Synthesis and anti-HIV activity of [ddN]-[ddN] dimers and benzimidazole nucleoside dimers

Article abstract of DOI:10.1002/cbdv.200800281

In an attempt to combine the HIV-inhibitory capacity of different 2′,3′-dideoxynucleoside (ddN) analogs, we have designed and synthesized several dimers of [AZT]-[AZT] and [AZT]-[d4T]. In addition, we also synthesized the dimers of 1-(1H-benzimidazol-1-yl

Full text of DOI:10.1002/cbdv.200800281

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Synthesis and Anti-HIV Activity of [ddN]-[ddN] Dimers and Benzimidazole
Nucleoside Dimers
by Guo-Rui Lia)1), Jun Liub)1), Qin Panb)1), Zhi-Bin Songa), Feng-Ling Luob), Shao-Ru Wanga),
Xiao-Lian Zhang*b), and Xiang Zhou*a)
a) College of Chemistry and Molecular Sciences, Wuhan University, Hubei, Wuhan, 430072, P. R. China
(phone: þ86-27-61056559; fax: þ86-27-87336380; e-mail: xzhou@whu.edu.cn)
b) State Key Laboratory of Virology, Department of Immunology, Hubei Province Key Laboratory of
Allergy and Immunology, Wuhan University School of Medicine, Wuhan, 430071, P. R. China
(phone: þ86-27-61056559; fax: þ86-27-87336380; e-mail: zhangxl65@whu.edu.cn)
In an attempt to combine the HIV-inhibitory capacity of different 2’,3’-dideoxynucleoside (ddN)
analogs, we have designed and synthesized several dimers of [AZT]-[AZT] and [AZT]-[d4T]. In
addition, we also synthesized the dimers of 1-(1H-benzimidazol-1-yl)-1-deoxy-b-d-ribofuranose. The in
vitro anti-HIVactivity of these compounds on a pseudotype virus, pNL4-3.Luc.R-E-, in the 293T cells has
been determined. Among these compounds, 2,2’-(propane-1,3-diyl)bis[1-(b-d-ribofuranosyl)-1H-benz-
imidazole] (3) showed the highest anti-HIV activity with similar effect as AZT.
Introduction. – Since the human immunodeficiency virus (HIV) was identified as
the etiological agent for AIDS in 1983 [1], many anti-HIV drugs have been discovered
and evaluated based on different targets, including nucleoside reverse-transcriptase
inhibitors (NRTIs), non-nucleoside reverse-transcriptase inhibitors (NNRTIs), pro-
tease inhibitors, entry inhibitors, and so on [2]. Among them, 2’,3’-dideoxynucleosides
(ddN) such as 3’-azido-2’,3’-dideoxythymidine (¼1-(3-azido-2,3-dideoxypentofurano-
syl)-5-methylpyrimidine-2,4(1H,3H)-dione; AZT; Fig. 1) [3], 2’,3’-didehydro-2’,3’-
dideoxythymidine
(¼1-[5-hydroxymethyl)-2,5-dihydrofuran-2-yl]-5-methylpyrimi-
dine-2,4(1H,3H)-dione; d4T; Fig. 1) [4] have been found to be effective in the
treatment of HIV-infected patients and are the early drugs approved by the FDA [5].
Unfortunately, drug resistance gradually emerges to these nucleoside based inhibitors
(nucleoside reverse-transcriptase inhibitors, NRTIs) [6], and unexpected side effects
become increasingly obvious [7]. So, there is still continued interest in developing novel
strategies and new drugs for the treatment of HIV/AIDS.
A combination of anti-HIV agents is now being one kind of clinical application as
therapeutic modalities to prevent emergence of virus-drug resistance [8]. Camarasa
and co-workers [9–11] have reported the synthesis and anti-HIV activity of a series of
heterodimers which combine an NRTI analogue and an NNRTI (non-nucleoside
reverse-transcriptase inhibitor) through appropriate spacers. In their studies, the nature
of the spacer and the position of the linker on the NRTI had little effect on the antiviral
activity. In contrast, the nature of the NRTI was shown to be important for the anti-
1
)
These authors contributed equally to this work.
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Fig. 1. Structures of AZT and d4T
HIV activities [11]. In order to fulfill the work in this field and evaluate the synergistic
effect of different 2’,3’-dideoxynucleoside (ddN) analogues, we have designed and
synthesized several dimers of the general formula [ddN]-spacer-[ddN] (Fig. 2). As ddN
inhibitors, we chose AZT and d4T (the drugs used in the clinical treatment of AIDS).
The ddN inhibitors were linked at the N(3) of the thymidine base of each compound by
an appropriate spacer. We also performed several modifications in the spacer to study
the effect of spacers on anti-HIV activity.
The derivatives of benzimidazole nucleoside have also been synthesized and exhibit
antiviral activities [12–14]. In this article, we describe the synthesis of dimers of
benzimidazole nucleoside (Fig. 2) for the first time and evaluated their anti-HIV
activities in vitro.
Fig. 2. Structures of 1’-deoxy-1’-(benzimidazol-1-yl)-b-d-ribofuranose and its dimers
Results and Discussion. – 1. Chemistry. The [ddN]-[ddN] dimers were synthesized
based on the method reported in [11], shown in Scheme 1. In this method, firstly AZT
was treated with the appropriate diiodoalkyl or dibromoalkyl reagent to yield the
nucleoside intermediate 4 and 5, then the nucleoside intermediate reacted with the ddN
(AZT, d4T) to give the N(3),N(3)-alkyl dimers 6–9. For the synthesis of [AZT]-
[AZT] dimer, we also tried a one-step method. Using this method, we synthesized
compound 10 in moderate yield.
The monomer 1-(benzimidazol-1-yl)-1-deoxy-b-d-ribofuranose was synthesized
according to the literature [15]. The compounds 2 and 3 were conveniently synthesized
in three steps (Scheme 2). The bis[benzimidazoles] 11 and 12 were synthesized by the
acid-catalyzed condensation of o-phenylenediamine with an appropriate biscarboxylic
acid. The synthesis of protected bisbenzimidazole nucleosides followed the glycosy-
lation procedure described in [15]. Refluxing bisbenzimidazoles with 2 equiv. of N,O-

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Scheme 1
a) K2CO3, acetone, (CH2)4I2 for 4, or K2CO3, acetone/DMF (1:1), BrCH2PhCH2Br for 5, reflux. b)
AZT, K2CO3, MeCN, reflux. c) d4T, K2CO3, MeCN, reflux. d) K2CO3, acetone, (CH2)3Br2, reflux.
bis(trimethylsilyl)acetamide and subsequent reaction of the persilylated base with
2 equiv. of 1,2,3,5-tetra-O-acetyl-b-d-ribofuranose in the presence of the Lewis acid
trimethylsilyl trifluoromethanesulfonate afforded the desired compound 13 and 14.
Deprotection of the acetylated nucleoside furnished the nucleosides 2 and 3.
The structures of all the target compounds were identified by 1H-NMR, 13C-NMR,
and high-resolution mass spectroscopy (HR-MS).
2. Anti-HIV Activity. The anti-HIV activity of these compounds was tested using a
pseudotype virus, pNL4-3.Luc.R-E-, in HEK 293T cells. The HIV-1 recombinant clone
has been widely investigated by biologists [16–18], and recently has also been used by
medicinal chemists for exploration of anti-HIV drugs [19].

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Scheme 2
a) 4n HCl, 1358. b) Conc. NH4OH. c) N,O-bis(trimethylsilyl)acetamide (BSA), MeCN. d) 1,2,3,5-tetra-
O-acetyl-b-d-ribofuranose (TAR), trimethylsilyl trifluoromethanesulfonate (TMSOTf), MeCN. e)
MeONa, MeOH.
In our study, an HIV pseudotype virus was produced by transfection of 293T cells
with plasmids pNL4-3.Luc.R-E- containing the env deficient HIV proviral genome and
an intact luciferase gene, and cotransfected with pGL4.73 [hRluc/SV40] as internal
control reporters. The replications of the HIV pseudotype in 293T cells were measured
through normalizing the transfection efficacy with luciferase and Renilla luciferase.
The relative luciferase activities represented the viral replication efficacy of HIV
pseudotype. To detect the effects of the different compounds on the HIV replication,
different compounds (with the same concentration 65 mm) were added to each HIV
proviral genome transfected cells, respectively. The preliminary results were given. As
shown in Fig. 3, for the [ddN]-[ddN] dimers, they all had some inhibition effects on the
HIV replications comparing with the control group without drugs but are less potent
than their parent compounds AZT and d4T (the result is consistent with the literature
[20]). What interests us most is, for the benzimidazole nucleosides, although the
monomer 1 and the dimer 2 showed 37% inhibition effect, the dimer 3, only different in
the length of the linker with 2, showed a ca. 60% inhibitory effect. It had a higher
activity than d4T and can inhibit the HIV replication almost at the same level as AZT.
To validate these results, we selected several compounds for further study. We
measured the expression of HIV p24 by ELISAusing anti-p24 monoclonal antibody. As
shown in Fig. 4, for the [ddN]-[ddN] dimer 7 and 9, the expression of HIV p24 was
higher than AZT and d4T. And the benzimidazole nucleoside dimer 3 showed lower
expression than d4T. The results were consistent with that of relative luciferase
activities.
In conclusion, we have synthesized the [ddN]-[ddN] dimers and several benzimi-
dazole nucleosides and tested their anti-HIV activity. The benzimidazole nucleoside
dimer 3 showed potent anti-HIV activity and has the potential as a new kind of anti-

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 3. Relationship between drugs and luciferase activity of the pseudotype reporter viruses. Blank refers
to no virus inoculated. The experiments have been repeated three times. Data are means of triplicate
wells of one representative experiment and the error bars represent the standard errors of the means.
Fig. 4. Relationship between drugs and p24 expression. Control refers to no drug added. P24 Expression
of control was arbitrarily assigned a value of 100%. Ratio of p24 expression was compared with that of
control. The experiments have been repeated three times. Data are means of triplicate wells of one
representative experiment and the error bars represent the standard errors of the means.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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HIVagent. Further structure modification on compound 3 and an in-depth study on the
HIV replication inhibitory activity of it are currently in progress.
Experimental Part
General. AZT and d4T were purchased from Tianzun Co. (P. R. China). All other chemicals and
solvents were commercially available. Anh. solvents (acetone, DMF, MeCN) were prepared by standard
methods. NMR Spectra: Varian Mercury-VX300 spectrometer, at 300 and 75 MHz, resp. MS: Bruker
Daltonics APE XII 47e and VG-707VHF mass spectrometer.
General Procedure for Preparation of 4 and 5. To a soln. of the nucleoside AZT in acetone was added
K2CO3 and (CH2)4I2 or BrCH2C6H4CH2Br (2 equiv.). The mixture was refluxed for 20 h. After
evaporation of the solvent, the residue was dissolved in AcOEt, washed with H2O, dried (Na2SO4),
filtered, and evaporated to dryness. The residue was purified by silica gel (SiO2) column chromatography
(CC) using a mixture of CHCl3/MeOH (30 :1, v/v) as the eluent to afford the compounds 4 and 5.
3’-Azido-3-N-(4-iodobutyl)-3’-deoxythymidine (¼1-(3-Azido-2,3-dideoxy-b-erythro-pentofurano-
syl)-3-(4-iodobutyl)-5-methylpyrimidine-2,4(1H,3H)-dione; 4). Yield: 650 mg (77%). Yellow syrup.
1H-NMR (CDCl3): 7.35 (s, HꢀC(6)); 6.04 (t, J¼6.0, HꢀC(1’)); 4.39 (m, HꢀC(3’)); 4.00–3.77 (m,
CH2(5’), HꢀC(4’), HOꢀC(5’), CH2N); 3.20 (t, J¼6.2, CH2I); 2.59–2.35 (m, CH2(2’));1.92 (s,
MeꢀC(5)); 1.88–1.83 (m, CH2); 1.74 (m, CH2). ESI-HR-MS: 472.0452 ([MþNa]þ , C14H20IN5NaO4þ ;
calc. 472.0458).
3’-Azido-3-N-(a-bromo-p-xylenyl)-3’-deoxythymidine (¼1-(3-Azido-2,3-dideoxy-b-erythro-pento-
furanosyl)-3-[4-(bromomethyl)benzyl]-5-methylpyrimidine-2,4(1H,3H)-dione; 5). Yield: 107 mg
(24%). White syrup. 1H-NMR (CDCl3): 7.46 (d, J¼8.1, 2 arom. H); 7.36 (s, HꢀC(6)); 7.32 (d, J¼8.1,
2 arom. H); 6.07 (t, J¼6.6, HꢀC(1’)); 5.09 (s, PhCH2); 4.46 (s, CH2I); 4.40–4.38 (m, HꢀC(3’)); 3.96–
3.80 (m, CH2(5’), HꢀC(4)); 2.64–2.41 (m, CH2(2’), HOꢀC(5’)); 1.93 (s, MeꢀC(5)).
General Procedure for Preparation of 6–9. To a soln. of the nucleoside intermediates 4 and 5 in dry
MeCN was added K2CO3 and ddN (AZT, d4T). The mixture was refluxed for 26 h. After evaporation of
the solvent, the residue was dissolved in AcOEt, washed with H2O, dried (Na2SO4), filtered, and
evaporated to dryness. The residue was purified by SiO2 CC using a mixture of CHCl3/MeOH 20 :1 as the
eluent.
[AZT]N3-(CH2)4-N3[AZT] (6). Yield: 514 mg (87%). White solid. 1H-NMR (CDCl3): 7.28 (s,
CH2(6)); 6.01 (t, J¼6.5, CH2(1’)); 4.45–4.39 (m, CH2(3’)); 4.01–3.78 (m, CH2(5’), HꢀC(4), CH2N);
2.66–2.38 (m, 2 CH2(2’)); 1.93 (s, 2 MeꢀC(5)); 1.68 (br., CH2CH2). 13C-NMR (CDC13): 163.5 (C¼O);
151.0 (C¼O); 135.1 (C(6)); 110.7 (C(5)); 87.9 (C(1’)); 84.7 (C(4’)); 62.2 (C(5’)); 60.1 (C(3’)); 41.3
(CH2N); 37.4 (C(2’)); 25.3 (CH2); 13.6 (MeꢀC(5)). ESI-HR-MS: 611.2297 ([MþNa]þ , C24H32N10NaO8þ ;
calc. 611.2302).
[AZT]N3-CH2C6H4CH2-N3[AZT] (7). Yield: 100 mg (71%). White solid. 1H-NMR (CDCl3): 7.38 (s,
4 arom. H); 7.30 (s, CH2(6)); 6.03 (t, J¼6.3, CH2(1’)); 5.06 (s, 2 C6H4CH2); 4.37 (m, CH2(3’)); 3.99–3.79
(m, 2 CH2(5’), 2 HꢀC(4)); 2.53–2.38 (m, 2 CH2(2’), 2 HOꢀC(5’)); 1.91 (s, 2 MeꢀC(5)). 13C-NMR
(CDC13): 163.0 (C¼O); 150.6 (C¼O); 135.8 (arom. C); 134.5 (C(6)); 129.0 (arom. C); 110.3 (C(5)); 87.1
(C(1’)); 84.2 (C(4’)); 61.7 (C(5’)); 59.6 (C(3’)); 43.9 (CH2N); 37.1 (C(2’)); 13.1 (MeꢀC(5)). ESI-HR-MS:
659.2297 ([MþNa]þ , C28H32N10NaOþ8 ; calc. 659.2302).
[AZT]N3-(CH2)4-N3[d4T] (8). Yield: 159 mg (86%). White solid. 1H-NMR (CDCl3): 7.36 (s,
HꢀC(6)-d4T); 7.27 (s, HꢀC(6)-AZT); 7.02 (br., HꢀC(1’)-d4T); 6.32 (d, J¼6.0, HꢀC(3’)-d4T); 5.98 (t,
J¼6.3, HꢀC(1’)-AZT); 5.87 (d, J¼5.4, HꢀC(2’)-d4T); 4.91 (br., HꢀC(4)-d4T); 4.43–4.39 (m,
HꢀC(3’)-AZT); 3.99–3.77 (m, 2 CH2(5’), HꢀC(4)-AZT, 2 CH2N); 2.65–2.37 (m, CH2(2’)-AZT); 1.92
(s, MeꢀC(5)); 1.89 (s, MeꢀC(5)); 1.68 (m, CH2CH2). 13C-NMR (CDC13): 163.9, 151.7, 150.9 (C¼O);
135.0 (C(6)); 126.6 (C(2’)); 110.3 (C(5)); 90.8 (C(3’)); 87.3 (C(1’)); 84.7 (C(4’)); 63.6, 61.9 (C(5’)); 60.1
(C(3’)); 41.2 (CH2N); 37.6 (C(2’)-AZT); 25.2 (CH2); 13.4 (MeꢀC(5)). ESI-HR-MS: 568.2126 ([Mþ
Na]þ , C24H31N7NaOþ8 ; calc. 568.2132).
[AZT]N3-CH2C6H4CH2-N3[d4T] (9). Yield: 165 mg (77%). White solid. 1H-NMR (CDCl3): 7.38 (s,
4 arom. H); 7.30 (s, CH2(6)); 7.02 (br, HꢀC(1’)-d4T); 6.29 (d, J¼6.0, HꢀC(3’)-d4T); 6.03 (t, J¼6.3,

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
CH2(1’)-AZT); 5.83 (d, J¼5.7, HꢀC(2’)-d4T); 5.06 (s, 2 CH2N); 4.37 (m, CH2(3’)-AZT); 3.99–3.79 (m,
2 CH2(5’), 2 HꢀC(4)-AZT); 2.53–2.38 (m, CH2(2’)-AZT); 1.90 (s, MeꢀC(5)); 1.87 (s, MeꢀC(5)).
13C-NMR (CDC13): 163.0, 150.6 (C¼O); 135.8 (C(6)); 134.5 (C(6)); 129.0 (C(2’)); 110.3 (C(5)); 87.1
(C(1’)); 84.2 (C(4’)); 61.7 (C(5’)); 59.6 (C(3’)); 43.9 (CH2N); 37.1 (C(2’)-AZT); 13.1 (MeꢀC(5)). ESI-
HR-MS: 659.2297 ([MþNa]þ , C28H32N10NaO8þ ; calc. 659.2302).
General Procedure for Preparation of [AZT]N3-(CH2)3-N3[AZT] (10). To a soln. of the nucleoside
AZT (267 mg, 1 mmol) in acetone (50 ml) was added K2CO3 (690 mg, 5 mmol) and (CH2)3Br2 (110 mg,
0.5 mmol). The mixture was refluxed for 72 h. After evaporation of the solvent, the residue was dissolved
in AcOEt (100 ml), washed with H2O (3ꢁ20 ml), dried over Na2SO4, filtered, and evaporated to
dryness. The residue was purified by SiO2 CC using a mixture of CHCl3/MeOH 20 :1 as the eluent to
afford 10 (200 mg, 70%). White syrup. 1H-NMR (CDCl3): 7.49 (s, CH2(6)); 6.06 (t, J¼6.2, CH2(1’)); 4.38–
4.32 (m, CH2(3’)); 3.95–3.74 (m, 2 CH2(5’), 2 HꢀC(4), 2 CH2N); 2.50–2.33 (m, 2 CH2(2’)); 1.92 (m,
CH2); 1.84 (s, 2 MeꢀC(5)). 13C-NMR (CDC13): 163.6 (C¼O); 151.0 (C¼O); 135.3 (C(6)); 110.5 (C(5));
87.8 (C(1’)); 84.8(C(4’)); 62.0 (C(5’)); 60.0 (C(3’)); 39.2 (CH2N); 37.4 (C(2’)); 26.3 (CH2); 13.5
(MeꢀC(5)). ESI-HR-MS: 597.2105 ([MþNa]þ , C23H30N10NaO8þ ; calc. 597.2146).
General Procedure for Preparation of 13 and 14. Under N2, to a suspension of bis[benzimidazole] in
MeCN was added N,O-bis(trimethylsilyl)acetamide (2.4 equiv.) and heated under reflux for 1 h. After
the mixture was cooled to r.t., 1,2,3,5-tetra-O-acetyl-b-d-ribofuranose (2 equiv.) in MeCN and
trimethylsilyl trifluoromethanesulfonate (2.5 equiv.) were added and heated under reflux for 4 h. The
mixture was concentrated under reduced pressure, the residue was purified by SiO2 CC using a mixture of
CHCl3/MeOH 20 :1 as the eluent to afford the compounds 13 and 14.
2,2’-(Methanediyl)bis[1-(2,3,5-tri-O-acetyl-b-d-ribofuranosyl)-1H-benzimidazole] (13). Yield:
306 mg (40%). Yellow solid. 1H-NMR (CDCl3): 7.75 (d, J¼7.5, 2 arom. H); 7.57 (d, J¼7.5, 2 arom.
H); 7.27 (m, 4 arom. H); 6.76 (d, J¼7.2, CH2(1’)); 5.56 (m, CH2(2’)); 5.44 (m, CH2(3’)); 4.87 (s, CH2); 4.41
(m, 2 CH2(5’), 2 HꢀC(4)); 2.20 (s, 2 MeCO); 2.18 (s, 2 MeCO); 0.81 (s, 2 MeCO). 13C-NMR (CDC13):
170.1, 169.6, 169.1 (C¼O); 149.1 (C(2)); 142.9, 132.9, 123.5, 123.1, 120.0, 112.0 (arom. C); 85.8 (C(1’));
79.2 (C(4’)); 70.0 (C(2’)); 69.2 (C(3’)); 63.0 (C(5’)); 29.9 (CH2); 20.7, 20.5, 18.1 (Me). ESI-HR-MS:
765.2609 ([MþH]þ , C37H41O14N4þ ; calc. 765.2614).
2,2’-(Propane-1,3-diyl)bis[1-(2,3,5-tri-O-acetyl-b-d-ribofuranosyl)-1H-benzimidazole] (14). Yield:
243 mg (31%). Yellow solid. 1H-NMR (CDCl3): 7.73 (d, J¼7.5, 2 arom. H); 7.60 (d, J¼7.8, 2 arom. H);
7.25 (m, 4 arom. H); 6.12 (d, J¼6.9, CH2(1’)); 5.58 (t, J¼6.9, CH2(2’)); 5.46 (dd, J¼4.2, 6.6, CH2(3’));
4.43 (d, J¼3.0, 2 CH2(5’)); 4.23 (m, 2 HꢀC(4)); 3.15 (m, 2 CH2); 2.59 (m, CH2); 2.22 (s, 2 MeCO); 2.15
(s, 2 MeCO); 1.86 (s, 2 MeCO). 13C-NMR (CDC13): 170.2, 169.5, 169.2 (C¼O); 154.0 (C(2)); 142.9,
132.6, 122.7, 119.4, 111.5 (arom. C); 86.3 (C(1’)); 79.8 (C(4’)); 70.9 (C(2’)); 69.3 (C(3’)); 62.9 (C(5’)); 26.4,
24.7 (CH2); 20.7, 20.5, 19.9 (Me). ESI-HR-MS: 793.2930 ([MþH]þ , C39H45O14N4þ ; calc. 793.2927).
General Procedure for the Preparation of Compounds 2 and 3. Compounds 13 and 14 were dissolved
in dry MeOH to which was added MeONa (20 equiv.). The soln. was stirred at r.t. for 48 h, and the
solvent was then removed under vacuum. The residue was suspended in H2O, and the suspension was
extracted with AcOEt. The combined org. extracts were dried over Na2SO4, filtered, and evaporated to
yield a white solid. The solid was purified by SiO2 CC using CHCl3/MeOH 8 :1 as the eluent to afford 2
and 3.
2,2’-Bis[1-(b-d-ribofuranosyl)-1H-benzimidazole] (2). Yield: 78 mg (80%). White powder.
1H-NMR (CDCl3/(D6)DMSO): 7.50 (d, J¼5.1, 2 arom. H); 7.18 (d, J¼5.1, 2 arom. H); 6.84 (m, 4
arom. H); 5.84 (d, J¼6.0, CH2(1’)); 4.51 (s, CH2); 4.17 (t, J¼6.8, CH2(2’)); 3.98 (m, CH2(3’)); 3.71 (m,
2 HꢀC(4)); 3.53 (m, 2 CH2(5’)). 13C-NMR ((D6)DMSO): 150.8 (C(2)); 142.8, 134.0, 123.1, 122.8, 119.3,
114.0 (arom. C); 89.4 (C(1’)); 86.5 (C(4’)); 73.0 (C(2’)); 70.1 (C(3’)); 61.9 (C(5’)); 28.2 (CH2). ESI-HR-
MS: 513.1981 ([MþH]þ , C25H29O8N4þ ; calc. 513.1981).
2,2’-(Propane-1,3-diyl)bis[1-(b-d-ribofuranosyl)-1H-benzimidazole] (3). Yield: 100 mg (88%).
1
White powder. H-NMR (CDCl3/(D6)DMSO): 7.68 (m, 2 arom. H); 6.82 (m, 4 arom. H); 6.69 (m, 2
arom. H); 6.00 (d, J¼6.0, CH2(1’)); 4.89 (br. s, 2 OH); 4.55 (m, CH2(2’)); 4.26 (m, CH2(3’)); 3.96 (m,
2 CH2); 3.76 (m, 2 HꢀC(4)); 3.20 (m, 2 CH2(5’)); 2.69 (m, CH2). 13C-NMR ((D6)DMSO): 160.2 (C(2));
148.0, 138.5, 127.3, 123.9, 118.5 (arom. C); 93.7 (C(1’)); 91.2 (C(4’)); 77.0 (C(2’)); 75.1 (C(3’)); 66.8
(C(5’)); 32.1, 30.5 (CH2). ESI-HR-MS: 541.2298 ([MþH]þ , C27H33O8N4þ ; calc. 541.2293).

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Cells. HEK293T Cell lines were cultured in Dulbecco modified Eagleꢂs medium supplemented with
10% fetal calf serum, 100 U of penicillin, and 100 mg of streptomycin per ml.
Plasmids. The plasmid pNL4-3.Luc.R-E- (NIH) was a non-infectious HIV-1 recombinant clone, into
which the firefly luciferase gene was inserted into the pNL4–3 nef and two frameshifts (5’ Env and Vpr
aa 26) rendered this clone Env- and Vpr- and allowed only a single cycle of replication. The plasmid
pGL4.73 [hRluc/SV40] (Promega) was intended for use as internal control reporters to co-transfect
HEK 293T cells with pNL4-3.Luc.R-E-.
Cells Transfection. Plate 2ꢁ105 cells per well in 500 ml Dulbecco modified Eagleꢂs medium without
antibiotics or serum in 24-well, sterile micro titer plates. The cells of each well were transfected with
0.8 mg of pNL4-3.Luc.R-E- and 0.1 mg phRL (Promega), and incubated with or without different
compounds for 48 h.
Luciferase and Renilla Luciferase Activity Assay. After 48 h, the growth medium was removed from
the cultured cells and gently applied a sufficient volume of PBS (500 ml) to rinse the cells on the bottom
of the culture platel. 100 ml 1X luciferase assay lysis buffer were added into each well and the cells were
harvested immediately. Fifty ml of luciferase assay reagent and 20 ml of cell lysate were added to the
luminometer tube. The mixture was stirred and the tube placed in a luminometer for luciferase activity
measurement. Fifty ml of renilla luciferase assay reagent and 20 ml of the same lysate sample were added
to the luminometer tube for renilla luciferase activity measurement.
Expressions of p24 Detection. HEK 293T Cells were transfected with plasmids and incubated with or
without different chemical compounds for 48 h as aforementioned. Then the cells were rinsed with PBS
and lysed with 1% Triton-100/PBS. Expressions of p24 were detected by enzyme-linked immunosorbent
assay using HIV-p24 diagnostic kit. Briefly, 50 ml of the cell lysats were incubated with each well of 96-
well plate coated with p24 MAb for 40 min at 378. The plate was then washed five times with wash buffer
and incubated with the polyclonal HRP-anti-p24 for 30 min at 378. After incubation, the plates were
washed again five times with wash buffer, and 100 ml of a 1:1 soln. of HRP substrate/H2O2 was added.
The substrate was incubated for 10 min at 378 before adding the stop solution, and the reading was done
at the absorbance 450 nm. Assays were performed in triplicate.
This work was supported by the grants from the 973 Program 2006CB504300, and Scientific
Research Grants from Hubei Province Science Technology Department (2006ABD007), Ministry of
Health of China, Key Infectious Diseases Projects (2008ZX10301), and the National Natural Science
Foundation of China (30370310, 20532020, and 30670098, 20621502). National Science Fund for
Distinguished Young Scholars (No. 20425206), the Cultivation Fund of the Key Scientific and Technical
Innovation Project, the Ministry of Education of China (No. 706040), the State Key Laboratory of Natural
and Biomimetic Drugs and State Key Laboratory of Applied Organic Chemistry.
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Received September 13, 2008