Synthesis and biological evaluation of novel β-carboline derivatives as Tat-TAR interaction inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.04.022

Four new β-carboline derivatives were synthesized bearing guanidinium group or amino group-terminated side chain targeting the TAR element. Compounds 5 and 6 with terminal guanidinium group showed inhibitory activities on Tat-TAR interaction as well as to HIV-1 in MT4 cells. Furthermore, capillary electrophoresis assay implied that compound 6 could not only bind to TAR but also hinder the Tat-TAR interaction.

Full text of DOI:10.1016/j.bmcl.2004.04.022

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3127–3130
Synthesis and biological evaluation of novel b-carboline derivatives
as Tat–TAR interaction inhibitors
a
Xiaolin Yu, Wei Lin, Jingyun Li and Ming Yang
a
b
a,
*
a
National Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, China
b
HIV/AIDS Laboratory, Institute of Military Medical Sciences, Beijing 100850, China
Received 2 March 2004; accepted 8 April 2004
Abstract—Four new b-carboline derivatives were synthesized bearing guanidinium group or amino group-terminated side chain
targeting the TAR element. Compounds 5 and 6 with terminal guanidinium group showed inhibitory activities on Tat–TAR
interaction as well as to HIV-1 in MT4 cells. Furthermore, capillary electrophoresis assay implied that compound 6 could not only
bind to TAR but also hinder the Tat–TAR interaction.
Ó 2004 Elsevier Ltd. All rights reserved.
HIV-1 gene expression is regulated through a complex
interplay of specific cis-acting elements within its long
terminal repeat (LTR) with host cell proteins or factors
as well as with its own accessory proteins. One of these
interactions is that HIV-1 regulatory protein Tat
stimulates transcriptional elongation by binding the
trans-activation response region (TAR), a 59-nucleotide
nine-rich motif (ARM), is responsible for Tat–TAR
5
specific interaction. Studies have shown that arginine
52 is largely responsible for the binding specificity and
thus for the conformational change in TAR RNA in
such a way that guanidinium group interacts with the
6
three-base bulge region of TAR RNA. Furthermore,
the observation that a single arginine residue (or argi-
nine amide) binds specifically to TAR and induces a
change in RNAconformation that largely mimics the
conformation of a portion of Tat–TAR complex sug-
gests a strategy that introducing additional functional-
ities around the arginine guanidinium group could
enhance the affinity and the specificity of drug–TAR
0
stem-loop found at the 5 untranslated end of all newly
1
transcribed HIV mRNAs. In the absence of Tat, tran-
scription does occur but yields only short polyadenyl-
2
ated transcripts. This interaction between TAR RNA
and Tat is essential for virus replication. Therefore,
blocking the Tat–TAR complex formation seems to be a
promising target for inhibiting virus multiplication.
7
interaction. And also many experiments provided evi-
dences that the high-affinity binding of small molecules
to the RNAtargets is governed by the electrostatic
interaction due to amino group, guanidinium group, or
Intensive research over the last decade on the transac-
tivation mechanism involving Tat–TAR interaction has
yielded significant biological and virological insights.
TAR RNA forms a stable hairpin structure with six
apical nucleotides (CUGGGA) and two stem regions,
upper and lower, separated by a bulge made of three
8
arginine residue. Our previous studies have indicated
that b-carboline derivatives could interact with nucleic
acids for their fused aromatic heterocycle ring struc-
tures, and that the optimized linker length and some
electronic substituents on the b-carboline ring are
3
unpaired nucleotides (UCU). Tat binds to this region of
the three-base bulge and recognizes both the identity
of adjacent Watson–Crick base pairs and the positions
9
important in the binding of nucleic acids.
4
of surrounding phosphate groups. HIV-1 Tat is a
protein with 86 amino acid residues, which includes a
basic region (amino acid residues 48–59), termed argi-
Based on the above idea, four new b-carboline deriva-
tives were designed bearing guanidinium group or
amino group-terminated side chain targeting the TAR
element. In order to probe whether the four compounds
could block the Tat-mediated transactivation, a tran-
sient co-transfection bioassay was performed by using a
reporter plasmid construct expressing the chloram-
phenicol acetyltransferase (CAT) under the control of
Keywords: Tat–TAR interaction; b-Carboline; Anti-HIV activity.
*
Corresponding author. Tel.: +86-10-82801569; fax: +86-10-828020-
2; e-mail: mingy@mail.bjmu.edu.cn
6
0
960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.022

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X. Yu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3127–3130
the HIV-1 LTR and a Tat expression vector. Further,
their anti-HIV-1 activity in MT4 cells was evaluated. In
addition, we used capillary electrophoresis (CE) to study
their binding specificity of TAR RNA in inhibiting the
Tat–TAR interaction.
1
00
9
0
0
8
70
6
5
4
3
0
0
0
0
The route used for the preparation of the compounds 3–
6
derivatives 1 and 2 were synthesized according to the
was carried out as outlined in Scheme 1. b-Carboline
20
10
0
10;11
procedure described in the literature.
Compounds 3
and 4 were obtained by b-carboline methyl ester reacted
with 1,3-propanediamine. Finally, coupling of b-carbo-
line carboxamide derivatives with S-methylisothiourea
yielded compounds 5 and 6. Molecules 3–6 were char-
Tat+TAR
TAR
Tat
3
4
5
6
Figure 1. Effects of compounds 3–6 on Tat-mediated transactivation in
93T cells.
2
1
12
acterized using MS, H NMR, and elemental analysis.
Activation of transcriptional elongation occurred fol-
lowing the recruitment of Tat to the transcription
machinery via a specific interaction with TAR RNA. To
evaluate the ability of these compounds to block Tat–
cated a significant stimulation of the basal level of
transcription of the HIV-1 LTR. The percent inhibition
of Tat-mediated transactivation of the HIV-1 LTR
varied with the individual compound. Compounds
bearing guanidinium group-terminated side chain
(compounds 5 and 6) were somewhat more inhibitory
than those with terminal amino group (compounds 3
and 4), thus the terminal guanidinium group did play an
important role in Tat–TAR interaction. Furthermore,
the substituted methyl group at C-1 position on the
b-carboline ring of the compounds (4 and 6) resulted in
the appearance of some more inhibitory activities. It is
likely that the increased molecular polarity induced by
the methyl group might give these compounds more
binding affinities to TAR RNA, especially in the com-
plicated eukaryotic system.
13
TAR interaction, we used a reporter gene-based assay.
93T cells were co-transfected with the reporter plas-
2
mids, pLTRCAT, which express CAT under the control
of the HIV-1 LTR and a Tat expression vector
pSVCMVTAT, along with the individual compound of
3
0 lM. None of the tested compounds at concentrations
up to 30 lM showed any significant cytotoxicity (data
not shown). Inhibitory effect of the tested compounds to
transactivation was measured by quantitatively deter-
mination of CAT expression 48 h after transfection by
colorimetric enzyme immunoassay. As seen from Figure
1, a substantial increase in the CAT activity upon co-
transfection with pSVCMVTAT and pLTRCAT indi-
COOH
COOH
b
a
NH2
NH
N
H
N
H
R
COOCH3
c
COOCH3
NH
N
N
H
R
N
H
R
1
2
: R=H
: R=CH
3
CONH(CH2)3NH2
d
N
N
H
R
R
3
4
: R=H
: R=CH
3
CONH(CH2)3NHC(=NH)NH2
e
N
N
H
5
: R=H
:R=CH
6
3
þ
Scheme 1. Reagents and conditions: (a) HCHO or CH
propanediamine, CHCl
4.8% for 6.
3
CHO, H ; (b) SOCl
2
, CH
3
OH; (c) S, xylene, dioxane; 59% for 1 and 71% for 2; (d) 1,3-
3
, MeOH, reflux, 8 h, 44.4% for 3 and 40.9% for 4; (e) S-methylisothiourea, 2 N NaOH, 4 °C to rt, reflux, 1 h, 51.6% for 5 and
6

X. Yu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3127–3130
3129
Since the four compounds showed inhibitory activities
on Tat–TAR interaction, they were expected to inhibit
virus replication. Thus the anti-HIV activity together
with the cytotoxic data of the four compounds was
evaluated in MT4 cells infected with the HIV-1IIIB
strain. Both compounds 5 and 6 showed inhibitory
activity to HIV-1IIIB cytopathic effects (CPE) of cell
fusion (EC50 P 50 lM) and without apparent cellular
toxicity (IC50 P 100–1000 lM), however, compounds 3
and 4 were neither cytotoxic nor inhibitory to HIV-1
than its complex forms because of the smaller charge to
mass ratio of the complexes. The binding of compound
6 with TAR RNA was observed as electrophoresis
mobility shift of TAR’s peak and the decrease of the
height of free compound’s peak in the presence of TAR.
Then the inhibition of compound 6 on the binding of
Tat–TAR complex was probed. Figure 2g showed the
height of Tat’s peak was higher than that of Figure 2f,
which implied that compound 6 could not only bind to
TAR but also hinder the Tat–TAR interaction.
14
(
Table 1).
All the experiments reported here showed that the newly
designed compounds 5 and 6 bearing guanidinium
group-terminated side chain could block the Tat–TAR
interaction and were active against HIV-1. These studies
provide a new idea for the design of new Tat–TAR
inhibitors. In addition, CE assay is an effective and
sensitive method with which to evaluate RNA–peptide
or RNA–drug interaction.
CE has become a powerful analytical technique in bio-
chemical studies, it has been used in the analysis of
RNA–protein interactions, here it has been used to
analyze the binding of compound 6 with HIV-1 TAR
RNAand its inhibition of the Tat–T AR interaction.
As shown in Figure 2, the well-shaped peak of com-
15
pound 6 with t ¼ 7:2 min was clearly visible under the
m
experimental condition used, so was Tat protein with
¼ 8:5 min. The migration time of free TAR RNA and
t
its complex form with compound 6 or Tat protein was in
m
Acknowledgements
the range of 28–30 min. Free TAR RNA migrated faster
We thank Prof. Dalong Ma, Prof. Xinxiang Zhang, Dr.
Xiaojian Yao and Mr. Chunhui Di for their excellent
technical assistance. This project was supported by the
National Natural Science Foundation of China (No.
20332010 and No. 30370323) and Special Fund for
Promotion Education, Ministry of Education of China
Table 1. Anti-HIV-1 activity and cytotoxicity data of compounds 3–6
determined in MT4 cells
a
b
Compound
EC50 (lM)
CC50 (lM)
(
No. 20030001041).
3
4
5
6
>1000
>100
P50
>1000
>100
>1000
>100
P50
References and notes
a
EC50: concentration required to protect cells against the cytopatho-
genicity of HIV by 50%.
1. Rana, T. M.; Jeang, K. Arch. Biochem. Biophys. 1999, 365,
175.
2. Turpin, J. A. Expert Rev. Anti-infect. 2003, 1, 97.
b
CC50: concentration required to inhibit uninfected cells proliferation
by 50%.
3
. (a) Aboul-ela, F.; Karn, J.; Varani, G. J. Mol. Biol. 1995,
53, 313; (b) Aboul-ela, F.; Varani, G. J. Mol.
Struct.-Theochem. 1998, 423, 29.
2
4
. Churcher, M.; Lamont, C.; Hamy, F.; Dingwall, C.;
Green, S. M.; Lowe, A. D.; Butler, P. J. G.; Gait, M. J.;
Karn, J. J. Mol. Biol. 1993, 230, 90.
5
6
. Verhoef, K.; Koper, M.; Berkhout, B. Virology 1997, 237,
2
28.
. (a) Watson, K.; Edwards, R. J. Biochem. Pharmacol. 1999,
8, 1521; (b) Calnan, B. J.; Biancalana, S.; Hudson, D.;
5
Frankel, A. D. Gene 1991, 5, 201.
. Nifosi, R.; Reyes, C. M.; Kollman, P. A. Nucl. Acids Res.
7
8
2
000, 28, 4944.
. (a) Peytou, V.; Condom, R.; Patino, N.; Guedj, R.;
Aubertin, A. M.; Gelus, N.; Bailly, C.; Terreux, R.;
Cabrol-Bass, D. J. Med. Chem. 1999, 42, 4042; (b) Mei, H.
Y.; Cui, M.; Heldsinger, A.; Lemrow, S. M.; Loo, J. A.;
Sannes-Lowery, K. A.; Sharmeen, L.; Czarnik, A. W.
Biochemistry 1998, 37, 14204.
9
. Xiao, S.; Lin, W.; Wang, C.; Yang, M. Bioorg. Med.
Chem. Lett. 2001, 11, 437.
0. Brossi, A.; Focella, A.; Teitel, S. J. Med. Chem. 1973, 16,
1
Figure 2. CE electropherograms of the studies on the binding of
compound 6 with TAR as well as inhibition of compound 6 on the
Tat–TAR interaction. (a) Compound 6 alone (100 lM); (b) Tat alone
418.
11. Lippke, K. P.; Schunack, W. G.; Wenning, W.; M u€ ller,
W. E. J. Med. Chem. 1983, 26, 499.
12. Melting points were determined on a XA-4 instrument and
(100 lM); (c) compound 6 þ Tat (100 lM, each); (d) TAR alone
(100 lM); (e) compound 6 þ TAR (100 lM, each); (f) Tat þ TAR
(100 lM, each); (g) compound 6 þ Tat þ TAR (100 lM, each).
1
are uncorrected. All H NMR spectra were taken on a
Varian Unity 300 NMR spectrometer. Chemical shifts (d)

3130
X. Yu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3127–3130
1
ꢀ1
for H spectra were expressed in ppm relative to tetra-
methylsilane (TMS) as an internal standard. Mass spectra
were measured on a VG-ZAB-HS spectrometer or an ABI
QSTAR spectrometer. Elemental analyses were deter-
mined on an Elementar Vario EL apparatus.
2
mycin (100 U mL ) at 37 °C in 5% CO containing
humidified air. The cells were seeded at a six-well plate
24 h prior to transfection, which was performed by
standard calcium phosphate co-precipitation techniques
with optimum amounts of the plasmids pLTRCAT and
pSVCMVTAT. Twenty four hours later, the culture
medium was removed and the cells were washed twice
with phosphate-buffered saline (PBS). Then the trans-
fected cells were added to fresh medium together with
diluted compounds of final concentration 30 lM, respec-
tively, and incubated for another 24 h. Forty eight hours
post-transfection, the cells were harvested and analyzed
for CAT activity using a commercial CAT ELISA kit
(Roche Molecular Biochemicals) in accordance with the
manufacturer’s protocol. All data were reported as a
percentage of CAT activity (ꢁSD). Results shown are
representative of three independent experiments.
1
H NMR, MS, elemental analysis, mp for representative
1
compounds: 3: H NMR (DMSO-d
6
): d 13.08 (s, 1H, NH),
8
.89 (d, J ¼ 7:2 Hz, 1H, 1-H), 8.80 (t, J ¼ 6:0 Hz, 1H, 4-
H), 8.39 (d, J ¼ 7:8 Hz, 1H, 8-H), 7.64 (m, 1H, 5-H), 7.57
(
3
d, J ¼ 9:0 Hz, 1H, 6-H), 7.29 (t, J ¼ 7:2 Hz, 1H, 7-H),
2 2 2
.38 (m, 2H, CH
), 2.60 (m, 2H, CH
), 1.63 (m, 2H, CH
MS (EI) (m/z): 269(M + H ); elemental analysis calcd (%)
);
þ
for C15
16 4
H N O: C 67.15, H 6.01, N 20.81; Found C 67.39,
1
H 6.46, N 20.34; mp: 218–222 °C. 4: H NMR (acetone-d
d 12.03 (s, 1H, NH), 8.77 (s, 1H, 4-H), 8.35 (d, J ¼ 7:8 Hz,
H, 8-H), 7.67 (d, J ¼ 7:8 Hz, 1H, 5-H), 7.59 (t,
6
):
1
J ¼ 7:8 Hz, 1H, 6-H), 7.30 (t, J ¼ 7:8 Hz, 1H, 7-H), 3.56
(
m, 2H, CH
2
), 3.33 (m, 2H, CH
2
), 2.83 (s, 3H, CH
3
), 1.90
); MS (EI) (m/z): 283(M + H) ; elemental
14. (a) Balzarini, J.; Pannecouque, C.; Clercq, E. D.; Pavlov,
A. Y.; Printsevskaya, S. S.; Miroshnikova, O. V.; Rezni-
kova, M. I.; Preobrazhenskaya, M. N. J. Med. Chem.
2003, 46, 2755; (b) Ayisi, N. K.; Nyadedzor, C. Antivir.
Res. 2003, 58, 25.
þ
(m, 2H, CH
analysis calcd (%) for C16
9.86; Found C 67.85, H 6.40, N 20.01; mp: 175–177 °C. 5:
H NMR (DMSO-d
2
18 4
H N O: C 68.09, H 6.38, N
1
1
6
): d 13.14 (s, 1H, NH), 8.92 (s, 1H, 1-
H), 8.81 (s, 1H, 4-H), 8.37 (d, J ¼ 8:1 Hz, 1H, 8-H), 7.64
15. Capillary electrophoresis assay: CE experiments were
carried out on a Beckman P/ACE 2100 capillary electro-
phoresis system using a Beckman 57 cm ꢂ 75 lm I.D. bare
fused-silica capillary, 50 cm to the UV detector. Phosphate
buffer (50 mM, pH 8.0) was used as running buffer.
Reverse polarity, a constant voltage of 11 kV, a tempera-
ture of 25 ꢁ 0.1 °C and pressure injection for 10 s at 20 psi
(1 psi ¼ 6894.76 Pa) were used. Absorption of samples was
detected at 210 nm. Prior to use, the capillary was pre-
treated with 0.2 M NaOH for 60 min, water for 30 min,
and finally with the running buffer until the baseline
become smooth. To decrease the adsorption of the
samples on the capillary wall through electrostatic inter-
action and to insure the reproducibility of migration time,
the capillary was washed between runs with 0.2 NaOH,
water, and running buffer each for 4 min orderly. Solutions
were filtered through a 0.22 lm PTFE membrane before
use.
(
d, J ¼ 8:1 Hz, 1H, 5-H), 7.58 (d, J ¼ 6:6 Hz, 6-H), 7.27 (t,
2
J ¼ 7:8 Hz, 1H, 7-H), 3.41 (m, 2H, CH
CH ), 1.75 (m, 2H, CH
elemental analysis calcd (%) for C16
H 5.22, N 21.93; Found C 50.51, H 5.24, N 22.30; mp:
), 3.11 (m, 2H,
þ
2
2
); TOF-MS: 311 (M + H );
18 6
H N OCl: C 50.24,
1
1
8
99–201 °C. 6: H NMR (DMSO-d
6
): d 12.03 (s, 1H, NH),
.66 (s, 1H, 4-H), 8.28 (d, J ¼ 7:2 Hz, 1H, 8-H), 7.64 (d,
J ¼ 8:4 Hz, 1H, 5-H), 7.53 (d, J ¼ 7:2 Hz, 1H, 6-H), 7.23
), 3.12 (m, 2H,
); TOF-MS: 325
OCl:
(
t, J ¼ 8:1 Hz, 1H, 7-H), 3.36 (m, 2H, CH
2
CH
(
2
), 2.82 (s, 3H, CH
3
), 1.69 (m, 2H, CH
2
þ
M + H ); elemental analysis calcd (%) for C17
H
20
N
6
C 51.39, H 5.04, N 21.16; Found C 51.21, H 5.30, N 21.27;
mp: 270–272 °C.
3. Transient transfection and CAT assays: 293T cells were
1
grown as monolayer in Dulbecco’s modified Eagle’s
medium (DMEM) (Gibco BRL) supplemented with 10%
ꢀ1
(
v/v) fetal calf serum, penicillin (100 U mL ), and strepto-