Design of new mononucleotide prodrugs: Aryl (SATE) phosphotriester derivatives

Article abstract of DOI:10.1081/NCN-100002302

Synthesis, biological activities and decomposition kinetics of novel phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing a S-tButyl-2-thioethyl (tBuSATE) group and L-tyrosinyl residues are reported. All the derivatives appeared to

Full text of DOI:10.1081/NCN-100002302

This article was downloaded by: [University of Cambridge]
On: 24 December 2014, At: 14:58
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lncn20
DESIGN OF NEW MONONUCLEOTIDE PRODRUGS: ARYL
(SATE) PHOSPHOTRIESTER DERIVATIVES
S. Peyrottes a , N. Schlienger a , T. Beltran a , I. Lefebvre a , A. Pompon a , G. Gosselin a ,
A.-M. Aubertin b , J.-L. Imbach a & C. Périgaud a
a Université Montpellier II , UMR 5625 CNRS - UM II, cc 008, place E. Bataillon,
Montpellier, cedex 05, 34095, France
b Université Louis Pasteur , INSERM U 74, 3 rue Koeberlé, Strasbourg, 67000, France
Published online: 07 Feb 2007.
To cite this article: S. Peyrottes , N. Schlienger , T. Beltran , I. Lefebvre , A. Pompon , G. Gosselin , A.-M. Aubertin , J.-L.
Imbach & C. Périgaud (2001) DESIGN OF NEW MONONUCLEOTIDE PRODRUGS: ARYL (SATE) PHOSPHOTRIESTER DERIVATIVES,
Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 315-321, DOI: 10.1081/NCN-100002302
To link to this article: http://dx.doi.org/10.1081/NCN-100002302
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 315–321 (2001)
DESIGN OF NEW MONONUCLEOTIDE
PRODRUGS: ARYL (SATE) PHOSPHOTRIESTER
DERIVATIVES
S. Peyrottes,1,∗ N. Schlienger,1 T. Beltran,1 I. Lefebvre,1
A. Pompon,1 G. Gosselin,1 A.-M. Aubertin,2 J.-L. Imbach,1
and C. Pe´rigaud1
1UMR 5625 CNRS - UM II, Universite´ Montpellier II, cc 008,
place E. Bataillon, 34095 Montpellier cedex 05, France
2INSERM U 74, Universite´ Louis Pasteur, 3 rue Koeberle´,
67000 Strasbourg, France
ABSTRACT
Synthesis, biological activities and decomposition kinetics of novel phosphotri-
ester derivatives of 3ꢀ-azido-2ꢀ,3ꢀ-dideoxythymidine (AZT) bearing a
S-tButyl-2-thioethyl (tBuSATE) group and L-tyrosinyl residues are reported.
All the derivatives appeared to be potent inhibitors of HIV-1 replication in var-
ious cell culture experiments. The proposed decomposition process of these
mixed phosphotriesters may involve successively an esterase and then a phos-
phodiesterase activation.
The antiviral activity of nucleoside analogues is closely related to their ability
to be phosphorylated intracellularly. Many strategies have been envisaged to mask
or reduce negative charges of nucleoside 5ꢀ-monophosphates, thereby forming more
lipophilic derivatives (pronucleotides) which would be expected to revert back to
the corresponding 5ꢀ-mononucleotides inside cells (1). Research progressed in this
way with the design of mononucleoside phosphotriester derivatives incorporating
two S-acyl-2-thioethyl (SATE) groups as biolabile phosphate protections (2). The
decomposition pathway of such pronucleotides involves two successive esterase
∗Corresponding author.
315
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

ORDER
REPRINTS
316
PEYROTTES ET AL.
activation steps (3). In vivo pharmacokinetic studies of bis(tBuSATE) phospho-
triesters seem to demonstrate that the second enzymate-mediated step could be
considered limiting (4,5). Therefore, we decided to explore mixed phosphotriester
derivatives bearing only one SATE chain and another moiety susceptible to be hy-
drolyzed selectively by an enzymatic system other than esterases. We previously
showed that the mononucleoside phenyl (tBuSATE) phosphotriester derivative of
AZT 1 (Fig.) leads in vitro to the selective intracellular release of the corresponding
5ꢀ-mononucleotide (6). Herein, we would like to report the synthesis and the eval-
uation of new phosphotriesters 2–4 bearing L-tyrosinyl residue as aryl substituent
(Fig.).
Figure 1. Structure of the studied arylphosphotriester derivatives of AZT.
Synthesis. The targeted compounds were obtained via a phosphoramidite approach
(Scheme 1).
The phosphitylating agent 5 bearing the SATE moiety and the desired ty-
rosinyl residue was obtained from commercially available bis(diisopropylamino)
chlorophosphine with subsequent coupling of the SATE chain (7) and then the aryl
precursor 6 in presence of 1H-tetrazole. The reaction of the tyrosinyl (tBuSATE)
phosphoramidite intermediate 5 with AZT, followed by in situ oxidation using
tert-butyl hydroperoxyde, afforded the fully protected phosphotriester derivatives
2. Treatment of compound 2 in various acidic conditions gave rise to partially
protected 3 and to the fully deprotected tyrosinyl (tBuSATE) phosphotriester deriva-
tive of AZT 4.
Antiviral evaluation. All the derivatives were evaluated for their in vitro anti-HIV
activity and were potent inhibitors of HIV-1 replication in cell culture experiments
(Table 1).
In contrast to AZT, the tyrosinyl (tBuSATE) phosphotriesters 2–4 exhibited
significant anti-HIV effects in thymidine kinase-deficient cells (CEM/TK−) with
EC50 values in the same range as the one observed for the pronucleotide 1. These
results suggest that the mixed aryl phosphotriesters 2–4 were able to deliver intra-
cellularly of the 5ꢀ-mononucleotide of AZT (AZTMP) and can be considered as
new pronucleotides.

ORDER
REPRINTS
ARYL (SATE) PHOSPHOTRIESTER DERIVATIVES
317
Scheme 1. Synthesis of tyrosinyl (tBuSATE) phosphotriester derivatives of AZT Reagents: (i)
(CH3)2NCH[OCH2C(CH3)3]2, tBuOH, toluene; (ii) tBuSATEOP[N(iPr)2]2, 1H-tetrazole, HN(iPr)2,
CH3CN; (iii) AZT, 1H-tetrazole, CH3CN, then tBuOOH, toluene; (iv) 30 % HCl, Et2O solution; (v)
10 % TFA, CH2Cl2, then HCl, dioxane solution.
Stability studies. Using a previously published procedure (3), the decomposition
pathways and kinetic data of compounds 2–4 were studied in total CEM-SS extracts
in order to mimic the conditions inside the cells. The metabolites formed during the
incubation of the phosphotriester were identified by HPLC-MS and by coinjection
with authentic samples. For this purpose, the intermediates 8, 10 and 11 (Schemes
2 and 3) were synthesized using a H-phosphonate approach (8).
These results indicate that the decomposition mechanism for the fully
protected phosphotriester 2 (Scheme 2) occurred following two concomitant
Table 1. Anti-HIV-1 Activity in Various Cell Lines of Mixed Aryl Phosphotriester
Derivatives 2–4 in Comparison to the Parent Nucleoside AZT and to the Phenyl
(tBuSATE) Phosphotriester Derivative 1 as Reference Pronucleotidea
CEM-SS
MT-4
CEM/TK−
b
c
b
c
b
c
Compound
EC50
CC50
EC50
CC50
EC50
CC50
AZT
0.003
0.002
0.034
0.004
0.006
>1
>10
>1
>10
>100
0.015
0.070
0.250
0.042
0.075
>1
>10
>1
>10
>10
>100
3.5
0.90
2.7
>100
>10
>1
>10
>100
1
2
3
4
29
aAll data represent average values for at least three separate experiments. The variation
of these results under standard operating procedures is below 10 %. bEC50: effective
concentration (µM) or concentration required to inhibit the replication of HIV-1 by 50%.
cCC50: cytotoxic concentration (µM) or concentration required to reduce the viability of
uninfected cells by 50%.

ORDER
REPRINTS
318
PEYROTTES ET AL.
esterase-mediated steps: (i) a main decomposition pathway (65%) resulting to the
loss of the tButyl group on the tyrosinyl residue, and leading to the phosphotri-
ester 7; (ii) hydrolysis of the tBuSATE chain giving rise to the formation of the
corresponding phosphodiester 8. The metabolites 7 and 8 were then converted to
the aryl phosphodiester 9, which was further hydrolyzed into AZTMP through
phosphodiesterase activity.
The two esterase-mediated steps were also observed for phosphotriester 3
in total CEM-SS cell extracts (Scheme 3). Nevertheless, compared to its analog
2, the major decomposition pathway (93%) of 3 corresponds to the loss of the
tBuSATE chain from 4 leading to phosphodiester 10. This is probably due to the
presence of the ionisable (at physiological pH) amino function in the vicinity of
the ester functionality. Finally, the metabolite 10 is directly converted into AZTMP
(phosphodiesterase activity).
Thedecompositionpathwayofthefullydeprotectedphosphotriester4resulted
successively in the removal of the SATE moiety (metabolite 11, Scheme 3) and then
of the tyrosinyl residue.
The half-lives of the mononucleoside phosphotriesters and the resulting meta-
bolites are summarized in Table 2. Comparison of the enzymatic stability of the
arylphosphodiester 10 and 11 (Scheme 3) demonstrated that the presence of a tButyl
ester functionality on the tyrosinyl residue of the phosphodiester derivatives seems
to modify the rate of the phosphodiesterase mediated step (Table 2). In order to
Scheme 2. Proposed decomposition mechanism of the fully protected derivative 2 in total CEM-SS
cell extracts.

ORDER
REPRINTS
ARYL (SATE) PHOSPHOTRIESTER DERIVATIVES
319
Scheme 3. Proposed decomposition mechanism of derivatives 3 and 4 in total CEM-SS cell extracts.
confirm that the conversion of the aryl phosphodiester metabolites to AZTMP was
not due to a chemical process, the stability of metabolite 11 was determined in
phosphate buffer (pH 7) and in denatured cell extract. No degradation of this entity
was observed after a week of incubation at 37◦C.
The comparison of kinetic data, especially the estimated half-life of AZTMP
formation, Log P values (which have been correlated with the passive diffusion of
compounds through cellular membranes) and EC50 in CEM-SS/TK− cells for the
studied compounds, indicates that the observed antiviral activity is closely related
to both parameters (Table 3).
It is known that a time-dependent relationship is required between the release
of the phosphorylated forms from the pronucleotide and expression of the cellular
target in order to observe an antiviral effect (2,9,10). Thus, derivative 4 presents
Table 2. Calculated Half-Lives of the Phosphotriester
Derivatives 2–4 and Their Metabolites in CEM-SS Cell
Extracts, and Corresponding Pseudo First-Order Rate
Constants
Compound
2
<10 min
0.061
Metabolites
8
40 min
0.017
7
28 min
0.025
9
4.3 h
0.0027
t1/2
k (min−1
)
)
3
20 min
0.035
10
3.9 h
0.0030
4
1.2 h
0.0093
11
25 h
0.00045
t1/2
k (min−1

ORDER
REPRINTS
320
PEYROTTES ET AL.
Table 3. Estimated Half-Lives of AZTMP
Formation, Apparent Water Octanol Partition
Coefficient (Log P) and EC50 in CEM/TK−
Cells
t1/2 of AZTMP
Formation
EC50 (µM) in
Log P
CEM/TK−
2
3
4
5.3 h
5.1 h
25 h
4.29
2.90
0.24
0.9
2.7
29
the weaker activity whereas fully protected 2 appeared the more potent, due to
favorable properties (fast release of AZTMP and high lipophilicity).
CONCLUSION
The present study demonstrates that aryl (tBuSATE) phosphotriester deriva-
tives of AZT 2–4 are able to deliver the parent 5ꢀ-mononucleotide efficiently and
they can be considered as novel pronucleotides. The proposed mechanism of de-
composition of these mixed phosphotriesters involves successively an esterase and
a phosphodiesterase step. The large number of chemical modifications which could
be envisaged on the aryl moiety enables the search for antiviral mononucleotide
prodrugs with a balance between aqueous solubility, lipophilicity, and enzymatic
stability in order to envisage further in vivo pharmacological studies. Work on this
area is currently in progress in our group.
ACKNOWLEDGMENTS
These investigations were supported by grants from the Agence Nationale de
Recherches sur le SIDA (ANRS) and the Fondation pour la Recherche Me´dicale
(FRM).
REFERENCES
1. Meier, C. Synlett 1998, 233–242.
2. Pe´rigaud, C.; Gosselin, G.; Imbach, J.-L. In Biomedical Chemistry/Applying Chemical
Principles to the Understanding and Treatment of Disease; Torrence, P. F., Ed.; John
Wiley & Sons Inc.: New York, 2000; Vol. 5, pp. 115–141.
3. Lefebvre, I.; Pe´rigaud, C.; Pompon, A.; Aubertin, A.-M.; Girardet, J.-L.; Kirn, A.;
Gosselin, G.; Imbach, J.-L. J. Med. Chem. 1995, 38, 3941–3950.
4. Placidi, L.; Pe´rigaud, C.; Cretton-Scott, E.; Gosselin, G.; Pierra, C.; Schinazi, R. F.;
Imbach, J.-L.; Sommadossi, J.-P. Antiviral Res. 1998, 37, 87.

ORDER
REPRINTS
ARYL (SATE) PHOSPHOTRIESTER DERIVATIVES
321
5. Martin, L. T.; Cretton-Scott, E.; Placidi, L.; Faraj, A.; Loi, A. G.; Schinazi, R. F.;
Mc Clure, H. M.; Gosselin, G.; Imbach, J.-L.; Sommadossi, J.-P. Nucleosides Nu-
cleotides, Nucleic Acids 2000, 19, 481–499.
6. Schlienger, N.; Peyrottes, S.; Aubertin, A.-M.; Gosselin, G.; Imbach, J.-L.; Pe´rigaud,
C. Nucleosides Nucleotides 1999, 18, 1025–1026.
7. Alvarez, K.; Vasseur, J.-J.; Beltran, T.; Imbach, J.-L. J. Org. Chem. 1999, 64, 6319–
6328.
8. Kuyl-Yeheskiely, E.; Tromp, C. M.; Van der Marel, G. A.; Van Boom, J. H. Tetrahedron
Lett. 1987, 28, 4461–4464.
9. Balzarini, J.; Naesens, L.; Aquaro, S.; Knispel, T.; Perno, C.; De Clercq, E.; Meier,
C. Mol. Pharmacol. 1999, 6, 1354–1361.
10. De Clercq, E. Int. Antiviral News 2000, 8, 53–56.

Request Permission or Order Reprints Instantly!
Interested in copying and sharing this article? In most cases, U.S. Copyright
Law requires that you get permission from the article’s rightsholder before
using copyrighted content.
All information and materials found in this article, including but not limited
to text, trademarks, patents, logos, graphics and images (the "Materials"), are
the copyrighted works and other forms of intellectual property of Marcel
Dekker, Inc., or its licensors. All rights not expressly granted are reserved.
Get permission to lawfully reproduce and distribute the Materials or order
reprints quickly and painlessly. Simply click on the "Request
Permission/Reprints Here" link below and follow the instructions. Visit the
U.S. Copyright Office for information on Fair Use limitations of U.S.
copyright law. Please refer to The Association of American Publishers’
(AAP) website for guidelines on Fair Use in the Classroom.
The Materials are for your personal use only and cannot be reformatted,
reposted, resold or distributed by electronic means or otherwise without
permission from Marcel Dekker, Inc. Marcel Dekker, Inc. grants you the
limited right to display the Materials only on your personal computer or
personal wireless device, and to copy and download single copies of such
Materials provided that any copyright, trademark or other notice appearing
on such Materials is also retained by, displayed, copied or downloaded as
part of the Materials and is not removed or obscured, and provided you do
not edit, modify, alter or enhance the Materials. Please refer to our Website
User Agreement for more details.
Order now!
Reprints of this article can also be ordered at
http://www.dekker.com/servlet/product/DOI/101081NCN100002302