Synthesis of novel 8-substituted carbocyclic analogs of 2′,3′-dideoxyadenosine with activity against hepatitis B virus

Article abstract of DOI:10.1081/NCN-120016613

Synthesis and antiviral activity of several new 8-substituted carbocyclic analogs of D-2′,3′-dideoxyadenosine are described. The new 8-substituted analogs were synthesized via lithiation of carbocyclic 2′,3′-dideoxyadenosine followed by quenching with ele

Full text of DOI:10.1081/NCN-120016613

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
SYNTHESIS OF NOVEL 8-SUBSTITUTED
CARBOCYCLIC ANALOGS OF 2′,3′-
DIDEOXYADENOSINE WITH ACTIVITY AGAINST
HEPATITIS B VIRUS
Kristjan S. Gudmundsson , Susan M. Daluge , Lynn D. Condreay & Lance C.
Johnson
To cite this article: Kristjan S. Gudmundsson , Susan M. Daluge , Lynn D. Condreay & Lance
C. Johnson (2002) SYNTHESIS OF NOVEL 8-SUBSTITUTED CARBOCYCLIC ANALOGS OF 2′,3′-
DIDEOXYADENOSINE WITH ACTIVITY AGAINST HEPATITIS B VIRUS, Nucleosides, Nucleotides
and Nucleic Acids, 21:11-12, 891-901, DOI: 10.1081/NCN-120016613
To link to this article: http://dx.doi.org/10.1081/NCN-120016613
Published online: 31 Aug 2006.
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©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.
NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 21, Nos. 11 & 12, pp. 891–901, 2002
SYNTHESIS OF NOVEL 8-SUBSTITUTED
CARBOCYCLIC ANALOGS OF 20,30-
DIDEOXYADENOSINE WITH ACTIVITY
AGAINST HEPATITIS B VIRUS
Kristjan S. Gudmundsson,1,* Susan M. Daluge,1 Lynn D. Condreay,2
and Lance C. Johnson2
1Division of Chemistry and
2Virology, GlaxoSmithKline, 5 Moore Drive,
Research Triangle Park, NC 27709
ABSTRACT
Synthesis and antiviral activity of several new 8-substituted carbocyclic
analogs of D-20,30-dideoxyadenosine are described. The new 8-substituted
analogs were synthesized via lithiation of carbocyclic 20,30-dideoxy-
adenosine followed by quenching with electrophiles. This methodology
allows for a divergent synthesis of a variety of 8-substituted analogs
from carbocyclic 20,30-dideoxyadenosine in high yields. 8-Methyl and
8-halogenated carbocyclic 20,30-dideoxyadenosine analogs showed 6–25
fold more activity against hepatitis B virus than the unsubstituted
carbocyclic D-20,30-dideoxyadenosine.
INTRODUCTION
20,30-Dideoxyribofuranosides have received considerable attention as
this class of nucleosides includes several compounds with useful clinical anti-
HIV activity (e.g., ddI and ddC).[1] The 2030-dideoxyribofuranosides suffer
from lack of stability of the glycosidic bond and 2030-dideoxyadenosine (ddA)
*Corresponding author. Fax: (919) 483-6053; E-mail: kristjan.s.gudmundsson@gsk.com
891
DOI: 10.1081=NCN-120016613
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

892
GUDMUNDSSON ET AL.
Figure 1.
also suffers from rapid deamination by adenosine deaminase. Several 20,30-di-
deoxyribofuranoside analogs substituted at the C-8 position of adenine have
been synthesized[2] and evaluated for hydrolytic[3] and enzymatic stability,[4]
but these analogs have not been reported to have significant antiviral acti-
vity.[5] The carbocyclic analogs of D- and L-ddA[6] and numerous other car-
bocyclic-20,30-dideoxynucleoside analogs[7] have been described and reported
to have antiviral activity, including activity against hepatitis B virus.[6,7] Since
carbocyclic and ribofuranoside analogs often have very different antiviral
activity, we became interested in the synthesis of 8-substituted carbocyclic D-
ddA analogs. These have, to the best of our knowledge, not been reported.
Herein we describe a facile route for the synthesis of several novel
8-substituted carbocyclic dideoxyadenosine analogs (1a-g) and their antiviral
activity against hepatitis B virus (HBV).
RESULTS AND DISCUSSION
We envisioned several potential routes toward the desired 8-substituted
carbocyclic dideoxyadenosine analogs as outlined in Sch. 1. Synthesis of
8-substituted derivatives by condensation of orthoformates and diamino-
pyrimidines to form the purine ring system is well precedented for carbo-
cyclic nucleosides,[6,7] but this route is only viable for a limited number of
8-substituted analogs.
As we desired a highly divergent route that could be used to add a
variety of functionality at the C-8 position we initially focused our attention
on C-8 halogenation of carbocyclic D-ddA. The 8-halogen could subse-
quently be replaced by addition-elimination or metal catalyzed coupling
reactions to give the desired 8-substituted compounds. Carbocyclic D-ddA
(2) was synthesized in a similar manner as described in the literature.[6]
Silylation of 2 using TBDMSCl and imidazole gave the silyl protected
carbocyclic dideoxyadenosine (3). Bromination of the C-8 position of 3
using N-bromosuccinimide in dichloromethane was successful, but gave a

CARBOCYCLIC ANALOGS OF ddA
893
Scheme 1.
relatively low and poorly reproducible yield (5-35%) of 4c. Attempts to
improve the yield by use of other solvents (such as N,N-dimethylforma-
mide, dioxane, acetonitrile) failed. Similarly, attempts to brominate the
unprotected carbocyclic dideoxyadenosine (2) using N-bromosuccinimide in
various solvents gave, again, low and poorly reproducible yield of the
desired 1c (5–15%).
We then decided to study lithiation of 3 followed by quenching of the
lithiated adenosine derivative with electrophiles using conditions similar to
those previously described for lithiation-electrophile quenching of 20,30-
dideoxyribofuranosyl derivatives.[2] We found that lithiation of 3 using
excess lithium diisopropylamide (LDA, 5 equiv) in tetrahydrofuran at
ꢀ78ꢁC followed by quenching with an electrophile (e.g., Br2) gave con-
sistently a good yield (e.g., 81% of the 8-bromo derivative, 4c) of the desired
products. For optimal yields, 5–6 equivalents of LDA were required,
fewer equivalents gave lower yield of the desired product and resulted in
recovery of more unreacted starting material. Other bases (such
as lithium tetramethylpiperidine, lithium bis(trimethylsilyl)amide) did not
improve the yield. These LDA lithiation-electrophile quenching conditions
were used for a variety of electrophiles to give good yields of 4a^d and 4f.
The 8-methylamine derivative 4e was obtained by an addition-elimination
reaction, whereby treatment of the 8-iododerivative 4d with methylamine at
130ꢁC gave 4e. The 8-phenyl derivative 4g was synthesized by palladium
catalyzed coupling of 4d and phenylboronic acid. Desilylation, using tetra-
butylammonium fluoride, gave the desired compounds 1a ^ g in excellent
overall yields.

894
GUDMUNDSSON ET AL.
Scheme 2. a, X ¼ CH3; b, X ¼ Cl; c, X ¼ Br; d, X ¼ I; e, X ¼ NHCH3; f, X ¼ CON(CH3)2; g,
X ¼ phenyl. (i) TBDMSCl (1.3 equiv), imidazole, DMF at RT; (ii) LDA (5 eq) at ꢀ 78ꢁC in
THF, then add electrophile (2–3 equiv); (iii) Treatment of 4d (X ¼ I) with CH3NH2 at 130ꢁC;
(iv) Treatment of 4d (X ¼ I) with PhB(OH)2 (1.4 equiv), Pd(PPh3)4 (0.3 equiv), Na2CO3 (aq, 2
equiv) in DME at 80ꢁC; (v) TBAF (2 equiv), THF, RT.
Several of the 8-substituted carbocyclic D-ddA analogs showed inter-
esting activity against HBV as outlined in Table 1.
Thus the 8-methyl analog (1a) and the 8-halogenated analogs (1b ^1d)
showed 6–25 fold better activity against HBV than the unsubstituted carbo-
cyclic D-ddA (2). The 8-methylamine derivative (1e) showed similar activity to
2, while the 8-dimethylcarboxamide (1f) and the 8-phenyl analog (1g) showed
no HBV activity, possibly due to the steric bulkiness of these substituents. For
all the 8-substituted carbocyclic analogs that showed activity against HBV,
the IC50 for activity was clearly delineated from cytotoxicity. None of the
carbocyclic D-ddA derivatives (1a ^1g) had activity against HIV-1 in MT-4

CARBOCYCLIC ANALOGS OF ddA
895
Table 1. Anti-HBV Activitya and Cytotoxicitya of Carbocyclic ddA (2) and 8-Substituted
Analogs in HepG2-2.2.15 Cells
Compound
Number
HBV Activityb
IC50 (mM)
HepG2 Toxicityb
Selectivity Index
(CC50=IC50)
CC50 (mM)
2
33
6
12
2
1.3
33
>200
>200
>200
48
>6
>33
>16
24
1a
1b
1c
1d
1e
1f
1g
9
7
>200
>200
>200
>6
>4
ꢀ
47
>200
aAssay conditions are described in the Experimental section.
bAntiviral 50% inhibitory concentration (IC50) and 50% cytotoxic concentration (CC50).
Results are averages from duplicate experiments.
lymphocytes that was separate from cytotoxicity.[8] Of particular interest was
the anti-HBV activity and selectivity of the 8-methyl (1a), 8-chloro (1b) and 8-
bromo (1c) substituted carbocyclic D-ddA derivatives. Additional analogs
related to these are being synthesized and studied.
EXPERIMENTAL
Chemistry
Nuclear magnetic resonance (NMR) spectra were obtained at 300 MHz
on Varian Unity Plus NMR spectrophotometer. The chemical shifts are
reported in parts per million (ppm) relative to tetramethylsilane as an internal
standard. Elemental analysis were performed by Atlantic Microlab Inc. Flash
column chromatography was performed using Merck Silica gel 60 (230–
400 mesh), and the stated solvent system under pressure. Mass spectra were
obtained on Micromass Platform mass spectrometers from Micromass Ltd.
Altrincham, UK, using Electrospray Ionization.
9-[(1S,3R)-3-({[tert-Butyl(dimethyl)silyl]oxy}methyl)cyclopentyl]-9H-
purin-6-amine (3). To a solution of carbocyclic D-ddA ([(1R,3S)-3-
(6-amino-9H-purin-9-yl)cyclopentyl]methanol (2), 1.0 g, 4.3 mmol) in
N,N-dimethylformamide (10 mL) was added imidazole (0.44 g, 6.4 mmol) and
t-butylchlorodimethylsilane (1.4 mL, 5.6 mmol). The resulting mixture was
stirred at room temperature for 12 h. Dichloromethane was added to the
reaction mixture and the organic phase washed with water and brine. The
organic phase was dried over magnesium sulfate. Filtration and concentration

896
GUDMUNDSSON ET AL.
followed by purification by flash chromatography (5% methanol in chloro-
form) gave 3 (1.4 g, 94%) as a white solid. 1H-NMR (CDCl3): d 8.28 (s, 1H),
7.88 (s, 1H), 5.70 (broad s, 2H), 4.84 (m, 1H), 3.60 (m, 2H), 1.6–2.4 (m, 7H),
0.84 (s, 9H), 0.0 (s, 6H); MS m=z 348 (M þ H). Anal. Calcd for
C17H29N5O2Si: C 58.75, H 8.41, N 20.15. Found, C 58.73, H 8.34, N 20.21.
[(1R,3S)-3-(6-Amino-8-methyl-9H-purin-9-yl)cyclopentyl]methanol
(1a). Diisopropylamine (2.0 mL, 14.4 mmol) was added to a solution of
n-butyllithium (9 mL of 1.6 M solution in hexane, 14.4 mmol) in tetra-
hydrofuran (20 mL) at ꢀ 78ꢁC. The resulting mixture was stirred for 5 min,
then a solution of compound 3 (1.0 g, 2.9 mmol) in dry tetrahydrofuran
(10 mL) was added. The dark reaction mixture was stirred for 30 min, then
iodomethane (0.5 mL, 7.3 mmol) was added. Reaction was stirred at ꢀ 78ꢁC
for additional 3 h, then quenched by addition of excess acetic acid. Ethyl
acetate and water are added to the reaction mixture and the phases were
separated. The ethyl acetate phase was washed with saturated aqueous
sodium bicarbonate, water and brine and dried over magnesium sulfate.
Filtration and concentration followed by purification by flash
chromatography (5% methanol in chloroform) gave 9-[(1S,3R)-3-({[tert-
butyl(dimethyl)silyl]oxy}methyl)cyclopentyl]-8-methyl-9H-purin-6-amine (4a,
0.50 g, 48%) as a solid. This solid (0.5 g, 1.4 mmol) was dissolved in tetra-
hydrofuran (10 mL) and to this solution was added tetrabutylammonium
fluoride (2.0 mL of 1 M solution in tetrahydrofuran, 2.0 mmol). The resulting
reaction was stirred at room temperature overnight, then concentrated in
vacuo. Purification by flash chromatography (5% methanol in chloroform)
1
gave 1a (230 mg, 67%) as a solid. H NMR (CDCl3): d 8.30 (s, 1H), 5.58
(broad s, 2H), 4.83 (m, 1H), 4.45 (m, 1H), 3.85 (m, 2H), 2.63 (s, 3H), 2.5 (m,
3H), 2.05 (m, 2H), 1.9 (m, 2H); MS m=z 248 (M þ H). Anal. Calcd for
C12H17N5Ox1=3 H2O: C 56.91, H 7.03, N 27.65. Found, C 56.94, H 6.94,
N 27.70.
[(1R,3S)-3-(6-Amino-8-chloro-9H-purin-9-yl)cyclopentyl]methanol
(1b). Diisopropylamine (0.73 g, 1.0 mL, 7.2 mmol) was added to a solution
of n-butyllithium (4.5 mL of 1.6 M solution in hexane, 7.2 mmol) in tetra-
hydrofuran (10 mL) at ꢀ 78ꢁC. The resulting mixture was stirred for 5 min
then a solution of compound 3 (0.5 g, 1.4 mmol) in dry tetrahydrofuran
(10 mL) was added. The dark reaction mixture was stirred for 30 min, then
hexachloroethane (1.0 g, 4.2 mmol) was added. Reaction was stirred at
ꢀ78ꢁC for additional 4 h, then quenched by addition of excess acetic acid.
Ethyl acetate and water are added to the reaction mixture and the phases
were separated. The organic phase was washed with water and brine and
dried over magnesium sulfate. Filtration and concentration followed by
purification by flash chromatography (10% methanol in chloroform) gave
4b (0.47 g, 86%) as a solid. This solid (0.47 g, 1.2 mmol) was dissolved in

CARBOCYCLIC ANALOGS OF ddA
897
tetrahydrofuran (10 mL) and to this solution was added tetrabutyl-
ammonium fluoride (3.0 mL of 1 M solution in tetrahydrofuran, 3.0 mmol).
The resulting reaction mixture was stirred at room temperature overnight,
then concentrated in vacuo. Purification by flash chromatography (10%
methanol in chloroform), followed by recrystallization from acetonitrile,
1
gave 1b (0.26 g, 79%) as a solid. H NMR (CDCl3): d 8.34 (s, 1H), 5.66
(broad s, 2H), 5.03 (m, 1H), 3.82 (m, 3H), 1.6–2.6 (m, 7H); MS m=z 268
(M þ H). Anal. Calcd for C11H14N5OCl: C 49.35, H 5.27, N 26.16. Found,
C 49.37, H 5.28, N 26.21.
[(1R,3S)-3-(6-Amino-8-bromo-9H-purin-9-yl)cyclopentyl]methanol
(1c). Treatment of 3 (0.5 g, 1.4 mmol) with lithium diisopropylamide (7.2
mmol, made in situ from n-butyllithium and diisopropylamine), followed by
addition of bromine (0.57 g, 0.19 mL, 3.6 mmol), gave 498 mg (81%, 98%
yield based on recovered starting material) of 4c as a yellow solid. This solid
(0.50 g, 1.2 mmol) was dissolved in tetrahydrofuran (10 mL) and to the
solution was added tetrabutylammonium fluoride (3.0 mL of 1 M solution,
3.0 mmol). The resulting solution was stirred at room temperature overnight,
then concentrated in vacuo. Purification by flash chromatography (10%
methanol in chloroform) gave 1c (0.34 g, 91%, 74% yield for the two step
synthesis from 3) as a solid. 1H NMR (CDCl3): d 8.19 (s, 1H), 6.03 (broad s,
2H), 4.88 (m, 1H), 3.76 (m, 2H), 1.7–2.5 (m, 7H); MS m=z 313 (M þ H).
Anal. Calcd for C11H14N5OBr x1=3CH3OH: C 42.16, H 4.79, N 21.69.
Found, C 42.13, H 4.69, N 21.48.
Alternatively, treatment of 3 (0.5 g, 1.44 mmol) with N-bromosuccini-
mide (0.76 g, 4.3 mmol) in dichloromethane (10 mL) at room temperature for
24 h also gave, after deprotection, 72 mg (16% yield for the two step synthesis
from 3) of 1c as a solid.
9-[(1S,3R)-3-({[tert-Butyl(dimethyl)silyl]oxy}methyl)cyclopentyl]-8-iodo-
9H-purin-6-amine (4d). Treatment of 3 (1.0 g, 2.9 mmol) with lithium
diisopropylamide (15 mmol, made in situ from n-butyllithium and diisopro-
pylamine) at ꢀ 78ꢁC in a similar fashion as described above, followed by
addition of iodine (1.9 g, 7.5 mmol) gave, after purification by flash chro-
matography (10% methanol in chloroform), 1.0 g (73%) yield of 4d as a solid
(100% yield based on recovered starting material). 1H NMR (CDCl3): d 8.16
(s, 1H), 5.60 (broad s, 2H), 4.75 (m, 1H), 3.63 (m, 2H), 1.7–2.5 (m, 7H), 0.84
(s, 9H), 0.0 (s, 6H); MS m=z 474 (M þ H). Anal. Calcd for C17H28N5OSiI: C
43.14, H 5.96, N 14.79. Found, C 43.52, H 5.97, N 14.72.
[(1R,3S)-3-(6-Amino-8-iodo-9H-purin-9-yl)cyclopentyl]methanol
(1d). Compound 4d (0.3 g, 0.63 mmol) was dissolved in tetrahydrofuran
(10 mL) and to this solution was added tetrabutylammonium fluoride
(1.0 mL of 1 M solution in tetrahydrofuran, 1.0 mmol). The resulting reaction

898
GUDMUNDSSON ET AL.
was stirred at room temperature overnight, then concentrated in vacuo.
Purification by flash chromatography (15% methanol in chloroform), fol-
lowed by recrystallization from acetonitrile, gave 0.16 g (71%) of 1d as a
solid. 1H NMR (d6-DMSO): d 8.07 (s, 1H), 7.33 (broad s, 2H), 4.80 (m, 1H),
4.62 (t, 1H), 3.47 (m, 2H), 1.6–2.2 (m, 7H); MS m=z 360 (M þ H). Anal.
Calcd for C11H14N5OI: C 36.79, H 3.93, N 19.50. Found, C 36.98, H 3.93,
N 19.24.
{(1R,3S)-3-[6-amino-8-(methylamino)-9H-purin-9-yl]cyclopentyl}methanol
(1e). Compound 4d (0.41 g, 0.87 mmol) was treated with methylamine in
methanol (10 mL of 2 M solution in methanol) at 130ꢁC in a steel vessel for
12 h. The reaction was cooled to room temperature and concentrated in
vacuo. Purification by flash chromatography (10% methanol in chloroform)
gave 0.33 g (quantitative yield) of 4e as a solid. Compound 4e (0.33 g,
0.87 mmol) was dissolved in tetrahydrofuran (10 mL) and to this solution
was added tetrabutylammonium fluoride (2.0 mL of 1 M solution in tetra-
hydrofuran, 2.0 mmol). The resulting reaction was stirred at room tem-
perature overnight, then concentrated in vacuo. Purification by flash
chromatography (20% methanol in ethyl acetate), followed by concentration
1
from dichloromethane, gave 0.16 g (70%) of 1e as a white solid. H NMR
(CDCl3): d 8.07 (s, 1H), 5.41 (m, 1H), 5.34 (broad s, 2H), 4.75 (m, 1H), 4.6
(broad s, 1H), 3.75 (d, 2H), 2.99 (d, 3H), 1.7–2.4 (m, 7H); MS m=z 263
(M þ H). Anal. Calcd for C12H18N6Ox0.3 CH2Cl2: C 51.33, H 6.51, N 29.20.
Found, C 51.49, H 6.69, N 29.50.
6-Amino-9-[(1S,3R)-3-(hydroxymethyl)cyclopentyl]-N,N-dimethyl-9H-
purine-8-carboxamide (1f). Treatment of 3 (0.5 g, 1.4 mmol) with lithium
diisopropylamide (7.5 mmol, made in situ from n-butyllithium and diisopro-
pylamine) at ꢀ78ꢁC in a similar fashion as described above, followed by
addition of dimethylcarbamyl chloride (0.3 mL, 3.0 mmol) gave, after pur-
ification by flash chromatography (10% methanol in chloroform), 0.26 g
(43%) yield of 4f as a solid. This solid (0.26 g, 0.62 mmol) was dissolved
in tetrahydrofuran (10 mL) and to this solution was added tetrabutyl-
ammonium fluoride (1.2 mL of 1 M solution in tetrahydrofuran, 1.2 mmol).
The resulting reaction was stirred at room temperature overnight, then
concentrated in vacuo. Purification by flash chromatography (20% methanol
1
in dichloromethane) gave 0.15 g (79%) of 1f as a white foam. H NMR
(CDCl3): d 8.37 (s, 1H), 5.95 (broad s, 2H), 5.05 (m, 1H), 4.1 (m, 1H), 3.80
(m, 2H), 3.21 (s, 6H), 1.7–2.4 (7H); MS m=z 305 (M þ H). Anal. Calcd for
C14H20N6O2x0.45 CH2Cl2: C 50.66, H 6.15, N 24.53. Found, C 50.56, H
6.52, N 24.54.
[(1R,3S)-3-(6-Amino-8-phenyl-9H-purin-9-yl)cyclopentyl]methanol
(1g). Compound 4d (0.7 g, 1.5 mmol) was dissolved in dimethoxyethane

CARBOCYCLIC ANALOGS OF ddA
899
(10 mL). To this solution was added phenylboronic acid (0.26 g, 2.1 mmol),
tetrakis(triphenylphosphine)palladium(0) (0.35 g, 0.4 mmol) and aqueous
sodium carbonate (0.37 g, 2.98 mmol in 2 mL of water). The resulting reac-
tion was heated at 80ꢁC for 12 h. This mixture was cooled to room tem-
perature, then water and ethyl acetate were added. The phases were separated
and the organic phase washed with water, brine and dried over magnesium
sulfate. The organic phase was filtered and concentrated to dryness invacuo.
Purification by flash chromatography (10% methanol in chloroform) gave
0.45 g (71%) of 4g as a solid. Compound 4g (0.45 g, 1.1 mmol) was dissolved
in tetrahydrofuran (10 mL) and to this solution was added tetrabutyl-
ammonium fluoride (2.0 mL of 1 M solution in tetrahydrofuran, 2.0 mmol).
The resulting reaction was stirred at room temperature overnight, then
concentrated invacuo. Purification by flash chromatography (10% methanol
1
in dichloromethane) gave 0.24 g (71%) of 1g as a solid. H NMR (d6-
DMSO): d 8.18 (s, 1H), 7.70 (m, 2H), 7.60 (m, 3H), 7.36 (broad s, 2H), 4.60
(m, 1H), 4.62 (t, 1H), 3.48 (m, 2H), 1.6–2.6 (m, 7H); MS m=z 310 (M þ H).
Anal. Calcd for C17H19N5Ox0.1 CH2Cl2: C 64.61, H 6.09, N 22.03. Found, C
64.56, H 6.08, N 21.92.
Antiviral (HBV) Assay
Antiviral potency and growth inhibition potential of compounds was
determined using the assay developed by Jansen et al.[9] Briefly, HepG2-
2.2.15 cells constitutively producing HBV[10] were seeded into 96 well
microtiter plates at a density of 56103 per well and growth medium con-
taining drug was replaced every other day for 9 days. Supernatants were then
collected and analyzed for HBV content. Samples were tested in conjunction
with both positive (.448 fg=mL plasmid DNA) and negative (RPMI medium
supplemented with 2 mM L-glutamine and 10% fetal calf serum) controls.
Data was normalized to non-drug treated cells, and expressed as a percent of
control for analysis.
Evaluation of toxicity (i.e., growth inhibition) was made by fixing
monolayers with 70% ethanol, and staining with bisbenzimide H33342 for
1 h at 37ꢁC. Fluorescence values of drug treated cells were compared to non-
drug treated cells and expressed as a percentage of control. HBV detection
(and hence efficacy determination) was performed by ‘‘capturing’’ virus from
supernatants on anti-HBsAg coated plates, washing, denaturing to release
HBV DNA, performing PCR with biotinylated primers, streptavidin capture
of biotinylated PCR products with concomitant probe hybridization, addi-
tion of substrate, and reading optical densities of the colorimetric reaction.
Dilutions of a standardized HBV-containing supernatant were included on
every plate, and HBV DNA concentrations of test wells were calculated from
this HBV standard curve.

900
GUDMUNDSSON ET AL.
REFERENCES
1. (a) Herdewijn, P.; Balzarini, J.; De Clercq, E.; Pauwels, R.; Baba, M.; Broder,
S.; Vanderhaeghe, H. J. Med. Chem. 1987, 30, 1270–1278; (b) Huryn, D.M.;
Okabe, M. Chem. Rev. 1992, 92, 1745–1768; (c) Mansour, T. S.; Storer, R.
Current Pharmaceutical Design 1997, 3, 227–264.
2. For C8 Bromination of Ribose and 20-deoxyribose Derivatives see: (a) Holmes,
R.E.; Robins, R.K. J. Am. Chem. Soc. 1964, 86, 1242–1245; (b) Srivastava,
P.C.; Nagpal, K.L. Experientia 1970, 26, 220; For Lithiation, Followed by
Electrophile Quenching for Furanose Derivatives see: (c) Hayakawa, H.; Tanaka,
H.; Sasaki, K.; Haraguchi, K.; Saitoh, T.; Takai, F.; Miyasaka, T. J. Hetero-
cycl. Chem. 1989, 26, 189–192; (d) Hayakawa, H.; Haraguchi, K.; Tanaka,
H.; Miyasaka, T. Chem. Pharm. Bull. 1987, 35, 72–79; (e) Tanaka, H.; Uchida,
Y.; Shinozaki, M.; Hayakawa, H.; Matsuda, A.; Miyasaka, T. Chem. Pharm.
Bull. 1983, 31, 787–790; (f) For Reviews see: Srivastava, P.C.; Robins, R.K.;
Meyer, R.B. Jr. Synthesis and Properties of Purine Nucleosides and Nucleo-
tides. In Chemistry of Nucleosides and Nucleotides; Townsend, Leroy B. Ed.;
Plenum Press, New York, 1988; 113–281.
3. Nair, V.; Buenger, G.S. J. Org. Chem. 1990, 55, 3695–3697.
4. Nair, V.; Buenger, G.S. Synthesis 1990, 962–966.
5. (a) Van Aerschot, A.A.; Mamos, P.; Weyns, N.J.; Ikeda, S.; De Clercq, E.; Her-
dewijn, P.A. J. Med. Chem. 1993, 36, 2938–2942; (b) Van Aerschot, A.; Herde-
wijn, P.; Balzarini, J.; Pauwels, R.; De Clercq, E. J. Med. Chem. 1989, 32,
1743–1749.
6. (a) Gala, D.; Schumacher, D.P. Synlett 1992, 1, 61–63; (b) Comin, M.J.;
Rodriguez, J.B. Anales de la Asociacion Quimica Argentina 1998, 86,
131–138; (c) Saksena, A.K.; Girijavallabhan, V.M.; Ganguly, A.K. Process
for Preparing Cyclopentyl Purine Derivatives. US 4999428, 1991; (d) Vince,
R.; Hua M.; Brownell, J.; Daluge, S.; Lee, F.; Shannon, W.M.; Lavelle,
G.C.; Qualls, J.; Weislow, O.S. Biochem. Biopharm. Res. Commun. 1988,
156, 1046–1053; (e) Wang, P.; Gullen, B.; Newton, M.G.; Cheng, Y.-C.; Schi-
nazi, R.F.; Chu, C.K. J. Med. Chem. 1999, 42, 3390–3399; (f) Wang, P.Y.;
Schinazi, R.F.; Chu, C.K. Bioorg. Med. Chem. Lett. 1998, 31, 1585–1588; (g)
Yoshio, T.; Takumi, H; Marumoto, R.; Yamamoto, N. Preparation of Carbo-
cyclic Derivatives of Purine Nucleosides as Virucides. EP 236935, 1987; (h)
Coward, J.K.; Slisz, E.P. J. Med. Chem. 1973, 16, 460–463; (i) For Synthesis
of Carbocyclic 20,30-dideoxydidehydroadenosine Derivatives, see: Daluge,
S.M. Synthesis and Antiviral Activity of (1R,4S)-4-(6-amino-9H-purin-9-yl)-
2-cyclopentene-1-methanol. WO 200047581, 2000.
7. (a) Agrofoglio, L.; Suhas, E.; Farese, A.; Condom, R.; Challand, R.; Earl,
R.A.; Guedj, R. Tetrahedron 1994, 50, 10,611–10,670; (b) Zhu, X.-F. Nucleo-
sides, Nucleotides and Nucleic Acids 2000, 19, 651–690; (c) Daluge, S.M.; Mar-
tin, M.T. et al. Nucleosides, Nucleotides and Nucleic Acids 2000, 19, 297–327
and references therein; (d) Tan, X.; Chu, C.K.; Boudinot, F.D. Advanced Drug
Delivery Reviews 1999, 39, 117–151; (e) Crimmins, M.T. Tetrahedron 1998,
54, 9229–9272.

CARBOCYCLIC ANALOGS OF ddA
901
8. Compounds were Assayed for HIV Activity in MT4 cells According to the
Method Described by: Averett, D.R. J. Virol. Methods 1989, 23, 263–276.
For all compounds IC50 > 10 mM.
9. Jansen, R.W.; Johnson, L.C.; Averett, D.R. Antimicrobial Agents and Che-
motherapy 1993, 37, 441–447.
10. Sells, M.A.; Chen, M.-L.; Acs, G. Proc. Natl. Acad. Sci. USA. 1987, 84,
1005–1009.
Received July 31, 2002
Accepted September 4, 2002