Synthesis and in vitro activity of D- and L-enantiomers of 5-(trifluoromethyl)uracil nucleoside derivatives

Article abstract of DOI:10.1081/NCN-100002502

Recently, β-L-nucleoside analogues have emerged as a new class of sugar modified nucleosides with potential antiviral and/or antitumoral activity. As a part of our ongoing research on this topic, we decided to synthesize 5-CF₃-β-L-dUrd (7), the hitherto unknown L-enantiomer of Trifluridine, an antiherpetic drug approved by FDA but only used in topical applications due to concomitant cytotoxicity. 5-CF₃-β-dUrd (7) as well as some other related L-nucleoside derivatives were stereospecifically prepared and tested in vitro against viral (HSV-1 and HSV-2) and human thymidine kinases (TK).

Full text of DOI:10.1081/NCN-100002502

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SYNTHESIS AND IN VITRO ACTIVITY OF D- AND L-
ENANTIOMERS OF 5-(TRIFLUOROMETHYL)URACIL
NUCLEOSIDE DERIVATIVES
Raul Salvetti a , Massimo Pregnolato b , Annalisa Verri , Federico Focher , Silvio Spadari ,
Arnaud Marchand c , Christophe Mathé c & Gilles Gosselin c
a Università di Pavia, V. Taramelli 12, Pavia, 27100, Italy
b Università di Pavia, V. Taramelli 12, Pavia, 27100, Italy
c UMR 5625 CNRS, Université Montpellier II, Montpellier, Cedex 5, 34095, France
Version of record first published: 07 Feb 2007.
To cite this article: Raul Salvetti , Massimo Pregnolato , Annalisa Verri , Federico Focher , Silvio Spadari , Arnaud
Marchand , Christophe Mathé & Gilles Gosselin (2001): SYNTHESIS AND IN VITRO ACTIVITY OF D- AND L-ENANTIOMERS OF 5-
(TRIFLUOROMETHYL)URACIL NUCLEOSIDE DERIVATIVES, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 1123-1125
To link to this article: http://dx.doi.org/10.1081/NCN-100002502
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1123–1125 (2001)
SYNTHESIS AND IN VITRO ACTIVITY
OF D- AND L-ENANTIOMERS OF
5-(TRIFLUOROMETHYL)URACIL
NUCLEOSIDE DERIVATIVES
Raul Salvetti,1 Massimo Pregnolato,1,∗ Annalisa Verri,2
Federico Focher,2 Silvio Spadari,2 Arnaud Marchand,3
Christophe Mathe´,3 and Gilles Gosselin3
1Universita` di Pavia, V. Taramelli 12, 27100 Pavia, Italy
2CNR, IGBE, via Abbiategrasso 207, 27100, Pavia, Italy
3UMR 5625 CNRS-Universite´ Montpellier II, 34095 Montpellier
Cedex 5, France
ABSTRACT
Recently, β-L-nucleoside analogues have emerged as a new class of sugar
modified nucleosides with potential antiviral and/or antitumoral activity. As a
part of our ongoing research on this topic, we decided to synthesize 5-CF3-β-
L-dUrd (7), the hitherto unknown L-enantiomer of Trifluridine, an antiherpetic
drug approved by FDA but only used in topical applications due to concomitant
cytotoxicity. 5-CF3-β-L-dUrd (7) as well as some other related L-nucleoside
derivatives were stereospecifically prepared and tested in vitro against viral
(HSV-1 and HSV-2) and human thymidine kinases (TK).
Trifluridine (ViropticTM) is an antiherpetic drug approved by the FDA in
1980 for the clinical treatment of primary keratoconjunctivitis and epithelial ker-
atitis (1). It is monophosphorylated by both cellular and HSV thymidine kinases,
and upon further phosphorylation by cellular nucleotide kinases, it inhibits viral
DNA replication by incorporation of its triphosphate form into DNA. On the other
hand, interactions of Trifluridine and its phosphorylated metabolites with cellu-
lar enzymes, including human thymidine kinase, thymidylate synthase, and DNA
∗Corresponding author.
1123
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

ORDER
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1124
SALVETTI ET AL.
polymerases are responsible for cellular toxicity. In order to design compounds with
reduced cytotoxic effects, we have carried out the synthesis of 5-CF3-β-L-dUrd
(7), the L-enantiomer of Trifluridine, as well as some other L-nucleoside deriva-
tives modified at the 2ꢁ and 3ꢁ positions of the sugar moiety. Condensation of 1,2-
di-O-acetyl-3,5-di-O-benzoyl-L-xylofuranose (2) (1) with commercially available
5-(trifluoromethyl)uracil under Vorbru¨ggen conditions gave 1-(2-O-acetyl-3,5-di-
O-benzoyl-β-L-xylo-furanosyl)-5-(trifluoromethyl)uracil (2) which was fully dea-
cylated to give β-L-xylofuranosyl-5-(trifluoromethyl)uracil 8 (Scheme 1). On the
other hand, regioselective 2ꢁ-O-deacylation of compound 2 afforded 1-(3,5-di-O-
benzoyl-β-L-xylo-furanosyl)-5-(trifluoromethyl)uracil (3), which was converted
via a radical reductive process (3) to compound 4, and upon deprotection, gave
1-(2-deoxy-β-L-threo-pentofuranosyl)-5-(trifluoromethyl)uracil (5). Selective 5ꢁ-
O-benzoylation of compound 5 provided the key-intermediate 6.
From 6, inversion of configuration at C-3ꢁ via a Mitsunobu reaction (4), and
subsequent debenzoylation with methanolic ammonia gave the target molecule
Scheme 1. Reagents and conditions: (a) silylated 5-CF3-uracil/TMSOTf/dichloroethane, r.t.;
=
(b) H2N-NH2.H2O/AcOH/pyridine, r.t.; (c) NH3/MeOH, r.t.; (d) DMAP/PhO(C S)Cl/CH3CN, r.t.;
then, (Me3Si)3SiH/AIBN/dioxane, reflux; (e) BzCl/pyridine, 0◦C; (f) DEAD/benzoic acid/THF, 0◦C;
(g) MsCl/TBAF/THF, r.t.; then, MeONa/MeOH, r.t.

ORDER
REPRINTS
5-(TRIFLUOROMETHYL)URACIL NUCLEOSIDE DERIVATIVES
1125
Table 1.
5-CF3-β-L-dUrd (7). Starting from the same key-intermediate 6, other modifica-
tions on 2ꢁ and 3ꢁ positions provided the corresponding 2ꢁ,3ꢁ-dideoxynucleoside 9
and the 2ꢁ,3ꢁ-unsaturated derivative 10.
The potential inhibitory effects of compounds 5, 7 and 8 versus their D-
nucleoside counterparts toward the thymidine kinases (5) of HSV-1 and HSV-2
have been evaluated. The IC50 values are reported in Table 1. All the nucleosides
(D and L) show higher affinity for HSV-1 TK with respect to HSV-2 TK. Further-
more, 5-CF3-β-L-dUrd (7) and commercially available Trifluridine show the best
inhibitory properties toward both viral enzymes. Regarding HSV-2 TK, Trifluri-
dine shows an IC50 value of 2 µM while 5-CF3-β-L-dUrd shows a higher value
(14.5 µM). On the other hand, against HSV-1 TK 5-CF3-β-L-dUrd (7) has an IC50
value of 0.5 µM which is comparable to the IC50 value (0.25 µM) found for Tri-
fluridine. This last result seems promising in term of potential antiherpetic activity
for 5-CF3-β-L-dUrd (7).
In order to investigate the potential antiherpetic activity of 7 and to study its
cellular effects of 5-CF3-β-L-dUrd (7), we have evaluated its inhibition of cellular
TK. Trifluridine shows an IC50 value of 2 µM while its L-enantiomer is not able
to inhibit cellular TK up to a concentration of 200 µM. This result appears very
interesting in terms of a lack of cellular toxicity for 5-CF3-β-L-dUrd (7). Based on
these results, the antiherpetic evaluation of 5-CF3-β-L-dUrd (7) in cell cultures is
warranted, and work on this topic is currently in progress in our laboratories.
REFERENCES
1. Carmine, A.A.; Brogden, R.N.; Heel, R.C.; Speight, T.M.; Avery, G.S. Drugs 1982,
23, 329–53.
2. Gosselin, G.; Bergogne, M.-C.; Imbach, J.-L. J. Heterocycl. Chem. 1993, 30, 1229–
1233.
3. Barton, D.H.R.; McCombie, S.W. J. Chem. Soc. Perkin Trans 1 1975, 1574–1585.
4. Hughes, D.L. Org. Prep. Proced. Int. 1996, 28, 127–164.
5. Spadari, S.; Maga, G.; Veni, A.; Focher, F. Exp. Opin. Invest. Drugs 1998, 7, 1285–
1300.

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