Synthesis and antiviral activity evaluation of novel 2-phenyl-4-(D-arabino-4′-cycloaminobutyl)triazoles: Acyclonucleosides containing unnatural bases

Article abstract of DOI:10.1016/S0968-0896(01)00349-2

Five 2-phenyl-4-(D-arabino-4′-cycloamino-3′-hydroxy-O-1′,2′-isopropylidenebutyl)-2H-1,2,3-triazoles, acyclonucleosides containing unnatural bases have been synthesised by opening of the epoxide ring of 2-phenyl-4-(D-arabino-3′,4′-epoxy-O-1′,2′-isopropylidenebutyl)-2H-1,2,3-triazole with the corresponding cyclic amine in 70-85% yields. The starting arabino-epoxytriazole was prepared in five steps starting from D-glucose in an overall yield of 15%. All the five triazolylacycllonucleosides were unambiguously identified on the basis of their spectral data. The structure of one of the intermediates, that is 2-phenyl-4-(D-arabino-1′,2′,3′,4′-tetrahydroxybutyl)-2H-1,2,3-triazole was confirmed by its X-ray crystallographic studies. These acyclonucleosides were subjected to antiviral evaluation in CEM-SS cell-based anti HIV assay with the lymphocytropic virus strains HIV-1_IIIB and HIV-1_RF. Copyright

Full text of DOI:10.1016/S0968-0896(01)00349-2

Bioorganic & Medicinal Chemistry 10 (2002) 963–968
Synthesis and Antiviral Activity Evaluation of Novel 2-Phenyl-4-
(D-arabino-40-cycloaminobutyl)triazoles: Acyclonucleosides
Containing Unnatural Bases
Himanshu,a Rahul Tyagi,a Carl E. Olsen,b William Errington,c
Virinder S. Parmara and Ashok K. Prasada,*
aDepartment of Chemistry, University of Delhi, Delhi-110 007, India
bChemistry Department, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, Frederiksberg C,
DK-1871 Copenhagen, Denmark
cDepartment of Chemistry, University of Warwick, Coventry CV47AL, UK
Received 11 July 2001; accepted 2 October 2001
Abstract—Five 2-phenyl-4-(d-arabino-40-cycloamino-30-hydroxy-O-10,20-isopropylidene-butyl)-2H-1,2,3-triazoles, acyclonucleosides
containing unnatural bases have been synthesised by opening of the epoxide ring of 2-phenyl-4-(d-arabino-30,40-epoxy-O-10,20-iso-
propylidenebutyl)-2H-1,2,3-triazole with the corresponding cyclic amines in 70–85% yields. The starting arabino-epoxytriazole was
prepared in five steps starting from d-glucose in an overall yield of 15%. All the five triazolylacyclonucleosides were unambiguously
identified on the basis of their spectral data. The structure of one of the intermediates, that is 2-phenyl-4-(d-arabino-10,20,30,40-tet-
rahydroxybutyl)-2H-1,2,3-triazole was confirmed by its X-ray crystallographic studies. These acyclonucleosides were subjected to
antiviral activity evaluation in CEM-SS cell-based anti HIV assay with the lymphocytropic virus strains HIV-1IIIB and HIV-1RF
# 2002 Elsevier Science Ltd. All rights reserved.
.
Introduction
deaminase and glycosidic cleavage by nucleoside phos-
phorylases is also a limiting factor in the development of
nucleoside-based antiviral agents.8,9 To overcome these
inactivation problems, a series of nucleoside analogues
were synthesised in which the cyclic carbohydrate moi-
ety was replaced by an acyclic side chain and these are
called acyclonucleosides.10ꢀ15 Acyclovir is one of the
most important compounds in this class and it has been
marketed as an antiherpes drug for over a decade.16
Acyclonucleoside derivatives having unnatural bases are
reported in the literature for their wide range of biolo-
gical activities. Taking lead from these reports, we have
synthesised triazolylacyclonucleosides having unnatural
bases and have evaluated them for antiviral activity in
standard CEM-SS cell-based microtiter anti- HIV assay
with two strains of HIV viruses.
For several years, there has been an intensive search for
drugs effective in the chemotherapy of viral diseases like
AIDS, herpes simplex and cytomegaloviruses.1ꢀ4 While
many antibacterial drugs have been developed over the
past 50 years, relatively few drugs are in clinical use as
antiviral agents at the present time.5 Most of these drugs
are analogues of naturally occurring nucleosides.6
Among these drugs, AZT is currently one drug that has
been shown to decrease the mortality and frequency of
opportunistic infections associated with AIDS. How-
ever, AZT is far from being the ideal antiviral agent,
since bone marrow toxicity and development of resis-
tant viruses can limit its usefulness as long term chemo-
therapeutic agent for AIDS and related complexities.7
Further, the stability of natural nucleosides and their
analogues towards the major pathways of nucleoside
inactivation, for example deamination by adenosine
Results and Discussion
Five triazolylacyclonucleosides, that is 2-phenyl-4-[d-
arabino-30-hydroxy-O-10,20-isopropylidene-40-(pyrrolidin-
1-yl)butyl]-2H-1,2,3-triazole (5), 2-phenyl-4-[d-arabino-
*Corresponding author. Tel.: +91-11-766-6555; fax: +91-11-766-
7206; e-mail: minuashok@now-india.net.in
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00349-2

964
?. Himanshu et al. / Bioorg. Med. Chem. 10 (2002) 963–968
30-hydroxy-O-10,20-isopropylidiene-40-(piperidin-1-yl)bu-
tyl]-2H-1,2,3-triazole (6), 2-phenyl-4-[d-arabino-40-(2000-
ethylpiperidin-1-yl)-30 -hydroxy-O-10,20-isopropylidene-
butyl]-2H-1,2,3-triazole (7), 2-phenyl-4-[d-arabino-40-
(4000-benzylpiperidin-1-yl)-30-hydroxy-O-10,20-isopropyl-
idenebutyl]-2H-1,2,3-triazole (8) and 2-phenyl-4-[d-ara-
bino-30-hydroxy-O-10,20-isopropylidene-40-(N-methylpip-
erazin-1-yl)butyl]-2H-1,2,3-triazole (9) were synthesised
by opening of the epoxide ring of 2-phenyl-4-(d-arabino-
30,40-epoxy-O-10,20-isopropylidenebutyl)-2H-1,2,3-triazole
(4) with pyrrolidine, piperidine, 2-ethylpiperidine, 4-
benzylpiperidine and N-methylpiperazine, respectively,
in 70–85% yields. All the five triazolylacyclonucleosides
5–9 were unambiguously identified on the basis of their
spectral data.
p-toluenesulfonylbutyl)-2H-1,2,3-triazole (2) in 90%
yield, which was characterised on the basis of its spec-
tral data. The tosylated triazolylsugar 2 was treated
with anhydrous acetone in the presence of anhydrous
FeCl3 to form exclusively the 10,20-isopropylidene
derivative 2-phenyl-4-(d-arabino-30-hydroxy-O-10,20-iso-
propylidene-40-O-p-toluenesulfonylbutyl)-2H-1,2,3-tria-
zole (3) in a regioselective fashion in 86% yield, which
on refluxing with sodium hydride in toluene resulted in
the formation of the epoxide 4; the formation of 30,40-
epoxide 4 also supports the O-10,20-isopropylidene pro-
tection in compound 3. The compounds 2–9 are new to
the literature as they have not been synthesised earlier.
X-ray crystallography
The epoxide 4, precursor of acyclonucleosides 5–9 was
synthesised in four steps starting from the conversion of
glucose to its phenylosazone, followed by oxidative
cyclisation of the osazone with 1% aqueous CuSO4 to
yield the intermediate compound 2-phenyl-4-(d-arabino-
10,20,30,40-tetrahydroxybutyl)-2H-1,2,3-triazole (1) in an
overall yield of 43% (Scheme 1).17 The structure of
triazolyl sugar 1 was established on the basis of spectral
data and by comparison of its mp and spectral data
with those reported in the literature.18ꢀ20 Finally, the
structure of compound 1 was confirmed by X-ray crys-
tallographic studies (Fig. 1). The selective tosylation of
compound 1 with one equivalent of TsCl in pyridine
afforded 2-phenyl-4-(d-arabino-10,20,30-trihydroxy-40-O-
The molecular structure of 2-phenyl-4-(d-arabino-
10,20,30,40-tetrahydroxybutyl)-2H-1,2,3-triazole (1) was
further confirmed by single crystal X-ray diffraction
study. The schematic representation of the molecular
structure of compound 1 is illustrated in Figure 1, bond
lengths [A] and bond angles [ꢁ] are given in Table 1 and
other crystallographic data are summarised in the
Experimental.
Antiviral activity evaluation
Triazolylacyclonucleosides 5–9 and their precursor 4
were evaluated for antiviral efficacy against two strains
of HIV-1(HIV-1IIIB and HIV-1RF) in the standard
CEM-SS cell-based anti-HIV assay (cf. Experimental).
In this assay system, none of these six compounds
reached an IC50 value at the highest test concentration
evaluated (100 mM), most of the compounds were cyto-
toxic at higher concentrations, except compound 4. The
overall assay performance was validated by the MOI-
sensitive positive control compound 30-azidothymidine
(AZT), exhibiting the expected level of antiviral activity
(IC50=0.003 mM against HIV-1IIIB and 0.006 mM
against HIV-1RF). Microscopic and macroscopic obser-
vations of the cells in each well of the microtiter plate
confirmed the efficacy and cytotoxicity results obtained
following staining of the cells with the MTS metabolic
dye. The data obtained in these assays is given in Table
2.
Scheme 1. Reagents and conditions: (i) C6H5NHNH2, 65–70 ꢁC; (ii)
.
1% aq CuSO4 5H2O, reflux; (iii) p-toluenesulfonyl chloride, pyridine,
25–27 ꢁC; (iv) acetone, anhydrous FeCl3, 25–27 ꢁC; (v) toluene, NaH,
reflux; (vi) cyclic amine, triethylamine, MeOH, 25–27 ꢁC.
Figure 1. Molecular structure of 2-phenyl-4-(d-arabino-10,20,30,40-tet-
rahydroxybutyl)-2H-1,2,3-triazole (1).

?. Himanshu et al. / Bioorg. Med. Chem. 10 (2002) 963–968
965
Table 1. Bond lengths and bond angles for the molecular structure of
2-phenyl-4-(d-arabino-10,20,30,40-tetrahydroxybutyl)-2H-1,2,3-triazole
light or on charring with 5% alcoholic H2SO4. Solvent
systems used for TLC were: A (methanol/chloroform,
1:9), B (methanol/chloroform, 1:99), C (methanol/
chloroform, 1:49) and D (ethyl acetate/petroleum ether,
1:9). All the organic solvents were dried and distilled
prior to their use. The antiviral efficacy and cellular
cytotoxicity of compounds 4–9 and AZT have been
evaluated at Southern Research Institute, MD, USA.
(1)a
Bond lengths (A)
O (1)–C (9)
Bond angles (ꢁ)
C (7)–N (1)–N (2)
1.430 (5)
1.435 (5)
1.339 (6)
1.333 (5)
1.329 (6)
1.387 (7)
1.371 (8)
1.380 (7)
1.490 (6)
1.512 (6)
103.5 (4)
123.8 (4)
104.8 (4)
119.5 (5)
118.0 (5)
119.2 (6)
119.5 (5)
108.1 (4)
131.1 (5)
110.2 (4)
108.3 (4)
113.5 (4)
109.1 (4)
111.1 (4)
114.5 (4)
121.6 (4)
121.4 (5)
119.1 (4)
121.4 (6)
120.4 (5)
109.0 (4)
120.7 (4)
107.4 (4)
112.3 (4)
110.5 (4)
109.9 (4)
114.6 (4)
O (3)–C (11)
N (1)–C (7)
N (2)–N (3)
N (3)–C (8)
C (1)–C (2)
C (3)–C (4)
C (5)–C (6)
C (8)–C (9)
C (10)–C (11)
N (3)–N (2)–C (1)
C (8)–N (3)–N (2)
C (6)–C (1)–N (2)
C (1)–C (2)–C (3)
C (3)–C (4)–C (5)
C (1)–C (6)–C (5)
N (3)–C (8)–C (7)
C (7)–C (8)–C (9)
O (1)–C (9)–C (10)
O (2)–C (10)–C (11)
C (11)–C (10)–C (9)
O (3)–C (11)–C (10)
O (4)–C (12)–C (11)
N (3)–N (2)–N (1)
N (1)–N (2)–C (1)
C (6)–C (1)–C (2)
C (2)–C (1)–N (2)
C (4)–C (3)–C (2)
C (6)–C (5)–C (4)
N (1)–C (7)–C (8)
N (3)–C (8)–C (9)
O (1)–C (9)–C (8)
C (8)–C (9)–C (10)
O (2)–C (10)–C (9)
O (3)–C (11)–C (12)
C (12)–C (11)–C (10)
2-Phenyl-4-(D-arabino-10,20,30-trihydroxy-40-O-p-toluene-
sulfonylbutyl)-2H-1,2,3-triazole (2). A solution of d-ara-
bino-tetrahydroxybutyltriazole17 (1, 795 mg, 3 mmol) in
pyridine (30 mL) was cooled to 0 ꢁC in ice-bath and p-
toluenesulfonyl chloride (572 mg, 3 mmol) added in
small lots under constant stirring. The reaction mixture
was stirred for 2.5 h at 25–27 ꢁC when TLC showed
complete conversion of the starting compound into a
fast moving product. The reaction was terminated by
pouring onto crushed ice and pyridine was neutralized
by dropwise addition of 2 N HCl affording pure 40-O-p-
toluenesulfonylated triazole 2 as a white amorphous
solid (1.1 g) in 90% yield, mp 105–106 ꢁC. Rf 0.32 (sol-
vent A); IR (Nujol): 3360 (OH), 2920, 1600, 1462, 1374,
O (2)–C (10)
O (4)–C (12)
N (1)–N (2)
N (2)–C (1)
C (1)–C (6)
C (2)–C (3)
C (4)–C (5)
C (7)–C (8)
C (9)–C (10)
C (11)–C (12)
1.447 (5)
1.425 (5)
1.341 (5)
1.422 (6)
1.370 (7)
1.389 (7)
1.385 (7)
1.399 (6)
1.521 (6)
1.511 (6)
1070, 968, 850 and 755 cmꢀ1 1H NMR (250 MHz,
;
DMSO-d6): d 2.39 (3H, s, CH3), 3.55 (1H, dd, J=1.7
and 8.9 Hz, C-40Ha), 3.87–3.90 (1H, m, C-40Hb),
4.05(1H, dd, J=6.5 and 9.6 Hz, C-30H), 4.26 (1H, d,
J=9.6 Hz, C-20H), 5.11 (1H, br s, C-10H), 7.36–7.40(1H,
m, C-400H), 7.45(2H, d, J=8.2 Hz, C-3000H and C-5000H),
7.54 (2H, m, C-200H and C-600H), 7.80 (2H, d,
J=8.2 Hz, C-2000H and C-6000H) and 7.97–7.99 (3H, m,
C-300H, C-500H and C-5H); 13C NMR (62.5 MHz,
DMSO-d6): d 20.90 (CH3), 65.16 and 68.21 (C-30 and C-
40), 73.46(C-20 and C-10), 118.22, 127.46, 127.78, 129.77
and 130.21(C-200, C-300, C-400, C-500, C-600, C-2000, C-3000, C-
5000 and C-6000), 132.56 and 135.43 (C-5 and C-4000),
139.43(C-100), 144.88(C-1000) and 152.86 (C-4); EIMS, m/
z (% rel. int.): 419 ([M]+, 5), 272 (20), 258 (90), 229
(15), 212 (85), 172 (100), 158 (30), 107 (25), 91 (80) and
77 (25).
aSymmetry transformations used to generate equivalent atoms; Num-
bering of atoms (cf. Fig. 1) does not correspond to the IUPAC
nomenclature.
Experimental
Melting points were determined on a Mettler FP 62
instrument and are uncorrected. The IR spectra were
recorded on a Perkin-Elmer RX1 FT-IR spectro-
1
photometer. The H NMR spectra were recorded either
on a Bruker AC-250 or Bruker Advance-300 spectro-
meter at 250 and 300 MHz, respectively. The 13C NMR
spectra were recorded either on a Bruker AC-250 or
Bruker Advance-300 spectrometer at 62.5 or 75.5 MHz,
respectively. TMS has been used as an internal standard
for both 1H and 13C NMR spectral recordings. The
chemical shift values are on d scale and the coupling
constants (J) are in Hz. EI mass spectra were recorded
on Jeol-DX 303 mass spectrometer at 70 eV. Analytical
TLC’s were performed on precoated Merck silica gel
60F254 plates and spots were detected either under UV
2-Phenyl-4-[D-arabino-30-hydroxy-O-10,20-isopropylidene-
40-O-p-toluenesulfonylbutyl]-2H-1,2,3-triazole (3). To a
suspension of 10,20,30-trihydroxy-40-O-p-toluenesulfony-
lated triazole 2 (838 mg, 2.0 mmol) in dry and distilled
acetone (50 mL), anhydrous FeCl3 (324 mg, 2.0 mmol)
was added and the reaction mixture was stirred at 25–
27 ꢁC for 25 h. On completion, the reaction was termi-
Table 2. Antiviral efficacy of epoxide 4 and acyclotriazolylnucleosides 5–9 against HIV-1IIIB and HIV-1RF virusesa,b
Antiviral efficacy versus HIV-1IIIB virus Antiviral efficacy versus HIV-1RF virus
Compd
CEM-SS/HIV-1IIIB
IC50 (mM)
CEM-SS
TC50 (mM)
Therapeutic
index
CEM-SS/HIV-1RF
IC50 (mM)
CEM-SS
TC50 (mM)
Therapeutic
index
4
5
6
7
>100
>100
>100
>100
>100
>100
0.003
>100
83.0
24.2
>100
2.7
—
—
—
—
—
—
>312.5
>100
>100
>100
>100
>100
>100
0.006
>100
72.8
18.3
66.0
2.5
—
—
—
—
—
—
>155.9
8
9
AZT
94.2
>1
66.8
>1
aAZT was evaluated in parallel as a relevant positive control compound.
bAll values are average of three measurements using six concentrations at half-log dilutions.

966
?. Himanshu et al. / Bioorg. Med. Chem. 10 (2002) 963–968
nated by addition of 10% aq solution of potassium
carbonate (10 mL). Acetone was removed under
reduced pressure and the brown syrup was extracted
with chloroform (3ꢂ20 mL). The organic layer was
separated, washed with water (2ꢂ50 mL), dried over
sodium sulphate and solvent removed under reduced
pressure. The residue thus obtained was subjected to
column chromatography on silica gel using petroleum
ether/ethyl acetate as eluent to obtain 3 as a colourless
oil (789 mg) in 86% yield. Rf 0.36 (solvent D); IR
(Nujol): 3320 (OH), 1600, 1499, 1463, 1372, 1217, 1071,
lowed by triethylamine (1 mL). The reaction mixture
was refluxed until TLC examination showed complete
conversion of the starting 4 into the product, the solvent
was removed under reduced pressure and the crude
product thus obtained was purified by column chroma-
tography on silica gel using chloroform as eluting sol-
vent to obtain compounds 5–9 as yellow coloured oils in
70 to 85% yields.
2-Phenyl-4-[D-arabino-30-hydroxy-O-10,20-isopropylidene-
40-(pyrrolidin-1-yl)butyl]-2H-1,2,3-triazole (5). It was
obtained as a light yellow oil (304 mg) in 85% yield. Rf
0.35 (Solvent B); IR (Nujol): 3364 (OH), 1599, 1498,
1460, 1375, 1248, 1217, 1067, 966, 888 and 758 cmꢀ1; 1H
NMR (300 MHz, CDCl3): d 1.51 and 1.52 (6H, 2s, 3H
each, 2ꢂCH3), 1.81–1.84 (4H, m, C-3000H and C-4000H),
2.66–2.87 (6H, m, C-40H, C-2000H and C-5000H), 4.02–4.04
(1H, m, C-30H), 4.13 (1H, br s OH), 4.28 (1H, t,
J=6.6 Hz, C-20H), 5.30 (1H, d, J=7.4 Hz, C-10H), 7.27–
7.36 (1H, m, C-400H), 7.44–7.49 (2H, m, C-200H and C-
600H), 7.84 (1H, s, C-5H) and 8.04–8.07 (2H, m, C-300H
and C-500H); 13C NMR (75.5 MHz, CDCl3): d 23.56 (C-
3000 and C-4000), 26.79 and 27.03 (2ꢂCH3), 54.25 (C-2000
and C-5000), 58.47 (C-40), 69.02 (C-30), 72.94 (C-20), 82.47
(C-10), 110.39 (C(CH3)2), 118.99 (C-300 and C-500), 127.56
(C-400), 129.26 (C-200 and C-600), 134.40 (C-5), 139.90 (C-
100) and 148.62 (C-4); EIMS, m/z (% rel. int.): 358
([M]+, 90), 343 (100), 312 (15), 283 (10), 273 (5), 243 (5),
227 (70), 186 (20), 158 (28), 135 (55), 114 (95) and 84
(25).
1
967, 844 and 755 cmꢀ1; H NMR (300 MHz, CDCl3): d
1.44 and 1.54 (6H, 2s, 3H each, (C(CH3)2), 2.48 (3H, s,
CH3), 3.66 (1H, dd, J=3.8 and 10.7 Hz, C-40Ha), 4.18
(1H, br d, J=10.7 Hz, C-40Hb), 4.81–4.86 (2H, m, C-
20H and C-30H), 4.92–4.96 (1H, m, C-10H), 7.26–7.49
(6H, m, Ar00-H and C-5H), 7.92 (2H, d, J=8.6 Hz, C-
3000H and C-5000H) and 8.04 (2H, d, J=8.6 Hz, C-2000H
and C-6000H); 13C NMR (75.5 MHz, CDCl3): d 22.16
(PhCH3), 25.07 and 26.41(2ꢂCH3), 73.46 (C-40), 78.20
(C-30), 81.71 (C-20 and C-10), 112.96 (C(CH3)2), 119.23,
127.42, 127.72, 129.58 and 130.59 (C-200, C-300, C-400, C-
500, C-600, C-2000, C-3000, C-5000 and C-6000), 136.44 (C-5),
and 140.22, 142.13, 145.47 and 147.16 (C-100, C-1000, C-4
and C-4000); EIMS, m/z (% rel. int.): 459 ([M]+, 5), 272
(18), 258 (90), 229 (10), 215 (24), 212 (85), 188 (15), 171
(100), 158 (30), 107 (25), 91 (80) and 77 (25).
2-Phenyl-4-[D-arabino-30,40-epoxy-O-10,20-isopropylidene-
butyl]-2H-1,2,3-triazole (4). To a solution of 30-hydroxy-
10,20-O-isopropylidene-40-p-toluenesulfonyltriazole (3,
690 mg, 1.5 mmol) in dry and distilled toluene (20 mL)
was added sodium hydride (40 mg, 1.0 mmol) and the
reaction mixture was refluxed in an oil bath for 2.5 h.
On completion, the reaction was terminated by addition
of methanol (10 mL), solvent removed under reduced
pressure and the residue obtained was purified by col-
umn chromatography on silica gel using petroleum
ether/ethyl acetate as eluting solvent to get the epoxide 4
as a colourless oil (258 mg) in 60% yield. Rf 0.42 (sol-
vent D); IR (Nujol): 1599, 1499, 1463, 1375, 1214, 1069,
2-Phenyl-4-[D-arabino-30 -hydroxy-O-10,20 -isopropyli-
diene-40-(piperidin-1-yl)butyl]-2H-1,2,3-triazole (6). It
was obtained as a light yellow oil (298 mg) in 80% yield.
Rf 0.35 (solvent B); IR (Nujol): 3362 (OH), 1599, 1498,
1
1458, 1365, 1248, 1217, 1067, 1000 and 888 cmꢀ1; H
NMR (300 MHz, CDCl3): d 1.42–1.45 (2H, m, C-4000H),
1.51 and 1.52 (6H, 2s, 3H each, 2ꢂCH3), 1.57–1.59 (4H,
m, C-3000H and C-5000H), 2.40–2.61 (6H, m, C-40H, C-
2000H and C-6000H), 3.20 (1H, br s, OH), 3.94–4.01 (1H,
m, C-30H), 4.25 (1H, dd, J=5.8 and 7.5 Hz, C-20H),
5.29(1H, d, J=7.5 Hz, C-10H), 7.31–7.36 (1H, m, C-
400H), 7.44–7.54 (2H, m, C-200H and C-600H), 7.83 (1H, s,
C-5H) and 8.04–8.07 (2H, m, C-300H and C-500H); 13C
NMR (75.5 MHz, CDCl3): d 23.97 and 25.73 (C-3000, C-
4000 and C-5000), 26.75 and 26.96 (2ꢂCH3), 54.62 (C-2000
and C-6000), 60.93 (C-40), 67.14 (C-30), 73.03 (C-20), 82.70
(C-10), 110.32 (C(CH3)2), 118.96 (C-300 and C-500), 127.48
(C-400), 129.19 (C-200 and C-600), 134.32 (C-5), 139.68 (C-
100) and 148.57 (C-4); EIMS, m/z (% rel. int.): 372
([M]+, 55), 357 (58), 343 (5), 279 (15), 243 (5), 215 (8),
186 (10), 167 (25), 149 (55), 128 (75) and 99 (100).
1
967, 887 and 755 cmꢀ1; H NMR (300 MHz, CDCl3): d
1.43 and 1.45 (6H, 2s, 3H each, 2ꢂCH3), 2.72–2.77 (2H,
m, C-40H), 3.18–3.21 (1H, m, C-30H), 4.19(1H, dd,
J=3.8 and 7.8 Hz, C-20H), 5.03 (1H, d, J=7.8 Hz, C-
10H), 7.19–7.25 (1H, m, C-400H), 7.33–7.38 (2H, m, C-
200H and C-600H), 7.74 (1H, s, C-5H) and 7.84 (2H, br d,
J=7.8 Hz, C-300H and C-500H); 13C NMR (75.5 MHz,
CDCl3): d 28.12 and 28.16 (2ꢂCH3), 45.91 (C-40), 52.34
(C-30), 73.82 (C-20), 81.74 (C-10), 112.07 (C(CH3)2),
120.31 (C-300 and C-500), 129.08 (C-400), 130.68 (C-200 and
C-600), 135.36 (C-5), 141.09 (C-100) and 148.98 (C-4);
EIMS, m/z (% rel. int.): 287 ([M]+, 67), 272 (70), 229
(95), 200 (52), 173 (50), 158 (80), 91 (62), 77 (80) and 59
(100).
2-Phenyl-4-[D-arabino-40 -(2000 -ethylpiperidin-1-yl)-30 -
hydroxy-O-10,20 -isopropylidenebutyl]-2H-1,2,3-triazole
(7). It was obtained as a light yellow oil (280 mg) in
70% yield. Rf 0.38 (solvent C); IR (Nujol): 3394 (OH),
1599, 1499, 1461, 1376, 1254, 1163, 1069, 966, 888 and
General procedure for the preparation of 2-phenyl-4-(D-
arabino-40-cycloamino-30-hydroxy-O-10,20-isopropylidene-
butyl)-2H-1,2,3-triazoles 5–9. To a solution of 30,40-
epoxytriazole 4 (287 mg, 1.0 mmol) in dry and distilled
methanol (10 mL) was added corresponding cyclic
amine, i.e., pyrrolidine, piperidine, 2-ethylpiperidine, 4-
benzylpiperidine or N-methylpiperazine (1.0 mmol), fol-
1
756 cmꢀ1; H NMR (300 MHz, CDCl3): d 0.85 (3H, t,
J=7.5 Hz, CH2CH3), 1.52 and 1.53 (6H, 2s, 3H each,
2ꢂCH3), 1.65–1.68 (8H, m, C-3000H, C-4000H, C-5000H and
CH2CH3), 2.41–2.59(5H, m, C-40H, C-2000H and C-
6000H), 3.68 (1H, br s, OH), 3.96–3.99 (1H, m, C-30H),

?. Himanshu et al. / Bioorg. Med. Chem. 10 (2002) 963–968
967
4.21–4.25 (1H, m, C-20H), 5.33 (1H, d, J=6.6 Hz, C-
10H), 7.27–7.35 (2H, m, C-200H and C-600H), 7.43–7.48
(1H, m, C-400H), 7.84 (1H, s, C-5H) and 8.06 (2H, br d,
J=7.9 Hz, C-300H and C-500H); 13C NMR (75.5 MHz,
CDCl3): d 11.09 (CH2CH3), 21.04, 21.41, 22.72 and
24.33 (C-3000, C-4000, C-5000, CH2CH3), 26.86 and 27.07
(C(CH3)2), 56.10 (C-2000), 49.77 (C-6000), 62.96 (C-40),
67.14 (C-30), 73.44 (C-20), 83.05 (C-10), 110.44 (C(CH3)2),
119.06 (C-300 and C-500), 127.57 (C-400), 129.83 (C-200 and
C-600), 134.43 (C-5), 139.99 (C-100) and 148.79 (C-4);
EIMS, m/z (% rel. int.): 385 (30), 371 (92), 313 (10), 279
(12), 188 (10), 149 (60), 126 (100) and 96 (47).
fractometer. Graphite monochromated Mo-Ka radia-
tion was used in all cases. The structure was solved
using SHELXTL-PLUS21 and refined with SHELXL-
96.22 The crystal data of compound 1 is given below.
2-Phenyl-4-(D-arabino-10,20,30,40-tetrahydroxybutyl)-2H-
1,2,3-triazole (1). C12H15N3O4, M=265.27, T=180(2)K,
l=0.71073 A. Monoclinic a=6.0354(6), b=4.7825(4),
c=21.723(2) A, b=93.702(2)ꢁ, V=625.71(10) A3, space
group P21, Z=2, Dx=1.408 Mg/m3, m=0.107 mmꢀ1
,
F(000)=280. Crystal size 0.50ꢂ0.08ꢂ0.08 mm; y range
for data collection 2.82–25.00ꢁ; index range ꢀ7ꢃhꢃ7,
ꢀ6ꢃkꢃ6, 26ꢃlꢃ28, reflections collected 3243, inde-
pendent reflections 2070 [R(int)=0.0598]; refinement
method full-matrix least squares on F2; data/restraints/
parameters 2070/1/176; goodness of fit on F2 0.895;
R(F)[I>2s(I)]=0.0632; wR(F2)=0.1357; largest diff.
2-Phenyl-4-[D-arabino-40 -(4000 -benzylpiperidin-1-yl)-30 -
hydroxy-O-10,20 -isopropylidenebutyl]-2H-1,2,3-triazole
(8). It was obtained as a light yellow oil (380 mg) in
80% yield. Rf 0.35 (solvent C); IR (Nujol): 3374 (OH),
1727, 1599, 1497, 1456, 1376, 1253, 1163, 1069, 967, 886
peak and hole 0.376 and ꢀ0.299 eAꢀ3
.
1
and 754 cmꢀ1; H NMR (300 MHz, CDCl3): d 1.43 and
1.44 (6H, 2s, 3H each, 2ꢂCH3), 1.53–1.60 (4H, m, C-
3000H and C-5000H), 1.87–1.97 (2H, m, CH2C6H5), 2.14–
2.22 (1H, m, C-4000H), 2.42–2.53 (4H, m, C-2000H and C-
6000H), 2.76–2.92 (2H, m, C-40H), 3.59 (1H, br s, OH),
3.88–3.94 (1H, m, C-30H), 4.16–4.20 (1H, dd, J=5.8
and 7.5 Hz, C-20H), 5.21 (1H, d, J=7.5 Hz, C-10H),
7.03–7.23 (5H, m, CH2C6H5), 7.25–7.28 (1H, m, C-
400H), 7.36–7.46 (2H, m, C-200H and C-600H), 7.76 (1H, s,
C-5H) and 7.96–7.99 (2H, m, C-300H and C-500H); 13C
NMR (75.5 MHz, CDCl3): d 25.79 and 26.01 (2ꢂCH3),
30.75 and 31.19 (C-3000, C-5000), 36.58 (C-4000), 41.96
(CH2C6H5), 51.57 and 54.41 (C-2000 and C-6000), 59.50 (C-
40), 66.18 (C-30), 71.99 (C-20), 81.57 (C-10), 109.34
(C(CH3)2), 117.92 (C-300 and C-500), 124.00 (C-40000),
124.89, 126.50, 127.21, 128.06 and 128.19 (C-200, C-20000,
C-30000, C-400, C-50000, C-600 and C-60000), 133.31 (C-5), and
138.78, 139.38 (C-100 and C-10000) and 147.45 (C-4);
EIMS, m/z (% rel. int.): 462 ([M]+, 20), 447 (15), 218
(18), 189 (100), 174 (10), 117 (10), 91 (65) and 70 (20).
Antiviral activity evaluation
Drug preparation. Compounds 4–9 were solubilised in
water to yield 40 mM stock solutions. All compounds
were tested at 100 mM concentration and five serial half-
logarithmic dilutions. AZT was used as a positive con-
trol antiviral compound.
Cell preparation. CEM-SS cells were passaged in T-75
flasks prior to use in the antiviral assay. On the day
preceeding the assay, the cells were split 1:2 to assure
they were in an exponential growth phase at the time of
infection. Total cell and viability quantification was
performed using a hemacytometer and trypan blue
exclusion. Cell viability was greater than 95% for the
cells to be utilized in the assay. The cells were resus-
pended at 5ꢂ104 cells/mL in tissue culture medium and
added to the drug-containing microtiter plates in a
volume of 50 mL.
2-Phenyl-4-[D-arabino-30-hydroxy-O-10,20-isopropylidene-
40-(N-methylpiperazin-1-yl)butyl]-2H-1,2,3-triazole (9).
It was obtained as a light yellow oil (272 mg) in 70%
yield. Rf 0.38 (solvent C); IR (Nujol): 3392 (OH), 1670,
1599, 1498, 1458, 1289, 1248, 1215, 1067, 1013, 888 and
758 cmꢀ1; 1H NMR (300 MHz, CDCl3): d 1.51 and 1.53
(6H, 2s, 3H each, 2ꢂCH3), 2.27 (3H, s, NCH3), 2.47–
2.66 (10H, m, C-40H, C-2000H, C-3000H, C-5000H and C-
6000H), 3.57 (1H, br s, OH), 3.96–3.99 (1H, m, C-30H),
4.27 (1H, t, J=7.3 Hz, C-20H), 5.29 (1H, d, J=7.6 Hz,
C-10H), 7.28–7.36 (1H, m, C-400H), 7.44–7.49 (2H, m, C-
200H and C-600H), 7.84 (1H, s, C-5H) and 8.04–8.06 (2H,
m, C-300H and C-500H); 13C NMR (75.5 MHz, CDCl3): d
26.77 and 26.98 (2ꢂCH3), 45.85 (NCH3), 55.05 (C-2000, C-
3000, C-5000 and C-6000), 60.11 (C-40), 67.26 (C-30), 72.84 (C-
20), 82.59 (C-10), 110.34 (C(CH3)2), 118.98 (C-300 and C-
500), 127.59 (C-400), 129.27 (C-200 and C-600), 134.34 (C-5),
139.88 (C-100) and 148.56 (C-4); EIMS, m/z (% rel. int.):
387 ([M]+, 50), 372 (45), 312 (4), 273 (4), 256 (5), 221
(10), 186 (15), 172 (5), 143 (75), 114 (100) and 98 (72).
Virus preparation. The virus used for the assay was the
lymphocytropic virus strains HIV-1IIIB and HIV-1RF
.
This virus was obtained from the NIH AIDS Research
and Reference Reagent Program and was grown in
CEM-SS cells for the production of stock virus pools. A
pre-titered aliquot of virus was removed from the free-
zer (ꢀ80 ꢁC) and allowed to thaw slowly to room tem-
perature in a biological safety cabinet. The virus was
resuspended and diluted into tissue culture medium
such that the amount of virus added to each well in a
volume of 50 mL was the amount determined to give
between 85 and 95% cell killing at 6 days post-infection.
TCID50 calculations by endpoint titration in CEM-SS
cells indicated that the multiplicity of infection of these
assays was approximately 0.01.
Plate format. Each plate contains cell control wells (cells
only), virus control wells (cells plus virus), drug cyto-
toxicity wells (cells plus drug only), drug colorimetric
control wells (drug only) as well as experimental wells
(drug plus cells plus virus). Samples were evaluated with
triplicate measurements using six concentrations at half-
log dilutions in order to determine IC50 values and to
measure cellular cytotoxicity, if detectable.
X-ray crystallography of triazolylacyclonucleoside 1.
The crystallographic measurements on butyltriazole 1
were made using a Siemens SMART area-detector dif-

968
?. Himanshu et al. / Bioorg. Med. Chem. 10 (2002) 963–968
MTS staining for cell viability. At assay termination,
the assay plates were stained with the soluble tetra-
zolium-based dye MTS (Cell Titer Reagent Promega) to
determine cell viability and quantify compound toxicity.
MTS is metabolised by the mitochondria enzymes of
metabolically active cells to yield a soluble formazan
product, allowing the rapid quantitative analysis of cell
viability and compound cytotoxicity. The MTS is a
stable solution that does not require preparation before
use. At termination of the assay, 20 mL of MTS reagent
was added per well. The wells were incubated overnight
for the HIV cytoprotection assay at 37 ꢁC. The incuba-
tion intervals were chosen based on empirically deter-
mined times for optimal dye reduction in each cell type.
Adhesive plate sealers were used in place of the lids, the
sealed plate was inverted several times to mix the solu-
ble formazan product and the plate was read spectro-
photometrically at 490 nm with a Molecular Devices
Vmax plate reader.
3. Wengel, J.; Schinazi, R. F.; Caruthers, M. H. Bioorg. Med.
Chem. 1995, 3, 1223.
4. Sekiyama, T.; Hatsuya, S.; Tanaka, Y.; Uduyama, M.;
Ono, N.; Iwayama, S.; Oikawa, M.; Suzuki, K.; Okunishi, M.;
Tsuji, T. J. Med. Chem. 1998, 41, 1248.
5. Hutchinson, D. W. TIMTECH 1990, 8, 348.
6. Nasr, M.; Litterst, C.; McGowan, J. Antiviral Res. 1990,
14, 125.
7. Fischl, M. A. AIDS 1989, 2 (Suppl. 1), 137.
8. Kafefuda, A.; Shuto, S.; Nagahata, T.; Seki, J.-I.; Sasaki,
T.; Matsuda, A. Tetrahedron 1994, 50, 10167.
9. Robins, M. J.; Zon, R.; Hansske, F.; Wnuk, S. F. Can. J.
Chem. 1997, 75, 762.
10. Prasad, A. K.; Trikha, S.; Parmar, V. S. Bioorg. Chem.
1999, 27, 135.
11. Chu, C. K.; Cutler, S. J. J. Heterocyclic Chem. 1986, 23,
289.
12. Parkanyi, C.; Yuan, H. L. J. Heterocyclic Chem. 1990, 27,
1409.
13. Pan, B.-C.; Chem, Z.-H.; Piras, G.; Dutschman, G. E.;
Rowe, E. C.; Cheng, Y.-C.; Chu, S.-H. J. Heterocyclic Chem.
1994, 31, 177.
14. Chen, W.; Flavin, M. T.; Filler, R.; Xu, Z.-Q. Perkin
Trans. 1, 1998, 3979.
Acknowledgements
15. Hirota, K.; Monguchi, Y.; Sajiki, H.; Sako, M.; Kitada,
Y. Perkin Trans. 1, 1998, 941.
16. Gao, H.; Mitra, A. K. Synthesis, 2000, 329.
17. Hann, R. M.; Hudson, C. S. J. Am. Chem. Soc. 1944, 66,
735.
18. Tipson, R. S. Methods in Carbohydrates Chemistry; Aca-
demic Press: New York, 1963; Vol. 2, p 16.
19. Raunak; Prasad, A. K.; Shakil, N. A.; Himanshu; Parmar,
V. S. Pure Appl. Chem. 2001, 73, 167.
The authors thank the Danish International Develop-
ment Agency (DANIDA, Denmark) and Council of
Scientific and Industrial Research (CSIR, New Delhi)
for financial assistance, and the Southern Research
Institute, MD, USA for their help in antiviral activity
evaluation of our compounds.
20. Prasad, A.K.; Himanshu; Bhattacharya, A.; Olsen, C.E.;
Parmar, V.S. Bioorg. Med. Chem. In press.
21. Sheldrick, G. M. SHELXTL-PLUS. Siemens Analytical
X-ray Instruments Inc.: Madison, WI, USA, 1990.
22. Sheldrick, G.M. SHELXL-96, Program for Crystal Struc-
ture Refinement; University of Gottingen: Germany, 1996.
References and Notes
1. Schinazi, R. F.; Mead, J. R.; Feorino, P. M. AIDS Res.
Hum. Retroviruses 1992, 8, 963.
2. Clercq, E. D. AIDS Res. Hum. Retroviruses 1992, 8, 119.