Evaluation of a New d4TMP Delivery System
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1421
Ta ble 2. Inhibitory Effects of cycloSal-d4TMPs 3a -h as Well
as Representative Salicyl Alcohols 6 on the Replication of
HIV-1 and HIV-2 and Toxicity in Wild-Type CEM/O Cells and
Mutant Thymidine Kinase-Deficient CEM/TK- Cells
conditions (data not shown). As for the corresponding
cycloSal-AZTMP derivatives, we observed a clear cor-
relation between the electronic properties caused by the
substituent of the salicyl alcohol and the hydrolysis half-
lives in different aqueous buffers.23a These results
clearly show that we are able to control the hydrolysis
kinetics of phosphotriesters 3 by varying the substitu-
ents in the aryl ring. From the results summarized in
Table 1, especially the donor-substituted cycloSal-
d4TMPs 3d ,f-h , which exhibited half-lives of 7-16 h
in phosphate buffer at pH 7.3 and high hydrolytic
stability at acidic conditions, seem to be interesting
candidates for biological evaluation. Furthermore, we
observed a change in the rate-limiting step in depen-
dence of the substituent. If an electron-withdrawing
substituent is placed in the aromatic ring of the starting
phosphotriesters 3, the initial cleavage to the phos-
phodiester 10 is the rate-limiting step, whereas in the
case of electron-donating substituents in 3 the second
step from diester 10 to d4TMP (2) is rate-determining.
All the mentioned results are in complete agreement
with the designed hydrolysis pathway via 2-hydroxy-
benzyl phosphodiester 10 to d4TMP (2) and salicyl
alcohols 6 (Scheme 4).
antiviral activity EC50 (µM)a
CEM/O
compd subst X (HIV-1)
CEM/O CEM/TK- cytotoxicity
(HIV-2)
(HIV-2)
CC50 (µM)b
3a
3b
3c
3d
3e
3f
3g
3h
6c
6d
6g
d4T (1)
AZT
5-NO2
5-Cl
5-H
0.29
0.42
0.28
0.18
0.17
0.18
0.087
0.09
0.40
1.40
0.62
0.70
0.42
0.34
0.12
0.17
40.0
2.67
0.50
0.80
0.17
0.18
0.093
0.08
ndc
75
49
47
39
35
38
21
21
5-OMe
3-OMe
5-Me
3-Me
3,5-Me
5-H
5-OMe >250
3-Me >250
0.18
0.007
>250
>250
>250
>250
0.26
>250
158
>250
56
ndc
ndc
15.0
0.006 >100
>100
a
b
50% effective concentration. 50% cytotoxic concentration.
c Not determined.
same time, this result made it clear that a second,
separate activation process for the delivery of the
nucleotide is obviously not required. However, this
experiment gives no further information if the reaction
proceeds via a benzyl carbocation or a Grob fragmenta-
tion (quinone methide). The 18O-isotope distribution
found for 3b was additionally verified in the hydrolysis
corresponding to the 3-methoxy-substituted cycloSal-
triester 3e.
Additionally, an interesting question arises concern-
ing the second cleavage step: is the second reaction an
intermolecular or intramolecular reaction? Thus, is
there a second nucleophilic attack of a water molecule
or a hydroxide at the phosphorus center or is the
intermediate phosphodiester cleaved via (i) a Grob
fragmentation to give a neutral quinone methide and
after water quenching the diol 6 or (ii) a spontaneous
C-O bond cleavage and a subsequent water quenching
of the formed ion pair?37 This question was addressed
by hydrolysis experiments of the 5-chloro-substituted
cycloSal-derivative 3b in [16O]water and [18O]water. In
the hydrolysis with [16O]water, we detected the inter-
mediate phosphodiester 10b (M ) 443/445 in 3:1 ratio
35Cl/37Cl), d4TMP (2) (M ) 303), and the diol 6 (M )
157/159 in 3:1 ratio 35Cl/37Cl) in the electrospray mass
spectra (negative mode) with the corresponding mass
peaks. The first reaction of the degradation is obviously
a nucleophilic attack at the phosphorus atom. Conse-
quently, in the experiment in [18O]water, the intermedi-
ate phosphodiester 10b should be detected with a mass
(M + 2 with a 3:1 ratio for 35Cl/37Cl). In the case of a
second nucleophilic attack at the phosphorus center, the
resulting d4TMP would have a mass (M + 4) and the
salicyl alcohol should have only a mass (M with a 3:1
ratio for 35Cl/37Cl) without 18O. If the reaction involves
a spontaneous C-O bond cleavage yielding d4TMP with
a mass (M + 2) and an intermediate benzyl cation/
quinone methide, which is rapidly quenched by water,
then the final salicyl alcohol should also have a mass
(M + 2 with a 3:1 ratio for 35Cl/37Cl). An alternative is
a nucleophilic reaction at the benzylic carbon atom
releasing d4TMP (M + 2) and directly the diol (M + 2).
Thus, the difference is a mass spectrometric analysis
of M + 4 and M in the former scenario or a situation
with M + 2 and M + 2 in the latter case for the
nucleotide and the salicyl alcohol, respectively. Run-
ning the reaction and analyzing the mixture by ESI
mass spectrometry, we observed the second situation
with both products bearing one 18O-isotope only. At the
Further hydrolysis studies were carried out in RPMI-
1640 culture medium with and without 10% heat-
inactivated FCS at 37 °C. This medium is normally
used for the cultivation of human T-lymphocytes used
in the in vitro HIV tests. The hydrolysis products in
RPMI-1640 medium were again exclusively d4TMP (2)
and the diols 6a -h , but the half-lives were slightly
shorter than in phosphate buffer. The shorter half-lives
in the RPMI-1640 medium most likely result from its
more basic pH value (pH 7.8). In the RPMI-1640
medium supplemented with 10% FCS, we also detected
the dephosphorylation of d4TMP to d4T by remaining
phosphatases in the serum. As judged from the ob-
tained half-lives, the donor-substituted derivatives of 3
should still be stable enough to serve as intracellular
depots of d4TMP (2). Finally, it should be mentioned
that we have not found any evidence for a contribution
of an enzymatic degradation of the cycloSal-phospho-
triesters in the RPMI-1640/FCS hydrolyses. This points
so far to a purely chemical degradation of our pro-
nucleotide. Additional hydrolysis studies in sera, plasma,
and cell extracts will be the subject of further work in
our laboratories and will be reported in due course.
An tivir a l Eva lu a tion . The cycloSal-ddAMP phos-
photriesters have already demonstrated the effective-
ness of our new pro-nucleotide system in anti-HIV
assays in rapidly dividing human T-lymphoblastic leu-
kemia cells (CEM/O).22 Moreover, particularly striking
was the structure-bioactivity correlation: The stronger
the electron-donating activity of the substituent, the
better was the antiviral activity against HIV-1 and
HIV-2 in CEM/O cells.22 These results confirmed the
highly selective intracellular delivery of ddAMP and
demonstrated the efficient ADA-bypass.

1422 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Meier et al.
Ta ble 3. Antiviral Evaluation of the Separated Diastereomers of cycloSal-d4TMPs 3 in Wild-Type CEM/O Cells and Mutant
Thymidine Kinase-Deficient CEM/TK- Cells
antiviral activity EC50 (µM)a
cytotoxicity
SI
compd
subst X
HPLCd
config
CEM/O (HIV-1)
CEM/O (HIV-2)
CEM/TK- (HIV-2)
CC50 (µM)b
valuec
3e
3e
3g
3g
3h
3h
1
3-OMe
3-OMe
3-Me
3-Me
3,5-Me
3,5-Me
slow
fast
slow
fast
slow
fast
Rp
Sp
Rp
Sp
Rp
Sp
0.14
0.47
0.08
0.42
0.093
0.50
0.18
0.17
1.03
0.067
1.1
0.17
0.80
0.26
0.12
9.67
0.063
0.70
0.08
0.38
15.0
38
84
11
76
18
22
56
320
9
190
108
218
57
4
a
b
50% effective concentration. 50% cytotoxic concentration. c Selectivity index: ratio of 50% cytotoxic concentration/50% effective
d
concentration. Eluting properties of the compound on reversed-phase HPLC column.
For these reasons, the parent nucleoside d4T (1) and
cycloSal-d4TMPs 3 were evaluated for their ability to
inhibit the replication of HIV-1 and HIV-2 in wild-type
CEM/O cells. Furthermore, the compounds were evalu-
ated in HIV-2-infected CEM/TK- cells. The use of the
TK-deficient cells should prove the TK-bypass. The test
compounds were free of the parent nucleoside 1, which
was verified by means of analytical HPLC. The results
obtained are displayed in Table 2.
d4TMP but completely inactive in TK- cells, the cy-
cloSal-d4TMP triesters are >1250 times more active in
the thymidine kinase-deficient CEM cell line. The
almost equal antiviral activity of the donor-substituted
phosphotriesters in the wild-type cells and the thymi-
dine kinase-deficient cells demonstrates the highly
selective intracellular delivery of d4TMP making these
compounds entirely independent of the TK. This clearly
proves the TK-bypass concept. The selectivity indices
(SI) of the cycloSal-d4TMPs 3b-h in CEM/TK--deficient
cells showed a 50-fold increase compared to that of the
parent nucleoside d4T, while the SI value in the wild-
type cells was approximately the same. In addition to
the cycloSal derivatives, three representative salicyl
alcohols 6 were tested in vitro. As shown in Table 2,
the diols used as masking group obviously did not
contribute to the antiviral effect, nor did they result in
any toxicity (EC50 and CC50 > 250 µM).
An interesting effect was observed for the antiviral
potency of the separated diastereomers of 3. The results
are summarized in Table 3. As can be seen, the “slow”-
eluting (Rp) diastereomer always proved much more
active than the “fast”-eluting (Sp) diastereomer. More-
over, in all cases the selectivity index was found to be
much higher for the Rp-stereoisomer compared to the
Sp-isomer. Again, the antiviral activity was retained
in the TK-deficient cells except for (Sp)-3-methoxy-
cycloSal-d4TMP (3e). One possible explanation for this
behavior could be a difference in hydrolysis stability
dependent on the configuration at the phosphorus
center. But only a 1.5-fold enhancement of hydrolysis
from the Rp- to the Sp-diastereomer was detected in
independent hydrolysis studies which seems not to be
sufficient to explain the above-examined difference in
bioactivity (data not shown). Other possibilities that
should also be taken into account are (i) a different
uptake of the cyclic phosphotriesters dependent on the
stereochemistry at the phosphorus atom and (ii) a
different enzymatic contribution in the hydrolysis of the
diastereomers of the phosphotriesters in the in vitro cell
system. How these effects influence the selectivity
index of the diastereomers remains unclear. Further
studies in order to explain the different properties of
the diastereomers are currently in progress in our
laboratories.
The parent nucleoside d4T (1) proved active against
HIV-1- and HIV-2-induced cytopathicity in CEM/O cells
(EC50 0.18 µM and 0.26 µM). However, as anticipated,
d4T (1) virtually lost its antiviral potential when evalu-
ated against HIV-2 in CEM/TK- cells due to the lack of
its bioactivating enzyme thymidine kinase (EC50 15.0
µM). As can be seen in Table 2, the cycloSal-d4TMPs
3c-h proved to be at least as active as d4T (1) against
HIV-1- as well as HIV-2-induced cytopathicity in the
wild-type cell line. Moreover, a correlation of the
antiviral activity with the electronic properties intro-
duced by the substituents and the hydrolytic stability
in the aqueous buffers and RPMI-1640 culture medium
was observed: the stronger the electron-donating activ-
ity of the substituent and the more stable the triester
(Table 1), the better is the antiviral activity against
HIV-1 and HIV-2 in CEM/O cells (Table 2). The
3-methyl- and the 3,5-dimethyl-cycloSal-d4TMPs (3g,h ,
respectively) exhibited at least equal or even a slightly
higher antiviral potency in the wild-type cell line than
d4T (1). As for d4T, all phosphotriesters 3 were slightly
less active against HIV-2-induced cytopathicity. It
should be mentioned that no correlation of the biological
activity with the lipophilicity of 3a -h (Pa values) could
be observed. Furthermore, particularly striking is the
complete retention of the biological activity in thymidine
kinase-deficient CEM/TK- cells for most of the com-
pounds. Only the most labile 5-nitro-cycloSal-d4TMP
(3a ) showed a considerable loss in antiviral activity
(Table 2). This loss in activity in the mutant TK- cell
line for 3a could be explained by the short half-life of
this acceptor-substituted derivative as compared to the
donor-substituted derivatives (having much longer half-
lives) in the buffer solutions (Table 2). Presumably, this
phosphotriester is predominantly hydrolyzed outside the
cells to give after dephosphorylation d4T (1), which
permeates into the cells by passive diffusion and is
rephosphorylated only in the wild-type cells. In con-
trast, the most active derivatives 3g,h were 180-fold
more active than d4T in the TK-deficient cells. As
compared to AZT, which is inherently more potent in
thymidine kinase-competent CEM cells than d4T or
Finally, the high biological activity of the donor-
substituted cycloSal-d4TMP derivatives was also ob-
served in MT-4 cells (Table 4) and Molt4/C8 cells (data
not shown). As in the in vitro tests in CEM cells, a
difference in biological activity with respect to the
configuration at the phosphorus atom was observed.

Synthesis, Evaluation of a New d4TMP Delivery System
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1423
Ta ble 4. Antiviral Evaluation of the cycloSal-d4TMPs 3 and
the Separated Diastereomers in MT-4 Cells
glacial acetic acid) by heating with a fan or a hot plate.
HPLC: (Merck-Hitachi) semipreparative HPLCsLiChroCART
250-10 containing LiChrospher 100 RP18 (10 µm); analytical
HPLCsLiChroCART 250-4 or 250-3 with LiChrospher 100
RP18 (5 µm). Gradient I (standard gradient): 18-100% CH3-
CN (0-16 min), 100% CH3CN (16-20 min), 18% CH3CN (19-
31 min); flow, 0.5 mL; UV detection at 265 nm. Gradient II:
0-100% CH3CN (0-18 min), 100% CH3CN (18.1-20 min), 0%
CH3CN (20.1-33 min); flow, 0.5 mL; UV detection at 265 nm.
Gradient III: 11-100% CH3CN (0-18 min), 100% CH3CN
(18-20 min), 11% CH3CN (20.1-32 min); flow, 0.5 mL; UV
detection at 265 nm. Distillation: Bu¨chi Kugelrohr apparatus.
antiviral activity EC50 (µM)a
compd subst X config HPLCb MT-4 (HIV-1) MT-4 (HIV-2)
3b
3f
3g
3g
3h
3h
1
5-Cl
Rp/Sp mixture
Rp/Sp mixture
Rp/Sp mixture
0.031
0.021
0.028
0.012
0.025
0.066
0.018
0.038
0.029
0.033
0.011
0.026
0.080
0.022
5-Me
3-Me
3-Me
3,5-Me Rp
3,5-Me Sp
Rp
slow
slow
fast
NMR. Spectra were recorded using 1H NMR: Bruker AMX
400 at 400 MHz or Bruker WH 270 at 270 MHz (DMSO as
internal standard). 13C NMR: Bruker AM 250 at 63 MHz
(CDCl3 or DMSO as internal standard). 31P NMR: Bruker
AMX 400 at 162 MHz (H3PO4 as external standard). All 1H
and 13C NMR chemical shifts (δ) are quoted in parts per million
(ppm) downfield from tetramethylsilane, (CD3)(CDH)SO being
set at δH 2.49 as a reference. The 31P NMR chemical shifts
are quoted in ppm using H3PO4 as external reference. The
spectra were recorded at room temperature, and all 13C and
31P NMR spectra were recorded in the proton-decoupled mode.
a
b
50% effective concentration. Eluting properties of the com-
pound on reversed phase HPLC column.
Again, the “slow”-eluting compound (Rp) was more
active than both the “fast”-eluting (Sp) diastereomer and
the mixture of the two diastereomers. This is shown
in the case of the 3,5-dimethyl-substituted derivative
3h , while the Rp-isomer of the 3-methyl-substituted
derivative 3g was even more active than the diastere-
omeric mixture (Table 4).
UV spectra were taken with
a Varian Cary 1E UV/vis
spectrophotometer. The infrared spectra were recorded with
a Perkin-Elmer 1600 series FT-IR spectrometer in KBr pellets.
Mass spectra were obtained with a Fisons electrospray VG
platform II spectrometer in negative mode (ESI-). The test
compounds were isolated as mixtures of diastereomers arising
from the mixed stereochemistry at the phosphate center. The
resulting lyophilized triesters did not give useful microana-
lytical data probably due to incomplete combustion of the
compounds but were found to be pure by rigorous HPLC
analysis (three different gradients), high-field multinuclear
NMR spectroscopy, and electrospray mass spectrometry.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e cy-
cloSa l-d 4TMP P h osp h otr iester s 3a -h . The reactions were
performed in an argon atmosphere under anhydrous condi-
tions. To a solution of d4T (1) (0.10 g, 0.37 mmol) in 6.0 mL
of CH3CN, cooled to 0 °C in ice/water, was added DIPEA (0.097
g, 2 equiv, 0.75 mmol). Then the chlorophosphanes 7 (2 equiv,
0.75 mmol) were added within 5 min, and the solutions were
stirred for a further 20 min for completion (TLC analysis). For
oxidation of the intermediate cyclic phosphites, tert-butyl
hydroperoxide (0.072 g, 2.1 equiv, 0.80 mmol) was added to
the reaction mixture at 0 °C. After the mixture stirred for 1
h and warmed to room temperature, the solvent was removed
under reduced pressure. The residues were purified twice by
chromatography on silica gel plates on a chromatotron first
using a gradient of CH3OH in ethyl acetate (0-30% methanol)
followed by a gradient of CH3OH in CH2Cl2 (0-20% methanol)
to yield the title compounds 3.
Con clu sion
In summary, as for the previously reported cycloSal-
ddAMP triesters, the described pro-nucleotide concept
is suitable to deliver d4TMP (2) selectively from cy-
cloSal-d4TMPs 3a -h by a controlled, nonenzymatic
mechanism at physiological pH according to the tandem
reaction. The half-lives of hydrolysis could be controlled
by the substituents in the aromatic ring. All compounds
exhibited considerably higher partition coefficients than
the parent nucleoside d4T (1). Antiviral evaluation of
the compounds proved that all cycloSal-d4TMP deriva-
tives exhibited high antiviral activity in the wild-type
CEM cells that exceeded in a few cases even the activity
of the parent nucleoside d4T. Most important is the
complete retention of the biological activity in thymidine
kinase-deficient CEM cells for most of the compounds
confirming the selective delivery of d4TMP (2) inside
the cells and the considerably higher selectivity indices
of some compounds. The presented phosphotriesters
will be investigated further in cell lines for their
hydrolysis behavior and monophosphate-releasing mech-
anism in order to explore this pro-nucleotide concept.
Exp er im en ta l Section
cyclo-(5-Nitr osa ligen yl)-5′-O-(2′,3′-d id eoxy-2′,3′-d id eh y-
d r oth ym id in yl)p h osp h a te (3a ): yield 55%; 1H NMR (400
MHz, DMSO-d6) δ 11.31 (s, 1H, NH), 8.30 (m, 1H, H4-aryl),
All experiments involving water-sensitive compounds were
conducted under scrupulously dry conditions (argon atmo-
sphere). 2′,3′-Dideoxy-2′,3′-didehydrothymidine (d4T) (1) was
prepared according to the method of Mansuri.9b
Solven ts. Anhydrous acetonitrile was obtained in a Sure/
Seal bottle from Fluka and stored over 4-Å molecular sieves;
ethyl acetate, methylene chloride, and methanol for chroma-
tography were distilled before use. Triethylamine was distilled
from CaH2 prior to use. The solvents for the HPLC were
obtained from Merck (acetonitrile, HPLC grade) and Riedel-
de-Haen (water, HPLC grade). Evaporation of solvents was
carried out on a rotary evaporator under reduced pressure or
using a high-vacuum pump.
Ch r om atogr aph y. Chromatotron (Harrison Research 7924)
and silica gel 60PF (Merck, gipshaltig) were used; UV detection
at 254 nm. Column chromatography: Merck silica gel 60 (40-
60 µm). 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 fluorescent indicator;
sugar-containing compounds were visualized with the sugar
spray reagent (0.5 mL of 4-methoxybenzaldehyde, 9 mL of
ethanol, 0.5 mL of concentrated sulfuric acid, and 0.1 mL of
4
8.24 (2 × d, 1H, H6, J HHCH3 ) 1.1 Hz), 7.37 (2 × d, 1H, H3-
3
4
aryl, J HH4 ) 9.0 Hz), 7.17 (2 × d, 1H, H6-aryl, J HH4 ) 1.3
3
4
Hz), 6.78 (2 × ddd, 1H, H1′, J HH2′ ) 3.5 Hz, J HH3′ ) 1.8 Hz,
4J HH4′ ) 1.6 Hz), 6.40 (2 × ddd, 1H, H3′, 3J HH2′ ) 5.9 Hz, 3J HH4′
) 3.5 Hz, 4J HH1′ ) 1.8 Hz), 6.03 (2 × ddd, 1H, H2′, 3J HH3′ ) 5.9
3
4
Hz, J HH1′ ) 3.5 Hz, J HH4′ ) 1.4 Hz), 5.34 (2 × ddd, 2H, CH2-
2
3
3
benzyl, J HH ) 14.4 Hz, J HP ) 17.4 Hz, J HP ) 6.6 Hz), 4.94
2
3
(m, 1H, H4′), 4.28 (2 × dddd, 2H, H5′, J HH ) 11.5 Hz, J HP
)
3
3
7.2 Hz, J HP ) 6.9 Hz, J HH4′ ) 2.5 Hz), 1.66 (2 × d, 3H, CH3-
thymine, J HH6 ) 1.1 Hz); 31P NMR (162 MHz, DMSO-d6) δ
4
-9.10, -9.35; Rf value 0.48 (CH2Cl2/MeOH, 9:1); analytical
HPLC tR 17.60 min (96%, gradient I).
cyclo-(5-Ch lor osa ligen yl)-5′-O-(2′,3′-d id eoxy-2′,3′-d id e-
h yd r oth ym id in yl)p h osp h a te (3b): yield 49%; 1H NMR (400
MHz, DMSO-d6) δ 11.34 (s, 1H, NH), 7.43 (m, 1H, H6), 7.16
3
(m, 2H, H6, H3-aryl), 6.79 (2 × ddd, 1H, H1′, J HH2′ ) 3.9 Hz,
4J HH3′ ) 1.7 Hz, 4J HH4′ ) 1.8 Hz), 6.39 (2 × ddd, 1H, H3′, 3J HH2′
3
4
) 5.9 Hz, J HH4′ ) 3.4 Hz, J HH1′ ) 1.7 Hz), 6.01 (2 × ddd, 1H,
3
3
4
H2′, J HH3′ ) 5.9 Hz, J HH1′ ) 3.9 Hz, J HH4′ ) 1.7 Hz), 5.43 (2

1424 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Meier et al.
2
3
3
× ddd, 2H, CH2-benzyl, J HH ) 14.4 Hz, J HP ) 17.3 Hz, J HP
(C1-aryl), 122.24 (C5-aryl), 117.02 (C6-aryl), 112.69 (C4-aryl),
109.66 (C5), 89.11 (C1′), 84.06 (C4′), 68.27 (C5′), 68.12 (CH2-
benzyl), 55.85 (OCH3), 11.69 (CH3-thymine); 31P NMR (162
MHz, DMSO-d6) δ -7.82, -7.81; MS (ESI-) m/z 421.5; UV λmax
(CH3CN) 266 nm; IR (KBr) ν 3448, 3190, 3060, 1686, 1490,
1467, 1284, 1243, 1113, 1023 cm-1; Rf value 0.51 (CH2Cl2/
MeOH, 9:1); analytical HPLC tR 14.37 min (96%), tR 18.85 min
(99%, gradient II), tR 17.03, 17.16 min (98%, gradient III).
2
) 6.3 Hz), 4.95 (m, 1H, H4′), 4.30 (2 × dddd, 2H, H5′, J HH
)
11.6 Hz, 3J HP ) 7.3 Hz, 3J HP ) 6.7 Hz, 3J HH4′ ) 2.3 Hz), 1.66 (2
4
× d, 3H, CH3-thymine, J HH6 ) 1.0 Hz); 13C NMR (63 MHz,
DMSO-d6) δ 163.74 (C4), 150.74 (C2), 135.69 (C2-aryl), 132.75
(C3′), 129.59 (C6), 128.30 (C6-aryl), 127.37 (C2′), 126.11 (C5-
aryl), 123.31 (C4-aryl), 120.11 (C1-aryl), 119.97 (C3-aryl),
109.68 (C5), 89.24 (C1′), 84.08 (C4′), 68.58 (C5′), 67.75 (CH2-
benzyl), 11.91 (CH3-thymine); 31P NMR (162 MHz, DMSO-d6)
δ -8.69, -8.75; MS (ESI-) m/z 425.4; UV λmax (CH3CN) 266
nm; IR (KBr) ν 3504, 3186, 3059, 1690, 1481, 1302, 1248, 1189,
1114, 1029 cm-1; Rf value 0.49 (CH2Cl2/MeOH, 9:1); analytical
HPLC tR 16.77 min (99%, gradient I), tR 19.97 min (98%,
gradient II), tR 18.73 min (98%, gradient III).
cyclo-(5-Meth ylsa ligen yl)-5′-O-(2′,3′-d id eoxy-2′,3′-d id e-
h yd r oth ym id in yl)p h osp h a te (3f): yield 53%; 1H NMR (250
MHz, DMSO-d6) δ 11.34 (2 × s, 1H, NH), 7.68 (m, 1H, H6),
7.18 (m, 1H, H6-aryl), 7.16 (m, 1H, H4-aryl), 7.00 (m, 1H, H3-
3
4
aryl), 6.78 (2 × ddd, 1H, H1′, J HH2′ ) 3.5 Hz, J HH3′ ) 1.9 Hz,
4J HH4′ ) 1.6 Hz), 6.37 (2 × ddd, 1H, H3′, 3J HH2′ ) 5.9 Hz, 3J HH4′
) 3.3 Hz, 4J HH1′ ) 1.9 Hz), 6.00 (2 × ddd, 1H, H2′, 3J HH3′ ) 5.9
cycloSa ligen yl-5′-O-(2′,3′-d id eoxy-2′,3′-d id eh yd r ot h y-
m id in yl)p h osp h a te (3c): yield 61%; 1H NMR (400 MHz,
DMSO-d6) δ 11.23 (s, 1H, NH), 7.36 (m, 1H, H4-aryl), 7.27 (2
3
4
Hz, J HH1′ ) 3.5 Hz, J HH4′ ) 1.6 Hz), 5.39 (2 × ddd, 2H, CH2-
2
3
3
benzyl, J HH ) 14.4 Hz, J HP ) 17.6 Hz, J HP ) 6.9 Hz), 4.94
(m, 1H, H4′), 4.28 (2 × dddd, 2H, H5′, J HH ) 11.3 Hz, J HP
4
2
3
× d, 1H, H6, J HHCH3 ) 1.6 Hz), 7.18 (m, 2H, H5-aryl, H6-
)
3
3
3
aryl), 7.10 (m, 1H, H3-aryl), 6.78 (2 × ddd, 1H, H1′, J HH2′
)
7.2 Hz, J HP ) 6.9 Hz, J HH4′ ) 2.5 Hz), 2.25 (s, 3H, CH3-C5-
aryl), 1.65 (2 × s, 3H, CH3-thymine); 13C NMR (63 MHz,
DMSO-d6) δ 163.73 (C4), 150.71 (C2), 147.42 (C2-aryl), 135.68
(C6), 133.73 (C3′), 132.84 (C5-aryl), 130.14 (C6-aryl), 127.28
(C2′), 126.18 (C4-aryl), 120.82 (C1-aryl), 117.83 (C3-aryl),
109.67 (C5), 89.18 (C1′), 84.11 (C4′), 68.43 (C5′), 68.23 (CH2-
benzyl), 20.14 (CH3-C5-aryl), 11.94 (CH3-thymine); 31P NMR
(162 MHz, DMSO-d6) δ -8.13, -8.20; MS (ESI-) m/z 405.4;
UV λmax (CH3CN) 265 nm; IR (KBr) ν 3168, 3038, 2929, 1696,
1498, 1466, 1307, 1207, 1115, 1036 cm-1; Rf value 0.52 (CH2-
Cl2/MeOH, 9:1); analytical HPLC tR 16.59 min (99%), tR 19.68
min (99%, gradient II), tR 18.28 min (98%, gradient III).
4
4
3.5 Hz, J HH3′ ) 1.9 Hz, J HH4′ ) 1.6 Hz), 6.37 (2 × ddd, 1H,
3
3
4
H3′, J HH2′ ) 5.9 Hz, J HH4′ ) 3.5 Hz, J HH1′ ) 1.9 Hz), 6.00 (2
3
3
4
× ddd, 1H, H2′, J HH3′ ) 5.9 Hz, J HH1′ ) 3.5 Hz, J HH4′ ) 1.6
2
3
Hz), 5.43 (2 × ddd, 2H, CH2-benzyl, J HH ) 14.5 Hz, J HP
)
3
16.8 Hz, J HP ) 5.5 Hz), 4.94 (m, 1H, H4′), 4.29 (2 × dddd,
2
3
3
3
2H, H5′, J HH ) 11.7 Hz, J HP ) 7.0 Hz, J HP ) 6.7 Hz, J HH4′
) 2.7 Hz), 1.64 (2 × d, 3H, CH3-thymine, J HH6 ) 1.6 Hz); 13
C
4
NMR (63 MHz, DMSO-d6) δ 163.74 (C4), 150.74 (C2), 149.54
(C2-aryl), 135.71 (C6), 132.82 (C3′), 129.91 (C6-aryl), 127.34
(C4-aryl), 126.20 (C2′), 124.49 (C5-aryl), 121.32 (C1-aryl),
118.12 (C3-aryl), 109.68 (C5), 89.20 (C1′), 84.12 (C4′), 68.39
(C5′), 68.28 (CH2-benzyl), 11.94 (CH3-thymine); 31P NMR (162
MHz, DMSO-d6) δ -8.22, -8.24; MS (ESI-) m/z 391.4; UV λmax
(CH3CN) 265 nm; IR (KBr) ν 3448, 3198, 3067, 1690, 1490,
1459, 1293, 1246, 1109, 1019 cm-1; Rf value 0.52 (CH2Cl2/
MeOH, 9:1); analytical HPLC tR 15.19 min (99%), tR 18.84 min
(99%, gradient II), tR 17.07 min (98%, gradient III).
cyclo-(3-m eth ylsa ligen yl)-5′-O-(2′,3′-d id eoxy-2′,3′-d id e-
h yd r oth ym id in yl)p h osp h a te (3g): yield 60%; 1H NMR (250
MHz, DMSO-d6) δ 11.34 (2 × s, 1H, NH), 7.25 (m, 1H, H4-
4
aryl), 7.18 (2 × d, 1H, H6, J HHCH3 ) 1.2 Hz), 7.16 (m, 2H,
3
H5-aryl, H6-aryl), 6.78 (2 × ddd, 1H, H1′, J HH2′ ) 3.5 Hz,
4J HH3′ ) 1.7 Hz, 4J HH4′ ) 1.8 Hz), 6.38 (2 × ddd, 1H, H3′, 3J HH2′
cyclo-(5-Meth oxysa ligen yl)-5′-O-(2′,3′-d id eoxy-2′,3′-d i-
d eh yd r oth ym id in yl)p h osp h a te (3d ): yield 73%; 1H NMR
(400 MHz, DMSO-d6) δ 11.27 (s, 1H, NH), 7.11 (2 × dd, 1H,
3
4
) 5.9 Hz, J HH4′ ) 3.5 Hz, J HH1′ ) 1.7 Hz), 6.01 (2 × ddd, 1H,
3
3
4
H2′, J HH3′ ) 5.9 Hz, J HH1′ ) 3.5 Hz, J HH4′ ) 1.7 Hz), 5.40 (2
2
3
3
× ddd, 2H, CH2-benzyl, J HH ) 14.3 Hz, J HP ) 17.6 Hz, J HP
4
3
H6, J HHCH3 ) 1.2 Hz), 6.97 (2 × d, 1H, H3-aryl, J HH4 ) 9.3
2
) 5.9 Hz), 4.94 (m, 1H, H4′), 4.28 (2 × dddd, 2H, H5′, J HH
)
3
4
Hz), 6.84 (2 × dd, 1H, H4-aryl, J HH3 ) 9.3 Hz, J HH6 ) 2.7
3
3
3
11.7 Hz, J HP ) 7.4 Hz, J HP ) 6.7 Hz, J HH4′ ) 2.7 Hz), 2.25
(s, 3H, CH3-C5-aryl), 1.65 (2 × s, 3H, CH3-thymine); 13C NMR
(63 MHz, DMSO-d6) δ 163.19 (C4), 150.18 (C2), 148.50 (C2-
aryl), 135.14 (C6), 132.76 (C3′), 130.46 (C4-aryl), 126.78 (C3-
aryl), 126.32 (C2′), 123.48 (C5-aryl), 123.15 (C6-aryl), 120.70
(C1-aryl), 109.12 (C5), 89.15 (C1′), 83.65 (C4′), 68.20 (C5′),
67.70 (CH2-benzyl), 14.22 (CH3-C3-aryl), 11.33 (CH3-thymine);
31P NMR (162 MHz, DMSO-d6) δ -7.48, -7.68; MS (ESI-) m/z
405.3; UV λmax (CH3CN) 265 nm; IR (KBr) ν 3448, 3189, 3049,
1690, 1473, 1288, 1190, 1113 cm-1; Rf value 0.51 (CH2Cl2/
MeOH, 9:1); analytical HPLC tR 16.25 min (99%), tR 19.40 min
(99%, gradient II), tR 18.09 min (98%, gradient III).
4
Hz), 6.80 (d, 1H, H6-aryl, J HH4 ) 2.7 Hz), 6.72 (2 × ddd, 1H,
3
4
4
H1′, J HH2′ ) 3.5 Hz, J HH3′ ) 1.9 Hz, J HH4′ ) 1.6 Hz), 6.30 (2
3
3
4
× ddd, 1H, H3′, J HH2′ ) 5.9 Hz, J HH4′ ) 3.5 Hz, J HH1′ ) 1.9
3
3
Hz), 5.94 (2 × ddd, 1H, H2′, J HH3′ ) 5.9 Hz, J HH1′ ) 3.5 Hz,
4J HH4′ ) 1.6 Hz), 5.32 (2 × ddd, 2H, CH2-benzyl, J HH ) 14.4
2
3
3
Hz, J HP ) 16.8 Hz, J HP ) 5.5 Hz), 4.87 (m, 1H, H4′), 4.20 (2
2
3
3
× dddd, 2H, H5′, J HH ) 11.6 Hz, J HP ) 7.3 Hz, J HP ) 6.6
Hz, 3J HH4′ ) 2.6 Hz), 3.66 (s, 3H, OCH3), 1.59 (2 × d, 3H, CH3-
thymine, J HH6 ) 1.2 Hz); 13C NMR (63 MHz, DMSO-d6) δ
4
163.75 (C4), 155.63 (C5-aryl), 150.73 (C2), 143.03 (C2-aryl),
135.71 (C6), 132.89 (C3′), 127.30 (C2′), 122.08 (C1-aryl), 119.07
(C3-aryl), 115.20 (C6-aryl), 110.81 (C4-aryl), 109.68 (C5), 89.18
(C1′), 84.13 (C4′), 68.29 (C5′), 68.27 (CH2-benzyl), 55.59
(OCH3), 11.94 (CH3-thymine); 31P NMR (162 MHz, DMSO-d6)
δ -8.08; MS (ESI-) m/z 421.5; UV λmax (CH3CN) 266 nm; IR
(KBr) ν 3448, 3164, 3036, 1685, 1498, 1458, 1288, 1198, 1115,
1027 cm-1; Rf value 0.50 (CH2Cl2/MeOH, 9:1); analytical HPLC
tR 15.19 min (99%), tR 19.24 min (99%, gradient II), tR 17.32
min (99%, gradient III).
cyclo-(3,5-Dim et h ylsa ligen yl)-5′-O-(2′,3′-d id eoxy-2′,3′-
d id eh yd r oth ym id in yl)p h osp h a te (3h ): yield 59%; 1H NMR
(400 MHz, DMSO-d6) δ 11.31 (2 × s, 1H, NH), 7.16 (2d, 1H,
4
H6, J HH ) 1.2 Hz), 7.03 (m, 1H, H4-aryl), 6.87 (m, 1H, H6-
3
4
aryl), 6.77 (2 × ddd, 1H, H1′, J HH2′ ) 3.5 Hz, J HH3′ ) 1.9 Hz,
4J HH4′ ) 1.6 Hz), 6.37 (2 × ddd, 1H, H3′, 3J HH2′ ) 5.9 Hz, 3J HH4′
) 3.5 Hz, 4J HH1′ ) 1.9 Hz), 6.00 (2 × ddd, 1H, H2′, 3J HH3′ ) 5.9
3
4
Hz, J HH1′ ) 3.5 Hz, J HH4′ ) 1.6 Hz), 5.34 (2 × ddd, 2H, CH2-
cyclo-(3-Meth oxysa ligen yl)-5′-O-(2′,3′-d id eoxy-2′,3′-d i-
d eh yd r oth ym id in yl)p h osp h a te (3e): yield 70%; 1H NMR
(400 MHz, DMSO-d6) δ 11.32 (s, 1H, NH), 7.17 (2 × d, 1H,
2
3
3
benzyl, J HH ) 14.4 Hz, J HP ) 17.6 Hz, J HP ) 6.4 Hz), 4.94
(m, 1H, H4′), 4.28 (2 × dddd, 2H, H5′, J HH ) 11.4 Hz, J HP
2
3
)
3
3
4
7.0 Hz, J HP ) 6.7 Hz, J HH4′ ) 2.7 Hz), 2.22 (s, 3H, CH3-C5-
aryl), 2.14 (2s, 3H, CH3-C3-aryl), 1.63 (2d, 3H, CH3-thymine,
4J HH6 ) 1.2 Hz); 13C NMR (68 MHz, DMSO-d6) δ 163.68 (C4),
150.68 (C2), 145.89 (C2-aryl), 135.64 (C6), 133.11 (C3′), 132.80
(C5-aryl), 131.41 (C6-aryl), 127.28 (C2′), 126.49 (C4-aryl),
123.69 (C3-aryl), 120.87 (C1-aryl), 109.64 (C5), 89.19 (C1′),
84.12 (C4′), 68.28 (C5′), 68.12 (CH2-benzyl), 20.09 (CH3-C5-
aryl), 14.70 (CH3-C3-aryl), 11.94 (CH3-thymine); 31P NMR (162
MHz, DMSO-d6) δ -7.32, -7.63; MS (ESI-) m/z 442.6; UV λmax
(CH3CN) 265 nm; IR (KBr) ν 3448, 3186, 3069, 1690, 1482,
1288, 1201, 1113, 1027 cm-1; Rf value 0.51 (CH2Cl2/MeOH,
H6, J HHCH3 ) 1.2 Hz), 7.11 (m, 2H, H5-aryl, H6-aryl), 6.83
(m, 1H, H4-aryl), 6.80 (2 × ddd, 1H, H1′, 3J HH2′ ) 3.4 Hz, 4J HH3′
) 1.7 Hz, 4J HH4′ ) 1.5 Hz), 6.36 (2 × ddd, 1H, H3′, 3J HH2′ ) 5.9
3
4
Hz, J HH4′ ) 3.3 Hz, J HH1′ ) 1.7 Hz), 6.00 (2 × ddd, 1H, H2′,
3J HH3′ ) 5.9 Hz, J HH1′ ) 3.4 Hz, J HH4′ ) 1.4 Hz), 5.43 (2 ×
3
4
2
3
3
ddd, 2H, CH2-benzyl, J HH ) 14.4 Hz, J HP ) 17.1 Hz, J HP
)
)
2
6.7 Hz), 4.97 (m, 1H, H4′), 4.29 (2 × dddd, 2H, H5′, J HH
3
3
3
11.5 Hz, J HP ) 7.1 Hz, J HP ) 6.8 Hz, J HH4′ ) 2.5 Hz), 3.32
(s, 3H, OCH3), 1.66 (2 × s, 3H, CH3-thymine); 13C NMR (63
MHz, DMSO-d6) δ 163.70 (C4), 150.68 (C2), 148.20 (C3-aryl),
138.90 (C2-aryl), 135.52 (C6), 132.79 (C3′), 127.28 (C2′), 124.57

Synthesis, Evaluation of a New d4TMP Delivery System
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1425
9:1); analytical HPLC tR 17.08 min (99%), tR 20.41 min (98%,
gradient II), tR 18.84 min (99%, gradient III).
cell culture infective dose (CCID50)/mL of cell suspension (1
CCID50 being the dose infective for 50% of the cell cultures).
Then 100 µL of the infected cell suspension was transferred
to microtiter plate wells, mixed with 100 µL of the appropriate
dilutions of compound, and further incubated at 37 °C. After
5 days, the number of viable cells was determined in a blood-
cell-counting chamber by Trypan blue staining for both virus-
infected and mock-infected cell cultures. The 50% effective
concentration (EC50) and the 50% cytotoxic concentration
(CC50) of the test compounds were defined as the compound
concentrations required to inhibit virus-induced cytopathicity
by 50% or to reduce the number of viable cells in mock-infected
cell cultures by 50%, respectively.
Deter m in a tion of th e P a r tition Coefficien ts. Pa values
were determined as follows: A sample of the compounds 3a -h
was dissolved in 1.0 mL of 1-octanol. To this solution was
added 1.0 mL of aqueous phosphate buffer solution (10 mmol,
pH 6.8). After the phases were mixed extensively for 10 min
(vortex) and separation by centrifugation (3 min at 9000 rpm),
aliquots of each phase were analyzed by analytical HPLC
(Merck LiChroCART column filled with LiChrospher 100
reversed-phase silica gel RP 18 (5 µm); gradient, 11-100%
CH3CN in water (0-18 min), 100% CH3CN (18-20 min), 11%
CH3CN (20-32 min); flow, 0.5 mL; UV detection at 265 nm).
By quantification of the peak areas, the Pa values were
obtained from the quotient.
Kin etic Da ta . (a ) All Aqu eou s Bu ffer s: Aqueous buffer
(1.0 mL) (30 mM TRIS buffer, pH 6.9; 30 mM sodium
phosphate buffer, pH 7.3; 30 mM sodium borate buffer, pH
8.9) was equilibrated at 37 °C and mixed with 0.5 mL of a
solution of 1.0 mg of compound 3a -h in 1.2 mL of H2O (∼1.9
mM) also equilibrated at 37 °C (final concentration of the
hydrolysis mixture: ∼0.63 mM). For the kinetic data, aliquots
(100 µL) of the hydrolysis mixture were stopped by the addition
of 5.0 µL of acetic acid and subsequently analyzed by analytical
HPLC (Merck LiChroCART column filled with LiChrospher
100 reversed-phase silica gel RP 18 (5 µm); gradient, 9-100%
CH3CN in 0.6 mM tetrabutylammonium phosphate, pH 3.8
(0-20 min) and 9% CH3CN (20-30 min); 0.5 mL flow; UV
detection at 265 nm). The degradation of 3 was followed by
quantification of the peak areas in the HPLC chromatograms.
The rate constants k were determined from the slope of the
logarithmic degradation curve of the title compounds. The
half-lives (t1/2) were calculated by using the rate constants k.
Ack n ow led gm en t. We gratefully acknowledge the
support by the Adolf-Messer-Stiftung, the Deutsche
Forschungsgemeinschaft (DFG Grant Me 1161/2-1), the
Fonds der Chemischen Industrie, and the Human
Capital & Mobility and Biomedical Research Pro-
gramme of the European Commission. We thank Mrs.
Ann Absillis for excellent technical help.
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, and 31P
NMR spectral data for the title compounds 3a -h (23 pages).
Ordering information is given on any current masthead page.
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(b) Rosw ell P a r k Mem or ia l In stitu te (RP MI)-1640
Cu ltu r e Med iu m Con ta in in g 10% Hea t-In a ctiva ted F CS:
This medium was used instead of aqueous buffers, and the
data were collected in the same way.
Ma ss Sp ectr om etr ic An a lysis. As for the kinetic studies,
∼0.63 mM solutions of the 5-chloro-cycloSal-d4TMP (3b) or
the 3-methoxy-cycloSal-d4TMP (3e) were prepared by solubi-
lizing the cycloSal derivative in 10 µL of acetonitrile, diluting
with HPLC-grade water (Riedel-de-Haen). The solution was
stored at 50 °C in a water bath. After several time intervals
aliquots were taken and analyzed by means of electrospray
mass spectroscopy run in the negative mode. The appropriate
mass peaks of the starting materials, the intermediate phos-
phodiesters, the salicyl alcohols, and d4TMP were detected
with their 35Cl/37Cl and 12C/13C isotope peaks. Subsequently,
the same concentrations were prepared in 10% normalized
[18O]water (Aldrich). Again, the hydrolysates were analyzed
by mass spectrometry, and the amounts of 18O-incorporation
were calulated.
An tir etr ovir a l Eva lu a tion . Human immunodeficiency
virus type 1 [HIV-1 (HTLV-IIIB)] was originally obtained from
a persistently HIV-infected H9 cell line as described previ-
ously38 which was kindly provided by Dr. R. C. Gallo (National
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prepared from the supernatants of HIV-1-infected MT-4 cells.
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(Pasteur Institute, Paris, France), and virus stocks were
prepared from the supernatants of HIV-2-infected MT-4 cells.
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Collection (Rockville, MD), and CEM/TK- cells were a gift from
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Stockholm, Sweden). CEM cells were infected with HIV as
previously described.39 Briefly, 4 × 105 cells/mL were infected
with HIV-1 or HIV-2 at ∼100 CCID50 (50% cell culture infective
dose)/mL of cell suspension. Then 100 µL of the infected cell
suspension was transferred to 96-well microtiter plate wells
and mixed with 100 µL of the appropriate dilutions of the test
compounds. After 4 days giant cell formation was recorded
microscopically in the HIV-infected cell cultures.
MT-4 cells (5 × 105 cells/mL) were suspended in fresh
culture medium and infected with HIV-1 at 100 times the 50%

1426 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Meier et al.
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(27) The Rp/Sp-configuration was assigned by the help of X-ray
analyses of both diastereomers of cyclo-(5-chlorosaligenyl)-(-)-
menthylmonophosphate used as model compound. The two
diastereomers of this model compound consistently exhibited
opposite Cotton effects in the CD spectra with respect to the
phosphorus configuration: whereas the Rp-isomer showed a
negative Cotton effect, the Sp-isomer showed a positive Cotton
effect at 224 nm. The attribution of configuration at the
phosphorus atom of the separated cycloSal-d4TMPs was carried
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