Synthesis of conformationally restricted 2′,3′-exo-Methylene carbocyclic nucleosides built on a bicyclo[3.1.0]hexane template

Article abstract of DOI:10.1016/S0968-0896(02)00063-9

A series of 2′,3′-exo-methylene carbocyclic nucleosides was synthesized as potential antiviral agents. These compounds were built on a bicyclo[3.1.0]hexane template that exhibits a rigid pseudoboat conformation and is capable of maintaining an identical conformation in solid state and in solution. The structures of the synthesized compounds were elucidated by NMR and X-ray crystallography. All the compounds were tested as anti-HIV and anti-HSV agents. The chemically synthesized 5′-triphosphate analogue of 7 was evaluated directly as a reverse transcriptase inhibitor.

Full text of DOI:10.1016/S0968-0896(02)00063-9

Bioorganic & Medicinal Chemistry 10 (2002) 2325–2333
Synthesis of Conformationally Restricted 20,30-exo-Methylene
Carbocyclic Nucleosides Built on a Bicyclo[3.1.0]hexane Template
Rashmi Gupta Bhushan and Robert Vince*
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA
Received 18 September 2001; accepted 9 January 2002
0
Abstract—A series of2 ,30-exo-methylene carbocyclic nucleosides was synthesized as potential antiviral agents. These compounds
were built on a bicyclo[3.1.0]hexane template that exhibits a rigid pseudoboat conformation and is capable of maintaining an
identical conformation in solid state and in solution. The structures of the synthesized compounds were elucidated by NMR and
X-ray crystallography. All the compounds were tested as anti-HIV and anti-HSV agents. The chemically synthesized 50-triphosphate
analogue of7 was evaluated directly as a reverse transcriptase inhibitor. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
furanose oxygen in carbocyclic nucleosides causes the
cyclopentane ring to adopt an unusual 10-exo con-
formation that is relatively far from the characteristic
North or South conformations observed in the typical
nucleosides.5
Carbocyclic nucleoside analogues in which a methylene
group replaces the O atom ofthe riboufranose ring
represent an extremely important class ofpotentially
active therapeutic agents. For example, carbocyclic
20,30-didehydro-20,30-dideoxyguanosine (carbovir) has
been identified as a potent and selective inhibitor of
HIV-1 replication and cytopathic effects in a variety of
human t-lymphoblastoid cell lines1,2 and has been
approved by the FDA as a drug for the treatment of
AIDS. In our ongoing search for antiviral nucleosides,
One ofthe strategies that can be used in carbocyclic
nucleosides to regain the ring pucker characteristic of
typical nucleosides is to construct them on a rigid
bicyclic system. We have synthesized a number of
purine carbocyclic nucleosides built on a rigid bicyclo
[3.1.0]hexane template. Since the bicyclo [3.1.0]hexane
template is known to exist rigidly in a pseudoboat
shape, these compounds are able to maintain identical
conformations in the solid state and in solution and
thereby help define the role ofsugar puckering in
nucleosides by stabilizing the active receptor bound
conformation. Since both a cyclopropane ring as well as
a C-C double bond have unsaturated properties these
nucleosides can also be expected to show anti-HIV
properties similar to carbovir.6
0
we have synthesized a series of2 ,30-exo-methylene car-
bocyclic nucleosides, where the sugar moieties are con-
formationally restricted due to the methylene group
fused between the 20 and 30 positions.
The sugar ring ofnucleosides and nucleotides equili-
brates in solution between two extreme forms of ring
pucker neighboring a 20-exo/30-endo (Northern) con-
formation and the opposite 20-endo/30-exo (Southern)
conformation resulting from gauche interactions
between the furan oxygen and the 20-and 30-hydroxyl
groups as well as the anomeric effect.3 In the solid state,
one ofthe two ofrms usually predominates and, simi-
larly when a nucleoside or nucleotide binds to its target
enzyme, only one ofthe two possible conofrmations
takes part in drug–receptor interactions.4 Loss ofthe
Most nucleoside analogues have to be converted to the
triphosphate form in order to exhibit biological activity.
However, the conformation required for triphosphate
production does not necessarily correlate with the con-
formational preference of reverse transcriptase.7 For
example, it has been proposed that the preference of
AZT for the extreme south conformation is responsible
for its potent anti-HIV activity.8 However, the anti-
podal north conformation has been suggested for the
interaction ofAZT-TP with its target enzyme, HIV
*Corresponding author. Tel.: +1-612-624-9911; fax: +1-612-624-
0139; e-mail: vince001@tc.umn.edu
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00063-9

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R. G. Bhushan, R. Vince / Bioorg. Med. Chem. 10 (2002) 2325–2333
reverse transcriptase.9 Marquez et al. have demon-
strated that AZT, by its flexible nature, can adopt a
south conformation required for phosphorylation by
kinase enzymes and subsequently switch to a north
conformation for interaction with reverse transcriptase.7
We have synthesized the triphosphate of 7 to study its
interaction with reverse transcriptase.
n-butanol to give the pyrimidylamino derivative 5. Ring
closure of 5 with triethyl orthoformate in the presence
ofHCl 11 gave (ꢀ)-cis-exo-2-(6-chloro-9H-purin-9-yl)-4-
(hydroxymethyl)-bicyclo[3.1.0] hexane (6). Treatment of
6 with liquid ammonia under high pressure gave the
adenine derivative,(ꢀ)-cis-exo-2-(6-amino-9H-purin-9-
yl)-4-(hydroxymethyl)-bicyclo[3.1.0] hexane (7).
The syntheses ofthe 2,6-disubstituted purine analogues
are shown in Scheme 2. Amine 4 was condensed with
2-amino-4,6-dichloropyrimidine to afford the pyrimid-
ylamino derivative, 8. This was then treated with
p-chlorobenzenediazonium chloride by the method of
Shealy and Clayton12 to give azopyrimidine 9. Azopyr-
imidine 9 was then reduced with zinc in the presence of
acetic acid2 and generated the amine 10. Subsequent
cyclization ofthe substituted pyrimidine with triethyl
orthoformate and HCl led to the formation of (ꢀ)-cis-
exo-2-(2-amino-6-chloro-9H-purin-9-yl)-4-(hydroxy-
methyl)-bicyclo[3.1.0]hexane (11).
Results and Discussion
Synthesis
To synthesize the target compounds (ꢀ) 20,30-dideoxy-
20,30-exo-methylene nucleosides, (ꢀ) 2-azabicyclo-
[2.2.1]hept-5-en-3-one 1 (commercially available) was
used as the starting material. Cyclopropanation of 1
using diazomethane and catalytic palladium acetate10
gave the desired cyclopropyl intermediate (ꢀ)-cis-exo-2-
(aza)-3-(oxo)-tricyclo[3.2.1.0]heptane, (2) as cyclopro-
panation took place from the less hindered side of the
lactam (Scheme 1). Acid-catalyzed hydrolysis of 2 with
1 N HCl solution gave (ꢀ)-cis-exo-2-(amino)-4-(car-
boxy)-bicyclo[3.1.0]hexane hydrochloride, (3). Reduc-
tion of 3 with lithium aluminum hydride (LAH) in
tetrahydrofuran (THF) led to the formation of the
amino alcohol, (ꢀ)-cis-exo-2(amino)-4-(hydroxymethyl)-
bicyclo[3.1.0]hexane (4). Once the carbocyclic sugar
moiety 4, was formed, it was coupled with 5-amino-4,6-
dichloropyrimidine in the presence oftriethylamine and
The 2-amino-6-chloropurine analogue was refluxed with
1 N HCl13 in order to obtain the guanine analogue, 12.
The diaminopurine analogue (13) was obtained by dis-
placing the 6-chloro group of 11 with liquid ammonia.
Displacement ofthe 6-chloro group of 11 with n-pro-
panol in the presence ofsodium hydride gave the cor-
responding propyl derivative (14). The corresponding
optically active (ꢁ) compounds (1a–14a) were also syn-
thesized using the same procedure (see Experimental).
Scheme 1. Reagents and conditions: (a) CH2N2, Pd(OAc)2, 24 h; (b) 1 N HCl, reflux 1.5 h; (c) LAH, THF, 24 h; (d) 5-amino-4,6-dichloropyrimidine,
Et3N, n-BuOH, reflux 24 h; (e) CH(OEt)3, concd HCl, 24 h; (f) liq NH3, 48 h.
Scheme 2. Reagents and conditions: (a) 2-amino-4,6-dichloropyrimidine, Et3N, n-BuOH, reflux 24 h; (b) p-chloroaniline, 3 N HCl, NaNO2,
CH3COOH, NaOAc, 24 h; (c) Zn, CH3COOH, EtOH, reflux, 5 h; (d) CH(OEt)3, concd HCl, 24 h.

R. G. Bhushan, R. Vince / Bioorg. Med. Chem. 10 (2002) 2325–2333
2327
Scheme 3. Reagents and conditions: (a) POCl3, PO(OMe)3, 0 ꢂC; (b) (Bu3NH)2H2P2O7ꢁ.
Synthesis ofthe triphosphate analogue was perofrmed
using a ‘one-pot’ approach by reacting the nucleoside 7
with phosphorus oxychloride and subsequently treating
the monophosphate intermediate with bis(tributyl-
ammonium) pyrophosphate to give the triphosphate 15
(Scheme 3).
4.74 ppm). Correlation was also seen between the C6-
0
Ha proton (0.34–0.30 ppm) and the C4 -H proton (1.94–
1.84 ppm) indicating that the cyclopropyl was below the
plane ofthe five-membered ring. The X-ray structure
(Fig. 2) further confirmed that the cyclopropyl group
was indeed below the plane ofthe cyclopentane ring.
Stereochemical assignments ofthe final compounds
were made on the basis of1-D and 2-D NMR spectro-
scopy and X-ray crystallography. The NOESY spectra
Biological Results and Conclusion
0
(Fig. 1) showed showed correlation between the C6 -Ha
Anti-HIV and anti-HSV activities ofthe synthesized
compounds have been evaluated. Preliminary data
0
proton (0.34–0.30 ppm) and the C2 -H proton (4.76–
Figure 1. 2-D NOESY ofcompound 7.

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R. G. Bhushan, R. Vince / Bioorg. Med. Chem. 10 (2002) 2325–2333
q; pentet, p; and multiplet, m. Fast-atom bombardment
(FAB) mass spectra (MS) were obtained on a VG
7070E-HF instrument. Optical rotations were measured
on a Rudolph polarimeter. Flash column chromato-
graphy was performed on Merck Science silica gel 60
(230–400) mesh. Thin layer chromatography (TLC)
was performed on Merck Science silica gel 60 F254
glass plates (0.25 mm thickness). Plates were visua-
lized by UV light, iodine vapor or anisaldehyde
solution.
Diazomethane
A Diazald distillation apparatus was fitted with a 250
mL round-bottomed distillation pot and a 250 mL
round-bottomed flask as a receiver. The reaction vessel
was heated with a water bath to 65–70 ꢂC, and the
receiver was cooled to ca. ꢁ40 ꢂC (controlled dry-ice/
acetone conditions). Potassium hydroxide (5.1 g, 178
mmol), 2-methoxyethylether (25 mL), water (10 mL)
and ether (10 mL) were taken in the distillation pot.
Diazald (10 g, 47 mmol) in ether (65 mL) was added
drop wise. Soon after the addition commenced, diazo-
methane in ether started to collect in the receiver.
After the final addition, 20 mL of ether was added
and allowed to distill over. This was repeated until
the distillate was colourless (usually only once).
Figure 2. X-ray crystal structure ofcompound 7.
showed that compounds 6 and 7 had an EC50 of0.1–1
mg/mL and compounds 12–14 had an EC50 of1–10 mg/
mL as compared to Acyclovir, a potent anti-herpes
compound which had an EC50 of0.1 mg/mL. No toxi-
city was observed for any of the compounds up to 100
mg/mL. None ofthe compounds showed significant
anti-HIV activity up to 100 mg/mL. The optically active
isomers also showed similar biological activities. The
triphosphate analogue 15 was evaluated against reverse
transcriptase and had an IC50 of36.39 mg/mL as com-
pared to dideoxy adenosine triphosphate, which had an
IC50 of0.023 mg/mL.
(ꢀ)-exo-7-Azatricyclo[3.2.1.0<2,4>]octan-6-one (2). A
solution of2-azabicyclo[2.2.1]hept-5-en-3-one (500 mg,
4.58 mmol) and palladium acetate (50 mg, 0.22 mmol)
in ether (10 mL) was stirred at room temperature for 15
min. To this solution CH2N2 in ether (80 mL) was
added dropwise over a 20-min period. The reaction
mixture was allowed to stir overnight at room tempera-
ture. It was then filtered over a pad ofCelite and con-
centrated. Purification ofthe residue by silica gel
chromatography using ethyl acetate/hexane (1:1) afforded
a yellow oil. The oil was recrystallized with dichloro-
methane–hexane to yield 294.6 mg (52.15%) of 2 as a
white solid: mp 82–83 ꢂC; Rf 0.3 (ethyl acetate). 1H
NMR (CDCl3, 300 MHz) d 6.3 (br, 1H, NH), 3.79–3.78
(d, 1H, H-1), 2.7 (d, 1H, H-5), 1.59–1.35 (m, 4H, H-4,
H-2, H-8, H-8), 1.29–1.25 (q, 1H, H-3), 0.82–0.75 (q,
1H, H-3). Anal. calcd for C7H9NO: C, 68.26; H, 7.36;
N, 11.37. Found: C, 68.32; H, 7.16; N, 11.57.
Based on the X-ray structure ofcompound 7, the angle
ofpseudorotation P was calculated as 91.89ꢂ which is
far different from the conventional ‘north’ or ‘south’
conformation, P=0ꢂ and P=180ꢂ respectively,
observed in typical nucleosides. The unusual conforma-
tion ofthese nucleosides may be responsible ofr their
lack ofsignificant activity against HIV as the compound
may not be in a suitable conformation to interact tightly
with reverse transcriptase, the target enzyme.
In conclusion, we have synthesized a series of2 ,30-exo-
methylene carbocyclic nucleosides and evaluated their
structure, conformation and biological activity.
0
(ꢁ)-exo-7-Azatricyclo[3.2.1.0<2,4>]octan-6-one (2a). The
reaction was repeated as above using (ꢁ) 2-azabicyclo
[2.2.1]hept-5-en-3-one (1.00 g, 9.16 mmol), palladium
acetate (1000 mg, 0.44 mmol) and CH2N2 in ether (160
mL). The reaction mixture was purified by silica gel
chromatography (ethyl acetate/hexane, 1:1) to give a
yellow oil. The oil was recrystallized with dichloro-
methane-hexane to yield 703.1 mg (62%) of 2a as a
white solid: mp 78–79 ꢂC; [a]D25 ꢁ217.793ꢂ (c 1, CH2Cl2).
Anal. calcd for C7H9NO: C, 68.26; H, 7.36; N, 11.37.
Found: C, 68.36; H, 7.13; N, 11.48.
Experimental
All reactions involving moisture sensitive reagents were
conducted in oven-dried glassware under nitrogen a
atmosphere. Solvents were dried when necessary. All
other chemicals and solvents were reagent grade unless
specified otherwise and were obtained from Aldrich
Chemical Company, Milwaukee, Wisconsin. Elemental
analyses were performed by M-H-W Laboratories,
Phoenix, AZ, USA. Melting points were determined on
a Mel-Temp II apparatus and are uncorrected. 1H
NMR spectra were recorded on Varian Unity 300,Var-
ian Unity 500, GE 300 and Varian Mercury 300 spec-
trometers and referenced to the solvent. Chemical shifts
are expressed in ppm. Peak multiplicities are abbre-
viated: broad, br; singlet, s; doublet, d; triplet, t; quartet,
(ꢀ)-cis-exo-2-(Amino)-4-(carboxy)-bicyclo[3.1.0]hexane
hydrochloride (3). A solution oflactam 2 (500 mg, 4.06
mmol) in 1 N HCl (15 mL) was heated under reflux
at 128 ꢂC for 1.5 h. The reaction mixture was then

R. G. Bhushan, R. Vince / Bioorg. Med. Chem. 10 (2002) 2325–2333
2329
concentrated to dryness under reduced pressure to
obtain a yellow oil. Addition ofacetone to the yellow oil
gave 882.3 mg (86.4%) of 3 as a white solid: mp 202–
(ꢁ)-cis-exo-2-[(5-Amino-6-chloro-4-pyrimidinyl)amino]-
4-(hydroxymethyl)-bicyclo[3.1.0]hexane (5a). Compound
4a (510 mg, 4.0157 mmol) was reacted with 5-amino-
4,6-dichloro-pyrimidine (987.85 mg, 6.023 mmol), tri-
ethylamine (1.12 mL) and 1-butanol (50 mL) as
above. Purification ofthe residue by silica gel chro-
matography using CHCl3 followed by chloroform/
methanol (30:1) afforded a yellow oil, which was
recrystallized using ethyl acetate–hexane to give a
206 ꢂC. H NMR (CD3OD, 300 MHz) d 3.73–3.70 (d,
1
1H, H-2), 3.01–2.98 (d, 1H, H-4), 1.99–1.79 (m, 3H,
H-3, H-3, H-1), 1.65–1.59 (q, 1H, H-5), 0.80–0.73 (q,
1H, H-6), 0.41–0.36 (q, 1H, H-6). Anal. calcd for
C7H12ClNO2: C, 47.33; H, 6.8; N, 7.88. Found: C,
47.51; H, 6.69; N, 7.89.
white solid, 591.4 mg (58%): mp 152–154 ꢂC; [a]D25
–
(ꢁ)-cis-exo-2-(Amino)-4-(carboxy)-bicyclo[3.1.0]hexane
hydrochloride (3a). Compound 2a (200 mg, 1.623
mmol) was heated under reflux with 1 N HCl as above
to yield a yellow oil, which on addition ofacetone
afforded a white solid (244.1 mg, 84.92%): mp 202–
206 ꢂC; [a]2D5 ꢁ52.104ꢂ (c 1, CH3OH). Anal. calcd for
C7H12ClNO2: C, 47.33; H, 6.8; N, 7.88. Found: C,
47.47; H, 6.90; N, 7.93.
8.016ꢂ (c 1, CH3OH). Anal. calcd for C11H15O ClN4:
C, 51.86; H, 5.93; N, 21.99. Found: C, 51.77; H,
6.14; N, 21.70.
(ꢀ)-cis-exo-2-(6-Chloro-9H-purin-9-yl)-4-(hydroxymethyl)-
bicyclo[3.1.0]hexane (6). A mixture ofcompound 5 (500
mg, 1.9646 mmol), triethyl orthoformate (10.7 mL), and
hydrochloric acid (12 N, 0.5 mL) was stirred overnight
at room temperature. The suspension was dried in
vacuo. Diluted hydrochloric acid (0.5 N, 15 mL) was
added and the mixture was stirred at room temperature
for 1 h. The mixture was neutralized to pH 8 with 1 N
sodium hydroxide and adsorbed onto silica gel. It was
packed into a column and eluted by chloroform/metha-
nol (20:1) to yield compound 6 as a white solid, 344.8
mg (66.3%): mp 176 ꢂC; TLC: Rf 0.26 (chloroform/
methanol, 10:1). 1H NMR (DMSO-d6, 300 MHz) d 8.79
(s, 1H, H-8), 8.74 (s, 1H, H-2), 4.92–4.90 (d, 1H, H-20),
4.54–4.50 (t, 1H, CH2OH, D2O exchangeable), 3.17–
3.11 (m, 1H, CH2OH), 2.96–2.89 (m, 1H, CH2OH),
2.11–2.06 (m, 1H, H-30), 1.99–1.92 (m. 2H, H-40, H-10),
1.84–1.79 (m, 1H, H-30), 1.66–1.60 (m, 1H, H-50), 0.77–
0.70 (m, 1H, H-60), 0.39–0.34 (q, 1H, H-60). Anal. calcd
for C12H13O ClN4: C, 54.44; H, 4.95; N, 21.16. Found:
C, 54.29; H, 4.75; N, 20.97.
(ꢀ)-cis-exo-2-(Amino)-4-(hydroxymethyl)-bicyclo[3.1.0]-
hexane (4). To lithium aluminum hydride (500 mg, 13.0
mmol) in anhydrous tetrahydrofuran (40 mL) was
added compound 3 the amino hydrochloride (577 mg,
3.2 mmol) at room temperature under N2. The reaction
mixture was stirred overnight and quenched with water
(3 mL). A white solid was filtered and the filtrate was
concentrated to dryness to obtain 4 as an oily residue,
(300 mg, 72.5%). This oil was then used for the next
step without any further purification. 1H NMR
(CD3OD, 300 MHz) d 3.60–3.56 (dd, 1H, CH2OH),
3.51–3.46 (dd, 1H, CH2OH), 3.33–3.31 (d, 1H, H-2),
2.18–2.15 (m, 1H, H-4), 1.73 (m, 1H, H–3), 1.29–1.24
(m, 3H, H-3, H-5, H-1), 0.47–0.45 (m, 1H, H-6), 0.1 (q,
1H, H-6).
(ꢁ)-cis-exo-2-(Amino)-4-(hydroxymethyl)-bicyclo[3.1.0]-
hexane (4a). The reaction was repeated as above using
compound 3a (908 mg, 5.129 mmol), lithium aluminum
hydride (1.00 g, 26.0 mmol) and tetrahydrofuran (70
mL). Compound 4a was obtained as an oil, 510.8 mg
(79%).
(ꢁ)-cis-exo-2-(6-Chloro-9H-purin-9-yl)-4-(hydroxymethyl)-
bicyclo[3.1.0]hexane (6a). Compound 5a (500 mg, 1.964
mmol) was reacted with triethyl orthoformate and
hydrochloric acid as above. Purification by a silica
gel column using an eluate ofchloroofrm/methanol
(30:1) gave a white solid 250 mg (50%): mp 158 ꢂC;
[a]2D5 ꢁ71.843ꢂ (c 1, CH3OH). Anal. calcd for C12H13O
ClN4: C, 54.44; H, 4.95; N, 21.16. Found: C, 54.36; H,
5.04; N, 21.08.
(ꢀ)-cis-exo-2-[(5-Amino-6-chloro-4-pyrimidinyl)amino]-
4-(hydroxymethyl)-bicyclo[3.1.0]hexane (5). To com-
pound 4, the amine alcohol (419.2 mg, 3.299 mmol)
were added 5-amino-4,6-dichloro-pyrimidine (811.60
mg, 4.9488 mmol), triethylamine (0.91 mL) and 1-buta-
nol (40 mL), and the mixture was heated under reflux
for 24 h. The volatile solvents were removed; the residue
was adsorbed onto silica gel and it was packed into a
column and eluted with chloroform followed by
chloroform/methanol (30:1). The product fractions were
collected and concentrated into a residue. The residue
was recrystallized using ethyl acetate to yield 338.6 mg
(40.3%) of 5 as a solid: mp 86–88 ꢂC; TLC: Rf 0.41
(chloroform/methanol, 10:1). 1H NMR (CD3OD,
300 MHz) d 7.75 (s, 1H, H-2), 4.37–4.35 (m, 1H, H-20),
3.57–3.54 (m, 2H, CH2OH), 2.18–2.13 (m, 1H, H-30),
1.81–1.74 (m, 1H, H-10), 1.62–1.57 (m, 1H, H-40), 1.48–
1.44 (m, 2H, H-50, H-30), 0.60–0.55 (m, 1H, H-60), 0.19–
0.15 (q, 1H, H-60). Anal. calcd for C11H15O ClN4: C,
51.86; H, 5.93; N, 21.99. Found: C, 51.80; H, 6.01; N,
21.80.
(ꢀ)-cis-exo-2-(6-Amino-9H-purin-9-yl)-4-(hydroxyme-
thyl)-bicyclo[3.1.0]hexane (7). Liquid ammonia was pas-
sed into a solution of 6 (300 mg, 1.133 mmol) in
methanol (5 mL) at ꢁ80 ꢂC in a bomb. The bomb was
sealed and heated at 75 ꢂC for 48 h. Ammonia and
methanol were evaporated. The residue was adsorbed
onto silica gel and it was packed into a column and
eluted with chloroform/methanol (10:1). The crude
product was recrystallized from ethanol to yield 194.7
mg (70.06%) of 7: mp 220 ꢂC; TLC: Rf 0.13 (chloro-
1
form/methanol, 10:1). H NMR (DMSO-d6, 300 MHz)
d 8.20 (s, 1H, H-8), 8.08 (s, 1H, H-2), 7.16 (br, 2H, NH2,
D2O exchangeable), 4.76–4.74 (d, 1H, H-20), 4.56–4.52
(t, 1H, CH2OH, D2O exchangeable), 3.30–3.11 (m, 1H,
CH2OH), 2.97–2.89 (m, 1H, CH2OH), 2.11–2.03 (m,
1H, H-30), 1.94–1.84 (m. 2H, H-40, H-10), 1.76–1.71 (m,
1H, H-30), 1.65–1.59 (m, 1H, H-50), 0.72–0.65 (q, 1H,

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R. G. Bhushan, R. Vince / Bioorg. Med. Chem. 10 (2002) 2325–2333
H-60), 0.34–0.30 (q, 1H, H-60). Anal. calcd for
C12H15ON5: C, 58.76; H, 6.16; N, 28.55. Found: C,
58.58; H, 6.31; N, 28.24.
7.70 (d, 2H, aromatic H’s), 7.58 (br, s, 2H), 7.54–7.51
(d, 2H, aromatic H’s), 4.76–4.72 (t, 1H, CH2OH, D2O
exchangeable), 4.52–4.46 (m, 1H, H-20), 3.48–3.46 (m,
1H, CH2OH), 3.45–3.41 (m, 1H, CH2OH), 2.09–2.06
(m, 1H, H-30), 1.7–1.6 (m, 1H, H-10), 1.55–1.50 (m, 1H,
H-40), 1.41–1.37 m, 2H, H-30, H-50), 0.50–0.46 (m, 1H,
H-60), 0.12–0.10 (m, 1H, H-60). Anal. calcd for
C17H18Cl2N6O: C, 51.91; H, 4.61; N, 21.36. Found: C,
51.83; H, 4.76; N, 21.30.
(ꢁ)-cis-exo-2-(6-Amino-9H-purin-9-yl)-4-(hydroxymethyl)-
bicyclo[3.1.0]hexane (7a). Compound 6a (270 mg, 1.02
mmol) was reacted with liquid ammonia as described
above. The residue was purified through a silica gel
column using an eluate ofchloroof rm/methanol (10:1)
to give a white solid, 355.7 mg (76.67%): mp 238 ꢂC;
[a]2D5ꢁ50.610ꢂ (c 1, CH3OH). Anal. calcd for
C12H15ON5: C, 58.76; H, 6.16; N, 28.55. Found: C,
58.66; H, 6.36; N, 28.40.
(ꢁ)-cis-exo-2-[(2-Amino-6-chloro-5-[(4-chlorophenyl)-
azo]-4-pyrimidinyl)amino]-4-(hydroxymethyl)-bicyclo[3.1.0]-
hexane (9a). A cold diazonium salt solution prepared as
described above was added to a mixture of 8a (400 mg,
1.574 mmol), acetic acid (8 mL), water (8 mL), and
sodium acetate trihydrate (3.2 g). Following the proce-
dure above, compound was obtained as a yellow pro-
duct, 402.4 mg (65%): mp 238 ꢂC; [a]D25 +125.06ꢂ (c
0.5, CH3OCH3). Anal. calcd for C17H18Cl2N6O: C,
51.91; H, 4.61; N, 21.36. Found: C, 51.76; H, 4.50; N,
21.19.
(ꢀ)-cis-exo-2-[(2-Amino-6-chloro-4-pyrimidinyl)amino]-
4-(hydroxymethyl)-bicyclo[3.1.0]hexane (8). To com-
pound 4 (428.2 mg, 3.37 mmol) was added 1-butanol
(40 mL), triethylamine (2.8 mL), and 2-amino-4,6-
dichloropyrimidine (829.84 mg, 5.06 mmol) and the
mixture was heated under reflux for 48 h. The solvent
was removed under reduced pressure and the residue
was purified through a silica gel column using an eluate
of chloroform followed by chloroform/methanol (20:1).
The product fractions were collected and concentrated
to give a white solid, which was then recrystallized using
dichloromethane–hexane to yield 605.2 mg (70.6%) of 8
as a white solid: mp 179–180 ꢂC; TLC: Rf=0.58 (di-
chloromethane/methanol, 9:1). 1H NMR (DMSO-d6,
300 MHz) d 7.1 (br, s, 1H, NH), 6.3 (br, s, 2H, NH2),
5.7 (s, 1H, H-1), 4.6 (m, 1H, H-20), 4.2 (t, 1H, CH2OH,
D2O exchangeable), 3.4 (m, 2H, CH2OH), 2.0 (m, 1H,
H-30), 1.7 (m, 1H, H-10), 1.5 (m, 1H, H-40), 1.36–1.31
(m, 2H, H-30, H-50), 0.5 (m, 1H, H-60), 0.01–0.00 (m,
1H, H-60). Anal. calcd for C11H15ClN4O.0.25H2O: C,
51.01; H, 6.03; N, 21.63. Found: C, 50.70; H, 6.22; N,
21.64.
(ꢀ)-cis-exo-2-[(2,5-Diamino-6-chloro-4-pyrimidinyl)-
amino]-4-(hydroxymethyl)-bicyclo[3.1.0]hexane (10). To
a suspension of 9 (1.227 g, 3.1221 mmol) in ethanol (92
mL) and water (41 mL) was added zinc dust (2.044 g,
31.2 mmol) and acetic acid (1 mL). The reaction mix-
ture was heated under reflux in nitrogen for 3 h. The
zinc was removed by filtration and the filtrate was con-
centrated to give a brown residue. The residue was then
adsorbed onto silica gel and it was packed into a col-
umn and eluted with chloroform/methanol (20:1). The
product fractions were concentrated to give a yellow oil.
Further purification from methanol–ether yielded com-
pound 10 as a white solid: 243.0 mg (64.7%); mp
ꢂ
1
178 C; TLC: Rf 0.29 (chloroform/methanol, 10:1). H
NMR (CD3OD, 300 MHz) d 4.37–4.35 (d, 1H, H-20),
3.59–3.56 (m, 2H, CH2OH), 2.19–2.12 (m, 1H, H-30),
1.84–1.74 (m, 1H, H-10), 1.58–1.53 (m, 1H, H-40), 1.43–
1.39 (m, 2H, H-30, H-50), 0.58–0.51 (m, 1H, H-60), 0.12–
0.08 (m, 1H, H-60). Anal. calcd for C11H16ClN5O: C,
48.98; H, 5.97; N, 25.96. Found: C, 49.23; H, 6.20; N,
25.61.
(ꢁ)-cis-exo-2-[(2-Amino-6-chloro-4-pyrimidinyl)amino]-
4-(hydroxymethyl)-bicyclo[3.1.0]hexane (8a). Compound
4a (462.3 mg, 3.6401 mmol) was reacted with 2-amino-
4,6-dichloropyrimidine (895 mg, 5.46 mmol), triethyla-
mine (3 mL) and 1-butanol (40 mL) as above. The resi-
due was purified through a silica gel column using an
eluate of chloroform followed by chloroform/methanol
(35:1) to give a white solid, 511.6 mg (55.5%): mp 68 ꢂC;
[a]2D5 ꢁ67.13ꢂ (c 0.5, CH3OH). Anal. calcd for
C11H15ClN4O: C, 51.86; H, 5.93; N, 21.99. Found: C,
51.73; H, 5.86; N, 22.02.
(ꢁ)-cis-exo-2-[(2,5-Diamino-6-chloro-4-pyrimidinyl)-
amino]-4-(hydroxymethyl)-bicyclo[3.1.0]hexane (10a). A
mixture of 9a (2.559 g, 6.513 mmol), zinc dust (4.259 g,
65 mmol), acetic acid (2.08 mL), water (100 mL) and
ethanol (180 mL) was heated under reflux in nitrogen
for 3 h and worked up as described above. The mixture
was then adsorbed onto silica gel and it was packed into
a column and eluted with chloroform/methanol (30:1).
The product fractions were concentrated to give a yel-
low oil. Further purification from methanol–ether yiel-
ded 10a as a white solid, 986.2 mg (58%); mp 186–
188 ꢂC; [a]D25 ꢁ22.04ꢂ (c 0.5, CH3OH). Anal. calcd for
(ꢀ)-cis-exo-2-[(2-Amino-6-chloro-5-[(4-chlorophenyl)-
azo]-4-pyrimidinyl)amino]-4-(hydroxymethyl)-bicyclo[3.1.0]-
hexane (9). A cold diazonium salt solution was prepared
from p-chloroaniline (345 mg, 2.7 mmol) in 3 N HCl (6
mL) and sodium nitrite (204 mg, 2.94 mmol) in water
(1.2 mL). This solution was added to a mixture of 8 (600
mg, 2.36 mmol), acetic acid (12 mL), water (12 mL), and
sodium acetate trihydrate (4.68 g). The reaction mixture
was stirred overnight at room temperature. The yellow
precipitate was filtered and washed with cold water until
neutral, and then it was air-dried in a fume hood to
yield, 638.1 mg (68.75%) ofcompound 9: mp 259 ꢂC;
TLC: Rf 0.6 (dichloromethane/methanol, 9:1). 1H NMR
(DMSO-d6, 300 MHz) d 10.36–10.34 (d, 1H, NH), 7.73–
.
C11H16ClN5O 0.5H2O: C, 47.40; H, 6.14; N, 25.11.
Found: C, 47.20; H, 6.25; N, 25.28.
(ꢀ)-cis-exo-2-(2-Amino-6-chloro-9H-purin-9-y]-4-(hydroxy-
methyl)-bicyclo[3.1.0]hexane (11). To a suspension of 10
(543 mg, 2.018 mmol) in triethyl orthoformate (11 mL)
was added hydrochloric acid (12 N, 0.5 mL) and the

R. G. Bhushan, R. Vince / Bioorg. Med. Chem. 10 (2002) 2325–2333
2331
mixture was stirred at room temperature overnight. The
suspension was dried in vacuo. Diluted hydrochloric
acid (0.5 N, 15 mL) was added to the residue and the
mixture was stirred at room temperature for 1 h. The
reaction mixture was then neutralized to pH 8 with 1 N
sodium hydroxide and concentrated to give a pale yel-
low residue. The residue was then adsorbed onto silica
gel and it was packed into a column and eluted with
chloroform/methanol (20:1) to chloroform/methanol
(15:1). The product fractions were concentrated to give
a white solid, 413.5 mg (73.4%). TLC: Rf 0.3 (chloro-
form/methanol; 10:1); mp 228–230 ꢂC. 1H NMR
(DMSO-d6, 300 MHz) d 8.20 (s, 1H, H-8), 6.89 (br, s,
2H, NH2, D2O exchangeable), 4.65–4.62 (m, 1H, H-20),
4.57–4.52 (m, 1H, CH2OH, D2O exchangeable), 3.29–
3.14 (m, 1H, CH2OH), 2.99–2.93 (m, 1H, CH2OH),
2.09–2.06 (m, 1H, H-30), 1.84–1.79 (m, 2H, H-40, H-10),
1.77–1.72 (m, 1H, H-30), 1.63–1.57 (m, 1H, H-50), 0.71–
0.67 (m, 1H, H-60), 0.33–0.28 (m, 1H, H-60). Anal. calcd
for C12H14ClN5O.0.6H2O: C, 49.61; H, 5.27; N, 24.09.
Found: C, 49.89; H, 5.01; N, 23.75.
was passed into a solution of 11 (150 mg, 0.5376 mmol)
in methanol (2 mL) at ꢁ80 ꢂC in a bomb. The bomb was
sealed and heated at 75 ꢂC for 48 h. The bomb was then
opened at ꢁ80 ꢂC and allowed to warm to room tem-
perature. Ammonia and methanol were evaporated. The
residue was adsorbed onto silica gel and it was packed
into a column and eluted with chloroform/methanol
(15:1) to chloroform/methanol (5:1). The product frac-
tions were concentrated to give a crude product, which
was recrystallized from ethanol to yield a white solid,
50.0 mg (36%); mp 240–242 ꢂC; TLC: Rf 0.18 (chloro-
1
form/methanol; 15:1). H NMR (DMSO-d6, 300 MHz)
d 7.77 (s, 1H, H-8), 6.61 (br, s, 2H, NH2, D2O
exchangeable), 6.61 (br, s, 2H, NH2, D2O exchange-
able), 4.58–4.53 (m, 2H, H-20, CH2OH, D2O
exchangeable), 3.19–3.14 (m, 1H, CH2OH), 2.98–2.96
(m, 1H, CH2OH), 2.046–2.04 (m, 1H, H-30), 1.79–1.74
(m, 2H, H-40, H-10), 1.70–1.65 (m, 1H, H-30), 1.61–1.58
(m, 1H, H-50), 0.66–0.64 (m, 1H, H-60), 0.27–0.26 (m,
0
.
1H, H-6 ). Anal. calcd for C12H16N6O 0.4H2O: C,
53.87; H, 6.33; N, 31.42. Found: C, 54.02; H, 6.12; N,
31.19.
(ꢁ)-cis-exo-2-(2-Amino-6-chloro-9H-purin-9-y]-4-(hydroxy-
methyl)-bicyclo[3.1.0]hexane (11a). A mixture of 10a
(200 mg, 0.74 mmol), triethyl orthoformate (5.4 mL)
and hydrochloric acid (12 N, 0.22 mL) was stirred at
room temperature overnight. The reaction mixture was
processed as described above to give a pale-yellow solid,
150 mg (75%), mp 190 ꢂC; [a]D25 ꢁ116.71 (c 0.5,
CH3OH). Anal. calcd for C12H14ClN5O: C, 51.52; H,
5.04; N, 25.03. Found: C, 51.43; H, 4.93; N, 24.91.
(ꢁ)-cis-exo-2-(2,6-Diamino-9H-purin-9-y]-4-(hydroxy-
methyl)-bicyclo [3.1.0]hexane (13a). Compound 11a
(150 mg, 0.537 mmol) was reacted with ammonia as
described above to yield white crystals of 13a, 75 mg
(53%); mp 160 ꢂC; MS (FAB-HR): 260 (M+), 261
(M++1); [a]2D5 ꢁ106.8ꢂ (c 0.5, CH3OH).
(ꢀ)-cis-exo-2-[2-Amino-6-propoxy-9H-purin-9-y]-4-(hy-
droxymethyl)-bicyclo [3.1.0]hexane (14). To compound
11 (100 mg, 0.3584 mmol) was added n-propanol (10
mL), sodium hydride (200 mg, 8.33 mmol) and heated
under reflux for 1 h. The reaction mixture was then
neutralized with acetic acid and concentrated to dryness
in vacuo. The residue was then partitioned between
ethyl acetate and water. The organic layer was dried
over Na2SO4 and concentrated. The residue was
adsorbed on silica gel and it was packed into a column
and eluted with chloroform/methanol (50:1) to chloro-
form/methanol (25:1). The product fractions were con-
(ꢀ)-cis-exo-2-(2-Amino-1,9-dihydro-6H-purin-6-one]-4-
(hydroxymethyl)-bicyclo [3.1.0]hexane (12). A solution
of 11 (100 mg, 0.3584 mmol) in 1 N HCl (10 mL) was
heated under reflux for 5 h. The reaction mixture was
then concentrated. The residue was then dissolved in
water and the solution was neutralized to pH 7 with 6 N
sodium hydroxide. A precipitate formed and the sus-
pension was refrigerated for 1 h. It was then filtered and
the solid was recrystallized using hot water to give a
white solid, 52.7 mg (56.27%); mp 292–294 ꢂC. 1H
NMR (DMSO-d6, 300 MHz) d 10.47 (s, br, 1H, OH,
D2O exchangeable), 7.75 (s, 1H, H-8), 6.40 (br, s, 2H,
NH2, D2O exchangeable), 4.57–4.51 (m, 2H, H-20,
CH2OH, D2O exchangeable), 3.27–3.15 (m, 1H, CH2OH),
2.99–2.93 (m, 1H, CH2OH), 2.07–2.01 (m, 1H, H-30),
1.82–1.72 (m, 2H, H-40, H-10), 1.65–1.62 (m, 1H, H-30),
1.60–1.56 (m, 1H, H-50), 0.69–0.62 (m, 1H, H-60), 0.28–
0.24 (m, 1H, H-60). Anal. calcd for C12H15N5O2: C,
55.16; H, 5.78; N, 26.80. Found: C, 55.20; H, 6.00; N,
26.66.
centrated to give
a crude product, which was
recrystallized from methanol-water to yield a white
solid, 34.8 mg (31.6%); mp 132–136 ꢂC; TLC: Rf 0.3
(chloroform/methanol; 15:1). 1H NMR (DMSO-d6,
300 MHz) d 7.92 (s, 1H, H-8), 6.36 (br, s, 2H, NH2,
D2O exchangeable), 4.60–4.53 (m, 2H, H-20, CH2OH,
D2O exchangeable), 4.32–4.27 (t, 2H, OCH2CH2),
3.20–3.13 (m, 1H, CH2OH), 2.98–2.92 (m, 1H,
CH2OH), 2.07–2.04 (m, 1H, H-30), 1.81–1.76 (m, 2H,
H-40, H-10), 1.73–1.71 (m, 2H, CH2CH2CH3), 1.68–1.67
(m, 1H, H-30), 1.62–1.57 (m, 1H, H-50), 0.95–0.90 (t,
3H, CH2CH3), 0.68–0.64 (m, 1H, H-60), 0.30–0.26 (m,
1H, H-60). Anal. calcd for C15H21N5O2.0.5H2O: C,
57.65; H, 7.09; N, 22.39. Found: C, 57.22; H, 7.19; N,
22.24.
(ꢁ)-cis-exo-2-(2-Amino-1,9-dihydro-6H-purin-6-one]-4-
(hydroxymethyl)-bicyclo [3.1.0]hexane (12a).
Com-
pound 11a (200 mg, 0.716 mmol) was hydrolyzed with
hydrochloric acid and purified as described above to yield
a white solid, 18 mg (9.6%); mp 304 ꢂC; [a]D25 ꢁ38.5ꢂ (c
0.25, DMSO). Anal. calcd for C12H15N5O2: C, 55.16; H,
5.78; N, 26.80. Found: C, 55.25; H, 5.66; N, 26.81.
(ꢁ)-cis-exo-2-[2-Amino-6-propoxy-9H-purin-9-y]-4-(hy-
droxymethyl)-bicyclo [3.1.0]hexane (14a). Compound
11a (150 mg, 0.537 mmol) was treated with sodium
hydride (300 mg, 12.49 mmol) and n-propanol (15
mL) as described above to give an oil, which was
(ꢀ)-cis-exo-2-(2,6-Diamino-9H-purin-9-y]-4-(hydroxy-
methyl)-bicyclo [3.1.0]hexane (13). Liquid ammonia

2332
R. G. Bhushan, R. Vince / Bioorg. Med. Chem. 10 (2002) 2325–2333
recrystallized from methanol–water to yield a white
solid, 60 mg (37%); mp 136–138 ꢂC; MS (FAB-HR):
303 (M+), 304 (M++1); [a]2D5 ꢁ122.32ꢂ (c 0.5,
CH3OH). Anal. calcd for C15H21N5O2. 1.25H2O: C,
55.28; H, 7.26; N, 21.49. Found: C, 55.43; H, 7.21; N,
21.79.
added to the wells. Some wells were used as virus controls
and left free of test compound. Other wells remained
free of virus to determine drug toxic effects against vero
cells. Acyclovir, a potent anti-HSV agent was used as
the positive control. The 96-well plates were incubated
for 3 days at 37 ꢂC in a humidified atmosphere contain-
ing 5% CO2 until maximum CPE were observed in the
virus control cultures. The cell monolayers were exam-
ined microscopically for virus-induced CPE and for
drug cytotoxicity. The IC50 value ofthe inhibitor is
defined as the concentration ofdrug that decreased the
number ofplaques 50% to that present in the com-
pound free control wells.
Bis(tributylammonium) pyrophosphate. A solution of
tetrasodium pyrophosphate decahydrate (6.3 g, 14.1
mmol) in 100 mL ofwater was passed through a Dowex
50X-8 (H+) column (25 g) directly into a solution of
tributylamine (67 mL, 28.1 mmol) in ethanol (200 mL).
The resulting solution was evaporated to an oil under
reduced pressure. This oil was triturated with diethyl
ether to give a colorless hygroscopic solid that was fur-
ther dried under reduced pressure. 5.12 g (65.8%). 31P
NMR (D2O, 300 MHz) d ꢁ10.05.
Reverse transcriptase assay
Purified recombinant HIV-1 reverse transcriptase was
obtained from the University of Alabama at Birming-
ham, Center for AIDS Research, Gene Expression Core
Facility (supported in part by the NIH Centers for
AIDS research program grant P30 AI27767). For the
study ofRNA transcription, HIV-1 reverse tran-
scriptase activity was measured in 50-mL reactions con-
taining 50 mM Tris (pH 8.0), 50 mM KCl, 10 mM
MgCl2, 4 mM b-mercaptoethanol, 3% glycerol, 1 mg/
mL bovine serum albumin, 3.33 mg/mL ofprimed 16S
rRNA from Escherichia coli, 10 mM dTTP, 10 mM
dGTP, 10 mM dCTP, and 0.25 mM [33P]dATP (the Km
(ꢀ)-cis-exo-2-(6-Amino-9H-purin-9-yl)-4-[(hydroxy)[hy-
droxy ( phosphonooxy ) - phosphinyl ]oxy]phosphinyloxy -
methyl]-bicyclo[3.1.0]hexane (15). To a suspension of
compound 7 (100 mg, 0.407 mmol) in dry trimethyl-
phosphate (2.8 mL) at 0 ꢂC was added phosphorus
oxychloride (161.36 mg, 1.054 mmol), and the mixture
was stirred at 0 C for 2 h. This reaction mixture was
then treated with a solution ofbis(tributylammonium)
pyrophosphate, (1.13 g, 2.038 mmol), and tributylamine
(0.32 g, 1.727 mmol) in dimethyl formamide (4.01 mL).
The mixture was stirred at 0 ꢂC for 15 min, and then
poured into ice-water (100 mL), neutralized with cold
aqueous triethylamine to pH 7, and lyophilized. The
semisolid was then dissolved in water (20 mL), and
applied to an ion-exchange column (DEAE Sephadex
A-25, HCOꢁ3 form, 20 g). The elution was performed by
a linear gradient ofwater (1.0 L) to 0.75 M triethyla-
mine bicarbonate (1.5 L) and monitored at 254 nm. The
fractions containing the triphosphate were combined,
and concentrated under reduced pressure, and then
lyophilized. The solid obtained was evaporated several
times with ethanol to remove the triethylammonium
bicarbonate, and then dried in vacuo to give compound
15, 132.1 mg. TLC: Rf 0.12 (isopropanol/ammonia/
concentration). The primer was annealed to the tem-
14
plate at a ratio of3 to 1 as described earlier.
After
incubation the DNA in each sample was precipitated
onto glass fiber filters using a 5% trichloroacetic acid
solution containing 10 mM pyrophosphate. These filters
were batch washed and counted for radioactivity.15
Assays were done in duplicate and the IC50 values were
reported.
Acknowledgements
We thank Dr. Lakshmi Akella for synthesizing some of
the intermediates, Jay Brownell for conducting the bio-
logical testing, and Dr. Maren Pink for the X-ray crystal
structure.
1
water, 7:1:2). H NMR (D2O, 300 MHz) d 8.11 (s, 1H,
H-8), 7.76 (s, 1H, H-2), 4.42–4.40 (d, 1H, H-20), 3.30–3.21
(m, 1H, CH2OH), 3.16–3.03 (m, 1H, CH2OH), 2.09–
2.01 (m, 1H, H-30), 1.71–1.61 (m, 1H, H-50), 1.48–1.45
(m. 2H, H-40, H-10), 1.33–1.27 (m, 1H, H-30), 0.48–0.42
(q, 1H, H-60), 0.01–0.00 (q, 1H, H-60); 31P NMR (1.6
mL Hepes Buffer, pH 7 containing 0.5 mL D2O and
0.05 mL EDTA, 150 mg/mL) d-4.63 (Pg);ꢁ9.69 (Pa),
ꢁ20.55 (Pb); MS (FAB-HR): 485.2 (M+), 486.0
(M++1), 484.2 (M+ꢁ1).
References and Notes
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Shannon, W. M.; Lavelle, G. C.; Qualls, J.; Weislow, O. S.;
Kiser, R.; Canonica, P. G.; Schultz, R. H.; Narayanan, V. L.;
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phys. Res. Commun. 1998, 156, 1046.
2. Vince, R.; Hua, M. J. Med. Chem. 1990, 33, 17.
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Nucleosides Nucleotides 1999, 18, 521.
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Org. Chem. 2000, 65, 2172.
6. Katagiri, N.; Yamatoya, Y.; Ishikura, M. Tetrahedron Lett.
1999, 40, 9069.
Antiviral test
The in vitro antiviral assays were carried out by virus-
induced cytopathogenic effects (CPE) inhibition studies.
Confluent monolayers ofArfican green monkey kidney
cells (Vero cell line) grown in minimal essential media
supplemented with 5% fetal calf serum in COSTAR 96-
well plates were infected with an innoculum of HSV-1
(strain F). Final concentrations of100 mg/mL followed
by 10-fold serial dilutions of the test compounds were

R. G. Bhushan, R. Vince / Bioorg. Med. Chem. 10 (2002) 2325–2333
2333
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