1,3-Dihydrobenzo[c]furan nucleoside analogues: Additional studies of the thymine derivative

Article abstract of DOI:10.1016/j.bmcl.2003.09.031

The detailed study of the 1,3-dihydrobenzo[c]furan derivative of thymine is reported. The lack of anti-HIV activity of this compound in cell culture experiments is shown to be related to the inability of the corresponding 5′-triphosphate derivative to int

Full text of DOI:10.1016/j.bmcl.2003.09.031

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4473–4475
1,3-Dihydrobenzo[c]furan Nucleoside Analogues: Additional
Studies of the Thymine Derivative
David Egron,a Christian Perigaud,a Gilles Gosselin,a,c Anne-Marie Aubertin,b
Abdesslem Faraj,c Majid Selouane,d Denis Posteld and Christophe Lend,*
a
´
´
UMR 5625 CNRS-UM II, Universite Montpellier II, cc 008, place E. Bataillon, 34095 Montpellier Cedex 5, France
´
b
´
Institut de Virologie de la Faculte de Medecine de Strasbourg, U. INSERM 74, 3 rue Koeberle, 67000 Strasbourg, France
c
´
Laboratoire Cooperatif Idenix CNRS-UM II, place E. Bataillon, 34095 Montpellier Cedex 5, France
d
´
Laboratoire des Glucides, Universite de Picardie Jules Verne, 80039 Amiens, France
Received 22 July 2003; revised 29 August 2003; accepted 1 September 2003
Abstract—The detailed study of the 1,3-dihydrobenzo[c]furan derivative of thymine is reported. The lack of anti-HIV activity of
this compound in cell culture experiments is shown to be related to the inability of the corresponding 50-triphosphate derivative to
interact efficiently with the reverse transcriptase.
# 2003 Elsevier Ltd. All rights reserved.
D4T (20,30-didehydro-20,30-dideoxythymidine, Fig. 1) is
used in the treatment of human immunodeficiency virus
(HIV) infection.1,2 Its mechanism of action is similar to
that of other nucleoside HIV-reverse transcriptase (RT)
inhibitors, and requires intracellular metabolization into
the corresponding 50-triphosphate derivative, which acts
as a competitive inhibitor for the RT reaction and
incorporation into viral DNA. In contrast to other
and also increase the chemical stability and lipophilicity
of the resulting compound. Herein, the synthesis, phys-
icochemical parameters and in vitro anti-HIV eval-
uation of the 1,3-dihydrobenzo[c]furan derivative of
thymine 1 (Fig. 1) are reported. Additional studies have
been carried out in order to explain the lack of anti-HIV
activity of this compound in cell culture experiments.
pyrimidine
nucleoside
analogues,
the
initial
Initially investigated in the case of uracil analogues,4,6
the most efficient synthesis of the 1,3-dihydro-
benzo[c]furan nucleoside 1 employed the Sharpless
asymmetric dihydroxylation methodology (Scheme 1).
Briefly, selective protection of one aldehyde function of
the o-phthalaldehyde 2 followed by homologation using
Wittig chemistry gave the styrene derivative 4. Asym-
metric dihydroxylation of 4 with AD-mix a afforded the
diol 5. Esterification of the primary hydroxyl group of 5
followed by deprotection of the aldehyde functionality
afforded the cyclic acetal 7 (a and b forms). This mix-
ture was then condensed with silylated thymine, and
deprotection of the resulting intermediates 8 and 9 gave
rise to the target nucleoside 1 and its a anomer 10 which
were separated by silica gel column chromatography.
phosphorylation of d4T, catalyzed by thymidine kinase,
is the rate-limiting step in this metabolic process.
Moreover, the introduction of the 20,30-double bond
results in increasing the lability of the glycosidic bond
and decreasing the lipophilicity compared to the corres-
ponding saturated 20,30-dideoxynucleoside series. Thus,
d4T penetrates into the central nervous system, an
important site of HIV replication, to a limited extent.3
Following several chemical strategies, we have pre-
viously reported the synthesis of 1,3-dihydro-
benzo[c]furan nucleoside analogues.4À6 In this new
series, the fusion of the benzene ring to the 20,30-posi-
tions of the sugar residue might preserve the biological
activity related to restricted nucleoside analogues such
as 20,30-didehydro-20,30-dideoxy-nucleoside derivatives,
The chemical stability of compound 1 was studied in
comparison to d4T (Table 1). In the three studied buf-
fers, hydrolysis of 1 and d4T gave rise to the formation
of thymine but the disappearance of the nucleoside
analogue 1 was significantly slower than that of d4T.
*Corresponding author. Tel.: +33-3-22-827476; fax: +33-3-22-
827568; e-mail: christophe.len@sc.u-picardie.fr
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.031

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D. Egron et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4473–4475
Figure 1.
Scheme 2. Reagents and conditions: (i) bis(S-pivaloyl-2-thioethyl)
N,N-diisopropylphosphoramidite, 1H-tetrazole, CH3CN then
tBuOOH, toluene; (ii) 2-chloro-4H-1,2,3-dioxaphosphorin-4-one, pyr-
idine, dioxane then bis(tri-n-butylammonium) pyrophosphate, DMF
then classical work-up.10
Table 1. Calculated half-lives (day) in three aqueous buffersa and
partition coefficients (log P)b of compound 1 in comparison to d4T
Compd
pH 7.3
pH 2.0
pH 1.2
log P
1
d4T
34
6
31
5
18
3
0.55Æ0.42
Scheme 1. Reagents and conditions: (i) propane-1,3-diol, PTSA, tolu-
ene; (ii) (C6H5)3CH3PBr, tBuOK, toluene; (iii) AD-mix a, tBuOH,
H2O; (iv) PivCl, (C2H5)3N, toluene; (v) HCl, MeOH; (vi) silylated
thymine, SnCl4, C2H4Cl2; (vii) NaOH, dioxane, H2O.
À0.77Æ0.18
aConcentration studied: 20 mM in Tris–HCl (pH 7.3), glycine-HCl
(pH 2.0) and KCl-HCl (pH 1.2).
bLog P determination was performed using log P dB 4.5 calculations
(ACD, Toronto, Canada).
Thus, the presence of the benzene ring as electron-with-
drawing group stabilizes the glycosidic bond compared
to the olefinic analogue. Moreover, introduction of the
aromatic residue in the 20,30-positions of d4T increases
the liphophilicity of the resulting structure to a positive
logarithmic value of the partition coefficient (Table 1).
12 was found to be inactive against wild HIV-1 RT up
to 100 mM, while d4T-TP, used as a positive control,
displayed a IC90 value of 0.1 mM. One explanation for
this result may be related to the steric effect of the ben-
zene ring which does not allow the access of the
triphosphate derivative to the enzyme nucleotide
binding site.
The 1,3-dihydrobenzo[c]furan derivative 1 was evaluated
for its inhibitory effect on the replication of HIV-1 in
human T4-lymphoblastoid cells, CEM-SS and MT-4.
This compound has been found inactive against HIV-1
replication at concentrations up to 10 mM. In order to
explain this lack of antiviral activity, the corresponding
mononucleoside phosphotriester 117 (Scheme 2) bearing
two S-pivaloyl-2-thioethyl (tBuSATE) groups has been
synthesized according to a published procedure.8 We
have previously demonstrated that in vitro a depen-
dence on kinase-mediated phosphorylation could be
overcome by the use of such kind of mononucleotide
prodrugs (pronucleotides). Thus, applied to d4T such
pronucleotide approach led to a 10-fold increase of the
anti-HIV activity of the parent nucleoside in cell culture
experiments.9 Unfortunately, the bis(tBuSATE) phos-
photiester 11 did not display in vitro anti-HIV activity.
In conclusion, despite better chemical stability and
lipophilicity of the benzo[c]furan derivative 1 compared
to d4T, the lack of activity of 1 cannot be overcome by
using a bis(SATE) pronucleotide approach. Further-
more, the inability of the triphosphate 12 to inhibit
HIV-1 RT restricts the utility of such 1,3-dihydro-
benzo[c]furan nucleosides as potential anti-HIV agents.
References and Notes
1. Cheer, S. M.; Goa, K. L. Drugs 2002, 62, 2667.
2. Hurst, M.; Noble, S. Drugs 1999, 58, 919.
3. Thomas, S. A.; Segal, M. B. Br. J. Pharmacol. 1998, 125,
49.
4. Ewing, D. F.; Fahmi, N.-E.; Len, C.; Mackenzie, G.;
Pranzo, A. J. Chem. Soc., Perkin Trans. 1 2000, 3561.
5. Ewing, D. F.; Fahmi, N.-E.; Len, C.; Mackenzie, G.;
Ronco, G.; Villa, P.; Shaw, G. Nucleosides & Nucleotides 1999,
18, 2613.
6. Selouane, A.; Vaccher, P.; Villa, P.; Postel, D.; Len, C.
Tetrahedron: Asymmetry 2002, 13, 407.
7. Selected data for cis-1-(1,3-dihydro-3-hydroxymethyle
benzo[c]furane) thymidin-50-yl bis(S-pivaloyl-2-thio-ethyl)
Consequently, the absence of antiviral activity of the
1,3-dihydrobenzo[c]furan derivative 1 could be due to
inadequate second and/or third phosphorylation steps,
or to inefficient inhibition of RT by its 50-triphosphate
form. In order to discriminate between these hypoth-
eses, the 50-triphosphate derivative 1211 was obtained
using the Ludwig–Eckstein methodology (Scheme 2).10
In an in vitro assay using poly(rA).oligo(dT)12-18 as
template primer and [3H]-TTP as substrate, compound

D. Egron et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4473–4475
4475
phosphate (11): 60% yield; 31P NMR (121 MHz, DMSO-d6) d
programmed over a 30 min period with a flow rate of 1 mL/
min).
8. Lefebvre, I.; Perigaud, C.; Pompon, A.; Aubertin, A.-M.;
Girardet, J.-L.; Kirn, A.; Gosselin, G.; Imbach, J.-L. J. Med.
Chem. 1995, 38, 3941.
9. Girardet, J.-L.; Perigaud, C.; Aubertin, A.-M.; Gosselin,
G.; Kirn, A.; Imbach, J.-L. Bioorg. Med. Chem. Lett. 1995, 5,
2981.
10. Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631.
11. Selected data for triphosphate 12: 10% yield; 31P NMR
(121 MHz, D2O) d À22 (dd, Pb,,JPbPa=18.2 Hz, JPbPg=19.4
Hz), À11.1 (d, Pa,JPbPa=18.2 Hz), À8.2 (d, Pg?,JPbPg=19.4
Hz), 1H NMR (300 MHz, D2O) d 7.57–7.28 (m, 5H, aromatic-
H, 10-H), 7.09 (s, 1H, 6-H), 5.56 (m, 1H, 30-H), 4.40–4.24 (m,
2H, 80-H and 800-H), 1.73 (s, 3H, Thy–CH3), 13C NMR
(75 MHz, D2O) d 166.7 (4-C), 152.6 (2-C), 138.8–134.6 (2s, 90-
C,100-C), 138.0 (6-C), 130.4, 129.4, 122.8, 122.7 (4s, aromatic
C), 112.1 (5-C), 87.7 (10-C), 83.3 (d, 30-C, JP-C=9.1 Hz), 67.4
(d, 80-C, JP-C=6.0 Hz), 11.5 (Thy-CH3), MS FAB<0 m/z 579
(MÀNa)À, 557 (MÀ2Na+H)À, 535 (MÀ3Na+2H)À, 513
(MÀ4Na+3H)À, HPLC rt=9.1 min (see ref 8 for conditions).
1
À0.66, H NMR (300 MHz, DMSO-d6) d 11.4 (bs, 1H, Thy–
NH), 7.53–7.32 (m, 4H, aromatic H), 7.30 (d, 1H, 10-H,
0
0
0
J1 À3 =1.4 Hz), 6.90 (d, 1H, 6-H, J=1.0 Hz), 5.44 (m, 1H, 3 -
H), 4.53–4.39 (m, 2H, 80-H and 800-H), 3.97–3.89 (m, 4H,
CH2O), 3.07–2.94 (m, 4H, CH2S), 1.67 (d, 3H, Thy–CH3,
J=0.7 Hz ), 1.16, 1.15 (2s, 18H, tBu), 13C NMR (75 MHz,
DMSO-d6) d 205.5, 205.7 (2s, C¼O), 163.7 (4-C), 151.0 (2-C),
138.4, 136.3 (2s, 40-C and 50-C), 135.7 (6-C), 129.7, 129.2,
122.7, 122.2 (4s, aromatic C), 110.2 (5-C), 86.4 (10-C), 81.4 (d,
30-C, JPÀC=8.4 Hz), 68.8 (d, 80-C, JP-C=5.7 Hz), 65.5, 65.4
(2d, CH2O, JP-C=5.6 Hz), 46.0 (s, (CH3)3C), 28.0, 27.9 (2d,
CH2S, JP-C=8.2 Hz), 26.8 ((CH3)3C), 11.9 (Thy–CH3), MS
FAB>0 m/z 1285 (2M+H)+, 643 (M+H)+, 145 (tBu-
COSCH2CH2)+, MS FAB<0 m/z 641 (MÀH)À, 497
(MÀtBuSATE)À, 385 (MÀNu)À, 125 (B)À, Anal calcd for
C28H39N2O9PS2: C 52.33, H 6.12, N 4.36. Found: C 51.83, H
6.07, N 4.30. HRMS calcd for C28H40N2O9PS2 (M+H)+
643.1913, Found 643.1946, UV (ethanol 95): lmax 263 nm, l
232 nm, HPLC rt=23.9 min (gradient from 0 to 80% aceto-
nitrile in 20 mM triethylammonium acetate buffer (pH 6.6),