Synthesis and primary evaluation of novel HIV-1 inhibitors

Article abstract of DOI:10.1080/15257770701527109

The overcoming of antiviral drug resistance is an important challenge in the treatment of HIV-1 infection. According to the theory of viral error catastrophe, slightly increasing the mutation rate could exceed the error threshold for viability of a viral population and kill it. Investigation of this mechanism could lead to the discovery of new antiviral agents capable of bypassing viral resistance. To this aim, we designed several modified nucleosides. We describe here the synthesis and partial evaluation of 8-amido-2′-deoxyadenosine. The supplementary amide group on the base should allow base-pairing with several natural nucleosides, thus creating supplementary mutations that would kill the virus. Copyright Taylor & Francis Group, LLC.

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Synthesis and Primary Evaluation of
Novel HIV-1 Inhibitors
b
a
a
Yazan El Safadi , Roland Marquet , Anne-Marie Aubertin
&
a
Valérie Vivet-Boudou
a
Architecture et Réactivité de l'ARN, UPR 9002 du CNRS, IBMC ,
Strasbourg, France
b
UMR 544 INSERM-Université Louis Pasteur, Institut de Virologie ,
Strasbourg, France
Published online: 10 Dec 2007.
To cite this article: Yazan El Safadi , Roland Marquet , Anne-Marie Aubertin & Valérie Vivet-Boudou
(2007) Synthesis and Primary Evaluation of Novel HIV-1 Inhibitors, Nucleosides, Nucleotides and
Nucleic Acids, 26:8-9, 1161-1165, DOI: 10.1080/15257770701527109
To link to this article: http://dx.doi.org/10.1080/15257770701527109
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1161–1165, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701527109
SYNTHESIS AND PRIMARY EVALUATION OF NOVEL
HIV-1 INHIBITORS
Yazan El Safadi and Roland Marquet 2 Architecture et R e´ activit e´ de l’ARN, UPR
9002 du CNRS, IBMC, Strasbourg, France
Anne-Marie Aubertin 2 UMR 544 INSERM-Universit e´ Louis Pasteur, Institut
de Virologie, Strasbourg, France
Val e´ rie Vivet-Boudou 2 Architecture et R e´ activit e´ de l’ARN, UPR 9002 du CNRS,
IBMC, Strasbourg, France
2
The overcoming of antiviral drug resistance is an important challenge in the treatment of HIV-1
infection.
According to the theory of viral error catastrophe, slightly increasing the mutation rate could ex-
ceed the error threshold for viability of a viral population and kill it. Investigation of this mechanism
could lead to the discovery of new antiviral agents capable of bypassing viral resistance.
To this aim, we designed several modified nucleosides. We describe here the synthesis and partial
ꢁ
evaluation of 8-amido-2 -deoxyadenosine. The supplementary amide group on the base should allow
base-pairing with several natural nucleosides, thus creating supplementary mutations that would
kill the virus.
Keywords HIV-1; antiviral drug resistance; viral error catastrophe
The overcoming of antiviral drug resistance is an important challenge in
the treatment of Human Immunodeficiency Virus type 1 (HIV-1) infection.
ꢁ
NRTIs used against HIV are modified nucleosides that lack the 3 -hydroxyl
group of the sugar moiety. They are phosphorylated and incorporated into
the elongating DNA strand by the viral reverse transcriptase (RT) and stop
DNA synthesis. These NRTIs are therefore referred to as “chain termina-
tors.” However, due to its exceptionally high mutation rate, HIV-1 develops
resistance. An important resistance mechanism simply consists of excising
the chain terminating nucleotide incorporated by RT once DNA synthesis is
blocked.
We would like to Thank Philippe Wolff for his help in preparing and purifying oligonucleotides.
This work is supported by the ANRS (Agence Nationale de Recherches sur le Sida), France.
Address correspondence to Val e´ rie Vivet-Boudou, Architecture et R e´ activit e´ de l’ARN, UPR 9002
du CNRS, IBMC, 15, rue Ren e´ Descartes, Strasbourg 67084, France. E-mail: V.vivet@ibmc.u-strasbg.fr
1
161

1
162
Y. El Safadi et al.
It, therefore, is necessary to investigate new HIV-1 inhibitors that act
against the virus with different mechanisms.
HIV-1 and other retroviruses exhibit mutation rates 1,000,000 times
greater than host organisms, thus constantly placing them close to the
threshold of “error catastrophe.” This threshold is the limit above which
viral replication stops due to an intolerable number of mutations. Thus,
even a slight increase in the mutation rate of the virus should be sufficient
to obliterate the viral population.[
1]
The goal of this work is to design novel inhibitors, with unmodified sugar
moieties, but with modified bases, that will be incorporated into the nascent
viral DNA and will create enough supplementary mutations to drive the virus
above its error threshold for viability.
This concept has recently been investigated in the context of HIV-1 and
[2]
could be of great utility in the treatment of AIDS. Mutagenic nucleosides
could also be used efficiently in combination with chain terminators. Recent
studies showed that HIV strains resistant to nucleoside reverse transcrip-
tase inhibitors exhibited no cross-resistance towards a mutagenic nucleoside
[3]
analogue.
CHEMISTRY
Our goal was to design and synthesize novel base-modified nucleoside
analogues using commercially available nucleosides. Referring to the struc-
ture of ribavirin, which is known to pair equally well with two natural
nucleotides,[ we decided to introduce an amide function on the base,
hoping that it will induce extra hydrogen bonds and render the modi-
fied nucleoside mutagenic. Based on this strategy several modified nucleo-
4]
ꢁ
sides were designed. Here, we focus on 8-amido-2 -deoxyadenosine, which
ꢁ
we synthesized starting with commercially available 2 -deoxyadenosine
(
Figure 1). The synthetic strategy was inspired by Matsuda’s synthesis of 8-
[5]
amidoadenosine and is based on the insertion of the amide by oxidation
of a cyano group.
ꢁ
The bromination of 2 -deoxyadenosine was carried out according to the
[6]
procedure described by Ben Gaied et al. and resulted in a very high yield
8
ꢁ
-bromo-2 deoxyadenosine. Thiolation of (1) also proceeded with excellent
yield. The sugar moiety was then protected by acetic anhydride under mild
condition (3), and the thiomethyl group was oxidized using potassium per-
ꢁ
manganate to give the protected 8-methylsulfonyl-2 -deoxyadenosine (4).
◦
This reaction was carried out at 0 C and needed close monitoring, since
excessive reaction times result in a slight yield drop. Substitution of the sul-
fonyl group by sodium cyanide was also a smooth reaction and resulted in
ꢁ
the protected 8-cyano-2 -deoxyadenosine (5) with a 95% yield. The last step
consisted in an alkaline hydrolysis of the cyano group with simultaneous

Novel HIV-1 Inhibitors
1163
ꢁ
FIGURE 1 Synthetic route to 8-amido-2 -deoxyadenosine.
cleavage of the sugar protecting groups. It was carried out in water, in pres-
ꢁ
ence of five equivalents of sodium hydroxide. After purification, 8-amido-2 -
deoxyadenosine (6) was obtained as a pure white powder.
The overall yield of this six-step synthesis was 27% (average yield of
8
0% per step). The poorest yield was that of the last reaction, a result of
the difficult purification of the final product, which has very low solubil-
ity. Matsuda used a 50/50 mixture of ethanol and water to recristallize 8-
amidoadenosine, and we found the use of pure water more efficient for the
deoxynucleoside.
ꢁ
ꢁ
The 8-amido-2 -deoxyadenosine-5 -triphosphate derivative was also
chemically synthesized, in order to perform in vitro RT incorporation tests
(
Figure 2).
Perfectly dry conditions and reagents were used to perform this
one-pot triphosphate synthesis. The first step consisted in forming the
monophosphate using phosphorous oxychloride in triethyl-phosphate. The
cyclic triphosphate was then formed using a bis-tri-n-butyl-ammonium-
pyrophosphate solution in DMF. The product was hydrolyzed to 8-amido-
ꢁ
ꢁ
2
-deoxyadenosine-5 -triphosphate with TEAB.

1
164
Y. El Safadi et al.
ꢁ
ꢁ
FIGURE 2 Chemical synthesis of 8-amido-2 -deoxyadenosine-5 -triphosphate.
The triphosphate derivative was purified using a C-18 reverse phase
preparative chromatography, with a pH 7.5-buffer solution of triethylamine
acetate and acetonitrile. Reverse phase chromatography was followed by an
ion exchange chromatography to replace ammonium cations by lithium
cations. Purity of the product was verified by 31P NMR, which clearly
showed 3 phosphorous atoms: (αP: (d, j = 18.4 Hz) −9.41 ppm, βP: (t,
p-p
p-p
J
p-p
= 18.8 Hz) −19.1 ppm and γ P: (d; J = 18.8 Hz) −3.80 ppm). UV spec-
ꢁ
trum proved the presence of the 8-amido-2 -deoxyadenosine (maximal ab-
sorption at 288 nm). Mass spectroscopy was also performed and interestingly
displayed 4 peaks corresponding to the triphosphate derivatives with 0, 1, 2,
and 3 lithium cations.
BIOLOGICAL TESTS
ꢁ
8
-Amido-2 -deoxyadenosine was tested on MT4 and CEM-SS cells and
−
4
showed no cytotoxicity at concentrations up to 1.10 M on noninfected
cells. In a preliminary test, antiviral activity was estimated by measuring
the RT activity in the cell culture supernatant. Five days after infection of
ꢁ
MT4 cells with HIV-1 IIIB and of CEM-SS cells with HIV-1 LAI, 8-amido-2 -
deoxyadenosine displayed no significant antiviral activity in this assay at con-
−
4
centrations up to 1 10 M. These results, however, preclude a mutagenic
activity of this compound, as several replication cycles might be required to
accumulate lethal mutations. Prolonged in vitro tests involving 10 to 12 se-
quential passages, followed by the sequencing of the viral genome need to
be performed in order to evaluate its mutagenic activity. Moreover, in vitro
tests did not tell whether the compound was phosphorylated and did not
give any direct information about the new base-pairing possibilities induced
by the modified base.
In order to answer these questions, other experiments are in progress.
First, we wish to evaluate the base pairing properties of the compound
with natural nucleosides. Oligonucleotides bearing the modified nucleoside
will be prepared using the phosphoramidite chemistry. Melting tempera-
ture (Tm) of duplexes containing different modified nucleoside/natural

Novel HIV-1 Inhibitors
1165
nucleoside base pairs will be measured. When compared to the natural du-
plexes Tm, they will inform us about the base-pairing abilities of the mod-
ified base. Second, we will also measure in vitro incorporation the modi-
ꢁ
fied nucleoside 5 -triphosphate by RT. These specific tests will help us to
ꢁ
understand the possible reason for the failure or success of 8-amido-2 -
deoxyadenosine in inhibiting HIV replication.
Several base-modified nucleosides have been designed, in order to in-
vestigate the lethal mutagenesis theory. According to this mechanism, HIV-
1
replication could be suppressed by a mutagenic nucleoside that drives
the virus over its error threshold for viability. Among those nucleosides,
ꢁ
8
-amido-2 -deoxyadenosine was readily prepared, starting from commer-
ꢁ
cially available 2 -deoxyadenosine. The six steps of the synthesis proceeded
smoothly, offering good yields, even though the last step was limiting, due
to the very poor solubility of the final compound. The 5 -triphosphate of the
ꢁ
modified nucleoside also was synthesized and purified.
ꢁ
8
-amido-2 -deoxyadenosine showed no activity on HIV-1 replication in
cell culture, but more tests need to be done, to determine its mutagenic
effect, its base pairing possibilities and its incorporation by RT.
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Anderson, J.P.; Daifuku, R.; Loeb, L.A. Viral error catastrophe by mutagenic nucleosides. Annu. Rev.
Microbiol. 2004, 58, 183–205.
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Harris, K.S.; Brabant, W.; Styrchak, S.; Gall, A.; Daifuku, R. KP-1212/1461, a nucleoside designed for
the treatment of HIV by viral mutagenesis. Antiviral Research 2005, 67, 1–9.
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Crotty, S.; Maag, D.; Arnold, J.J.; Zhong, W.; Lau, J.Y.N.; Hong, Z.; Andino, R.; Cameron, C.E. The
broad spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nature Medicine 2000,
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Gaied, N.B.; Glasser, N.; Ramalanjaona, N.; Beltz, H.; Wolf, P.; Marquet, R.; Burger, A.; Mely, Y. 8-vinyl-
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deoxyadenosine, an alternative analog to 2 -deoxyribosyl-2-aminopurine with improved properties.
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