Synthesis and anti-HIV-1 integrase activity of modified dinucleotides

Article abstract of DOI:10.1016/j.ejmech.2009.09.007

The synthesis of a series of thirty-eight new modified dinucleotides and dinucleotide conjugate analogues of d-^5′ApC^3′ is described. The inhibitory activity of these compounds toward HIV-1 integrase was examined in enzymatic assays using the natural dinucleotide as a reference. Among the compounds, a perylene-dinucleotide conjugate has shown a two micromolar anti-integrase activity due to the presence of both the intercalator and the dinucleotide.

Full text of DOI:10.1016/j.ejmech.2009.09.007

European Journal of Medicinal Chemistry 44 (2009) 5029–5044
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-HIV-1 integrase activity of modified dinucleotides
a
a
a
b
a
a
´
Yves Aubert , Marcel Chassignol , Victoria Roig , Gladys Mbemba , Julien Weiss , Herve Meudal ,
**
Jean-François Mouscadet b , Ulysse Asseline
,
a,
*
a Centre de Biophysique Mole´culaire CNRS UPR 4301, affiliated with the University of Orle´ans and with INSERM Rue Charles Sadron, 45071 Orle´ans Cedex 02, France
b LBPA, UMR CNRS 8113, ENS Cachan, 61 avenue du pre´sident Wilson, 94235 Cachan, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a series of thirty-eight new modified dinucleotides and dinucleotide conjugate
0
0
analogues of d-5 ApC3 is described. The inhibitory activity of these compounds toward HIV-1 integrase
was examined in enzymatic assays using the natural dinucleotide as a reference. Among the compounds,
a perylene-dinucleotide conjugate has shown a two micromolar anti-integrase activity due to the
presence of both the intercalator and the dinucleotide.
Received 10 March 2009
Received in revised form
30 June 2009
Accepted 4 September 2009
Available online 9 September 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
HIV-1 integrase inhibitors
Modified dinucleotides
Intercalator-dinucleotide conjugates
1. Introduction
Among the different classes of integrase inhibitors, dinucleotide
analogs have proven to be potent competitors of the double-stranded
The efficient replication of retrovirus HIV-1 relies on the virally
encoded protein integrase [1–5]. This enzyme is necessary for the
first two steps of DNA integration: 30-processing in which two
nucleotides are removed from each 30-end of the viral DNA, and
strand transfer in which each 30-processed viral DNA end is
attached to the DNA host-cell. As a result, HIV-1 integrase has
attracted intense effort towards the development of potent inhib-
itors. Such efforts have led to the development of different classes
of compounds, [6–10] with one of them, Raltegravir (Isentress@)
a selective strand transfer inhibitor, approved by the FDA [11] and
a few currently under clinical trials [12,13]. However, resistance has
already been observed in patients treated with Raltegravir, leading
to virologic failure. There is therefore an ongoing constant need for
the development of new inhibitors [14–17].
oligonucleotide integrase substra0te [18].0 The monophosphorylated
0
0
0
0
natural dinucleotides p-d-5 ApC3 , p-d-5 ApT3 et p-d-5 CpT3 have
been shown to be moderate inhibitors of both the processing and
strand transfer steps [19]. Modified dinucleotides resistant to
nucleases with similar inhibitory effects have also been synthesized
[20,21] while others have exhibited more specific inhibition for the
strand transfer process [22]. In the quest for new dinucleotides with
nuclease stability and improved anti-integrase activities, we have
0
0
developed different sets of modified dinucleotide 5 CpA3 . This
dinucleotide was chosen as a scaffold in order to maintain structural
features for recognition since it is preserved at the end of the LTR
sequences after the processing step [1]. Modifications in dinucleo-
tides make them more resistant towards nucleases. The sugar ring,
the internucleotidic linkage, and the cytosine base have been
modified. Intercalating agents and varied ligands were also attached
to either the 50- or the 30-ends of the dinucleotide and the deoxyri-
bose and phosphodiester groups were replaced by a polymethylene
chain to attach the nucleic bases. We report here their synthesis and
anti-integrase activity. The different sets of modified dinucleotides
synthesized are listed in Fig. 1
Abbreviations: HIV-1, Human Immunodeficiency virus type 1; IN, Integrase;
DNA, Deoxyribonucleic Acid; FDA, Food and Drug Administration; IC 50, half
maximal inhibitory concentration is a measure of the effectiveness of a compound
0
0
inhibiting a biochemical or biological function; p-d-5 ApC3 , 50-monophosphorylated
dinucleotide phosphodiester involving 20-deoxyadenosine and 20-deoxycytidine; p-
0
0
d-5 ApT3 , 50-monophosphorylated dinucleotide phosphodiester involving 20-deox-
0
0
yadenosine and thymidine; p-d-5 CpT3
,
50-monophosphorylated dinucleotide
2. Chemistry
0
0
phosphodiester involving 20-deoxycytidine and thymidine; d-5 CpA3 , dinucleotide
phosphodiester involving 20-deoxycytidine and 20-deoxyadenosine.
* Corresponding author. Tel.: þ33 2 38 25 55 97; fax: 33 2 38 63 15 17.
** Corresponding author. Tel.: þ33 1 47 40 76 75; fax: 33 1 47 40 76 71.
E-mail addresses: mouscadet@lbpa.ens-cachan.fr (J.-F. Mouscadet), ulysse.
asseline@cnrs-orleans.fr (U. Asseline).
0
0
2.1. The natural dinucleotide d-5 CpA3
1
0
0
The natural dinucleotide
D
-d-5 CpA3 1 (Figs. 1 and 2), used as
a reference for evaluating the anti-integrase properties of the new
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.007

5030
Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
D-d-5' [N-4-guanidinoalkyl]CpA3'
5'
C
A
5'
5'
OH
C
HO
OH
9-11
L-d-5'CpA3'
2
L-d-5'ApC3'
3
O
O
O
D-d-5' [N-4-aminoalkyl]CpA3'
B
4-8
A
O
O
O
O
O
A
C
O
E
F
P
O
R1-p-d-5'CpA3'
23-31
D-d-5'CpA3'
1
D-d-3'CpA3'
12
O
A
P
C
D-d-5'CpA5'
13
O
P
O
3'
O
3'
O
O
O
O
O
H
3'
D
D
G
D
OH
OH
D-d-5'CpA3'-p-R2
32-37
d-5'CA3'
1
L-d-5'AC3'
38, 39
D-
L-d-5'CA3'
14, 17, 20
15, 18, 21
16, 19, 22
3
2
Fig. 1. Sets of modified dinucleotides: A: sugar modification; B: base modification; C:
internucleotidic polarity changes; D: internucleotidic linkage modification; E: 50-
conjugates; F: 30-conjugates; G: interbase linkage.
Fig. 2. Structures of dinucleotides 1–3.
30-H-phosphonate derivatives were then reacted with the conve-
nient nucleosides to give the dinucleoside H-phosphonates that
were oxidized and then deprotected, as reported for0 the preparation
compounds reported in this study, was obtained in solution via
H-phosphonate chemistry [23]. Briefly, the commercially available
H-phosphonate derivative of 20-deoxycytidine, protected at the 50-
hydroxyl function with a dimethoxytrityl group and at the base
exocyclic amine function with a benzoyl group, and 20-deoxy-
adenosine, protected at the base exocyclic amine and 30-hydroxyl
functions with benzoyl groups, were condensed in the presence of
pivaloyl chloride. The fully protected dinucleoside H-phosphonate
thus obtained was oxidized by iodine treatment in a pyridine/H2O
mixture and then deprotected using reported procedures [24]. We
chose this strategy because the dinucleoside-H-phosphonate
derivative can be used as a precursor for the preparation of dinu-
cleotides with varied internucleotidic linkages [25–27], as outlined
in section 2.5.
0
0
0
of compound 1, to give the
L
-d-5 CpA3 2 and
L
-d-5 ApC3 3 dinucle-
otides. The structures of these deprotected dinucleotide compounds
were confirmed by 1H NMR and mass spectrometry analysis.
2.3. N-4-alkylation of the 20-deoxycytidine
N-4 alkylation of the 20-deoxycytidine was performed in order to
allow additional interactions between the dinucleotide and the
integrase (Scheme 1). As substituent groups we chose aliphatic bis-
aminated polymethylene linkers with different lengths (from 2 to 6
methylene groups). Alkylation of the 20-deoxycytidine was per-
formed following previously reported procedures starting from the
triazolo derivative of 20-deoxyuridine 40 [29]. Reaction with the
monotritylated linkers 41–45, followed by 30-desilylation led to
mononucleotides 46–50. The latter were transformed into their
phosphoramidite derivatives 51–55 that were reacted with the
conveniently protected 20-deoxyadenosine. After oxidation, depro-
tection and purification following classical procedures, the alkylated
dinucleotides 4–8 were obtained with good yields. The guanidinium
derivatives 9–11 of the amino-alkylated dinucleotides 6–8 involving
the tetra, penta and hexamethylene linkers were also obtained
following a procedure adapted from our previous work [30].
2.2. Dinucleotides 2 and 3 with L
-20-deoxyribose units
To obtain our first modification, we chose to investigate the
influence of the sugar by replacing the natural -sugar by its corre-
spon0ding 0 -enantiomer (Fig. 2). The dinucleotide phosphodiesters
-d-5 CpA3 2 and
from commercially available unprotected
strategies adapted from those commonly reported in the D-series.
Briefly, the exocyclic amino functions of the
-20-deoxycytidine and
the
-20-deoxyadenosine were benzoylated and the 50-hydroxyl
D
L
0
0
L
L
-d-5 ApC3 3 were synthesized in solution starting
-dC and -dA using
L
L
L
L
function dimethoxytritylated [24]. One half of each compound was
then transformed into its H-phosphonate derivative using 2-chloro-
5, 6-benzo-1, 3, 2-dioxaphosphorin-4-one [28]. The second half of
each compound was 30-benzoylated and then detritylated to afford
2.4. Dinucleotides 12 and 13 with modified internucleotidic linkage
polarities
In order to evaluate the i0nfluence of the internucleotidic
0
0
0
the
L
-nucleosides with free 50-hydroxyl functions. The nucleoside
polarity, the dinucleotides
D
-d-3 CpA3 12 and
D
-d-5 CpA5 13 were
N
N
H
N
NH2
H
H
H
H
N-(CH2)nNH2
N
N
O
N
N-(CH2)nNHMMTr
N
N-CH2)nN-C
N-(CH2)nNHMMTr
N
+
-
NH2
N
DMTrO
40
O
N
Cl
O
N
N
HO
O
O
N
HO
DMTrO
DMTrO
O
O
O
O
O
O
a
c
d
e
b
OTBDMS
+
NH2
NH2
N
N
O
O
O
N
N
N
N
OH
N
MMTr-HN(CH2)nNH2
-
-
P
O
O
P
P
O
N
O
O
OCH2CH2CN
46 n = 2, 47 n = 3
48 n = 4, 49 n = 5
50 n = 6
N
41 n = 2, 42 n = 3
43 n = 4, 44 n = 5
45 n = 6
O
O
51 n = 2, 52 n = 3
53 n = 4, 54 n = 5
55 n = 6
OH
OH
9 n = 4
10 n = 5
11 n = 6
4 n = 2, 5 n = 3
6 n = 4, 7 n = 5
8 n = 6
Scheme 1. Synthesis of the dinucleotides 4–11. Reagents and conditions. a: CH3CN, rt, 15 h. b: 1 M tetrabutylammonium fluoride in THF, 2 h, rt. c: 2-cyanoethyldiisopropyl-
chlorophosphoramidite, diisopropylethylamine, dichloromethane. d: 4-N-benzoyl-30-benzoyl-20-deoxyadenosine, Te; oxidation step; deprotection and purification steps. e: 1H-
pyrazole-1-carboxamidine hydrochloride, 1 M aqueous Na2CO3; purification.

Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
5031
3'
-
-
5'
C
C
S
O
HO
5'
C
O
HO
O
O
Nu
Nu
P
P
Nu
Nu
O
HO
O
O
O
14-16
A
O
O
O
A
A
P
O
P
1, 12, 13
O
O
a
O
b
O
O
P
O
5
'
O
O
OH
H
P
O
O
O
H
3'
OH
3'
Nu'
Nu'
D- d-5'CA3'
1
3'
D-d- CA
3'
D-d-5'CA5'
13
12
dinucleoside-H-phosphonates
Fig. 3. Structures of dinucleotides 12 and 13 with modified internucleotidic linkage
polarities.
with 5'-3', 3'-3' and 5'-5' polarities
c
synthesized (Fig. 3). As for the preparation of the natural dinucle-
NH2
0
0
otide D
-d-5 CpA3 1, the synthesis was performed via H-phospho-
(CH2)4
nate chemistry because of the possibility of the internucleotidic
linkage modification when using this strategy (Scheme 2). The
conveniently protected monomers were obtained as follows:
20-deoxycytidine and 20-deoxyadenosine protected by a dimethox-
ytrityl group on their 50-hydroxyl functions and benzoyl groups at
both their exocyclic amine and their 30-hydroxyl functions were
detritylated. An H-phosphonate group was incorporated at the
50-position of the 20-deoxycytidine as reported above and the latter
was reacted with the 20-deoxyadenosine derivative involving the
free 50-hydroxyl group to give the dinucleotide H-phosphonate
N
H
Nu
Nu
P
O
17-19
d
NH2
H
N-C
+
NH2
-
(CH2)4 Cl
N
H
30
Bz 30
P
Nu
Nu
derivative
phonate
D
-d- CBzp[H]A
. The modified dinucleotide H-phos-
0
0
-d-5 CBzp[H]ABz 5 was obtained by a reaction between the
O
20-22
D
30 H-phosphonate derivative of the 50-O-dimethoxytrityl-4-N-
benzoyl 20-deoxycytidine and the 30-hydroxyl function of the 50-O-
dimethoxytrityl-6-N-benzoyl-20-deoxyadenosine in the presence
of pivaloyl chloride. After the oxidation step, deprotection and
Scheme 2. General scheme for the internucleotidic bond modification. The inter-
nucleotidic H-phosphonate linkage can be converted into phosphodiester (route a),
phosphorothioate (route b), phosphoramidate (route c). The amino-ending linkers can
be guanidylated (route d). Nu0 stands for the protected nucleosides and Nu for the
deprotected ones.
0
0
purification led to the dinucleotides
D
D
-d-3 CpA3 12 and
0
0
-d-5 CpA5 13.
inside oligonucleotides. For these reasons we decided to try
obtaining the above-mentioned dinucleotides as pure isomers by
reversed-phase separation and were successful for all the dinu-
cleotides except for 16 obtained as an isomer mixture.
2.5. Modifications of the internucleotidic linkage
0
0
The phosphodiester bond of the natural dinucleotide
D
-d-5 CpA3
0
0
0
0
1 and the unnatural dinucleotides D D
-d-3 CpA3 12 and -d-5 CpA5 13
(Scheme 2) were replaced by a phosphorothioate group 14–16. An
amino-ending linker was also attached to the internucleotidic
position by the intermediate of a phosphoramidate linkage 17–19.
2.6. Covalent attachment of varied0 ligands to either the 50- or the
0
30-endofthedinucleotide
D
-d-5 CpA3 23–37 (seeTable 2 forstructures)
Finally,
a
guanidylated linker was also attached to the
In order to induce additional interactions with the integrase,
various ligands such as linkers with terminal hydroxyl or amino
functions, or intercalating groups were attached to either the 50- or
internucleotidic position 20–22. The syntheses of the dinucleotides
14–22 were performed by modification of the dinucleoside H-
0
0
0
0
30
30
phosphonates
D
-d-5 CBzp[H]ABz
,
D
-d-3 CBz[H]pABz
and DD
-
the 30-end of the dinucleotide -d-5 CpA3 . Two strategies can be
D
0
50
d-5 CBzp[H]ABz
in accordance with literature reports and our
used. In the first, the ligands are directly incorporated via the use of
modified supports involving either them (30-conjugates 32–34) or
their phosphoramidite (24, 28, 31) or H-phosphonate (30) deriva-
tives. The ligands must withstand the chemical conditions required
for both the dinucleotide synthesis and deprotection steps. In the
second strategy, a post-synthetic specific reaction is performed
between convenient groups, incorporated at preselected positions
in both the ligands and the deprotected dinucleotide. In this study,
for the preparation of conjugates 23, 25–27, 29, and 35–37 we
chose to use the reaction between halogenoalkyl linkers attached
to the ligands and a phosphorothioate group incorporated in either
the 50-end 0 (56) 0 or the 30-end (57) of the dinucleotide phos-
phodiester5 CpA3 (Scheme 3). The 50 and 30-phosphorothioate
incorporations were performed following our previously published
procedures [31].
previous results. Part of each dinucleoside-H-phosphonate was
separately transformed into dinucleoside phosphorothioate by
treatment with S8 in a pyridine/CS2 mixture to give, after depro-
tection and purification, the dinucleotide phosphorothioate 14–16
[25]. The remaining part of each pure dinucleoside-H-phosphonate
was
separately
d-5 DMTrCBzp*[NH(CH2)4NHMMTr]A
with 1,4-diaminobutane, protected at one amino function with
monomethoxytrityl group 43, in CCl4/pyridine mixture,
transformed
into
the
dinucleoside
0
Bz30
by amidative oxidation
a
a
following a procedure adapted from our previously published work
[26,27], to give, after deprotection and reversed-phase purification,
dinucleosides 17–19. Dinucleosides 20–22 involving the guanidy-
lated linkers were obtained by treatment of dinucleotides 17–19
involving the amino linker with 1H-pyrazole-1-carboxamidine as
mentioned above [30]. The replacement of the internucleotidic
phosphodiester bond by either a phosphorothioate or a phosphor-
amidate bond induced the formation of isomer pairs that have been
shown to possess different biochemical properties when included
2.6.1. 50-conjugates 23–31
2.6.1.1. Bis-aminoalkylated-dinucleotide conjugate 23. In order to
allow possible ionic interactions with integrase, a triamine linker

5032
Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
O-
Table 1
O-
Structures and activities of the dinucleotides 1–22.
-
S-P-5'CA3' + RX
R- S-P-5'CA3'
O
Compounds
IC 50 (mM)
0
0
1
2
3
D
-d-5 CpA3
340
O
0
0
0
L
-d-5 CpA3
ꢀ340
56
23, 25-27, 29
0
L
-d-5 ApC3
ꢀ340
0
0
D
-d-5 [N-4-aminoalkyl]CpA3
O-
O-
4
>1000
>1000
>1000
>340
>340
>340
>340
>340
ꢀ340
ꢀ340
ꢀ340
ꢀ340
110
5
6
7
8
5'CA3'-P-S-R
5'CA3'-P-S- + RX
O
35-37
O
57
0
0
9
D
-d-5 [N-4-guaridiroalkyl]CpA3
10
11
12
13
14
15
16
17
18
19
20
21
22
Scheme 3. General scheme for 50-conjugation (top) and 30-conjugation (bottom) via
phosphothiolodiester linkage.
0
0
0
D
D
D
D
D
D
D
D
D
D
D
-d-3 CpA3
0
-d-5 CpA5
0
0
0
-d-5 Cp(S)A3
0
0
was attached to the 50-end of the dinucleotide
D
-d-5 CpA3 as
0
-d-3 Cp(S)A3
follows. Bistrifluoroacetylated diethylenetriamine [32] 58 was
reacted with 1,3-diiodopropane to give the 1,7-bis trifluoroacetyl-
4-(3-iodopropyl)hepta-1,4,7-triazane 59 (Scheme 4). The latter was
then reacted with 50-thiophosphorylated dinucleotide S-p- CpA
56 to give, after deprotection, the modified dinucleotide 23
involving a linker ending with two amino groups.
50
50
-d- Cp(S)A
0
0
0
0
-d-5 Cp(amino)A3
ꢀ340
0
-d-3 Cp(amino)A3
102
0
-d-5 Cp(amino)A5
100
50
30
0
0
0
0
-d-5 Cp(guanidino)A3
ꢀ340
0
-d-3 Cp(guanidino)A3
100
0
-d-5 Cp(guanidino)A5
100
2.6.1.2. Intercalator-dinucleotide conjugates 24–31. Intercalating
agents are planar molecules with fused aromatic rings [33]. In
addition to their intercalating properties these compounds are
lipophilic so that they are able to display different interactions with
theintegrase. We surmised that intercalator-dinucleotide conjugates
would be able to interact in two different ways [34]. The dinucleotide
would interact specifically with the integrase active site, while the
intercalatorwould be involved in additionalinteractionsnearby with
otherparts of theenzyme. Thedifferentintercalatingagents linkedto
with 1,3-dibromopropane in acetonitrile to give the new acridine
derivative 62.
2.6.1.2.2. Anthraquinone derivatives. We chose to attach a series of
three different anthraquinones to the 50-end of the dinucleotide in
order to display varied interactions between the dinucleotide-
intercalator conjugates and the target. The first one, involving an
iodopentyl linker attached to the position 1 via an amide function
67, was obtained by reaction of the 1-aminoanthraquinone 63 with
6-bromohexanoyl chloride in the presence of pyridine (Scheme 6).
Halide exchange was then performed by NaI treatment to give the
anthraquinone with the iodopentyl linker 67. The attachment of the
same linker to position 1 of the 1,4-diaminoanthraquinone 64 was
performed following the same strategy except that an excess of
anthraquinone was used. The two anthraquinone derivatives 67
and 68 were reacted with the 50-thiophosphorylated dinucleotide
56 to give the anthraquinone-dinucleotide conjugates 26 and 27.
The third anthraquinone derivative involves two hexamethylene
0
0
the 50-end of the natural phosphodiester dinucleotide
D
-d-5 CpA3
were acridine, anthraquinones, thiazole orange and perylene (see
Table 1). Depending on their structures, these molecules exhibit
different areas and lipophilic properties, thus offering a wide variety
of possible interactions with the integrase.
2.6.1.2.1. Acridine
derivatives. 6-Chloro-2-methoxy-9-amino-
acridine was attached either via a neutral or a positively charged
linker. The linkage of the acridine derivative by a hexamethylene
tether was performed on solid-phase by using its phosphoramidite
derivative, obtained as previously reported [35]. After synthesis,
the deprotection step, performed with a sodium hydroxide solution
(0.4 N) in a MeOH/H2O mixture in order to avoid the cleavage of the
bond between the acridine ring and the linker, led to the acridine-
dinucleotide conjugate 24. Acridine was also attached to the 50-end
of the dinucleotide via a new linker involving a positively charged
group. The presence of this charge precluded the preparation of the
phosphoramidite derivative. For this reason the linkage of this new
acridine-linker derivative to the dinucleotide was performed by the
reaction of the 50-thiophosphorylated dinucleotide 56 with the
bromoalkylated linker attached to the acridine derivative [31]. The
synthesis of the new acridine-linker compound was performed
following a two-step procedure (Scheme 5). First, 6, 9-dichloro-2-
methoxyacridine 60 and 3-dimethylaminopropylamine were
reacted in phenol to give the 6-chloro-2-methoxy-9-(3–3–dime-
thylaminopropyl)-amino acridine 61. The latter was then reacted
Cl
N
Cl
N
H
CH3
CH3
a
N-(CH2)3-N
Cl
OCH3
OCH3
61
60
Cl
N
CH
-
3
H
Br
+
(CH ) -Br
N-(CH ) -N
-
2 3
2 3
CH3
H
H
a
OCH3
[F3C-C-N-CH2CH2]2-NH
[F3C-C-N-CH2CH2]2-N(CH2)3I
62
O
O
59
Scheme 5. Synthesis of the acridine derivative 62 with a positively charged linker
Reagents and conditions. a: 3-dimethylaminopropylamine (2 eq), phenol, 100 ꢁC, 2 h.
b: 1,3-dibromopropane (2 eq), CH3CN, 80 ꢁC, 2 h.
58
Scheme 4. Synthesis of the protected bis-aminoalkylated linker 59

Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
5033
O
H
O
O
O
O
O
O
NH2
X
N-C-(CH2)5-Br
HO-C
O
C-OH
O
a
O
O
69
X
65, X = H
66, X = NH
a
2
O
H
63, X = H
64, X = NH2
b
DMTrO(CH2)6-N-C
C-OH
H
N-C-(CH2)5-I
O
O
70
O
O
c
b
67, X = H
68, x = NH2
X
O
H
H
DMTrO(CH2)6-N-C
C-N-(CH2)6-OH
Scheme 6. Synthesis of the anthraquinone-linker derivatives 67 and 68 Reagents and
conditions. a: 6-bromohexanoylchloride (1. 2 eq), pyridine ( ), benzene, reflux 2 h for
compound 63 (4 h for compound 64); b: NaI (5 eq), NaHCO3 (5eq), acetone, reflux 8 h.
O
O
71
3
O
d
H
H
O
OCH2CH2CN
linkers attached to the 2 and 7 positions via amide linkages
(Scheme 7). 2,7-Biscarboxyanthraquinone 69 was obtained as
previously reported [36]. At this stage, the bisfunctionalization by
two aminohexanol linkers was performed via amide bond forma-
tion. In order to allow covalent attachment of the anthraquinone
derivative to only one dinucleotide, both linkers were selectively
protected on their hydroxyl functions by a dimethoxytrityl and
a tert-butyldiphenylsilyl group, respectively. The dimethoxytrity-
lated linker was attached to the 2,7-biscarboxyanthraquinone
derivative after activation with carbonyldiimidazole in the presence
of a catalytic amount of hydroxybenzotriazole and triethylamine to
give 70. After purification of the monoderivatized anthraquinone
70, the silylated linker was attached following the same strategy.
The silyl group was removed by tetrabutylammonium fluoride
treatment to give the bis substituted anthraquinone 71. The
phosphoramidite derivative 72 was obtained by treatment of 71
with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the
presence of N,N-diisopropylethylamine. After reaction of the
phosphoramidite 72 with the free hydroxyl function of the dinu-
cleotide bound to the support followed by iodine oxidation, the
anthraquinone-dinucleotide conjugate 28 was obtained after
removal of the protective groups and reversed-phase purification.
C-N-(CH2)6-O-P
DMTrO(CH2)6-N-C
N
O
O
72
O
Scheme 7. Synthesis of the anthraquinone-linker derivative 72 Reagents and condi-
tions. a: Pyr, NEt3, hydroxybenzotriazole (catalytic amount), 1,10-carbonyldiimidazole
(1. 2 eq), rt, 2 h; 1-O-(4,40-dimethoxytrityl)-6-hexylamine (0. 9 eq), rt, 20 h. b: Pyr,
NEt3, hydroxybenzotriazole (catalytic amount), 1,10-carbonyldiimidazole (1. 2 eq), rt,
2 h; 1-O-(tert-butyl-diphenylsilanyl)-6-hexylamine (0. 9 eq), rt, 20 h. c: 1 M tetrabu-
tylammonium fluoride in THF (5 eq), rt, 1 h. d: ClCH2-CH2Cl, DIEA (3.5 eq), 2-cya-
noethyl-N,N-diisopropylchlorophosphoramidite (1. 4 eq), 75 min.
For both conjugates the deprotection step was achieved by
concentrated aqueous ammonia treatment.
2.6.2. 30-conjugates 32–37
2.6.2.1. Dinucleotide with hydroxyl- and aminoalkyl linkers
32–34. The synthesis of the dinucleotide-linker conjugates 32–34
was performed via phosphoramidite chemistry on solid-phase
using modified supports releasing, after the deprotection step, the
linkers with the desired functionalities. The modified support
releasing the aminohexyl linker was obtained as previously
reported [40]. The modified supports releasing the dinucleotides
with hydroxypropyl and the hydroxydodecyl linkers were obtained
in a four-step process from 1,3-propanediol and 1,12-dodecanediol
following described procedures [41,42].
2.6.1.2.3. Thiazole orange derivative. Acridine and anthraquinone
are intercalating molecules composed of three fused rings. Thiazole
orange, composed of a benzothiazole and a quinolinium nuclei
linked via a monomethine bond, offers many more possibilities of
interaction with the integrase. Thiazole orange was attached to the
50-end of the dinucleotide via a phosphothiolodiester and a hepta-
methylene linker 29. The synthesis of the thiazole orange linker
derivative was performed as previously reported [37].
2.6.2.2. Dinucleotide-acridine conjugates 35–37. The acridine was
also linked to the 30-end of the dinucleotide via a phosphothiolo-
diester linkage and either a tri- or a hexamethylene linker 35, 36.
These syntheses were performed by the reaction between the
dinucleotide bearing
a
30-terminal thiophosphate group 57,
2.6.1.2.4. Perylene derivatives. Perylene is composed of five fused
rings. It is therefore the most lipophilic among the intercalators
used in this study. In order to provide it with different possibilities
of interaction with the enzyme, the perylene was attached to the 50-
end of the dinucleotide either via a phosphodiester or to the
obtained using a previously reported support [31], and acridine
derivatives involving bromoalkyl linkers. The latter were obtained
using a procedure adapted from one of our previous reports [37].
The acridine derivative 62 involving the quaternary ammonium
group was also linked to the 30-end of the dinucleotide via a phos-
phothiolodiester linkage 37.
b
-anomeric position of a 20-deoxyribose following procedures
adapted from our previous work [38,39]. The synthesis of the
dinucleotide conjugate 30 involving the perylene attached to the
ß-anomeric position of a 20-deoxyribose unit added to the 50end of
the dinucleotide was performed on solid-phase by using the
H-phosphonate derivative of the perylene sugar unit [38]. As
reported for the acridine-dinucleotide conjugate 24, the synthesis
of the perylene-dinucleotide conjugate 31, involving an eighth-
atom linker, was performed via phosphoramidite chemistry [39].
2.7. Replacement of the two 20-deoxyribose units and 0the
0
phosphod0iester linkage of the dinucleotides
D
-d-5 CpA3
0
and D
-d-5 TpG3 by a polymethylene linker 38, 39
As shown on a CPK model, the two 20-deoxyribose units and the
phosphodiester linkage can be replaced by a hexamethylene linker,

5034
Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
NHBz
N
O(CH2)2-
NO2
O(CH2)2-
N
N
NO2
N
NHBz
N
N
N
H
N
N
N
N
H
a
N
N
(CH2)6Br
N
+
NH
iBu
73
+
a
N
N
NH
iBu
77
(CH2)6Br
78
Br-(CH2)6-Br
Br-(CH2)6-Br
74
75
74
b
b
O
O
O
NHBz NHBz
O
N
NH2
N
N
NH2
N
N
H3C
H3C
NH
NH
N
N
N
N
N
N
c
N
N
N
NH
NH
iBu
c
NH
NH2
N
N
N
O
O
N
N
N
O
N
O
(CH2)6
(CH2)6
79
(CH2)6
76
(CH2)6
38
39
Scheme 8. Synthesis of the compound 38 Reagents and conditions. a: K2CO3 (1eq);
1,6-dibromohexane (5 eq); 60 ꢁC, 10 h. b: 4-N-benzoylcytosine (1. 2 eq), K2CO3 (1.1 eq),
DMF, 55 ꢁC, 8 h, then 20 ꢁC, 12 h. c: NH4OH (28%) (12 mL), MeOH (1.5 mL), 20 ꢁC, 72 h.
Scheme 9. Synthesis of the compound 39. Reagents and conditions. a: K2CO3 (1eq);
1,6-dibromohexane (4 eq); 60 ꢁC, 11 h. b: Thymine (1.15 eq), DBU (1.73 eq), Py, rt, 24 h.
c: NH4OH (28%) (4 mL), 50 ꢁC, 15 h.
Acridine-linker
Thiazole orange-linker
Perylene-linker
Strand transfer
products
21-mer substrate
3’- processing product
24
29
31
150
100
50
29
24
Acridine-linker
Thiazole orange-
linker
100
50
0
0
10-8 10-7 10-6 10-5 10-4 10-3
[compound] (mol.L-1)
10-8 10-7 10-6 10-5 10-4 10-3
[compound] (mol.L-1)
31
24
100
50
0
100
50
0
Perylene-linker
29
31
10-8 10-7 10-6 10-5 10-4 10-3
[compound] (mol.L-1)
10-8 10-7 10-6 10-5 10-4 10-3
[compound] (mol.L-1)
Fig. 4. Anti-integrase activities of conjugates 24, 29 and 31 compared to those of the corresponding intercalator-linker derivatives. Upper panels: PAGE analysis of integrase overall
activity for 1 hr at 37 ꢁC, in the presence of increasing drug concentration (left to right, 100 nM IN þ 10 mM EDTA, 300 nM, 1
mM, 3mM, 10mM, 30mM, 100mM, 300mM, no drug).

Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
5035
Table 2
function by an isobutyryl group and a p-nitrophenylethyl moiety at
position 6 of 77 [43] was reacted with 1, 6- dibromohexane 74 to
give the alkylated guanine 78. The latter was then reacted with
thymine to give the protected compound 79 and, after depro-
tection, compound 39.
Structures and activities of the dinucleotide conjugates 23–37.
Compounds
IC 50 (mM)
R1-p-5'CpA3'-p-R2
R1-
R2
3. Pharmacology
H2N(CH2)2
N(CH2)3-S
23
H
H2N(CH2)2
786
The integration of pro-viral cDNA into host DNA involves two
reactions catalyzed by the viral enzyme integrase: 30end processing
of the viral DNA ends and strand transfer [1–10]. In the cytoplasm of
the infected cells, the viral RNA genome is reverse transcribed into
double stranded viral DNA. Then, integrase binds to both extrem-
ities of the viral cDNA, forming with other various viral and cell
proteins the pre-integration complex. Inside this complex, the
enzyme removes the dinucleotide GT from the two 30- ends of the
linear viral DNA (processing reaction) leading to overhanging CA
ends. Then the pre-integration complex is transported from the
cytoplasm to the nucleus of the infected cells were the 30-processed
ends of the viral DNA attack phosphodiester bonds on both strands
of the target DNA simultaneously resulting in the incorporation of
viral DNA into host cell DNA (strand transfer reaction). These two
catalytic reactions (30-terminal processing and strand transfer) can
be reproduced in vitro by using recombinant integrase and
synthetic DNA duplexes mimicking the terminal sequences of viral
DNA as substrate. The anti-integrase activity of potent inhibitors
directed towards these two catalytic steps can be evaluated in vitro
by monitoring the amount of processed and strand–transfer
products by polyacrylamide gel electrophoresis analysis (See Fig. 4).
Cl
H
24
N
(CH2)6-O
13.2
N
H
OCH3
Cl
N
CH
+
-
3X
H
N
(CH2)3-N-(CH2)3-S
25
H
H
>100
CH3
OCH3
H
O
O
N-C-(CH2)5-S
26
27
X = H
O
13.9
13.4
X = NH2
X
H
H
O
HO(CH2)6-N-C
C-N-(CH2)6 -O
28
29
O
O
H
21
O
+
H3C
N
(CH2)7-S
N
H
H
x
S
65.5
30
O-(CH2)3
HO
0
0
O
Dinucleotide analogues of the preserved d-5 -CA-3 at the ends of
the viral DNA processed strands can possibly inhibit the integrase
activity by competing with the viral duplex target.
30
31
O
1.8
H
H
(CH )- (CH ) -O
N-
2
2 6
4. Results and discussion
32
33
34
H
(CH2)3-OH
>100
>100
>100
Each modified dinucleotide 2–39 was screened for inhibitory
activity against HIV-1 integrase. Inhibitory activities were studied
in a full activity assay monitoring simultaneously both 30processing
and strand transfer activities. The unmodified dinucleotide 1 was
also tested for comparative activity. The results can be separated
into two parts. Among the modified dinucleotides 2–22, only five
compounds are slightly more efficient (three to four-fold) inhibitors
H
H
(CH2)12-OH
(CH2)6-NH2
OCH3
35
36
n = 3
H
H
S-(CH2)n
N
N
H
n = 6
>100
>100
Cl
OCH3
than the unmodified dinucleotide 1 (IC 50 ¼ 340
mM) (Table 1). All
CH3
+
-
these compounds are involving two modifications with respect to
the dinucleotide 1 used as a reference: a change of the internu-
cleotide bond polarity and the replacement of the phosphodiester
linkage. They are: 16 with a 50–50 polarity and a phosphorothioate
linkage; 19 and 22 with a 50–50 polarity and a phosphoramidate
internucleotidic linkage and 18 and 21 with a 30–30 polarity and
a phosphoramidate internucleotidic linkage. The other changes of
X
37
S-(CH2)3-N-(CH2)3
CH3
H
N
N
H
Cl
the dinucleotide structure such as the replacement of the
D
-20-
while the nucleic bases cytosine and adenine are able to stack
deoxyribose by the
L
-20-deoxyribose (compounds 2 and 3) and the
together in a man0ner similar to the one they adopt in the natural
alkylation of the cytidine (compounds 4–11) did not improve the
inhibitory effect of the dinucleotide. In the dinucleotide conjugate
series 23–37 (Table 2) two types of behaviour could be observed.
50-Conjugation (compounds 23–31) led to an increase in the
inhibitory effect, except for compounds 23, involving at the 50-end
a branched linker with two amino groups, as compared to the
activity of the unmodified dinucleotide 1. On the contrary, no
improvement of the dinucleotide potency was observed by 30-
conjugation (compounds 32–37). The intercalators linked to the 50-
end of the dinucleotide were acridine, anthraquinones, thiazole
orange and perylene (compounds 24–31, Table 2) that exhibit
different areas and lipophilic properties, thus offering a wide
variety of possible interactions with the integrase. Two of them
0
dinucleotide
D
-d-5 CpA3 . We therefore chose to tether the cytosine
and the adenine with a hexamethylene linker attached to position 1
of the cytosine and position 9 of the adenine to give compound 38.
In the same way, the two 20deoxyribose units and the phospho-
0
0
diester linkage of the dinucleotide D
-d-5 TpG3 (complementary to
the CA dinucleotide on the double-stranded proviral DNA) were
also replaced by a hexamethylene linker to afford compound 39.
The synthesis was performed as reported in Schemes 8 and 9. 6-N-
Benzoyladenine 73 was reacted with 1, 6-dibromohexane 74 to give
the base-linker 75. The latter was reacted with 4-N-benzoylcyto-
sine to give the protected compound 76 that was debenzoylated to
give 38. In the same manner, guanine protected at the amino

5036
Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
acridine and perylene were attached to the dinucleotide via
different linkers. The strongest inhibitory effect was observed with
thiazole orange and perylene as well as those of the corresponding
intercalator-linker derivatives indicated that only the activity of the
conjugate involving the perylene attached to the terminal phos-
phate was due to the presence of both the dinucleotide and the
intercalator. The activities of the other conjugates were similar to
those of the corresponding intercalators. Removal of both the
20-deoxyribose units and internucleotidic phosphodiester linkage
led to inactive compounds.
the perylene-dinucleotide conjugate 31 (IC 50 ¼ 1.8
other conjugates 24 and 26–30 exhibited IC 50 values between 13.2
and 66 M. The linkage of thiazole orange, involving a benzothia-
zolium and a lepidinium nuclei, (compound 29) led to a weaker
inhibitory effect (IC 50 ¼ 65.5 M) than that of intercalators with
three fused aromatic rings such as acridine (compound 24, IC
50 ¼ 13.2 M) and anthraquinones (compound 26, IC 50 ¼ 13.9 and
M). In the anthraquinone-dinucleotide
mM), while
m
m
m
compound 27, IC 50 ¼ 13.4
m
6. Experimental
series, the addition of an hydroxyhexyl chain on the intercalator in
position opposite to the attachment site of the linker (compound
6.1. General methods
28) led to a slightly reduced activity (IC 50 ¼ 21
mM). In these
conjugates the intercalators were attached via flexible poly-
methylene chains involving five to seven methyl groups. For a given
intercalator the inhibitory effect was dependent on the linker
structure. The dinucleotide-acridine conjugate 24 involving
All chemicals were used as obtained unless otherwise stated.
50-O-(4,40-Dimethoxytrityl)–4–N-benzoyl-20-deoxycytidine,
50-O-
(4,40-Dimethoxytrityl)–6–N-benzoyl-20-deoxyadenosine, 50-O-(4,40-
Dimethoxytrityl)–4–N-benzoyl-20-deoxycytidine-30-H-phosphonate,
50-O-(4,40-Dimethoxytrityl)–6–N-benzoyl-20-deoxyadenosine-30-H-
a neutral hexamethylene chain (IC 50 ¼ 13.2
mM), was at least 7-
fold more efficient than the conjugate 25 with the acridine attached
via a bulky positively charged linker. The inhibitory activities of the
perylene-dinucleotide conjugates 30 and 31 were also dependent
on their structures. The attachment of the perylene via a phos-
phodiester bond and a nine atom linker (compound 31) led to more
phosphonate, b-L b-L
-20-deoxycytidine, -20-deoxyadenosine, adenine,
thymine, cytosine and guanine were purchased from Distribio.
1-Aminoanthraquinone, 1,4-diaminoanthraquinone, 6-bromohex-
anoyl chloride, sodium iodide, benzoyl anhydride, benzoyl chloride,
diethanolamine, tert-butyldiphenylsilyl chloride, trimethylsilyl-
chloride, isobutyryl chloride, 1,3-diiodopropane, 1,6-dibromohexane,
imidazole, phosphorus oxychloride, monomethoxytrityl chloride,
dimethoxytrityl chloride, diethylenetriamine, ethyltrifluoroacetate,
3-bromopropanol,1,3-dibromopropane, carbon tetrachloride, ethyl-
potent compound (IC 50 ¼ 1.8
methylene tether to the
-anomeric position of an additional 20-
deoxyribose (compound 30, IC 50 ¼ 30 M). The comparison of the
mM) than its linkage via a three
b
m
inhibitory activities of conjugates 24, 29 and 31 and those of the
corresponding intercalator-linker derivatives (Fig. 4) showed that
the activity of conjugates 24 and 29 were similar to those of the
corresponding linker-intercalator compounds while that of 31 was
due to the presence of both the dinucleotide and the intercalator.
These results indicated that for conjugates 24 and 29, intercalation
probably occurred. However, the dinucleotide moiety was not in
position favourable to alter efficiently the integrase activity while
for the conjugate 31, both the perylene unit and the dinucleotide
were involved in the inhibitory effect. The removal of both the
20-deoxyribose units and internucleotidic phosphodiester linkage
led to inactive compounds 38 and 39.
enediamine,
1,3-diaminopropane,
1,4-diaminobutane,1,5-dia-
minopentane, 1,6-diaminohexane, 6-aminohexanol, 1,3-propanediol,
1,12-dodecanediol, sodium iodide, phenol, 2-methoxy-6,9-dichlor-
oacridine, diisopropylethylamine, sodium sulphate, 3-N,N-dimethy-
laminopropylamine, acetic anhydride, triphenylphosphine, carbon
tetrabromide,
pivaloyl
chloride,
2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite, aluminium chloride, iodine,
sodium hydrogenocarbonate were purchased from Aldrich. Acetic
acid, triethylamine and sodium sulfate were purchased from Merck,
2-chloro-5, 6-benzo-1,3,2-dioxaphosphorin-4-one from Fluka, pyri-
dine, dimethylformamide and dichloromethane from SDS, and
acetonitrile from Labo-Standa. Analytical thin-layer chromatography
(TLC) was performed on precoated alumina plates (Merck silica gel
60F 254 ref. 5554). For flash chromatography, Merck silica gel 60
(70–230 mesh) (ref. 7734) or Merck neutral alumina (Merck 5550)
were used. Chelex 100 (100–200 mesh) resinwas from Biorad. All4,40-
dimethoxytrityl-containing substances were identified as orange-
colored spots on TLC plates by spraying with a 10% perchloric acid
solution. Amino-containing substances were identified as purple-
colored spots on TLC plates by spraying with a 0.1% ninhydrin solution
in n-butanol. Oligonucleotide syntheses were performed on an
Expedite Nucleic Acid Synthesis system 8909 from Perseptive Bio-
systems. Reversed-phase chromatography analysis was performed on
a600E(SystemController)equippedwithaphotodiodearraydetector
5. Conclusion
We report here 0the 0synthesis and anti-integrase activity of 38
new dinucleotide 5 CA3 analogues. The modified compounds can
be classified into two series. The first one concerns the replacement
of the D L
- 20-deoxyribose by its enantiomer, the N4 alkylation of the
cytosine, the modification of the internucleotidic linkage polarity
and the replacement of the phosphodiester linkage by phosphor-
othioate and phosphoramidate bonds. Only a few compounds
exhibited a moderate increase in the inhibitory effect as compared
to that of the parent unmodified dinucleotide. In the second series,
different linkers, ending with either hydroxyl or amino functions,
and intercalators (acridine via neutral and positively charged
linkers, three anthraquinone analogs, thiazole orange and two
perylene derivatives) were attached to either the 50- or the 30-end
of the natural dinucleotide. In the dinucleotide conjugate series two
types of behaviour can be observed. 50-Conjugation led to an
increase in the inhibitory effect (IC 50 values ranging from 1.8 to
(Waters 990) using
a Lichrospher 100 RP18 (5 mm) column
(125 mm ꢂ 4 mm) from Merck with a linear gradient of CH3CN in
0.1 M aqueous triethylammonium acetate, pH 7, with a flow rate of
1 mL/min. Reversed-phase chromatography purifications were per-
formed on a 600 E (System Controller) equipped with a UV detector
(VARIAN UV 50) using a Lichrospher 100 RP18 (10 mm) column
66
a positively charged linker, as compared to the activity of the
M.). On the contrary, no
improvement in the dinucleotide inhibitory effect was observed by
30-conjugation. The strongest inhibitory effect was observed with
the perylene-dinucleotide conjugate involving the longer linker
m
M), except for the compounds involving amino groups or
(250 mm ꢂ 10 mm) from Merck with a linear gradient of CH3CN in
0.1 M aqueous triethylammonium acetate, pH 7, with a flow rate of
4 mL/min. Mass analysis ion-molecular weights of the oligonucleo-
tides was performed by Electrospray Mass Spectroscopy using
a Quattro II (Micromas) instrument.1H NMR spectrawere recorded on
a Varian Unity 500 Spectrometer. Absorption spectra were recorded
with a Uvikon 860 spectrophotometer. The concentrations were
determined using the following molar absorption coefficients [44]
unmodified dinucleotide. (IC 50 ¼ 340
m
attached to the terminal phosphate (IC 50 ¼ 1.8
mM). A comparison
of the inhibitory activities of the conjugates involving acridine,

Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
5037
3260 ¼ 20 000 Mꢃ1 cmꢃ1 for dinucleotides 1–23, 32–34 and for
compounds 38 and 39. For the dinucleotide intercalator conjugates
L-20-Deoxycytidylyl-(30 / 50)–20–deoxyadenosine 21H NMR
(500 MHz, D2O):
1.59 [m, 1H, H-20(C)], 2.23 [m, 1H, H-20(C)], 2.60
d
24–31 and 35–37 the 3260 values were considered to be the sum of the
3
[m, 1H, H-20(A)], 2.87 [m, 1H, H-20(A)], 3.58–3.70 [m, 2 H, H-
50,50(C)], 4.04 [m, 3H, H-40(C), H-50, 50(A)], 4.23 [m, 1H, H-40(A)],
4.53 [m, 1H, H-30(C)], 4.78 [m, 1H, H-30(A)], 5.92 [d, J ¼ 7.53 Hz, 1H,
H-5(C)], 5.99 [t, J ¼ 6.94 Hz, 1H, H-10(C)], 6.42 [t, J ¼ 6.64 Hz, 1H, H-
10(A)], 7.50 [d, J ¼ 7.56 Hz, 1H, H-6(C)], 8.16 (s, 1H, H-2(A)], 8.40 [s,
1H, H-8(A)]. Mass analysis: (ESI) calcd for C19H25N8O9P (M-H)ꢃ:
539.1, found 539.1.
values of the dinucleotide and the intercalator molecules as follows:
acridine-dinucleotide conjugates: 24, 25, and 35–37 3260 ¼ 65
000 Mꢃ1 cmꢃ1; anthraquinone-dinucleotide conjugate 26, 3260 ¼ 86
400 Mꢃ1 cmꢃ1; anthraquinone-dinucleotide conjugate 27, 3260 ¼ 75
400 Mꢃ1 cmꢃ1; anthraquinone-dinucleotide conjugate 28, 3260 ¼ 87
700 Mꢃ1 cmꢃ1; thiazole orange-dinucleotide conjugate 29, 3260 ¼ 27
000 Mꢃ1 cmꢃ1; perylene-dinucleotide conjugates 30 and 31, 3260 ¼ 60
L-20-Deoxyadenosinyl-(30 / 50)–20–deoxycytidine 31H NMR
(500 MHz, D2O): d
2.23 [m, 1H, H-20(C)], 2.36 [m, 1H, H-20(C)], 2.67–
000 Mꢃ1 cmꢃ1
.
2.81 [m, 2H, H-20,20(A)], 3.82 [m, 2H, H-50,50(C)], 4.05–4.21 [m, 3H,
H-50,50(A), H-40(C)], 4.31 [m, 1H, H-40(A)], 4.52 [m, 1H, H-30(C)], 4.87
[m, 1H, H-30(A)], 5.80 [d, J ¼ 7.56 Hz,1H, H-5(C)], 6.18 [t, J ¼ 6.53 Hz,
1H, H-10(A)], 6.33 [t, J ¼ 6.76 Hz,1H, H-10(C)], 7.75 [d, J ¼ 7.52 Hz,1H,
H-6(C)], 8.09 [s, 1H, H-2(A)], 8.21 [s, 1H, H-8(A)]. Mass analysis (ESI)
for C19H25N8O9P (M-H): 539.1, found: 539.1.
6.2. Chemistry
6.2.1. 20-Deoxycytidylyl-(30 / 50)–20–deoxyadenosine 1
50-O-(4,40-Dimethoxytrityl)–4–N-benzoyl-20-deoxycytidine, 30-
H-phosphonate, triethylammonium salt (1 mmol) and 30-O-
benzoyl-6-N-benzoyl-20-deoxyadenosine
(0.98 mmol)
were
solubilized in anhydrous pyridine (10 mL) and pivaloylchloride
(3 mmol) was added dropwise. After a 10 min reaction under stir-
ring, the mixture was diluted with CH2Cl2 (50 mL). Then, the
organic phase was washed with a 1 M solution of NaHCO3 (50 mL),
dried over Na2SO4 and concentrated to dryness. The purification
and isomer separation were performed by silica gel chromatog-
6.2.3. Synthesis of the dinucleotides involving the alkylated
cytosine (Scheme 1)
6.2.3.1. General procedure for the monomethoxytritylation of the bis-
amine 41–45. Bis-amines were dried separately by co-evaporation
with pyridine, dissolved with pyridine and monomethoxytrityl
chloride (0.1 eq) was added. After a 21 h reaction, the mixture was
diluted with CH2Cl2, washed with a 0.5 M aqueous NaHCO3 solution,
dried over Na2SO4, filtered and concentrated to dryness. The
orange-colored oils were purified by flash chromatography using
a CH2Cl2/CH3OH/NEt3 (98:2:1, v/v/v) mixture as eluent to give the
monotritylated bisamines 41–45 with yields ranging from 90 to 95%.
raphy (on Merck 60H, 15
then AcOEt/Acetone/CH3COOH (90:10:0.2, v/v/v) as eluent. Then,
an iodine solution (30 mg, 118 mol) in a pyridine/H2O (10:1, v/v)
mixture (2.54 mL) was added to the fully protected dinucleoside H-
phosphonate (80 mol) in anhydrous pyridine (1.4 mL) under
mm) using AcOEt/CH3COOH (99.8:0.2, v/v)
m
m
stirring at rt. After 15 min, a solution of Na2SO3 (1% in water) was
added to the reaction mixture until decoloration. The reaction
mixture was diluted with CH2Cl2 (10 mL) and the organic phase
washed with a 0.2 M aqueous solution of NaHCO3 (10 mL), dried
over Na2SO4, filtered, and concentrated to dryness. The residue was
dissolved with an 80% acetic acid solution in water (2.5 mL). After
10 min, the orange solution was concentrated to dryness and the
residue dissolved with a concentrated (28%) aqueous ammonia
solution. The suspension was maintained for 6 h at 50 ꢁC. After
removal of the ammonia, the water solution was washed with ethyl
acetate (3 ꢂ 10 mL) and filtered. Purification was performed by
reversed-phase chromatography on a Lichrospher Merck 100 RP 18
6.2.3.2. General procedure for the preparation of the alkylated 20-
deoxycytidine derivatives 46–50. The triazolo derivative of the 4,
40-dimethoxytrityl-20-deoxyuridine protected at the 30-end with
a
a
tertbutyldimethylsilyl group 40 was obtained following
previously reported procedure [29]. The latter (355 mg,
0.51 mmol) and the selected monomethoxytritylated bisamine
41–45 (3 eq) were dissolved in CH3CN (4 mL). After a 15 h reac-
tion, the mixture was concentrated to dryness and the residue
dissolved with CH2Cl2. The organic phase was washed with water,
dried and concentrated to dryness and the residue purified by
flash chromatography using a CH2Cl2/CH3OH/NEt3 (98:1:1, v/v/v)
mixture as eluent to give the fully protected 4-N-alkylated-20-
deoxyuridine derivatives. The latter (0.44 mmol) were then dis-
solved with a 1 M tetrabutyl ammonium fluoride solution in THF
(5 mL). After a 2 h reaction at rt, the mixture was concentrated to
dryness. The oily residue was dissolved with CH2Cl2 (50 mL),
washed with a 0.5 M aqueous NaHCO3 solution, dried, filtered and
concentrated to dryness. The orange-colored oil was purified by
flash chromatography using a CH2Cl2/CH3OH (98:2, v/v) mixture
as eluent to give the alkylated derivatives 46–50 with yields
ranging from 42 to 68%.
column (10
a 0.1 M triethylammonium buffer, pH 7, and a flow rate of 4 mL/min.
1H NMR (500 MHz, D2O): 1.58 [m, 1H, H-20(C)], 2.23 [m, 1H, H-
mm, 250 mm 10 mm) with a linear gradient of CH3CN in
d
20(C)], 2.60 [m, 1H, H-20(A)], 2.87 [m, 1H, H-20(A)], 3.63 (m, 2H, H-
50,50(C), 4.04 (m, 3H, H-50, 50(A) H-40(C), 4.23 [m, 1H, H-40(A)], 4.52
[m, 1H, H-30(C)], 4.78 (m, 1H, H-30(A), 5.93 [d, J ¼ 7.55 Hz, 1H, H-
5(C)], 5.99 [dd, J ¼ 7.71 Hz, J ¼ 7.74 Hz, 1H, H-10(C)], 6.48 [t,
J ¼ 6.64 Hz, 1H, H-10(A)], 7.86 [d, J ¼ 7.56 Hz, 1H, H-6(C)], 8.17 [s, 1H,
H-2(A)], 8.40 [s, 1H, H-8(A)]. Mass analysis (ESI) calcd for
C19H25N8O9P (M-H)ꢃ: 539.1, found: 539.3.
50-O-(4,40-Dimethoxytrityl)-4-N-[2-(4-monomethoxytritylam
6.2.2. Dinucleotides 2 and 3 involving
L
-20-deoxyribose units
-dA nucleosides, the
ino)ethyl]-20-deoxycytidine 461H NMR (500 MHz, CDCl3):
d 2.25
Starting from commercial -dC and
L
L
(m, 1H, H-20), 2.55 (m, 1H, H-20), 3.39–3.62 (m, 6H,
H-50,50, 2CH2), 3.78 (s, 9H, 3 OCH3), 4.02 (m, 1H,H-40), 4.50 (m, 1H,
H-30), 5.21 (m, 1H, OH-30), 5.36 (d, J ¼ 6.93 Hz, 1H, H-5), 6.34 (m, 1H,
H-10), 6.78–6.87 (m, 5H, ArH), 7.16–7.37 (m, 16H, ArH), 7.39–7.47
(m, 6H, ArH), 7.84 (d, J ¼ 6.58 Hz, 1H, H-6). Mass analysis (ESI) calcd
for C52H52N4O7 (M þ H)þ: 845.4, found:845.3.
synthesis of these dinucleotides involving regular internucleotidic
phosphodiester linkage was performed in solution via the H-
phosphonate chemistry. The nucleoside protection (4,40-dime-
thoxytrityl for the 50-hydroxyl function and benzoyl group for 30-
hydroxyl function and exocyclic amine on cytosine and adenine)
was achieved as reported for the protection of the corresponding
50-O-(4,40-Dimethoxytrityl)-4-N-[3-(4-monomethoxytritylam
natural
D
-nucleosides The nucleoside-H-phosphonate derivatives
ino)propyl]-20-deoxycytidine 471H NMR (500 MHz, CDCl3):
d 1.73
were obtained following a reported procedure using 2-chloro-5, 6-
benzo-1, 3, 2-dioxaphosphorin-4-one as a phosphitilating agent
[28]. The oxidation, deprotection and purification steps were per-
formed as described above for the preparation of compound 1.
(m, 2H, CH2), 2.24 (m, 1H, H-20), 2.56 (m, 1H, H-20), 3.38–3.51
(m, 2H, H-50,50), 3.54–3.67 (m, 4H, 2CH2), 3.76 (s, 9H, 3 OCH3),
4.02 (m, 1H, H-40), 4.49 (m, 1H, H-30), 5.29 (d, J ¼ 7.29 Hz, 1H, H-5),
5.75 (m, 1H, OH-30), 6.34 (m, 1H, H-10), 6.78–6.86 (m, 5H, ArH),

5038
Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
7.15–7.47 (m, 22H, ArH), 7.77(d, J ¼ 7.24 Hz, 1H, H-6). Mass analysis
(ESI) calcd for C53H54N4O7 (M þ H)þ: 859.4, found: 859.4.
50-O-(4,40-Dimethoxytrityl)-4-N-[4-(4-monomethoxytritylam
under stirring at 50 ꢁC for 6 h. After removal of the ammonia, the
residue was dissolved with water (4 mL) and the solution washed
with ethyl acetate and filtered. Purification was performed by
reversed-phase chromatography on a Lichrospher Merck 100 RP 18
ino)butyl]-20-deoxycytidine 481H NMR (500 MHz, CDCl3):
d 1.58
(m, 4H, 2CH2), 2.24 (m, 1H, H-20), 2.55 (m, 1H, H-20), 3.37–3.56 (m,
6H, H-50,50, 2CH2), 3.80 (s, 9H, 3 ꢁOCH3), 4.01 (m, 1H, H-40), 4.49
(m,1H, H-30), 4.92 (m, 1H, OH-30), 5.25 (d, J ¼ 7.16 Hz, 1H, H-5), 6.34
(m, 1H, H-10), 6.79–6.87 (m, 5H, ArH), 7.15–7.48 (m, 22H, ArH), 7.77
column (10
m
m, 250 mm ꢂ 10 mm) with a linear gradient of CH3CN
in a 0.1 M triethylammonium buffer, pH 7, and a flow rate of 4 mL/
min to give the dinucleotides 4–8.
4-N-(2-Aminoethyl)–20–deoxycytidylyl-(30/ 50)–20–deoxyade
(d, J ¼ 7.20 Hz, 1H, H-6). Mass analysis (ESI) calcd for C54H56N4O
nosine 41H NMR (500 MHz, D2O): 1.63 [m, 1H, H-20(C)], 2.27 [m,
d
7
(M þ H)þ: 873.4, found: 873.4.
1H, H-20(C)], 2.59 [m, 1H, H-20(A)], 2.86 [m, 1H, H-20(A)], 3.27 (m,
4H, 2CH2), 3.60–3.75 [m, 2H, H-50,50(C), 4.03 (m, 1H, H-40(C)], 4.07
[m, 2H, H-50, 50(A)], 4.24 [m, 1H, H-40(A)], 4.55 [m,1H, H-30(C)], 4.75
[m,1H, H-30(A)], 5.93 [d, J ¼ 7.55 Hz, 1H, H-5(C)], 6.01 [dd,
J ¼ 7.80 Hz, J ¼ 6.10 Hz, 1H, H-10(C)], 6.43 [t, J ¼ 6.68 Hz, 1H, H-
10(A)], 7.80 [d, J ¼ 7.28 Hz, 1H, H-6(C)], 8.17 [s, 1H, H-2(A)], 8.42 [s,
1H, H-8(A)]. Mass analysis (ESI) calcd for C21H30N9O9P (M þ H)þ:
584.2, found: 584.3.
50-O-(4,40-Dimethoxytrityl)-4-N-[5-(4-monomethoxytritylam
ino)pentyl]-20-deoxycytidine 491H NMR (500 MHz, CDCl3):
d 1.39
(m, 2H, CH2), 1.47–1.63 (m, 4H, 2CH2), 2.25 (m, 1H, H-20), 2.55 (m,
1H, H-20), 3.37–3.56 (m, 6H, H-50,50, 2CH2), 3.80 (s, 9H, 3 ꢁOCH3),
4.01 (m, 1H, H-40), 4.49 (m, 1H, H-30), 4,74 (m, 1H, OH-30), 5.28 (d,
J ¼ 7.08 Hz, 1H, H-5), 6.34 (m, 1 H, H-10, 6.79–6.87 (m, 5H, ArH),
7.15–7.48 (m, 22H, ArH), 7.79 (d, J ¼ 7.08 Hz, 1H, H-6). Mass analysis
(ESI) calcd for C55H58N4O7 (M þ H)þ: 887.4, found: 887.4.
4-N-(3-Aminopropyl)–20–deoxycytidylyl-(30/ 50)-20-deoxyade
50-O-(4,40-Dimethoxytrityl)-4-N-[6-(4-monomethoxytritylam
nosine 51H NMR (500 MHz, D2O): 1.64 [m, 1H, H-20(C)], 1.96 (m,
d
ino)hexyl]-20-deoxycytidine 501H NMR (500 MHz, CDCl3):
d
1.32
2H, CH2), 2.26 [m,1H, H-20(C)], 2.59 [m,1H, H-20(A)], 2.85 [m,1H, H-
20(A)], 3.03 [m, 4H, 2CH2), 3.60–3.75 (m, 2H, H-50,50(C)], 4.02 [m,1H,
H-40(C)), 4.07 (m, 2H, H-50, 50(A)], 4.23 [m, 1H, H-40(A)], 4.55 [m,1H,
H-30(C)], 4.75 [m, 1H, H-30(A)], 5.88 [d, J ¼ 7.59 Hz,1H, H-5(C)], 6.01
[dd, J ¼ 7.62 Hz, J ¼ 6.29 Hz, 1H, H-10(C)], 6,44 [t, J ¼ 6.67 Hz, 1H, H-
10(A)], 7.42 [d, J ¼ 7.62 Hz, 1H, H-6(C)], 8.17 [s, 1H, H-2(A)], 8.42 [s,
1H, H-8(A)]. Mass analysis (ESI) calcd for C22H32N9O9P (M þ H)þ:
598.2, found: 598.3.
(m, 4H, 2CH2), 1.56 (m, 4H, 2CH2), 2.26 (m, 1H, H-20), 2.56 (m, 1H, H-
20), 3.38–3.53 (m, 6H, H-50,50, 2CH2), 3.80 (s, 9H, 3 OCH3), 4.01 (m,
1H, H-40), 4.49 (m, 1H, H-30), 4.73 (m,1H, OH-30), 5.29 (d, J ¼ 7.17 Hz,
1H, H-5), 6.34 (m, 1H, H-10), 6.79–6.86 (m, 5H, ArH), 7.15–7.49 (m,
22H, ArH), 7.79 (d, J ¼ 7.11 Hz, 1H, H-6). Mass analysis (ESI) calcd for
C56H60N4O7 (M þ H)þ: 901.5, found: 901.4.
6.2.3.3. General procedure for the preparation of phosphoramidites
51–55
4-N-(4-Aminobutyl)-20-deoxycytidylyl-(30/ 50)-20-deoxyade
nosine 61NMR (500 MHz, D2O): 1.59–1.76 [m, 5H, H-20(C), 2CH2)],
d
The corresponding phosphoramidite derivatives were obtained
following a classical procedure using the commercially available
phosphitilating reagent 2-cyanoethyldiisopropylchloro-phosphor-
amidite. Compounds 46–50 (0.23 mmol) were dried by co-evapo-
ration with anhydrous pyridine (5 mL) then with anhydrous CH3CN
(5 mL, three times) and left in a dessicator under vacuum overnight.
The next day, the dessicator was filled with argon before its
opening. The residue was dissolved with 1,2-dichloroethane (5 mL)
and diisopropylethylamine (0.16 mL, 0.12 g, 0.92 mmol) was added
and then 2-cyanoethyldiisopropylchlorophosphoramidite (0.
077 mL, 82 mg, 0.35 mmol) dropwise under stirring at rt. After a 1 h
reaction, the mixture was diluted with cold ethyl acetate (30 mL).
The organic phase was washed with a 0.5 M aqueous sodium
bicarbonate solution (20 mL) and a 3 M aqueous sodium chloride
solution (10 mL), dried and concentrated to dryness. The residue
was purified on a silica gel column using CH2Cl2/AcOEt/NEt3
(70:30:1, v/v/v) mixture as eluent. Yields 86–93%.
2.24 [m, 1H, H-20(C)], 2.59 [m, 1H, H-20(A)], 2.85 [m, 1H, H-20(A)],
3.05 (m, 4H, 2CH2), 3.60–3.71 [m, 2H, H-50,50(C)], 4.01 [m, 1H, H-
40(C)], 4.07 [m, 2H, H-50, 50(A)], 4.23 [m,1H, H-40 (A)], 4.54 [m,1H, H-
30(C)], 4.78 [m, 1H, H-30(A)], 5.83 [d, J ¼ 7.60 Hz, 1H, H-5(C)], 6.02
[dd, J ¼ 7.71 Hz, J ¼ 6.18 Hz, 1H, H-10(C)], 6.43 [t, J ¼ 6.64 Hz, 1H, H-
10(A)], 7.37 [d, J ¼ 7.62 Hz, 1H, H-6(C)], 8.15 [s, 1H, H-2(A)], 8.42 [s,
1H, H-8(A)]. Mass analysis (ESI) calcd for C23H34N9O9P (M þ H)þ:
612.2, found: 612.2.
4-N-(5-Aminopentyl)-20-deoxycytidylyl-(30/ 50)-20-deoxyaden
osine 71H NMR (500 MHz, D2O):
d 1.44 (m, 2H, CH2), 1.64 (m, 4H,
2CH2), 1.71 [m, 1H, H-20(C)], 2.23 [m, 1H, H-20(C)], 2.60 [m, 1H, H-
20(A)], 2.85 [m, 1H, H-20(A)), 3.0 (m, 4H, 2CH2), 3.60–3.72 [m, 2H, H-
50,50(C)], 4.01 [m, 1H, H-40(C)], 4.07 [m, 2H, H-50,50(A)], 4.23 [m, 1H,
H-40(A)], 4.54 [m, 1H, H-30(C)], 4.78 [m, 1H, H-30(A)], 5.82 [d,
J ¼ 7.57 Hz,1H, H-5(C)], 6.02 [m, 1H, H-10(C))], 6.43 [t, J ¼ 6.61 Hz,
1H, H-10(A)], 7.36 [d, J ¼ 7.61 Hz, 1H, H-6(C)], 8.14 [s, 1H, H-2(A)],
8.41 [s, 1H, H-8(A)]. Mass analysis (ESI) calcd for C24H36N9O9P
(M þ H)þ: 626.2, found: 626.4.
6.2.3.4. General procedure for the preparation of the modified
dinucleotides involving the alkylated 20- deoxycytidine derivatives
4–8
4-N-(6-Aminohexyl)-20-deoxycytidylyl-(30/ 50)-20-deoxyaden
osine 81H NMR (500 MHz, D2O):
d 1.40 (m, 4H, 2CH2), 1.56–170 [m,
5H, 2CH2, H-20(C)], 2.23 [m, 1H, H-20(C)], 2.59 [m, 1H, H-20(A)], 2.85
[m, 1H, H-20(A)], 2.99 (m, 4H, 2CH2), 3.60–3.71 [m, 2H, H-50,50(C)],
4.01 [m, 1H, H-40(C)], 4.07 [m, 2H, H-50,50(A)], 4.23 [m, 1H, H-40(A)],
4.54 [m, 1H, H-30(C)], 4.77 [m, 1H, H-30(A)], 5.81 [d, J ¼ 7.60 Hz, 1H,
H-5(C)], 6.02 [dd, J ¼ 7.66 Hz, J ¼ 6.23 Hz, 1H, H-10(C)], 6.42 [t,
J ¼ 6.63 Hz, 1H, H-10(A)], 7.35 [d, J ¼ 7.63 Hz, 1H, H-6(C)], 8.13 [s, 1H,
H-2(A)], 8.41 [s, 1H, H-8(A)]. Mass analysis (ESI) calcd for
C25H38N9O9P (M þ H)þ: 640.3, found: 640.3.
In a round-stopped flask containing 30O-benzoyl-4-N-benzoyl-
20-deoxyadenosine (20 mg, 35.5
mmol), a 0.5 M solution of tetrazole
in CH3CN (2 mL) and a 0.059 M solution in CH3CN of the phos-
phoramidite derivative of the selected 4-N-alkylated-20-deoxy-
cytidine (35.4
mmol, 0.6 mL) were simultaneously added. After
a 15 min reaction, 2.5 mL of the iodine solution used on the
synthesizer were added. Then, after a 5 min reaction, the iodine
excess was reduced by adding a 10% aqueous solution of Na2S2O3.
Next, the mixture was diluted with CH2Cl2 (15 mL) and the organic
phase washed with a 0.5 M aqueous solution of NaHCO3 (10 mL),
filtered, dried and concentrated to dryness. The residue was dis-
solved with an 80% acetic acid solution in water (1 mL). After
10 min, the orange solution was concentrated to dryness and the
residue dissolved with a concentrated (28%) aqueous ammonia
solution (6 mL) and MeOH (3 mL). The mixture was maintained
6.2.3.5. Preparation of the dinucleotides involving guanidylated
cytosine 9–11
Modified dinucleotides 6–8 (50 DO, 2.5
mmol) and 1H-pyrazole-
1-carboxamidine hydrochloride (30 mg, 205
mmol) were dissolved
in 1 M aqueous Na2CO3 (1.8 mL). After an 18 h reaction, the crude
guanidylated dinucleotides were purified by reversed-phase

Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
5039
chromatography. The hydrophobicity of the guanidylated dinucle-
6.2.5. General procedure for the preparation of the modified
dinucleosides involving the phosphorothioate linkage 14–16
(Scheme 2)
otides 9–11 showed a slight increase as compared to that of the
corresponding aminated compounds 6–8. After reversed-phase
purification, the modified dinucleotides were obtained with 64–
68% yields and their mass was confirmed by ESI mass spectrometry.
4-N-(4-Guanidinobutyl)-20-deoxycytidylyl-(30/ 50)-20-deoxyade
nosine 9 Mass analysis (ESI) calcd for C24H36N11O9P (M-H)ꢃ: 652.2,
found: 652.3.
4-N-(5-Guanidinopentyl)-20-deoxycytidylyl-(30/ 50)-20-deoxyad
enosine 10 Mass analysis (ESI) calcd for C25H38N11O9P (M-H)ꢃ: 666.3,
found: 666.3.
4-N-(6-Guanidinohexyl)-20-deoxycytidylyl-(30/ 50)-20-deoxyad
enosine 11 Mass analysis (ESI) calcd for C26H40N11O9P (M-H)ꢃ: 680.3,
found: 680.3.
Elemental sulphur (40 mg) and each fully protected dinucleo-
side H-phosphonate with 50–30, 50–50 and 30–30 polarities (80
mol)
m
were dissolved in a pyridine/CS2 (40:60, v/v) mixture (2.5 mL) at rt.
After 2 h, the reaction mixture was concentrated to dryness,
deprotected and purified as reported above for dinucleotides 1, 12
and 13.
20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine phosphorothio
ate 14 Mass analysis (ESI) calcd for C19H25N8O8PS (M-H)ꢃ: 555.1
found: 555.1.
20-Deoxycytidylyl-(50 / 50)-20-deoxyadenosine phosphorothio
ate 15 Mass analysis (ESI) calcd for C19H25N8O8PS (M-H)ꢃ: 555.1,
found: 555.2.
6.2.4. Preparation of the dinucleosides 12 and 13 involving the
phosphodiester linkage with modified polarities
20-Deoxycytidylyl-(30 / 30)-20-deoxyadenosine phosphorothio
ate 16 Mass analysis (ESI) calcd for C19H25N8O8PS (M-H)ꢃ: 555.1,
found: 555.0.
0
0
0
0
The synthesis of the dinucleotide 5 CpA5 and 3 CpA3 involving
internucleotidic linkages with modified polarities was also per-
formed in solution via H-phosphonate chemistry. The fully pro-
6.2.6. General procedure for the preparation of the modified
dinucleotides involving the 1, 4-diaminobutane linker
attached to the internucleotidic linkage 17–19 (Scheme 2)
0
0
tected dinucleotide-H-phosphonate 5 Cp(H)A5 was obtained by
reaction of commercially available 50-O-(4,40-dimethoxytrityl)-4-N-
benzoyl-20-deoxycytidine-30-H-phosphonate, (triethylammonium
50-O-(4,40-dimethoxytrityl-6-N-benzoyl-20-deoxy-
A solution of N-monomethoxytritylbutanediamine (0.75 mmol)
in anhydrous pyridine was added to each fully protected dinu-
cleoside H-phosphonate with 50–30, 50–50 and 30–30 polarities
(0.142 mmol) dissolved in CCl4 (1.5 mL) at rt. After a 15 h reaction,
the mixture was concentrated to dryness, purified by flash chro-
matography using a CH2Cl2/MeOH (95:5, v/v) mixture as eluent.
The obtained dinucleotides were deprotected and purified as
reported above for dinucleotides 1, 12 and 13.
salt)
adenosine in the presence of pivaloyl chloride. Purification was
performed by silica gel chromatography (on Merck 60 H, 15 m)
and
m
using AcOEt/Acetone/CH3COOH (60:40:0.2, v/v/v)0 as eluent. The
0
fully protected dinucleotide-H-phosphonate 3 Cp(H)A3 was
obtained by using commercially available 4-N-benzoyl- 30-O-
benzoyl-20-deoxycytidine and 6-N-benzoyl-30-O-benzoyl-20-deox-
yadenosine and proceeded as follows. The 50-H-phosphonate
derivative of 4-N-benzoyl-30-O-benzoyl-20-deoxycytidine was
obtained following a reported procedure using 2-chloro-5,6-benzo-
1,3,2-dioxaphosphorin-4-one as phosphitilating agent. The 50-H-
phosphonate derivative was then reacted with the 50-hydroxyl
function of the 6-N-benzoyl-30-O-benzoyl-20-deoxyadenosine in
the presence of pivaloylchloride. Purification was also performed by
20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine-N-(4-aminobut
yl)phosphoramidate 17 (50 / 30)-Cp[NH(CH2)4NH2]A. Mass anal-
ysis (ESI) calcd for C23H35N10O8P (M þ H)þ: 611.2. found: 609.3.
20-Deoxycytidylyl-(50 / 50)-20-deoxyadenosine-N-(4-aminobut
yl)phosphoramidate 18 (50 / 50)- Cp[NH(CH2)4NH2]A. Mass anal-
ysis (ESI) calcd for C23H35N10O8P (M þ H)þ: 611.2. found: 611.2.
20-Deoxycytidylyl-(30 / 30)-20-deoxyadenosine-N-(4-aminobut
yl)phosphoramidate 19 (30 / 30)-Cp[NH(CH2)4NH2]A. Mass anal-
ysis (ESI) calcd for C23H35N10O8P (M þ H)þ: 611.2. found: 611.3.
silica gel chromatography (on Merck 60 H, 15 mm) using AcOEt/
CH3COOH (99.8:0.2, v/v/v) as eluent. Then, the oxidation step was
performed as described above for the preparation of compound 1.
0
0
The deprotection of the dinucleotide-phosphodiester 5 CpA5 was
achieved as reported for the natural dinucleotide 1 while for the
6.2.7. General procedure for the preparation of the modified
dinucleotides involving the guanidylated diaminobutane linker
attached to the internucleotidic position 20–22
0
0
dinucleotide-phosphodiester 3 CpA3 , only the ammonia treatment
was required. Purification of the crude dinucleotides 12 and 13 was
also achieved as reported for0compound 1.
20-Deoxycytidylyl-(50 / 5 )-20-deoxyadenosine 12 1H NMR
(500 MHz, D2O):d
2.04 [m, 1H, H-20(C)], 2.31 (m, 1H, H-20(C)), 2.61
Starting from dinucleotides 17–19, the procedure was identical
to the one reported for 0the preparation of dinucleotides 9–11.
20-Deoxycytidylyl-(3 / 50)-20-deoxyadenosine-N-(4-guanidino
butyl)phosphoramidate 20 (50 / 30)-Cp[NH(CH2)4NH-Gua]A: Mass
analysis (ESI) calcd for C24H37N12O8P (M þ H)þ: 653.3. found: 653.2.
20-Deoxycytidylyl-(50 / 50)-20-deoxyadenosine-N-(4-guanidino
butyl)phosphoramidate 21 (50 / 50)- Cp[NH(CH2)4NH-Gua]A: Mass
analysis (ESI) calcd for C24H37N12O8P (M þ H)þ: 653.3. found: 654.2.
20-Deoxycytidylyl-(30 / 30)-20-deoxyadenosine-N-(4-guanidino
butyl)phosphoramidate 22 (30 / 30)-Cp[NH(CH2)4NHGua]A: Mass
analysis (ESI) calcd for C24H37N12O8P (M þ H)þ: 653.3. found: 653.3.
[m,1H, H-20 (A)], 2.83 [m, 1H, H-20 (A)], 3.89–4.00 [m,2H, H-50,50
(C)], 4.07 [m, 3H, H-50,50,(A), H-40 (C)], 4.24 [m,1H, H-40(A)], 4.37 [m,
1H, H-30 (C)], 4.72 [m, 1H, 30(A)], 5.63 [d, J ¼ 7.51 Hz, 1H, H-5(C)],
6.12 [t, J ¼ 6.56 Hz, 1H, H-10(C)], 6.40 [t, J ¼ 6.64 Hz, 1H, H-10(A)],
7.52 [d, J ¼ 7.54 Hz, 1H, H-6(C)], 8.14 [s, 1H, H-2(A)], 8.30 [s, 1H, H-
8(A)]. Mass analysis (ESI) calcd for C19H25N8O9P (M-H)ꢃ: 539.1,
found: 539.2.
20-Deoxycytidylyl-(30 / 30)-20-deoxyadenosine
(500 MHz, D2O):
131H
2.37 [m, 1H, H-20(C)], 2.64 [m, 1H, H-20(C)], 2.74
NMR
d
[m, 1H, H-20(A)], 2.91 [m, 1H, H-20(A)], 3.84 [m, 4H, H-50,50(C), H-
50,50(A)], 4.27 [m, 1H, H-40(C)], 4.38 [m, 1H, H-40(A)], 4.83 [m, 1H, H-
30(C)], 4.99 [m, 1H, H-30(A)], 6.06 [d, J ¼ 7.57 Hz, 1H, H-5(C)], 6.29 [t,
J ¼ 6.83 Hz, 1H, H-10(C)], 6.48 [dd, J ¼ 8.21 Hz, J ¼ 6.01 Hz, 1H, H-
10(A)], 7.86 [d, J ¼ 7.64 Hz, 1H, H-6(C)], 8.20 [s, 1H, H-2(A)], 8.32 [s,
1H, H-8(A)]. Mass analysis (ESI) calcd for C19H25N8O9P (M-H)ꢃ:
539.1, found: 539.3.
6.2.8. Synthesis of the conjugates
6.2.8.1. Synthesis of the modified dinucleotide 23
The synthesis of the dinucleotide 50-phosphorothioate S-
0
0
p-5 CpA3 56 was performed on solid-phase following a procedure

5040
Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
adapted from our previous reports [31]. 67 DO (21%). Mass analysis
(ESI) calcd for C19H26N8O11P2S (M-H)ꢃ: 635.1. found: 635.3.
and lyophilised. The solid was dissolved with a 15 mM solution of
18-crown-6 in MeOH (1 mL) and a 10 mM solution of 2-methoxy-6-
chloro-9-(3-bromopropylamino)acridine [or 2-methoxy-6-chloro-
9-(6-bromohexylylamino)acridine] in MeOH (1 mL) was added.
The reaction mixture was protected from the light and maintained
under stirring for 24 at rt. A 1 M solution of phosphate buffer pH 6
(1.5 mL) was added and the mixture was extracted with CH2Cl2. The
aqueous solution was concentrated and purified by reversed-phase
chromatography.
Synthesis of the 1,7-bis-trifluoroacetyl- 4-(3-iodopropyl)hepta-
1,4,7-triazane linker 59 (Scheme 4) 1,7-bis-Trifluoroacetyl-hepta-
1,4,7-triazane [27] (2 g, 0. 24 mmol), diiodopropane (4.3 g,
0.72 mmol), and NEt3 (1 mL, 0.36 mmol) were dissolved with
CH3CN and the mixture was heated at 65 ꢁC for 16 h. Then, the
mixture was concentrated to dryness and the residue purified by
chromatography on a silica gel column using a CH2Cl2/acetone/H2O
(95:5:1 to 85:15:1, v/v/v) mixture as eluent. White solid. 0.6 g
20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine-30-[3-(6-
chloro-2-methoxy-9-acridinylamino)propyl]thiophosphate 35 15
OD (23%). Mass analysis (ESI) calcd for C36H41ClN10O12P2S (M-H)ꢃ:
933,2, found 933.3.
(27%). 1H NMR (500 MHz, CDCl3):
d 1.89 (m, 2H, CH2CH2I), 2.60 (t,
J ¼ 6.7 Hz, 2H, NCH2(CH2)2I) 2.68 (t, J ¼ 5.8 Hz, 4H, NCH2), 3.18 (t,
J ¼ 6.4 Hz, 2H, ICH2), 3.46 (m, 4H, HNCH2), 6.90 (s br, 2H, NH). Mass
analysis (ESI) for C11H16F6IN3O2. (M þ H)þ: 464.0, found 464.1.
Synthesis of the modified dinucleotide 23 Compound 59 (35 mg,
20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine-30-[6-(6-
chloro-2-methoxy-9-acridinylamino)hexyl]thiophosphate 36 6 OD
(9%). Mass analysis (ESI) calcd for C39H47ClN10O12P2S (M-H)ꢃ:
975.2, found, 975.4.
75 mmol) was added to the dinucleotide 56 (7.8 mmol, 150 OD) and
then 18-crown-6 (30 mg) in MeOH (1.5 mL). After 7 h stirring at
40 ꢁC, aqueous ammonia (28%) solution was added and the mixture
was left to stand at rt for 48 h. The mixture was then concentrated
to dryness. The residue was dissolved with water (2 ml). The
aqueous phase was extracted with CH2Cl2 and the crude dinucle-
otide 23 purified by reversed-phase chromatography. Mass analysis
(ESI) calcd for C26H43N11O11P2S (M-H)ꢃ: 778.2 found: 778.3.
6.2.8.2.3. Synthesis of the dinucleotide acridine conjugates 25 and
37
6.2.8.2.3.1. Synthesis of the acridine linker derivative 62
6-Chloro-2-methoxy-9-(3-dimethylaminopropylamino)-
acridine 61 6,9-Dichloro-2-methoxyacridine (0.5 g, 1.8 mmol), 3-
dimethylaminopropylamine (0.45 mL, 3.58 mmol) and phenol (2 g)
were heated at 100 ꢁC for 2 h. After cooling to rt, MeOH (2.5 mL)
was added and the resulting mixture poured into a 2 M NaOH water
solution (20 mL). The solid obtained was filtered, dried and purified
6.2.8.2. Synthesis of the dinucleotide-acridine conjugates 24, 25
and 35–37 (Scheme 3)
6.2.8.2.1. 20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine-50-[6-(6-
chloro-2-methoxy-9-acridinylamino) hexyl] phosphate 24
The synthesis of the dinucleotide acridine conjugate 24 was
performed via phosphoramidite chemistry following our previ-
ously reported procedure [35]. Mass analysis (ESI) calcd for C39H46
Cl N10O13P2 (M-H)ꢃ: 959.3. found: 959.1 and 961.2.
by flash chromatography on a neutral alumina column (63–200 mm,
activity 1) using a CH2Cl2/MeOH, (98:2, v/v) mixture as eluent.
Yellow solid. 368 mg (60%). 1H NMR (500 MHz, CDCl3): 1.91 (m, 2H,
CH2), 2.41 (s, 6H, N(CH3)2), 2.65 (t, J ¼ 5.50 Hz, 2H, CH2N), 3.97 (s,
3H, OCH3), 4.0 (m, 2H, CH2NH), 7.22 (dd, J ¼ 9.28 Hz, J ¼ 2.03 Hz, 1H,
H-7), 7.30 (d, J ¼ 2.56 Hz, 1H, H-1), 7.40 (dd, J ¼ 9.37 Hz, J ¼ 2.62 Hz,
1H, H-3), 7.97 (d, J ¼ 9.36 Hz, 1H, H-4), 8.02 (d, J ¼ 1.93 Hz, 1H, H-5),
8.09 (d, J ¼ 9.29 Hz, 1H, H-8). Mass analysis (ESI) calcd for
C19H22ClN3O (M þ H)þ: 344.2, found: 344.3, 346.3 (M þ Hþ2)þ.
3-Bromopropyl-3-(6-chloro-2-methoxy-9-acridinylamino)pro-
pyldimethylammonium bromide 62 (Scheme 5) Compound 61
(100 mg, 0.291 mmol), 1,3-dibromopropane (0.6 mL, 5.79 mmol)
and CH3CN (7 mL) were heated at 80 ꢁC for 2 h. Then, the mixture
was concentrated to dryness and the residue dissolved with water
(20 mL). The excess of 1, 3-dibromopropane was removed by
extraction with CH2Cl2 (4 ꢂ15 mL). The aqueous solution was
lyophilised and the residue purified by flash chromatography on
6.2.8.2.2. Synthesis of the dinucleotide-acridine conjugates 35 and
36
0
0
The synthesis of the dinucleotide 30-phosphorothioate 5 CpA3 p-
S 57 was performed on solid-phase using our previously reported
support [31] 94 OD (3 mg, 63%). Mass analysis (ESI) calcd for
C19H26N8O11P2S (M-H)ꢃ: 635.1, found: 635.3. The synthesis of 6-
chloro-2-methoxy-9-(6-bromohexylamino)acridine was obtained
as previously reported [37]. The synthesis of the 6-chloro-2-
methoxy-9-(3-bromopropylamino)acridine
was
performed
following the same strategy. 6-Aminohexanol was replaced by 3-
aminopropanol.
a neutral alumina column (63–200
MeOH, (97:3, v/v) mixture as eluent. Yellow solid. 108 mg (79%). 1H
NMR (500 MHz, D2O): 1.78 (m, 2H, CH2), 1.88 (m, 2H, CH2), 2.94 (s,
mm, activity 1) using a CH2Cl2/
6-Chloro-2-methoxy-9-(3-hydroxypropylamino)acridine 1H NMR
(500 MHz, DMSO-d6):
d
1.87 (m, 2H, CH2), 3.29 (s, 1H, NH), 3.53 (m,
d
2H,CH2N), 3.84 (m, 2H, CH2O), 3.93 (s, 3H,OCH3), 4.65 (t, J ¼ 4.84 Hz,
1H, OH), 7.32(dd, J ¼ 9.30 Hz, J ¼ 2.10 Hz,1H, H-3), 7.42 (dd, J ¼ 9.33 Hz,
J ¼ 2.62 Hz, 1H, H-7), 7.60 (d, J ¼ 2.51 Hz, 1H, H-5), 7.84 (d, J ¼ 9.32 Hz,
1H, H-4), 7.87 (d, J ¼ 2.10 Hz, 1H,H-1), 8.36 (d, J ¼ 9.33 Hz, 1H, H-8).
Mass analysis (ESI) calcd for C17H17ClN2O2 (M þ H)þ: 317.1, found:
317.1, 319.1 (M þ Hþ2)þ.
6H, N(CH3)2), 3.11 (m, 2H, CH2), 3.20 (m, 2H, CH2), 3.42 (t,
J ¼ 5.79 Hz, 2H, CH2Br), 3.51 (t, J ¼ 5.71 Hz, 2H, CH2N), 3.85 (s, 3H,
OCH3), 6.75 (m, 1H, ArH), 7.07 (m, 1H, ArH), 7.19 (m, 1H, ArH), 7.45
(m, 2H, ArH), 7.58 (m 1H, ArH). Mass analysis (ESI) calcd for
C22H28BrClN3O Mþ: 464.1, found: 464.2, 466.1 (M þ 2)þ, 468.2
(M þ 4)þ.
6-Chloro-2-methoxy-9-(3-bromopropylamino)acridine 1H NMR
(500 MHz, CDCl3):
d
2.51 (m, 2H, CH2), 3.63 (t, J ¼ 6.06 Hz, 2H,
6.2.8.2.3.2. Synthesis of the dinucleotide acridine conjugates 25 and
37
CH2Br), 3.89 (s, 1H, NH), 4.02 (s, 3H, OCH3), 4.08 (t, J ¼ 6.88 Hz, 2H,
CH2N), 7.23 (m, 1H, H-3), 7.28 (m, 1H, H-7), 7.38 (d, J ¼ 2.44 Hz, 1H,
H-5), 7.92 (d, J ¼ 9.31 Hz, 1H, H-4), 8.08 d, J ¼ 2.04 Hz, 1H, H-1), 8.10
(d, J ¼ 9.28 Hz, 1H, H-8). Mass analysis (ESI) calcd for C17H16BrClN2O
(M þ H)þ: 379.0, found: 379.0, 381.2 (M þ Hþ2)þ, 383.0
(M þ Hþ4)þ.
The dinucleotide 50-phosphorothioate 56 (14 OD, 0.70
dinucleotide 30-phosphorothioate 57, 24 OD, 1.2
mol) was dis-
mmol) (or
m
solved with an 18.9 mM solution of 18-crown-6 in MeOH (12 eq)
and a 21.1 mM solution of 62 in MeOH (7 eq) was added. The
reaction mixture was protected from the light and maintained
under stirring for 40 h at rt. Then, the solution was concentrated
and a 1 M solution of phosphate buffer pH 6 (1.5 mL) was added
Synthesis of the dinucleotide acridine conjugates 35 and 36 A
water solution (1 mM, 1 mL) of the dinucleotide 30-phosphor-
othioate was passed over Chelex 100 resin (100–200 mesh, Biorad)

Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
5041
and the mixture was extracted with CH2Cl2. The aqueous solution
was concentrated and purified by reversed-phase chromatography.
(0.4 g, 1 mmol), NaI (0.75 g, 5 mmol), NaHCO3 (0.42 g, 5 mmol) and
acetone (25 mL) were refluxed for 14 h. Then, the mixture was
concentrated to dryness and the residue dissolved with CH2Cl2
(60 mL). The organic phase was washed with H2O (3 ꢂ 20 mL),
dried over Na2SO4 and concentrated. The residue was purified on
a silica gel column using a CH2Cl2/MeOH (99:1 to 98.5:1.5, v/v)
mixture as eluent. Purple solid. 0.42 g (70%). 1H NMR (500 MHz,
Compound 25: 25 OD (59%)
lmax ¼ 266, 344, 424 and 446 nm.
Mass analysis (ESI) calcd for C41H53ClN11O12P2S, Mþ: 1020.3, found
1019.4. Compound 37: 15 OD (21%) lmax ¼ 266, 345, 423 and
446 nm. Mass analysis (ESI) calcd for C41H53ClN11O12P2S, Mþ:
1020.3, found 1020.4.
DMSO-d6)
d 1.42 (m, 2H, CH2), 1.66 (m, 2H, CH2), 1.81 (m, 2H, CH2),
6.2.8.3. Synthesis of the dinucleotide anthraquinone conjugates
26 –28
2.42 (t, J ¼ 6.75 Hz, 2H, CH2C ¼ O), 3.28 (t, J ¼ 6.75 Hz, 2H, CH2I),
7.23 (d, J ¼ 9 Hz, 1 H, H-3), 7.74–7.88 (m, 2 H, H-6, H-7), 8.11 (m, 2H,
H-5, H-8) 8.64 (d, J ¼ 9.5 Hz, 1H, H-2), 12.13 (s, 1H, NH). Mass
analysis (ESI) m/z calcd for C20H19IN2O3. (M þ H)þ ¼ 464.0, found
464.1.
6.2.8.3.1. Synthesis of the anthraquinone-linker derivatives 67 and
68 (Scheme 6)
1-(6-Bromohexanoylamino)anthracene-9,10-dione 65 6-Bro-
mohexanoylchloride (0.8 mL, 5.4 mmol) was added dropwise,
over 15 min to a refluxed mixture of 1-aminoanthraquinone 63
(1 g, 4.5 mmol) and pyridine (catalytic amount) in anhydrous
benzene (25 mL). Refluxing conditions were maintained for 2 h.
The reaction mixture was cooled to rt and filtered. The residue
was washed with CH2Cl2 and the organic phase concentrated. The
yellow residue was dissolved with CH2Cl2 and the organic phase
was washed with NaHCO3 and then sat. NaCl, was dried over
Na2SO4 and concentrated. The residue was purified on a silica gel
column using a CH2Cl2/MeOH (99:1 to 98.5/1.5, v/v) mixture as
eluent. Yellow solid. 1.7 g (94%). 1H NMR (500 MHz, DMSO-d6):
6.2.8.3.2. Synthesis of anthraquinone-linker derivative 72;
(Scheme 7)
Anthracene- 2,7- dicarboxylic acid -9,10-dione 69 was obtained
as previously reported [22]. 1H NMR (500 MHz, DMSO-d6)
d 8.29 (d,
J ¼ 8.5 Hz, 2H, H-3, H-6), 8.38 (d, J ¼ 8.5 Hz, 2H, H-4, H-5), 8.65 (s,
2H, H-1, H-8). Mass analysis (ESI) calcd for C16H8O6. (M-
H)ꢃ ¼ 295.0, found 294.6.
1-O-Tertbutyldiphenylsilanyl-6-hexylamine 1H NMR (500 MHz,
CDCl3): d 1.06 [s, 9H, C(CH3)3], 1.26–1.46 (m, 8H, CH2, NH2), 1.58 (m,
2H, CH2), 2.68 (t, J ¼ 7 Hz, 2H, CH2NH2), 3.67 (t, J ¼ 6.5 Hz, 2H,
CH2OSi), 7.36–7.45 (m, 6H, ArOSi), 7.68 (m,4H, ArOSi). Mass analysis
(ESI) for C22H33NOSi. (M þ H)þ ¼ 357.2, found 356.4.
d
1.48 (m,2H, CH2), 1.70 (m, 2H, CH2), 1.86 (m, 2H, CH2), 2.53 (t,
J ¼ 7 Hz, 2H, CH2C ¼ O), 3.54 (t, J ¼ 6 Hz, 2H, CH2Br), 7.81–7.88 (m,
4H, H-3, H-4, H-6, H-7), 8.15 (d, J ¼ 7 Hz, 1H, H-8), 8.21 (d,
J ¼ 7 Hz, 1H, H-5), 8.95 (d, J ¼ 9 Hz, 1H, H-2), 12.0 (s, 1H, NH). Mass
analysis (ESI) m/z calcd for C20H18BrNO3, (M þ H)þ: 400.0, found
400.1.
1-O-(4,40-Dimethoxytrityl)-6-hexylamine 1H NMR (500 MHz,
CDCl3):
d 1.26–1.48 (m, 6H, CH2), 1.58–1.75 (m, 4H, CH2, NH2), 2.68
(t, J ¼ 7.1 Hz, 2H, NCH2), 3.05 (t, J ¼ 6.6 Hz, 2H, CH2ODMTr), 3.80 (s,
6H, OCH3), 6.81–6.85 (m, 4H, DMTr), 7.18–7.46 (m, 9H, DMTr). Mass
analysis (ESI) for C27H33NO3, (M þ H)þ: 420.6, found 420.4.
1-(6-Iodohexanoylamino)anthracene-9-10-dione 67 1-(6-Bro-
mohexanoylamino)anthracene-9-10-dione 65 (0.4 g, 1 mmol), NaI
(0.75 g, 5 mmol), NaHCO3 (0.42 g, 5 mmol) and acetone (25 mL)
were refluxed for 8 h. Then, the mixture was concentrated to
dryness and the residue dissolved with CH2Cl2 (60 mL). The organic
phase was washed with H2O, dried over Na2SO4 and concentrated.
The residue was purified on a silica gel column using a CH2Cl2/
MeOH (98:2, v/v) mixture as eluent. Yellow solid. 0.42 g (95%).
1H NMR (500 MHz, DMSO-d6): 1.44 (m, 2H, CH2), 1.70 (m, 2 H,
CH2), 1.87 (m, 2H, CH2), 2.53 (t, J ¼ 7 Hz, 2H, CH2C ¼ O), 3.28 (t,
J ¼ 7 Hz, 2H, CH2I), 7.82–7.89 (m, 4H, H-3, H-4, H-6, H-7), 8.17 (d,
J ¼ 7 Hz, 1H, H-8), 8.24 (d, J ¼ 7 Hz, 1H, H-5), 8.97 (d, J ¼ 9 Hz, 1H,
H-2), 12.0 (s, 1H, NH). Mass analysis (ESI) m/z calcd for C20H18INO3.
(M þ H)þ: 448.0, found 448.1.
2-N-[6-O-(4,40-Dimethoxytrityl)hexyl] anthracene-2-carbox-
amide-7-carboxylic acid-9,10-dio-ne 70 To 69 (0.38 g, 1.3 mmol),
dried by co-evaporation with anhydrous pyridine (three times), and
dissolved with anhydrous pyridine (15 mL) were added NEt3
(1 mL), hydroxybenzotriazole (10 mg) and carbonyldiimidazole
(0.26 g, 1.6 mmol). After 2 h of stirring at rt, 1-O-(4,40-dimethoxy-
trityl)-6-hexylamine (0.49 g, 1.17 mmol) was added and the stirring
was maintained for 20 additional h. Then, the mixture was
concentrated to dryness and the residue was purified on a silica gel
column using a CH2Cl2/MeOH/NEt3 (98:2:0.5, v/v/v) mixture as
eluent. Yield: 0.36 g (40%). 1H NMR (500 MHz, DMSO-d6):
d 1.29–
1.40 (m, 4H, CH2), 1.52–1.60 (m, 4H, CH2), 2.95 (t, J ¼ 6.4 Hz, 2H,
CH2O DMTr), 3.29 (m, 2H, NCH2), 3.72 (s, 6H, OMe), 6.87 (d,
J ¼ 8.8 Hz, 4H, DMTr), 7.18–7.37 (m, 9H, DMTr), 8.23–8.37 (m, 4H, H-
3, H-6, H-4, H-5), 8.68 (dd, J ¼ 6.6 Hz, J ¼ 1.4 Hz, 2 H, H-1, H-8), 8.93
(t, J ¼ 5.5 Hz, 1H, NH). Mass analysis (ESI) m/z calcd for C43H39NO8.
(M-H)ꢃ ¼ 696.3, found 696.2.
4-Amino-1-(6-bromohexanoylamino)anthracene-9,10-dione 66
6-Bromohexanoylchloride (0.4 mL, 2.5 mmol) was added dropwise,
over 15 min, to a refluxed mixture of 1-4-diaminoanthraquinone
(2 g, 8.4 mmol) and pyridine (catalytic amount) in anhydrous
benzene (50 mL). Refluxing conditions were maintained for 4 h.
The reaction mixture was cooled to rt and filtered. The residue was
washed with toluene and the organic phase concentrated. The
purple residue was dissolved with CH2Cl2 and the organic phase
was washed with NaHCO3 and then sat. NaCl, dried over Na2SO4
and concentrated. The residue was purified on a silica gel column
using a CH2Cl2/MeOH (98:2 to 97.5/2.5, v/v) mixture as eluent.
2-N-[6-O-(4,40-Dimethoxytrityl)hexyl]-7-N-(6-hydroxyhex-
yl)anthracene-2,7-dicarboxamide-9,10-dione 71 The synthesis
was performed as reported for 70 except that compound 69 was
replaced
by
2-{[u
-O-(4,40-dimethoxytrityl)-hexylamido]-7-
carboxylic acid}-anthracene-9-10-dione (0.35 g, 0.5 mmol) and 1-
O-(4,40-dimethoxytrityl)-6-hexylamine by 1-O-(tertbutyldiphe-
nylsilanyl)-6-hexylamine. Yellow product. Yield: 0.45 g (85%). The
compound (0.40 g, 0.38 mmol) was dissolved in a 1 M tetrabuty-
lammonium fluoride solution in THF (2 mL) and THF (5 mL) and
the mixture was stirred for 1 h at rt. The solution was concen-
trated and the residue dissolved with CH2Cl2. The organic phase
was washed with H2O, dried over Na2SO4, filtered and concen-
trated to dryness. The residue was purified on a silica gel column
using a CH2Cl2/MeOH (97:3 to 93:7, v/v) mixture as eluent. Yellow
Purple solid. 0.8 g (80%). 1H NMR (500 MHz, DMSO-d6):
d 1.46 (m, 2
H, CH2), 1.68 (m, 2H, CH2), 1.85 (m, 2H, CH2), 2.46 (t, J ¼ 7 Hz, 2H,
CH2C ¼ O), 3.53 (t, J ¼ 6.5 Hz, 2 H, CH2Br), 7.28 (dd, J ¼ 9 Hz, J ¼ 1 Hz,
1H, H-3), 7.85 (m, 2H, H-6, H-7), 8.17 (dd, J ¼ 7.5 Hz, J ¼ 1 Hz, 2H, H-
5, H-8), 8.69 (dd, J ¼ 10 Hz, J ¼ 0.5 Hz, 1H, H-2) 12.29 (s, 1H, NH).
Mass analysis (ESI) m/z calcd for C20H19BrN2O3, (M þ H)þ: 415.1,
found 415.2.
solid. Yield: 0.27 g, (85%). 1H NMR (500 MHz, CDCl3):
d 1.40–1.70
4-Amino-1-(6-iodohexanoylamino)anthracene-9,10-dione 68
4-Amino-1-(6-bromohexanoylamino)anthracene-9-10-dione 66
(m, 16 H, CH2), 3.05 (s br, 2H, CH2O), 3.50 (m, 4H, NCH2), 3.75 (m,
8H, OCH3, CH2OH), 6.50–6.70 (m, 2H, HNCO), 6.80–7.40 (m, 13H,

5042
Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
DMTr), 8.17 (d, J ¼ 7.5 Hz, 2H, H-3, H-6), 8.25 (t, J ¼ 7.5 Hz, 2H, H-4,
H-5), 8.45 (dd, J ¼ 8 Hz, J ¼ 1.5 Hz, 2H, H-1, H-8). Mass analysis
(ESI) m/z calcd for C49H52N2O8, (M-H)ꢃ: 795.4, found 795.4.
2-N-[6-O-(4,40-Dimethoxytrityl)hexyl]-7-N-{6-O-[(2-cyanoeth
oxy)-N,N0-diisopropylamino-phosphoramidite]hexyl}anthracene-
2,7-dicarboxamide-9,10-dione 72
synthesizer) was added. After 2 min, the support was washed with
CH3CN (3 ꢂ 1 mL). The support-bound conjugate was treated with
aqueous ammonia solution (28%) (4 mL) for 24 h at rt. Then, the
solution was concentrated to dryness and the residue dissolved with
a 70% acetic acid aqueous solution (3 mL). After a 30 min reaction at
rt, the mixture was concentrated to dryness. The residue was dis-
solved with water (4 mL) and the mixture extracted with CH2Cl2
(3 ꢂ 5 mL). The crude anthraquinone dinucleotide was purified by
reversed-phase chromatography as indicated above. 16 OD. 20-
Deoxycytidylyl-(30Ò50)-20-deoxyadenosine-50-{6-[7-N-(6-hydroxyh
Compound 71 (0.26 g, 0.32 mmol) was dried by co-evaporation
with anhydrous pyridine (3 ꢂ 5 mL) and left in a dessicator under
vacuum overnight. The next day, the dessicator was filled with
argon before being opened. The residue was dissolved with 1,2-
dichloroethane (10 mL) and diisopropylethylamine (0.172 g,
1.33 mmol), then 2-cyanoethyldiisopropylchloro-phosphoramidite
(0.11 g, 0.47 mmol) was added dropwise under stirring at rt. After
75 min, silica gel TLC analysis using an EtOAc/acetone/TEA
mixture as eluent (75:25:5, v/v/v) showed a nearly complete
reaction with the formation of two new spots corresponding to
both isomers of the phosphoramidite derivative. The reaction
mixture was diluted with cold ethyl acetate (30 mL) previously
washed with a cold aqueous 5% sodium carbonate solution. The
organic phase was washed with a cold 10% aqueous sodium
carbonate solution (10 mL) and with a cold saturated aqueous
sodium chloride solution (10 mL), dried over Na2SO4 and
concentrated to dryness. The residue was purified on a silica gel
column using AcOEt/acetone/NEt3 (75:25:5, v/v/v) mixture as
eluent. Yellow oil. Yield: 0.27 g, (83%). 1H NMR (500 MHz, CDCl3):
exyl)-2-anthracenyl-2,7-dicarboxamide-9,10-dione]hexyl}
phos-
phate 28. Mass analysis (ESI) calcd for C47H58N10O17P2 (M þ H)þ:
1097, found 1096.5.
6.2.8.4. Synthesis of the dinucleotide-perylene conjugates 30 and
31
The synthesis was performed as previously reported for the
preparation of oligonucleotide-perylene conjugates involving the
same perylene-linker units [38,39]. The deprotection step by
a concentrated aqueous ammonia solution was adapted to the
sequence. The mixture was heated at 50 ꢁC for 8 h. The purification
was performed by reversed-phase chromatography.
20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine-50-{10-O-[3-(3-
perylenyl)propyl]-20-deoxy-30-ribosyl}phosphate 30 19 OD. Mass
analysis (ESI) calcd for C47H50N8O15P2 (M þ H)þ: 1029.3, found
1029.8.
d
1.18 (d, J ¼ 6.8 Hz, 6H, HC(CH3)2), 1.37–1.50 (m, 8H, CH2), 1.61–
1.75 (m, 8H, CH2), 2.67 (t, J ¼ 6.4 Hz, 2H, OCH2CH2CN), 3.07 (t,
J ¼ 6.6 Hz, 2H, CH2ODMTr), 3.52 (m, 4 H, HN-CH2), 3.79 (s, 6H,
OCH3), 3.57–3.93 [m, 6H, HC(CH3)2, OCH2CH2CN, OCH2(CH2)5NH],
6.47 –6.60 (m, 2H, HNCO), 6.82 (m, 4H, DMTr), 7.18–7.46 (m, 9H,
DMTr), 8.26–8.31 (m, 2H, H-3, H-6), 8.38 (m,2H, H-4, H-5), 8.58
(dd, J ¼ 11.3 Hz, J ¼ 1.4 Hz, 2H, H-1, H-8). 31P NMR (CDCl3):
147.27 ppm. Mass analysis (ESI) m/z calcd for C58H69N4O9P, (M-
H)ꢃ 995.5, found 995.4.
20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine-50-[6-(3-per-
ylenylmethylamino)hexyl] phosphate 31 18 OD. Mass analysis (ESI)
calcd for C46H52N9O12P2 (M þ H)þ: 984.32, found 982.3.
6.2.8.5. Synthesis of the modified dinucleotide 32–34 The
synthesis was performed via phosphoramidite chemistry on
modified supports [41,42].
20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine-30-(3-hydrox-
ypropyl) phosphate 32 Mass analysis (ESI) calcd for C22H32N8O13P2
(M-H)ꢃ: 677.2, found: 677.2. 0
6.2.8.3.3. Synthesis of the dinucleotide-anthraquinone conjugates
26 and 27
20-Deoxycytidylyl-(30 / 5 )-20-deoxyadenosine-30-(12-hydrox-
ydodecyl) phosphate 33 Mass analysis (ESI) calcd for
C31H50N8O13P2 (M-H)ꢃ: 803.3, found: 803.2.
The dinucleotide 50-phosphorothioate 56. (30 OD, 1.5
mmol) was
20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine-30-(6-amino-
hexyl) phosphate 34 Mass analysis (ESI) calcd for C25H39N9O12P2
(M-H)ꢃ: 718.2, found: 718.4.
dissolved with a 20 mM solution of 18-crown-6 in MeOH (0.8 mL)
and a 20 mM solution of compound 67 or 68 in DMF (0.2 mL, 4
mmol) was added. The reaction mixture was protected from the
light and maintained under stirring for 6 h at 40 ꢁC. Then, the
solution was concentrated and the residue dissolved with water
(3 mL). The mixture was extracted with CH2Cl2 (3 ꢂ 3 mL). The
aqueous solution was concentrated and purified by reversed-phase
chromatography.
6.2.9. Synthesis of 38 and 39
6.2.9.1. 9-[6-(1-Cytosinyl)hexyl]adenine 38 (Scheme 8)
9-(6-Bromohexyl)-6-N-benzoyladenine75 6-N-benzoyladenine
73 (0.6 g, 2.5 mmmol) was dried by co-evaporation with anhydrous
CH3CN (3 ꢂ15 mL), dissolved with anhydrous CH3CN (25 mL) and
potassium carbonate (0.34 g, 2.5 mmol) was added under magnetic
stirring at rt. Then 1, 6-dibromohexane 74 (1.6 mL, 10 mmol) was
added under an inert atmosphere and the mixture heated at 60 ꢁC
for 10 h. The mixture was concentrated to dryness and the residue
dissolved with CH2Cl2 (50 mL). The organic phase was washed with
ice-cold H2O (15 mL) dried over Na2SO4 and concentrated. The
residue was dissolved with CH2Cl2 (minimum amount) and the
solution was added dropwise to pentane under vigorous stirring.
The sediment was purified by silica gel chromatography with
a CH2Cl2/acetone (95:5 to 80:20, v/v) mixture as eluent. White
20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine-50-[6-(1-anthra
cenyl-9,10-dione)amino-6-oxohexyl] thiophosphate 26 17 OD.
Mass analysis (ESI) calcd for C39H43N9O14P2S (M þ H)þ: 955.8,
found 955.1.
20-Deoxycytidylyl-(30 / 50)-20-deoxyadenosine-50-[6-(4-ami
no-1-anthracenyl-9-10-dione)amino-6-oxohexyl] thiophosphate
27 15 OD. Mass analysis (ESI) calcd for C39H44N10O14P2S (M þ H)þ:
971.2, found 970.3.
6.2.8.3.4. Synthesis of the dinucleotide-anthraquinone 28
0
0
Trityl-off dinucleotide 5 CpA3 (1
mmol) bound to the support was
dried in a dessicator under vacuum overnight. The next day, the
dessicator was filled with argon before being opened. A 0.5 M solu-
tion of tetrazole in CH3CN (0.8 mL) and a 0.12 M solution of the
phosphoramidite derivative 72 (0.3 mL) were added to the dinucle-
otide and left to react for 1½ h at rt. The support was washed with
CH3CN (3 ꢂ 3 mL). A 0.1 M iodine solution (1 mL) (used on the
solid. 250 mg (25%). 1H NMR (500 MHz, CDCl3):
d 1.43 (m, 2H, CH2),
1.53 (m, 2H, CH2), 1.86 (m, 2H, CH2), 1.98 (m, 2H, CH2), 3.40 (t,
J ¼ 6.66 Hz, 2H, CH2Br), 4.30 (t, J ¼ 7.23 Hz, 2H, NCH2), 7.50–7.64 (m,
3H, ArH), 8.03 (m, 3H, H-2, ArH), 8.82 (s, 1H, H-8), 9.02 (s br, 1H,
NH). Mass analysis (ESI.) m/z calcd for C18H20BrN5O. (M þ H)þ:
402.1, found 402.1 and 404.2.

Y. Aubert et al. / European Journal of Medicinal Chemistry 44 (2009) 5029–5044
5043
9-[6-(1-Cytosinyl)hexyl]adenine 38 Compound 75 (0.15 g,
6.3. Biological studies
0.37 mmol) and 4-N-benzoylcytosine (97 mg, 0.45 mmol) were
dried with CH3CN separately.4-N-benzoylcytosine was dissolved
with dry DMF (8 mL), then K2CO3 (56 mg, 0.41 mmol) was added.
The mixture was stirred at 30 ꢁC for 10 min and compound 75
dissolved in DMF (4 mL) was added. The mixture was heated at
55 ꢁC for 8 h under stirring and the stirring was maintained for 12 h
at 20 ꢁC. Then, the mixture was concentrated and the residue was
purified on preparative TLC using CH2Cl2/EtOH (97:3, v/v) twice and
then (95:5, v/v) three times as eluent. Yield: 70 mg (35%).
Compound 76 (53 mg, 0.1 mmol) was reacted with concentrated
aqueous NH4OH (12 mL) and MeOH (1.5 mL) for 72 h at 20 ꢁC. The
mixture was concentrated to 9 mL and extracted with CH2Cl2. The
aqueous solution was concentrated and purified by reversed-phase
chromatography using a CH3CN (5 to 27% over 30 min) gradient in
a 0.05 M TEAA, pH 7, buffer. Yield: 320 OD, 18%. 1H NMR (500 MHz,
Activity assays were carried out for 1 hr at 37 ꢁC in a buffer
containing 10 mM HEPES (pH 7.2), 1 mM DTT, 7.5 mM MgCl2 in the
presence of 12.5 nM 21-mer double-stranded DNA substrate and
100 nM recombinant HIV-1 integrase as previously described [45].
Products were separated by electrophoresis in denaturing 18%
acrylamide/urea gels. Gels were analysed with
a Molecular
Dynamics STORM phosphoimager and quantified with Image
QuantTM 4.1 software. Inhibition in the presence of drugs was
expressed as the fractional product in percent of the control
without drug. Inhibition curves were fitted using Graphpad Prism 5
software.
Acknowledgements
DMSO-d6): d 1.25 (m, 4H, CH2), 1.52 (m, 2H, CH2), 1.80 (m, 2H, CH2),
This work was supported by grants from the Agence Nationale
de la Recherche contre le SIDA (ANRS). We thank the ‘‘Plateforme
de Spectrometrie de masse’’ of the ‘‘Centre de Biophysique
3.58 (t, J ¼ 7.14 Hz, 2H, NCH2), 4.11 (t, J ¼ 7.06 Hz, 2H, NCH2), 5.60 (d,
J ¼ 7.11 Hz, 1H, H-50), 7.15 (s, 1H, H-2), 7.52 (d, J ¼ 7.11 Hz,1H, H-60),
8.13 (s, 1H, H-8). Mass analysis (ESI.) m/z calcd for C15H20N8O.
(M þ H)þ ¼ 329.2, found 329.2. lmax ¼ 263 nm.
´
´
Moleculaire’’ for analysis facilities; C. Bure and G. Gabant for
running the mass spectrometers.
6.2.9.2. 9-[6-(1-Thymyl)hexyl]guanine 39 (Scheme 9)
References
9-(6-Bromohexyl)-2-N-isobutyryl-6-O-[2-(4-nitrophenyl)ethyl]
guanine 78 2-N-Isobutyryl-O-6-[2-(4-nitrophenyl)ethyl]guanine
77 (400 mg, 1.08 mmol) [43], 1,6-dibromohexane 74 (1.38 g,
5.43 mmol), K2CO3 (156 mg, 1.13 mmol) and CH3CN (50 mL) were
heated at 60 ꢁC for 11 h. Then, the mixture was concentrated to
dryness and the residue dissolved with CH2Cl2 (50 mL). The organic
phase was washed with H2O, dried and concentrated to dryness.
The residue was purified by silica gel chromatography with
increasing concentrations of MeOH in CH2Cl2 (0 to 10%). White
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d 1.19 (m, 2H, CH2),
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1H NMR (500 MHz, CDCl3):
d
1.27 (d, J ¼ 6.88 Hz, 6H, CH(CH3)2),
1.34 (m, 4H, 2CH2), 1.68 (m, 2H, CH2), 1.93 (m, 5H, CH2, CH3(T)), 2.79
(sp, J ¼ 6.87 Hz, 1H, CH(CH3)2), 3.67 (t, J ¼ 7.26 Hz, 2H, CH2N(T)),
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8(G)), 9.50 (bs, 1H, NH), 9.90 (bs, 1H, NH), 12.23 (bs, 1H, NH). Mass
analysis (ESI) calcd for C20H27N7O4 (M þ H)þ: 430.2, found:
430.3.6.2.9.1.3.3 9-[6-(1-Thymyl)hexyl]guanine 39
Compound 79 (20 mg, 46.6 mmol) was dissolved in concentrated
aqueous ammonia (28%) (4 mL). After a 15 h reaction at 50 ꢁC, the
mixture was concentrated to dryness and the crude 39 was purified
by recristallization with a H2O/CH3CN (9:1, v/v) mixture. White
solid, 9 mg (53%).1H NMR (500 MHz, DMSO-d 6):
d 1.23 (m, 4H,
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J ¼ 7.25 Hz, 2 H, CH2N(G)), 4.14 (t, J ¼ 6.94 Hz, 2H, CH2N(T)), 6.07 (s,
2H, NH2), 7.50 s, 1H, H-6(T)), 7.89 (s, 1H, H-8 (G)), 10.68 (s, 1H, NH),
11.16 (s, 1H, NH). Mass analysis (ESI) calcd for C16H21N7O3 (M þ H)þ:
360.2, found: 360.3.
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