Inhibition of HIV-1 integrase-catalysed reaction by new DNA minor groove ligands: The oligo-1,3-thiazolecarboxamide derivatives

Article abstract of DOI:10.1016/S0223-5234(00)01181-8

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the retrovirus, responsible for catalysing the insertion of the viral genome into the host cell chromosome. For this reason it provides an attractive target for antiviral drug design. We synthesized a series of novel thiazole (Tz)-containing oligopeptides (TCOs; oligo-1,3-thiazolecarboxamides), specifically interacting within the minor groove of DNA. The oligocarboxamide derivatives contained 1-4 Tz rings and different N- and C-terminal groups. The effect of these oligocarboxamides on the HIV-1 IN-catalysed reaction was investigated. Some of the compounds were able to inhibit the reaction. The inhibitory effect of the TCOs increased with the number of Tz units. The structure of various additional positively and/or negatively charged groups attached to the N- and C-termini of TCOs had a pronounced effect on their interaction with the DNA substrate complexed to IN. Modified TCOs having a better affinity for this complex should provide a rationale for the design of drugs targeting the integration step. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)01181-8

Eur. J. Med. Chem. 35 (2000) 989–1000
´
© 2000 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(00)01181-8/FLA
Original article
Inhibition of HIV-1 integrase-catalysed reaction by new DNA minor groove
ligands: the oligo-1,3-thiazolecarboxamide derivatives
Vladimir A. Ryabinina, Alexander N. Sinyakova, Vaea Richard de Soultraitb, Anne Caumontb,
Vincent Parissib, Olga D. Zakharovac, Elena L. Vasyutinac, Ekaterina Yurchenkoc,
Roman Bayandinc, Simon Litvakb, Laura Tarrago-Litvakb*, Georgy A. Nevinskyc
aInstitute of Molecular Biology, Koltsovo, Novosibirsk region 633159, Russia
bUMR 5097 CNRS, Universite´ Victor Segalen Bordeaux 2, IFR 66 ‘‘Pathologies Infectieuses’’, 146, rue Le´o Saignat,
33076 Bordeaux cedex, France
cNovosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8, Lavrentiev,
Novosibirsk 630090, Russia
Received 14 March 2000; revised 27 June 2000; accepted 11 July 2000
Abstract – Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the retrovirus,
responsible for catalysing the insertion of the viral genome into the host cell chromosome. For this reason it provides an attractive
target for antiviral drug design. We synthesized a series of novel thiazole (Tz)-containing oligopeptides (TCOs; oligo-1,3-thiazolecar-
boxamides), specifically interacting within the minor groove of DNA. The oligocarboxamide derivatives contained 1–4 Tz rings and
different N- and C-terminal groups. The effect of these oligocarboxamides on the HIV-1 IN-catalysed reaction was investigated.
Some of the compounds were able to inhibit the reaction. The inhibitory effect of the TCOs increased with the number of Tz units.
The structure of various additional positively and/or negatively charged groups attached to the N- and C-termini of TCOs had a
pronounced effect on their interaction with the DNA substrate complexed to IN. Modified TCOs having a better affinity for this
´
complex should provide a rationale for the design of drugs targeting the integration step. © 2000 Editions scientifiques et me´dicales
Elsevier SAS
HIV-1 / integrase / thiazole-containing oligopeptides / inhibition
1. Introduction
(IN), and DNA sequences located in the U3 and U5
regions at the ends of the viral long terminal repeats
(reviewed in [4]). The integration reaction has been
analysed in vivo and in vitro and it has been shown
that it proceeds in three steps [5–7]. The first step is 3%
processing: two nucleotides (GT) from the 3%-ends of
each strand of linear viral DNA are removed by IN
leaving at the viral 3%-ends the conserved CA dinucle-
otide. The second step or strand transfer, is a con-
certed cleavage ligation reaction during which IN
makes staggered cuts in the target DNA and ligates
the recessed 3%-OH ends of the viral DNA to the
overhanging 5%-phosphate ends of the target DNA at
the cleavage site. The product of this reaction is a
gapped intermediate. The last step is 5%-end joining, in
Replication of retroviruses depends on the integra-
tion of a double stranded DNA copy of the retroviral
genome into the host cell nuclear genome [1–3]. Inte-
gration requires the virus-encoded protein, integrase
Abbre6iations: HIV-1, human immunodeficiency virus type 1; IN,
integrase; RT, reverse transcriptase; LTR, long terminal repeat; TCO,
thiazole-containing oligopeptides; MGL, minor groove ligand; Tz,
thiazole; IC50, the concentration giving 50% inhibition of the enzyme
activity.
* Correspondence and reprints: Laboratoire REGER, UMR 5097-
CNRS, 146, rue Le´o Saignat, 33076 Bordeaux cedex, France. Tel.:
+33 (0)5 5757 1740; fax: +33 (0)5 5757 1766
E-mail address: laura.litvak@reger.u-bordeaux2.fr (L. Tarrago-
Litvak).

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V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
which the integration process is completed by re-
moval of the two unpaired nucleotides at the 5%-ends
of the viral DNA and repair of the gaps between the
viral and target DNA sequences. Since IN is essential
for productive retroviral infection, the enzyme is a
potential target for antiviral chemotherapeutic inter-
vention. In contrast to human immunodeficiency
virus type 1 (HIV-1) protease and reverse transcrip-
tase, for which an extensive number of chemical in-
hibitors have been described, there are few reports
about selective inhibitors of IN.
Most of the therapeutic agents used against HIV-1
induce the emergence of resistance strains. That
is why one of the most important lines in the phar-
macology concerning HIV-1 is focused on the devel-
opment of new, or on the modification of already
existing drugs. Among the new and promising anti-
virals tools are compounds which interact reversibly
with nucleic acids. Minor groove binding molecules
are capable of binding within the minor groove
of double-stranded B-DNA. Such molecules con-
tain several small aromatic rings such as pyrrole,
furan or benzene connected by bonds with torsional
freedom.
The prototype minor groove binders are the natu-
rally occurring oligopeptide antibiotics netropsin and
distamycin. Distamycin was first isolated from Strep-
tomyces distallicus [8] and later synthesized [9–11]. It
inhibits both Gram-positive and Gram-negative bac-
teria and replication of viral DNA (vaccinia, herpes
simplex virus) [12]. The synthesis of certain analogues
of distamycin, containing either additional pyrrole
units or derivatives in which the pyrrole rings were
replaced by benzene, pyridine, thiophene, thiazole
(Tz), imidazole, pyrazole, or triazole has been re-
ported [13–18]. These compounds were used as anti-
tumour or antiviral drugs [12, 19] or as tools to study
the modulation of enzymes [20–22].
The development of methods to synthesize minor
groove molecules capable of binding with virtually
any DNA sequence increased the interest for such
compounds [23, 24]. The heteroanalogues of dis-
tamycin and netropsin that have been mostly synthe-
sized are those in which one of the methylpyrrole
units was substituted with one Tz. A dipeptide com-
posed exclusively of thiazolecarboxamide fragments
has been described [25]. Sulfonated and phospho-
nated distamycin derivatives capable of suppressing
HIV-1 replication are of potential therapeutic impor-
tance [26]. Of obvious interest are the analogues of
distamycin and netropsin in which the N-
methylpyrrole fragment is substituted with a Tz cycle,
since these compounds are likely to possess different
site-specificity compared to oligopyrrolecarboxam-
ides. Due to the low reactivity of the key oligomer
2-amino-4-carbonic acid-1,3-Tz, there are few studies
concerning the minor groove binders containing the
Tz fragment in their structure [27–30]. Studies re-
lated to the site-specificity of
a Tz-containing
oligopeptide (TCO) analogue of netropsin demon-
strated that this compound bound to 4–5 base pairs
of the minor groove. There are also reports showing
the influence of the methyl substituents of a Tz-con-
taining lexitropsin on the mode of binding to DNA
[28].
We recently synthesized a series of new lex-
itropsins, the TCOs. These compounds are deriva-
tives of oligocarboxamides containing 1–4 Tz rings
and different N- and C-terminal groups. We previ-
ously showed that TCOs were able to inhibit the
polymerization reaction catalysed by HIV-1 RT [31].
Similar to distamycin, the TCOs were good inhibitors
when reverse transcription was done in the presence
of a DNA–DNA template-primer. But in contrast to
distamycin, the TCOs were also able to inhibit the
reaction with RNA·DNA or DNA·RNA template-
primers. The inhibition obtained with the TCOs
showed that these compounds were more powerful
and versatile inhibitors of the RT-dependent poly-
merization than the natural distamycin.
IN binds to specific sequences located on both
extremities of the DNA on the HIV-1 LTRs. The
retroviral LTR that recognizes a DNA binding site
on IN contains an AT-rich sequence. This sequence
has been exploited as a possible target for DNA
minor groove binders, since the DNA minor groove
may fit aromatic molecules better at AT-rich regions.
Here we addressed the question whether TCOs can
interact with the proviral DNA complexed to HIV-1
IN. If this was the case, the newly synthesized drugs
will be targeting different sites of the HIV-1 life cycle:
reverse transcription and integration. Thus, attacking
the virus on these sites together can prove effective
for combination therapy. Therefore, we analysed the
effect of a series of oligothiazolecarboxamides on the
reaction catalysed by HIV-1 IN and compared their
effect with that of distamycin, a natural oligopeptide,
containing three pyrrole rings, which is a DNA mi-
nor groove ligand.

V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
991
Figure 1. Scheme of the synthesis.

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V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
2. Chemistry
25 mL). The yield of product 5 was 12.6 g (72%).
1H-NMR (CDCl3) l 9.46 (bs, 1H), 7.27–7.40 (m, 15H),
All thiazolecarboxamide analogues were synthe-
sized by chain extension with 2-triphenylmethyl-
amino-4-carboxy-1,3-Tz (see figure 1). The structures
of the initial monomers and oligomers synthesized
were confirmed chromatographically and by 1H-
NMR spectra. The concentrations of TCOs were de-
termined spectrophotometrically using the following
extinction coefficients: Tz1-derivatives=12.0/mM cm
at lmax=252 nm; Tz2-derivatives=21.1/mM cm at
7.27 (s, 1H).
2.3. 1,2,3-Benzotriazol-1-yl-2-triphenylmethylamino-
1,3-thiazol-4-carboxylate (6)
Compound 5 (7.20 g, 18.6 mmol) was rapidly stirred
with 1-hydroxy-1,2,3-benzotriazole (2.52 g, 18.6 mmol)
in dry dichloromethane (200 mL), and DCC (3.90 g,
19.0 mmol) was added to the mixture in small portions
at room temperature. After stirring the mixture for 4 h,
the precipitated urea was filtered off and the solution
was poured into hexane (200 mL). The precipitate was
collected and dried in vacuo over SiO2. The resulting
crude product 6 (9.0 g, 96%) was sufficiently pure and
could be used directly for synthesis. 1H-NMR (CDCl3) l
7.65 (s, 1H), 7.28–7.65 (m, 4H), 7.32 (s, 15H), 7.08 (s,
1H).
l
l
l
max=264 nm; Tz3-derivatives=32.0/mM cm at
max=287 nm; Tz4-derivatives=37.0/mM cm at
max=292 nm.
TLC analysis was carried out on precoated plates
of silica gel 60F254 (Merck). Purification was per-
1
formed on silica gel (40–60 mm). H-NMR spectra
were recorded with a Bruker AM-200 apparatus. Re-
verse phase HPLC was performed with Altex dual-
pump system and 2.5×25 cm with Diasorb 130 C16T
(BioChemMack). All the chemicals used were ob-
tained from Fluka. EDTA-bisanhydride was prepared
according to [32].
2.4. Ethyl-2-[2-(triphenylmethylamino)-1,3-thiazol-4-
carboxamido]-1,3-Tz-4-carboxylate (7a)
A mixture of compound 4 (1.83 g, 10.6 mmol), com-
pound 6 (5.35 g, 10.6 mmol) and NEt3 (2.6 mL, 18
mmol) in dry DMF (25 mL) was stirred for 1 h at 50 °C.
The greenish mixture was allowed to react at 50 °C
without stirring for 16 h. The mixture was evaporated
under reduced pressure. The residue was purified by
column chromatography and the resulting homogeneous
product 7a was eluted by chloroform/hexane (1:1) (5.0
Matrix-assisted, laser desorption/ionization time-
of-flight mass spectrometry (MALDI TOF-MS) was
performed at the Institute of Molecular Biology
(Moscow).
2.1. Ethyl 2-amino-1,3-Tz-4-carboxylate (4)
This compound was prepared as described in refer-
ence [33]. After recrystallization from EtOH the yield of
1
g, 88%). H-NMR (CDCl3) l 10.40 (bs, 2H), 7.81 (s,
1H), 7.31 (s, 15H), 7.26 (s, 1H), 6.60 (s, 1H), 4.38 (q,
2H, J=7.0 Hz), 1.37 (t, 3H, J=7.0 Hz).
1
pure compound was 62%. H-NMR (CDCl3) l 7.42 (s,
1H), 7.02 (br s, 2H), 4.26 (q, 2H, J=7.0 Hz), 1.29 (t,
3H, J=7.0 Hz).
2.5. Ethyl-2-[2-[2-(triphenylmethylamino)-1,3-
thiazol-4-carboxamido]-1,3-thiazol-4-carboxamido]-
1,3-Tz-4-carboxylate (7b)
2.2. 2-Triphenylmethylamino-4-carboxy-1,3-Tz (5)
A mixture of compound 4 (7.4 g, 43 mmol) and
triphenylmethyl chloride (12.0 g, 43 mmol) was dis-
solved in 60 mL of dry pyridine and stirred for 16 h at
room temperature. The precipitate was filtered off,
washed with pyridine (2×10 mL), and the filtrate was
evaporated under reduced pressure up to 40–60 mL.
Ten grams of NaOH in 100 mL of 60% EtOH was
added to the residue. The mixture was stirred until
precipitate formation and left at room temperature for
20 h. The mixture was cautiously acidified with acetic
acid to pH 5–6. The precipitate was collected by filtra-
tion, washed with water (10×50 mL) and acetone (2×
Compound 7a (2.6 g, 4.8 mmol) dissolved in CHCl3
(30 mL) was treated with TFA (15 mL) for 1 h at room
temperature. The mixture was evaporated under reduced
pressure and 20 mL of water was added to the residue.
The precipitate was filtered, washed with 5% NaHCO3
(20 mL), rinsed with water (4×20 mL), dried on filter,
washed with ether and dried in vacuo over SiO2. The
resulting amine (1.03 g), compound 6 (1.82 g, 3.5 mmol)
and NEt3 (0.7 mL, 5.0 mmol) in dry DMF (5 mL) was
stirred for 1 h at 50 °C. The greenish mixture was
allowed to react at 50 °C without stirring for 16 h. The
mixture was evaporated under reduced pressure. The

V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
993
residue was purified by column chromatography using
chloroform as eluent giving the homogeneous product
7b (1.76 g, 55%). 1H-NMR ((CD3)2CO) l 10.90 (bs, 1H),
9.80 (bs, 1H), 8.14 (s, 1H), 8.21 (s, 1H), 8.04 (s, 1H),
7.25–7.56 (m, 16H), 4.32 (q, 2H, J=7.0 Hz), 1.34 (t,
3H, J=7.0 Hz).
was filtered off, the filtrate was washed with water
(2×50 mL) and then evaporated. The residue was
dissolved in ethanol (150 mL), and a mixture of NaOH
(27 g) and H2O (30 mL) was added dropwise at room
temperature. The mixture was stirred for 4 h at room
temperature, evaporated to 100 mL, water (200 mL) was
added to the residue and the mixture was cautiously
acidified with acetic acid to pH 5–6. The product was
extracted with CHCl3 (2×100 mL), the organic layer
was washed with water (3×200 mL) and dried over
Na2SO4. After evaporation, practically pure 9 was ob-
2.6. Ethyl-2-[2-[2-[2-(triphenylmethylamino)-1,3-
thiazol-4 carboxamido]-1,3-thiazol-4-carboxamido]-
1,3-thiazol-4-carboxamido]-1,3-Tz-4-carboxylate (7c)
1
Compound 7b (0.88 g, 1.3 mmol) in CHCl3 (15 mL)
was treated with TFA (7 mL) for 1 h at room tempera-
ture. The mixture was evaporated under reduced pres-
sure. Ten millilitres of water were added to the residue.
The precipitate was filtered, washed with 5% NaHCO3
(5 mL), rinsed with plenty of water, dried on filter,
washed with ether (4×5 mL) and dried in vacuo over
SiO2. The resulting amine (0.51 g), compound 6 (1.55 g,
3.1 mmol) and NEt3 (0.6 mL, 4.2 mmol) in dry DMF (6
mL) were stirred for 1 h at 50 °C. The greenish mixture
was allowed to react without stirring at 50 °C for 16 h.
The mixture was evaporated under reduced pressure.
The residue was purified by column chromatography
using chloroform/DMF (9:1) as eluent giving the homo-
genous product 7c (0.49 g, 48%). 1H-NMR (CDCl3-
(CD3)2SO=1:1) l 7.84 (s, 1H), 7.81 (s, 1H), 7.68 (s,
1H), 7.62 (s, 1H), 7.05–7.25 (m, 16H), 4.17 (q, 2H,
J=7.0 Hz), 1.20 (t, 3H, J=7.0 Hz).
tained (24.8 g, 65%). H-NMR ((CD3)2CO) l 7.12–7.52
(m, 15H), 2.26 (t, 2H, J=7.0 Hz), 2.10 (t, 2H, J=7.0
Hz), 1.28–1.67 (m, 6H).
2.9. 1,2,3-Benzotriazol-1-yl-6-
triphenylmethylaminohexylcarboxylate (10)
DCC (3.70 g, 17.9 mmol) in small portions was added
to compound 9 (6.36 g, 17.0 mmol) and 1-hydroxy-
1,2,3-benzotriazole (2.42 g, 17.9 mmol) in dry
dichloromethane (100 mL) at room temperature. After
the mixture was stirred for 3 h, the precipitated urea was
filtered off. Filtrate was evaporated, dried in vacuo over
SiO2. The resulting crude product 10 (8.27 g, 99%) was
sufficiently pure and could be used directly for synthesis.
1H-NMR ((CD3)2CO) l 7.12–7.91 (m, 19H), 2.25 (t, 2H,
J=7.0 Hz), 2.16 (t, 2H, J=7.0 Hz), 1.28–1.67 (m, 6H).
2.10. 6-Triphenylmethylaminohexylcarboxy deri6ati6es
11a–d
2.7. Ethyl ester-6-aminohexanoic acid hydrochloride (8)
Compound 7a–c (1.0 mmol) was dissolved in the
mixture of CHCl3 (15 mL) and TFA (7 mL). After 1 h
at room temperature the mixture was evaporated under
reduced pressure, and water (10 mL) was added to the
residue. The precipitate was filtered, washed with 5%
NaHCO3 (5 mL), rinsed with plenty of water, dried on
filter, washed with ether (4×10 mL) and dried in vacuo
over SiO2. The resulting amine, 1.0 mmol compound 4
or compound 10 (1.02 g, 2.1 mmol) and NEt3 (0.3 mL)
in dry DMF (6 mL) was stirred for 10 h at 60 °C. The
brownish mixture was evaporated under reduced pres-
sure. The residue was purified by column chromatogra-
phy using chloroform as eluent. The yield of pure
compounds 11a, 11b, 11c, 11d was 85, 76, 58 and 63%,
respectively.
Thionyl chloride was added dropwise to the solution
of 6-aminohexanoic acid (13.1 g, 100 mmol) in dry
ethanol (120 mL) at temperature 0–5 °C. The mixture
was stirred for 1 h at room temperature, filtered off and
the filtrate was evaporated. The crude product was
recrystallized from EtOH. It was homogeneous accord-
ing to chromatography. The yield of pure compound 8
was 18.6 g (95%). 1H-NMR ((CD3)2CO) l 7.05–7.43 (m,
15H), 4.02 (q, 2H, J=7.0 Hz), 2.19 (t, 2H, J=7.0 Hz),
2.08 (t, 2H, J=7.0 Hz), 1.28–1.60 (m, 6H), 1.20 (t, 3H,
J=7.0 Hz).
2.8. 6-Triphenylmethylaminohexanoic acid (9)
Triphenylmethyl chloride (26.8 g, 97 mmol) was
added to a solution of compound 8 (18.5 g, 95 mmol)
and NEt3 (30 mL) in dry CH2Cl2 at 0 °C. The mixture
was stirred for 3 h at room temperature. The precipitate
1H-NMR of compound 11a ((CD3)2CO) l 11.07 (bs,
1H), 8.02 (s, 1H), 7.1–7.5 (m, 15H), 4.30 (q, 2H, J=7.0
Hz), 2.91 (bs, 2H), 2.57 (t, 2H, J=7.0 Hz), 1.46–1.80
(m, 6H), 1.32 (t, 3H, J=7.0 Hz).

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V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
1H-NMR of compound 11b ((CD3)2CO) l 11.29 (bs,
1H), 7.96 (s, 1H), 7.86 (s, 1H), 7.15–7.55 (m, 15H), 3.54
(bt, 2H), 2.64 (bs, 2H), 2.49 (bt, 2H), 2.17 (s, 6H),
1.10–2.10 (m, 10H).
1H), 10.77 (bs, 1H), 8.09 (s, 1H), 8.01 (s, 1H), 7.1–7.5
(m, 16H), 4.29 (q, 2H, J=7.0 Hz), 2.93 (bs, 2H), 2.58 (t,
2H, J=7.0 Hz), 1.49–1.79 (m, 6H), 1.33 (t, 3H, J=7.0
Hz).
1H-NMR of compound 13c ((CD3)2CO) l 8.00 (s,
1H), 7.90 (s, 1H), 7.85 (s, 1H), 7.15–7.55 (m, 15H), 3.58
(b.quin, 2H), 2.60 (bm, 2H), 2.33 (bm, 2H), 2.07 (s, 6H),
1.10–2.10 (m, 10H).
1H-NMR of compound 11c ((CD3)2CO) l 11.44 (bs,
1H), 10.85 (bs, 2H), 8.19 (s, 1H), 8.10 (s, 1H), 8.03 (s,
1H), 7.1–7.5 (m, 16H), 4.30 (q, 2H, J=7.0 Hz), 2.90
(bs, 2H), 2.60 (t, 2H, J=7.0 Hz), 1.30–1.84 (m, 6H),
1.33 (t, 3H, J=7.0 Hz).
1H-NMR of compound 13d (DMF-d7) l 8.40 (s, 1H),
8.31 (s, 1H), 8.28 (s, 1H), 8.10 (s, 1H), 7.1–7.5 (m, 15H),
3.83 (bs, 2H), 2.55 (bs, 2H), 2.30 (bs, 2H), 2.13 (s, 6H),
1.1–2.0 (m, 10H).
1H-NMR of compound 11d (DMF-d7) l 11.30 (bs,
1H), 10.60 (bs, 1H), 8.44 (s, 1H), 8.39 (s, 1H), 8.30 (s,
1H), 8.20 (s, 1H), 7.1–7.5 (m, 16H),4.35 (q, 2H, J=7.0
Hz), 2.90 (bs, 2H), 2.60 (t, 2H, J=7.0 Hz), 1.20–1.90
(m, 6H), 1.36 (t, 3H, J=7.0 Hz).
1H-NMR of compound 14a (CCl4) l 10.78 (bs, 1H),
7.82 (bs, 1H), 7.74 (s, 1H), 7.20–7.50 (m, 15H), 3.27 (s,
3H), 2.80 (bt, 2H), 2.35 (bt, 2H), 2.01 (bm, 2H), 1.77
(bm, 4H).
1H-NMR of compound 14b ((CD3)2CO) l 11.49 (bs,
1H), 10.62 (bs, 1H), 8.35 (bs, 1H), 7.78 (bs, 1H), 7.75 (s,
1H), 7.15–7.50 (m, 15H), 3.25 (s, 3H), 2.75 (t, 2H), 2.33
(t, 2H), 1.90–2.10 (m, 2H), 1.70–1.80 (m, 4H).
1H-NMR of compound 14c (DMF-d7) l 8.38 (s, 1H),
8.03 (bs, 1H), 7.83 (s, 1H), 7.15–7.55 (m, 15H), 2.61 (t,
2H), 2.11 (s, 3H), 2.09 (t, 2H), 1.3–1.8 (m, 6H).
2.11. Preparation of acids 12a–d
4 M NaOH (10 mL) was added to the solution of
ethyl ester (11a–d) (3 mmol) in ethanol (20 mL). The
mixture was stirred for 12 h at 20 °C, diluted with water
(30 mL) and cautiously acidified with HCl to pH 5–6.
The precipitated acid was collected, washed with water
(6×10 mL) and dried in vacuo over SiO2. The resulting
acids (90–95%) were sufficiently pure and could be used
directly for synthesis.
2.13. 1-Triphenylmethyl-1,6-diaminohexane (15)
Triphenylmethyl chloride (6.6 g, 24 mmol) in 50 mL
dry CH2Cl2 was added to a solution of 1,6-diaminohex-
ane (16.6 mg, 140 mmol) in dry CH2Cl2 (100 mL). The
mixture was stirred for 4 h at 20oC. The product was
purified by column chromatography using chloroform/
EtOH/NEt3 (94:5:1) as eluent. The yield of homoge-
neous compound 15 was 2.73 g (7.7 mmol, 32%).
1H-NMR ((CD3)2CO) l 7.10–7.60 (m, 15H), 3.09 (t,
7.84, 2H, J=7.2 Hz), 2.10 (t, 7.84, 2H, J=7.2 Hz), 1.52
(bm, 4H), 1.32 (bm, 4H).
2.12. N,N-dimethyl-1,3-propylendiamine deri6ati6es
(13a–d) and methylamine deri6ati6es
DCC (0.4 g, 2.0 mmol) in small portions were added
to the mixture of acid 12a–c (1.0 mmol), 1-hydroxy-
1,2,3-benzotriazole (0.3 g, 2.0 mmol) in dry DMF (3
mL) at room temperature. After stirring for 2 h, N,N-
dimethyl-1,3-diaminopropane (0.2 mL) or methylamine
hydrochloride (0.4 g, 6 mmol) and NEt3 (5 mL) were
added and the reaction mixture was left for 12 h. The
precipitate was filtered off, washed with CH2Cl2 (5 mL).
Combined filtrates diluted with CHCl3 (50 mL), were
washed with 5% NaHCO3 (2×15 mL), water (3×25
mL), dried over Na2SO4 and evaporated. The yield of
products 13a, 13b, 13c, 13d and 14a, 14b, 14c after
column chromatography (0–20% gradient of EtOH in
CHCl3/NEt3 (95:5)) was 60, 49, 55, 35, 65, 52 and 40%,
respectively.
2.14. Preparation of compound 16
To the mixture of acid 12c (145 mg, 0.23 mmol),
1-hydroxy-1,2,3-benzotriazole (40 mg, 0.26 mmol) in dry
CH2Cl2 (5 mL) was added DCC (54 g, 0.26 mmol) at
room temperature. After stirring for 4 h, the amine 15
(93 mg, 0.26 mmol) and 70 mL NEt3 were added and the
reaction mixture was boiled for 4 h. The yield of pure
compound 16 after column chromatography (0–5% gra-
dient of ethanol in chloroform) was 187 mg (0.19 mmol,
84%).
1H-NMR of compound 13a ((CD3)2CO) l 8.10 (bs,
1H), 7.87 (s, 1H), 7.15–7.55 (m, 15H), 3.54 (bt, 2H),
2.64 (bs, 2H), 2.49 (bt, 2H), 2.17 (s, 6H), 1.10–2.10 (m,
10H).
1H-NMR ((CD3)2CO) l 11.46 (bs, 1H), 10.50 (bs,
1H), 7.84 (s, 1H), 7.10–7.50 (m, 30H), 7.98 (s, 1H), 3.40
(q, 2H, J=6.5 Hz), 2.84 (t, 2H, J=7.0 Hz), 2.64 (t, 2H,
1H-NMR of compound 13b ((CD3)2CO) l 8.17 (bs,

V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
995
J=7.0 Hz), 2.14 (t, 2H, J=7.0 Hz), 0.94–1.90 (m,
14H).
2.63 (t, 2H, J=7.0 Hz), 2.54 (t, 2H, J=7.0 Hz), 2.18 (s,
6H), 2.11 (t, 2H, J=7.0 Hz), 1.40–1.90 (m, 8H).
1H-NMR of compound V ((CD3)2SO) l 12.40 (bs, 1H),
12.00 (bs, 1H), 8.19 (s, 1H), 7.88 (s, 1H), 3.48 (t, 2H,
J=7.0 Hz), 3.02 (t, 2H, J=7.0 Hz), 2.52 (t, 2H, J=7.0
Hz), 2.14 (t, 2H, J=7.0 Hz), 1.20–1.80 (m, 14H).
1H-NMR of compound VI ((CD3)2SO) l 12.43 (bs,
1H), 11.95 (bs, 1H), 8.33 (s, 1H), 8.00 (bs, 1H), 7.85 (s,
1H), 4.30 (bs, 2H), 2.81 (s, 3H), 2.16 (t, 2H, J=7.0 Hz),
1.68 (m, 4H, J=7.0 Hz), 1.39 (m, 4H, J=7.0 Hz).
1H-NMR of compound VII ((CD3)2SO) l 12.57 (bs,
1H), 12.43 (bs, 1H), 11.95 (bs, 1H), 8.34 (s, 1H), 8.33 (s,
1H), 8.00 (bs, 1H), 7.85 (s, 1H), 4.40 (bs, 2H), 2.79 (s,
3H), 2.12 (t, 2H, J=7.0 Hz), 1.61 (m, 4H, J=7.0 Hz),
1.35 (m, 4H, J=7.0 Hz).
2.15. Preparation of compound 17
DCC (93 g, 0.45 mmol) was added to the mixture of
acid 12d (260 mg, 0.30 mmol), 1-hydroxy-1,2,3-benzotri-
azole (69 mg, 0.45 mmol) in dry DMF (5 mL) at room
temperature. After stirring for 4 h, NH2CH2CH-
(OH)CH2OH (120 mg, 1.30 mmol) was added and the
reaction mixture was left for 4 h at 60 °C. The yield of
pure compound 17 after column chromatography (0–
15% gradient of EtOH in CHCl3/NEt3 (95:5)) was 110
mg (0.12 mmol, 40%).
1H-NMR ((CD3)2CO) l 11.48 (bs, 1H), 10.75 (bs,
2H), 10.30 (bs, 1H), 8.18 (s, 1H), 8.09 (bs, 1H), 8.02 (s,
1H), 8.01 (s, 1H), 7.82 (s, 1H), 7.10–7.50 (m, 15H), 4.51
(bs, 1H), 3.88 (bs, 1H), 3.60 (m, 4H), 2.60 (t, 2H, J=7.0
Hz), 2.15 (t, 2H, J=7.0 Hz), 1.70 (m, 4H), 1.45 (m,
2H).
1H-NMR of compound 19a (D2O) l 7.69 (s, 1H), 3.00
(t, 2H, J=7.0 Hz), 2.86 (s, 3H), 2.51 (t, 2H, J=7.0 Hz),
1.69 (bruin, 4H, J=7.0 Hz), 1.43 (bruin, 4H, J=7.0 Hz).
2.17. EDTA deri6ati6es VIII, IX, X
Compounds 18b or 18c or 20 (0.2 mmol), after evapo-
ration with pyridine (2×5 mL), were mixed with dry
DMF (10 mL), EDTA-anhydride (2 mmol) and pyridine
(0.8 mL). The mixture was stirred for 5 h at room
temperature and evaporated under reduced pressure.
The residue was purified by reverse phase HPLC (0–
15% gradient of CH3CN, 0.1% TFA). The yield of
2.16. Preparation of compounds I–IX
Compounds 13, 14, 16 or 17 (100 mg) were dissolved
in the mixture of CHCl3 (2 mL) and TFA (1 mL) and
after 1 h at room temperature, the mixture was evapo-
rated under reduced pressure and washed with ether
(4×5 mL), dissolved in 0.1% TFA and purified by
HPLC (0–15% gradient CH3CN, 0.1% TFA 50–80%).
The yield of homogeneous products I, II, III, IV, V, VI,
VII after column chromatography (0–20% gradient of
EtOH in CHCl3/NEt3 (95:5)) was 80, 75, 71, 50, 51, 60
and 55%, respectively.
compounds VIII, IX,
X was 33, 39 and 40%,
respectively.
1H-NMR of compound VIII ((CD3)2CO) l 8.10 (s,
1H), 7.84 (s, 1H), 3.90 (bm, 14H), 3.50 (t, 2H, J=7.0
Hz), 3.10 (t, 2H, J=7.0 Hz), 3.07 (s, 6H), 2.60 (t, 2H,
J=7.0 Hz), 2.10 (bm, 2H), 1.60–1.80 (m, 4H), 1.40–
1.50 (m, 2H).
1H-NMR of compound I ((CD3)2SO) l 11.48 (bs,
1H), 7.84 (s, 1H), 3.56 (t, 2H, J=7.0 Hz), 2.64 (t, 2H,
J=7.0 Hz), 2.53 (t, 2H, J=7.0 Hz), 2.20 (s, 6H), 2.10
(t, 2H, J=7.0 Hz), 1.40–1.90 (m, 8H).
1H-NMR of compound IX ((CD3)2CO) l 8.12 (s, 1H),
8.07 (s, 1H), 7.88 (s, 1H), 3.91 (bm, 14H), 3.47 (t, 2H,
J=7.0 Hz), 3.07 (t, 2H, J=7.0 Hz), 3.04 (s, 6H), 2.57
(t, 2H, J=7.0 Hz), 2.11 (bm, 2H), 1.61–1.85 (bm, 4H),
1.41–1.55 (bm, 2H).
1H-NMR of compound II ((CD3)2SO) l 12.30 (bs,
1H), 11.48 (bs, 1H), 7.98 (s, 1H), 7.84 (s, 1H), 3.54 (t,
2H, J=7.0 Hz), 2.65 (t, 2H, J=7.0 Hz), 2.53 (t, 2H,
J=7.0 Hz), 2.17 (s, 6H), 2.11 (t, 2H, J=7.0 Hz),
1.40–1.90 (m, 8H).
1H-NMR of compound X ((CD3)2CO) l 8.14 (s, 1H),
7.93 (s, 1H), 3.90 (bm, 24H), 3.50 (bt, 2H, J=7.0 Hz),
3.18 (t, 2H, J=7.0 Hz), 2.83 (t, 2H, J=7.0 Hz), 2.67 (t,
2H, J=7.0 Hz), 1.20–1.80 (m, 14H).
1H-NMR of compound III ((CD3)2SO) l 12.40 (bs,
2H), 11.48 (bs, 1H), 7.99 (s, 1H), 7.98 (s, 1H), 7.84 (s,
1H), 3.55 (t, 2H, J=7.0 Hz), 2.65 (t, 2H, J=7.0 Hz),
2.52 (t, 2H, J=7.0 Hz), 2.18 (s, 6H), 2.10 (t, 2H, J=7.0
Hz), 1.40–1.90 (m, 8H).
2.18. Preparation of compounds XI–XIII
1H-NMR of compound IV ((CD3)2SO) l 12.40 (bs,
2H), 12.20 (bs, 1H), 11.48 (bs, 1H), 8.01 (s, 1H), 7.99 (s,
1H), 7.98 (s, 1H), 7.84 (s, 1H), 3.56 (t, 2H, J=7.0 Hz),
DCC (93 mg, 0.45 mmol) was added to the mixture of
acids 12b–d (0.30 mmol), 1-hydroxy-1,2,3-benzotriazole
(69 mg, 0.45 mmol) in dry DMF (5 mL) at room

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V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
temperature. After stirring for
4
h, 3-amino-1,2-
X
MALDI TOF-MS (monoisotopic) 1030.7
(1030.4 calcd for M+H).
MALDI TOF-MS (monoisotopic) 457.2
(457.1 calcd for M+H).
MALDI TOF-MS (monoisotopic) 583.2
(583.1 calcd for M+H).
MALDI TOF-MS (monoisotopic) 709.2
(709.1 calcd for M+H).
propanediol (120 mg, 1.30 mmol) was added and the
reaction mixture was left for 4 h at 60 °C. Reaction
mixtures were purified by column chromatography (0–
15% gradient of EtOH in CHCl3/NEt3 (95:5)). The
triphenylmethyl derivatives obtained were dissolved in a
mixture of CH2Cl2 (2 mL) and TFA (1 mL) and after 1
h at room temperature, the mixture was evaporated
under reduced pressure and washed with ether (4×5
mL), dissolved in 0.1% TFA and purified by HPLC.
The yield of pure homogeneous compounds XI, XII
and XIII after HPLC (0–90% gradient of CH3CN, 0.1%
TFA) was 55, 50 and 40%, respectively.
XI
XII
XIII
3. Results and discussion
A series of oligo-1,3-thiazolcarboxamide derivatives
with different numbers of thiazolcarboxamide units in
the peptide and substituents in the side chain was
synthesized. The structures of the Tz containing
oligopeptides are given in figure 2. All the compounds
synthesized (I–XIII) were homogeneous according to
the data of reverse phase chromatography, nuclear
magnetic resonance (NMR) spectroscopy and mass
spectrometry.
1H-NMR of compound XI ((CD3)2SO) l 12.45 (bs,
1H), 11.95 (bs, 1H), 8.30 (s, 1H), 8.05 (bs, 1H), 7.88 (s,
1H), 4.43 (bm, 1H), 4.30 (bs, 2H), 3.34 (m, 3H), 3.07 (m,
1H), 2.15 (t, 2H, J=7.0 Hz), 1.68 (m, 4H, J=7.0 Hz),
1.39 (m, 4H, J=7.0 Hz).
1H-NMR of compound XII ((CD3)2SO) l 12.62 (bs,
1H), 12.41 (bs, 1H), 11.95 (bs, 1H), 8.32 (s, 1H), 8.30 (s,
1H), 8.02 (bs, 1H), 7.87 (s, 1H), 4.41 (bs, 3H), 4.30 (bs,
2H), 3.32 (m, 3H), 3.05 (m, 1H), 2.13 (t, 2H, J=7.0
Hz), 1.65 (m, 4H, J=7.0 Hz), 1.36 (m, 4H, J=7.0 Hz).
1H-NMR of compound XIII ((CD3)2CO) l 11.48 (bs,
1H), 10.75 (bs, 2H), 10.30 (bs, 1H), 8.18 (s, 1H), 8.09
(bs, 1H), 8.02 (s, 1H), 8.01 (s, 1H), 7.82 (s, 1H), 4.51 (bs,
1H), 3.88 (bs, 1H), 3.60 (m, 4H), 2.60 (t, 2H, J=7.0
Hz), 2.15 (t, 2H, J=7.0 Hz), 1.70 (m, 4H), 1.45 (m,
2H).
To analyse the interaction of the TCOs with HIV-1
IN we used the 3%-end processing reaction catalysed
by the enzyme. The reaction was assayed with a
double stranded oligonucleotide derived from the
HIV-1 U5 end of the LTR, containing [32P]-labeled
GT nucleotides in the 3%-end. Sequence-specific re-
moval from the 3%-end generated a radiolabeled dinu-
cleotide product detected following electrophoresis
(figure 3A). In parallel, the reaction products were
analysed by counting the remaining labeled material
in the acid-insoluble fraction after dinucleotide cleav-
age (figure 3B). As the measurements using both
determinations matched very well, the second method
was used to estimate the effect of TCOs on the
IN-catalysed reaction. Figure 3 illustrates the results
obtained with one of the TCOs, compound III. With
increasing concentrations of this compound, a dose-
dependent inhibition of the 3%-processing reaction was
obtained with an IC50 value of 2.3 mM. All TCOs
present similar patterns of inhibition, although with
different efficiencies. The IC50 values for all com-
pounds analysed are given in table I. Distamycin, the
natural oligopeptide containing three pyrrole rings,
was used as a reference of DNA minor groove binder.
The IC50 value for distamycin, obtained under the
same experimental conditions, is also reported in table
I. TCOs containing 3 or 4 Tz units (compounds III
and XIII) were able to suppress the IN activity as
efficiently as distamycin. A very efficient inhibitor of
MALDI TOF-MS for the different compounds was
the following:
I
MALDI TOF-MS (monoisotopic) 342.3
(342.2 calcd for M+H).
II
MALDI TOF-MS (monoisotopic) 468.3
(468.2 calcd for M+H).
III
IV
V
MALDI TOF-MS (monoisotopic) 594.3
(594.2 calcd for M+H).
MALDI TOF-MS (monoisotopic) 720.2
(720.2 calcd for M+H).
MALDI TOF-MS (monoisotopic) 482.1
(482.2 calcd for M+H).
VI
VII
VIII
IX
MALDI TOF-MS (monoisotopic) 397.1
(397.1 calcd for M+H).
MALDI TOF-MS (monoisotopic) 523.2
(523.1 calcd for M+H).
MALDI TOF-MS (monoisotopic) 742.2
(742.3 calcd for M+H).
MALDI TOF-MS (monoisotopic) 868.3
(868.2 calcd for M+H).

V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
997
Figure 2. Structures of the TCOs I–XIII.

998
V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
tives. As shown in figure 4, the IC50 values for TCOs
of different length correlated with the number of Tz
units in the oligopeptides. The logarithmic depen-
dences of the IC50 values on the number of Tz units
were close to linear (figure 4), suggesting that each
monomer of the TCO molecule contributed in an
additive form to the interaction with the nucleotide
units of DNA.
An interesting point is the influence of the amines,
that are positively charged at physiological pH, on the
inhibitory activity of the molecules. TCOs having the
same number of Tz units, but additional positively
charged groups attached to the N- and C-termini
were more efficient inhibitors of the IN-dependent
reaction. In contrast, the introduction of EDTA
(compounds VIII, IX and X) or diol-groups (com-
pounds XI, XII and XIII) into the oligopeptides led to
higher IC50 values. Such decrease on the affinity of
TCOs towards DNA complexed with IN may be
explained by the negative charges of EDTA- and the
diol-groups, and/or by the electrostatic repulsion be-
tween TCOs and the internucleoside phosphate
groups of DNA.
Figure 3. Inhibition on the IN-catalysed 3%-processing reac-
tion by oligopeptide III. A: Reaction products were analysed
by urea-polyacrylamide gel electrophoresis. The complete re-
action mixture containing the labeled substrate was incubated
for 30 min at 30 °C in the absence of IN (lane S) or in the
presence of 40 nM IN (lane IN). Lanes 1–9: same as lane IN,
but in the presence of 0.7, 1, 3, 10, 30, 50, 70, 90 and 100 mM
of compound III, respectively. Arrows indicate the migration
of the labeled oligonucleotides containing 21 and 2 nucle-
otides. B: Reaction products were determined by measuring
the TCA-precipitable radioactivity remaining after IN-depen-
dent processing. Results are expressed as relative activity,
100% corresponding to IN activity in the absence of the
inhibitor.
the IN-catalysed reaction was compound IV with an
IC50 value about one order of magnitude lower than
that of distamycin.
Next we analysed the relationship between the in-
hibitory efficiency and the length of the TCO deriva-
Figure 4. Logarithmic dependence of the IC50 values upon the
number of Tz units in the structure of TCOs: compounds I, II,
III and IV ( ) and XI, XII and XII (ꢀ).

V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
999
Table I. Concentration of TCOs inhibiting the IN 3%-processing activity by 50% (IC50).
Compound
Structures of TCOs
IC50 (mM)
I
NH2(CH2)5CO-[Tz]-NH(CH2)3N(CH3)2
NH2(CH2)5CO-[Tz]2-NH(CH2)3N(CH3)2
NH2(CH2)5CO-[Tz]3-NH(CH2)3N(CH3)2
NH2(CH2)5CO-[Tz]4-NH(CH2)3N(CH3)2
NH2(CH2)5CO-[Tz]2-NH(CH2)6NH2
NH2(CH2)5CO-[Tz]2-NHCH3
10009200
3097
II
III
IV
V
VI
VII
VIII
IX
X
290.3
0.390.05
3898
70915
992
8609150
10930
300970
350950
3098
2.290.4
3.590.5
NH2(CH2)5CO-[Tz]3-NHCH3
EDTA-NH(CH2)5-CO-[Tz]2-NH(CH2)3-N(CH3)2
EDTA-NH(CH2)5-CO-[Tz]3-NH(CH2)3-N(CH3)2
EDTA-NH(CH2)5-CO-[Tz]2-NH-EDTA
NH2(CH2)5CO-[Tz]2-NH CH2CH(OH)CH2OH
NH2(CH2)5CO-[Tz]3-NH CH2CH(OH)CH2OH
NH2(CH2)5CO-[Tz]4-NH CH2CH(OH)CH2OH
Distamycin A
XI
XII
XIII
Since the affinities of TCOs depended most on their
length, it can be hypothesized that DNA complexed
with IN interacted mainly with the Tz monomers, and
that such interactions were responsible for the effi-
ciency of the inhibition by oligo-1,3-thiazolecarbox-
amide derivatives. At the same time, it cannot be
excluded that the additional groups attached to the
N- and C-termini of TCOs may influence the interac-
tions either with the enzyme or 6ia the enzyme on
their interaction with DNA.
heating for 2 min at 90 °C and slow cooling. This
ds-DNA was labeled at the 3%-end with [a-32P]dGTP and
[a-32P]TTP in the presence of the exonuclease-free
Klenow fragment of E. coli DNA polymerase.
IN activity was measured by the 3%-end processing
reaction. The standard reaction mixture (20 mL) con-
tained 20 mM Hepes (pH 7.5), 10 mM DTT, 0.1 mM
EDTA, 4 mM NaCl, 7.5 mM MnCl2, 0.05% NP 40, 3.5
nM ds [32P]-substrate. The reaction mixture was incu-
bated for different times (2–60 min) at 30 °C in the
presence of 10–40 nM IN. The reaction products of
IN-dependent [32P](GT) dinucleotide removal were esti-
mated using two methods: a) separation on 12% poly-
acrylamide gel electrophoresis in the presence of 7 M
urea, or b) by measuring the label remaining in the
acid-insoluble material by the utilization of dry What-
man 3MM filters (15×15 mm) presoaked in 5%
trichloroacetic acid (TCA). Further treatment of the
filters was carried out as described in [37]. All measure-
ments were carried out within linear ranges of the time
dependence of product accumulation and IN concentra-
tion. The IC50 values were determined at substrate con-
centration around 3 times the Km. The data of 3–4
experiments were averaged. The error was within 10–
30%.
4. Experimental protocol
4.1. Reagents
Reagents were purchased from Merck, Sigma, Phar-
macia and Euromedex. All oligodeoxynucleotides
(ODNs) were synthesized as described in [34] and their
concentration was determined according to [35].
4.2. Enzymes
Recombinant HIV-1 IN, expressed in yeast, was ob-
tained and purified as previously described [36].
4.3. Enzyme assays
5. Conclusions
Double stranded DNA substrate for 3%-end processing
was prepared by annealing the 19-mer ODN (5%-GT-
GTGGAAAATCTCTAGCA) with the 21-mer comple-
mentary strand (5%-ACTGCTAGAGATTTTCCA) by
A set of thiazolecarboxamide derivatives able to
interact with the minor groove of DNA was synthe-
sized and its effect was evaluated on the HIV-1 IN-

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V.A. Ryabinin et al. / European Journal of Medicinal Chemistry 35 (2000) 989–1000
catalysed reaction. Some TCOs were efficient in-
hibitors of this reaction: compound IV, for example,
with an IC50 of 0.3 mM. These first generation of
TCOs will serve as leads provided that additional
chemical groups allow to increase their inhibitory
power.
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In addition, as we previously showed that short
oligonucleotides of specific sequence were efficient
inhibitors of HIV-1 IN [36], we can suppose that such
oligonucleotides conjugated to the TCOs described in
this work could offer a unique opportunity for ratio-
nal design of new IN-targeting drugs. Further work is
being performed to synthesize such derivatives.
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Acknowledgements
[22] Chen A.Y., Yu C., Gatto B., Liu L.F., Proc. Natl. Acad. Sci.
USA 90 (1993) 8131–8135.
This work was supported in part by the Russian Fund
for Basic Research (RFBR 00-04-49058), INTAS-RFBR
(95-IN-RU-1214), PICS-RFBR (571), the French
Agency for Research against AIDS (ANRS), the Poˆle
Me´dicament d’Aquitaine, the CNRS and the Universite´
Victor Segalen Bordeaux 2. We thank S.A. Grachev for
MALDI TOF-mass spectrometry.
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