Design and synthesis of guanidinoglycosides directed against the TAR RNA of HIV-1

Article abstract of DOI:10.1002/hlca.200390213

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of the Tat protein with the transactivation responsive region (TAR) RNA. Tat-TAR RNA Interaction is mediated by a short arginine-rich domain of the protein. Disruption of this interaction could, in theory, create a state of complete viral latency. Here, four novel 6-amino-6-deoxytrehalose guanidinoglycoside derivatives (10 and 13-15) as target molecules have been designed to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. They were obtained by coupling 6-amino-6-deoxy-α,α-trehalose (6) with the protected amino acids, deprotection by catalytic hydrogenation, followed by guanidinylated with S-methylisothiourea sulfate. Their abilities to inhibit Tat-TAR RNA interaction were determined by a Tat-dependent HIV-1 long terminal repeats (LTR)-driven chloramphenicol acetyltransferase (CAT) assays.

Full text of DOI:10.1002/hlca.200390213

Helvetica Chimica Acta Vol. 86 (2003)
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Design and Synthesis of Guanidinoglycosides Directed against the TAR
RNA of HIV-1
by Min Wanga), Peng-Fei Tub), Zhi-Dong Xua), Xiao-Lin Yua), and Ming Yang*a)
a) National Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, P.R.
China (phone: 8610-62091569; fax: 8610-62092264; e-mail: yangm@mail.bjmu.edu.cn)
b) School of Pharmaceutical Sciences, Peking University, Beijing 100083, P.R. China
Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of the Tat
protein with the transactivation responsive region (TAR) RNA. Tat-TAR RNA Interaction is mediated by a
short arginine-rich domain of the protein. Disruption of this interaction could, in theory, create a state of
complete viral latency. Here, four novel 6-amino-6-deoxytrehalose guanidinoglycoside derivatives (10 and 13
15) as target molecules have been designed to bind to TAR RNA for blocking the interaction of Tat-TAR RNA.
They were obtained by coupling 6-amino-6-deoxy-a,a-trehalose (6) with the protected amino acids,
deprotection by catalytic hydrogenation, followed by guanidinylated with S-methylisothiourea sulfate. Their
abilities to inhibit Tat-TAR RNA interaction were determined by a Tat-dependent HIV-1 long terminal repeats
(LTR)-driven chloramphenicol acetyltransferase (CAT) assays.
Introduction. Human immunodeficiency virus type 1 (HIV-1) gene expression is
regulated by a number of virus-encoded proteins, including the transactivator of
transcription (Tat) protein that plays a critical role in virus replication [1]. Tat is
responsible for high levels of expression from the viral long terminal repeat (LTR)
promoter, involving the interaction of Tat with the viral RNA at a specific region called
transactivation responsive element (TAR). Discovery of therapeutic agents that
disrupt the Tat-TAR interaction, therefore, would provide a strategy for inhibiting
HIV-1 replication.
TAR RNA comprises the first 59 nucleotides at the nascent end of the retrovirus
RNA. It forms a hairpin stem-loop structure containing a six-nucleotide loop (residues
30 35), a three-nucleotide pyrimidine bulge (residues 23 25), and two single-
nucleotide bulges (residues 5 and 17). The Tat-binding region of TAR centers on the
three-nucleotide bulge [2]. It is also known that the binding domain of the Tat protein is
an arginine-rich sequence (residues 49 57: ArgLysLysArgArgGlnArgArgArg).
Arginine 52 in the protein is the only sequence contact mediating the complex
formation between Tat and TAR [3], and its guanidino group interacts with residue
U23 of the three-nucleotide bulge.
With structural information from the HIV-1 Tat-TAR interaction, attempts to
produce antiviral peptide or peptidomimetic substances that inhibit Tat binding to TAR
element have been 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 [4][5]. Tat-mimetic compounds ALX40-4C [6] and CGP64222 [7] that
target TAR RNA, demonstrate pronounced antiviral activity.

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Helvetica Chimica Acta Vol. 86 (2003)
Among natural RNA-binding molecules, aminoglycoside antibiotics have interest-
ing properties that make them similar to peptide RNA-binders. They may inhibit Tat
binding to TAR RNA in the mm range of concentration [8]. Recent studies have shown
that the conjugates of aminoglycosideÀarginine display high affinity for TAR RNA in
vitro in the range of 20 400n m [9]. These molecules were biologically evaluated, and
R3G and R4K exhibited antiviral activity in vitro in mm concentrations, with undetected
cytotoxicity at the active concentrations (CC50 > 3940 mm for R3G and 1130 mm for
R4K) [10][11].
The above prompted us to design novel compounds capable of binding to TAR
RNA, by combining a carbohydrate skeleton similar to aminoglycoside antibiotics with
side chains of variable length bearing a guanidine or an arginine moiety, resembling
peptide side chains. Here, we report the synthesis of 6-amino-6-deoxy-a,a-trehalose
guanidinoglycoside derivatives 10 and 13 15, and the inhibition of Tat-TAR
interaction by Tat-dependent HIV-1 LTR-driven chloramphenicol acetyltransferase
(CAT) assays [12 14].
Results and Discussion.
The syntheses of the 6-amino-6-deoxy-a,a-trehalose
guanidinoglycoside derivatives were carried out in three principal steps. First, 6-amino-
6-deoxy-a,a-trehalose (6) as a precursor was synthesized. Second, coupling of the
protected amino acids and the precursor 6, followed by deprotection, formed amide
molecules 10 12 with variable-length side chains containing an amino group or an
arginine residue. Third, conversion of the amino groups to guanidino groups yielded the
final compounds 13 15.
6-amino-6-deoxy-a,a-trehalose (6) was synthesized according to the procedures
described in [15 17] with some improvements (Scheme 1). a,a-Trehalose dihydrate
(1) was dehydrated directly in DMF instead of pyridine, and then brominated with Ph3P
and N-bromosuccinimide (NBS) to give predominantly the 6-bromo-6-deoxy-a,a-
trehalose (2), which, on conventional acetylation, afforded the 2,3,4,2',3',4',6'-hepta-O-
acetyl-6-bromo-6-deoxy-a,a-trehalose (3) in 58% yield. In the 13C-NMR spectrum of
3, the signal due to C(6) was shifted from 61.0to 30.4 ppm, indicating the presence of a
Br-atom at C(6). The 1H-NMR spectrum showed seven single peaks for O-Ac groups at
1.99 2.12 ppm, with the corresponding resonance at 169.5 170.6 ppm (CO groups of
Ac) and 20.6 20.9 ppm (Me groups of Ac) in the 13C-NMR spectrum. The replace-
ment reaction of compound 3 with NaN3 in DMF provided the 2,3,4,2',3',4',6'-hepta-O-
acetyl-6-azido-6-deoxy-a,a-trehalose (4) in 74% yield. The chemical shift of C(6) was
shifted to 50.9 ppm, strongly supporting the fact that the Br-atom was replaced by
azide. Deacetylation of compound 4 with MeONa in MeOH afforded 6-azido-6-deoxy-
a,a-trehalose (5) [18] in 78% yield. Analysis by 1H- and 13C-NMR spectra showed no
signals for O-Ac groups, confirming the removal of Ac groups. To avoid high pressure
and to decrease reaction time, compound 5 was reduced with NH2NH2 in the presence
of 20% Pd(OH)2/C catalyst to give the 6-amino-6-deoxy-a,a-trehalose (6) [19] in 68%
yield. The 13C-NMR spectrum of 6 revealed an upfield shift of C(6) (from 50.9 to
41.1 ppm), indicating that reduction occurred under this reaction.
Compounds 10 12 were obtained in two steps (Scheme 2). The protected amino
acids were activated with N,N'-dicyclohexylcarbodiimide (DCC) and coupled with
compound 6 in DMF to lead to compounds 7 9, in 39 61% yields. (Benzyloxy)car-

Helvetica Chimica Acta Vol. 86 (2003)
2639
Scheme 1
a) Ph3P, NBS, DMF, 48 h. b) Ac2O, pyridine, 24 h. c) NaN3, DMF, 958, 14 h. d) NaOMe, MeOH, 16 h. e)
NH2NH2, 20% Pd(OH)2/C, MeOH, reflux, 6 h.
bonyl (Cbz) and NO2 groups were used for protection of the amino and guanidino
functions, respectively. The 1H-NMR spectra of 7, 8, and 9 revealed the presence of the
1H signals of (benzyloxy)carbonyl group at 7.25 7.36 ppm (arom. H) and 5.02
5.15 ppm (CH2 groups of Cbz), respectively. All of the characteristic a,a-trehalose
1H signals, in particular the anomeric H-atoms (as doublets at 5.07 ppm), were
observed. In addition, the appearance of the signals of the amide C-atoms at 172.6
174.9 ppm (NHCO) and 158.5 159.2 ppm (CO of Cbz), those of the (benzyloxy)-
carbonyl group C-atoms at 128.8 138.4 ppm (arom. C) and 67.4 67.9 ppm (CH2 of
Cbz), and those of the a,a-trehalose anomeric C-atoms at 95.2 95.5 ppm confirmed
the structures of compounds 7, 8, and 9. Deprotection was realized by catalytic
hydrogenation in the presence of 10% Pd/C to give compounds 10 12 in 77 84%
1
1
yields. The disappearance of the H and 13C signals of Cbz groups in the H- and
13C-NMR spectra of compounds 10 12, and appearance of the signal of the guanidino
C-atom at 159.4 ppm in the 13C-NMR spectrum of compound 10 verified the success of
this reaction.
Attempts to prepare guanidinoglycoside derivatives with N,N'-Bis(benzyloxycar-
bonyl)-S-methylisothiourea in DMF were unsuccessful [20]. Compounds 6, 11, and 12

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Helvetica Chimica Acta Vol. 86 (2003)
Scheme 2
a) DCC, DMF, MeOH, 08 ! r.t., 16 h. b) H2, 10% Pd/C, MeOH. c) S-Methylisothiourea sulfate, NH3 ¥ H2O, 858,
48 h.
were successfully guanidinylated with S-methylisothiourea sulfate at 858 for 48 h. The
crude products were subjected to reversed-phase C-18 chromatography and to a
subsequent Sephadex LH-20 column to provide guanidinoglycoside derivatives 13 15,
in 42 55% yields (Scheme 2). The guanidino 13C signals at 160.4, 158.1, and 157.4 ppm
were detected in the 13C-NMR spectra.
The inhibitory activities of Tat-TAR RNA interaction by target compounds 10, 13,
14, and 15 were examined by using Tat-dependent HIV-1 LTR-driven CAT gene-
expression colorimetric enzyme assays. Human embryonic kidney cells (293T) were co-
transfected with plasmid pCepIII-CAT containing the HIV-1 LTR linked to a CAT
reporter gene and plasmid pSV2-Tat expressing Tat protein, and CAT protein

Helvetica Chimica Acta Vol. 86 (2003)
2641
Figure. Effect of compounds 6, 10, 13, 14, and 15 on Tat-dependent HIV-1 LTR-driven CAT expression
expression is dependent on Tat. As shown in the Figure, the decreased CATactivities in
the presence of target compounds 10, 13, 14, and 15 show that they compete with Tat for
TAR RNA binding and lead to inhibition of Tat function in vivo. Compared with the
amino precursor 6, compounds 10, 13, 14, and 15 are more potent inhibitors of Tat-TAR
RNA interaction. These results indicate that the compounds comprised of a
carbohydrate core with side chains of variable length bearing a guanidino group may
serve as specific inhibitors of Tat binding to TAR RNA. The CAT activity of
compounds 13, 14, 15, and 10 decreased from 69.7 to 41.3 with the side-chain length
increased at 3 mm concentration; compounds 10 and 15 reduced CATactivity more than
50%. Based on these findings, we can conclude that not only a carbohydrate core
containing a guanidino group facilitates the binding to TAR RNA, but a longer chain,
which connects the core and the charge-bearing moiety, is also beneficial.
Experimental Part
General. All solvents were dried and distilled prior to use. Evaporations were carried out under reduced
pressure at a bath temp. of < 458. TLC: precoated silica-gel 6 0 F254 plates; detection by irradiation with UV light
(254 nm), by absorption of I2 vapor, or by spraying with 10% H2SO4 in EtOH and ninhydrin. Column
chromatography (CC): silica gel (200 300 mesh; Qingdao Chemical Co.). M.p.: Electrothermal XT-4 digital
melting-point apparatus; uncorrected. Optical rotations: Perkin-Elmer 243B polarimeter. NMR Spectra: Varian
VXR-300 instrument; d in ppm with Me4Si as an internal standard (ˆ0ppm), J in Hz. FAB-MS: ZAB-HS
instrument; in m/z.
2,3,4,2',3',4',6'-Hepta-O-acetyl-6-bromo-6-deoxy-a,a-trehalose (3). a,a-Trehalose dihydrate (10.0 g,
26 mmol) was dehydrated by dissolving in DMF (250ml) and concentrating the resulting soln. to 150ml. The
concentrated soln. was cooled with an ice bath to 08 and N-bromosuccinimide (NBS; 9.4 g, 52 mmol) was added
with stirring. After 0.5 h, Ph3P (13.6 g, 52 mmol) was added gradually, and the mixture was stirred for another
48 h at r.t. MeOH (150ml) was added to decompose excess reagent, and the solvents were removed under
reduced pressure and lyophilized to give a yellow solid. H2O was added, and the soln. was extracted with CHCl3.
The combined org. phases were concentrated and lyophilized to give a white solid product of which the chief
component was compound 2. The product was acetylated with Ac2O (100 ml) in pyridine (350 ml) without

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Helvetica Chimica Acta Vol. 86 (2003)
purification. After 24 h at r.t., the soln. was poured slowly into ice-water (2 l), and the resulting precipitate was
filtered. The solid was washed with 5% NaHCO3, then H2O, and lyophilized. The product was purified by CC
(CH2Cl2/AcOEt 4 :1), and 3 (10.72 g, 58%) was isolated. White powder. M.p. 117.0 118.08 ([17]: 119 1208).
[a]D ˆ ‡176 (c ˆ 0.84, CHCl3). 1H-NMR (CDCl3): 5.50 5.41 ( m, 2 HÀC(6')); 5.29 (dd, J ˆ 3.6, 2 HÀC(1));
5.13 5.09 (m, 2 HÀC(5)); 5.04 4.90 (m, HÀC(6)); 4.21 4.10( m, 2 HÀC(3)); 4.09 3.96 (m, 2 HÀC(2));
3.38 3.25 (m, 2 HÀC(4)); 2.12 (s, MeCO); 2.09 (s, MeCO); 2.05 (s, MeCO); 2.04 (s, MeCO); 2.01 (s, MeCO);
2.00 (s, MeCO); 1.99 (s, MeCO). 13C-NMR (CDCl3): 170.6, 169.9, 169.8, 169.6, 169.5 (CO); 92.2, 92.1, 71.1, 70.1,
‡
69.8, 69.7, 69.6, 69.3, 68.5, 68.2, 61.7, 30.4 (sugar ring C); 20.9, 20.6 (MeCO). FAB-MS: 700.6 (C26H36BrO17, [M ‡
‡
H] ; calc. 700.4).
2,3,4,2',3',4',6'-Hepta-O-acetyl-6-azido-6-deoxy-a,a-trehalose (4). A mixture of 3 (9.10g, 13 mmol) and
NaN3 (2.54 g, 39 mmol) in DMF (100 ml) was heated for 20 h at 958. After cooling, the insoluble material was
filtered off. The filtrate was concentrated under reduced pressure to afford a syrup. CHCl3 and H2O were added,
and the org. layer was dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was
further purified by CC (CH2Cl2/AcOEt 4 :1), and 4 was obtained (6.40g, 74%). White crystals. M.p. 119.0
1
120.08 ([17]: 119 120.58). [a]D ˆ ‡117 (c ˆ 0.96, CHCl3). H-NMR (CDCl3): 5.49 5.41 (m, 2 HÀC(6')); 5.29
(dd, J ˆ 3.6, 2 HÀC(1)); 5.08 4.92 (m, 2 HÀC(5), 2 HÀC(6)); 4.25 4.05 (m, 2 HÀC(3)); 4.04 3.95
(m, 2 HÀC(2)); 3.36 3.11 (m, 2 HÀC(4)); 2.09 (s, MeCO); 2.06 (s, MeCO); 2.05 (s, MeCO); 2.03 (s, MeCO);
2.02 (s, MeCO); 2.01 (s, MeCO); 2.00 (s, MeCO). 13C-NMR (CDCl3): 170.6, 170.0, 169.9, 169.7, 169.6, 169.5
(CO); 92.8, 92.4, 69.9, 69.8, 69.7, 69.6, 68.4, 68.2, 61.7, 50.9 (sugar ring C): 20.6 (MeCO). FAB-MS: 662.0
‡
‡
(C26H36N3O17, [M ‡ H] ; calc. 662.6).
6-Azido-6-deoxy-a,a-trehalose (5). To a suspension of 4 (5.95 g, 9 mmol) in MeOH (120ml), a catalytic
amount of MeONa was added, and the mixture was stirred for 14 h at r.t. The soln. was neutralized by addition
of Amberlite IR-120 (H ) resin. The resin was washed with MeOH, and the combined soln. was concentrated
‡
under reduced pressure. Purification of the residue by CC (CHCl3/MeOH 1.5 :1) afforded 5 (2.57 g, 78%).
White powder. M.p. 192.0 194.2 8. [a]D ˆ ‡149 (c ˆ 0.81, MeOH). 1H-NMR (CD3OD): 5.08 (dd, J ˆ 3.6,
2 HÀC(1)); 3.80 3.71 ( m, 2 HÀC(6'), 2 HÀC(5)); 3.66 3.60( m, 2 HÀC(6)); 3.48 3.23 (m, 2 HÀC(3),
2 HÀC(2), 2 HÀC(4)). 13C-NMR (CD3OD): 95.3; 95.1; 74.5; 74.2; 73.9; 73.1; 72.9; 72.6; 71.8; 62.6; 52.6 (sugar
‡
‡
ring C). FAB-MS: 368.2 (C12H22N3O10, [M ‡ H] ; calc. 368.3).
6-Amino-6-deoxy-a,a-trehalose (6). A soln. of 5 (2.39 g, 6.5 mmol) in MeOH (50ml) was degassed by
evacuating the flask and backfilling with N2. NH2NH2 (0.55 ml, 16.6 mmol) was added, followed immediately by
20% Pd(OH)2/C (130mg), and the mixture was heated at reflux for 8 h. The soln. was cooled to r.t., filtered, and
concentrated under reduced pressure. The residue was purified with Sephadex LH-20 resin with 5% MeOH and
1
lyophilized to afford 6 (1.51 g, 68%). White powder. M.p. 2038 (dec.) [a]D ˆ ‡127 (c ˆ 0.95, H2O). H-NMR
(D2O): 5.04 (dd, J ˆ 3.6, 2 HÀC(1)); 3.86 3.70( m, 2 HÀC(6'), 3.68 3.56 (m, 2 HÀC(5), 2 HÀC(6)); 3.53
3.46 (m, 2 HÀC(3)); 3.31 3.17 (m, 2 HÀC(2), 2 HÀC(4)). 13C-NMR (D2O): 94.1; 94.0; 73.1; 72.8; 72.6; 72.0;
‡
‡
71.6; 71.3; 70.2; 68.8; 61.0; 41.1 (sugar ring C). FAB-MS: 342.1 (C12H24NO10, [M ‡ H] ; calc. 342.3).
6-({Na-[(Benzyloxy)carbonyl]-Nw-nitro-l-arginyl}amino)-6-deoxy-a,a-trehalose (7). DCC (270mg,
1.32 mmol) was added to 10ml of MeOH soln. containing Na-benzyloxycarbonyl-Nw-nitroarginine (470mg,
1.32 mmol). After stirring for 30min at 0 8, a soln. of 6 (150mg, 0.44 mmol) in 5 ml of DMF was added dropwise.
The mixture was allowed to slowly warm to ambient temp. and stirred for an additional 16 h. Precipitated N,N'-
dicyclohexylurea (DCU) was removed by filtration, and the filtrate was concentrated under reduced pressure.
The residue was purified by chromatography (silica gel; CHCl3/MeOH 1.5 :1) to afford 7 (110mg, 39%).
Colorless powder. M.p. 144.0 145.2 8. [a]D ˆ ‡155 (c ˆ 0.92, MeOH). 1H-NMR (CD3OD): 7.35 7.25
(m, 5 arom. H); 5.15 5.02 (m, 4 H, CH2(Cbz) and 2 HÀC(1) overlap); 4.12 (t, J ˆ 5.2, a-CH); 3.88 3.82
(m, 2 HÀC(6')); 3.79 3.73 (m, 2 HÀ(5)); 3.68 3.57 (m, 2 HÀC(6)); 3.53 3.45 (m, 2 HÀC(3)); 3.44 3.42
(m, 2 HÀC(2)); 3.40 3.29 ( m, 2 HÀC(4)); 3.13 (t, J ˆ 9.0, d-CH2); 1.66 (br., b-CH2, g-CH2). 13C-NMR
(CD3OD): 174.9 (NHCO); 160.9 (NO2C); 158.5 (CO(Cbz)); 138.0, 129.5, 129.1, 129.0 (arom. C); 95.3, 95.2,
74.5, 74.2, 74.1, 73.9, 73.5, 73.2, 72.0, 71.9, 62.6, 41.8 (sugar ring C); 67.9 (CH2(Cbz)); 56.2 (a-CH); 41.5 (d-CH2);
‡
‡
30.5 (b-CH2); 26.0( g-CH2). FAB-MS: 677.0(C 26H41N6O15, [M ‡ H] ; calc. 677.6).
6-[N-({[(Benzyloxy)carbonyl]amino}butanoyl)amino]-6-deoxy-a,a-trehalose (8). From 4-{[(benzyloxy)-
carbonyl]}butanoic acid (313 mg, 1.32 mmol) by a method analogous to that for 7: 8 (150mg, 61%). Colorless
powder. M.p. 113.0 114.0 8. [a]D ˆ ‡136 (c ˆ 0.84, MeOH). 1H-NMR (CD3OD): 7.33 7.26 (m, 5 arom. H);
5.07 (d, J ˆ 3.6, 2 HÀC(1)); 5.05 (s, CH2(Cbz)); 3.88 3.81 (m, 2 HÀC(6')); 3.77 3.73 (m, 2 HÀC(5)); 3.68
3.62 (m, 2HÀC(6)); 3.51 3.47 (m, 2 HÀC(3)); 3.44 3.42 (m, 2 HÀC(2)); 3.39 3.27 (m, 2 HÀC(4)); 3.12
(q, J ˆ 7.2, a-CH2), 2.23 (t, J ˆ 7.5, g-CH2); 1.76 (m, J ˆ 7.2, b-CH2). 13C-NMR (CD3OD): 176.2 (NHCO); 159.0
(CO(Cbz)); 138.4, 129.5, 129.0, 128.8 (arom. C); 95.3, 95.2, 80.0, 74.6, 74.1, 73.9, 73.2, 72.0, 71.9, 62.6, 41.4 (sugar

Helvetica Chimica Acta Vol. 86 (2003)
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‡
‡
ring C); 67.4 (CH2(Cbz)); 41.2 (g-CH2); 34.1 (a-CH2); 27.4 (b-CH2). FAB-MS: 561.0(C 24H37N2O13, [M ‡ H] ;
calc. 561.6).
6-({N-[(Benzyloxy)carbonyl]glycyl}amino)-6-deoxy-a,a-trehalose (9). From N-[(benzyloxy)carbonyl]gly-
cine (276 mg, 1.32 mmol) by a method analogous to that for 7: 9 (139 mg, 59%). Colorless powder. M.p. 124.0
125.48. [a]D ˆ ‡158 (c ˆ 0.83, MeOH). 1H-NMR (CD3OD): 7.36 7.29 (m, 5 arom. H); 5.10( s, CH2(Cbz)); 5.07
(d, J ˆ 3.6, 2 HÀC(1)); 3.92 3.81 (m, 2 HÀC(6')); 3.77 3.74 (m, 2 HÀC(5)); 3.71 3.59 (m, 2 HÀC(6)); 3.52
3.45 (m, 2 HÀC(3)); 3.34 3.27 (m, 2 HÀC(2), 2 HÀC(4)); 3.14 (t, J ˆ 9.9, CH2). 13C-NMR (CD3OD): 172.6
(NHCO); 159.2 (CO(Cbz)); 138.0, 129.5, 129.1, 129.0, 128.9 (arom. C); 95.5, 95.3, 74.5, 74.3, 73.9, 73.4, 73.3, 73.2,
‡
‡
71.9, 62.6, 44.9 (sugar ring C); 67.4 (CH2(Cbz)); 41.5 (a-CH2). FAB-MS: 533.0(C 22H33N2O13, [M ‡ H] ; calc.
533.5).
6-[(l-Arginyl)amino]-6-deoxy-a,a-trehalose (10). Compound 7 (100 mg, 0.15 mmol) was dissolved in 15 ml
of MeOH and hydrogenated in the presence of 10% Pd/C (16 mg) at 0.4 Mpa for 48 h. The mixture was then
filtered through Celite, and the solid was washed thoroughly with MeOH and H2O. The filtrate was then
concentrated. Purification of the residue on a Sephadex LH-20 column (50 Â 1 cm, eluted with 5% MeOH)
afforded, after lypophilization, 10 (57 mg, 77%). White powder. M.p. 154.0 156.0 8. [a]D ˆ ‡147 (c ˆ 0.92,
H2O). 1H-NMR (D2O): 5.00 (dd, J ˆ 3.6, 2 HÀC(1)); 3.84 (t, J ˆ 6.4, a-CH); 3.77 3.69 (m, 2 HÀC(6'),
2 HÀC(5), 2 HÀC(6)); 3.65 3.31 (m, 2 HÀC(3)); 3.28 (t, J ˆ 9.3, d-CH2), 3.20 3.08 ( m, 2 HÀC(2),
2 HÀC(4)); 1.78 1.73 (m, b-CH2); 1.53 (m, g-CH2). 13C-NMR (D2O): 172.9 (NHCO); 159.4 (guanidino C);
96.2, 96.1, 75.2, 75.0, 74.2, 74.1, 73.8, 73.7, 73.1, 72.4, 63.2, 43.2 (sugar ring C); 55.8 (a-CH); 42.7 (d-CH2); 31.0( b-
‡
‡
CH2); 26.5 (g-CH2). FAB-MS: 498.0(C 18H36N5O11, [M ‡ H] ; calc. 498.5).
6-[(4-Aminobutanoyl)amino]-6-deoxy-a,a-trehalose (11). Compound 8 (100 mg, 0.18 mmol) was dissolved
in 15 ml of MeOH and hydrogenated in the presence of 10% Pd/C (16 mg) at atmospheric pressure for 5 h. The
mixture was treated and purified as described above for 10 to provide 11 (64 mg, 84%). White powder. M.p.
122.4 124.38. [a]D ˆ ‡163 (c ˆ 0.85, H2O). 1H-NMR (D2O): 4.98 (dd, J ˆ 3.6, 2 HÀC(1)); 3.70 3.62
(m, 2 HÀC(6'), 2 HÀC(5), 2 HÀC(6)); 3.49 3.43 (m, 2 HÀC(3)); 3.29 3.25 (m, 2 HÀC(2)); 3.17 3.10
(m, 2 HÀC(4)); 2.84 (t, J ˆ 7.5, a-CH2); 2.23 (t, J ˆ 7.5, g-CH2); 1.78 (m, J ˆ 7.5, b-CH2). 13C-NMR (D2O): 177.9
(NHCO), 96.1, 95.9, 75.3, 75.0, 74.9, 73.9, 73.8, 73.4, 72.4, 63.2, 42.5 (sugar ring C); 41.6 (g-CH2); 35.2 (a-CH2);
‡
‡
26.0( b-CH2). FAB-MS: 427.0(C 16H31N2O11, [M ‡ H] ; calc. 427.4).
6-Deoxy-6-(glycylamino)-a,a-trehalose (12). According to a method analogous to that for 11, 9 (100 mg,
0.19 mmol) was hydrogenated to afford 12 (61 mg, 82%). White powder. M.p. 115.0 117.0 8. [a]D ˆ ‡140( c ˆ
0.80, H2O). 1H-NMR (D2O): 4.99 (d, J ˆ 3.6, 2 HÀC(1)); 3.71 3.60( m, 2 HÀC(6'), 2 HÀC(5), 2 HÀC(6));
3.55 3.44 (m, 2 HÀC(3), 2 HÀC(2)); 3.35 3.25 (m, 2 HÀC(4)); 3.16 (t, J ˆ 9.3, CH2). 13C-NMR (D2O): 171.3
(NHCO), 96.2, 96.0, 75.2, 75.0, 74.9, 73.7, 73.3, 72.3, 63.1, 43.4 (sugar ring C); 42.5 (a-CH2). FAB-MS: 399.0
‡
‡
(C14H27N2O11, [M ‡ H] ; calc. 399.3).
6-Deoxy-6-guanidino-a,a-trehalose (13). To a soln. of the S-methylisothiourea sulfate (245 mg, 1.76 mmol)
in H2O (5 ml) was added the conc. aq. NH3 (0.20 ml) with stirring at 08. After 1 h, 6 (150mg, 0.44 mmol) was
added. The mixture was stirred at 858 for 48 h. The solvent was concentrated under reduced pressure, and the
residue was applied to reversed-phase C-18 chromatography (30 Â 1 cm, eluted with H2O). The product-
containing fraction was further purified on a Sephadex LH-20 column (50 Â 1 cm, eluted with 5% MeOH).
After concentration of the eluate, the residue was dissolved in a small amount of H2O. The frozen aq. soln. was
lyophilized to give 13 (93 mg, 55%). Colorless powder. M.p. 122.0 124.0 8. [a]D ˆ ‡139 (c ˆ 0.84, H2O).
1H-NMR (D2O): 5.03 (dd, J ˆ 3.6, 2 HÀC(1)); 3.80 3.60 ( m, 2 HÀC(6'), 2 HÀC(5), 2 HÀC(6)); 3.53 3.46
(m, 2 HÀC(3)); 3.42 3.32 (m, 2 HÀC(2)); 3.25 (t, J ˆ 9.6, 2 HÀC(4)). 13C-NMR (D2O): 160.4 (guanidino C);
‡
96.3, 96.1, 75.2, 75.0, 74.9, 73.7, 73.6, 73.5, 73.3, 72.3, 63.2, 44.7 (sugar ring C). FAB-MS: 384.0 (C13H26N3O10,
‡
[M ‡ H] ; calc. 384.4).
6-Deoxy-6-{[(guanidino)acetyl]amino}-a,a-trehalose (14). From 12 (50mg, 0.13 mmol) by a method
analogous to that for 13: 14 (25 mg, 45%). Colorless powder. M.p. 214.08 (dec.). [a]D ˆ 162 (c ˆ 0.92, H2O).
1H-NMR (D2O): 4.98 (dd, J ˆ 3.6, 2 HÀC(1)); 3.71 3.60( m, 2 HÀC(6'), 2 HÀC(5), 2 HÀC(5)); 3.55 3.44
(m, 2 HÀC(3), 2 HÀC(2)); 3.35 3.25 (m, 2 HÀC(4)); 3.16 (t, J ˆ 9.3, CH2). 13C-NMR (D2O): 170.9 (NHCO);
158.1 (guanidino C); 94.0, 93.8, 73.2, 72.9, 72.8, 71.8, 71.6, 71.5, 71.2, 70.2, 61.0, 44.3 (sugar ring C); 40.4 (a-CH2).
‡
‡
FAB-MS: 441.0(C 15H29N4O11, [M ‡ H] ; calc. 441.4).
6-Deoxy-6-[(4-guanidinobutanoyl)amino]-a,a-trehalose (15). From 11 (50mg, 0.12 mmol) by a method
analogous to that for 13: 15 (23 mg, 42%). Colorless powder. M.p. 176.0 178.0 8. [a]D ˆ ‡142 (c ˆ 0.86, H2O).
1H-NMR (D2O): 4.98 (dd, J ˆ 3.6, 2 HÀC(1)); 3.70 3.57 ( m, 2 HÀC(6'), 2 HÀC(5), 2 HÀC(6)); 3.49 3.44
(m, 2 HÀC(3)); 3.29 3.22 (m, 2 HÀC(2)); 3.18 3.11 (m, 2 HÀC(4)); 3.04 (t, J ˆ 7.2, a-CH2); 2.19 (t, J ˆ 7.5, g-
CH2); 1.71 (m, J ˆ 7.2, b-CH2). 13C-NMR (D2O): 176.5 (NHCO); 157.4 (guanidino C); 93.9, 93.7, 73.2, 72.9, 72.8,

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Helvetica Chimica Acta Vol. 86 (2003)
71.8, 71.7, 71.6, 71.2, 70.2, 61.1, 40.9 (sugar ring C); 40.4 (g-CH2); 33.1 (a-CH2); 25.0( b-CH2). FAB-MS: 469.4
‡
‡
(C17H33N4O11, [M ‡ H] ; calc. 469.5).
Tat-Dependent HIV-1 LTR-Driven CAT Assays. 293T Cells were maintained in Dulbecco×s modified Eagle
medium (DMEM; GIBCO BRL) supplemented with 10% fetal calf serum and antibiotics at 378 in 5% CO2 in
an incubator. Cells were seeded into 6-well plates the day prior to transfection. 293T cells were co-transfected
with plasmid pCepIII-CAT containing the HIV-1 LTR linked to a CAT reporter gene and plasmid pSV2-Tat
expressing Tat protein in a ratio of 1:1 by the calcium phosphate method. After transfection for 24 h at 378, the
medium was discarded and replaced with fresh medium containing the tested compounds (final concentration
30 mm). All tested compounds were cultured for an additional 24 h. CAT Expression was determined with a
commercial CAT ELISA kit (Boehringer Mannheim).
The project was supported by the National Natural Science Foundation of China and Special Fund for
Promotion Education, Ministry of Education of China.
REFERENCES
[1] S. M. Kingsman, A. J. Kingsman, Eur. J. Biochem. 1996, 240, 491.
[2] J. Karn, M. A. Graeble, Trends Genet. 1992, 8, 365.
[3] J. Tao, A. D. Frankel, Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1571.
[4] X. Wang, I. Huq, T. M. Rana, J. Am. Chem. Soc. 1997, 119, 6444.
[5] N. Tamilarasu, I. Huq, T. M. Rana, J. Am. Chem. Soc. 1999, 121, 1597.
[6] W. A. O×Brien, M. Sumner-Smith, S. H. Mao, S. Sadeghi, J. Q. Zhao, I. S. Chen, J. Virol. 1996, 70, 2825.
[7] F. Hamy, E. R. Felder, G. Heizmann, J. Lazdins, F. Aboul-Ela, G. Varani, J. Karn, T. Klimkait, Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 3548.
[8] H. Y. Mei, A. A. Galan, N. S. Halim, D. P. Mack, D. W. Moreland, K. B. Sanders, H. N. Truong, A. W.
Czarnik, Bioorg. Med. Chem. Lett. 1955, 5, 2755.
[9] A. Litovchick, A. G. Evdokimov, A. Lapidot, Biochemistry 2000, 39, 2838.
¬
[10] C. Cabrera, A. Gutierrez, J. Blanco, J. Barretina, A. Litovchick, A. Lapidot, A. G. Evdokimov, B. Clotet,
¬
J. A. Este, Hum. Retroviruses 2000, 16, 627.
¬
¬
[11] C. Cabrera, A. Gutierrez, J. Barretina, J. Blanco, A. Litovchick, A. Lapidot, B. Blotet, J. A. Este, Antiviral
Res. 2002, 53, 1.
[12] I. Choudhury, J. Wang, S. Stein, A. Rabson, M. J. Leibowitz, J. Gen. Virol. 1999, 80, 777.
[13] N. Tamilarasu, I. Huq, T. M. Rana, Bioorg. Med. Chem. Lett. 2001, 11, 505.
[14] I. Huq, Y. Ping, N. Tamilarasu, T. M. Rana, Biochemistry 1999, 38, 5172.
[15] S. Hanessian, M. M. Ponpipom, P. Lavallee, Carbohydr. Res. 1972, 24, 45.
[16] A. Liav, M. B. Goren, Carbohydr. Res. 1980, 87, 287.
[17] S. Hanessian, P. Lavallee, J. Antibiot. 1972, 25, 683.
[18] W. A. Greenberg, E. Scott Priestley, P. S. Sears, P. B. Alper, C. Rosenbohm, M. Hendrix, S. C. Hung, C. H.
Wong, J. Am. Chem. Soc. 1999, 121, 6524.
[19] A. A. Malik, S. B. Preston, T. G. Archibald, M. P. Cohen, K. Baum, Synthesis 1989, 450.
[20] N. S. Chandrakumar, Synth. Commun. 1996, 26, 2613.
Received January 21, 2003