The importance of the 4'-hydroxyl hydrogen for the anti-trypanosomal and antiviral properties of (+)-5'-noraristeromycin and two 7-deaza analogues

Article abstract of DOI:10.1016/S0968-0896(98)00036-4

(+)-5'-Noraristeromycin (1) has shown significant antiviral activity while its 7-deaza analogue 2 is an anti-trypanosomal candidate. To determine the relevance of the 4'-hydroxyl hydrogen in these activities, a derivative of 1 (that is, 3) where the C-4' hydroxyl hydrogen has been replaced by a methyl group has been prepared beginning with palladium (0) mediated coupling of the sodium salt of N⁶-benzoyladenine (9) and (1S,4R)-4-methoxy-2-cyclopenten-1-yl acetate (5). The synthesis of compound 5 is described from (1S,4R)-1-[(tert-butyldimethylsilyl)oxy]-4-hydroxycyclopent-2-ene (6) in three steps. Analogous preparations of the 7-deaza and 8-aza-7-deaza derivatives of 3 related to 2 (that is, 4 and 12) are also reported. The new derivatives (3, 4, and 12) failed to show improved antiviral activity. Compound 12 was the only derivative with some anti-trypanosomal activity, giving 40% inhibition of growth at 100μM against bloodstream forms of a Typanosoma brucei brucei isolate in a standard in vitro screen. This study indicated that the C-4'-hydroxyl hydrogen plays a role in the medicinal properties of 1 and 2. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00036-4

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 797±801
The Importance of the 40-Hydroxyl Hydrogen for the
Anti-Trypanosomal and Antiviral Properties of
(+)-50-Noraristeromycin and Two 7-Deaza Analogues
Katherine L. Seley,a Stewart W. Schneller,a,* Erik De Clercq,b
Donna Rattendi,c Schenella Lane,c Cyrus J. Bacchic and Brent Korbad
aDepartment of Chemistry, Auburn University, Auburn, AL 36849-5312, USA
bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
cHaskins Laboratories and Department of Biology, Pace University, New York, NY 10038, USA
dGeorgetown University Medical Center, Division of Molecular Virology and Immunology, Rockville, MD 20852, USA
Received 6 November 1997; accepted 18 December 1997
AbstractÐ(+)-50-Noraristeromycin (1) has shown signi®cant antiviral activity while its 7-deaza analogue 2 is an anti-
trypanosomal candidate. To determine the relevance of the 40-hydroxyl hydrogen in these activities, a derivative of 1
(that is, 3) where the C-40 hydroxyl hydrogen has been replaced by a methyl group has been prepared beginning with
palladium (0) mediated coupling of the sodium salt of N6-benzoyladenine (9) and (1S,4R)-4-methoxy-2-cyclopenten-1-yl
acetate (5). The synthesis of compound 5 is described from (1S,4R)-1-[(tert-butyldimethylsilyl)oxy]-4-hydroxy-
cyclopent-2-ene (6) in three steps. Analogous preparations of the 7-deaza and 8-aza-7-deaza derivatives of 3 related to 2
(that is, 4 and 12) are also reported. The new derivatives (3, 4, and 12) failed to show improved antiviral activity.
Compound 12 was the only derivative with some anti-trypanosomal activity, giving 40% inhibition of growth at
100 mM against bloodstream forms of a Typanosoma brucei brucei isolate in a standard in vitro screen. This study
indicated that the C-40-hydroxyl hydrogen plays a role in the medicinal properties of 1 and 2. # 1998 Elsevier Science
Ltd. All rights reserved.
Introduction
In the search for new medicinal agents derived from
nucleosides, recent successes with analogues in the L-
con®guration have been surprising due to their un-
natural structure.1 Our investigations have uncovered
two L-like carbocyclic nucleosides with promising activ-
ity: (+)-50-noraristeromycin (1) with potency towards
hepatitis B virus2 and (+)-7-deaza-50-noraristeromycin
(2) with e€ectiveness as an anti-trypanosomal candi-
date.3 To explore the structural features of 1 and 2 that
are necessary for their respective activities, attention has
focused on the role of the C-40 hydroxyl hydrogen by
Chemistry
considering the methyl derivatives (3 and 4). The results
of this investigation are described.
To achieve the desired compounds 3 and 4, sights were
®rst set on the preparation of the cyclopentene deriva-
tive 5 for coupling4 with an appropriate heterocyclic
base. Scheme 1 shows that the synthesis of 5 began with
*Corresponding author.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00036-4

798
K. L. Seley et al./Bioorg. Med. Chem. 6 (1998) 797±801
Scheme 1. Reaction conditions: (a) MeI, KOH, DMSO; (b) Bu4NF; (c) Ac2O.
methylation of the known5 silylated derivative 6 to give
7. Desilylation of 7 provided 8, which was then acetyl-
ated to 5.
Anti-HBV Results
Modi®cation of 1 by the replacement of the C-40
hydrogen by a methyl group (compound 3) reduced
anti-HBV activity (see Table 1). Further modi®cations
of 1 to either the 7-deaza (4) or the 8-aza-7-deaza (12)
derivatives completely abolished anti-HBV activity
(Table 1). However, the replacement of the C-40 hydrogen
by a methyl group (compounds 3 and 12) signi®cantly
reduced cytotoxicity to human hepatoblastoma cells.
With 5 available, its coupling with the sodium salt of
N6-benzoyladenine (9) provided 10 (Scheme 2). Deben-
zoylation of 10 to 11 followed by glycolization yielded
(+)-3. Synthesis of 4 and its 8-aza analogue6 12, was
accomplished in a similar manner, but utilizing the
sodium salt of 4-chloropyrrolo[2,3-d]pyrimidine7 (13) or
the sodium salt of 4-methoxypyrazolo[3,4-d]pyrimidine8
(14), respectively, for the initial palladium coupling step
to 15 and 16, respectively (Scheme 2). Ammonolysis and
glycolization a€orded the desired 4 and 12.
Anti-Trypanosomal Results
Compounds 3, 4, and 12 were tested for in vitro growth
inhibition of two trypanosome isolates in a standard
screen. Trypanosoma brucei brucei Lab 110 EATRO is
a standard isolate used for routine testing, while Try-
panosoma brucei rhodesiense KETRI 243 is a clinical
isolate from East Africa. Although none of the com-
pounds were highly e€ective, with activities of only 9%
and 22% (at 100 mM) being noted for compounds 3 and
4, respectively, compound 12 was 40% inhibitory to T.
b. brucei growth at the same concentration. Compound
12 was only minimally active (16%) against the T. b.
Antiviral Results
Compounds 1, 2, 3, 4, and 12 were assayed against
herpes simplex virus type 1 (strain KOS) and type 2
(strain g), vaccinia virus, and vesicular stomatitis virus
in human embryonic skin-muscle (E6SM) ®broblasts,
vesicular stomatitis virus in HeLa cells, parain¯uenza
type 3 virus, reovirus type 1, and Sindbis virus in Vero
cells, human immunode®ciency virus type 1 (strain IIIB)
and type 2 (strain ROD) in CEM cells, and cytomegalo-
virus (strains AD-169 and Davis) and varicella-zoster
virus (strains Oka and YS) in human embryonic lung
(HEL) cells. Whereas compound 1 showed activity
against vaccinia, vesicular stomatitis, parain¯uenza-3,
and reo-1 at a 50% inhibitory concentration (IC50) of
0.7, 2, 0.2 and 7 mg/mL, no antiviral activity was noted
with compounds 2, 3, 4 and 12 up to the highest con-
centrations tested (50 mg/mL in HEL, 250 mg/mL in
CEM, >400 mg/mL in the other cells).
Table 1. HBV activity in 2.2.15 cells
Compd
CC50(mM)
EC50(mM)
EC90(mM)
1
466‹20
277‹21
>1000
1.4‹0.1
>100
41‹4.3
>10
9.6‹0.8
>100
119‹10
>10
2
3
4
12
666‹31
>1000
>10
>10
Scheme 2. Reaction conditions: (a) NH4OH:MeOH; (b) NH3 in MeOH; (c) OsO4/60% aq 4-methylmorpholine N-oxide.

K. L. Seley et al./Bioorg. Med. Chem. 6 (1998) 797±801
799
A
rhodesiense isolate, with compounds 3 and 4 showing
even less activity (12% and 11%, respectively).
(1S,4R)-4-Methoxy-2-cyclopenten-1-yl acetate (5).
solution of 7 (8.93 g, 38.9 mmol) and Bu4NF (1.0 M in
THF, 82 mL, 82 mmol) was stirred at room temperature
for 3 h. The solvent was evaporated under reduced
pressure and the residue was puri®ed via column chro-
matography eluting with hexane:EtOAc (5:1, followed
by 2:1) to a€ord 3.5 g (78%) of 8 as a colorless syrup,
which was used directly in the next step; 1H NMR
(CDCl3) d 1.57 (dt, 1H), 2.65 (dt, 1H), 3.06 (br, 1H),
Conclusion
The biological results presented herein show that repla-
cement of the 40-hydroxyl hydrogen of 1 with a methyl
group leads to diminished antiviral and anti-trypanoso-
mal activity. This could be due to intolerable steric fac-
tors introduced by the methyl substituent or loss of the
proton that has a necessary biological function.
3.34 (s, 3H), 4.24 (q, 1H), 4.60 (q, 1H), 6.01 (d, 2H); 13
C
NMR (CDCl3) d 40.10, 56.08, 74.50, 83.33, 133.17,
137.42.
To a chilled stirring solution of 8 (3.5 g, 30.52 mmol) in
dry CH2Cl2 (150 mL) was added pyridine (3.5 g,
45.5 mmol), dimethylaminopyridine (0.05 g), and acetic
anhydride (4.5 g, 45.5 mmol). The reaction mixture was
stirred overnight at room temperature, at which point it
was treated with saturated NaHCO3 solution (50 mL)
and stirred vigorously for 15 min. The organic layer was
separated and washed with ice-cold 1 N HCl (200 mL),
brine (2Â200 mL), dried (MgSO4) and evaporated under
reduced pressure. The residue was puri®ed via column
chromatography eluting with hexane:EtOAc (19:1) to
give 2.65 g (55%) of 5 as a yellow syrup; 1H NMR
(CDCl3) d 1.66 (dt, 1H), 2.03 (s, 3H), 2.74 (p, 1H), 3.35
(s, 3H), 4.32 (t, 1H), 5.51 (t, 1H), 6.05 (dd, 2H); 13C
NMR (CDCl3) d 20.93, 36.75, 56.07, 76.49, 82.84,
132.85, 135.65, 170.58. Calcd for C8H12O3: C, 61.70; H,
7.71. Found: C, 61.59, H, 7.77.
Experimental
Melting points were recorded on a Meltemp II melting
point apparatus and are uncorrected. Combustion ana-
lyses were performed by M-H-W Laboratories (Phoenix
AZ, USA). 1H and 13C spectra were recorded on a Bruker
AC 250 spectrometer (operated at 250 and 62.5 MHz,
respectively) all referenced to internal tetramethylsilane
(TMS) at 0.0 ppm. The spin multiplicities are indicated
by the symbols s (singlet), d (doublet), t (triplet), q
(quartet), p (pentet), m (multiplet) and br (broad).
Optical rotations were measured on a JASCO DIP-370
polarimeter. Reactions were monitored by thin-layer
chromatography (TLC) using 0.25 mm Whatman Dia-
mond silica gel 60-F254 precoated plates with visualiza-
tion by irradiation with a Mineralight UVGL-25 lamp
or exposure to iodine vapor. Column chromatography
was performed on Whatman silica, 230±400 mesh, 60 A
and elution with the indicated solvent system. Yields
refer to chromatographically and spectroscopically (1H
and 13C NMR) homogeneous materials.
(1S,4R)-4-Methoxy-1-(6-amino-9H-purin-9-yl)cyclopent-
2-ene ((±)-11). To a solution of N6-benzoyladenine
(2.02 g, 8.41 mmol) in dry DMSO (30 mL) was added
NaH (0.23 g, 8.62 mmol, 95%) to generate 9. The mix-
ture was stirred at room temperature under an argon
atmosphere for 30 min. Tetrakis(triphenylphosphine)-
palladium (0.61 g, 0.53 mmol), Ph3P (0.23 g, 0.88 mmol)
and a solution of 5 (1.2 g, 7.65 mmol) in dry THF
(30 mL) was added.9 The mixture was stirred at 55 ꢀC
for 2 days. The volatiles were evaporated under reduced
pressure, and the residue was slurried in CH2Cl2 and
®ltered. The ®ltrate was washed with brine and evapo-
rated. The residue was puri®ed via column chromato-
graphy eluting with EtOAc:MeOH (19:1, followed
by 9:1) to a€ord 3.8 g of 10 as a gum, which was
used directly in the next reaction, without further
puri®cation.
(1S,4R)-1-[(tert-Butyldimethylsilyl)oxy]-4-methoxycyclo-
pent-2-ene (7). To a suspension of crushed KOH (23.0 g,
0.41 mol) in DMSO (500 mL) that had been stirring at
room temperature for 15 min was added 65 (22.0 g,
0.10 mol) followed immediately by the dropwise addi-
tion of MeI (10.21 g, 0.21 mol). The reaction mixture
was then stirred at room temperature for 4 h, after
which it was poured onto ice and extracted with CH2Cl2
(3Â500 mL). The organic layers were combined and
washed with brine, dried (MgSO4), and evaporated. The
residue was puri®ed via column chromatography eluting
with hexane/EtOAc (10:1, followed by 4:1) to a€ord
8.93 g (38%) of 7 as a colorless syrup; 1H NMR
(CDCl3) d 0.08 (s, 6H), 0.86 (s, 9H), 1.57 (dt, 1H), 2.65
(dt, 1H), 3.33 (s, 3H), 4.26 (t, 1H), 4.67 (t, 1H), 5.93 (m,
2H); 13C NMR (CDCl3) d 4.67, 18.10, 25.84, 40.83,
55.67, 74.84, 83.04, 132.39, 137.54. Calcd. for C12H24
O2Si: C, 63.28; H, 10.54. Found: C, 63.49, H, 10.39.
A solution of 10 (3.8 g, 11.3 mmol) in NH4OH:H2O (1:1,
100 mL) was sealed in a steel vessel and heated at 110 ꢀC
for 2 days. The vessel was cooled to 0 ꢀC, and the sol-
vents removed under reduced pressure. The residue was
then puri®ed via column chromatography, eluting with
EtOAc:MeOH (19:1, followed by 10:1). Fractions con-
taining product were combined and evaporated to give

800
K. L. Seley et al./Bioorg. Med. Chem. 6 (1998) 797±801
1.65 g (42% in two steps) of 11 as a white crystalline
solid, mp 155±156 ꢀC; [a]23 73.40ꢀ (c 0.20, MeOH);
in a steel vessel and heated at 110 ꢀC for 3 days. The
vessel was cooled to 0 ꢀC, and the solvents removed
under reduced pressure. The residue was then puri®ed
via column chromatography, eluting with CH2Cl2:
MeOH (98:2). Fractions containing product were com-
bined and evaporated to give 1.48 g (80%) of 17 as a
white sticky foam; 1H NMR (DMSO-d6) d 1.73±1.82
(dt, 1H), 2.87±2.99 (dt, 1H), 3.40 (s, 3H), 4.44 (br, 1H),
5.34 (br, 2H), 5.86 (br, 1H), 6.01 (dd, 1H), 6.27 (dd,
1H), 6.36 (d, 1H), 7.10 (d, 1H), 8.33 (s, 1H); 13C NMR
(DMSO-d6) d 38.55, 56.63, 56.76, 83.87, 98.07, 103.21,
122.35, 133.92, 135.25, 150.01, 151.65, 156.75. Calcd for
C12H14N4O: C, 62.76; H, 6.10; N, 24.22. Found: C,
62.49; H, 6.15; N, 24.52.
D
1H NMR (DMSO-d6) d 1.80 (dt, 1H), 2.87 (m, 1H), 3.30
(s, 3H), 4.44 (t, 1H), 5.47 (t, 1H), 6.23 (dt, 2H), 7.20 (br,
2H), 7.94 (s, 1H), 8.15 (s, 1H); 13C NMR (DMSO-d6) d
37.94, 55.94, 56.51, 83.14, 118.83, 132.77, 135.81,
138.55, 149.10, 152.38, 155.95. Calcd for C11H13N5O: C,
57.31; H, 5.64; N, 30.16. Found: C, 57.27; H, 5.73; N,
30.20.
(1S,2S,3S,4R)-4-Methoxy-1-(6-amino-9H-purin-9-yl)cyclo-
pentane-2,3-diol ((+)-3). To a solution of 11 (0.85 g,
3.66 mmol) in THF:H2O:acetone (75 mL, 1:1:1) was
added OsO4 (0.1 g) and 4-methylmorpholine N-oxide
(1 mL). The mixture was stirred at room temperature
overnight until TLC (EtOAc:MeOH, 5:1) showed no
remaining starting material. The solvent was evapo-
rated, and the residue was puri®ed via column chroma-
tography, eluting with EtOAc:MeOH (9:1). Fractions
containing product were combined and evaporated to
a€ord 0.46 g (47%) 3 as a white solid, mp 230±231 ꢀC;
(1S,2S,3S,4R)-4-Methoxy-1-(4-aminopyrrolo[2,3-d]pyr-
imidin-7-yl)cyclopentane-2,3-diol ((+)-4). To a solution
of 17 (1.5 g, 6.48 mmol) in THF:H2O (40 mL, 3:1) was
added OsO4 (0.125 g) and 4-methylmorpholine N-oxide
(1.5 mL). The mixture was stirred at room temperature
for two days until TLC (CH2Cl2:MeOH, 95:5) showed
no remaining starting material. The solvent was evapo-
rated, and the residue was puri®ed via column chroma-
tography, eluting with CH2Cl2:MeOH (95:5). Fractions
containing product were combined and evaporated, and
the residue dissolved in MeOH and treated with de-
colorizing charcoal, ®ltered and the ®ltrate evaporated
to a€ord an o€ white solid. This was recrystallized in
MeOH:EtOAc to a€ord 0.26 g (15%) of 4 as a white
solid, mp 176±177 ꢀC; [a]24D +32.2ꢀ (c 0.20, MeOH); 1H
NMR (DMSO-d6) d 1.65±1.76 (m, 1H), 2.45±2.57 (m,
1H), 3.29 (s, 3H), 3.58 (t, 1H), 3.89 (br, 1H), 4.21±4.30
(dt, 1H), 4.90 (q, 1H), 4.94 (t, 1H), 5.56 (d, 1H), 6.55 (d,
1H), 6.94 (br, 2H), 7.18 (d, 1H), 8.03 (s, 1H); 13C NMR
(DMSO-d6) d 32.96, 56.40, 57.91, 73.14, 74.05, 83.46,
119.25, 139.92, 149.72, 152.07, 155.95. Calcd for
C12H16N4O3: C, 54.72; H, 6.08; N, 21.12. Found: C,
54.68; H, 6.25; N, 21.09.
[a]21 +38.4ꢀ (c 0.20, DMF); 1H NMR (DMSO-d6) d
D
1.99 (m, 1H), 2.57 (m, 1H), 3.30 (s, 3H), 3.57 (q, 1H),
3.91 (d, 1H), 4.43 (q, 1H), 4.64 (q, 1H), 4.96 (d, 1H),
5.06 (d, 1H), 7.17 (br, 2H), 8.12 (s, 1H), 8.14 (s, 1H); 13
C
NMR (DMSO-d6) d 32.96, 56.40, 57.91, 73.14, 74.05,
83.46, 119.25, 139.92, 149.72, 152.07, 155.95. Calcd for
C11H15N5O3: C, 49.98; H, 5.68; N, 26.31. Found: C,
49.78; H, 5.89; N, 26.33.
(1S,4R)-4-Methoxy-1-(4-chloropyrrolo[2,3-d]pyrimin-7-yl)-
cyclopent-2-ene (15). To a solution of 4-chloropyrrolo-
[2,3-d]pyrimidine7 (2.85 g, 18.56 mmol) in dry DMSO
(30 mL) was added NaH (0.48 g, 19.0 mmol, 95%) to
give 13. The mixture was stirred at room temperature
under an argon atmosphere for 30 min. To this was
added tetrakis(triphenylphosphine)palladium (1.35 g,
1.17 mmol), Ph3P (0.51 g, 1.94 mmol) and a solution of 5
(2.68 g, 17.09 mmol) in dry THF (30 mL).3 The mixture
was stirred at 55 ꢀC for 2 days. The volatiles were eva-
porated under reduced pressure, and the residue was
slurried in CH2Cl2 and ®ltered. The ®ltrate was washed
with brine and evaporated. The residue was puri®ed via
column chromatography eluting with hexane/EtOAc
(9:1, followed by 5:1) to a€ord 2.08 g (48.5%) of 15 as a
colorless syrup; 1H NMR (CDCl3) d 1.77±1.85 (dt, 1H),
2.87±2.99 (p, 1H), 3.41 (s, 3H), 4.47 (m, 1H), 5.93 (m,
1H), 6.02 (dd, 1H), 6.33 (d, 1H), 6.60 (d, 1H), 7.40 (d,
1H), 8.63 (s, 1H); 13C NMR (CDCl3) d 38.21, 56.80,
57.05, 83.61, 99.94, 117.65, 126.95, 133.18, 135.95,
150.38, 150.48, 151.88. Calcd for C12H12ClN3O: C, 57.90;
H, 4.82; N, 16.76. Found: C, 57.86; H, 4.55; N, 16.77.
(1S,4R)-4-Methoxy-1-(4-methoxypyrazolo[3,4-d]pyrimin-
7-yl)cyclopent-2-ene (16). To a solution of 4-methoxy-
pyrazolo[3,4-d]pyrimidine8 (3.12 g, 20.78 mmol) in dry
DMSO (30 mL) was added NaH (0.54 g, 21.4 mmol,
95%) to give 14. The mixture was stirred at room tem-
perature under an argon atmosphere for 30 min. To this
solution was added tetrakis(triphenylphosphine)palla-
dium (1.51 g, 1.31 mmol), Ph3P (0.57 g, 2.17 mmol) and
a
solution of 5 (3.0 g, 19.13 mmol) in dry THF
(30 mL).10 The mixture was stirred at 55 ꢀC for 2 days.
The volatiles were evaporated under reduced pressure,
and the residue was slurried in CH2Cl2 and ®ltered. The
®ltrate was washed with brine and evaporated. The
residue was puri®ed via column chromatography eluting
with hexane:EtOAc (9:1, followed by 4:1) to a€ord
2.03 g (43%) of 16 as white crystals, mp 81±82 ꢀC; 1H
NMR (CDCl3) d 2.10±2.20 (dt, 1H), 2.91±3.03 (dt, 1H),
(1S,4R)-4-Methoxy-1-(4-aminopyrrolo[2,3-d]pyrimin-7-yl)-
cyclopent-2-ene (17). A solution of 15 (2.0 g, 7.98 mmol)
in saturated methanolic ammonia (150 mL) was sealed

K. L. Seley et al./Bioorg. Med. Chem. 6 (1998) 797±801
801
3.41 (s, 3H), 4.15 (s, 3H), 4.56 (t, 1H), 5.91 (m, 1H), 6.05
(dt, 1H), 6.30 (dt, 1H), 8.07 (s, 1H), 8.56 (s, 1H); 13C
NMR (CDCl3) d 37.16, 54.10, 56.27, 59.95, 83.67,
102.86, 131.67, 132.69, 135.09, 154.27, 154.96, 163.96.
Calcd for C12H12N3ClO: C, 57.90; H, 4.82; N, 16.76.
Found: C, 57.86; H, 4.55; N, 16.77.
maintained under conditions that were identical to those
used for the antiviral assays.11
Anti-trypanosomal
The compounds were dissolved in medium and added
aseptically. Coulter counts were taken at 72 h as descri-
bed previously.3,10
(1S,2S,3S,4R)-4-Methoxy-1-(4-aminopyrazolo[3,4-d]pyr-
imidin-7-yl)cyclopentane-2,3-diol ((+)-12). A solution of
16 (1.8 g, 7.28 mmol) in saturated methanolic ammonia
(150 mL) was sealed in a steel vessel and heated at
110 ꢀC for 3 days. The vessel was cooled to 0 ꢀC, and the
solvents removed under reduced pressure. The residue
was then puri®ed via column chromatography, eluting
with CH2Cl2:MeOH (95:5). Fractions containing pro-
duct were combined and evaporated to give 1.3 g (77%)
of 18 as a gum, which was used directly without char-
acterization.
Acknowledgements
The assistance of Dr Jiri Zemlicka of the School of
Medicine, Wayne State University in obtaining the
optical rotation data is much appreciated. This investi-
gation received ®nancial support from the Department
of Health and Human Services (U19-A131718) and
from the UNDP/World Bank/World Health Organiza-
tion Special Programme for Research and Training in
Tropical Diseases (TDR) grant 950594 (C.J.B.). This
assistance is gratefully acknowledged. The anti-HBV
assays were supported by contract NO1-AI-45195
between the National Institutes of Allergy and Infec-
tious Disease and Georgetown University and this
assistance is also much appreciated. These investigations
were also supported in part by the Belgian Fonds voor
Geneeskundig Weteusehappelyk Onderzoek and the
Biomedical Research Programme of the European
Commission. We thank Anita Van Lierde, Frieda De
Meyer, Anita Camps, Katrin Saelen, Ann Absillis and
Ria Van Berwaer for excellent technical assistance.
To a solution of 18 (1.3 g, 6.0 mmol) in THF:H2O
(50 mL, 10:1) was added OsO4 (0.125 g) and NMO
(2 mL). The mixture was stirred at room temperature
overnight. The solvent was evaporated, and the residue
was puri®ed via column chromatography, eluting with
CH2Cl2:MeOH (95:5, followed by 9:1). Fractions con-
taining product were combined and evaporated to
a€ord a colorless syrup, which hardened upon cooling.
This solid was then recrystallized in MeOH to a€ord
0.26 g (77% in two steps) 12 as a white solid, mp 128±
129 ꢀC; [a]24 +29.6ꢀ (c 0.20, MeOH); 1H NMR
D
(DMSO-d6) d 1.78±1.88 (m, 1H), 2.41±2.53 (m, 1H),
3.27 (s, 3H), 3.30 (br, 1H), 3.59 (m, 1H), 3.89 (br, 1H),
4.33 (q, 1H), 4.89 (dd, 1H), 4.98 (q, 1H), 7.66 (br, 2H),
8.11 (s, 1H), 8.14 (s, 1H); 13C NMR (DMSO-d6) d 32.99,
56.56, 59.18, 73.18, 74.73, 83.26, 100.36, 132.17, 153.61,
References and Notes
1. For a leading reference, see Furman, P. A.; Wilson, J. E.;
Reardon, J. E.; Painter, G. R. Antiviral Chem. Chemother.
1996, 6, 345, and references cited therein.
.
155.70, 158.11. Calcd for C11H15N5O3 1.0 MeOH: C,
2. Seley, K. L.; Schneller, S. W.; Korba, B. Nucleosides
Nucleotides, 1997, 16, 2095.
49.63; H, 5.88; N, 25.54. Found: C, 49.86; H, 5.62; N,
25.38.
3. Seley, K. L.; Schneller, S. W.; Rattendi, D.; Bacchi, C. J. J.
Med. Chem. 1997, 40, 622.
Antiviral
4. Deardor€, D. R.; Linde, R. G. II; Martin, A. M.; Shulman,
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Antiviral assays were carried out in the systems descri-
bed previously.9
5. Nokami, J.; Matsuura, H.; Nakasima, K.; Shibata, S. Chem.
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6. Seley, K. L.; Schneller, S. W.; Rattendi, D.; Lane, S.;
Bacchi, C. J. J. Med. Chem. 1997, 40, 625.
Anti-HBV
7. Davoll, J. J. Chem. Soc. 1960, 131.
Analysis of anti-HBV activity and associated toxicity
were performed following 9 days of consecutive daily
administration of the test compounds to con¯uent cul-
tures of the HBV-producing cell line, 2.2.15, as pre-
viously described.11 Anti-HBV activity was determined
by blot hybridization analysis of extracellular HBV vir-
ion DNA in culture medium.11 Cytotoxicity was deter-
mined by uptake of neutral red dye on cultures
8. Robins, R. K. J. Am. Chem. Soc. 1956, 78, 784.
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