α,α-Trehalose derivatives bearing guanidino groups as inhibitors to HIV-1 Tat-TAR RNA interaction in human cells

Article abstract of DOI:10.1016/j.bmcl.2004.02.073

Replication of HIV-1 requires specific interactions of Tat protein with TAR RNA. Disruption of Tat-TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. The core molecule 6,6 ^′-diamino-6,6^′-dideoxy-α,α- trehalose was obtained from selective bromination of, α,α-trehalose at C-6,6^′, followed by acetylation, azide displacement, deacetylation, and reduction. Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated the novel α,α-trehalose derivatives. Their abilities to inhibit Tat-TAR RNA interaction in human cells were determined by a Tat-dependent HIV-1 LTR-driven CAT assays.

Full text of DOI:10.1016/j.bmcl.2004.02.073

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2585–2588
a,a-Trehalose derivatives bearing guanidino groups as inhibitors to
HIV-1 Tat–TAR RNA interaction in human cells
Min Wang,a,b Zhidong Xu,a,b Pengfei Tu,a Xiaolin Yu,a Sulong Xiaoa and Ming Yanga,*
aNational Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, PR China
bDepartment of Chemical Pharmaceutics, Heibei University of Science and Technology, Shijiazhuang 050018, PR China
Received 7 January 2004; accepted 21 February 2004
Abstract—Replication of HIV-1 requires specific interactions of Tat protein with TAR RNA. Disruption of Tat–TAR RNA
interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for
blocking the interaction of Tat–TAR RNA. The core molecule 6,60-diamino-6,60-dideoxy-a,a-trehalose was obtained from selective
bromination of, a,a-trehalose at C-6,60, followed by acetylation, azide displacement, deacetylation, and reduction. Coupling of the
core molecule with the protected amino acid, then deprotection and guanidinylation generated the novel a,a-trehalose derivatives.
Their abilities to inhibit Tat–TAR RNA interaction in human cells were determined by a Tat-dependent HIV-1 LTR-driven CAT
assays.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Based on the structural information from HIV-1 Tat–
TAR interaction, Tat-antagonistic compounds that
inhibit Tat binding to TAR element were undertaken by
a number of researchers. Tat-derived basic peptides as
well as the oligocarbamates and oligoureas bind to TAR
RNA specifically with high affinities in vitro.11;12 Tat-
mimetic compounds ALX40-4C and CGP64222 that
target TAR RNA, display efficient suppression of HIV-1
replication.13;14
Human immunodeficiency virus type 1 (HIV-1) gene
expression depends on the interaction of the viral reg-
ulatory protein, trans-activator of transcription (Tat)
with the trans-activation responsive region (TAR) RNA,
a 59-base stem-loop structure located at the 50-end of all
nascent HIV-1 transcripts.1–3 Therefore, a molecule that
interrupts Tat–TAR interaction must work as an
antagonist to the replication of HIV-1. It could be
developed into therapeutic agents.
Among natural RNA-binding molecules, aminoglyco-
side antibiotics have interesting properties that make
them similar to peptide RNA binders. They may com-
petitively block the binding of the Tat protein to TAR
RNA.15 However, these molecules lack site specificity to
the TAR RNA; therefore, new compounds with selec-
tive binding to the TAR must be designed and synthe-
sized. Recent studies have shown that the conjugates of
aminoglycoside–arginine and guanidinylated derivatives
of aminoglycoside enhance the affinity and selectivity to
the HIV-1 TAR as compared to the parent aminogly-
coside.16–18
HIV-1 TAR contains a six-nucleotide loop, a three-
nucleotide pyrimidine bulge and two single-nucleotide
bulges. The trinucleotide bulge containing residues U23,
C24, and U25 is essential for high affinity and specific
binding of the Tat protein.4;5 The TAR RNA binding
domain of the Tat protein is known as an arginine-rich
sequence (RKKRRQRRR, residues 49–57).6–8 Arginine
residue at position 52 is the only sequence-specific con-
tact mediating the complex formation between Tat and
TAR, and its guanidino group interacts with the residue
U23 of the trinucleotide bulge.9;10
The above study prompts us to design and prepare novel
compounds by combining a carbohydrate skeleton
similar to aminoglycoside antibiotics with side chains of
variable length bearing guanidino group or an arginine
moiety to inhibit HIV Tat–TAR interaction selectively.
Here we report the synthesis of 6,60-diamino-6,60-dide-
oxy-a,a-trehalose derivatives bearing guanidino groups
Keywords: a,a-Trehalose; Guanidino groups; Tat–TAR interaction.
* Corresponding author. Tel.: +86-10-8280-1569/2087; fax: +86-10-
8280-2062; e-mail addresses: yangm@mail.bjmu.edu.cn; yangm@
bjmu.edu.cn
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.073

2586
M. Wang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2585–2588
(10 and 13–15) and the inhibition to Tat–TAR RNA
interaction by Tat-dependent HIV-1 long terminal
repeats (LTR)-driven chloramphenicol acetyltransferase
(CAT) assays.
nidino functions, respectively. Deprotection was realized
using catalytic hydrogenation in the presence of 10% Pd/
C to give compounds 10–12, with 73.2–77.4% yields.
The disappearance of the proton and carbon signals of
benzyloxycarbonyl groups in the 1H and 13C NMR
spectra of compounds 10–12 and appearance of guani-
dino carbon at 157.3 ppm in the 13C NMR spectrum of
target compound 10 verified the success of this reac-
tion.23
2. Results and discussion
Scheme 1 outlines the preparation of 6,60-diamino-6,60-
dideoxy-a,a-trehalose (6). Compound 6 was synthesized
using previously described procedures with some
improvements.19;20 a,a-trehalose dihydrate (1) was
dehydrated directly in DMF instead of pyridine, and
then brominated with 4.3 equiv of Ph3P and NBS, to
give predominantly the 6,60-dibromo-6,60-dideoxy-a,a-
trehalose (2), which on conventional acetylation affor-
ded the 2,3,4,20,30,40-hexa-O-acetyl-6,60-dibromo-6,60-
dideoxy-a,a-trehalose (3). The displacement reaction of
compound 3 with NaN3 in DMF provided the
2,3,4,20,30,40-hexa-O-acetyl-6,60-diazido-6,60-dideoxy-a,a-
trehalose (4). Deacetylation of compound 4 with
MeONa in MeOH afforded 6,60-diazido-6,60-dideoxy-
a,a-trehalose (5).21 In order to avoid high pressure and
decrease reaction time, the azide was reduced with
H2NNH2 in the presence of 20% Pd(OH)2/C catalyst to
give the 6,60-diamino-6,60-dideoxy-a,a-trehalose (6).22
In the guanidinylation reaction, a number of reagents
were explored. The most commonly used reagent N,N0-
bis(benzyloxycarbonyl)-S-methylisothiourea, failed.24;25
Compounds 6, 11, and 12 were successfully guanidinyl-
ated with S-methylisothiourea sulfate at 85 °C for 48 h.
The crude products were subjected to reversed-phase
chromatography and subsequent Sephadex LH-20 col-
umn to provide the target compounds 13–15, with 44.4–
47.8% yields (Scheme 2). The guanidino carbon signals
at 158.2, 157.4, and 158.1 ppm were detected in the 13C
NMR spectra, respectively.23
The activities of target compounds 10 and 13–15 to in-
hibit Tat–TAR RNA interaction in human cells were
examined by using Tat-dependent HIV-1 LTR-driven
CAT gene expression colorimetric enzyme assays.26–28
Human embryonic kidney cells (293T) were maintained
in Dulbeccoꢀs modified Eagle medium (DMEM) sup-
plemented with 10% fetal calf serum and antibiotics at
37 °C in 5% CO2 in a incubator. Cells were seeded into
six-well plates the day prior to transfection. 293T cells
were co-transfected with plasmid pCepIII-CAT con-
taining the HIV-1 LTR linked to a CAT reporter gene
and plasmid pSV2-Tat expressing Tat protein in a ratio
A general synthesis for compounds 10–12 was per-
formed according to the following procedure (Scheme
2). The protected amino acids were activated with N,N0-
dicyclohexylcarbodiimide (DCC) and coupled with
compound 6 in DMF to lead to compounds 7–9, with
46.1–52.8% yields. Benzyloxycarbonyl (Cbz) and nitro
groups were used for protection of the amino and gua-
Br
OH
Br
O
O
AcO
AcO
HO
HO
O
HO
HO
OAc
O
OH
OH
b
a
O
O
OH
OAc
OH
O
O
O
OAc
OH
OH
Br
HO
Br
AcO
HO
HO
2
3
1
NH2
N3
N3
O
O
O
HO
HO
AcO
AcO
HO
HO
OH
O
OAc
O
OH
e
d
c
O
OH
OAc
OH
O
O
O
OH
OAc
OH
H2N
N3
N3
HO
AcO
HO
6
4
5
Scheme 1. Reagents and conditions: (a) Ph3P, NBS, DMF, 48 h; (b) Ac2O, pyridine, 24 h; (c) NaN3, DMF, 95 °C, 27 h; (d) NaOMe, MeOH, 17 h;
(e) H2NNH2, 20% Pd(OH)2/C, MeOH, reflux, 8 h.

M. Wang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2585–2588
2587
NHR
O
HO
HO
OH
O
f
6
OH
R
O
7
8
9
COCH(NH)(Cbz)CH2CH2CH2NHCNH(NH)(NO2)
COCH2CH2CH2NH(Cbz)
OH
COCH2NH(Cbz)
RHN
HO
NHR1
O
HO
HO
OH
7
8
9
g
O
OH
R1
O
10 COCH(NH2)CH2CH2CH2NHC(NH)NH2
11 COCH2CH2CH2NH2
12 COCH2NH2
OH
R1HN
HO
NHR2
O
HO
HO
6
h
OH
12
11
O
OH
R2
O
13 C(NH)NH2
14 COCH2NHC(NH)NH2
15 COCH2CH2CH2NHC(NH)NH2
OH
R2HN
HO
Scheme 2. Reagents and conditions: (f) DCC, DMF, MeOH, 0 °C to rt, 16 h; (g) H2, 10% Pd/C, MeOH; (h) S-methylisothiourea sulfate, NH3ÆH2O,
85 °C, 48 h.
of 1:1 by the calcium phosphate method. After trans-
fection for 24 h at 37 °C, the medium was discarded and
replaced with fresh medium containing the tested com-
100
100
91.0
80
60
40
20
0
pounds (at final 30 lM concentrations). All tested
compounds were cultured for an additional 24 h. CAT
expression was determined using a commercial CAT
ELISA kit.
69.5
55.3
48.6
44.2
As shown in Figure 1, the decreased CAT activities in
the presence of target compounds 10 and 13–15 show
that they competed with Tat for TAR RNA binding and
led to inhibition of Tat function in vivo. Compared with
the amino precursor 6, compounds 10 and 13–15 were
more potent to inhibit Tat–TAR RNA interaction. The
result presented here suggest that the compounds com-
prised of a carbohydrate core with side chains of vari-
able length bearing a guanidino group, may serve as a
specific inhibitor of Tat binding to TAR RNA. This
may be ascribed to the strong basicity of the guanidino
groups. They are fully protonated and positively
charged under physiological conditions, providing a
favorable electrostatic environment for interaction with
nucleic acids. Both the electrostatic and potential to
form hydrogen bonds are essential for the compound
interaction with TAR. The CAT activity of compounds
13–15 and 10 decreased from 69.5 to 44.2 as the side
chain length increased at 3 lM concentrations, and
Tat+TAR
6
13
14
15
10
Figure 1. Inhibition to Tat–TAR RNA interaction by compounds 6,
10, and 13–15 in 293T cells.
compounds 10 and 15 reduced CAT activity more than
50%. Based on this, we can find that not only a carbo-
hydrate core containing a guanidino group facilitates the
binding to TAR RNA, but a longer chain, connecting
the core and the charge-bearing moiety, is also beneficial.
Hence the most potent compound inhibiting Tat–TAR
interaction in 293T cells was considered to be the 6,60-
diamino-6,60-dideoxy-a,a-trehalose-arginine conjugate 10.

2588
M. Wang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2585–2588
17. Hamasaki, K.; Ueno, A. Bioorg. Med. Chem. Lett. 2001,
11, 591.
3. Conclusion
We have designed and synthesized four 6,60-diamino-
6,60-dideoxy-a,a-trehalose derivatives bearing guanidino
groups. All the target compounds exhibited inhibiting
activities to Tat–TAR RNA interaction in 293T cells,
and the derivative bearing arginine moiety was the most
potent agent in this series.
18. Litovchick, A.; Evdokimov, A. G.; Lapidot, A. FEBS
Lett. 1999, 445, 73.
19. Hanessian, S.; Lavallee, P. J. Antibiot., Sec. A 1972, 25,
683.
20. Birch, G.; Richardson, A. C. Carbohydr. Res. 1968, 8, 411.
21. Greenberg, W. A.; Scott Priestley, E.; Sears, P. S.; Alper,
P. B.; Rosenbohm, C.; Hendrix, M.; Hung, S. C.; Wong,
C. H. J. Am. Chem. Soc. 1999, 121, 6527.
22. Malik, A. A.; Preston, S. B.; Archibald, T. G.; Cohen, M.
P.; Baum, K. Synthesis 1989, 450.
23. Compound 10: mp 156.0–158.0 °C. ½aꢀ +179 (c 0.87,
Acknowledgements
D
1
H2O). H NMR (D2O) d 4.90 (d, 2H, J 3.6 Hz, H-1), 3.81
(m, 2H, a-CH), 3.66–3.10 (m, 12H, H-6, H-5, H-60, H-3,
H-2, and H-4), 3.04 (t, 4H, J 8.4 Hz, d-CH2), 1.51 (m, 4H,
c-CH2), 1.46 (m, 4H, b-CH2). 13C NMR (D2O) d 176.8
(NH–CO), 157.3 (guanidino C), 94.5, 94.1, 72.9, 71.9,
71.6, 71.3 (sugar C), 67.4 (Cbz-CH2), 54.7 (a-CH), 41.3
(C-6), 40.4 (d-CH2), 31.4 (b-CH2), 24.8 (c-CH2).
FABMS: m=z 653.0 [M+H]þ. Anal. Calcd for
C24H48N10O11Æ2HClÆ2H2O: C, 37.84; H, 7.15; N, 18.39.
Found: C, 37.92; H, 7.23; N, 18.47. Compound 13: mp
This work was supported by the National Natural Sci-
ence Foundation of China (No. 30370323) and Doctoral
Program Foundation of China (No. 20030001041).
References and notes
1. Dayton, A. I.; Sodroski, J. G.; Rosen, C. A.; Goh, W. C.;
Haseltine, W. A. Cell 1986, 44, 941.
226.0 °C (decomp.). ½aꢀ +147 (c 0.85, H2O). 1H NMR (D2
D
O) d 5.00 (d, 2H, J 3.6 Hz, H-1), 3.78–3.73 (m, 2H, H-6),
3.69–3.63 (m, 2H, H-5), 3.49–3.39 (m, 4H, H-60 and H-3),
3.31–3.26 (m, 2H, H-2), 3.24–3.17 (t, 2H, J 9.0 Hz, H-4).
13C NMR (D2O): d 158.2 (guanidino C), 94.3, 72.9, 72.6,
71.4, 71.2 (sugar C), 42.5 (C-6). FABMS: m=z 425.0
[M+H]þ. Anal. Calcd for C14H28N6O9ÆH2SO4Æ2H2O: C,
30.11; H, 6.14; N, 15.05. Found: C, 30.05; H, 6.10; N,
2. Rosen, C. A.; Sodroski, J. G.; Goh, W. C.; Dayton, A. I.;
Loppke, J.; Haseltine, W. A. Nature 1986, 319, 555.
3. Fisher, A. G.; Feinberg, M. B.; Josephs, S. F.; Harper,
M. E.; Marselle, L. M.; Reyes, G.; Gonda, M. A.;
Aldovini, A.; Debouck, C.; Gallo, R. C.; Wong-Staal, F.
Nature 1986, 320, 367.
4. Jones, K. A.; Peterlin, B. M. Annu. Rev. Biochem. 1994,
63, 717.
15.18. Compound 14: mp 214.0 °C (decomp.). ½aꢀ +157 (c
D
0.83, H2O). 1H NMR (D2O) d 4.93 (d, 2H, J 3.6 Hz, H-1),
3.85 (m, 2H, H-6) 3.67–3.60 (m, 4H, H-5, and H-60), 3.45–
3.40 (m, 4H, H-2, and H-3), 3.34–3.29 (m, 2H, H-4), 3.15
(t, 4H, J 9.3 Hz, CH2). 13C NMR (D2O) d 171.0 (NH–
CO), 158.1 (guanidino C), 94.4, 94.2, 73.0, 71.8, 71.6, 71.2
(sugar C), 44.3 (a-CH2), 40.5 (C-6). FABMS: m=z 539.5
[M+H]þ. Anal. Calcd for C18H34N8O11ÆH2SO4Æ2H2O: C,
32.14; H, 5.99; N, 16.66. Found: 32.07; H, 5.73; N, 16.72.
5. Emerman, M.; Guyader, M.; Montagnier, L.; Baltimore,
D.; Muesing, M. A. EMBO J. 1987, 6, 3755.
6. Ruben, S.; Perkins, A.; Purcell, R.; Joung, K.; Sia, R.;
Burghoff, R.; Haseltine, W. A.; Rosen, C. A. J. Virol.
1989, 63, 1.
7. Hauber, J.; Malim, M. H.; Cullen, B. R. J. Virol. 1989, 63,
1181.
8. Weeks, K. M.; Ampe, C.; Schultz, S. C.; Steitz, T. A.;
Crothers, D. M. Science 1990, 249, 1281.
9. Calnan, B. J.; Tidor, B.; Biancalana, S.; Hudson, D.;
Frankel, A. D. Science 1991, 252, 1167.
10. Karn, J.; Graeble, M. A. Trends Genet. 1992, 8, 365.
11. Wang, X.; Huq, I.; Rana, T. M. J. Am. Chem. Soc. 1997,
119, 6444.
12. Tamilarasu, N.; Huq, I.; Rana, T. M. J. Am. Chem. Soc.
1999, 121, 1597.
13. OꢀBrien, W. A.; Sumner-Smith, M.; Mao, S. H.; Sadeghi,
S.; Zhao, J. Q.; Chen, I. S. Y. J. Virol. 1996, 70, 2825.
14. Hamy, F.; Felder, E. R.; Heizmann, G.; Lazdins, J.;
Aboul-Ela, F.; Varani, G.; Karn, J.; Klimkait, T. Proc.
Natl. Acad. Sci. U.S.A. 1997, 94, 3548.
15. Mei, H. Y.; Galan, A. A.; Halim, N. S.; Mack, D. P.;
Moreland, D. W.; Sanders, K. B.; Truong, H. N.; Czarnik,
A. W. Bioorg. Med. Chem. Lett. 1995, 5, 2755.
16. Litovchick, A.; Evdokimov, A. G.; Lapidot, A. Biochem-
istry 2000, 39, 2838.
Compound 15: mp 185 °C (decomp.). ½aꢀ +172 (c 0.96,
D
H2O). 1H NMR (D2O) d 4.92 (d, 2H, J 3.6 Hz, H-1), 3.67–
3.60 (m, 4H, H-6, and H-5), 3.46–3.39 (m, 4H, H-60, and
H-3), 3.29–3.22 (m, 2H, H-2), 3.17–3.17 (m, 2H, H-4), 3.03
(t, 4H, J 7.2 Hz, a-CH2), 2.18 (t, 4H, J 7.5 Hz, c-CH2),
1.75 (m, 4H, J 7.5 Hz, b-CH2). 13C NMR (D2O) d 176.5,
(NH–CO), 157.4 (guanidino C), 93.9, 73.0, 71.8, 71.6, 71.3
(sugar C), 41.0 (C-6), 40.4 (c-CH2), 33.1 (a-CH2), 25.0 (b-
CH2). FABMS: m=z 595.4 [M+H]þ. Anal. Calcd for
C22H42N8O11ÆH2SO4Æ3H2O: C, 35.39; H, 6.75; N, 15.01.
Found: C, 35.21; H, 6.89; N, 14.90.
24. Su, W. G. Synth. Commun. 1996, 26, 407.
25. Chandrakumar, N. S. Synth. Commun. 1996, 26, 2613.
26. Huq, I.; Ping, Y.; Tamilarasu, N.; Rana, T. M. Biochem-
istry 1999, 38, 5172.
27. Choudhury, I.; Wang, J.; Stein, S.; Rabson, A.; Leibowitz,
M. J. J. Gen. Virol. 1999, 80, 777.
28. Litovchick, A.; Lapidot, A.; Eisenstein, M.; Kalinkovich,
A.; Borkow, G. Biochemistry 2001, 40, 15612.