Synthesis of oxathiolane imidazole nucleosides

Article abstract of DOI:10.1016/S0014-827X(02)01287-9

Nucleosides have been of great interest since their strong antiviral activities were discovered. 1,3-Oxathiolane ring system has been known for many years, but it is in recent years that the ring has been used as the sugar ring in nucleoside analogs (Synthesis (1991) 1046; J. Am. Chem. Soc. 113 (1991) 9377; Tetrahedron Lett. 35 (1994) 4739). Besides, bredinin is a natural nucleoside antibiotic with imidazole moiety and there are some other studies reported on nucleosides with the imidazole group (Biorg. Med. Chem. 7 (1999) 481; Biorg. Med. Chem. 7 (1999) 1617; Nucleosides Nucleotides 18 (1999) 331). These findings make the imidazole group interesting as the base of a nucleoside. In this study, in order to find out the structure-activity relationships of L-oxathiolanyl nucleosides, L-oxathiolanyl imidazole nucleosides 7 and 8 were synthesized, via novel intermediates 2-6, which were then tested for anti-HIV activity (Antivir. Res. 1-11 (1994) 25) in human peripheral blood mononuclear (PBM) cells, the synthesized nucleosides did not show significant activity up to 100 μM against HIV-1.

Full text of DOI:10.1016/S0014-827X(02)01287-9

Il Farmaco 57 (2002) 993ꢁ997
/
www.elsevier.com/locate/farmac
Synthesis of oxathiolane imidazole nucleosides
Ayse Kocabalkanli a,ꢀ, Raymond F. Schinazi b
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Istanbul, 34452 Beyazit-Istanbul, Turkey
b Emory University School of Medicine, VA Medical Center, Decatur, GA 30033, USA
Received 27 April 2002; received in revised form 4 May 2002; accepted 25 May 2002
Abstract
Nucleosides have been of great interest since their strong antiviral activities were discovered. 1,3-Oxathiolane ring system has been
known for many years, but it is in recent years that the ring has been used as the sugar ring in nucleoside analogs (Synthesis (1991)
1046; J. Am. Chem. Soc. 113 (1991) 9377; Tetrahedron Lett. 35 (1994) 4739). Besides, bredinin is a natural nucleoside antibiotic with
imidazole moiety and there are some other studies reported on nucleosides with the imidazole group (Biorg. Med. Chem. 7 (1999)
481; Biorg. Med. Chem. 7 (1999) 1617; Nucleosides Nucleotides 18 (1999) 331). These findings make the imidazole group interesting
as the base of a nucleoside. In this study, in order to find out the structureꢁ
oxathiolanyl imidazole nucleosides 7 and 8 were synthesized, via novel intermediates 2ꢁ
activity (Antivir. Res. 1ꢁ11 (1994) 25) in human peripheral blood mononuclear (PBM) cells, the synthesized nucleosides did not
/
activity relationships of
L-oxathiolanyl nucleosides, L-
/6, which were then tested for anti-HIV
/
show significant activity up to 100 mM against HIV-1.
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Nucleosides; Oxathiolane; Imidazole; Synthesis; Antiviral activity
1. Introduction
In this study the
methyl-a- -[1,3]oxathiolan-5-yl)imidazole
mide (7) and 1-(2-hydroxymethyl-b-
L-like nucleosides, 1-(2-hydroxy-
L
4-carboxa-
Belleau et al. reported the synthesis and anti-HIV
L-[1,3]oxathiolan-
5-yl)imidazole 4-carboxamide (8) were synthesized via
activity of an unnatural class of nucleosides, (9)-BCH-
/
novel intermediates 2ꢁ6 and evaluated for anti-HIV
/
189 in which C-3? position had been replaced by a sulfur
atom [1]. It was of interest to synthesize the pure
enantiomers of BCH-189 (Fig. 1), since it was being
tested clinically on patients with AIDS. Chu et al.
activity in human peripheral blood mononuclear (PBM)
cells infected with HIV-1 strain LAI the synthesized
nucleosides did not show significant activity up to 100
mM against HIV-1.
synthesized (ꢂ)-BCH-189 starting from D-mannose
/
[2], but this synthesis had many steps and consequently
the yield was low. Chu et al. in another study, obtained
this same compound from 1,6-thioanhydro-D-galactose
2. Chemistry
by a more efficient and shorter route [3], which enabled
the examination of the structure-activity relationships of
the [1,3]oxathiolanyl nucleosides as anti-HIV agents. In
(2R,
acid-(
5R)-5-Hydroxy-[1,3]oxathiolane-2-carboxylic
) menthyl ester (1), prepared by the method given
L
general, b-
biologically active isomers, but when the four possible
isomers of BCH-189 were evaluated the -like nucleo-
-like nucleoside [4,5].
D isomers of nucleosides were found to be the
by Jin et al. [6], was treated with HCl/MeOH to give
menthyl 5-methoxy-[1,3]oxathiolane-2-carboxylate (2)
L
side was more potent than the
D
ꢀ Corresponding author
E-mail address: aysekocab@yahoo.com (A. Kocabalkanli).
Fig. 1.
´
0014-827X/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 1 2 8 7 - 9

994
A. Kocabalkanli, R.F. Schinazi / Il Farmaco 57 (2002) 993ꢁ997
/
[7]. Compound 2 in THF (tetrahydrofurane) was
reduced with LAH (lithium aluminium hydride) in
THF to 5-methoxy-2-hydroxymethyl-[1,3]oxathiolane
(3) [8]. A solution of 3 and pyridine in CH2Cl2 was
treated with benzoyl chloride to obtain 5-methoxy-2-
benzoylmethyl-[1,3]oxathiolane (4) [3,7]. Imidazole 4-
carboxylic acid methyl ester was heated with (NH4)2SO4
and HMDS (hexamethyldisilazane) and after removal of
HMDS, 4 in CH2Cl2 and TMSOTf (trimethylsilyl
triflate) was added to this residue to give methyl 1-(2-
benzoyloxymethyl-b (and a)-L-[1,3]oxathiolan-5-yl)imi-
dazole 4-carboxylate (5 and 6, respectively) [4,7].
Compound 5 (or 6) in saturated NH3/MeOH was stirred
at 90 8C to gain 1-(2-hydroxymethyl-b (or a)-L-[1,3]ox-
athiolan-5-yl)imidazole 4-carboxamide (7 or 8, respec-
tively).
and menthyl). FAB-MS, m/z 303 [Mꢂ
H]ꢂ, 165, 139,
119, 83. Anal. Calc. for C15H26O4S: C, 59.57; H, 8.67.
Found: C, 59.59; H, 8.70%.
/
3.2. 5-Methoxy-2-hydroxymethyl-[1,3]oxathiolane (3)
[8]
To a suspension of LAH (1.602 mmol, 60.88 mg) and
13 ml of THF was added dropwise a solution of 2 (0.801
mmol, 241.8 mg) in 7 ml of THF and stirred under
nitrogen for 2 h in ice bath. Then LAH was quenched
with 50 ml of MeOH and the mixture was filtered
through Celite pad, evaporated to dryness under re-
duced pressure, and purified by silica gel column
chromatography (20:80 EtOAcꢁ
yellow oil, as a mixture of a and b anomers: H NMR
(CDCl3): d 5.49ꢁ5.33 (m, 2H, H-2 and H-5), 3.89ꢁ3.76
(m, 2H, H-2?), 3.44, 3.41 (2ꢃs,3H,CH3), 3.26ꢁ3.07 (m,
2H, H-4), 2.43, 2.09 (2ꢃ
s, 1H, OH). 13C NMR
(CDCl3): d 106.82 (CH3), 84.55 (CH2ꢀOH), 64.32,
55.32, 38.52 (Cꢀoxathiolane). Anal. Calc. for
/
C6H14) to give 3, a
1
/
/
/
/
3. Experimental
/
/
M.p.s were determined on a Mel-temp II melting
point apparatus in open capillaries and are uncorrected.
Elemental analyses were performed by Atlantic Micro-
/
C5H10O3S: C, 39.98; H, 6.71. Found: C, 39.99; H,
6.79%.
1
lab Inc., Norcross, GA. H and 13C NMR spectra were
obtained on a Bruker AMX spectrophotometer at 400
MHz, using Me4Si as the internal standard and CDCl3
as the solvent. FAB MS were recorded on a Micromass
Autospec high resolution mass spectrometer. Optical
rotations were determined on a JASCO DIP-370 Digital
Polarimeter. UV spectra were determined on a Beckman
DU 650 spectrophotometer. TLC was performed on
Uniplates (silica gel) purchased from Analtech Co.
Column chromatography was performed using either
3.3. 5-Methoxy-2-benzoyloxymethyl-[1,3]oxathiolane
(4) [3,7]
A solution of 3 (1.798 mmol, 270 mg) and 0.5 ml of
pyridine in 6 ml of CH2Cl2 was treated with BzCl (2.158
mmol, 0.25 ml) at 0 8C and the mixture was stirred at
r.t. for 1 h. The reaction mixture was quenched with ice
while stirring for another 20 min, washed with H2O,
saturated NaHCO3 and brine, dried on MgSO4 and
purified by silica gel column chromatography (10:90
Silica Gel-60 (220ꢁ
or Silica Gel G (TLC gradeꢀ
column chromatography.
/
440 mesh) for flash chromatography
/440 mesh) vacuum flash
EtOAcꢁ
(CDCl3): d 8.18ꢁ
(m, 2H, H-2 and H-5), 4.56ꢁ
3.42 (2ꢃs, 3H, CH3), 3.24ꢁ
NMR (CDCl3): d 165.94, 165.91 (Cꢁ
130.64, 129.63, 128.76 (Cꢀphenyl), 107.44, 106.43
(CH3), 83.03, 80.96 (CH2OBz), 66.02, 55.17, 37.95,
37.63 (Cꢀoxathiolane). FAB-MS m/z: 255 [Mꢂ
H]ꢂ,
223, 195, 105. Anal. Calc. for C12H14O4S×0.1C6H14: C,
57.56; H, 5.90. Found: C, 57.38, H, 5.50%.
/
C6H14) to give 4, a yellow oil. 1H NMR
7.44 (m, 5H, H-phenyl), 5.64ꢁ5.41
4.41 (m, 2H, H-2?), 3.44,
3.09 (m, 2H, H-4). 13C
O), 134.43, 133.07,
/
/
/
3.1. Menthyl 5-methoxy-[1,3]oxathiolane-2-carboxylate
/
/
(2) [7]
/
/
(2R,
acid-(
5R)-5-hydroxy-[1,3]oxathiolane-2-carboxylic
) menthyl ester (2.71 mmol, 780 mg) which was
L
/
/
prepared according to the method given by Jin et al. [6]
was treated with HCl/MeOH solution (1%, 3.6 ml) at
room temperature (r.t.) for 3 h. The mixture was
quenched with 0.7 ml of pyridine and concentrated to
dryness under reduced pressure. Residue was purified by
/
3.4. Methyl 1-(2-benzoyloxymethyl-b-
[1,3]oxathiolan-5-yl)imidazole 4-carboxylate (5) and
methyl 1-(2-benzoyloxymethyl-a- -[1,3]oxathiolan-5-
L-
silicagel column chromatography (5:95 EtOAcꢁ
to give 2, a yellow oil, as a mixture of a and b anomers:
1H NMR (CDCl3): d 5.59, 5.52 (2ꢃ
s, 2H, H-2), 4.78ꢁ
4.68 (m, 1H, H-5), 3.53, 3.43 (2ꢃs, 3H, CH3O), 3.27ꢁ
3.07 (m, 2H, H-4), 1.70ꢁ1.01 (m, 9H, H-menthyl), 1ꢁ
0.96 (m, 7H, H of CHꢀ(CH3)2), 0.85ꢁ0.82 (s, 3H, CH3
of menthyl). 13C NMR (CDCl3): d 169.49 (Cꢁ
O),
107.97 (CH3O), 55.78, 47.40, 41.07, 37.78, 34.52,
31.77, 26.39, 23.61, 22.37, 21.15, 16.50 (Cꢀoxathiolane
/
C6H14)
L
yl)imidazole 4-carboxylate (6) [4,7]
/
/
/
/
A mixture of imidazole 4-carboxylic acid methyl ester
(1.966 mmol, 247.95 mg), (NH4)2SO4 (9.93 mg) and
HMDS (10 ml) was heated at 140 8C for 18 h. HMDS
was removed in vacuo. First, 4 (1.966 mmol, 500 mg) in
20 ml of CH2Cl2, and then TMSOTf (1.6 ml) was added
to the residue. The mixture was stirred at r.t. for 6 h,
/
/
/
/
/
/

A. Kocabalkanli, R.F. Schinazi / Il Farmaco 57 (2002) 993ꢁ
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997
995
then poured into saturated NaHCO3 and stirred for 30
min. Aqueous layer was washed with CH2Cl2 and
combined organic layers were washed with H2O, dried
on MgSO4, filtered through Celite pad, evaporated and
(m, 1H, H-2?). 13C NMR (CDCl3): d 161.48 (Cꢁ
139.21, 138.98, 133.27, 133.13, 124.62, 124.55, 88.24,
/
O),
87.30, 87.28, 87.18, 64.66. FAB-MS m/z: 230 [Mꢂ
/
H]ꢂ.
Anal. Calc. for C8H11N3O3S: C, 41.91; H, 4.84; N,
18.33. Found: C, 41.86; H, 4.98; N, 18.13%.
Compound 6 was dissolved in saturated NH3/MeOH
(5 ml) and stirred 12 h in sealed bomb at 90 8C. After
reaction all solvent evaporated to dryness and coevapo-
rated with MeOH to give crude compound 8 which is
purified by PTLC (0.5% MeOHꢁ
spot was collected to give compound 5 as an oil which
was crystallized with MeOHꢁether to give white solid
(70 mg, 20.1%): m.p. 80ꢁ81 8C; UV (MeOH) lmax 230.5
nm. [a]2D5 34.198 (c, 1.60, MeOH); 1H NMR (CDCl3):
d 8.07ꢁ8.03 and 7.49ꢁ7.45 (m, 5H, H-phenyl), 7.79 (s,
1H, H-5) 7.62ꢁ7.58 (m, 1H, H-2), 7.12ꢁ7.11 (d, Jꢅ4.93
Hz, 1H, H-1?), 5.80 (dd, Jꢅ4.2 Hz, 1H, H-4?), 4.57 (dd,
Jꢅ6.3 Hz, 1H, H-5?), 4.47 (dd, Jꢅ4.1 Hz, 1H, H-5?),
3.88 (s, 3H, CH3), 3.75 (dd, Jꢅ5.2 Hz, 1H, H-2?), 3.28
(dd, Jꢅ
4.5 Hz, 1H, H-2?). 13C NMR (CDCl3): d
166.00, 160.77 (CꢁO), 139.87, 138.32, 133.50, 129.24,
128.58, 127.38 (Cꢀphenyl and imidazole), 88.30 (CH3),
84.19 (CH2OBz), 64.66, 51.79, 39.86 (Cꢀoxathiolane).
FAB-MS m/z: 349 [Mꢂ
H]ꢂ. Anal. Calc. for
0.1MeOH: C, 55.00; H, 4.70, N, 7.97.
/CHCl3). The bottom
/
/
ꢄ
/
/
/
purified by silica gel column chromatography (CHCl3ꢁ
/
/
/
/
MeOH, 9:1), (20 mg, 43.5%): m.p. 173ꢁ175 8C; UV
/
/
(H2O) lmax 207.0 (o 24 044) 237.0 (o 9154) (pH 2), 207.5
(o 15 624) 238.5 (o 22 125) (pH 7), 207.0 (o 10 297) 237.0
/
/
1
/
(o 13 658) (pH 11); [a]2D5 31.788 (c 0.78, MeOH); H
NMR (CDCl3): d 7.97 (s, 1H, H-5), 7.94 and 7.92 (2s,
/
/
2H, NH2), 7.84 (s, 1H, H-2), 6.95 (t, Jꢅ
1?), 5.47 (t, 1H, H-4?), 5.17 (t, Jꢅ3.5 2H, H-5?), 3.54 (m,
1H, OH) 3.23 (m, Jꢅ5.3 Hz, 1H, H-2?), 3.20 (m, 1H, H-
2?). 13C NMR (CDCl3): d 163.38 (Cꢁ
O), 140.52, 134.03,
130.61, 129.98, 90.12, 88.28, 65.48. FAB-MS m/z: 230
[Mꢂ MeOH: C,
H]ꢂ. Anal. Calc. for C8H11N3O3S×
/3.6 Hz, 1H, H-
/
/
/
/
/
/
C16H16N2O5S×
/
Found: C, 55.39, H, 4.69, N, 8.01%.
/
/
The upper spot was collected to give compound 6 as
41.79.; H, 5.05; N 17.83. Found: C, 41.74; H, 5.02; N,
an oil which was crystallized with MeOHꢁ
white solid (45 mg, 12.9%): m.p. 71ꢁ
(MeOH) lmax 232 nm. [a]D25
30.798 (c, 1.00, MeOH);
1H NMR (CDCl3): d 8.19 (s, 1H, H-5), 8.09ꢁ
8.07 and
7.62ꢁ7.58 (m, 5H, H-phenyl), 7.78 (s, 1H, H-2), 6.82
(dd, Jꢅ3.6 Hz, 1H, H-1?), 5.57 (dd, Jꢅ3.5 Hz, 1H, H-
4?), 4.77 (dd, Jꢅ5.3 Hz, 1H, H-5?), 4.70 (dd, Jꢅ3.5 Hz,
1H, H-5?), 3.87 (s, 3H, CH3), 3.72 (dd, Jꢅ5.3 Hz, 1H,
H-2?), 3.20 (dd, Jꢅ
3.5 Hz, 1H, H-2?). 13C NMR
(CDCl3): d 166.04, 160.59 (CꢁO), 137.26, 133.42,
129.78, 129.41, 128.53, (Cꢀphenyl and imidazole),
88.30 (CH3), 84.19 (CH2OBz), 64.66, 51.79, 39.86 (Cꢀ
oxathiolane). FAB-MS m/z: 349 [Mꢂ
H]ꢂ. Anal. Calc.
/
ether to give
73 8C; UV
17.64%.
/
ꢂ
/
/
4. Antiviral assay
/
/
/
Human PBM cells (obtained from Atlanta Red Cross)
were isolated by FicollꢁHypaque discontinuous gradi-
ent centrifugation from healthy seronegative donors.
Cells were stimulated with phytohemagglutinin A
/
/
/
/
/
/
(Difco, Sparks, MD) for 2ꢁ3 days prior to use. HIV-1/
/
/
LAI obtained from the Centers for Disease Control and
Prevention (Atlanta, GA) was used as the standard
reference virus for the antiviral assays. Infections were
done in bulk for 1 h, either with 100 TCID 50/1ꢃ
cells for a flask (T25) assay or with 200 TCID 50/6ꢃ
/
/
for C16H16N2O5S: C, 55.16; H, 4.63, N, 8.04. Found: C,
55.42, H, 4.75, N, 7.99%.
/
107
105
/
cells/well for a 24 well plate assay. Cells were added to a
plate or flask containing a 10-fold serial dilution of the
test compound. Assay medium was RPMI-1640 supple-
mented with heat inactivated 16% fetal bovine serum,
3.5. 1-(2-Hydroxymethyl-b-
yl)imidazole 4-carboxamide (7) and 1-(2-
hydroxymethyl-a-L-[1,3]oxathiolan-5-yl)imidazole 4-
carboxamide (8)
L-[1,3]oxathiolan-5-
1.6 mM
L-glutamine, 80 IU/ml penicillin, 80 mg/ml
streptomycin, 0.0008% DEAE-Dextran, 0.045% sodium
bicarbonate, and 26 IU/ml recombinant interleukin-2
(Chiron Corp, Emeryville, CA). AZT was used as a
positive control for the assay. Untreated and uninfected
PBM cells were grown in parallel at equivalent cell
concentrations as controls. The cell cultures were
Compound 5 was dissolved in saturated NH3/MeOH
(5 ml) and stirred 12 h in sealed bomb at 90 8C. After
reaction all solvent evaporated to dryness and coevapo-
rated with MeOH to give crude compound 7 which is
purified by silica gel column chromatography (CHCl3ꢁ
/
MeOH, 9:1), (35 mg, 76.1%): m.p. 164ꢁ166 8C; UV
/
maintained in a humidified 5% CO2ꢁair at 37 8C for
/
(H2O) lmax 231.0 (o 73 641) 276.5 (o 14 762) (pH 2),
202.5 (o 10 903) 238.5 (o 13 152) (pH 7), 210.5 (o 10 277)
5 days and supernatants were collected for reverse
transcriptase (RT) activity.
239.0 (o 12 510) (pH 11); [a]D25
ꢄ
/
64.748 (c 0.37, MeOH);
1H NMR (CDCl3): d 8.22 (s, 1H, H-5), 7.97 (s, 1H, H-
2), 7.85 and 7.64 (2s, 2H, NH2), 6.84 (t, Jꢅ3.6 Hz, 1H,
H-1?), 5.47 (t, 1H, H-4?), 5.21 (t, Jꢅ3.5 Hz, 2H, H-5?),
3.73 (bs, 1H, OH) 3.55 (m, Jꢅ5.3 Hz, 1H, H-2?), 3.21
Supernatants were centrifuged at 12,000 rpm for 2 h
to pellet the virus. The pellet was solubilized with
vortexing in 100 ml virus solubilization buffer containing
0.5% Triton X-100, 0.8 M NaCl, 0.5 mM phenylmethyl-
sulfonyl fluoride, 20% glycerol, and 0.05 M Tris, pH 7.8.
/
/
/

996
A. Kocabalkanli, R.F. Schinazi / Il Farmaco 57 (2002) 993ꢁ997
/
Ten microliters of each sample were added to 75 ml RT
reaction mixture (0.06 M Tris, pH 7.8, 0.012 M MgCl2,
0.006 M dithiothreitol, 0.006 mg/ml poly (rA)n oligo
(dT)12ꢁ18, 96 microg/ml dATP, and 1 mM of 0.08 mCi/
Structures of the novel compounds were determined
by the elemental analyses and their formulae were
1
confirmed by UV, H and 13C NMR and MS spectro-
metric data.
3
ml H-thymidine triphosphate (Moravek Biochemicals,
In the 1H NMR spectrum of compound 2, the signals
of the CH3O group were observed at 3.43 and 3.53 ppm
as two singlets due to two anomers, a and b; and the
Brea, CA) and incubated at 37 8C for 2 h. The reaction
was stopped by the addition of 100 ml 10% trichlor-
oacetic acid containing 0.05% sodium pyrophosphate.
The acid insoluble product was harvested onto filter
paper using a Packard Harvester (Meriden, CT), and the
RT activity was read on a Packard Direct Beta Counter
(Meriden, CT). The RT results were expressed in counts
per minute (CPM) per milliliter. The antiviral 50%
effective concentration (EC50) and 90% effective con-
centration (EC90) were determined from the concentra-
tion-response curve using the median effect method [7].
signals due to menthyl group were observed at 1.70ꢁ
1.01, 1.00ꢁ0.96 and 0.85ꢁ0.82 ppm. These signals
disappeared in the H NMR spectrum of compound 3
and signals at 3.89ꢁ3.76 ppm due to methylene and 2.43
/
/
/
1
/
and 2.09 ppm due to the OH group were observed which
confirmed the reduction of compound 2. In the 1H
NMR spectrum of compound 4, the signal due to OH
group disappeared and the signal due to benzoyl group
1
7.44 ppm. In the H NMR spectrum
appeared at 8.18ꢁ
/
of compound 5 (and compound 6) the signals due to
C2ꢀH and C5ꢀH of the imidazole moiety could be
observed at 7.62ꢁ7.58 (7.78; 6) and 7.79 (8.19; 6) ppm;
the protons of the oxathiolane moiety were observed as
a doublet for CH2ꢀCH ꢀO at 7.12ꢁ7.11 (as a double
doublet at 6.82; 6); and another double doublet for Sꢀ
CH at 5.80 (5.57; 6); as two double doublets for SꢀCH2
at 3.75 and 3.28 (at 3.72 and 3.20; 6) and as two double
doublets for CH2ꢀOBz at 4.57 and 4.47 (4.77 and 4.70;
6) ppm. These protons absorbed as double doublets
because of the rigidity of the ring. The protons of Sꢀ
OBz were diastereotropic and therefore
4.1. Cytotoxicity assays
/
/
/
The compounds were evaluated for their potential
toxic effects on uninfected PHA-stimulated human
PBM cells, in CEM (T-lymphoblastoid cell line obtained
from American Type Culture Collection, Rockville,
MD.) and Vero (African green monkey kidney) cells.
PBM cells were obtained from whole blood of healthy
seronegative donors (HIV-1 and hepatitis B virus) by
single-step Ficoll-Hypaque discontinous gradient cen-
trifugation. Log phase Vero, CEM and PHA-stimulated
/
/
/
/
/
/
/
CH2 and CH2ꢀ
/
human PBM cells were seeded at a density of 5ꢃ
/
103,
gave peaks at different values which splits each other. In
1
the H NMR spectra of the compounds 7 and 8, the
2.5ꢃ
/
103 and 5ꢃ104 cells/well, respectively. All of the
/
cells were plated in 96-well cell culture plates containing
ten-fold serial dilutions of the test drug. The cultures
were incubated for 3, 4, and 5 days for Vero, CEM, and
peaks due to CH3 and phenyl were replaced by the NH2
(at 7.85 and 7.64 ppm; 7 and 7.94 and 7. 92 ppm; 8 as
two singlets) and OH (at 3.73 ppm as a broad singlet; 7
and 3.54 ppm as a multiplet; 8) peaks.
PBM cells, respectively in a humidified 5% CO2ꢁair at
/
37 8C. At the end of incubation, MTT tetrazolium dye
solution (Cell titer 96†, Promega, Madison, WI) was
added to each well and incubated overnight. The
reaction was stopped with stop solubilization solution
(Promega, Madison, WI). The plates were incubated for
5 h to ensure that the formazan crystals were dissolved.
The plates were read at a wavelength of 570 nm using an
ELISA plate reader (Bio-tek instruments, Inc., Wi-
nooski, VT, Model # EL 312e). The 50% inhibition
concentration (IC50) was determined from the con-
The structures of the synthesized compounds were
also confirmed by the data provided by 13C NMR
spectra; the peaks due to the entering groups could be
observed, while the peaks due to the leaving groups
disappeared in the spectra.
The FAB-MS spectra of the synthesized compounds
showed the [Mꢂ
H]ꢂ peaks, but in the spectrum of
compound 3, the compound fragmented so fast, that the
/
mentioned peak could not be observed.
Optical rotations of b and a anomers 5, 6 and 7, 8
/
centrationꢁ
/
response curve using the median effect
were determined to be ꢄ
/
34.19, ꢂ
/
30.79 and ꢄ
/
64.74, ꢂ
method [7].
31.788, respectively. Since the b and a anomers are both
mixtures of the two enantiomers which could not be
separated by column chromatography, they do not
rotate the plane polarized light equally.
In the uv spectra of compounds 5, 6, 7 and 8, the lmax
were determined to be 230.5, 232, 231 and 207 nm,
respectively.
As a result of this antiviral assay, compounds 7 and 8
did not show significant activity up to 100 mM against
HIV-1. The compounds were found out to be toxic in
the various mammalian cells up to 100 mM (Fig. 1).
5. Results and discussion
In this study, novel oxathiolane imidazole nucleosides
were synthesized with the expectation of gaining com-
pounds with anti-HIV activity. Compounds 7 and 8
were tested for anti-HIV activity in human PBM cells,
Compounds 7 and 8 were synthesized as described in
Section 2 and Scheme 1.

A. Kocabalkanli, R.F. Schinazi / Il Farmaco 57 (2002) 993ꢁ
/
997
997
Scheme 1.
[(ꢂ
from
/
)-(2S, 5R)-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine]
and they were found not to possess significant activity
D
-mannose and its anti-HIV activity, J. Org. Chem. 56
up to 100 mM against HIV-1. Nucleosides always have
been challenging structures with anti-HIV activity and
structural modification of 7 and 8 may still lead to
promising compounds.
(1991) 6504.
[3] L.S. Jeong, A.J. Alves, S.W. Carrigan, O.K. Hea, J.W. Beach, C.K.
Chu, An efficient synthesis of enantiomerically pure (ꢂ
1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine [(ꢂ)-BCH-189]
from -galactose, Tetrahedron Lett. 33 (1992) 595.
/)-(2S, 5R)-
/
D
[4] J.W. Beach, L.S. Jeong, A.J. Alves, D. Pohl, O.K. Hea, C.-N.
Chang, S.-L. Doong, R.F. Schinazi, Y.-L. Cheng, C.K. Chu,
Acknowledgements
Synthesis of enantiomerically pure (2?R, 5?S)-(ꢄ)-1-[2-(hydroxy-
/
methyl)oxathiolan-5-yl]cytosine as a potent antiviral agent against
hepatitis B virus (HBV) and human immunodeficiency virus (HIV),
J. Org. Chem. 57 (1992) 2217.
A. Kocabalkanli acknowledges the support of the
NATO Science Fellowship Program of The Scientific
and Technical Research Council of Turkey (TUBITAK)
and extends her special thanks to Dr C.K.Chu for his
collaboration. R.F. Schinazi is supported in part by the
Department of Veterans Affairs and NIH grant AI-
32351 and AI-41980.
[5] R.F. Schinazi, C.K. Chu, A. Peck, A. McMillan, R. Mathis, D.
Cannon, L.-S. Jeong, J.W. Beach, W.-B. Choi, S. Yeola, D.C.
Liotta, Activity of the four optical isomers of 2?,3?-dideoxy-3?-
thiacytidine (BCH-189) against HIV-1 in human lymphocytes,
Antimicrob. Agents Chemother. 36 (1992) 672ꢁ676.
/
[6] H. Jin, M.A. Siddiqui, C.A. Evans, H.L.A. Tse, T.S. Mansour,
M.D. Goodyear, P. Ravenscroft, C.D. Beels, Diastereoselective
synthesis of the potent antiviral agent (ꢄ)-2?-deoxy-3?-thiacytidine
/
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