Synthesis and antiviral activity of ethidium-arginine conjugates directed against the TAR RNA of HIV-1

Article abstract of DOI:10.1021/jm980728e

The regulatory protein Tat is essential for viral gene expression and replication of the human immunodeficiency virus type 1 (HIV-1). Tat transactivates the HIV-1 long terminal repeat (LTR) via its binding to the transactivation responsive element (TAR) and increases the viral transcription. Studies have shown that the binding of arginine and arginine derivatives induces a conformational change of the TAR RNA at the Tat-binding site. The unpaired A17 residue delimits a small cavity which constitutes a receptor site for small molecules, especially for ethidium bromide. These binding characteristics have prompted us to design a series of ethidium- arginine conjugates capable of interacting with the TAR RNA. Here we report the synthesis of six ethidium derivatives equipped with arginine side chains. These molecules were biologically evaluated, and two compounds (17 and 20) exhibited in vitro anti-HIV-1 activity at micromolar concentration, without toxicity (up to 100 μM concentration). Melting temperature studies indicated that the most active molecule (20) bound strongly to TAR in vitro. RNase protection experiments agreed with the molecular modeling studies which suggested that the ethidium moiety of 20 was inserted next to the A17 residue while the arginine side chain occupied the pyrimidine bulge.

Full text of DOI:10.1021/jm980728e

4042
J . Med. Chem. 1999, 42, 4042-4053
Syn th esis a n d An tivir a l Activity of Eth id iu m -Ar gin in e Con ju ga tes Dir ected
Aga in st th e TAR RNA of HIV-1
Vale´rie Peytou,† Roger Condom,*,† Nadia Patino,† Roger Guedj,† Anne-Marie Aubertin,‡ Nathalie Gelus,§
Christian Bailly,§ Raphae¨l Terreux,| and Daniel Cabrol-Bass|
Laboratoire de Chimie Bioorganique, CNRS ESA 6001, Universite´ de Nice Sophia-Antipolis, F-06108 Nice Cedex 2, France,
INSERM-U74, Universite´ Louis Pasteur, Institut de Virologie, 3 rue Koeberle´, F-67000 Strasbourg, France, Laboratoire de
Pharmacologie Antitumorale du Centre Oscar Lambret, INSERM-U524, IRCL, Place de Verdun, F-59045 Lille, France, and
LARTIC, Universite´ de Nice Sophia-Antipolis, F-06108 Nice Cedex 2, France
Received December 23, 1998
The regulatory protein Tat is essential for viral gene expression and replication of the human
immunodeficiency virus type 1 (HIV-1). Tat transactivates the HIV-1 long terminal repeat (LTR)
via its binding to the transactivation responsive element (TAR) and increases the viral
transcription. Studies have shown that the binding of arginine and arginine derivatives induces
a conformational change of the TAR RNA at the Tat-binding site. The unpaired A17 residue
delimits a small cavity which constitutes a receptor site for small molecules, especially for
ethidium bromide. These binding characteristics have prompted us to design a series of
ethidium-arginine conjugates capable of interacting with the TAR RNA. Here we report the
synthesis of six ethidium derivatives equipped with arginine side chains. These molecules were
biologically evaluated, and two compounds (17 and 20) exhibited in vitro anti-HIV-1 activity
at micromolar concentration, without toxicity (up to 100 µM concentration). Melting temperature
studies indicated that the most active molecule (20) bound strongly to TAR in vitro. RNase
protection experiments agreed with the molecular modeling studies which suggested that the
ethidium moiety of 20 was inserted next to the A17 residue while the arginine side chain
occupied the pyrimidine bulge.
In tr od u ction
Lys Arg Arg Gln Arg Arg Arg), is involved in TAR RNA
binding.8 While lysine residues contribute to electro-
static interactions, the arginine at position 52 is the only
sequence contact mediating the complex formation
between Tat and TAR.9 HIV-1 TAR is a sequence of 59
nucleotides located downstream from the transcription
initiation site in the LTR.10,11 It forms a hairpin stem-
loop structure consisting of base-pair interactions be-
tween nucleotides +1 to +59. It contains a 6-nucleotide
loop (residues 30-35) and a 3-nucleotide pyrimidine
bulge (residues 23-25) (Chart 1).12-14 That the bulge
is essential for high affinity and specific binding of the
Tat protein is evidenced by a number of studies.
Acquired immune deficiency syndrome (AIDS) is the
final stage resulting from a gradual but accelerating
decline in immune competence characterized by immune
dysfunction and depletion of CD4 T helper T-cells. The
causative agent of AIDS is the human immunodeficiency
virus type 1 (HIV-1), discovered in 1983.1 The gene
expression is regulated by a number of virus-encoded
proteins, including the transactivator of transcription
(Tat) protein which plays a determinant role in virus
replication.2 Tat is responsible for high levels of expres-
sion from the viral long terminal repeat (LTR) promoter
involving the interaction of Tat with the viral RNA at
a specific region, called transactivation responsive ele-
ment (TAR).3,4 Tat also acts by increasing the initiation
level of the LTR transcription.5 The goal of HIV thera-
peutics is to attempt to stop or at least to slow HIV
disease progression. The TAR RNA represents an at-
tractive therapeutic target for the design of antiviral
drugs.
First, dynamic molecular modeling studies have
shown a conformational change of TAR between the free
and the Tat-bound form of the RNA.15 Studies suggested
that U23, C24, and U25 are stacked on A22 in the
absence of Tat and that the arrangement of the 3-nucle-
otide bulge is disrupted in the presence of Tat or a single
arginine: U23 moves in direct proximity to G26, and the
two other nucleotides become unstacked.16-18 Tat binds
to TAR precisely in the region of the bulge UCU of TAR
via an arginine residue and recognizes both the identity
of adjacent Watson-Crick base pairs and the positions
of surrounding phosphate groups.19-22 On the basis of
NMR spectra of the truncated TAR sequence associated
with derivatives of arginine or derivatives of Tat, a
structural model of the complex was proposed.17,23-25
This model has shown that residues U23, A27, and G26
interact with the guanidinium group of arginine 52.
Thus, a large part around the bulge is critical for
formation of the Tat-TAR complex. Second, it is im-
HIV-1 Tat is an 86-amino acid protein produced from
a doubly spliced mRNA formed by joining an exon
preceding the env gene with the second exon within env.
Mutational and NMR analyses have revealed different
structural and functional domains.6,7 One of the do-
mains, the basic region (residues 48-57: Gly Arg Lys
* Corresponding author: Prof. Roger Condom. Tel: (+33) (0)4 92 07
61 52. Fax: (+33) (0)4 92 07 61 51. E-mail: condom@unice.fr.
† Laboratoire de Chimie Bioorganique.
‡ Institut de Virologie.
§ Laboratoire de Pharmacologie Antitumorale.
| LARTIC.
10.1021/jm980728e CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/21/1999

TAR Binding Ethidium-Arginine Conjugates
Ch a r t 1. Structure of the TAR RNA of HIV-1
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4043
Ch a r t 2. Intercalation of the Potential Inhibitors
Directed Against the TAR RNA of HIV-1
portant to note that a single arginine residue in solution
binds to the bulge of TAR with millimolar affinity.26
Third, a number of RNA-interacting compounds includ-
ing groove binders, classical and threading intercalators,
unfused aromatic intercalators, and agents with mixed
binding modes have been identified.27 Some classical
intercalating agents, such as ethidium bromide, were
found to bind more tightly to RNA than to DNA.27 With
DNA, ethidium exhibits a slight preference for binding
to pyrimidine(3′-5′)purine sites and particularly to CpG
steps.28-31 With different RNA hairpin segments, ethid-
ium also binds preferentially to CpG sites proximal to
a bulge.32,33 More recent studies have revealed that,
upon binding to TAR, ethidium intercalates near the
unpaired A17 residue.34,35 Molecular studies have shown
that the amino groups at the 3 and 8 positions of
ethidium are critical components in ethidium interac-
tions with nucleic acids and that the geometry of the
phenanthridine ring system is nearly ideal for maximiz-
ing favorable contacts with the nucleic acid bases that
form the top and the bottom of the intercalation site.36
Therefore, the development of agents which may inter-
fere with the protein-RNA complex formation is a
potential strategy for controlling the proliferation of
HIV.37-49
The capacity of ethidium to form stable complexes
with RNA including TAR, on the one hand, and the fact
that arginine and arginine derivatives bind to the
pyrimidine bulge of TAR, on the other hand, prompted
us to design bifunctional molecules capable of binding
to both binding sites of TAR. For this purpose, we
synthesized a series of molecules containing: (i) an
arginine residue which would bind near the U23-C24-
U25 bulge, (ii) an ethidium moiety which would inter-
calate between the base pairs C18-G44 and C19-G43,
close to the A17 bulge, and (iii) a spacer arm connecting
the two RNA-binding parts of the molecule (Chart 2).
Spacer arms of variable length were prepared in order
to determine the optimal distance needed to allow the
two parts of the molecules to interact with TAR. The
arginine residue was attached to the 3′ (meta) position
of the ethidium chromophore. Previous studies have
shown that substitutions at the meta position cause
minimal interference with the intercalation binding
mode.50
The synthesis and the anti-HIV-1 activity of a series
of six molecules against different cell lines in vitro are
presented here. Two of them (17 and 20) exhibited in
vitro anti-HIV-1 activity at micromolar concentration
without toxicity (up to 100 µM concentration). The
binding to TAR of the most active compound 20 was
investigated using melting temperature and RNase
footprinting experiments, and the drug-TAR RNA
complex formation was also studied by molecular mod-
eling.
Ch em istr y
The synthesis of the ethidium derivatives was carried
out in three principal steps. First, a modified ethidium
molecule (8) was synthesized. We introduced a carboxyl
function to the meta position of the 6-phenyl group for
use in anchoring an arginine-terminated chain via an
amide linkage. Second, we synthesized a series of
molecules (9-11) containing an amino group and an
arginine residue separated by a spacer. Third, coupling
of these two key molecules followed by deprotection of
the guanidinium ion yielded the final compounds 15-
20 (Scheme 1).
Carboxyethidium derivative 8 was synthesized using
previously described procedures50-53 with some modi-
fications. 2-Aminobiphenyl (1) was acylated with 3-cy-
anobenzoyl chloride (2) in chlorobenzene to give amide
3, which was then cyclized to give phenanthridine 4 by
refluxing with phosphoryl chloride in nitrobenzene. The
use of tin(IV) chloride to catalyze the reaction provided
4 in good yield.54 Hydrolysis of the nitrile of the
phenanthridine 4 in 75% H2SO4 gave carboxylic acid

4044 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Sch em e 1. Route Leading to Potential Inhibitors
Peytou et al.
derivative 5. Quaternization of phenanthridine 5 was
carried out using dimethyl sulfate, which also methy-
lated the carboxyl group to lead to methyl ester 6, in
the form of a very hygroscopic oil. 6 was refluxed in a 6
N HCl aqueous solution and gave the carboxylic acid
derivative 7, in chloride ion form. Finally, 7 was reduced
using iron powder in the presence of 1 N HCl in water-
ethanol mixture, yielding the key ethidium chloride
derivative 8. Purification following the reduction step
was quite difficult and required repeated runs through
an Amberlite XAD-2 resin column.
vated using DCC/HOSu and coupled in situ with the
H-Arg(Pmc)OH to lead to acid 24. Then, the carboxyl
function was methylated by adding methyl iodide and
cesium carbonate to give methyl ester 25. Deprotection
of the primary amine of 25 was realized using catalytic
hydrogenation in the presence of 10% Pd/C to produce
the first arginine-terminated chain 9, for linking with
the carboxyethidium 8. The second arginine-terminated
chain 10 was prepared following the same route starting
from N-(benzyloxycarbonyl)caproic acid. Intermediate
compounds 26 and 27 were isolated at each step of the
synthesis. Finally, coupling of N-(benzyloxycarbonyl)-
caproic acid with 9 gave methyl ester 28 which was
Compounds 9-11 were obtained in three steps
(Scheme 2). N-(Benzyloxycarbonyl)-â-alanine was acti-

TAR Binding Ethidium-Arginine Conjugates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4045
Ta ble 1. Yields of the Deprotection Step Leading to
Compounds 15-23
Sch em e 2. Route Leading to the Arginine-Linking
Chains
chain length
ester form
acid form
side product
n ) 2, m )0
n ) 5, m ) 0
n ) 5, m ) 1
15, 40%
16, 37%
17, 39%
18, 21%
19, 15%
20, 23%
21, 25%
22, 28%
23, 24%
ethidium-arginine conjugates compared with an al-
ready “active” compound. The results are given in Table
2.
Compounds 15, 16, 18, and 19, which all contain a
short spacer [NH(CH2)2CO for 15 and 18, NH(CH2)5CO
for 16 and 19], were devoid of anti-HIV-1 activity at
concentrations up to 100 µM (or at nontoxic concentra-
tions), in infected CEM-SS, MT4, and PBMC cell lines.
In sharp contrast, under the same conditions, com-
pounds 17 and 20 containing the longest spacer
[NH(CH2)5CONH(CH2)2CO] reduced viral multiplication
by 50% at a micromolar concentration without apparent
cellular toxicity up to 100 µM concentration, on cultures
of CEM-SS and PBMC cells acutely infected by HIV-1.
However, on cultures of MT4 cells the anti-HIV-1
activity of 17 was strongly diminished. On the basis of
the promising biological data obtained with 20, the most
active conjugate in the series, the interaction of this
drug with TAR was investigated using complementary
biochemical methods as well as molecular modeling.
Experiments were performed by Dr. Pierre Charneau,
from Institut Pasteur, France, concerning the intercala-
tion of compounds 17 and 20 with TAR. They have
shown that, first, these compounds interact with TAR
at the second stage of the cellular infection and, second,
they reach the inside of the cells.
catalytically hydrogenated to led to the third arginine-
terminated chain 11.
Next, the modified ethidium chloride 8 was joined to
each of the three arginine-linking chains via amide
linkages to yield, after further reactions, three target
inhibitor molecules containing two binding sites (15-
17). First, protected compounds 12-14 were prepared
by condensation of the carboxyl group of the modified
ethidium chloride 8 and the primary amino groups of
each of the three different arginine-terminated chains
9-11. The DCC/HOBt coupling method was used to
obtain compounds 12-14 in good yield (78-89%). Then,
the NG-2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc)
groups (protecting the guanidine side-chain function of
arginine) were removed using trifluoroacetic acid (TFA)
or 50% TFA in dichloromethane (DC) at room temper-
ature.56,57 Although the Pmc groups were cline removed,
the TFA/DC (1:1) reagent resulted in partial solvolysis
of the methyl carboxylate groups. Thus, in addition to
obtaining the three target inhibitor molecules 15-17,
the corresponding fully deprotected derivatives 18-20
were also obtained. In addition to these compounds, side
products with a trifluoroacetamido group at the 7
position of phenanthridine appeared (compounds 21-
23). HPLC was used to monitor the deprotection steps
to minimize the yield of side products. The three
products obtained during each deprotection reaction
were separated by semipreparative HPLC. Yields for the
deprotection reactions are given in Table 1. Molecules
15-20 were characterized using mass spectrometry
(electrospray method (ESI)), proton and carbon NMR
data, and infrared spectrometry. Purity of all com-
pounds was determined by HPLC.
Dyn a m ic Molecu la r Mod elin g Stu d ies. A molec-
ular modeling study was conducted to support the
design of this inhibitory family. The three-dimensional
structures of (i) Tat protein-TAR RNA complex, (ii) free
TAR RNA, and (iii) TAR RNA with an arginine residue
were used. The coordinates were obtained from the
Protein Data Bank.
The Tat protein-TAR RNA interaction site is located
between the C19 and A22 nucleotides, in the major
groove of the TAR RNA. Analysis of the structure of the
TAR RNA-arginine residue complex showed that argi-
nine interacted with the bases of the bulge region. This
interaction induced rotations of these bases which had
several effects. The first one was to fold the TAR RNA
structure, and the second one allowed the interaction
of arginine with the C29 and G36 bases. This new
interaction site is located deeply in the structure of TAR
RNA. This latest position obtained by molecular simula-
tion was in accordance with NMR data. Moreover,
analysis of the TAR RNA-ethidium 8 complex con-
firmed that ethidium 8 could insert between two pairs
of RNA nucleotides and that the most energetically
favorable intercalation was between GC and CG base
pairs. TAR RNA has two GC and CG base-pair sites.
The first one, G28-C37 and C29-G36, is located in the
high region of TAR, but this part of the structure is
deformed and obstructed by the free loop. Therefore,
ethidium intercalation was not possible at this site.
Differently, the second one, G43-C19 and G44-C18, is
located in a pocket formed by the major groove and
extended by the A17 externalized nucleotide.
Resu lts a n d Discu ssion
In Vitr o Biologica l Eva lu a tion . The anti-HIV-1
activity of compounds 15-20 and ethidium 8 (used as
a reference) was evaluated in infected CEM-SS, MT4,
and PBMC cells. Arginine and ethidium compounds
were included in the biological evaluation testing. As
arginine was devoid of activity, we have chosen to keep
ethidium as a reference. So, we have shown that an
increased viral inhibition was measured with some

4046 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Peytou et al.
Ta ble 2. Anti-HIV-1 Activity of Compounds 15-20 (ethidium 8 as a reference) on Infected and Uninfected CEM-SS, MT4, and
PBMC Cell Linesa
CEM-SS
IC5050 (µM)
MT4
PBMC
evaluated molecules
8, Eth-COOH (ref)
15, Eth (n ) 2, m ) 0), ArgOMe
16, Eth (n ) 5, m ) 0), ArgOMe
17, Eth (n ) 5, m ) 1), ArgOMe
18, Eth (n ) 2, m ) 0), ArgOH
19, Eth (n ) 5, m ) 0), ArgOH
20, Eth (n ) 5, m ) 1), ArgOH
CC50 (µM)
IC50 (µM)
CC50 (µM)
IC50 (µM)
CC50 (µM)
95
83
>100
>100
85
>100
>100
>100
>100 (38%)
>100
>100
11
42
21
20
78
89
8.7
73
44
47
>100
>100
>100
>100
>100
60
>100 (45%)
47
59
>100
>100
>100
>100
>100
>100
4.6
>100
>100
>100 (40%)
2.4
2.5
a
When the inhibition did not reach 50%, the measured value is indicated in parentheses.
Interaction between TAR RNA and compounds 15-
20 was then modeled. In the first phase, the arginine
residue drives the molecule to the TAR RNA-arginine
interaction site. Arginine interacted with the bulge
bases and induced a conformational change of the bulge.
The structure of TAR RNA folded up and the arginine
residue interacted with C29 and G36. At this stage of
the simulation, the tested molecule was roughly in a
linear conformation. In the second phase, the compound
folded up and the ethidium moiety interacted with the
RNA backbone. In the third phase, the ethidium part
slipped on the RNA ribbon and the molecule was
roughly parallel to the major groove. Analysis of con-
formers resulting from dynamic simulations in aqueous
phase showed that the major groove was wide enough
to accommodate the linking spacer insertion. Energetic
and geometric studies of the complex with the linking
spacer in the major groove showed that there was no
close contact; on the contrary, heteroatoms of the linking
spacer created stabilizing interactions with the RNA
ribbon. These interactions contributed to locking the
major groove. At this stage, the length of the linking
spacer was critical because if this one was too short,
the ethidium moiety could not reach the intercalation
site. Different simulations showed that the linking
spacer of molecules 15, 16, 18, and 19 was too short for
ethidium intercalation, while molecules 17 and 20 had
the optimal linking spacer length. Figure 1 shows the
final complex between the molecule 20 and the TAR
RNA as obtained from the insertion mechanism hypoth-
esis. Ethidium deformation was consistent with inter-
F igu r e 1. Molecular model of interaction between compound
20 and the TAR RNA of HIV-1. The arginine part is indicated
in blue, the linker in yellow, and the ethidium moiety in red.
The TAR RNA ribbon is represented as a large pink line.
calation simulations performed with a 4-base-pair RNA
duplex.57
Meltin g Tem p er a tu r e Stu d ies. We compared the
ability of ethidium 8 and compound 20 to alter the
thermal denaturation profile of TAR RNA. Representa-
tive melting curves for the free RNA and its complexes
with the two drugs are shown in Figure 2.
RNa se P r otection Exp er im en ts.58 The location of
the drug binding site on the TAR RNA was investigated
by RNase footprinting using three complementary en-
donucleases: (i) RNase A which preferentially cuts the
single-stranded bulge and loop sequences of TAR rather
than the two double-stranded stem regions of the RNA,
(ii) RNase T1 which cuts selectively at GpN in single-
stranded regions, and (iii) RNase V1 which cleaves RNA
predominantly at double-stranded regions. Typical cleav-
age patterns obtained with TAR cut by the three
enzymes in the absence and presence of 20 at concen-
trations ranging from 0.1 to 2 µM are shown in Figure
3. The alteration of the cleavage profiles are indicated
together with their locations on the TAR sequence.
The sites where the enzyme cleavage is modified in
the presence of the drug can be divided into two
groups: (1) the modification around the A17 unpaired
The helix-to-coil transition occurs at about 54 °C in
the absence of drugs. The Tm is shifted to higher
temperature with both compounds but the extent of
stabilization is markedly different. For a drug/RNA ratio
> 0.1, larger ∆Tm values (Tm complex - Tm RNA) were
measured with drug 20 and then with ethidium 8. For
example, at a drug/TAR ratio of 0.4, ∆Tm values of 11.5
and 7.5 °C were determined with 20 and 8, respectively.
The data strongly suggest that the presence of the
arginine side chain increases significantly the stability
of the drug-RNA complex. In other words, the cationic
tail seems to play the expected role, reinforcing the
interaction with the RNA target.

TAR Binding Ethidium-Arginine Conjugates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4047
is reduced in the presence of 20. A drug-induced
cleavage inhibition can also be detected at A27 and G36
with RNases V1 and T1, respectively. The strong
cleavage at the loop by RNase T1 remains unaffected
in the presence of the drug. These experiments suggest
that the small A bulge around position 17 and/or the
upper pyrimidine Tat bulge around position 40 can serve
as a binding site for the drug. Although we cannot
identify the exact position of the drug-bound molecules,
it is gratifying that the footprinting experiments are in
agreement with the modeling data presented above. The
model for which the ethidium moiety is inserted at the
dinucleotide step G43pG44‚C18pC19 and the arginine
extends into the pyrimidine bulge is fully consistent
with the experimental probing experiments. The reduc-
tion in cleavage intensity at G44 seen with RNase T1
may reflect the intercalation of the ethidium chro-
mophore, whereas the cleavage inhibition at the UC site
facing the bulge would account for the insertion of the
arginine moiety into the major groove cavity at the Tat
binding site.
Con clu sion
F igu r e 2. (A) Thermal denaturation curves for TAR in the
absence and presence of ethidium 8 and compound 20 at a
drug/RNA ratio of 0.4. The indicated Tm values (°C) were
obtained from first-derivative plots. (B) Variation of ∆Tm as a
function of the drug/RNA ratio.
In this study, we have shown that rationally designed
ethidium-arginine conjugates could act as efficient anti-
HIV-1 agents providing that the linker chain between
the two RNA-binding entities was sufficiently long. For
the most active molecule in the series (20), the distance
between the +N-CH3 of the ethidium moiety and the
guanidinium cationic end of arginine is about 19 Å.
Apparently, this much distance is required for the drug
to bind at both RNA-binding sites. Analogues of 20 are
now being synthesized.
residue and (2) the protected cleavage sites at or near
the pyrimidine bulge. The experiments with RNase T1
show that the drug-induced reduction in cleavage
intensity at G44 is accompanied by an enhanced cutting
at U46. The extents of enhanced cleavage and protection
are proportional to the drug concentration. In the same
region, cleavage at A20 by RNase V1 is markedly
increased when the drug is bound. The RNase A
cleavage at the UC site opposite the pyrimidine bulge
Exp er im en ta l Section
Gen er a l. Reactions were carried out under a nitrogen
atmosphere, unless otherwise indicated. All solvents were
F igu r e 3. RNase footprinting of compound 20 on TAR. The concentration (µM) of the drug is shown at the top of the appropriate
gel lanes. Control tracks labeled “Ct” contained no drug. The sites of cleavage protection and enhancement are indicated on the
gels and are reported on the sequence of TAR.

4048 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Peytou et al.
distilled and dried before use, using standard procedures.
Thin-layer chromatography (TLC) was run on precoated silica
gel plates (Merck 60F 254). Chromatography was carried out
on columns packed with silica gel (Merck 60, 230-400 mesh)
or on Amberlite XAD-2 resin (20-60 mesh). Melting points
were determined using a Bu¨chi capillary instrument and are
uncorrected. Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker AC 200 instrument, at 200 MHz. Mass
spectra were recorded by direct injection on a INCOS 500E
Finnigan MAT spectrometer (chemical ionization or electronic
impact, at 70 eV) or a TSQ 7000 Finnigan MAT spectrometer
(positive electrospray), via an atmospheric pressure ionization
source. Infrared spectra were recorded on a Bruker IFS 45
spectrometer (B) or on a Perkin Elmer Pargon 1000 FT-IR
spectrometer (PE). Samples were prepared in KBr disks.
Microanalyses were performed in the Vernaison-France Analy-
ses Laboratory (CNRS). HPLC analyses and purifications were
performed on a Merck L6200 (M) or on a Hewlett Packard
Series 1100 (HP) equipped with UV detectors (at 280 nm), or
on a Waters 600 (W) equipped with a diode array detector,
using a Lichrospher 100 RP-18 column as support.
d6) δ 166 (COOH), 149 (C-NO2), 148 (C-NO2), 144 (C-
COOH); IR (B) 3600-3000 (OH), 1616 (CO), 1515 (NO2), 1345
(NO2); MS m/z ) 390 (M + H+). Anal. (C20H11N3O6‚HCl)
Calcd: C 56.47, H 2.85, N 9.88. Found: C 56.75, H 2.93, N
9.40. HPLC (W) 280 nm, elution gradient CH3CN/H2O (20:80
to 50:50) for 30 min, tR ) 7.84 min.
9-(3-Met h oxyca r b on ylp h en yl)-2,7-d in it r o-10-m et h yl-
p h en a n th r id in e Hem isu lfa te (6). A solution of 5 (5.3 g,
13.67 mmol) in dimethyl sulfate (40 mL) was refluxed for 3 h.
After cooling, addition of diethyl ether (40 mL) resulted in the
formation of a dark yellow resin. The residual yellow solution
was filtered and the resin dissolved in hot ethanol and then
concentrated under reduced pressure to afford 6 as a dark
yellow hygroscopic oil which was used without further puri-
1
fication: TLC (Hex/AcOEt, 3:7) Rf ) 0.90; H NMR (DMSO-
d6) δ 9.60 (2H, m, H-3 and H-4), 9.43 (1H, bs, H-1), 8.98 (2H,
m, H-2 and H-5), 8.52 (1H, bs, H-4′), 8.46 (1H, d, H-3′, J ) 7.6
Hz), 8.25 (1H, bs, H-6), 8.19 (1H, d, H-1′, J ) 7.6 Hz), 8.07
(1H, t, H-2′, J ) 7.6 Hz), 4.50 (3H, s, CH3-N+), 3.92 (3H, bs,
COOMe).
9-(3-Ca r boxyp h en yl)-2,7-d in itr o-10-m eth ylp h en a n th r i-
d in e Ch lor id e (7). A mixture of 6 (6.3 g, 13.61 mmol) and 6
N HCl (100 mL) was refluxed for 4 h. After cooling, the reaction
mixture was concentrated under reduced pressure to afford a
brown material. This solid was dissolved in a minimum volume
of water and applied to an Amberlite XAD-2 resin column (300
g) which was then washed with H2O (4 L). Subsequent elution
with 50% aqueous methanol yielded orange fractions which
were concentrated and dried under reduced pressure to afford
7 as an orange resin (69% yield): TLC (Hex/AcOEt, 3:7) Rf )
0.65 (yellow); 1H NMR (DMSO-d6) δ 8.47 (1H, d, H-3, J ) 8.9
Hz), 8.44 (1H, d, H-4, J ) 7.9 Hz), 8.23 (1H, d, H-2, J ) 8.9
Hz), 8.01 (1H, bs, H-4′), 7.91 (1H, d, H-3′, J ) 7.5 Hz), 7.79
(1H, d, H-1, J ) 2.2 Hz), 7.73 (1H, d, H-5, J ) 7.9 Hz), 7.65
(2H, m, H-6 and H-1′), 7.54 (1H, t, H-2′, J ) 7.5 Hz), 2.83 (3H,
s, CH3-N+); 13C NMR (DMSO-d6) δ 166 (COOH), 149 (CdN+),
146 (C-NO2), 145 (C-NO2), 143 (C-COOH), 51 (CH3-N+); IR
(PE) 3600-2600 (OH), 1643 (CO), 1515 (NO2), 1341 (NO2); MS
m/z ) 404.0 (M+); HPLC (W) 280 nm, elution gradient CH3CN/
H2O (1% TFA) (20:80 to 50:50) for 30 min, tR ) 17.32 min.
1-(3-Cyan oben zam ido)-3,8-din itr obiph en yl (3). 1-Amino-
3,8-dinitrobiphenyl (1) (5 g, 19.66 mmol) and m-benzoyl
chloride (2) (3.74 g, 22.61 mmol) were refluxed in chloroben-
zene (50 mL) for 2 h. After cooling, the reaction mixture was
concentrated under reduced pressure and the residue was
triturated with chloroforme/diethyl ether/hexane to obtain a
white precipitate. After filtration, the product was recrystal-
lized from glacial acetic acid to give pure 3 (91% yield): TLC
(Hex/AcOEt, 1:1) Rf ) 0.46; mp ) 221-223 °C; 1H NMR
(DMSO-d6) δ 10.65 (1H, bs, NH), 8.48 (1H, d, H-1, J ) 2.3
Hz), 8.30 (2H, d, H-5 and H-6, J ) 8.8 Hz), 8.27 (1H, bs, H-4′),
8.25 (1H, dd, H-2, J ) 8.5 Hz, J ) 2.3 Hz), 8.09 (1H, d, H-3′,
J ) 8.4 Hz), 8.05 (1H, d, H-1′, J ) 8.4 Hz), 7.81 (2H, d, H-4
and H-7, J ) 8.8 Hz), 7.78 (1H, d, H-3, J ) 8.5 Hz), 7.72 (1H,
t, H-2′, J ) 8.4 Hz); 13C NMR (DMSO-d6) δ 163 (CO-NH),
147 (C-NO2), 146 (C-NO2), 111 (CN); IR (B) 3399 (NH), 2230
(CN), 1688 (CO), 1522 (NO2), 1345 (NO2); MS m/z ) 389 (M +
H+). Anal. (C20H12N4O5) Calcd: C 61.84, H 3.12, N 14.43.
Found: C 61.82, H 3.03, N 13.87. HPLC (W) 280 nm, elution
CH3CN/H2O (70:30), tR ) 5.00 min.
9-(3-Ca r b oxyp h en yl)-2,7-d ia m in o-10-m et h ylp h en a n -
th r id in e Ch lor id e (8). A solution of 7 (3.7 g, 8.41 mmol) in
1 N HCl was refluxed for 4 h. After cooling to ambient
temperature, H2O (19 mL), ethanol (106 mL), and Fe (4.25 g,
76.53 mmol) were added in the order specified with vigorous
stirring and the resulting mixture was refluxed for 4 additional
hours. The hot mixture was filtered through Celite, and the
filtrate was reduced under reduced pressure. The concentrated
solution was then applied to an Amberlite XAD-2 resin column
(200 g) which was then washed with H2O (2 L). Subsequent
elution with 50% aqueous methanol yielded red fractions which
were concentrated and dried under reduced pressure to afford
8 as a purple powder (55% yield): TLC (Hex/AcOEt, 3:7) Rf )
0.65 (red); mp ) 258-260 °C; 1H NMR (DMSO-d6) δ 8.58 (1H,
d, H-3, J ) 9.4 Hz), 8.53 (1H, d, H-4, J ) 9.2 Hz), 8.18 (1H, d,
H-3′, J ) 7.5 Hz), 8.02 (1H, bs, H-4′), 7.63 (1H, t, H-2′, J ) 7.5
Hz), 7.50 (2H,m, H-5 and H-1′), 7.30 (2H, m, H-1 and H-2),
6.58 (2H, bs, NH2R), 6.35 (1H, d, H-6, J ) 2.0 Hz), 5.99 (2H,
bs, NH2â), 3.94 (3H, s, CH3-N+); 13C NMR (DMSO-d6) δ 167
(COOH), 159 (CdN+), 150 (C-NH2), 147 (C-NH2), 141 (C-
COOH), 48 (CH3-N+); IR (PE) 3600-2600 (OH + 2NH2), 1643
(CO); MS m/z ) 344.1 (M+). Anal. (C21H18N3O2Cl) Calcd: C
66.47, H 4.79, N 11.08. Found: C 66.17, H 4.72, N 10.90. HPLC
(W) 288 nm, elution gradient CH3CN/H2O (1% TFA) (20:80 to
50:50) for 20 min, tR ) 11.48 min.
9-(3-Cyan oph en yl)-2,7-din itr oph en an th r idin e (4). 3 (6.63
g, 16.20 mmol) and POCl3 (2.08 mL, 22.68 mmol) in nitroben-
zene (40 mL) were refluxed for 1 h; then tin chloride (0.38 mL,
3.24 mmol) was added. After 2 h, the solution was cooled and
nitrobenzene was removed by distillation. The residue was
triturated with boiling anhydrous ethanol (120 mL) for 10 min.
After filtration, the solid product was washed again with hot
anhydrous ethanol to yield a beige powder 4 (98% yield): TLC
(Hex/AcOEt, 7:3) Rf ) 0.60; mp ) 302-304 °C; 1H NMR
(DMSO-d6) δ 9.38 (1H, d, H-4, J ) 8.9 Hz), 9.29 (1H, d, H-3,
J ) 9.1 Hz), 8.98 (1H, d, H-1, J ) 2.3 Hz), 8.95 (1H, dd, H-5,
J ) 8.9 Hz, J ) 2.2 Hz), 8.80 (1H, d, H-6, J ) 2.2 Hz), 8.64
(1H, dd, H-2, J ) 9.1 Hz, J ) 2.3 Hz), 8.40 (1H, bs, H-4′), 8.26
(1H, d, H-3′, J ) 7.7 Hz), 8.23 (1H, d, H-1′, J ) 7.7 Hz), 7.97
(1H, t, H-2′, J ) 7.7 Hz); 13C NMR (DMSO-d6) δ 149 (C-NO2),
148 (C-NO2), 113 (CN); IR (B) 2227 (CN), 1518 (NO2), 1343
(NO2); MS m/z ) 371 (M + H+). Anal. (C20H10N4O4) Calcd: C
64.85, H 2.72, N 15.14. Found: C 64.14, H 2.74, N 15.87. HPLC
(W) 280 nm, elution CH3CN/H2O (70:30), tR ) 8.08 min.
9-(3-Ca r boxyp h en yl)-2,7-d in itr op h en a n th r id in e (5). A
solution of 4 (6 g, 16.20 mmol) in 75% H2SO4 (25 mL) was
heated at 130 °C for 2 h. After cooling, the reaction mixture
was poured onto crushed ice, adjusted to pH 8.5 with NH4OH,
and then strongly acidified with concentrated HCl. The
precipitate was filtered and washed with water until neutral
pH. After drying, compound 5 was obtained as a beige-yellow
solid (88% yield): TLC (Hex/AcOEt, 3:7) Rf ) 0.30; mp ) 271-
N-r-(â-Ala n yl)-N-(2,2,5,7,8-p en ta m eth ylch r om a n -6-su l-
fon yl)a r gin in e Meth yl Ester (9). 25 (100 mg, 0.15 mmol)
and 10% Pd/C (16 mg, 0.15 mmol) were placed, at 0 °C, in
methanol. The reaction mixture was placed under hydrogen
flow for 3 h. After removal of Pd/C through Celite, methanol
was removed under reduced pressure and trituration with
diethyl ether afforded 9 as white crystals (99% yield). This
product was used without further purification: mp ) 130-
132 °C; 1H NMR (CD3OD) δ 4.25 (1H, m, CH), 3.55 (3H, s,
1
273 °C; H NMR (DMSO-d6) δ 9.21 (1H, d, H-4, J ) 9.1 Hz),
9.12 (1H, d, H-3, J ) 9.1 Hz), 8.80 (1H, d, H-1, J ) 2.2 Hz),
8.77 (1H, d, H-6, J ) 2.1 Hz), 8.70 (1H, dd, H-5, J ) 9.1 Hz,
J ) 2.1 Hz), 8.45 (1H, dd, H-2, J ) 9.1 Hz, J ) 2.2 Hz), 8.33
(1H, bs, H-4′), 8.19 (1H, d, H-3′, J ) 7.5 Hz), 7.79 (1H, d, H-1′,
J ) 7.5 Hz), 7.63 (1H, t, H-2′, J ) 7.5 Hz); 13C NMR (DMSO-

TAR Binding Ethidium-Arginine Conjugates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4049
OMe), 3.05 (2H, m, CH2-1), 2.80 (2H, m, CH2-5), 2.55 (2H, m,
CH2-2), 2.45 (6H, s, 2CH3(o)), 2.30 (2H, m, CH2â), 2.00 (3H, s,
CH3(m)), 1.75-1.45 (6H, m, CH2-3, CH2-4 and CH2R), 1.20 (6H,
m, 2CH3gem).
) 893.5 (M+); HPLC (HP) 288 nm, elution gradient CH3CN/
H2O (1‰ TFA) (20:80 to 100:0) for 30 min, tR ) 29.89 min.
9-[3-[[[[N-(2,2,5,7,8-P en ta m eth ylch r om a n -6-su lfon yl)-
ar gin in e m eth yl ester ]-â-alan yl]am in ocapr oyl]keto]ph en -
yl]-2,7-d ia m in o-10-m eth ylp h en a n th r id in e Ch lor id e (14).
This was prepared from 8 (60 mg, 0.16 mmol) and 11 (205
mg, 0.32 mmol) by a method entirely analogous to that for 12
above, and 14 was isolated as a purple resin (87% yield). This
product was used without further purification: 1H NMR
(DMSO-d6) δ 8.82 (1H, bs, NHa), 8.63 (2H, m, H-3 and H-4),
8.25 (2H, m, H-3′ and H-4′), 7.84 (3H, m, H-1′, H-2′ and H-5),
7.67 (1H, bs, NHb), 7.32 (3H, m, H-1, H-2 and NHc), 6.72 (3H,
m, NHd, NHe and NHf), 6.48 (2H, bs, NH2R), 6.34 (1H, s, H-6),
6.00 (2H, bs, NH2â), 4.16 (1H, m, CH), 3.98 (3H, s, CH3-N+),
3.52 (3H, s, OMe), 3.22 (4H, m, CH2-1 and CH2-4), 3.01 (2H,
m, CH2-8), 2.60-2.40 (10H, m, 2CH3(o), CH2-5 and CH2â), 2.28
(2H, m, CH2-3), 2.02 (3H, s, CH3(m)), 1.95-1.15 (18H, m, 3CH2-
2, CH2-6, CH2-7, CH2R and 2CH3gem); IR (PE) 3600-3000
(2NH2 + 6NH), 1642 (CO), 1625 (2CO), 1109 (COMe); MS m/z
) 964.5 (M+); HPLC (HP) 288 nm, elution gradient CH3CN/
H2O (1% TFA) (20:80 to 100:0) for 30 min, tR ) 29.45 min.
9-[3-[[(Ar gin in e m eth yl ester )-â-a la n yl]k eto]p h en yl]-
2,7-d ia m in o-10-m eth ylp h en a n th r id in e Ch lor id e (15)
a n d 9-[3-[(Ar gin yl-â-a la n yl)k eto]p h en yl]-2,7-d ia m in o-10-
m eth ylp h en a n th r id in e Ch lor id e (18). 12 (173 mg, 0.20
mmol) was added to 50% trifluoroacetic acid in dichlo-
romethane (DC) (4 mL). HPLC was used to monitor the process
and to stop the reaction as soon as the side product appeared
(about 1.5 h 30). The reaction mixture was concentrated under
reduced pressure, without heating, then repeatedly dissolved
in DC, and concentrated again several times. The product was
further purified by reverse-phase semipreparative HPLC
(CH3CN/H2O (1% TFA) 20:80, gradient 100:0 for 60 min).
Three compounds were isolated: the ester 15, the acid 18, and
the side product 21 in 40%, 21%, and 25% yields, respectively,
as purple resins for 15 and 21 and an orange resin for 18.
N-r-(Am in oca pr oyl)-N-(2,2,5,7,8-pen ta m eth ylch r om a n -
6-su lfon yl)a r gin in e Meth yl Ester (10). This was prepared
from 27 (70 mg, 0.10 mmol) by a method analogous to that for
9. Compound 10 was collected as white crystals (98% yield).
This product was used without further purification: mp )
135-137 °C; 1H NMR (CDCl3) δ 6.80 (2H, m, NH2a), 6.20 (3H,
m, NHc, NHd and NHe), 4.50 (1H, m, CH, m), 3.70 (3H, s,
OMe), 3.15 (4H, m, CH2-1 and CH2-6), 2.50 (10H, m, CH2-3,
2CH3(o) and CH2â), 2.30 (2H, m, CH2R), 2.05 (3H, s, CH3(m)),
1.60 (4H, m, CH2-4 and CH2-5), 1.50 (6H, m, 3CH2-2), 1.30
(6H, m, 2CH3gem).
N-r-[(Am in oca p r oyl)-â-a la n yl]-N-(2,2,5,7,8-p en ta m eth -
ylch r om a n -6-su lfon yl)a r gin in e Meth yl Ester (11). This
was prepared from 28 (586 mg, 0.75 mmol) by a method
analogous to that for 9, except the product was crystallized
from dichloromethane/diethyl ether. Compound 11 was iso-
lated as white crystals (96% yield). This product was used
without further purification: mp ) 126-128 °C; 1H NMR
(CDCl3) δ 6.80 (2H, m, NH2a), 6.20 (4H, m, NHc, NHd, NHe
and NHf), 4.50 (1H, m, CH), 3.70 (3H, s, OMe), 3.15 (4H, m,
CH2-1 and CH2-8), 3.05 (2H, m, CH2-4), 2.50 (12H, m, CH2-3,
CH2-5, 2CH3(o) and CH2â), 2.30 (2H, m, CH2R), 2.05 (3H, s,
CH3(m)), 1.60 (4H, m, CH2-6 and CH2-7), 1.50 (6H, m, 3CH2-
2), 1.30 (6H, m, 2CH3gem).
9-[3-[[[N-(2,2,5,7,8-P en tam eth ylch r om an -6-su lfon yl)ar gi-
n in e m eth yl ester ]-â-a la n yl]k eto]p h en yl]-2,7-d ia m in o-10-
m eth ylp h en a n th r id in e Ch lor id e (12). 8 (100 mg, 0.26
mmol), 1-hydroxybenzotriazole (35.5 mg, 0.26 mmol), and 9
(276.5 mg, 0.52 mmol) were dissolved in dimethylformamide
(DMF), at 0 °C. After 10 min, 1,3-dicyclohexylcarbodiimide
(54.3 mg, 0.26 mmol) was added and the reaction mixture was
stirred for 18 h. DMF was removed under reduced pressure,
and the residue was washed with 5% aqueous NH4OH solu-
tion, then with 1 N HCl aqueous solution, and finally with
ethyl acetate. There was a solid product left, which was
dissolved in methanol. This organic layer was dried on MgSO4
and then concentrated under reduced pressure. The product
was purified by passing through an Amberlite XAD-2 column
(50 g) with elution with a 5% methanol in water (500 mL)-
100% methanol gradient. Red fractions were concentrated
under reduced pressure to give 12 as a purple resin (89%
yield). This product was used without further purification: 1H
NMR (DMSO-d6) δ 8.99 (1H, bs, NHa), 8.66 (2H, m, H-3 and
H-4), 8.20 (2H, m, H-3′ and H-4′), 7.82 (3H, m, H-1′, H-2′ and
H-5), 7.34 (2H, m, H-1 and H-2), 7.20-6.90 (4H, m, NHb, NHc,
NHd and NHe), 6.54 (2H, bs, NH2R), 6.35 (1H, s, H-6), 6.04
(2H, bs, NH2â), 4.16 (1H, m, CH), 3.99 (3H, s, CH3 -N+), 3.50
(5H, m, OMe and CH2-1), 2.99 (2H, m, CH2-5), 2.60-2.45 (8H,
m, 2CH3(o) and CH2-2), 2.04 (3H, s, CH3(m)), 1.80-1.15 (14H,
m, CH2-3, CH2-4, CH2R, CH2â and 2CH3gem); IR (PE) 3600-
3000 (2NH2+5NH), 1643 (CO), 1627 (2CO), 1112 (COMe); MS
m/z ) 851.4 (M+); HPLC (HP) 288 nm, elution gradient
CH3CN/H2O (1% TFA) (20:80 to 100:0) for 30 min, tR ) 29.18
min.
9-[3-[[[N-(2,2,5,7,8-P en tam eth ylch r om an -6-su lfon yl)ar gi-
n in e m eth yl ester ]a m in oca p r oyl]k eto]p h en yl]-2,7-d ia m i-
n o-10-m eth ylp h en a n th r id in e Ch lor id e (13). This was
prepared from 8 (100 mg, 0.26 mmol) and 10 (295 mg, 0.52
mmol) by a method analogous to that for 12 above, and 13
was isolated as a purple resin (78% yield). This product was
used without further purification: 1H NMR (DMSO-d6) δ 8.82
(1H, bs, NHa), 8.64 (2H, m, H-3 and H-4), 8.22 (2H, m, H-3′
and H-4′), 7.83 (3H, m, H-1′, H-2′ and H-5), 7.55-7.15 (6H,
m, H-1, H-2, NHb, NHc, NHd and NHe), 7.00 (2H, bs, NH2R),
6.56 (2H, bs, NH2â), 6.35 (1H, s, H-6), 4.18 (1H, m, CH), 3.98
(3H, s, CH3-N+), 3.58 (3H, s, OMe), 3.16 (2H, m, CH2-1), 3.00
(2H, m, CH2-6), 2.60-2.45 (8H, m, 2CH3(o) and CH2-3), 2.12
(2H, m, CH2â), 2.02 (3H, s, CH3(m)), 1.80-1.00 (18H, m, 3CH2-
2, CH2-4, CH2-5, CH2R and 2CH3gem); IR (PE) 3600-3000
(2NH2 + 5NH), 1640 (CO), 1625 (2CO), 1109 (COMe); MS m/z
Com p ou n d 15: 1H NMR (D2O) δ 8.60 (2H, m, H-3 and H-4),
8.07 (1H, d, H-3′, J ) 7.6 Hz), 7.65 (4H, m, H-1′, H-2′, H-4′
and H-5), 7.35 (2H, m, H-1 and H-2), 6.72 (1H, s, H-6), 4.25
(1H, m, CH), 4.07 (3H, s, CH3-N+), 3.73 (3H, s, OMe), 3.48
(2H, m, CH2-1), 2.99 (2H, m, CH2-5), 2.55 (2H, m, CH2-2),
1.80-1.30 (4H, m, CH2-3 and CH2-4); IR (PE) 3600-3000
(3NH2 + 4NH), 1642 (CO), 1627 (2CO), 1112 (COMe); MS m/z
) 585.3 (M+). Anal. (C31H37N8O4Cl) Calcd: C 59.97, H 6.01, N
18.06. Found: C 60.35, H 6.08, N 17.79. HPLC (HP) 288 nm,
elution by gradient CH3CN/H2O (1% TFA) (20:80 to 100:0) for
30 min, tR ) 13.92 min.
Com p ou n d 18: 1H NMR (D2O) δ 8.59 (2H, m, H-3 and H-4),
8.07 (1H, d, H-3′, J ) 7.5 Hz), 7.78 (4H, m, H-1′, H-2′, H-4′
and H-5), 7.51 (2H, m, H-1 and H-2), 6.73 (1H, s, H-6), 4.28
(1H, m, CH), 4.14 (3H, s, CH3-N+), 3.66 (2H, m, CH2-1), 2.88
(2H, m, CH2-5), 2.61 (2H, m, CH2-2), 1.80-1.45 (4H, m, CH2-3
and CH2-4); IR (PE) 3600-2600 (OH + 3NH2 + 4NH), 1697
(CO), 1625 (2CO); MS m/z ) 571.3 (M+). Anal. (C30H35N8O4Cl)
Calcd: C 59.38, H 5.82, N 18.48. Found: C 59.21, H 5.78, N
18.79. HPLC (HP) 288 nm, elution by gradient CH3CN/H2O
(1% TFA) (20:80 to 100:0) for 30 min, tR ) 9.06 min.
Com p ou n d 21: 1H NMR (DMSO-d6) δ 9.00-7.00 (15H, m,
aromatic, NHa, NHb, NHc, NHd and NH2e), 6.80 (2H, bs,
H
NH2R), 6.50 (1H, s, H-6), 6.32 (1H, s, NHâ-COCF3), 4.26 (1H,
m, CH), 4.14 (3H, s, CH3-N+), 3.58 (3H, s, OMe), 3.48 (2H, m,
CH2-1), 3.09 (2H, m, CH2-5), 2.51 (2H, m, CH2-2), 1.80-1.35
(4H, m, CH2-3 and CH2-4); MS m/z ) 681.3 (M+). Anal.
(C33H36N8O5F3Cl) Calcd: C 55.29, H 5.07, N 15.64. Found: C
55.87, H 5.05, N 16.02. HPLC (HP) 288 nm, elution by gradient
CH3CN/H2O (1% TFA) (20:80 to 100:0) for 30 min, tR ) 25.91
min.
9-[3-[[(Ar gin in e m eth yl ester )am in ocapr oyl]keto]ph en -
yl]-2,7-d ia m in o-10-m et h ylp h en a n t h r id in e
Ch lor id e
(16) a n d 9-[3-[[(Ar gin in yl)a m in oca p r oyl]k eto]p h en yl]-
2,7-diam in o-10-m eth ylph en an th r idin e Ch lor ide (19). This
was prepared from 13 (195 mg, 0.21 mmol) by a method
analogous to that for 15 above, and the three compounds: the
ester 16, the acid 19, and the side product 22 were collected

4050 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Peytou et al.
in 37%, 15%, and 28% yields, respectively, as purple resins
for 16 and 22 and an orange resin for 19.
Calcd: C 58.92, H 5.96, N 15.87. Found: C 58.63, H 6.00, N
16.07. HPLC (HP) 288 nm, elution by gradient CH3CN/H2O
(1% TFA) (20:80 to 100:0) for 30 min, tR ) 27.63 min.
Com p ou n d 16: 1H NMR (D2O) δ 8.45 (2H, m, H-3 and H-4),
8.04 (1H, m, H-3′), 7.73 (3H, m, H-1′, H-2′ and H-4′), 7.37 (3H,
m, H-1, H-2 and H-5), 6.54 (1H, s, H-6), 4.05 (4H, m, CH and
CH3 -N+), 3.59 (3H, s, OMe), 3.30 (2H, m, CH2-1), 3.02 (2H,
m, CH2-6), 2.55 (2H, m, CH2-3), 1.85-1.20 (10H, m, 3CH2-2,
CH2-4 and CH2-5); IR (PE) 3600-3000 (3NH2 + 4NH), 1641
(CO), 1627 (2CO), 1110 (COMe); MS m/z ) 627.3 (M+). Anal.
(C34H43N8O4Cl) Calcd: C 61.60, H 6.54, N 16.91. Found: C
61.43, H 6.49, N 16.93. HPLC (HP) 288 nm, elution by gradient
CH3CN/H2O (1% TFA) (20:80 to 100:0) for 30 min, tR ) 17.66
min.
N-r-[N-Ben zyloxyca r bon yl)-â-a la n yl]-N-(2,2,5,7,8-p en -
ta m eth ylch r om a n -6-su lfon yl)a r gin in e (24). N-(Benzyloxy-
carbonyl)-â-alanine (460 mg, 2.06 mmol) and N-hydroxysuc-
cinimide (335 mg, 3.09 mmol) were dissolved in DMF with
stirring, at 0 °C. After 15 min, 1,3-dicyclohexylcarbodiimide
(425 mg, 2.06 mmol) was added. The reaction mixture was
stirred at room temperature for 12 h; then, at 0 °C, H-
Arg(Pmc)OH (1 g, 2.27 mmol) was added. The solution was
stirred for 12 additional hours, at room temperature, and DMF
was removed under reduced pressure. The residue was dis-
solved in 10% NaHCO3 aqueous solution and then washed with
ethyl acetate. The basic aqueous solution was acidified with
citric acid (to pH 3), and the product was extracted with ethyl
acetate. Organic layers were pooled, washed with water, then
dried on MgSO4, and concentrated under reduced pressure.
Compound 24 was isolated as a colorless resin (92% yield).
This product was used without further purification: TLC
(AcOEt/MeOH, 7:3) Rf ) 0.30; 1H NMR (CDCl3) δ 7.25 (5H,
Ph(Z), s), 6.80 (1H, d, NHb, J ) 2.0 Hz), 6.20 (3H, bs, NHc,
NHd and NHe), 5.50 (1H, bs, NHa), 5.00 (2H, s, CH2(Z)), 4.45
(1H, m, CH), 3.35 (2H, m, CH2-1), 3.10 (2H, m, CH2-5), 2.60-
2.45 (8H, m, 2CH3(o) and CH2-2), 2.35 (2H, m, CH2â), 2.00 (3H,
s, CH3(m)), 1.80-1.35 (6H, m, CH2-3, CH2-4 and CH2R), 1.25
(6H, m, 2CH3gem).
N-r-[N-(Ben zyloxyca r bon yl)-â-a la n yl]-N-(2,2,5,7,8-p en -
ta m eth ylch r om a n -6-su lfon yl)a r gin in e Meth yl Ester (25).
24 (1.38 g, 2.14 mmol) and cesium carbonate (697 mg, 2.14
mmol) were dissolved in DMF (40 mL). After stirring at room
temperature for 10 min, methyl iodide (0.15 mL, 2.35 mmol)
was added and stirring was continued for 12 additional hours.
DMF was removed under reduced pressure, the residue was
dissolved in 10% Na2CO3 aqueous solution, and the product
was extracted with ethyl acetate. Organic layers were pooled,
washed with NaCl aqueous solution, then dried on MgSO4,
and concentrated under reduced pressure. The product was
purified by chromatography on a silica gel column, with elution
by 100% ethyl acetate. After trituration with anhydrous
diethyl ether, white crystals of 25 were obtained (56% yield).
This product was used without further purification: TLC
(AcOEt, 100%) Rf ) 0.22; mp ) 175-177 °C; 1H NMR (CDCl3)
δ 7.25 (5H, s, Ph(Z)), 6.80 (1H, d, NHb, J ) 2.0 Hz), 6.20 (3H,
bs, NHc, NHd, NHe), 5.50 (1H, bs, NHa), 5.00 (2H, s, CH2(Z)),
4.45 (1H, m, CH), 3.65 (3H, s, OMe), 3.35 (2H, m, CH2-1), 3.10
(2H, m, CH2-5), 2.60-2.45 (8H, m, 2CH3(o) and CH2-2), 2.35
(2H, m, CH2â), 2.00 (3H, s, CH3(m)), 1.80-1.35 (6H, m, CH2-
3, CH2-4 and CH2R), 1.25 (6H, m, 2CH3gem).
Com p ou n d 19: 1H NMR (D2O) δ 8.46 (2H, m, H-3 and H-4),
8.04 (1H, m, H-3′), 7.73 (3H, m, H-1′, H-2′ and H-4′), 7.38 (3H,
m, H-1, H-2 and H-5), 6.45 (1H, s, H-6), 4.05 (4H, m, CH and
CH3-N+), 3.30 (2H, m, CH2-1), 3.01 (2H, m, CH2-6), 2.30 (2H,
m, CH2-3), 1.90-1.40 (10H, m, 3CH2-2, CH2-4 and CH2-5); IR
(PE) 3600-2600 (OH + 3NH2 + 4NH), 1700 (CO), 1627 (2CO);
MS m/z ) 613.3 (M+). Anal. (C33H41N8O4Cl) Calcd: C 61.08,
H 6.32, N 17.27. Found: C 61.32, H 6.39, N 17.58. HPLC (HP)
288 nm, elution by gradient CH3CN/H2O (1% TFA) (20:80 to
100:0) for 30 min, tR ) 12.79 min.
Com p ou n d 22: 1H NMR (DMSO-d6) δ 9.00-7.00 (15H, m,
aromatic, NHa, NHb, NHc, NHd and NH2e), 6.77 (2H, bs,
H
NH2R), 6.43 (1H, s, H-6), 6.32 (1H, s, NHâ-COCF3), 4.16 (4H,
m, CH and CH3-N+), 3.60 (3H, s; OMe), 3.28 (2H, m, CH2-1),
3.02 (2H, m, CH2-6), 2.30 (2H, m, CH2-3), 1.85-1.25 (10H, m,
3CH2-2, CH2-4 and CH2-5); MS m/z ) 723.6 (M+). Anal.
(C36H42N8O5F3Cl) Calcd: C 59.72, H 5.85, N 15.49. Found: C
60.08, H 5.89, N 15.77. HPLC (HP) 288 nm, elution by gradient
CH3CN/H2O (1% TFA) (20:80 to 100:0) for 30 min, tR ) 28.83
min.
9-[3-[[[(Ar gin in e m eth yl ester )-â-alan yl]am in ocapr oyl]-
keto]ph en yl]-2,7-diam in o-10-m eth ylph en an th r idin e Ch lo-
r id e (17) a n d 9-[3-[[(Ar gin in yl-â-a la n yl)a m in oca p r oyl]-
k e t o]p h e n yl]-2,7-d ia m in o-10-m e t h ylp h e n a n t h r id in e
Ch lor id e (20). This was prepared from 14 (120 mg, 0.12
mmol) by a method analogous to that for 15 above, and the
three pure compounds: the ester 17, the acid 20, and the side
product 23 were isolated in 39%, 23%, and 24% yields,
respectively, as purple resins for 17 and 23 and an orange resin
for 20.
Com p ou n d 17: 1H NMR (D2O) δ 8.39 (2H, m, H-3 and H-4),
8.04 (1H, d, H-3′, J ) 7.6 Hz), 7.77 (2H, m, H-2′ and H-4′),
7.64 (1H, m, H-1′), 7.37 (3H, m, H-1, H-2 and H-5), 6.55 (1H,
s, H-6), 4.18 (1H, m, CH), 4.05 (3H, m, CH3-N+), 3.65 (5H, m,
OMe and CH2-1), 3.30 (2H, m, CH2-4), 3.14 (2H, m, CH2-8),
2.34 (2H, m, CH2-5), 2.18 (2H, m, CH2-3), 1.90-1.35 (10H, m,
3CH2-2, CH2-6 and CH2-7); IR (PE) 3600-3000 (3NH2 + 5NH),
1642 (CO), 1628 (3CO), 1109 (COMe); MS m/z ) 698.4 (M+).
Anal. (C37H48N9O5Cl) Calcd: C 60.54, H 6.60, N 17.19.
Found: C 60.32, H 6.65, N 17.51. HPLC (HP) 288 nm, elution
by gradient CH3CN/H2O (1% TFA) (20:80 to 100:0) for 30 min,
tR ) 16.98 min.
N-r-[N-(Ben zyloxycar bon yl)am in ocapr oyl]-N-(2,2,5,7,8-
p en ta m eth ylch r om a n -6-su lfon yl)a r gin in e (26). This was
prepared from N-(benzyloxycarbonyl)aminocaproic acid (2 g,
7.54 mmol) by a method analogous to that for 24, and
compound 26 was isolated as a white resin (70% yield). This
product was used without further purification: TLC (AcOEt/
MeOH, 7:3) Rf ) 0.22; 1H NMR (CDCl3) δ 7.30 (5H, s, Ph(Z)),
6.20 (1H, d, NHb, J ) 2.0 Hz), 6.00 (3H, bs, NHc, NHd and
NHe), 5.0 (3H, m, NHa and CH2(Z)), 4.50 (1H, m, CH), 3.15
(4H, m, CH2-1 and CH2-6), 2.50 (10H, m, CH2-3, 2CH3(o) and
CH2â), 2.30 (2H, m, CH2R), 2.05 (3H, s, CH3(m)), 1.70 (6H, m,
3CH2-2), 1.60 (4H, m, CH2-4 and CH2-5), 1.30 (6H, m,
2CH3gem).
Com p ou n d 20: 1H NMR (D2O) δ 8.38 (2H, m, H-3 and H-4),
8.04 (1H, d, H-3′, J ) 7.6 Hz), 7.78 (2H, m, H-2′ and H-4′),
7.64 (1H, m, H-1′), 7.37 (3H, m, H-1, H-2 and H-5), 6.52 (1H,
s, H-6), 4.18 (1H, m, CH), 4.05 (3H, m, CH3-N+), 3.65 (5H, m,
OMe and CH2-1), 3.30 (2H, m, CH2-4), 3.14 (2H, m, CH2-8),
2.34 (2H, m, CH2-5), 2.18 (2H, m, CH2-3), 1.90-1.35 (10H, m,
3CH2-2, CH2-6 and CH2-7); IR (PE) 3600-2600 (OH + 3NH2
+ 4NH), 1700 (CO), 1627 (3CO); MS m/z ) 684.4 (M+). Anal.
(C36H46N9O5Cl) Calcd: C 60.06, H 6.44, N 17.52. Found: C
60.24, H 6.39, N 17.27. HPLC (HP) 288 nm, elution by gradient
CH3CN/H2O (1% TFA) (20:80 to 100:0) for 30 min, tR ) 13.36
min.
N-r-[N-(Ben zyloxycar bon yl)am in ocapr oyl]-N-(2,2,5,7,8-
p en ta m eth ylch r om a n -6-su lfon yl)a r gin in e Meth yl Ester
(27). This was prepared from 26 (345 mg, 0.50 mmol) by a
method analogous to that for 25, except the chromatography
column eluant was now 10% methanol in ethyl acetate.
Compound 27 was obtained as white crystals (55% yield). This
product was used without further purification: TLC (AcOEt,
Com p ou n d 23: 1H NMR (DMSO-d6) δ 9.00-7.00 (16H, m,
aromatic, NHa, NHb, NHc, NHd, NHe and NH2f), 6.75 (2H, bs,
H
1
NH2R), 6.42 (1H, s, H-6), 6.35 (1H, s, NHâ-COCF3), 4.16 (1H,
m, CH), 4.05 (3H, m, CH3-N+), 3.65 (5H, m, OMe and CH2-1),
3.30 (2H, m, CH2-4), 3.14 (2H, m, CH2-8), 2.34 (2H, m, CH2-
5), 2.18 (2H, m, CH2-3), 1.90-1.30 (10H, m, 3CH2-2, CH2-6
and CH2-7); MS m/z ) 794.5 (M+). Anal. (C39H47N9O6F3Cl)
100%) Rf ) 0.42; mp ) 140-142 °C; H NMR (CDCl3) δ 7.30
(5H, s, Ph(Z)), 6.20 (1H, d, NHb, J ) 2.0 Hz), 6.00 (3H, bs,
NHc, NHd and NHe), 5.0 (3H, m, NHa and CH2(Z)), 4.50 (1H,
m, CH), 3.70 (3H, s, OMe), 3.15 (4H, m, CH2-1 and CH2-6),
2.50 (10H, m, CH2-3, 2CH3(o) and CH2â), 2.30 (2H, m, CH2R),

TAR Binding Ethidium-Arginine Conjugates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4051
2.05 (3H, s, CH3(m)), 1.70 (6H, m, 3CH2-2), 1.60 (4H, m, CH2-4
and CH2-5), 1.30 (6H, m, 2CH3gem).
cells were cultured in the presence of different concentrations
of tested compounds for 7 days, after virus incubation in cell
cultures. The production of virus HIV-1 was measured by
quantification of the reverse transcriptase activity in the
culture supernatant. Cytotoxicity of the drugs was measured
after incubation for 7 days in their presence using the
colorimetric MTT test. When the inhibition did not reach 50%,
the measured value is indicated in parentheses.
N-r-[[N-(Ben zyloxyca r bon yl)a m in oca p r oyl]-â-a la n yl]-
N-(2,2,5,7,8-p en t a m et h ylch r om a n -6-su lfon yl)a r gin in e
Meth yl Ester (28). N-(Benzyloxycarbonyl)aminocaproic acid
(288 mg, 1.09 mmol), 1-hydroxybenzotriazole (142 mg, 1.31
mmol), 9 (572 mg, 1.09 mmol), and 1,3-dicyclohexylcarbodi-
imide (225 mg, 1.09 mmol) were dissolved in DMF (30 mL),
at 0 °C. The reaction mixture was stirred, at room tempera-
ture, for 18 h. The DCU precipitate was filtered and DMF
removed under reduced pressure. The residue was dissolved
in ethyl acetate. Organic layer was washed with 10% NaHCO3
aqueous solution, then with 10% citric acid aqueous solution,
and with water, finally dried on MgSO4, and concentrated
under reduced pressure. The product was purified by chro-
matography on a silica gel column, with elution by 10%
methanol in ethyl acetate, and compound 28 was isolated as
a colorless oil (70% yield). This product was used without
Dyn a m ic Molecu la r Mod elin g St u d ies. The three-
dimensional structures of (i) Tat protein-TAR RNA complex,
(ii) free TAR RNA, and (iii) TAR RNA with an arginine residue
were found in the Protein Data Bank. All molecular modeling
studies except the arginine-TAR RNA approach were per-
formed using the Insight II molecular modeling package.
Energy was computed with Cff91 as force field, and minimiza-
tion was performed using the conjugate gradient algorithm
with 9000 iteration steps or until convergence (defined as an
energy gradient of 0.0001 kcal/mol or less). The geometric
constraints were created with the specific command “gener-
icdist” of the discover module. The annealing was defined by
50 cycles of 3000 minimization steps followed by 5000 steps
of molecular dynamic with a temperature of 300 K. For the
arginine-TAR RNA approach, the Sybyl package was used.
Interaction energy was calculated by the MM2 force field and
minimization performed using the conjugate gradient algo-
rithm with 5000 iteration steps. Visualization of different
interactions between arginine and TAR RNA was made with
the Sybyl program and all others with the Insight II program.
All calculations were done on a IRIS indigo 2 R10.000 of the
Centre de Mode´lisation et d′Imagerie Mole´culaire of UNSA.
In Vitr o Tr a n scr ip tion . Synthetic oligonucleotides corre-
sponding to the wild-type or the bulge-deleted (∆U) TAR
sequences were cloned between HindIII and EcoRI sites of the
pUC19 plasmid. After digestion with EcoRI, the RNA was
transcribed as a runoff product of 60 nucleotides from the T3
RNA polymerase promoter. In each case the transcript in-
cluded an additional G residue on the 3′-end derived from the
EcoRI cleavage site. Transcription reaction was performed in
buffer containing 40 mM Tris-HCl, pH 7.4, 25 mM NaCl, 16
mM MgCl2, 10 mM DTT, and 1 mM NTPs. The reaction was
initiated by addition of 10 µg linearized plasmid DNA template
and 40 units T3 RNA polymerase and incubated for 2 h at 37
°C. Nucleic acids were then fractionated on a 10% (w/v)
polyacrylamide gel containing 8 M urea in TBE buffer (89 mM
Tris-borate, pH 8.3, 10 mM EDTA). After electrophoresis, the
RNA was eluted in water for 18 h at 4 °C and precipitated
with ethanol. The RNA was resuspended in water to give a
500 µM stock solution (ꢀ260/phosphate ) 10 688 M-1 cm-1). For
the footprinting experiments, the RNA was 3′-end labeled with
[32P]cytidine biphosphate and T4 RNA ligase and then purified
again from a 10% denaturing acrylamide gel. RNA solutions
were prepared with doubly distilled sterile water to prevent
from nuclease contamination. Tubes and tips were treated with
1% diethyl pyrocarbonate (DEPC from Sigma).
1
further purification: TLC (AcOEt, 100%) Rf ) 0.21; H NMR
(CDCl3) δ 7.30 (5H, s, Ph(Z)), 6.20 (2H, m, NHb and NHc),
6.00 (3H, bs, NHd, NHe and NHf), 5.0 (3H, m, NHa and
CH2(Z)), 4.50 (1H, m, CH), 3.70 (3H, s, OMe), 3.35 (2H, m,
CH2-8), 3.15 (4H, m, CH2-1 and CH2-4), 2.50 (12H, m, CH2-3,
CH2-5, 2CH3(o) and CH2â), 2.30 (2H, m, CH2R), 2.05 (3H, s,
CH3(m)), 1.60 (4H, m, CH2-6 and CH2-7), 1.50 (6H, m, 3CH2-
2), 1.30 (6H, m, 2CH3gem).
Cells a n d Vir u ses. CEM-SS and MT4 cells were cultured
in RPMI medium supplemented with 10% fetal calf serum
(heated at 56 °C for 30 min). CEM-SS cells were obtained from
Peter Nara through AIDS Research and Reference Reagent
Program, Division of AIDS, NIAd, NIH. HIV-1 LAI and HIV
IIIB were produced in CEM-SS cells as previously reported.59
An ti-HIV-1 Activity a n d Cytotoxicity Mea su r em en ts
in Vitr o on CEM-SS In fected a n d Un in fected Cells. The
effect of the compounds on the replication of HIV-1 LAI in
acutely infected CEM-SS cells was evaluated using a procedure
previously described59 with slight modifications. The rate of
virus production was measured by quantification of the reverse
transcriptase activity associated with the virus particles
released in the culture supernatant. Briefly, cells were infected
with TCID50; after a 30-min adsorption period, unbound
particles were removed and cells were cultured in the presence
of different concentrations of tested compounds for 5 days
before determining rates of virus production. Three measure-
ments were carried out for each concentration of each com-
pound. The 50% inhibitory concentration of virus replication
(IC50) was derived from the computer-generated median plot
of the dose-effect data.60 The cytotoxicity of the molecules for
uninfected cells was measured after an incubation of 5 days
in the presence of various drug concentrations, using a
colorimetric assay (MTT test).61 Again, three measurements
were carried out for each concentration of each compound. The
50% cytotoxicity concentration (CC50) at which OD540 was
reduced by one-half was calculated using the program men-
tioned above. The entire experiment was repeated twice.
An ti-HIV-1 Activity a n d Cytotoxicity Mea su r em en ts
in Vitr o on MT4 In fected a n d Un in fected Cells. The anti-
HIV-1 test based on MT4 infected cells was previously
described.62,63 Measurements were performed in triplicate for
each concentration of each compound, and the entire experi-
ment was repeated twice. Briefly, in MT4 cells, the determi-
nation of the antiviral activity of the tested compounds was
based on a reduction of HIV-1-induced pathogenicity, the
metabolic activity of the cells being measured by the ability
of mitochondrial dehydrogenases to reduce 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan.
When the inhibition did not reach 50%, the measured value
is indicated in parentheses.
Meltin g Tem p er a tu r e Stu d ies. Absorption spectra were
recorded on a Uvikon 943 spectrophotometer. The 12-cell
holder was thermostated with a Neslab RTE 111 cryostat.
Drug-RNA complexes were prepared by adding aliquots of a
concentrated CGP40336A solution to a RNA solution at
constant concentration (usually 20 µM) in BPE buffer, pH 7.1
(6 mM Na2HPO4, 2 mM Na2H2PO4, 1 mM EDTA). A heating
rate of 1 °C/min was used, and data points were collected every
30 s. The temperature inside the cuvette was monitored by
using a thermocouple. The absorbency at 260 nm was mea-
sured over the range 25-90 °C in 1-cm path length reduced
volume quartz cells. The “melting” temperature Tm was taken
as the midpoint of the hyperchromic transition determined
from first-derivative plots. The reproducibility of the Tm
measurements was (0.5 °C.
An ti-HIV-1 Activity a n d Cytotoxicity Mea su r em en ts
in Vitr o on P BMC In fected a n d Un in fected Cells. The
techniques used for measuring inhibition of virus multiplica-
tion were previously described.59-61 Measurements were per-
formed in triplicate for each concentration of each compound,
and the entire experiment was repeated twice. Briefly, PBMC
RNa se F ootp r in tin g. Samples of the labeled RNA frag-
ment were incubated with a buffered solution containing the
desired drug concentration. After 20-min incubation at 4 °C
to ensure equilibration, the digestion was initiated by addition
of the RNase (A, T1, or V1) solution. The RNases (Pharmacia

4052 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Peytou et al.
(16) Wang, Z. Y.; Rana, T. M. RNA conformation in the Tat-TAR
complex determined by site-specific photocross-linking. Biochem-
istry 1996, 35, 6491-6499.
(17) Aboul-Ela, F.; Karn, J .; Varani, G. The structure of the human
immunodeficiency virus type-1 TAR RNA reveals principles of
RNA recognition by Tat protein. J . Mol. Biol. 1995, 253, 313-
332.
(18) Sumner-Smith, M.; Roy, S.; Barnett, R.; Reid, L. S.; Kuperman,
R.; Delling, U.; Sonenberg, N. Critical chemical features in trans-
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(19) Pritchard, C. E.; Grasby, J . A.; Hamy, F.; Zacharek, A. M.; Singh,
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Biotech, Saclay, France) were used according to the supplier’s
recommended protocol in the activity buffer provided. After
1-min incubation at room temperature, the reaction was
stopped by freeze-drying and samples were lyophilized. The
RNA in each tube was resuspended in 5 µL of formamide-TBE
loading buffer, denatured at 90 °C for 4 min, and then chilled
in ice for 4 min prior to loading on to a 0.3-mm thick, 10%
polyacrylamide gel containing 8 M urea and TBE buffer (89
mM Tris base, 89 mM boric acid, 2.5 mM Na2EDTA, pH 8.3).
After 2-h electrophoresis at 1500 V, the gel was soaked in 10%
acetic acid for 10 min, transferred to Whatman 3MM paper,
dried under vacuum at 80 °C, and then analyzed on the
Phosphorimager (Molecular Dynamics). Each resolved band
on the autoradiograph was assigned to a particular bond
within the RNA fragment by comparison of its position relative
to sequencing standards generated by treatment of the RNA
with diethyl pyrocarbonate followed by aniline-induced cleav-
age at the modified bases (A track).
(20) Hamy, F.; Asseline, U.; Grasby, J .; Iwai, S.; Pritchard, C.; Slim,
G.; Butler, P. J . G.; Karn, J .; Gait, M. J . Hydrogen-bonding
contacts in the major groove are required for human immuno-
deficiency virus type-1 Tat protein recognition of TAR RNA. J .
Mol. Biol. 1993, 111-123.
(21) Calnan, B. J .; Tidor, B.; Biancalana, S.; Hudson, D.; Frankel,
A. D. Arginine-mediated RNA recognition: the arginine fork.
Science 1991, 1167-1171.
(22) Delling, U.; Roy, S.; Sumner-Smith, M.; Barnett, R.; Reid, L.;
Rosen, C. A.; Sonenberg, N. The number of positively charged
amino acids in the basic domain of Tat is critical for trans-
activation and complex formation with TAR RNA. Proc. Natl.
Acad. Sci. U.S.A. 1991, 6234-6238.
(23) Tao, J .; Chen, L.; Frankel, A. D. Dissection of the proposed base
triple in HIV TAR RNA indicates the importance of the Hoogs-
teen interaction. Biochemistry 1997, 3491-3495.
(24) Puglisi, J . D.; Tan, R.; Calnan, B. J .; Frankel, A. D.; Williamson,
J . R. Conformation of the TAR-arginine complex by NMR.
Science 1992, 76-80.
Ack n ow led gm en t. We are grateful to J . M. Guigo-
nis for doing the mass spectrometry analyses. We
express our warmest thanks to Dr. R. A. Earl for reading
the manuscript and for helpful suggestions. Thanks are
also due to Dr. T. T. Tran for helpful discussions and
suggestions. This work was supported by grants from
ANRS (Agence Nationale de Recherches sur le SIDA)
and SIDACTION (Fondation pour la Recherche Me´di-
cale).
(25) Puglisi, J . D.; Chen, L.; Frankel, A. D.; Williamson, J . R. Role
of RNA structure in arginine recognition of TAR RNA. Proc.
Natl. Acad. Sci. U.S.A. 1993, 3680-3684.
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