Synthesis and antiviral activity of a series of new cyclohexenyl nucleosides.

Abstract

A series of new cyclohexenyl nucleosides is synthesized by coupling the heterocyclic bases with a protected cyclohexenyl precursor under Mitsunobu conditions. The compounds were evaluated for their antiviral and cytostatic activity. Pronounced activity against herpes simplex virus type 1 and type 2 was observed for the 2,6-diaminopurine analogue.

Full text

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Antiviral Chemistry & Chemotherapy 14:31–37
1©2003 International Medical Press 0956-3202/02/$17.00
The development of new nucleoside analogues as antiviral
agents has remained an attractive research field. Due to
their hydrolytic stability, carbocyclic nucleosides have taken
a particular place in the design process of new antiviral
agents (Marquez, 1996). Most of these compounds are
cyclopentane derivatives and less work has been done on
conformationally more rigid carbocyclic analogues such as
cyclohexane nucleosides. Some of these cyclohexane nucle-
osides were synthesized in the past but they were devoid of
antiviral activity (Schaeffer et al., 1964, 1968; Pérez-Pérez,
1995; Mikhailov et al., 1996; Maurinsh et al., 1997).
However, the more flexible cyclohexene nucleosides look
more promising. Indeed, introduction of a double bond
into the cyclohexane ring could facilitate the phosphoryla-
tion of the nucleoside analogue and their eventual incorpo-
ration in DNA and, hence, lead to antiviral activity (Wang
et al., 2000). A cyclohexene ring is more flexible than a
cyclohexane ring and is more prone to conformational
changes that might be needed for substrate/inhibitor
recognition during enzymatic reactions.
Therefore, cyclohexenyl guanine (Cycl-G) 1 and cyclo-
hexenyl adenine (Cycl-A) 2 were synthesized and their
biological activities were investigated (Wang et al., 2000).
In particular, D-cyclohexenyl-G has found to exhibit
potent and selective activity against herpes viruses [herpes
simplex virus type 1 (HSV-1) and type 2 (HSV-2), varicel-
la-zoster virus (VZV), cytomegalovirus (CMV)], in analo-
gy with that of the known antiviral drugs acyclovir and
ganciclovir (Wang et al., 2000). This activity could be
explained by the intercellular phosphorylation of Cycl-G to
its triphosphate in virus-infected cells, as deduced from the
low activity of cyclohexenyl G against thymidine kinase-
deficient (TK–) viral strains (Figure 1).
These results prompted us to synthesize other cyclohex-
enyl nucleosides (3–10) in order to study the effects of base
modification on antiviral activity and toxicity. We recently
reported a straightforward procedure for obtaining the
racemic 1 using as the key step a Diels-Alder reaction
(Wang et al., 2001). The compounds envisaged here were
obtained analogously as racemic mixtures, as separation of
the (+) and (–) enantiomers proved tedious. In case of a
positive biological evaluation, the synthesis of the separate
enantiomers could be envisaged.
Materials and methods
Chemistry
For all reactions, analytical grade solvents were used. All
moisture-sensitive reactions were carried out in oven-
dried glassware (100°C) under a nitrogen atmosphere.
Anhydrous solvent 1,4-dioxane was refluxed on sodi-
um/benzophenone and distilled. Melting points were
determined in capillary tubes with a Büchi SMP-20 cap.
apparatus and were uncorrected.
mined with a Varian Unity 500 MHz spectrometer with
tetramethylsilane (TMS) as internal standard for
NMR spectra and a 200 MHz Varian Gemini apparatus
was used for 13C NMR determination with DMSO-d6
(39.6 ppm) or CDCl3(76.9 ppm) as internal standard for
the 13C NMR spectra (s, singlet; d, doublet; dd, double
doublet; t, triplet; br s, broad singlet; br d, broad doublet;
m, multiplet). Exact mass measurements were performed
on a quadrupole time-of-flight mass spectrometer (Q-
Tof-2, Micromass, Manchester, UK) equipped with a
standard electrospray-ionization (ESI) interface; samples
were infused in i-PrOH/H2O 1:1 at 3 µl/min. Precoated
1H NMR was deter-
1H
Synthesis and antiviral activity of a series of new
cyclohexenyl nucleosides
Ping Gu1, Jordi Morral1, Jing Wang1, Jef Rozenski1, Roger Busson1, Arthur Van Aerschot1,
Erik De Clercq2and Piet Herdewijn1*
1Laboratory of Medicinal Chemistry and 2Laboratory of Virology and Chemotherapy, Rega Institute for
Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
*Corresponding author: Tel: +32 1633 7387; Fax: +32 1633 7340; E-mail: piet.herdewijn@rega.kuleuven.ac.be
A series of new cyclohexenyl nucleosides is syn-
thesized by coupling the heterocyclic bases with a
protected cyclohexenyl
Mitsunobu conditions. The compounds were eval-
uated for their antiviral and cytostatic activity.
Pronounced activity against herpes simplex virus
precursor under
type 1 and type 2 was observed for the 2,6-
diaminopurine analogue.
Keywords: synthesis, antiviral, cyclohexenyl nucle-
osides
Introduction

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2©2003 International Medical Press
aluminum sheets (Fluka Silica gel/TLC-cards, 254 nm)
were used for TLC; the spots were examined with UV
light, or sprayed with sulfuric acid/anisaldehyde or 1%
potassium permanganate solution. Column chromatogra-
phy was performed on ICN silica gel 63-200 60 Å.
Elemental analyses were done at the University of
Konstanz, Germany, and were in agreement with calculat-
ed values within 0.4% error margin. The names of the
compounds accorded to the rules of IUPAC and were
checked with a nomenclature program (ACD-Labs,
Version 4.08, Sept. 1999, Adv. Chem. Dev., Inc.,Toronto,
Canada).
(±)-(4aR,7R,8aS)-2-phenyl-4a,7,8,8a-tetrahydro-4H-
1,3-benzodioxin-7-ol (11)
The preparation of this compound has been described
previously (Wang et al., 2001).
(±)-1-[(1S,4R,5S)-5-hydroxy-4-(hydroxymethyl)-2-cyclo-
hexen-1-yl]-2,4(1H,3H)-pyrimidinedione (3)
A suspension of 1.73 g (8 mmol) of N3-benzoyluracil
(12), 0.93 g (4 mmol) of (±)-(4aR,7R,8aS)-2-Phenyl-
4a,7,8,8a-tetrahydro-4H-1,3-benzodioxin-7-ol (11), 1.15
g (8 mmol) sodium benzoate and 2.62 g (10 mmol) triph-
enylphosphine in 100 ml of anhydrous dioxane was stirred
under nitrogen. A solution of 1.60 ml (10 mmol) of
diethylazodicarboxylate (DEAD) in 40 ml anhydrous
dioxane was slowly added over a period of 3 h. The mix-
ture was further stirred overnight at room temperature
and filtered, and the filtrate was distilled in vacuo to
remove the solvent. The residue was dissolved in 100 ml
of methanol saturated with ammonia and stirred
overnight at room temperature. Evaporation and coevap-
oration with methanol left colourless oil that was purified
on silica gel EtOAc/n-Hexane 5–40% (Rf=0.4).
Compound 21 was obtained as a white solid 0.51 g (1.6
mmol, yield 36%).
Compound 21 (300 mg,0.91 mmol) was treated with 20
ml of 80% trifluoroacetic acid solution at room temperature
for 2 days. After evaporation and coevaporation with
toluene and methanol, the residue was dissolved in 10 ml
water and extracted with ether. The water layer was con-
centrated and the resulting white solid was purified by col-
umn chromatography (CH3OH/EtOAc 0–8%, Rf=0.3).
Crystallization from CH3OH/EtOAc afforded 3 as a white
crystal (138 mg, 0.55 mmol, overall yield 23%).
mp.171°C;1H NMR (DMSO-d6) δ 1.78 (m,2H,H-2′,
2′′, 2.08 (dt, 1H, J=2.5 Hz, 5.5 Hz, H-4′), 3.51 (m, 2H, -
CH2OH), 3.72 (m, 1H, H-3′), 4.70 (br s, 1H, -CH2OH),
4.78 (br s, 1H, 3′-OH), 5.10 (m, 1H, H-1′), 5.54 (d, 1H,
J=8.0 Hz, H-5), 5.61 (ddd, 1H, J=2.5 Hz, 3.5 Hz, 10.0 Hz,
H-6′), 5.98 (ddd, 1H, J=2.0 Hz, 3.0 Hz, 10.2 Hz, H-5′),
7.44 (d, 1H, J=8.0 Hz, H-6), 11.28 (br s, 1H, -NH);13C
NMR (DMSO-d6) δ 34.6 (C-2′), 46.0 (C-4′), 50.1 (C-1′),
61.7 (-CH2OH), 63.1 (C-3′), 101.1 (C-5), 125.3 (C-6′),
134.8 (C-5′), 142.6 (C-6), 151.1 (C-2), 163.6 (C-4) ppm.
HRMS calcd. for C11H15N2O4(M+H)+: 239.1032, found
239.1019.
P Gu et al.
1 Cycl-G 2 Cycl-A
( ± )( ± )
N
N
NH
N
O
HO
HO
N
N
N
N
HO
HO
HO
N
NH
X
O
O
HO
N
N
X
O
HO
HO
N
N
N
N
HO
HO
( ± )
3: X=H
4: X=I
5: X=F
6: X=CH3
7: X=H
8: X=F
9: X=CH3
10
NH2
NH2
NH2
NH2
NH2
Figure 1. Chemical structures of cyclohexenyl nucleosides

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Antiviral Chemistry & Chemotherapy 14:1
3
Antiviral activity of a series of new cyclohexenyl nucleosides
(±)-1-[(1S,4R,5S)-5-hydroxy-4-(hydroxymethyl)-2-cyclo-
hexen-1-yl]-5-iodo-2,4(1H,3H)-pyrimidinedione (4)
Starting with 0.46 g (2 mmol) of compound 11, 1.36 g (4
mmol) of N3-benzoyl-5-iodouracil (13), 1.0 g (4 mmol)
triphenylphosphine, 0.58 g (4 mmol) of sodium benzoate
and 0.72 ml (4 mmol) of DEAD in 20 ml anhydrous diox-
ane, using the same procedure as described for 3, after
purification by column chromatography (CH3OH/CH2Cl2
0–10%, Rf=0.5), 203 mg (0.56 mmol, overall yield 29%) of
compound 4 was obtained.
mp.207°C;1H NMR (DMSO-d6) δ 1.81 (m,2H,H-2′,
2′′), 2.10 (m, 1H, H-4′), 3.59 (m, 2H, -CH2OH), 3.75 (m,
1H,H-3′),4.72–4.83 (m,2H,-CH2OH,3′-OH),5.08 (m,
1H, H-1′), 5.65 (ddd, 1H, J=2.2 Hz, 3.9 Hz, 10.0 Hz, H-
6′), 6.03 (ddd, 1H, J=1.7 Hz, 3.2 Hz, 10.0 Hz, H-5′), 7.86
(s, 1H, H-6), 11.61 (br s, 1H, -NH);13C NMR (DMSO-
d6) δ 34.5 (C-2′), 46.1 (C-4′), 50.7 (C-1′), 61.3 (-
CH2OH), 62.8 (C-3′), 68.2 (C-5), 124.9 (C-6′), 135.4 (C-
5′), 146.7 (C-6), 150.6 (C-2), 160.7 (C-4) ppm. HRMS
calcd. for C11H14IN2O4
(M+H)+: 365.0000, found
365.0013.
(±)-5-fluoro-1-[(1S,4R,5S)-5-hydroxy-4-(hydrox-
ymethyl)-2-cyclohexen-1-yl]-2,4(1H,3H)-pyrimidine-
dione (5)
Following the same procedure as described for 3, and start-
ing with 0.93 g (4 mmol) of 11, 1.87 g (8 mmol) of N3-
benzoyl-5-fluorouracil (14), 2.62 g (10 mmol) of triph-
enylphosphine, 1.15 g (8 mmol) of sodium benzoate and
1.60 ml (10 mmol) of DEAD in 40 ml anhydrous dioxane,
550 mg of compound 23 was obtained. After purification
by column chromatography (CH3OH/CH2Cl20–10%,
Rf=0.3), the desired fluorouracil derivative 5 was obtained
as a white crystal in 44% yield (112 mg, 0.44 mmol).
mp. 208°C;1H NMR (DMSO-d6) δ 1.74–1.84 (m, 2H,
H-2′, 2′′), 2.08 (m, 1H, H-4′), 3.50–3.58 (ddd, 2H, J=5.1
Hz, 10.5 Hz, 22.5 Hz, -CH2OH), 3.79 (m, 1H, H-3′),
4.71 (t, 1H, J=5.3 Hz, -CH2OH), 4.76 (d, 1H, J=4.4 Hz,
3′-OH), 5.09 (m, 1H, H-1′), 5.62 (ddd, 1H, J=2.2 Hz, 3.4
Hz, 10.0 Hz, H-6′), 5.95 (ddd, 1H, J=1.9 Hz, 3.2 Hz, 10.0
Hz, H-5′), 7.73 (d, 1H, J=7.1 Hz, H-6), 11.81 (br s, 1H, -
NH);13C NMR (DMSO-d6) δ 34.1 (C-2′), 46.0 (C-4′),
50.5 (C-1′), 61.5 (-CH2OH), 62.9 (C-3′), 125.1 (C-6′),
126.8 127.5 (C-6), 135.2 (C-5′), 137.4 142.0 (C-5), 149.7
(C-2), 157.6 (C-4) ppm. HRMS calcd. for C11H14FN2O4
(M+H)+: 257.0938, found 257.0900.
(±)-1-[(1S,4R,5S)-5-hydroxy-4-(hydroxymethyl)-2-cyclo-
hexen-1-yl]-5-methyl-2,4(1H,3H)-pyrimidinedione (6)
To a solution of 0.83 g (3.6 mmol) of (±)-(4aR,7R,8aS)-2-
Phenyl-4a,7,8,8a-tetrahydro-4H-1,3-benzodioxin-7-ol
(11), 1.64 g (7.2 mmol) of N3-benzoylthymine (15), 1.04 g
(7.2 mmol) of sodium benzoate and 1.88 g (7.2 mmol) of
triphenylphosphine in 100 ml of anhydrous dioxane,a solu-
tion of 1.41 ml (7.2 mmol) of DIAD in 30 ml was drop
wise added under nitrogen environment. The mixture was
kept stirring overnight at room temperature. The reaction
was filtered and evaporated. The crude 20 was directly
treated with 100 ml saturated ammonia/methanol solution
for 6 h. After evaporation and coevaporation with
methanol, a colourless oil of thymine analogue 24 was
obtained and it was further treated with 40 ml 80% triflu-
oroacetic acid water solution for 2 days. Workup purifica-
tion on silica gel (CH3OH/CH2Cl20–10%, Rf=0.3) and
recrystallization from CH3OH/EtOAc yielded a white
crystal 6 (144 mg, 0.57 mmol, overall yield 16%).
mp. 227°C;1H NMR (DMSO-d6) δ 1.73 (d, 3H, J=1.0
Hz, -CH3), 1.77 (br t, 2H, J=5.7 Hz, H-2′, 2′′), 2.09 (m,
1H, H-4′), 3.54 (m, 2H, -CH2OH), 3.79 (m, 1H, H-3′),
4.76 (t, 1H, J=5.2 Hz, -CH2OH), 4.80 (d, 1H, J=4.2 Hz,
3′-OH), 5.10 (m, 1H, H-1′), 5.60 (ddd, 1H, J=2.2 Hz, 3.4
Hz, 10.0 Hz, H-6′), 5.96 (ddd, 1H, J=2.1 Hz, 3.0 Hz, 10.1
Hz, H-5′), 7.31 (d, 1H, J=1.2 Hz, H-6), 11.31 (br s, 1H, -
NH);13C NMR (DMSO-d6) δ 12.2 (-CH3), 34.4 (C-2′),
46.1 (C-4′), 49.6 (C-1′), 61.7 (-CH2OH), 63.1 (C-3′),
108.7 (C-5), 125.7 (C-6′), 134.6 (C-5′), 138.4 (C-6),
151.1 (C-2), 164.3 (C-4) ppm. HRMS calcd. for
C12H17N2O4(M+H)+: 253.1188, found 253.1180.
(±)-4-Amino-1-[(1S,4R,5S)-5-hydroxy-4-(hydrox-
ymethyl)-2-cyclohexen-1-yl]-2(1H)-pyrimidinone (7)
A premixed solution of 1,2,4-triazole (309 mg, 4.48 mmol)
and phosphoroxychloride (120 µl, 1.28 mmol) in 30 ml
dried pyridine was added to 210 mg (0.64 mmol) of (±)-1-
[(4aR,7S,8aS)-2-phenyl-4a,7,8,8a,-tetrahydro-4H-1,3-
benzodioxin-7-yl]-4-amino-2(1H)-pyrimidinone (21).
The mixture was stirred at room temperature. After 16 h,
the mixture was cooled to 0°C in an ice bath and ammonia
gas was bubbled for 12 min and the reaction was left for 10
min further at room temperature. Evaporation and coevap-
oration with toluene and methanol, a yellow syrup was
obtained which was further treated with 80% trifluo-
roacetic acid solution (30 ml) for 2 days at room tempera-
ture. The reaction mixture was concentrated, and coevapo-
rated with toluene and methanol. The residue was chro-
matographed on silica gel (CH3OH/CH2Cl25–20%,
Rf=0.5) to yield 7 (62.4 mg, 0.26 mmol, overall yield 41%)
as a light yellow solid.
mp.129°C;1H NMR (DMSO-d6) δ 1.78 (m,2H,H-2′,
2′′), 2.08 (m, 1H, H-4′), 3.48–3.56 (ddd, 2H, J=5.4 Hz,
10.6 Hz, 21.4 Hz, -CH2OH), 3.68 (m, 1H, H-3′), 4.67 (br
s,1H,-CH2OH),4.74 (br s,1H,3′-OH),5.15 (m,1H,H-
1′), 5.58 (ddd, 1H, J=2.2 Hz, 3.7 Hz, 10.0 Hz, H-6′), 5.82
(d, 1H, J=7.5 Hz, H-5), 6.00 (ddd, 1H, J=2.0 Hz, 3.2 Hz,
10.0 Hz, H-5′), 7.58 (d, 1H, J=7.3 Hz, H-6), 7.64, 8.02 (br

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4©2003 International Medical Press
d, 2H, -NH2);13C NMR (DMSO-d6) δ 34.8 (C-2′), 46.2
(C-4′), 51.0 (C-1′), 61.7 (-CH2OH), 62.9 (C-3′), 93.5 (C-
5), 125.2 (C-6′), 134.9 (C-5′), 144.7 (C-6), 152.8 (C-2),
163.3 (C-4) ppm. HRMS calcd. for C11H16N3O3(M+H)+:
238.1192, found 238.1179.
(±)-4-Amino-5-fluoro-1-[(1S,4R,5S)-5-hydroxy-4-
(hydroxymethyl)-2-cyclohexen-1-yl]-2(1H)-pyrimidi-
none (8)
Following the procedure used for preparation of 7, and
started from 172 mg (0.5 mmol) of (±)-1-[(4aR,7S,8aS)-
2-phenyl-4a,7,8,8a-tetrahydro-4H-1,3-benzodioxin-7-
yl]-5-fluoro-2,4 (1H,3H)-pyrimidinone (23), 1,2,4-tria-
zole (242 mg, 3.5 mmol), phosphoroxychloride (93 µl, 1
mmol) in 30 ml dry pyridine, the 5-fluorocytosine ana-
logue was obtained (59.9 mg, 0.2 mmol) in an overall yield
of 47%.
mp. 264°C;1H NMR (DMSO-d6) δ 1.71–1.82 (m, 2H,
H-2′, 2′′), 2.05 (m, 1H, H-4′), 3.56 (m, 2H, -CH2OH),
3.70 (m,1H,H-3′),4.68 (t,1H,J=5.1 Hz,-CH2OH),4.70
(d, 1H, J=4.4 Hz, 3′-OH), 5.09 (m, 1H, H-1′), 5.60 (ddd,
1H, J=2.2 Hz, 3.9 Hz, 10.0 Hz, H-6′), 5.98 (m, 1H, H-5′),
7.37, 7.59 (br d, 2H, -NH2), 7.60 (d, 1H, J=7.1 Hz, -NH);
13C NMR (DMSO-d6) δ 35.0 (C-2′),46.3 (C-4′),51.0 (C-
1′), 61.4 (-CH2OH), 62.5 (C-3′), 125.4 (C-6′), 127.5 (C-
6), 127.9 (C-5′), 135.4 (C-5), 154.0 (C-2), 157.4 (C-4)
ppm. HRMS calcd. for C11H15FN3O3(M+H)+: 256.1097,
found 256.1080.
(±)-4-Amino-1-[(1S,4R,5S)-5-hydroxy-4-(hydrox-
ymethyl)-2-cyclohexen-1-yl]-5-methyl-2(1H)-pyrimidi-
none (9)
Starting with (±)-1-[(4aR,7S,8aS)-2-phenyl-4a,7,8,8a-
tetrahydro-4H-1,3-benzodioxin-7-yl]-5-methyl-
2,4(1H,3H)-pyrimidinone (24) (148 mg, 0.44 mmol),
1,2,4-triazole (240 mg, 3.48 mmol), phosphoroxychloride
(81 µl, 0.86 mmol) and 15 ml dry pyridine, using the same
procedure as described for 7, and purified on silica gel
(CH3OH/CH2Cl25–20%, Rf=0.4), a white solid was
obtained, which was crystallized from CH2Cl2/CH3OH
(60 mg, 0.2 mmol, overall yield 55%).
mp. 277°C;
2H, H-2′, 2′′), 1.83 (s, 3H, -CH3), 2.09 (m, 1H, H-4′),
3.54 (m, 2H, -CH2OH), 3.70 (m, 1H, H-3′), 4.70–4.78
(br,2H,-CH2OH,3′-OH),5.08 (m,1H,H-1′),5.58 (ddd,
1H, J=2.1 Hz, 3.6 Hz, 10.0 Hz, H-6′), 5.96 (ddd, 1H,
J=2.6 Hz, 3.0 Hz, 10.2 Hz, H-5′), 7.48 (d, 1H, J=1.2 Hz,
H-6), 8.07–8.57 (br d, 2H, -NH2);13C NMR (DMSO-d6)
δ 12.7 (-CH3), 34.4 (C-2′), 46.2 (C-4′), 50.9 (C-1′), 61.7
(-CH2OH), 62.9 (C-3′), 101.3 (C-5), 125.4 (C-6′), 135.0
(C-5′), 142.6 (C-6), 151.6 (C-2), 162.1 (C-4) ppm.
HRMS calcd. for C12H18N3O3(M+H)+: 252.1348, found
252.1330.
1H NMR (DMSO-d6) δ 1.76–1.80 (mt,
(±)-(1S,2R,5S)-5-(2,6-diamino-9H-purin-9-yl)-2-
(hydroxymethyl)-3-cyclohexen-1-ol (10)
To a mixture of (±)-(4aR,7R,8aS)-2-phenyl-4a,7,8,8a-
tetrahydro-4H-1,3-benzodioxin-7-ol (11) 500 mg (2.2
mmol), 2-amino-6-chloropurine (821 mg, 4.8 mmol) and
triphenylphosphine (1.15 g, 4.4 mmol) in dry dioxane 30
ml under N2, at room temperature, was slowly added a
solution of DIAD (0.86 ml, 4.4 mmol) in dry dioxane 20
ml over a period of 3 h.The reaction mixture was stirred at
room temperature for 2 days, filtered and concentrated.
The residue was submitted to column chromatography
(CH3OH/CH2Cl21%, Rf=0.3), a mixture of compound 25
and triphenylphosphine oxide was obtained. Due to the
difficult separation, we decided to deprotect 25 before final
purification.
A solution of impure compound 25 (about 1.4 g) in 100
ml methanol saturated with ammonia is heated in a Parr
pressure reactor for 8 h at 100°C. After evaporation, the
obtained residue was purified by flash chromatography
(CH3OH/CH2Cl20–8%, Rf=0.3) to give 270 mg (0.74
mmol) of 26 as yellow syrup. The benzylidene moiety was
removed with 80% trifluoroacetic acid solution.After evap-
oration and coevaporation with toluene and methanol, the
residue was purified by
(CH3OH/CH2Cl25–20%, Rf=0.5) to give diaminopurine
analogue 10 (107 mg, 0.39 mmol, overall yield 18%), which
was crystallized from CH2Cl2/CH3OH to give a white
crystal.
mp. 268°C;1H NMR (DMSO-d6) δ 1.87 (m, 1H, H-
2′), 1.97 (dt, 1H, J=4.0 Hz, 13.2 Hz, H-2′′), 2.12 (m, 1H,
H-4′), 3.52–3.66 (m, 3H, -CH2OH, H-3′), 4.70 (t, 1H,
J=5.2 Hz, -CH2OH), 4.76 (d, 1H, J=5.4 Hz, 3′-OH), 4.99
(m, 1H, H-1′), 5.77 (ddd, 1H, J=2.5 Hz, 3.9 Hz, 10.0 Hz,
H-6′), 5.83 (br s, 2H, -NH2), 5.98 (ddd, 1H, J=1.6 Hz, 2.9
Hz, 9.9 Hz, H-5′), 6.68 (br s, 2H, -NH2) 7.60 (s, 1H, H-
8);13C NMR (DMSO-d6) δ 35.9 (C-2′), 46.6 (C-4′), 47.9
(C-1′), 61.6 (-CH2OH), 62.8 (C-3′), 113.7 (C-5), 125.2
(C-6′), 133.7 (C-5′), 136.1 (C-8), 151.5 (C-4), 156.4 (C-
6) 160.5 (C-2) ppm. HRMS calcd. for C12H17N6O2
(M+H)+: 277.1413, found 277.1416.
flash chromatography
Virology
Antiviral activity determinations against herpesviruses were
performed in either E6SM or HEL cell cultures as previous-
ly described (De Clercq et al.,1980).The origin of the virus-
es, HSV-1 (strains KOS, F and McIntyre), TK–HSV-1
(strain KOS ACVr), HSV-2 (strains G, 196 and Lyons),
VZV (strains OKA and YS), TK–VZV (strains 07-1 and
YS-R), vaccinia virus (VV), vesicular stomatitis virus (VSV)
and CMV (strains AD169 and Davis) have been reported
(De Clercq et al., 1986). The assays for evaluating activity
P Gu et al.

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Antiviral Chemistry & Chemotherapy 14:1
5
Antiviral activity of a series of new cyclohexenyl nucleosides
against human herpesvirus 6 (HHV-6) have been described
(De Clercq et al.,2001).The cytotoxicity measurements were
based on microscopically visible alteration of normal cell
morphology (E6SM) or inhibition of normal cell growth
(HEL) as previously described (De Clercq et al., 1981).
Results
(±)-(4aR,7R,8aS)-2-phenyl-4a,7,8,8a-tetrahydro-4H-1,3-
benzodioxin-7-ol (11) is the key intermediate for the syn-
thesis of cyclohexenyl nucleoside. A straightforward
approach to synthesize compound 11 was recently reported
(Wang et al., 2001). This involved a Diels-Alder cyclo-
addition reaction to build up the six-membered ring skele-
ton, a Fraser-Reid reductive rearrangement and protection
of the diol with a benzylidene group. This approach was
followed for the synthesis of 11, which was then used for
base introduction.
For the synthesis of the uracil analogue 3, a Mitsunobu
reaction was employed by using 1 eq. of compound 11, 2 eq.
of N3-benzoyluracil 12, 2 eq. of triphenylphosphine (Ph3P),
2 eq. of sodium benzoate (PhCOONa), and 2 eq. of diethyl
azodicarboxylate (DEAD) or diisopropyl azodicarboxylate
(DIAD) in dioxane (Figure 2).The benzoyl group of 17 was
removed by treatment with saturated ammonia/methanol
and the benzylidene group was deprotected with 80% triflu-
oroacetic acid at room temperature. The uracil derivative 3
was obtained in 23% yield and, likewise, following the same
procedure, starting from 13, 14 and 15, compounds 4, 5 and
6 were obtained in 29, 44 and 16% overall yield, respectively.
Mitsunobu reaction using either N3-benzoylated
thymine or uracil, or N-benzoylcytosine afforded mainly
the O2-substituted nucleosides. Only by addition of sodi-
um benzoate the desired pyrimidine analogues could be
obtained in moderate yield. The Mitsunobu conditions
used here were somewhat different (Varasi et al., 1987;
Hughes et al., 1988) from the standard conditions (Jenny
et al., 1991), as we were unable to obtain the desired N1-
substituted pyrimidine nucleoside analogues using these
standard conditions.
The cytosine, 5-methylcytosine and 5-fluorocytosine
analogues 7, 8 and 9 were obtained from their uracil coun-
terparts (21, 23 and 24, respectively) via the 4-triazolyl-
pyrimidinone intermediates (Krug et al., 1989) (Figure 3).
Therefore, compounds 21, 23 and 24 were treated with
phosphorous oxychloride and 1, 2, 4-triazole, and subse-
quently treated with bubbling ammonia gas through the
reaction mixture for 10–15 min. After chromatographic
purification, the benzylidene group was removed with
80% trifluoroacetic acid solution to yield 7, 8 and 9 in
overall yield of 15, 17 and 20%, respectively (starting from
compound 11).
2-Amino-6-chloropurine was introduced under
Mitsunobu conditions to afford 25. This compound could
be separated from its N-7 substituted isomer using column
chromatography. Treatment of 25 with methanol saturated
with ammonia in a Parr pressure reactor at 100°C
(Verheggen et al., 1995) gave, after removal of the benzyli-
dene moiety, the desired 2,6-diaminopurine derivative 10
in combined yield of 18% (Figure 4).
12: X=H
13: X=I
14: X=F
15: X=CH3
3: X=H
4: X=I
5: X=F
6: X=CH3
17: X=H, R=Bz
18: X=I, R=Bz
19: X=F, R=Bz
20: X=CH3, R=Bz
21: X=H, R=H
22: X=I, R=H
23: X=F, R=H
24: X=CH3, R=H
ii
i
iii
HO
N
NH
X
HO
( ± )
O
O
Ph
O
N
O
R
N
X
( ± )
O
O
Ph
+
11
O
O
N
XBz
N
H
OH
( ± )
O
O
Figure 2. Synthesis of compounds 3, 4, 5 and 6
i) Ph3P, PhCOONa, DEAD or DIAD, Dioxane, r.t. 2 days; ii) NH3/CH3OH, 12 h; iii) TFA/H2O (80%).

Page 6

6©2003 International Medical Press
Discussion
The newly synthesized compounds 4, 7, 8, 9 and 10 were
investigated for their inhibitory effect on the cytopatho-
genicity of HSV-1, TK–HSV-1, HSV-2, VV and VSV in
human embryonic lung (HEL) cell cultures. Compounds 3,
5 and 6 were tested against HSV-1, HSV-2 and VSV in
human embryonic skin muscle (E6SM) fibroblast cell cul-
tures (Table 1).The antiviral activity was compared with that
of known and approved antiviral drugs, one with a pyrimi-
dine base moiety (brivudin) and two with a purine base moi-
ety (acyclovir,ganciclovir).The sources of the viruses and the
methodology used to monitor antiviral activity have been
described previously (De Clercq et al., 1980, 1986).
While compounds 3–5 did not display any meaningful
activity, the analogues 6–10 demonstrated significant
activity against HSV-1 and HSV-2. Of these compounds,
10 effected a 50% reduction of the cytopathogenicity
induced by HSV-1 at a concentration of 1.4 µM.However,
none of them approached the activity level of the refer-
ence compounds, brivudin, acyclovir or ganciclovir. The
activity of the deoxycytidine analogues 7, 8 and 9 against
HSV-1 and HSV-2 was very similar. The diaminopurine
derivative 10 is more potent against HSV-1 than against
HSV-2. As the compounds were less active against the
TK–acyclovir-resistant (ACVr) strain, intracellular phos-
phorylation by the virus-induced thymidine kinase must
play an important role in their metabolic activation.
Compounds were not active against VV at 210 µM. None
of the compounds proved cytotoxic at a concentration up
to 1000 µM, as monitored by microscopically detectable
alteration of normal cell morphology. Compounds 3 and 6
were also evaluated for their activity against VZV and
CMV. However, they exhibited no inhibitory effect on the
cytopathogenicity of VZV (whether TK+or TK-) or CMV
in HEL cells (data not shown). Compounds 3, 4 and 8–10
were not active against human herpesvirus type 6A or 6B
in human T-lymphoblast HSB-2 and Molt-3 cells (De
Clercq et al., 2001) at concentrations up to 20 µM (data
not shown).
From these results it is clear that only the diaminogua-
nine derivative, which can be considered as a precursor of
cyclohexenyl-G, is of interest because of its moderate anti-
HSV activity. Further research is needed to unravel its
P Gu et al.
21: X=H, R=H
23: X=F, R=H
24: X=CH3, R=H
7: X=H
8: X=F
9: X=CH3
i ii, iii
Ph
O
O
N
O
N
R
X
( ± )
O
O
N
O
N
N
N
N
X
Ph
O
( ± )
HO
HO
N
N
O
X
( ± )
NH2
Figure 3. Synthesis of compounds 7, 8 and 9
i) POCl3, 1,2,4-1H-triazole, pyridine; ii) NH3; iii) TFA/H2O (80 %).
Ph
N
H
N
N
N
CI
Ph
O
N
CI
N
N
HO
HO
N
N
N
N
N
O
O
O
OH
( ± )
( ± )( ± )
+
11
25 10
i
ii, iii
NH2
NH2
NH2
NH2
Figure 4. Synthesis of compound 10
i) Ph3P, PhCOONa, DIAD, Dioxane, r.t. 2 days; ii) NH3/CH3OH, Parr bomb, 100°C, 14 h; iii) TFA/H2O (80%).

Page 7

Antiviral Chemistry & Chemotherapy 14:1
7
Antiviral activity of a series of new cyclohexenyl nucleosides
mode of action and to evaluate the individual (+) and (–)
enantiomers of compound 10.
Acknowledgements
We thank A Marchand and I Lagoja for their helpful sugges-
tions and L Baudemprez for NMR analysis.We also thank A
Van Lierde and F De Meyer for their excellent technical assis-
tance and C Biernaux for her dedicated editorial help.
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Table 1. Antiviral activity against HSV-1, HSV-2, VV and VSV and cytotoxicity of compound 3–10
MIC (µM)
Compound3456789 10 D-Cycl G * AcyclovirGanciclovir
Cell virus
HSV-1 (KOS)
HSV-1 (F)
HSV-1 (McIntyre)
HSV-1 (TK–KOS ACVr) 1007
HSV-2 (G)
HSV-2 (196)
HSV-2 (Lyons)
VV
VSV
MCC
E6SM
1007
ND§
ND
HEL
>220
220
659
1098
659
659
659
>220
>220
≥1098
E6SM
936
ND
ND
ND
>312
ND
ND
>312
>312
≥1561
E6SM
190
ND
ND
ND
190
ND
ND
>1585
>1585
>1585
HEL
40
13
202
67
40
67
67
1011
>1686 >1567
>1686 >1567
HEL
63
38
38
940
13
38
188
>1567
HEL
191
64
191
955
64
191
191
>1592
>1592
>1592
HEL
1.4
2.3
6.9
174
35
35
35
869
>290
≥1448
E6SM
0.01
0.01
0.01
1.37
0.18
0.25
0.25
ND
ND
>1442
HEL
0.4
0.6
0.6
213
0.4
0.4
0.4
>1775
>1775
>1775
HEL
0.02
<0.01
0.01
3.1
<0.01
0.03
0.01
>392
>392
>392
1007
ND
ND
>1679
>1679
>1679
MIC, minimum inhibitory concentration, or concentration required to reduce virus-induced cytopathogenicity by 50%; MCC, minimal cytotox-
ic concentration, or concentration required to cause a microscopically detectable alteration of normal cell morphology.
* Literature data taken from reference (Wang et al., 2000). ND, not determined.
Received 9 December 2002; accepted 11 February 2003