S-acyl-2-thioethyl aryl phosphotriester derivatives of AZT: Synthesis, antiviral activity, and stability study

Article abstract of DOI:10.1021/jm021016y

The synthesis, antiviral activity, and stability study of phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing modified L-tyrosinyl residues are reported. These compounds were obtained via phosphoramidite (P^III) chemistr

Full text of DOI:10.1021/jm021016y

782
J . Med. Chem. 2003, 46, 782-793
S-Acyl-2-Th ioeth yl Ar yl P h osp h otr iester Der iva tives of AZT: Syn th esis,
An tivir a l Activity, a n d Sta bility Stu d y
Suzanne Peyrottes,† Gae¨lle Coussot,† Isabelle Lefebvre,† J ean-Louis Imbach,† Gilles Gosselin,†
Anne-Marie Aubertin,‡ and Christian Pe´rigaud*,†
UMR 5625 CNRS-UMII, Universite´ Montpellier II, Case Courrier 008, Place Euge`ne Bataillon,
34095 Montpellier Cedex 05, France, and Laboratoire de Virologie de la Faculte´ de Me´decine, Unite´ 74 I.N.S.E.R.M.,
Universite´ L. Pasteur, 3, Rue Koeberle´, 67000 Strasbourg, France
Received August 2, 2002
The synthesis, antiviral activity, and stability study of phosphotriester derivatives of 3′-azido-
2′,3′-dideoxythymidine (AZT) bearing modified L-tyrosinyl residues are reported. These
compounds were obtained via phosphoramidite (PIII) chemistry from the appropriate aryl
precursors. All the derivatives were evaluated for their in vitro anti-HIV activity, and they
appeared to be potent inhibitors of HIV-1 replication in various cell culture experiments, with
EC50 values between the micro- and nanomolar range, especially in thymidine kinase deficient
(TK-) cells, showing their ability to act as mononucleotide prodrugs. The proposed decomposition
process of these mixed mononucleoside aryl phosphotriesters successively involves an esterase
and a phosphodiesterase hydrolysis.
In tr od u ction
Resu lts a n d Discu ssion
Ch em istr y. A simple retrosynthetic route to the
target compounds is presented in Scheme 1. Briefly,
the mixed phosphotriester derivatives 2-8 could be
obtained by in situ oxidation of the corresponding
phosphite triesters containing AZT. Thus, the mixed
phosphoramidite agents bearing the two phosphate
protections consist of subsequent coupling of the tBu-
SATE chain moiety and the appropriate tyrosinyl
residue on the commercially available bis(diisopropyl-
amino) chlorophosphine. The choice of the protecting
groups borne by the L-tyrosinyl analogues should be
done thoughtfully. Indeed, the cleavage conditions of
such groups have to be compatible with the stability of
the final derivatives, which possess base and nucleophile
sensitive functions (thioester, phosphotriester). Conse-
quently, we decided to use acid-labile protecting groups
(tert-butyloxycarbonyl, tBoc, and isopropylidene) and/
or groups susceptible to being hydrolyzed by an enzy-
matic system during biological studies (i.e., acetyl group)
to keep the integrity of the final compounds.
The tyrosinol precursor 10 (Scheme 2) was obtained
in two steps starting from the commercially available
N-R-Boc tyrosine methyl ester. The ester was reduced
with NaBH4/LiCl6,7 to the primary alcohol 9 and then
converted to the oxazolidine 10 by treatment with 2,2-
dimethoxypropane in the presence of a catalytic amount
of p-toluenesulfonic acid7,8 in acetone. The reaction
conditions of the reductive step had to be forced (over-
night, solvent reflux) in order to increase the yield.
Indeed, it has previously been demonstrated that esters
bearing phenoxy groups are not efficiently reduced.9 We
assume that the free phenol function of tyrosine could
interfere with sodium borohydride, forming various
alkoxy hydroborates and uselessly consuming the re-
agent.
In the past decade, an increasing number of research
groups have focused their attention on the study of
mononucleotide prodrugs, namely, pronucleotides.1-4
Such pronucleotides were designed to give rise to the
intracellular delivery of 5′-mononucleotides, the latter
being further metabolized to the corresponding mono-
nucleoside triphosphate analogue in order to exert
antiviral activity.
In this area, we have recently reported the potentiali-
ties of mixed mononucleoside phosphotriester deriva-
tives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing
one S-pivaloyl-2-thioethyl (tBuSATE) group and an aryl
residue as biolabile phosphate protection.5 In this previ-
ous study, we have demonstrated that the antiviral
activity of the related mononucleotide aryl (tBuSATE)
phosphotriester derivatives could be influenced by the
ionizable, polar, or lipophilic nature of the aryl substit-
uents. In the case of the tyrosinyl (tBuSATE) phospho-
triester derivative (Figure 1, compound 1) the presence
of the zwitterion, especially the free carboxylate func-
tion, hindered the cellular passive diffusion of the
compound and led to a loss of the antiviral activity in
CEM thymidine kinase-deficient (TK-) cells. In this
respect, we decided to design novel mixed phosphotri-
ester derivatives of AZT incorporating polar but not
anionic residues in order to study their influence
regarding anti-HIV activities, kinetic decomposition
parameters, and lipophilicity. Herein, the synthesis and
the study of derivatives where the carboxylate group of
L-tyrosine has been replaced by an alcohol (Figure 1,
compounds 3 and 4) or an amide (Figure 1, compounds
5-8) function are reported.
* To whom correspondence should be addressed. Phone: 33(0)-
467144776. Fax: 33(0)467042029. E-mail: perigaud@univ-montp2.fr.
† Universite´ Montpellier II.
The 1H NMR spectral data of the N-R-(Boc)oxazolidine
derivative of L-tyrosine 10 were complex regarding the
‡ Universite´ L. Pasteur.
10.1021/jm021016y CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/01/2003

Phosphotriester Derivatives of AZT
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 783
F igu r e 1. Structures of the studied phosphotriester derivatives of AZT.
Sch em e 1. Retrosynthetic Route to the Desired Derivatives 2-8
Sch em e 2. Synthesis of the Tyrosinol Precursor, 10a
Scheme 3 shows the pathway used for the preparation
of tyrosinamide precursors 13 and 17. Attempts to
directly use the commercially available N-R-acetyltyro-
sinamide or N-R-Boc-tyrosinamide 14 in the phosphora-
midite coupling reaction (Scheme 4) only gave rise to
byproducts; therefore, we decided to introduce Boc
protecting groups on the amide function. The same
synthetic steps were applied on both N-R-acetyl and Boc
derivatives. Because the introduction of the Boc protect-
ing group could not be carried out selectively on the
amide in the presence of the free phenol, the phenoxy
group has to be transiently masked with a tert-butyl-
(dimethyl)silyl group,10 giving rise to compounds 11 and
15. These O-protected derivatives were then succes-
sively treated by Boc anhydride in the presence of DIEA
and then by TBAF solution, yielding either the N,N-R-
acetyl-Boc-N′,N′-diBoc-tyrosinamide 13 or to the N-R-
Boc-N′,N′-diBoc-tyrosinamide 17.
a
Reagents: (i) NaBH4, LiCl, THF reflux; (ii) (CH3O)2C(CH3)2,
pTs acid cat., acetone.
presence of rotamers, which slowly interconvert at room
temperature (20 °C), since, for example, two multiplets
were observed for the H-R of the amino acid.8 When the
NMR spectrum was recorded at 55 °C, we obtained a
simplified spectrum. The same doubling effect was
observed for the corresponding phosphotriester 2, which
exhibits four signals in 31P NMR spectrum (δ -5.11,
-5.22, -5.34, -5.36 ppm in CDCl3) with a ratio of 1:1:
2:2 instead of the two expected (ratio, 1:1) because of
the presence of diastereoisomers on the phosphorus
stereocenter. Furthermore, this splitting effect disap-
peared after the selective hydrolysis of the N,O-acetal,
giving rise to phosphotriesters 3 and 4, which showed
only two signals in 31P NMR (δ -5.17 and -5.55 ppm
in CDCl3, with a ratio of 1:1), thus confirming the
presence of a mixture of rotamers and not of the
racemization process.
Synthesis of the desired mixed aryl phosphotriester
derivatives was accomplished via PIII (phosphoramidite)
chemistry (Scheme 4), previously described for the
tyrosinyl (tBuSATE) phosphotriester derivative of AZT.5
The tyrosinyl precursors 10, 13, and 17 were condensed
in the presence of 1H-tetrazole with the SATE phos-
phorobisamidite reagent.11 Then, reaction of the ad-
equate tyrosinyl (tBuSATE) phosphoramidite interme-
diates 18a -c with AZT, followed by in situ oxidation
using tert-butyl hydroperoxide, gave rise to the fully

784 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Peyrottes et al.
Sch em e 3. Synthesis of the Tyrosinamide Precursors 13 and 17a
a
Reagents: (i) TBDMSiCl, imidazole, pyridine; (ii) (Boc)2O, DMAP, DIEA, CH2Cl2; (iii) TBAF, THF.
Sch em e 4. General Approach for the Synthesis of Aryl Phosphotriester Derivatives 2-8a
a
Reagents: (i) tBuSATEO-P(NiPr2)2, 1H-tetrazole, HN(iPr)2, CH3CN; (ii) AZT, 1H-tetrazole, CH3CN, then tBuOOH, toluene; (iii) various
acidic treatments.
protected phosphotriester derivatives 2, 5, and 6 in 82%,
60%, and 63% overall yields, respectively.
phosphorus atom. The diastereomeric ratio (1:1) was
determined by either HPLC or 31P NMR.
Treatment of compounds 2, 5, and 6 in various acidic
conditions allowed us to obtain the partially protected
entities 3 and 8, and also the fully deprotected aryl
(tBuSATE) phosphotriester derivatives of AZT 4 and 7.
Compound 2 was reacted with 1 equiv of p-toluene-
sulfonic acid in order to remove selectively the isopro-
pylidene group and to give rise to the N-R-Boc-tyrosinol
analogue 3. The fully deprotected phosphotriester 4
could be obtained from 2 using a saturated solution of
dry HCl in ether (35% in weight), leading directly to
the hydrochloride salt, in 75% yield. Finally, the removal
of the tBoc group from both 5 and 6 was carried out
using trifluoroacetic acid/dichloromethane solution (10%
in volume) in time-controlled reactions, and phospho-
triesters 7 and 8 could be isolated in good yields after
their conversion to the corresponding hydrochloride
salts.
An tivir a l Activity. The inhibitory effects on the
HIV-1 replication of the mixed phosphotriesters 2-8
were evaluated in three cell culture systems (Table 1)
in comparison to the parent nucleoside AZT and to the
reference mixed pronucleotide 1. In human T4 lympho-
blastoid CEM-SS and MT-4 cells, all compounds dis-
played activity against HIV-1 that was comparable to
that of the parent nucleoside analogue AZT, with 50%
effective concentration (EC50) about submicromolar
concentration range. In contrast to AZT, the tyrosinyl
(tBuSATE) phosphotriesters 2-8 exhibited significant
anti-HIV effects in CEM/TK- cells with EC50 values in
micromolar range, demonstrating the successful release
of the corresponding 5′-mononucleotide (AZTMP) inside
infected cells. Indeed, this cell line, highly deficient in
cytosolic TK, should be considered as an ideal in vitro
system to investigate the antiviral potency of thymine
containing nucleotide analogues that may release the
corresponding 5′-mononucleotide into cells.3 The differ-
Compounds 2-8 were isolated as diastereomeric
mixtures resulting from the stereochemistry on the

Phosphotriester Derivatives of AZT
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 785
Sch em e 5. Synthesis of Aryl Phosphodiester Derivatives 4a and 8a a
a
Reagents: (i) H-phosphonate monoester of AZT (triethylammonium form), PivCl, C5H5N, CH3CN, then I2, H2O, then Dowex Na+; (ii)
dry HCl in ether, then Dowex Na+.
Ta ble 1. Anti-HIV-1 Activitya (µM) in Three Cell Culture
same range. These results suggested that the prodrug
activation kinetics of such aryl phosphotriesters also
Systems and Apparent Partition Coefficients (log Papp) of Mixed
Aryl Phosphotriester Derivatives 2-8 in Comparison to the
played an important role in the observed in vitro anti-
Parent Nucleoside AZT and the Tyrosinyl (tBuSATE)
Phosphotriester Derivative 1 of AZT as a Reference
HIV activity.
Pronucleotide
St a b ilit y St u d ies. With a previously published
procedure,14 the decomposition pathways and kinetic
data of compounds 1, 4, 7, and 8 were studied at 37 °C,
in total CEM-SS extracts in order to mimic the behavior
of such pronucleotides inside the cells, as well as in
culture medium (RPMI1640 containing 10% heat inac-
tivated fetal calf serum) to evaluate the stability of the
studied phosphotriesters in the extracellular medium
used during cell culture antiviral evaluation. The me-
tabolites formed during the incubation of the phospho-
triesters were identified by HPLC-ESI-MS coupling
experiments and/or co-injection with authentic samples.
Thus, aryl phosphodiesters 4a and 8a were obtained
using standard H-phosphonate chemistry (Scheme 5).
The H-phosphonate monoester derivative of AZT15 was
coupled with the aryl precursor 10 or 13 in the presence
of pivaloyl chloride,16,17 followed by in situ oxidation
using iodine and water. The resulting aryl phosphodi-
esters 4b and 8b were treated with a saturated solution
of dry HCl in ether (35% in weight) and led to 4a and
8a as hydrochloride forms.
The rate constants of disappearance and the half-lives
of the studied phosphotriesters were calculated accord-
ing to pseudo-first-order kinetic models and optimized
using mono- or polyexponential regressions. These
kinetic models were in accordance with the experimen-
tal data. Best-fit computed curves are shown in Figure
2, and the deduced half-lives of the starting materials
and intermediates in various media are given in Tables
2 and 3.
These experiments allowed us to propose a decompo-
sition mechanism for the tyrosinol phosphotriester 4
(Scheme 6) similar to the one previously published for
the tyrosinyl entity 1.18 In both media (cell extracts and
culture medium), the first step is an esterase-mediated
activation with the loss of the tBuSATE chain, leading
to the formation of the corresponding aryl phosphodi-
ester derivative of AZT 4a . This metabolite is then a
substrate for a second enzymatic activity that gives rise
to the formation of AZTMP. As expected, compound 4
metabolized very slowly (half-life of about a week) in
culture medium compared to cell extracts.
In cell extracts, the decomposition pathways observed
for the tyrosinamide phosphotriester derivatives 7 and
8 were more complex, involving two concomitant hy-
drolysis reactions in the first step (Scheme 6). For
compound 7, the main decomposition way (93%) results
CEM-SS
MT-4
CEM/TK-
b
c
b
c
b
c
compd EC50 CC50 EC50 CC50 EC50 CC50
log Papp
AZT 0.003 >100 0.015 >100 >100 >100 0.06 ( 0.003
1
2
3
4
5
6
7
8
0.006 >100 0.075
>10
>10
>10
29 >100 0.25 ( 0.015
0.031
0.017
>10 0.098
>10 0.044
9
4
7
4
6
4
8
>10 3.7d
>10 2.5d
0.007 >100 0.018 >100
>100 0.64 ( 0.02
0.049
0.015
0.002
0.082
>10 0.034
>10 0.032
>10 0.016
>10 0.066
>10
>10
>10
>10
>10 5d
>10 5.8d
>10 1.27 ( 0.005
>10 1.52 ( 0.006
a
All data represent average values of at least three separate
experiments. The variation of these results under standard
b
operating procedures is below (10%. EC50: effective concentra-
tion or concentration required to inhibit the replication of HIV-1
by 50%. c CC50: cytotoxic concentration or concentration required
d
to reduce the viability of uninfected cells by 50%. Calculated
values. The log P determination was performed using Advanced
Chemistry Development (Toronto, Canada) log P dB 4.5 calcula-
tions.
ences observed in the anti-HIV activities of the mixed
phosphotriester derivatives of AZT in the TK+ and TK-
cell lines may be explained with regard to the particular
metabolism of this nucleoside analogue and to the
kinetic parameters involved in the decomposition of each
derivative.12,13
The log P value for each target compound was either
calculated or obtained experimentally (Table 1). As
expected, the replacement of the carboxylate functional-
ity on the tyrosinyl residue and the presence of protect-
ing groups on the amino function increased the lipophi-
licity of the resulting phosphotriester in a stepwise
range of 0-6 log P values.
Comparison between anti-HIV activities in CEM/TK-
cells and apparent partition coefficients of phosphotri-
ester derivatives 1-8 (Table 1) showed that lipophilicity
is not the only factor involved in the observed in vitro
biological effect. Indeed, the replacement of the car-
boxylate by polar but not anionic groups such as alcohol
or amide functions allowed us to obtain water-soluble
pronucleotides without drastically losing the antiviral
activity. Indeed, the tyrosinol analogue 4 exhibited a
low log P value (log Papp ) 0.64, Table 1) but retained
the antiviral activity in the thymidine-kinase-deficient
cell line compared to derivative 1 (log Papp ) 0.25, Table
1). Despite marked differences in their lipophilicity, the
EC50 values for the phosphotriesters 2-8 are in the

786 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Peyrottes et al.
F igu r e 2. Decomposition kinetics in total CEM-SS cell extracts at 37 °C of phosphotriesters 1 ([), 4, 7, and 8 (2) at 50 µM initial
concentration and their corresponding metabolites phosphodiesters 1a (/), 4a , 7a , 8a (9) and AZTMP (b).
Ta ble 2. Calculated Half-Lives of the Phosphotriester
Derivatives 1, 4, 7, 8 and Their Metabolites in CEM-SS Cell
Extracts and Corresponding Pseudo-First-Order Rate
Constantsa
Ta ble 3. Calculated Half-Lives of the Phosphotriester
Derivatives 1, 4, 7, and 8 in Culture Medium
compound
1
4
7
8
compound
metabolite
1
1a
t1/2 (day)
metabolite observed
13.9
AZTMP
7.4
4a
7.7
AZTMP
5.5
AZTMP
t1/2 (h)
1.2
0.0096
25
k (min-1
)
)
0.00046
rise to the formation of the corresponding phosphodi-
ester 7a . The resulting metabolites 1 and 7a were then
converted to the tyrosinyl phosphodiester 1a , which
was further hydrolyzed into AZTMP. These two ways
of hydrolysis were also observed during the study of
the N-R-acetyltyrosinamide phosphotriester 8 in total
CEM-SS cell extracts (Scheme 6). Nevertheless, com-
pared to its analogue 7, the major decomposition
pathway (>95%) for the N-R-acetyl derivative 8 corre-
sponds to the removal of the tBuSATE chain and
formation of phosphodiester 8a . In this case, metabolite
1a was not observed and 8a was directly converted into
AZTMP. In the culture medium, the decomposition
patterns were similar for phosphotriesters 7 and 8, the
corresponding phosphodiesters could not be detected
(shorter half-life and/or insufficient accumulation), and
only the formation of AZTMP was observed.
compound
metabolite
4
4a
t1/2 (h)
2.5
0.0046
43.1
0.00027
k (min-1
compound
metabolite
7
1 (93%)
7a (7%)
1a
t1/2
k (min-1
23 min
0.030
2.3 h
0.005
6 h
0.0019
33.5 h
0.00034
)
metabolite
compound
8
1 (<5%)
ND
8a (>95%)
t1/2 (h)
k (min-1
1.9
0.0061
13.1
0.00088
)
a
All data represent average values for three separate experi-
ments, and the accuracy was lower than 10%.
in the hydrolysis of the amide function to the corre-
sponding acid and leads to phosphotriester 1 (as il-
lustrated by the HPLC chromatogram and correspond-
ing MS spectra, Figure 3), whereas the minor way
corresponds to the loss of the tBuSATE chain, giving
Because the behavior of the tyrosinamide phospho-
triesters (7 and 8) was puzzling, we attempted to
determine which kind of enzymatic system could be
involved in the hydrolysis of the amide bond. Examina-
tion of the literature19-21 showed that several enzymes,

Phosphotriester Derivatives of AZT
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 787
F igu r e 3. HPLC chromatogram and related ESI-MS spectra obtained after incubation of phosphotriester 7 (50 µM initial
concentration) in CEM-SS cell extracts at 37 °C for 75 min.
Sch em e 6. Proposed Decomposition Mechanism for Aryl Phosphotriester Derivatives 1, 4, 7, and 8 in Total CEM-SS
Cell Extracts
called amidases, from various organisms could be
responsible for the hydrolysis of aliphatic and aromatic
amides to the corresponding carboxylic acids and am-
monia. However, in the amidase family, only the EC
3.5.1.4 from Pseudomonas aeruginosa was commercially
available and catalyzed the hydrolysis of small-chain
aliphatic amides. Indeed, this enzyme was not able to
accept phosphotriester 7 as the substrate. We investi-
gated the behavior of 7 in the presence of various
inhibitors of the amidase family such as metal ions
(Hg2+ and Zn2+) and phenylmethanesulfonyl fluoride
(PMSF).22-25 The enzyme activity responsible for the
decomposition of 7 in 1 was completely inhibited by
these metal ions and no degradation of 7 was observed
after 3 h of incubation in cell extracts preincubated with
1 mM of either HgCl2 or ZnCl2, indicating that this

788 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Peyrottes et al.
Ta ble 4. Calculated Half-Lives of the Phosphodiester
Derivatives 1a , 4a , and 8a in Cell Extracts (CE) and
Heat-Inactivated Cell Extracts
column (Nucleosil, C18, 150 mm × 4.6 mm, 5 µm), equipped
with a prefilter, and a precolumn (Nucleosil, C18, 5 µm) were
used. Detection was monitored at 267 nm. Nucleotidic deriva-
tives were eluted using a linear gradient of 0-80% acetonitrile
in 20 mM triethylammonium acetate buffer (pH 6.9) over 30
min at 1 mL/min flow rate. For amino acids, eluents used
contained 0.1% aqueous TFA, and the compounds were eluted
with a linear gradient of 0-90% acetonitrile over 30 min at 1
mL/min flow rate. Electrospray ionization mass spectrometry
(ESI-MS) was performed using a SSQ 7000 single quadrupole
mass spectrometer (Finnigan, San J ose, CA) in the negative-
ion mode with a spray voltage at - 4.5 kV. The capillary
temperature was maintained at 250 °C. Nitrogen served both
as sheath gas (operating pressure of 80 psi) and as auxiliary
gas with a flow rate of 15 units. Under these conditions, full-
scan data acquisition was performed from m/z 200 to 800 in
centroid mode and using a cycle time of 1.0 s. AZT was from
Brantford Chemicals Inc., bis(N,N-diisopropylamino)chloro-
phosphine was purchased from Aldrich, N-R-tert-butoxycar-
bonyl-L-tyrosine methyl ester, and N-R-acetyl-L-tyrosinamide
were from Novabiochem. S-(2-Hydroxyethyl) thiopivaloate14
and O-(S-pivaloyl-2-thioethyl) N,N,N′,N′-tetraisopropylphos-
phorodiamidite11 were synthesized following previously pub-
lished procedures.
compound
1a
4a
68
8a
19.3
CE (h)
12.8
CE inactivateda
stable
stable
stable
stable
stable
CE preincubated with EDTAa
6.9 daysb
a
Stable means no degradation observed after 5 days of incuba-
b
tion. The half-life corresponds to the conversion of the amide
function of 8a in acid (putative amidase activity). The resulting
N-R-acetyltyrosinyl phosphodiester is then stable.
activity was highly sensitive to such sulfhydryl reagents.
However, only partial inhibition was observed with
PMSF, a well-known serine protease inhibitor. In this
case, the half-life of 7 was 2.5 h instead of 23 min.
The half-lives of the mononucleoside phosphotriesters
and the resulting metabolites are summarized in Tables
2 and 3. In cell extracts, the rate of the tBuSATE moiety
loss was similar for all phosphotriesters (between 2 and
3 h). However, comparison of the enzymatic stability of
the aryl phosphodiesters 1a , 4a , and 8a (Scheme 6)
demonstrated that the nature of the functionality on the
aryl residue (amino acid, amino alcohol or amino amide)
of the phosphodiester derivatives seems to influence the
hydrolysis rate of the phosphodiesterase-mediated step
(Table 2). In this respect, we studied the behavior of
1a , 4a , and 8a in cell extracts, either heat-inactivated
in order to confirm that their conversion into AZTMP
was not due to chemical process or preincubated with
EDTA, which is known as an inhibitor of type I
phosphodiesterase.26 The corresponding kinetic data
(Table 4) were in agreement with the fact that decom-
position of the studied phosphodiesters was under
enzymatic control, which is likely to be due to type I
phosphodiesterase activity.
Biologica l Meth od s. The origin of the viruses and the
techniques used for measuring inhibition of virus multiplica-
tion have previously been described.14
Sta bility Stu d ies. HPLC analyses were carried out using
an improved “on-line ISRP cleaning method”.14 Briefly, the
crude sample (80 µL, initial concentration 50 µM) was injected
onto the precolumn, and the eluent was 20 mM triethylam-
monium acetate buffer (pH 6.9, 5 min). Elution of the analytes
was done using a linear gradient of 0% to 80% acetonitrile over
30 min at 1 mL/min flow rate. The decomposition products
were identified by HPLC-MS after calibration and/or by co-
injection with authentic samples (AZTMP, AZT, phosphotri-
ester 1, phosphodiesters 1a , 4a , and 8a ). For each incubation
time, the calculation of the relative concentration of each
species was related to the peak areas. These data were
considered as the experimental data. The rate constants for
disappearance and the half-lives (kinetic data) of the phos-
photriester and phosphodiester derivatives were calculated
according to pseudo-first-order kinetic models and optimized
using mono- or polyexponential regressions.
Tota l CEM-SS Cell Extr a cts. CEM-SS cell extracts were
prepared, and stability studies were performed according to
published procedures.14 When needed, CEM-SS cell extracts
were inactivated 24 h at 56 °C or preincubated for 10 min with
4 mM EDTA and 30 min with 1 mM HgCl2, ZnCl2, or PMSF.
P a r tition Coefficien t. The apparent partition coefficients
(log Papp) were evaluated for their distribution between 1-oc-
tanol (puriss, Fluka) and potassium phosphate buffer (0.02 M,
pH 7.2) using an adapted shake-flask technique.28 Determina-
tions were performed in triplicate for each compound. For the
HPLC conditions, see the General Methods.
Con clu sion
The present study demonstrates that aryl (tBuSATE)
phosphotriester derivatives of AZT 2-5 are able to allow
the efficient intracellular delivery of the parent 5′-
mononucleotide and can be considered as pronucle-
otides. The proposed mechanism for the decomposition
of these mixed phosphotriesters involves successively
an esterase and a phosphodiesterase hydrolytic step.
The large number of chemical modifications that could
be envisaged on the aryl moiety opens the way to the
search of antiviral mononucleotide prodrugs with an
adequate balance among aqueous solubility, lipophilic-
ity, and enzymatic stability in order to envisage further
in vivo pharmacological studies. In this respect, the
structural features of the aryl phosphotriester 4, 7, and
8 appear as promising phosphate protecting groups in
view of anti-HIV activity and physicochemical properties
of the resulting pronucleotides. Further work on this
topic is currently in progress.
N-(ter t-Bu toxyca r bon yl)-L-tyr osin -ol, 9. Boc-Tyr-OMe
(1.47 g, 5 mmol) was dissolved in dry THF (15 mL), and lithium
chloride (0.85 g, 4 equiv) and sodium borohydride (0.76 g, 4
equiv) were added. The mixture was refluxed for 72 h, the
solution was cooled to room temperature, and methanol (5 mL)
was added. After the mixture was stirred for 12 h at room
temperature, an aqueous acetic acid solution (10%, v/v) was
added to lower pH (∼pH 5). The mixture was diluted with ethyl
acetate (100 mL), and the resulting solution was washed with
water (50 mL). The aqueous layer was extracted three times
with ethyl acetate. The combined organic layers were dried
over Na2SO4 and filtered, and the solvent was removed in
vacuum. Purification of the residue by column chromatography
on silica gel, eluting with a stepwise gradient of MeOH in CH2-
Cl2 (2:98), afforded compound 9 as a white foam that was
recrystallized from chloroform, 73% yield. Rf ) 0.23 (MeOH/
Exp er im en ta l Section
Gen er a l Meth od s. Unless otherwise stated, material and
methods used were as previously described.27 Analytical HPLC
studies were carried out on an Alliance 2690 system (Waters,
Milford, MA) equipped with a model 996 photodiode detector
and a Millenium data workstation. A reverse-phase analytical
CH2Cl2 5:95); mp 120 °C, lit. 112-114 °C;29 [R]20 -26° (c 1.0,
D
EtOH), lit. -23° (c 1.2, EtOH);7 1H NMR (CDCl3, 200 MHz) δ
7.10-6.90 (m, 3H, OHech, J ) 8.4, Hδ), 6.72 (d, 2H, J ) 8.4,

Phosphotriester Derivatives of AZT
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 789
Hꢀ), 5.11 (d, 1H, J ) 8.4, NHech), 3.76-3.42 (m, 4H, CH2OH,
HR), 2.69 (d, 2H, J ) 6.5, Hâ), 1.39 (s, 9H, C(CH3)3); 13C NMR
(CDCl3) δ 155.5, 153.7 (Cq, CON), 129.3 (Cγ), 128.1 (Cδ), 114.5
(Cꢀ), 79.0 (C(CH3)3), 63.3 (C-OH), 52.9 (CR), 35.6 (Câ), 28.3
(C(CH3)3); MS FAB+ m/z 535 (2M + H)+, 268 (M + H)+, 212
(M - tBu + H)+; MS FAB- m/z 533 (2M - H)-, 266 (M - H)-;
HPLC tR 15.3 min; UV (EtOH) λmax 225 nm (ꢀ 7370). Anal.
(C14H21NO4) C, H, N.
and the oily residue was purified by column chromatography
on silica gel, eluting with pentane/Et2O (0-10%). An amount
of 1.33 g (52%) of an oil was obtained. Rf ) 0.56 (Et2O/pentane
20:80); [R]20D +1.9 (c 1.1, EtOH); 1H NMR (DMSO-d6, 200 MHz)
δ 7.04 (d, 2H, J ) 8.4, Hδ), 6.78 (d, 2H, J ) 8.4, Hꢀ), 5.58-5.49
(m, 1H, HR), 3.35-3.21 (m, 1H, partly covered by HDO peak,
Hâa), 2.98-2.83 (dd, 1H, J ) 13.8 and 8.4, Hâb), 2.24 (s, 3H,
CH3acetyl), 1.45 (s, 18H, 2C(CH3)3Boc), 1.40 (s, 9H, C(CH3)3Boc),
0.94 (s, 9H, SiC(CH3)3), 0.17 (s, 6H, Si(CH3)2); 13C NMR
(DMSO-d6) δ 172.6 (CONamido), 171.0 (COCH3), 154.5 (Cq),
152.4, 150.0 (CONBoc), 131.5 (Cγ), 131.1 (Cꢀ), 120.3 (Cδ), 85.6,
84.5 (C(CH3)3Boc), 59.7 (CR), 34.8 (Câ), 27.9 (2s, C(CH3)3Boc), 26.9
(CH3CO), 26.4 (C(CH3)3), 18.8 (SiC(CH3)3), -3.7 (Si(CH3)); MS
FAB+ m/z 659 (M + Na)+, 637 (M + H)+, 537 (M - Boc + 2H)+;
MS FAB- m/z 1170 (2M - Boc - H)-, 535 (M - Boc)-. Anal.
(C32H52N2O9Si) C, H, N.
N-(ter t-Butoxycar bon yl)-2,2-dim eth yl-4-(4-h ydr oxyph en-
yl)-1,3-oxa zolid in e, 10. N-R-tert-Butoxycarbonyl-L-tyrosinol
(0.78 g, 2.9 mmol) was dissolved in dry acetone (20 mL), and
2,2′-dimethoxypropane (0.725 mL, 2 equiv) and p-toluene-
sulfonic acid (0.055 g, 0.1 equiv) were added. The mixture was
refluxed for 4 h. The solution was concentrated, and the
residue was dissolved in dichloromethane (25 mL). The organic
layer was washed with NaHCO3 solution (10% w/v, 20 mL).
The aqueous layer was extracted three times with CH2Cl2, the
combined organic layers were dried over Na2SO4 and filtered,
and the solvent was removed in vacuum. Purification of the
residue by column chromatography on silica gel, eluting with
a stepwise gradient of MeOH (0-1%) in CH2Cl2, afforded
compound 10 (0.70 g) as a white foam, 78% yield. Rf ) 0.51
N-r-(Acetyl-ter t-bu toxyca r bon yl)-N,N-d i(ter t-bu toxy-
ca r bon yl)-L-tyr osin a m id e, 13. TBAF (1.1 M THF solution,
1.9 mL, 1.1 equiv) was added dropwise to a solution of 12 (1.25
g, 1.9 mmol) in anhydrous THF (25 mL) at 0 °C. After 30 min
of stirring, the reaction mixture was diluted with ethyl acetate
and washed with saturated aqueous NaCl solution and water.
The organic layer was dried over Na2SO4 and concentrated,
and the oily residue was purified by flash column chromatog-
raphy on silica gel, eluting with pentane/Et2O (10-30%). An
(MeOH/CH2Cl2 5:95); mp 107 °C, lit. 106-107 °C 7; [R]20
D
-32.6° (c 0.98, EtOH), lit. -28° (c 1.28, HCCl3).8 Because of
the presence of conformer mixture, 1H NMR spectra were
processed at two different temperatures (20 and 55 °C) and
compared. 1H NMR (CDCl3, 200 MHz, 20 °C) δ 7.00-6.95 (2d,
2H, J ) 8.2, Hδ), 6.75-6.67 (2d, 2H, J ) 8.1, Hꢀ), 4.01-3.97,
3.88-3.85 (2m, 2 × 0.5H conformers, HR), 3.70-3.69 (2s, 2H,
CH2O), 3.20-2.98 (dd, 1H, J ) 13.0, 10.8, Hâa), 2.71-2.53 (dd,
1H, J ) 13.0, 10.6, Hâb), 1.47, 1.41 (2d, 6H, J ) 11.2, C(CH3)2),
1.45 (s, 9H, C(CH3)3); 1H NMR (CDCl3, 200 MHz, 55 °C) δ
7.11-7.00 (d, 2H, J ) 8.2, Hδ), 6.82-6.70 (d, 2H, J ) 8.4, Hꢀ),
4.11-3.97 (br s, 1H, HR), 3.85, 3.72 (2s, 2H, CH2O), 3.06 (d,
1H, J ) 12.4, Hâa), 2.70, 2.55 (2d, 1H, J ) 13.2, 10.2, Hâb),
1.57, 1.49 (2s, 6H, C(CH3)2), 1.53 (s, 9H, C(CH3)3); 13C NMR
(CDCl3) δ 155.1, 152.5 (Cq, CON), 130.9 (Cγ), 130.4 (Cδ), 115.9
(Cꢀ), 94.3 (C(CH3)2), 80.8 (C(CH3)3), 66.3 (CH2O), 59.7 (CR), 38.5
(Câ), 28.9 (C(CH3)3), 27.6, 24.3 (C(CH3)2); MS FAB+ m/z 922
(3M + H)+, 615 (2M + H)+, 308 (M + H)+, 252 (M - tBu +
2H)+; MS FAB- m/z 920 (3M - H)-, 613 (2M - H)-, 306 (M -
H)-, 250 (M - tBu)-; HPLC tR 22.7 min; UV (EtOH) λmax 226
nm (ꢀ 8660). Anal. (C17H25NO4) C, H, N.
amount of 0.78 g (79%) of an oil was obtained. Rf ) 0.59 (Et2O);
1
[R]20 - 105 (c 1, EtOH); H NMR (CDCl3, 200 MHz) δ 7.00
D
(d, 2H, J ) 8.4, Hδ), 6.6 (d, 2H, J ) 8.4, Hꢀ), 5.65-5.61 (m,
1H, HR), 3.37-3.21 (dd, 1H, J ) 13.9 and 6.3, Hâa), 3.01-2.95
(dd, 1H, J ) 13.9 and 8.1, Hâb), 2.29 (s, 3H, CH3acetyl), 1.42 (s,
18H, 2C(CH3)3Boc), 1.39 (s, 9H, C(CH3)3Boc); 13C NMR (CDCl3)
δ 173.2 (CONamido), 171.3 (COCH3), 155.2 (Cq), 152.6, 150.0
(CONBoc), 131.3 (Cγ), 129.6 (Cꢀ), 115.6 (Cδ), 85.2, 84.8 (C(CH3)3Boc),
60.2 (CR), 35.2 (Câ), 28.0, 27.9 (2s, C(CH3)3Boc), 26.8 (CH3CO);
MS FAB+ m/z 523 (M + H)+; MS FAB- m/z 1043 (2M - H)-,
521 (M - H)-, 321 (M - 2Boc + H)-; HPLC tR 29.2 min. Anal.
(C26H38N2O9) C, H, N.
N-r-ter t-Bu toxyca r bon yl-L-tyr osin a m id e, 14. Boc-Tyr-
OMe (0.88 g, 3 mmol) was dissolved in methanolic ammonia
(10 mL), and the mixture was stirred for 60 h at 5 °C. The
solvents were removed in vacuum. Purification of the residue
by crystallization from ethyl acetate afforded the title com-
pound 14 (0.75 g) as white solid in 90% yield. Rf ) 0.41 (MeOH/
CH2Cl2 1:9); mp 160.5-161.0 °C; [R]20D +1.8 (c 1.0, EtOH); 1H
NMR (DMSO-d6, 200 MHz) δ 9.19 (s, 1H, NHech), 7.33 (s, 1H,
CONH2a), 7.06-7.01 (m, 3H, OHech, Hδ), 6.99-6.62 (m, 3H,
CONH2b, Hꢀ), 4.06-3.96 (m, 1H, HR), 2.88-2.78 (m, 1H, Hâa),
2.67-2.54 (m, 1H, Hâb), 1.31 (s, 9H, C(CH3)3); 13C NMR
(DMSO-d6) δ 174.9, 174.8 (2s, CON), 156.3, 156.1 (Cq), 130.9
(Cγ), 129.1 (Cδ), 115.7, 115.5 (Cꢀ), 79.0 (C(CH3)3), 56.6 (CR), 37.5
(Câ), 28.9 (C(CH3)3); MS FAB+ m/z 561 (2M + H)+, 281 (M +
H)+, 225 (M - tBu + H)+; MS FAB- m/z 559 (2M - H)-, 279
(M - H)-; HPLC tR 13.1 min. Anal. (C14H20N2O4) C, H, N.
N -r-(Ace t yl)-O-(t er t b u t yl(d im e t h yl)silyl)-L-t yr osin -
a m id e, 11. tert-Butyl(dimethyl)silyl chloride (0.88 g, 1.2 equiv)
was added to a solution of N-R-(acetyl)-L-tyrosinamide (1 g,
4.5 mmol) and imidazole (0.74 g, 2.4 equiv) in dry pyridine
(10 mL). After 4 h of stirring, the reaction mixture was diluted
with ethyl acetate and poured into a saturated NaHCO3
aqueous solution. The aqueous layer was extracted with ethyl
acetate, and the combined extracts afforded after evaporation
a white solid that was recrystallized from hexanes/ethyl
acetate (1:9) to afford 1.40 g (93%) of white crystals. Rf ) 0.45
(MeOH/CH2Cl2 10:90); mp 173 °C; [R]20 +6.7 (c 1.05, EtOH);
D
N-r-(ter t-Bu toxycar bon yl)-O-(ter t-bu tyl(dim eth yl)silyl)-
L-tyr osin a m id e, 15. tert-Butyl(dimethyl)silyl chloride (0.45
g, 1.1 equiv) was added to a solution of 14 (0.75 g, 2.7 mmol)
and imidazole (0.40 g, 2.2 equiv) in dry pyridine (10 mL). After
4 h of stirring, the reaction mixture was diluted with ethyl
acetate and poured into a saturated NaHCO3 aqueous solution.
The aqueous layer was extracted with ethyl acetate, and the
combined organic layers gave a crude product purified by
column chromatography using pentane/Et2O (10-60%). Re-
sulting fractions afforded after evaporation a white solid that
was recrystallized in hexanes/ethyl acetate (1:9) to afford 0.74
1H NMR (DMSO-d6) δ 7.82 (d, 1H, J ) 8.5, NHamino), 7.24 (s,
1H, NHamido), 6.94 (d, 2H, J ) 8.5, Hδ), 6.85 (s, 1H, NHamido),
6.56 (d, 2H, J ) 6.7, Hꢀ), 4.24-4.17 (m, 1H, HR), 2.78-2.73
(dd, 1H, J ) 13.8 and 4.7, Hâa), 2.52-2.45 (dd, 1H, J ) 13.8
and 9.7, Hâb), 1.59 (s, 3H, CH3acetyl), 0.77 (s, 9H, SiC(CH3)3),
0.00 (s, 6H, Si(CH3)2); 13C NMR (DMSO-d6) δ 174.2 (CONamido),
169.8 (COCH3), 154.3 (Cq), 131.9 (Cγ), 131.0 (Cꢀ), 120.2 (Cδ),
54.8 (CR), 37.8 (Câ), 26.4 (C(CH3)3), 23.3 (CH3CO), 18.8
(SiC(CH3)3), -3.7 (Si(CH3)2); MS FAB+ m/z 673 (2M + H)+,
337 (M + H)+; MS FAB- m/z 671 (2M - H)-, 335 (M - H)-;
HPLC tR 15.3 min. Anal. (C17H28N2O3Si) C, H, N.
g (70%) of white crystals. Rf ) 0.29 (Et2O); mp 94.1 °C; [R]20
D
N-r-(Acetyl-ter t-bu toxycar bon yl)-O-(ter t-bu tyl(dim eth -
yl)silyl)-N,N-d i(ter t-bu toxyca r bon yl)-L-tyr osin a m id e, 12.
To a solution of 11 (1.34 g, 4 mmol) in dichloromethane (25
mL) at room temperature was added (iPr)2NEt (4.1 mL, 6
equiv), DMAP (0.122 g, 0.25 equiv), and di(tert-butyl) dicar-
bonate (3.45 mL, 3.75 equiv). After overnight stirring, the
reaction mixture was diluted with dichloromethane and
washed with 1 M aqueous KHSO4 and 1 M aqueous NaHCO3.
The organic layer was dried over Na2SO4 and concentrated,
+1.3 (c 1.1, EtOH); 1H NMR (DMSO-d6) δ 7.17 (s, 1H, NHamino),
6.96 (d, 2H, J ) 8.4, Hδ), 6.84 (s, 1H, NHamido), 6.62-6.56 (m,
3H, NHamido, Hꢀ), 3.91-3.85 (m, 1H, HR), 2.74-2.69 (dd, 1H, J
) 13.8 and 4.0, Hâa), 2.51-2.44 (dd, 1H, J ) 13.6 and 9.7, Hâb),
1.14 (s, 9H, (CH3)3Boc), 0.78 (s, 9H, SiC(CH3)3), 0.00 (s, 6H, Si-
(CH3)2); 13C NMR (DMSO-d6) δ 174.6 (CONamido), 156.0 (CON-
Boc), 154.3 (Cq), 132.0 (Cγ), 131.1 (Cꢀ), 120.2 (Cδ), 115.6 (Cꢀ),
78.6 (C(CH3)3Boc), 56.5 (CR), 37.7 (Câ), 29.0 (C(CH3)3Boc), 26.4
(SiC(CH3)3), 18.8 (SiC(CH3)3), -3.7 (Si(CH3)2); MS FAB+ m/z

790 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Peyrottes et al.
789 (2M + H)+, 417 (M + Na)+, 395 (M + H)+, 339 (M - tBu
+ 2H)+; MS FAB- m/z 787 (2M - H)-, 393 (M - H)-; HPLC tR
28.4 min. Anal. (C20H34N2O4Si) C, H, N.
29.0 (d, J P-C ) 10.4, CH2S), 27.8, 27.3 (C(CH3)2, 2 × C(CH3)3),
24.9 (d, J P-C ) 6.8, NCH(CH3)2), 24.8 (d, J P-C ) 7.5, NCH-
(CH3)2); 31P NMR (CDCl3) δ 147.0, 146.9, 146.8; MS FAB+ m/z
599 (M + H)+, 499 (M - Boc + 2H)+; MS FAB- m/z 597 (M -
H)-, 513 (M - tBu)-, 497 (M - Boc)-, 413 (M - tBu - Boc)-.
Anal. (C30H51N2O6PS) C, H, N.
N-r-(ter t-Bu toxycar bon yl)-N,N-di(ter t-bu toxycar bon yl)-
O-(ter t-b u t yl(d im et h yl)silyl)-L-t yr osin a m id e, 16. To a
solution of 15 (0.83 g, 2.1 mmol) in dichloromethane (20 mL)
at room temperature was added (iPr)2NEt (1.45 mL, 4 equiv),
DMAP (0.044 g, 0.17 equiv), and di(tert-butyl) dicarbonate
(1.21 mL, 2.5 equiv). After overnight stirring, the reaction
mixture was diluted with dichloromethane and washed with
1 M aqueous KHSO4 and 1 M aqueous NaHCO3. The organic
layer was dried over Na2SO4 and concentrated, and the oily
residue was purified by flash column chromatography on silica
gel, eluting with pentane/Et2O (0-10%). An amount of 1.09 g
O-[N-(Acetyl-Boc)-L-tyr osin yl-N,N-d i-Boc-a m id e]-O-(S-
p iva loyl-2-th ioeth yl)-N,N-d iisop r op yl P h osp h or a m id ite,
18b. Yield, 65%; Rf ) 0.45 (Et3N/EtOAc/cyclohexane 1:1:8); 1H
NMR (CDCl3) δ 7.10-6.96 (2 m, 4H, Hδ, Hꢀ), 5.78-5.64 (m,
1H, HR), 3.95-3.53 (m, 4H, 2 × N-CH, P-OCH2), 3.50-3.37
(m, 1H, Hâb), 3.16-3.02 (m, 3H, CH2S, Hâa), 2.37 (s, 3H,
CH3acetyl), 1.74-1.44 (m, 27H, C(CH3)3Boc), 1.28-1.16 (m, 21H,
-C(CH3)3SATE, N-CH(CH3)2); 13C NMR (CDCl3) δ 206.7 (COS),
172.6, 171.2 (CON), 153.7 (Cq), 152.6, 150.0 (CONBoc), 131.8
(Cγ), 131.1 (Cꢀ), 120.1 (Cδ), 84.9 (C(CH3)3Boc), 63.0, 62.9 (d, J P-C
) 17.6, POCH2), 60.0 (CR), 46.8 (C(CH3)3SATE), 43.9, 43.8 (d,
J P-C ) 13.0, NCH(CH3)2), 35.2 (Câ), 30.4, 30.3 (d, J P-C ) 7.4,
CH2S), 28.0-27.8 (C(CH3)3Boc), 27.3 (C(CH3)3SATE), 26.8 (CH3acetyl),
25.0 (d, J P-C ) 6.7, NCH(CH3)2), 24.8 (d, J P-C ) 7.7, NCH-
(CH3)2); 31P NMR (CDCl3) δ 146.9, 146.8; MS FAB+ m/z 996
(85%) of an oil was obtained. Rf ) 0.67 (Et2O); [R]20 + 3.4 (c
D
1.2, EtOH); 1H NMR (DMSO-d6, 200 MHz) δ 7.34-7.13 (m,
3H, NH, Hδ), 6.76 (d, 2H, J ) 8.4, Hꢀ), 5.11 (m, 1H, HR), 2.83-
2.59 (m, 2H, partly covered by DMSO peak, Hâ), 1.49 (s, 18H,
(CH3)3Boc-amido), 1.31 (s, 9H, C(CH3)3Boc-amino), 0.94 (s, 9H, SiC-
(CH3)3), 0.17 (s, 6H, Si(CH3)2); 13C NMR (DMSO-d6) δ 174.6
(CON), 156.2, 154.6 (Cq), 149.6 (CONBoc), 131.3 (Cγ), 131.1 (Cꢀ),
120.3 (Cδ), 85.9, 80.7 (C(CH3)3Boc), 56.5 (CR), 36.3 (Câ), 28.9 (s,
C(CH3)3Boc), 28.0, 26.4 (C(CH3)3), 18.8 (SiC(CH3)3), -3.7 (Si-
(CH3)); MS FAB+ m/z 617 (M + Na)+, 595 (M + H)+, 517 (M -
Boc + Na + H)+; MS FAB- m/z 593 (M - H)-, 493 (M - Boc)-.
Anal. (C30H50N2O8Si) C, H, N.
(M + H)+; MS FAB- m/z 994 (M - H)-. Anal. (C39H64N3O11
PS, 20% oxidized form) C, H, S.
-
O-[N-(Boc)-L-tyr osin yl N,N-di-Boc-am ide]-O-(S-pivaloyl-
2-th ioeth yl)-N,N-diisopr opyl P h osph or am idite, 18c. Yield,
60%; Rf ) 0.47 (Et3N/EtOAc/cyclohexane 1:1:8); 1H NMR
(CDCl3) δ 7.63 (s, 1H, NH), 7.10-6.94 (m, 4H, Hδ, Hꢀ), 5.05-
4.98 (m, 1H, HR), 3.83-3.66 (m, 4H, 2 × NCH, P-OCH2), 3.47-
3.38 (m, 1H, Hâa), 3.16-3.10 (m, 3H, CH2S, Hâb), 1.51 (s, 9H,
C(CH3)3Boc), 1.44, 1.43 (2s, 18H, C(CH3)3Boc), 1.29-1.16 (m, 21H,
-C(CH3)3SATE, N-CH(CH3)2); 13C NMR (CDCl3) δ 206.7 (COS),
168.6 (CONamido), 153.9 (Cq), 151.9, 149.7 (CONBoc), 131.3, 130.9
(Cγ, Cδ), 120.3 (Cꢀ), 84.5, 83.1 (C(CH3)3Boc), 62.9 (d, J P-C ) 17.5,
POCH2), 62.1 (CR), 46.9 (C(CH3)3SATE), 43.9 (d, J P-C ) 12.9,
NCH(CH3)2), 34.9 (Câ), 30.3 (d, J P-C ) 7.2, CH2S), 28.4-27.8
(C(CH3)3Boc), 27.3 9 (C(CH3)3SATE), 25.0 (d, J P-C ) 6.8, NCH-
(CH3)2), 24.9 (d, J P-C ) 7.6, NCH(CH3)2); 31P NMR (CDCl3) δ
146.9, 146.8; MS FAB+ m/z 772 (M + H)+, 686 (M - tBuCO +
2H)+; MS FAB- m/z 802 (M sulfurated - H)-, 770 (M - H)-.
Anal. (C37H62N3O10PS) C, H. N: calcd 5.44; found 5.96.
Gen er a l P r oced u r e for th e P r ep a r a tion of P h osp h o-
tr iester s 2, 5, a n d 6. To a solution of AZT (0.093 g, 0.35 mmol)
in dry CH3CN (3 mL) containing 3 Å molecular sieves (0.5 g)
was added 1H-tetrazole (0.098 g, 1.40 mmol, 4 equiv) and
dropwise a solution of the appropriate phosphoramidite 18a ,
18b, or 18c (0.42 mmol, 1.2 equiv) in dry CH3CN (1.5 mL).
The mixture was stirred for 1 h, tert-butyl hydroperoxide (0.28
mL, 0.84 mmol, 3 M in toluene, 2.4 equiv) was added, and the
solution was further stirred for 1 h. The mixture was diluted
with CH2Cl2 and washed successively with aqueous Na2S2O3
(10%, w/v) and water, and the organic layer was dried over
Na2SO4, filtered, and evaporated to dryness under reduced
pressure.
O-[N-Boc-2,2′-d im eth yl-4-(4-h yd r oxyp h en yl)-1,3-oxa zo-
lid in e]-O-(S-p iva loyl-2-th ioeth yl)-3′-a zid o-3′-d eoxyth ym i-
d in -5′-yl P h osp h a te, 2. Column chromatography of the
residual syrup on silica gel using chloroform as eluent afforded
phosphotriester 2 (82%) as a white foam. Rf ) 0.47 (MeOH/
CH2Cl2 5:95); 1H NMR (CDCl3, 200 MHz) δ 8.69, 8.59 (2s, 1H,
NHAZT), 7.40-7.12 (m, 5H, H-6, Hδ, Hꢀ), 6.28-6.20 (m, 1H,
H-1′), 4.41-4.38 (m, 3H, H-3′, H-5′, H-5′′), 4.28-4.15 (m, 2H,
POCH2), 4.07 (m, 2H, Ha, H-4′), 3.84-3.69 (m, 2H, CH2OTyr),
3.19-3.04 (m, 3H, Hâa, CH2S), 2.74-2.61 (dd, J ) 10.3 and
13.1 Hz, 1H, Hâb), 2.45-2.31 (m, 2H, H-2′, H-2′′), 1.90 (s, 3H,
CH3AZT), 1.54-1.48 (m, 15H, C(CH3)2, C(CH3)3Boc), 1.24 (2s, 9H,
C(CH3)3SATE); 13C NMR (CDCl3) δ 206.0 (COS), 163.7 (C-4),
155.2 (CON), 152.6, 152.1 (Cq), 150.4 (C-2), 136.5, 135.5 (C-6),
131.3-130.6 (Cγ, Cδ), 120.4 (Cꢀ), 112.0 (C-5), 94.6-94.0
(C(CH3)2), 85.2 (C-1′), 82.5 (d, J P-C ) 7.9, C-4′), 80.9-80.1
(C(CH3)3), 67.6-67.4 (C-5′, POCH2), 66.4, 66.2 (CH2OTyr), 60.4
(C-3′), 59.4 (CR), 46.2 (C(CH3)3SATE), 39.2 (Câ), 38.0 (C-2′), 30.2-
28.8 (CH2S, C(CH3)2, C(CH3)3Boc), 27.7 (C(CH3)3SATE), 12.9, 12.8
(2s, CH3AZT); 31P NMR (CDCl3) δ -5.11, -5.22, -5.34, -5.36;
MS FAB+ m/z 781 (M + H)+, 725 (M - tBu + 2H)+, 681 (M -
N-r-(ter t-Bu toxycar bon yl)-N,N-di(ter t-bu toxycar bon yl)-
L-tyr osin a m id e, 17. TBAF (1 M THF solution, 0.55 mL, 1.1
equiv) was added dropwise to a solution of 16 (0.3 g, 0.5 mmol)
in anhydrous THF (5 mL) at 0 °C. After 30 min of stirring,
the reaction mixture was diluted with ethyl acetate and
washed with saturated aqueous NaCl solution and water. The
organic layer was dried over Na2SO4 and concentrated, and
the oily residue was purified by column chromatography on
silica gel, eluting with Et2O/pentane (10-30%). An amount of
0.24 g (91%) of an oil were obtained. Rf ) 0.61 (Et2O); [R]20
-
D
1
9 (c 1.1, EtOH); H NMR (CDCl3) δ 7.61 (s, 1H, OH), 6.94 (d,
2H, J ) 8.4, Hδ), 6.69 (d, 2H, J ) 8.4, Hꢀ), 6.39 (bs, 1H, NH),
4.94-4.91 (m, 1H, HR), 3.34-3.29 (dd, 1H, J ) 14.2 and 5.2,
Hâ), 3.03-2.97 (dd, 1H, J ) 14.3 and 9.8, Hâ), 1.41 (s, 9H,
C(CH3)3Boc), 1.34 (s, 18H, 2C(CH3)3Boc); 13C NMR (CDCl3) δ
168.9 (CONamido), 155.6 (Cq), 151.9, 149.8 (CONBoc), 131.0 (Cγ),
128.8 (Cꢀ), 115.9 (Cδ), 84.8, 83.3 (C(CH3)3Boc), 62.2 (CR), 34.8
(Câ), 28.4, 28.2 (2s, C(CH3)3Boc); MS FAB+ m/z 961 (2M + H)+,
861 (2M - Boc + 2H)+, 481 (M + H)+; MS FAB- m/z 959 (2M
- H)-, 479 (M - H)-, 379 (M - Boc)-. Anal. (C24H36N2O8) H,
N. C: calcd 59.98; found 59.30.
Gen er a l P r oced u r e for th e P r ep a r a tion of P h os-
p h or a m id ites, 18a -c. To a solution of the appropriate
tyrosinyl precursor 10, 13, or 17 (0.50 mmol) in dry CH3CN
(5 mL) containing 3 Å molecular sieves (0.5 g) was added at 0
°C tBuSATE phosphorodiamidite (0.29 g, 0.75 mmol, 1.5
equiv), diisopropylamine (0.14 mL, 1.00 mmol, 2 equiv), and
1H-tetrazole (0.07 g, 1.00 mmol, 2 equiv). After being stirred
for 2 h at room temperature, the reaction mixture was diluted
with acid-free EtOAc, washed with brine and water, dried with
Na2SO4, filtered, and concentrated in vacuum. Purification of
the residue by flash column chromatography on silica gel,
eluting with EtOAc/cyclohexane (1:9, v/v) containing 1% of
triethylamine, afforded a diastereoisomeric mixture (1:1) of the
desired phosphoramidite as a colorless oil.
O-[N-Boc-2,2′-d im eth yl-4-(4-h yd r oxyp h en yl)-1,3-oxa zo-
lid in e]-O-(S-p iva loyl-2-th ioeth yl)-N,N-d iisop r op yl P h os-
p h or a m id ite, 18a . Yield, 78%; Rf ) 0.64 (Et3N/EtOAc/
1
cyclohexane 1:1:8); H NMR (CDCl3) δ 7.07-6.88 (m, 4H, Hδ,
Hꢀ), 4.02, 3.85 (2m, 2 × 0.5H, two conformers, HR), 3.80-3.59
(m, 6H, 2 × N-CH, P-OCH2, CH2OTyr), 3.09-2.94 (m, 3H,
CH2S, Hâa), 2.55-2.49 (m, 1H, Hâb), 1.65, 1.64 (2s, 3H,
C(CH3)2), 1.56-1.38 (m, 12H, C(CH3)2, C(CH3)3), 1.19-1.09 (m,
21H, C(CH3)3SATE, N-CH(CH3)2); 13C NMR (CDCl3) δ 206.8
(COS), 153.7, 152.6, 152.2 (CON, Cq), 130.7,130.5 (Cγ, Cδ),
120.5 (Cꢀ), 94.4, 93.9 (C(CH3)2), 80.5, 80.1 (C(CH3)3Boc), 66.4,
66.2 (CH2-OTyr), 63.0 (d, J P-C ) 17.7, POCH2), 59.7 (CR), 46.8
(C(CH3)3SATE), 43.9 (d, J P-C ) 12.8, NCH(CH3)2), 39.3, 38.2 (Câ),

Phosphotriester Derivatives of AZT
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 791
(m, 3H, H-4′, H-5′, H-5′′), 3.92 (bs, 1H, HR), 3.71 (t, 2H, Hâa
Boc + 2H)+; MS FAB- m/z 1560 (2M)-, 779 (M - H)-, 635 (M
- SATE)-, 490 (M - Tyr)-; HPLC tR 31.4, 30.7 min; UV
(EtOH) λmax 265 nm (ꢀ 8960). Anal. (C34H49N6O11PS) C, H, N.
,
Hâb), 2.84 (2s, 2H, CH2OTyr), 2.32 (m, 2H, H-2′, H-2′′), 1.61 (d,
J ) 25.3, 3H, CH3AZT), 1.36 (s, 15H, C(CH3)2, C(CH3)3Boc); 13C
NMR (D2O) δ 166.0 (s, C-4), 154.2 (CON), 152.0, 151.5 (2s,
C-2, Cq), 138.6 (C-6), 135.7 (Cγ), 127.1 (Cꢀ), 120.8 (Cδ), 112.0
(C-5), 95.0 (C(CH3)2), 85.2 (C-1′), 83.0 (C-4′), 82.0 (C(CH3)3),
65.1 (C-5′), 65.0 (CH2OTyr), 60.5 (C-3′), 58.9 (CR), 38.2 (Câ), 35.7
(C-2′), 27.1-25.7 (C(CH3)3Boc), 23.6-22.1 (C(CH3)2), 12.1 (CH3AZT);
31P NMR (D2O) δ -4.15; MS FAB+ m/z 659 (M + H)+; MS FAB-
m/z 1043 (2M - Na - H)-; HPLC tR 22.6 min; UV (EtOH) λmax
265 nm (ꢀ 8320); HRMS (C27H37N6NaO10P) calcd 659.2206,
found 659.2186.
O-[N-(Acetyl-Boc)-L-tyr osin yl-N,N-d i-Boc-a m id e]-O-(3′-
a zid o-3′-d eoxyth ym id in -5′-yl) P h osp h a te, Sod iu m Sa lt,
8b. Yield, 41%; Rf ) 0.53 (MeOH/CH2Cl2 2:8); 1H NMR (D2O,
200 MHz) δ 7.30 (s, 1H, H-6), 6.95 (s, 4H, Hꢀ, Hδ), 6.00 (t, J )
6.6, 1H, H-1′), 4.37 (bs, 2H, H-3′, HR), 4.11-3.86 (m, 3H, H-4′,
H-5′, H-5′′), 2.83, 2.79 (2m, 2H, 2Hâ), 2.37 (s, 2H, H-2′, H-2′′),
1.83 (s, 3H, CH3acetyl), 1.69 (s, 3H, CH3AZT), 1.32 (d, 9H,
C(CH3)3BocAmino), 1.18 (s, 18H, C(CH3)3BocAmido); 13C NMR (D2O)
δ 171.7 (CON), 170.4 (COacetyl), 164.7 (C-4), 150.7 (C-2), 149.6
(Cq), 136.4 (C-6), 1.32.5 (Cγ), 121.2 (Cδ), 120.5 (Cꢀ), 111.8 (C-
5), 84.8 (C-1′), 83.7 (C-4′), 82.9 (C(CH3)3), 66.4 (C-5′), 61.2 (C-
3′), 59.7 (CR), 36.4, 36.2 (C-2′, Câ), 26.3-25.8 (C(CH3)3Boc), 24.9
(CH3acetyl), 12.3 (CH3AZT); 31P NMR (D2O) δ -4.28; MS FAB+
m/z 874 (M + H)+, 774 (M - Boc + 2H)+, 674 (M - 2Boc +
3H)+, 574 (M - 3Boc + 2H)+; FAB- m/z 850 (M - Na - H)-,
750 (M - Boc- Na)-; HPLC tR 22.5 min; UV (EtOH) λmax 265
nm (ꢀ 10010); HRMS (C36H49N7NaO15P) calcd 873.7797, found
873.7801.
Gen er a l P r oced u r e for th e Acid ic Dep r otection of
Com p ou n d s 2, 5, 6, 4b, a n d 8b. Phosphotriester or phos-
phodiester (0.2 mmol) was dissolved in dioxane (1 mL), and a
solution of hydrogen chloride in diethyl ether (2 mL, 35%) was
added at 0 °C. The mixture was stirred at room temperature
for 1 or 2 h. The solvent was removed in vacuum, and the
residue is either subjected to silica gel column chromatography
for phosphotriester derivatives or reverse-phase C18 column
chromatography, eluting with a linear gradient of CH3CN in
H2O (0-25%) for phosphodiesters. The desired fractions were
evaporated and passed through an ion-exchange column
(Dowex Na+) for the corresponding phosphodiesters. The
residue was lyophilized in dioxane/water (1:4) to afford the title
compound as a white powder.
O-[N-Boc-2-a m in o-4-(4-h yd r oxyp h en yl)p r op a n ol]yl-O-
(S-p iva loyl-2-t h ioet h yl)-3′-a zid o-3′-d eoxyt h ym id in -5′-yl
P h osp h a te, 3. Purification of the residue by column chroma-
tography on silica gel, eluting with MeOH/HCCl3 (2:98),
afforded phosphotriester 3 (56%) as well as phosphotriester 4
(35%). Rf ) 0.42 (MeOH/CH2Cl2 8:92); 1H NMR (CDCl3) δ 7.28,
7.24 (2s, 1H, H-6), 7.13-7.02 (m, 4H, Hδ, Hꢀ), 6.17-6.11 (m,
1H, H-1′), 4.97, 4.91 (2s, 1Hech, NHTyr), 4.42-4.35 (m, 1H, H-3′),
4.30-4.25 (m, 2H, H-5′, H-5′′), 4.21-4.10 (m, 2H, POCH2),
4.00-3.96 (m, 1H, H-4′), 3.74 (br s, 1H, HR), 3.55, 3.44 (2m,
2H, CH2OTyr), 3.10-3.02 (m, 2H, CH2S), 2.76 (m, 2H, Hâ),
2.57-2.55 (m, 1Hech, OHTyr), 2.43-2.32, 2.29-2.15 (2m, 2H,
H-2′, H-2′′), 1.76, 1.75 (2s, 3H, CH3AZT), 1.37, 1.36 (2s, 9H,
C(CH3)3Boc), 1.16, 1.15 (2s, 9H, C(CH3)3SATE); 13C NMR (CDCl3)
δ 206.1 (COS), 164.0 (C-4), 156.4 (CON), 150.6-149.1 (Cq, C-2),
136.1 (Cγ), 135.8 (C-6), 131.1 (Cδ), 120.3 (Cꢀ), 111.9 (C-5), 85.4
(C-1′), 82.7-82.5 (C-4′), 77.6 (C(CH3)3), 67.5-67.4 (C-5′, POCH2),
64.2 (CH2OTyr), 60.4 (C-3′), 53.9 (CR), 47.0 (C(CH3)3SATE), 46.4
(Câ), 37.8 (C-2′), 28.9-28.8 (CH2S, C(CH3)3Boc), 27.7 (C(CH3)3SATE),
12.9, 12.8 (2s, CH3AZT); 31P NMR (CDCl3) δ -5.17, -5.55; MS
FAB+ m/z 741 (M + H)+, 685 (M - tBu + 2H)+, 641 (M - Boc
+ 2H)+; MS FAB- m/z 1480 (2M)-, 739 (M - H)-, 595 (M -
SATE)-, 490 (M - Tyr)-; HPLC tR 25.4 min; UV (EtOH) λmax
265 nm (ꢀ 9860). Anal. (C31H45N6O11PS) C, H, N.
O-[N-(Boc)-L-tyr osin yl N,N-di-Boc-am ide]-O-(S-pivaloyl-
2-th ioeth yl)-3′-a zid o -3′-d eoxyth ym id in - 5′-yl P h osp h a te,
5. Column chromatography of the residual syrup on silica gel
using chloroform as eluent afforded phosphotriester 5 (63%)
as a white foam. Rf ) 0.39 (MeOH/CH2Cl2 5:95); 1H NMR
(CDCl3) δ 8.67 (s, 1H, NHAZT), 7.57 (s, 1H, NHTyr), 7.33, 7.27
(2d, 1H, J ) 1.0, H-6), 7.15 (m, 2H, Hδ), 7.05 (m, 2H, Hꢀ), 6.18-
6.12 (m, 1H, H-1′), 4.99-4.94 (m, 1H, HR), 4.37-4.23 (m, 3H,
H-3′, H-5′, H-5′′), 4.18-4.10 (m, 2H, POCH2), 3.98-3.95 (m,
H, H-4′), 3.43-3.38 (m, 1H, Hâa), 3.09-2.97 (m, 3H, CH2S, Hâb),
2.36 and 2.22 (2m, 2H, H-2′, H-2′′), 1.83, 1.82 (2s, 3H, CH3AZT),
1.41 (s, 9H, C(CH3)3Boc), 1.37 (s, 18H, C(CH3)3Boc), 1.16, 1.15
(2s, 9H, C(CH3)3SATE); 13C NMR (CDCl3) δ 205.9 (COS), 168.7
(CONamido), 163.8 (C-4), 152.0 (Cq), 150.5, 149.8 (CON), 149.3
(C-2), 135.6 (C-6), 131.6 (Cγ), 130.5 (Cδ), 120.2 (Cꢀ), 112.0 (C-
5), 85.3 (C-1′), 84.6, 83.2 (C(CH3)3Boc), 82.5 (C-4′), 67.6-67.4
(C-5′, POCH2), 61.7 (C-3′), 60.5 (CR), 47.0 (C(CH3)3SATE), 37.8
(C-2′), 35.2 (Câ), 30.2 (CH2S), 28.4, 28.2 (C(CH3)3Boc), 27.7
(C(CH3)3SATE), 12.9, 12.8 (2s, CH3AZT); 31P NMR (CDCl3) δ -5.3,
-5.4; MS FAB+ m/z 954 (M + H)+, 854 (M - Boc + 2H)+, 754
(M - 2Boc + 3H)+, 654 (M - 3Boc + 4H)+; MS FAB- m/z 1906
(2M)-, 952 (M - H)-, 808 (M - SATE)-; HPLC tR 30.1 min;
UV (EtOH) λmax 264 nm (ꢀ 9340). Anal. (C41H60N7O15PS) C, H,
N, P.
O-[N-(Acetyl-Boc)-L-tyr osin yl-N,N-d i-Boc-a m id e]-O-(S-
pivaloyl-2-th ioeth yl)-3′-azido-3′-deoxyth ym idin -5′-yl P h os-
p h a te, 6. Column chromatography of the residual syrup on
silica gel using chloroform as eluent afforded phosphotriester
1
6 (60%) as a white foam. Rf ) 0.39 (MeOH/CH2Cl2 5:95); H
NMR (CDCl3, 200 MHz) δ 8.70 (s, 1H, NHAZT), 7.34, 7.30 (2d,
1H, H-6, J ) 1.04 and 1.27), 7.22-7.03 (m, 4H, Hδ, Hꢀ), 6.19-
6.14 (m, 1H, H-1′), 5.61-5.58 (m, 1H, HR), 4.36-4.21 (m, 3H,
H-3′, H-5′, H-5′′), 4.16-4.10 (m, 2H, POCH2), 3.99-3.98 (m,
1H, H-4′), 3.48-3.43 (m, 1H, Hâb), 3.09-3.03 (m, 2H, CH2S),
2.93-2.89 (m, 1H,Hâa), 2.40-2.17 (m, 5H, CH3acetyl, H-2′, H-2′′),
1.84 (s, 3H, CH3AZT), 1.42 (m, 27H, C(CH3)3Boc), 1.15, 1.14 (2s,
9H, C(CH3)3SATE); 13C NMR (CDCl3) δ 206.0 (COS), 172.6
(CONamido), 170.8 (COacetyl), 163.9 (C-4), 152.4 (Cq), 150.5 (CON),
149.9 (C-2), 135.6 (C-6), 131.8 (Cγ), 130.5 (Cꢀ), 120.0 (Cδ), 112.0
(C-5), 85.2 (C-1′), 85.0 (C(CH3)3Boc), 82.5 (C-4′), 67.6-67.4 (C-
5′, POCH2), 60.5 (C-3′), 59.9 (CR), 47.0 (C(CH3)3SATE), 35.7 (Câ),
37.8 (C-2′), 30.3 (CH2S), 28.1-27.7 (C(CH3)3Boc, C(CH3)3SATE),
26.8 (CH3acetyl), 12.9, 12.8 (2s, CH3AZT); 31P NMR (CDCl3) δ
-5.30, -5.37; MS FAB+ m/z 996 (M + H)+; MS FAB- m/z 994
(M - H)-; HPLC tR 33.1 min; UV (EtOH) λmax 264 nm (ꢀ 9940).
Anal. (C43H62N7O16PS) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of P h osp h od i-
ester s 4b a n d 8b. AZT-HPM15 (triethylammonium form, 0.086
g, 0.2 mmol) and tyrosine precursor (1.1 equiv) were coevapo-
rated in dry pyridine and dissolved in a mixture of anhydrous
pyridine/acetonitrile (1:1, 1.5 mL). To this stirred solution was
added dropwise pivaloyl chloride (0.033 mL, 1.3 equiv). After
30 min, iodine (0.152 g, 3 equiv) and water (0.040 mL, 10
equiv) were successively added and the reaction was allowed
to proceed for a further 15 min. Then, an aqueous solution of
Na2S2O4 (0.1 M, 10 equiv) was added until the reaction mixture
became colorless. The mixture was diluted with dichlo-
romethane, and the organic layer was washed with a TEAB
solution (0.5 M, 30 mL). The organic solution was dried over
Na2SO4 and concentrated. The oily residue was purified on a
silica gel column using dichloromethane and methanol (0-
20%) as eluent. The residue was dissolved in a water/ethanol
mixture (5:2) and filtrated on an ion-exchange resin (Dowex
Na+), and the collected fractions were concentrated, dissolved
in dioxane/water (1:1), and lyophilized in order to obtained the
title compound as a sodium salt.
O-[2-Am in o(ch lor ydate)-4-(4-h ydr oxyph en yl)pr opan ol]-
yl-O-(S-p iva loyl-2-th ioeth yl)-3′-a zid o-3′-d eoxyth ym id in -
5′-yl P h osp h a te, 4. Purification of the residue by column
chromatography on silica gel, eluting with MeOH/HCCl3 (1:
9), gave the phosphotriester 4 (75%) as a white foam. Rf )
O-[N-Boc-2,2′-d im eth yl-4-(4-h yd r oxyp h en yl)-1,3-oxa zo-
lidin e]-O-(3′-azido-3′-deoxyth ym idin -5′-yl) P h osph ate, So-
d iu m Sa lt, 4b. Yield, 53%; Rf ) 0.58 (MeOH/CH2Cl2 2:8); 1H
NMR (D2O, 200 MHz) δ 7.40 (d, J ) 26.0, 1H, H-6), 7.00 (s,
4H, Hꢀ, Hδ), 6.10 (bs, 1H, H-1′), 4.30 (s, 1H, H-3′), 4.15-4.10
1
0.15 (MeOH/CH2Cl2 15:85); H NMR (DMSO-d6) δ 7.27, 7.26

792 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Peyrottes et al.
(2s, 1H, H-6), 7.08, 6.95 (2d, 2 × 2H, J ) 8.4, Hδ, Hꢀ), 5.95-
5.90 (m, 1H, H-1′), 5.13 (bs, 1H, OHTyr), 4.30-4.24 (m, 1H,
H-3′), 4.17-4.08 (m, 2H, H-5′, H-5′′), 3.98-3.92 (m, 2H,
POCH2), 3.80 (m, 1H, H-4′), 3.30-3.27 (m, 2H, CH2OTyr), 3.14
(m, 1H, partly covered by HDO peak, HR), 2.94-2.89 (m, 2H,
CH2S), 2.70-2.59 (2m, 2H, 2 × Hâ), 2.26-2.09 (2m, 2H, H-2′,
H-2′′), 1.52 (2s, 3H, CH3AZT), 0.95 (2s, 9H, C(CH3)3); 13C NMR
(DMSO-d6) δ 205.9 (COS), 164.5 (C-4), 151.22 (C-2), 149.7 (d,
J P-C ) 6.5, Cq), 136.8 (C-6), 134.7 (Cγ), 131.6 (Cδ), 120.8 (d,
J P-C ) 4.4, Cꢀ), 110.8 (C-5), 84.6 (C-1′), 81.9 (d, J P-C ) 7.7,
C-4′), 68.2 (C-5′), 67.3 (d, J P-C ) 5.6, POCH2), 60.7-60.4 (m,
C-3′, CH2OTyr), 54.5 (CR), 46.6 (C(CH3)3SATE), 36.4 (C-2′), 34.9
(Câ), 28.9 (d, J P-C ) 7.7, CH2S), 27.7 (C(CH3)3SATE), 12.9, 12.8
(2s, CH3AZT); 31P NMR (DMSO-d6) δ -5.3, -5.5; MS FAB+ m/z
641 (M - HCl)+ ; MS FAB- m/z 675 (M - H)-, 639 (M - HCl)-,
490 (M - HCl - Tyr)-, 495 (M - HCl - SATE)-; HPLC tR
(DMSO-d6, 81 MHz) δ -5.35, -5.46; MS FAB+ m/z 696 (M +
H)+ ; MS FAB- m/z 1389 (2M - H)-, 694 (M - H)-; HPLC tR
24.5 min; UV (EtOH) λmax 265 nm (ꢀ 9800). Anal. (C28H38N7O9-
PS‚0.5H2O) C, H, N.
O-[N-(Acet yl)-L-t yr osin a m id e]yl-O-(3′-a zid o-3′-d eoxy-
th ym id in -5′-yl) P h osp h a te, Sod iu m Sa lt, 8a . Yield, 43%.
Rf ) 0.53 (MeOH/CH2Cl2 2:8); 1H NMR (D2O, 200 MHz) δ 7.3
(s, 1H, H-6), 6.9 (s, 4H, Hꢀ, Hδ), 6.0 (t, J ) 6.6, 1H, H-1′), 4.3
(s, 2H, H-3′, HR), 4.1 (m, 1H, H-5′), 4.0 (m, 2H, H-4′, H-5′′),
2.8-2.7 (m, 2H, Hâ), 2.4 (t, J ) 6.4, 2H, H-2′, H-2′′), 1.8 (s,
3H, CH3acetyl), 1.6 (s, 3H, CH3AZT); 13C NMR (D2O) δ 177, 175
(CON), 163 (C-4), 153, 152 (C-2, Cq), 139 (C-6), 134 (s, Cγ),
132 (Cꢀ), 122 (Cδ), 117 (C-5), 86 (C-1′), 84 (C-4′), 66 (C-5′), 61
(s, C-3′), 56 (CR), 37 (C-2′, Câ), 23.0 (CH3acetyl), 12.5 (s, CH3AZT);
31P NMR (D2O) δ -4.06; MS FAB+ m/z 1147 (2M + H)+, 574
(M + H)+, 552 (M - Na + 2H)+, 307 (M - AZT + 2H)+; FAB-
m/z 1101 (2M - 2Na - H)-, 572 (M - H)-, 550 (M - Na -
H)-; HPLC tR 14.3 min; UV (EtOH) λmax 268 nm (ꢀ 14 300);
HRMS (C21H26N7NaO9P) calcd 574.1427, found 574.1436.
22.6 min; UV (EtOH) λmax 265 nm (ꢀ 9300). Anal. (C26H38
-
ClN6O9PS) H. C calcd 46.12, found 46.62. N: calcd 12.41, found
11.34.
O-[2-Am in o(ch lor ydate)-4-(4-h ydr oxyph en yl)pr opan ol]-
yl-O-(3′-a zid o-3′-d eoxyt h ym id in -5′-yl) P h osp h a t e, So-
d iu m Sa lt, 4a . Yield, 41%. Rf ) 0.53 (MeOH/CH2Cl2 2:8); 1H
NMR (D2O, 200 MHz) δ 7.2 (s, 1H, H-6), 6.9 (s, 4H, Hꢀ, Hδ),
5.9 (bs, 1H, H-1′), 4.2 (m, 1H, H-3′), 4.0-3.9 (m, 3H, H-4′, H-5′,
Ack n ow led gm en t. These investigations were sup-
ported by grants from CNRS and Fondation pour la
Recherche Me´dicale (FRM).
H-5′′), 3.5 (d, J ) 11.32, 1H, CH2OTyr), 3.3 (m, 2H, CH2OTyr
,
HR), 2.6 (m, 2H, Hâ), 2.2 (s, 2H, H-2′, H-2′′), 1.4 (s, 3H, CH3AZT);
13C NMR (D2O) δ 162.1 (C-4), 148.6-147.9 (C-2, Cq), 134.3 (C-
6), 128.6 (Cγ), 127.1 (Cꢀ), 117.9 (Cδ), 108.6 (C-5), 85 (C-1′), 80.7
(C-4′), 65 (C-5′), 60 (CH2OTyr), 59.3 (C-3′), 55 (CR), 35,7 (C-2′),
35 (Câ), 11.4 (CH3AZT); 31P NMR (D2O) δ -4.02; MS FAB+ m/z
519 (M + H)+, 497 (M - Na + 2H)+; FAB- m/z 991 (2M - Na
- H)-, 495 (M - Na - H)-; HPLC tR 13.7 min; UV (EtOH)
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λmax 265 nm (ꢀ 9200); HRMS (C19H25N6NaO8P) calcd 519.1369,
found 519.1368.
O-[L-Tyr osin a m id e]yl-O-(S-p iva loyl-2-th ioeth yl)-3′-a zi-
d o-3′-d eoxyt h ym id in -5′-yl P h osp h a t e, 7. Purification of
the residue by column chromatography on silica gel, eluting
with a gradient of MeOH in HCCl3 (0-5%) gave the trifluoro-
acetate form of phosphotriester 7 (80%) as a white foam. Rf )
0.21 (MeOH/CH2Cl2 15:85); 1H NMR (DMSO-d6) δ 7.72 (s, 1H,
NHech), 7.47, 7.45 (2s, 1H, H-6), 7.38 (s, 1H, NHech), 7.26-7.12
(m, 5H, NHech, Hδ, Hꢀ), 6.16-6.10 (m, 1H, H-1′), 4.49-4.36 (m,
1H, H-3′), 4.35-4.26 (m, 2H, H-5′, H-5′′), 4.17-4.12 (m, 2H,
POCH2), 4.01 (bs, 1H, H-4′), 3.74 (bs, 1H, HR), 3.13-3.11 (m,
2H, CH2S), 3.04-2.98 (m, 2H, Hâ), 2.44-2.31 (2m, 2H, H-2′,
H-2′′), 1.73, 1.72 (2s, 3H, CH3AZT), 1.17, 1.12 (2s, 9H, C(CH3)3);
13C NMR (DMSO-d6) δ 205.9 (COS), 164.5 (C-4), 151.2
(C-2), 149.7 (d, J P-C ) 6.6, Cq), 136.7 (C-6), 134.3 (Cγ), 131.7
(Cδ), 120.6 (Cꢀ), 110.8 (C-5), 84.6 (C-1′), 81.9 (d, J P-C ) 7.9,
C-4′), 68.1 (C-5′), 67.2 (POCH2), 60.6 (C-3′), 55.0 (CR), 46.8
(C(CH3)3SATE), 46.5 (Câ), 36.4 (C-2′), 28.9 (d, J P-C ) 7.5, CH2S),
27.7 (C(CH3)3SATE), 12.9 (2s, CH3 AZT); 31P NMR (DMSO-d6) δ
-5.3, -5.5; MS FAB+ m/z 654 (M + H)+, 510 (M - SATE +
2H)+; MS FAB- m/z 766 (M + CF3CO2-)-, 652 (M - H)-, 490
(M - Tyr)-, 403 (M - AZT)-; HPLC tR 21.7 min; UV (EtOH)
λmax 265 nm (ꢀ 8750). Anal. (C28H37F3N7O11PS) C, H, N, S.
O-[N-(Acet yl)-L-t yr osin a m id e]yl-O-(S-p iva loyl-2-t h io-
eth yl)-3′-a zid o-3′-d eoxyth ym id in -5′-yl P h osp h a te, 8. Pu-
rification of the residue by column chromatography on silica
gel, eluting with MeOH/HCCl3 (1:9), gave the phosphotriester
8 (40%) after lyophilization in dioxane as a white powder. Rf
) 0.22 (MeOH/CH2Cl2 1:9); 1H NMR (DMSO-d6) δ 8.06 (bs,
1H, NHTyr), 7.47 (m, 2H, H-6, NHamido), 7.30-6.98 (m, 5H,
NHamido, Hδ, Hꢀ), 6.18-6.10 (m, 1H, H-1′), 4.52-4.23 (m, 4H,
H-3′, H-5′, H-5′′, HR), 4.22-4.09 (m, 2H, POCH2), 4.06-3.97
(m, 1H, H-4′), 3.18-3.05 (m, 2H, CH2S), 3.02-2.65 (2m, 2H,
Hâ), 2.46-2.28 (m, 2H, H-2′, H-2′′), 1.76, 1.74 (2s, 3H, CH3AZT),
1.17, 1.16 (2s, 9H, C(CH3)3); 13C NMR (DMSO-d6) δ 206.1
(COS), 174 (CON), 165.0 (C-4), 151.3 (C-2), 149.5 (d, J P-C
6.5, Cq), 136.8 (C-6), 134.5 (Cγ), 132.0 (Cδ), 121.5 (d, J P-C
)
)
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4.4, Cꢀ), 110.4 (C-5), 85.4 (C-1′), 81.6 (d, J P-C ) 7.6, C-4′), 67.4
(C-5′), 65.9 (POCH2), 60.7 (m, C-3′), 55.4 (CR), 45.7 (C(CH3)3SATE),
48.2 (C-2′), 43.8 (Câ), 28.6 (d, J P-C ) 7.7, CH2S), 27.5
(C(CH3)3SATE), 23.6 (CH3acetyl), 12.7 (2s, CH3AZT); 31P NMR

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