Identification of a novel family of nucleosides that specifically inhibit HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/S0960-894X(01)00635-7

N-3-Benzyloxycarbonylmethyl- and N-3-carboxymethyl-TBDMS-substituted nucleosides were synthesized and evaluated for activity against HIV replication. It was found that the N-3-carboxymethyl-TBDMS-substituted nucleosides were specific inhibitors of HIV-1 replication. They should be considered as members of a novel and original class of NNRTIs.

Full text of DOI:10.1016/S0960-894X(01)00635-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 3085–3088
Identification of a Novel Family of Nucleosides That Specifically
Inhibit HIV-1 Reverse Transcriptase
Cristina Chamorro,a Esther Lobaton,a Marıa-Cruz Bonache,a Erik De Clercq,b
Jan Balzarini,b Sonsoles Velazquez,a Ana San-Felixa and Marıa-Jose Camarasaa,*
a
´
´
Instituto de Quımica Medica (C.S.I.C.), Juan de la Cierva 3, 28006-Madrid, Spain
bRega Institute for Medical Research, K.U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Dedicated to Professor Erik De Clercq on his 60th Birthday
Received 22 June 2001; revised 3 September 2001; accepted 24 September 2001
Abstract—N-3-Benzyloxycarbonylmethyl- and N-3-carboxymethyl-TBDMS-substituted nucleosides were synthesized and evaluated
for activity against HIV replication. It was found that the N-3-carboxymethyl-TBDMS-substituted nucleosides were specific inhi-
bitors of HIV-1 replication. They should be considered as members of a novel and original class of NNRTIs. # 2001 Elsevier
Science Ltd. All rights reserved.
HIV treatment and efficient virus suppression can only
be achieved by administration of a concomitant combi-
nation of several drugs to HIV-infected individuals.
Highly active antiretroviral therapy (HAART)1 usually
consists of at least three, if not more, different anti-HIV
drugs. However, HIV chemotherapy has to be improved
since residual virus replication is still ongoing under
HAART and the compounds can select for drug-resis-
tant virus strains. Thus, novel strategies are required
that better suppress or limit the accumulation of resis-
tance mutations in viruses perhaps by, among many
other approaches, a faster switching to other drug regi-
mens. Therefore, further development of different types
of drugs that may be of use for multiple drug-combina-
tion chemotherapy of HIV is needed.
the polymerase active site.9ꢀ11 Within NNRTIs, TSAO-
12 is the prototype of a peculiar group of specific HIV-
T
1 RT inhibitors, developed in our laboratories.12,13
Structurally, TSAO derivatives are highly functionalized
nucleosides. These compounds exert their unique selec-
tivity for HIV-1 through a specific interaction with the
HIV-1 RT that, unlike the interaction of the nucleoside
type of RT inhibitors (i.e., AZT, ddI, d4T, etc.), is
noncompetitive with regard to the natural substrates.14
TSAO derivatives are so far the only family of nucleo-
sides (with an intact sugar moiety) that inhibit HIV-1
replication in a specific manner. TSAO derivatives select
for HIV-1 resistance through an unique mutation
(E138K) in HIV-1 RT.15ꢀ17
Following with our efforts to seek novel leads targeted
at the HIV-1 RT as specific HIV-1 RT inhibitors, we
have identified a novel family of nucleosides that specifi-
cally inhibit HIV-1 reverse transcriptase. In this letter, we
disclose our synthetic efforts and biological evaluation.
A key target in the search for effective drugs useful for
AIDS therapy is reverse transcriptase (RT).2,3 A num-
ber of inhibitors of HIV RT have been developed.4ꢀ6
Among them, the nonnucleoside RT inhibitors
(NNRTIs) represent a group of highly potent and spe-
cific inhibitors of HIV-1 replication.7,8 They interact
noncompetitively with the enzyme at an allosteric non-
substrate binding site that is close to but distinct from
The synthetic routes for the novel specific nucleosides
are shown in Schemes 1–3. Reaction of 1-[20,50- or 30,50-bis-
O-(tert-butyldimethylsilyl)-b-d-ribofuranosyl]thymine18 (1
or 2), 1-[20,30,50-tri-O-(tert-butyldimethylsilyl)-b-d-ribo-
furanosyl]thymine19 (3) or 1-[50-O-(tert-butyldimethyl-
silyl)-b-d-ribofuranosyl]thymine20 (4) with benzyl-
bromoacetate, in acetone in the presence of K2CO3
*Corresponding author. Tel.: +34-915622900; fax: +34-915644853;
e-mail: mj.camarasa@iqm.csic.es
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00635-7

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C. Chamorro et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3085–3088
(Scheme 1) gave the corresponding N-3-substituted
derivatives 5 (90% yield), 6 (73% yield), 7 (71% yield)
and 8 (40% yield). Catalytic hydrogenation (H2, Pd/C)
of 5–8 gave the corresponding free acid derivatives 9
(82% yield), 10 (85% yield), 11 (76% yield) and 12
(88% yield), respectively.
toluene/acetonitrile (1:1), followed by treatment with
tributyltin hydride (Bu3SnH) in the presence of a,a0-
azobis(isobutyronitrile), gave the 30-deoxy derivative 17
in 59% yield. Further treatment of 17 with (H2, Pd/C)
gave the target compound 18 in 85% yield.
The 20-deoxy derivative 23 (Scheme 3) was prepared
from thymidine as follows: Treatment of 30,50-bis-O-
(tert-butyldimethylsilyl)thymidine22 (19) with benzyl
bromoacetate in acetone, in the presence of K2CO3,
gave the N-3-substituted derivative 21 (97% yield)
which upon catalytic hydrogenation (H2, Pd/C) gave the
20-deoxy analogue 23 (81% yield). Finally, the 20,30-
dideoxy derivative 24 was prepared in 82% yield from 50
-O-(tert-butyldimethylsilyl)-20,30-dideoxythymidine23 (20)
following a similar procedure.
Treatment of 5 with 80% aqueous acetic acid (Scheme
2) gave the 50-deprotected analogue 13 (60% yield).
Treatment of 5 with tetrabutylammonium fluoride gave
the fully deprotected compound 15 (75% yield). Catalytic
hydrogenation (H2, Pd/C) of 13 or 15 gave the carboxy-
methyl derivatives 14 (74%) and 16 (94%), respectively.
The 30 or 20-deoxy- and 20,30-dideoxy analogues 18, 23
and 24 were also prepared. Compound 18 was synthe-
sized by radical deoxygenation from 5, following the
method of Barton and McCombie.21 Thus, reaction of 5
with N,N0-(thiocarbonyl)diimidazole (Scheme 2) in
Table 1 summarizes the results of the biological evalua-
tion of compounds 5–18 and 21–24, expressed as EC50
values or compound concentration required to inhibit
virus-induced cytopathicity in CEM cell cultures by
50%. The antiviral data on TSAO-T and AZT are also
reported as reference compounds.
Whereas several compounds inhibited HIV-1 replication
in the lower micromolar concentration range, none of
the compounds proved active against HIV-2 at subtoxic
concentrations (Table 1). Therefore, the active com-
pounds should be considered as specific inhibitors of
HIV-1 replication.
Beside of one exception (13), none of the N-3-benzyl
oxycarbonylmethyl derivatives showed antiviral activity
whereas the majority of the N-3-carboxymethyl derivatives
were endowed with anti HIV-1 activity.
Interestingly, optimal anti HIV-1 activity was achieved
with those N-3-carboxymethyl thymine nucleosides that
contained a TBDMS group at the 50-position together
with one TBDMS group either at 20 or 30 of the
(deoxy)ribose moiety (9, 10, 18 and 23).
Scheme 1. Preparation of nucleosides 5–12.
Scheme 2. Preparation of nucleosides 13–18.
Scheme 3. Preparation of nucleosides 21–24.

C. Chamorro et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3085–3088
3087
Table 1. Inhibitory effect of nucleosides 5–18, 21–24, TSAO-T and
AZT against HIV-1 and HIV-2 replication in CEM cellsa
active N-3-carboxymethyl nucleoside analogues here
described (i.e., 9, 10, 18) keep full inhibitory activity
against mutant HIV-1 reverse transcriptase that contain
the Glu138Lys mutation (Table 2). These findings are in
agreement with our observation that the novel nucleo-
side analogues keep inhibitory potential against a
mutant HIV-1/138Lys strain in cell culture. Indeed,
compounds 9, 10 and 18 showed an EC50 against HIV-
1/138Lys of 2.0, 8.2 and 4.0 mM, respectively. Our
results indicate that the novel nucleoside analogues
interact differently with HIV-1 RT than the TSAO
derivatives, and thus, N-3-carboxymethyl-TBDMS-sub-
stituted nucleoside analogues should be considered as
members of a novel and original class of NNRTIs.
b
c
Compds
EC50b (mM)
HIV-1
EC50 (mM)
HIV-2
CC50 (mM)
5
6
7
8
>10
>50
>10
>10
4.5ꢂ2.1
1.8ꢂ2.1
32ꢂ10.6
117ꢂ41.6
4.0ꢂ0.0
20ꢂ7.1
>250
>10
>10
>50
>10
>10
25.7ꢂ2.8
57.1ꢂ24.3
102ꢂ9.8
30ꢂ1.5
23.0ꢂ2.4
29.3ꢂ10.7
>125
9
10
11
12
13
14
15
16
17
18
21
22
23
24
TSAO-T
AZT
>10
>125
>250
>10
>250
>250
>250
>2
>10
>10
>50
>10
>250
26ꢂ1.4
>250
>250
>250
21.6ꢂ8.7
20.7ꢂ0.2
56.4ꢂ27.5
>250
>250
>2
4.5ꢂ0.7
Acknowledgements
ꢁ10
>250
5.0ꢂ1.4
20.3ꢂ0.4
We thank Ann Absillis and Lizette van Berckelaer for
excellent technical assistance. We also thank the Ministery
of Education of Spain for grants to C.C., E.L. and
M.C.B and GlaxoSmithKline S.A. for an award to
M.C.B. The Spanish CICYT (project SAF2000-0153-
C02-01), the Comunidad de Madrid (Project 08.2/0044/
2000), the European Commission (Project QLK2-CT-
2000-00291) and FORTIS/ISEP are also acknowledged
for financial support.
>250
>250
>20
0.004ꢂ0.001
>250
0.06ꢂ0.01
0.003ꢂ0.002
14ꢂ2
6.0ꢂ0.1
aData represent the mean values for three independent experiments.
b50% Effective concentration, or compound concentration required to
inhibit HIV-induced cytopathicity by 50%.
c50% Cytotoxic concentration, or compound concentration required
to reduce the viability of mock-infected cells by 50%.
Table 2. Sensitivity of HIV-1 wild-type Glu138 and mutant Glu138-
Lys recombinant RTs to the inhibitory effect of 9, 10, 18 and TSAO-
Ta
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