Adenine and deazaadenine nucleoside and deoxynucleoside analogues: inhibition of viral replication of sheep MVV (in vitro model for HIV) and bovine BHV-1.

Article abstract of DOI:10.1016/S0968-0896(02)00131-1

A series of N(6)-cycloalkyl-2',3'-dideoxyadenosine derivatives has been prepared by coupling of 2,6-dichloropurine to protected 2,3-dideoxyribose, followed by reaction with appropriate cycloalkylamines. Synthesized compounds, along with other purine nucleoside analogues previously synthesized in our laboratory, have been tested for their antiviral activity against Bovine herpesvirus 1 (BHV-1) and sheep Maedi/Visna Virus (MVV), the latter being an in vitro and in vivo model of Human Immunodeficiency Virus (HIV). All compounds showed good antireplicative activity against MVV, with the N(6)-cycloheptyl-2',3'-dideoxyadenosine (5b) being the most active [effective concentration (EC(50)) causing 50% reduction of cytopatic effects (CPE)=27 nM]. All compounds showed also a from low to very low cell toxicity, resulting in a cytotoxic dose 50 (CD(50))/EC(50) ratio in some cases higher than 1000.

Full text of DOI:10.1016/S0968-0896(02)00131-1

Bioorganic & Medicinal Chemistry10 (2002) 2973–2980
Adenine and Deazaadenine Nucleoside and Deoxynucleoside
Analogues: Inhibition of Viral Replication of Sheep MVV
(In Vitro Model for HIV)and Bovine BHV-1
Daniela Salvatori,a Rosaria Volpini,b Silvia Vincenzetti,a Alberto Vita,a
Stefano Costanzi,b Catia Lambertucci,b Gloria Cristallib and Sauro Vittorib,*
a
`
Dipartimento di Scienze Veterinarie, Universita di Camerino, Italy
Dipartimento di Scienze Chimiche, Universita di Camerino, Italy
b
`
Received 19 February2002; accepted 9 April 2002
Abstract—A series of N6-cycloalkyl-20,30-dideoxyadenosine derivatives has been prepared by coupling of 2,6-dichloropurine to
protected 2,3-dideoxyribose, followed by reaction with appropriate cycloalkylamines. Synthesized compounds, along with other
purine nucleoside analogues previously synthesized in our laboratory, have been tested for their antiviral activity against Bovine
herpesvirus 1 (BHV-1) and sheep Maedi/Visna Virus (MVV), the latter being an in vitro and in vivo model of Human0Immuno-
deficiencyVirus (HIV). All compounds showed good antireplicative activityagainst MVV, with the N6-cycloheptyl-20,3 -dideoxy-
adenosine (5b) being the most active [effective concentration (EC50) causing 50% reduction of cytopatic effects (CPE)=27 nM]. All
compounds showed also a from low to very low cell toxicity, resulting in a cytotoxic dose 50 (CD50)/EC50 ratio in some cases higher
than 1000. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
bythe human and animal herpesviruses for their simi-
5,6
larityin the biological and pathogenetic behaviour.
In spite of the numerous efforts to fight the viral dis-
eases byvaccination, viral infections are still widespread
and represent a continuous threat to the welfare of
humans and animals. In the last decades, beside the
development of new methods in the preparation of
highlysophisticated immunizing products, a different
strategyhas been proposed to fight virus infections, that
is the use of antiviral drugs.
For this study, we selected the MVV as a model for HIV
and BHV-1 as a model for the human herpesviruses.
The MVV is associated with chronic respiratorydisease
and/or mastitis and neurological distress in sheep and is
responsible of serious economic losses when a flock
becomes infected.7 BHV-1 is considered one of the most
widespread pathogenic agents of cattle and effects of
infection caused bythe virus usuallyvaryfrom rela-
tivelymild illness to severe respiratorydisease, but
could result also in a number of other clinical condi-
tions, such as conjunctivitis, genital lesions, abortions,
enteritis and encephalitis. For this reason the persistence
of the virus in herds represents a continuous threat to
the welfare of the animals and the cause of significant
economic losses for cattle industry.8,9 In addition, ser-
ious problems to human health are caused byhuman
herpesviruses, which mainly cause systemic, eye, and
genital tract infections, leading even to the development
of cancer.10
The most appropriate and convenient wayfor testing
the potential drugs for their antiviral activityappears to
be the use of animal models, of course where a close
analogyexists between viruses which are pathogenic for
humans and those which affect animals. One example is
represented bytwo lentiviruses: the first is identified as
the Human ImmunodeficiencyVirus (HIV, causative
agent of AIDS) and the second as the Maedi Visna
Virus (MVV) of sheep.1,2 The two viruses appear to be
strikinglysimilar as far as biochemical and genomic
properties are concerned.3,4 A further example is given
Most of the antiviral drugs that have been licensed so
far for clinical use are members of the class of purine
and pyrimidine nucleoside analogues, and a number of
*Corresponding author. Tel.: +39-07-3740-2266; fax: +39-07-3740-
2295; e-mail: sauro.vittori@unicam.it
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00131-1

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D. Salvatori et al. / Bioorg. Med. Chem. 10 (2002) 2973–2980
the most promising inhibitors of retrovirus replication
are 2030-dideoxyanalogues.11 In a previous paper we
demonstrated the anti-HIV activityof N6-cycloalkyl
derivatives of 1-deazaadenine nucleosides.12 Further-
more, our research group has a long expertise in syn-
thesizing and testing purine nucleoside analogues, such
as deazaadenosine derivatives,13ꢀ16 N6-substituted deri-
vatives,17 20-deoxy,12,18 30deoxy,19,20 and 20,30-dideoxy12
analogues of adenosine, and various combinations of
the above modifications.12,17
Based on the fact that, as stated previously, many of the
known nucleoside inhibitors of viral replication are
dideoxypurine nucleosides, and the fact that we
demonstrated the anti-lenti virus (anti-HIV) activityof
N6-cycloalkyl derivatives of 1-deazaadenine nucleo-
sides,12 in the present studywe undertook the synthesis
of 2,6-dichloro-20,30-dideoxypurine riboside 4 which is a
versatile intermediate for the introduction of N6-sub-
stituent on dideoxypurine nucleosides (Scheme 1). This
dichloro derivative was obtained bycoupling 2,6-
dichloropurine to protected 2,3-dideoxyribose. From
this compound we synthesized four N6-cycloalkyl deri-
vatives 5a, 5b, 6a, 6b, bearing small (C3) or large (C7)
carbon ring on N6, with or without a chlorine atom in
the 2 position.
The synthesized compounds have been tested for their
antireplicative activityon BHV-1 and on MVV, toge-
ther with numerous related nucleosides, both from our
chemical library( 7–23, 28–31) or from commercial
sources (24–27) (Fig. 1). All compounds have also been
Scheme 1. Synthesis of 2,6-dichloro-9-(2,3-dideoxy-a-d-glycero-
pentofuranosyl)-9H-purine and 2,6-dichloro-9-(2,3-dideoxy-b-d-gly-
ceropentofuranosyl)-9H-purine: (i) HMDS, (NH4)2SO4, Á; (ii)
CH3CN, TMSTf.
Figure 1. Structure of tested compounds.21ꢀ23

D. Salvatori et al. / Bioorg. Med. Chem. 10 (2002) 2973–2980
2975
tested for their cytotoxicity on the cell lines used for
virus replication.
The site of dideoxyribosylation was assigned to be N-9
1
on the purine ring based on UV and H NMR spectral
data. A strong NOE effect was observed on H-8 upon
irradiation of H-10, clearlyexcluding N-3 as the site of
ribosylation.
Compounds 24 and 26 are substrates of Adenosine
Deaminase (ADA), enzyme that converts adenine
nucleosides into 6-hydroxypurine nucleosides by hydro-
lytic deamination.24,25 Theyhave been tested in all
experiments both in absence and in presence of erythro-
2-hydroxy-3-nonyladenine (EHNA), a reference ADA
inhibitor, to assess the influence of deamination on their
biological activity.
Furthermore, the UV spectra of compounds 3 and 4, at
different pH value, were essentiallyindistinguishable
and almost identical with that of 2,6-dichloro-9-
methylpurine, previously reported in the literature,
while being rather different from that of 2,6-dichloro-7-
methylpurine.30
Compound
4 was reacted with the appropriate
Results and Discussion
Chemistry
cycloalkylamine and then, without purification of the
protected intermediate, with methanol saturated with
ammonia. After usual work up and chromatographic
purification compounds 5a and 5b were obtained in
good yields (Scheme 2).
The synthesis of the N6-cycloalkyl-20,30-dideox-
adenosine derivatives 5a, 5b, 6a, 6b was accomplished as
reported in Schemes 1 and 2.
Catalytic dehalogenation of 2-chloroderivatives 5a and
5b was accomplished byshaking the nucleoside, dis-
solved in ethanol, in an atmosphere of hydrogen in the
presence of a catalytic amount of palladium on carbon.
After purification the 2-unsubstituted nucleosides 6a
and 6b were obtained with yield of 73 and 63%,
respectively.
Commerciallyavailable 2,6-dichloropurine ( 1) was
refluxed in hexamethyldisilazane (HMDS) added of a
catalytic amount of ammonium sulfate; after work up in
drycondition, the silylated derivative was coupled with
2,3-dideoxy-1-O-methyl-5-O-(4-methylbenzoyl)-d-glycero-
pentofuranoside (2)26 in CH2Cl2, using TMS–triflate as
catalyst. After chromatographic purification the two
anomers a (3) and b (4) were obtained in 3:2 ratio,
respectively(Scheme 1). Similar coupling reactions have
been alreadyreported in literature, but from differently
protected sugar, with diverse procedure and lower
yield.27,28
Pharmacology
Synthesized compounds, along with other purine
nucleosides previouslysynthesized in our laboratory( 7–
23, 28–31), with some structurallyrelated reference
compounds (24–27) known for their antiviral and/or
cytotoxic activity, and with reference anti-herpesviruses
(Ganciclovir) or anti-lentiviruses (3TC) drugs (Fig. 1),
have been tested on BHV-1 (strain IBR-LA), grown on
bovine kidneycell line MDBK, and on MVV (strain
KV1772)31 replicating on sheep chondrocytes G81092.
For both viruses, EC50 assays and titre reduction assays
have been performed. All compounds have also been
tested for their median cytotoxic dose (CD50) on the cell
lines used for virus replication.
The anomeric configuration of compounds 3 and 4 was
assigned applying NOE difference spectroscopy.
Saturation of H-10 signal of 4 yielded 1% NOE of the
H-40 signal, establishing b-d configuration, while
saturation of H-10 signal of 3 gave no effect on the H-40
signal, establishing a-D configuration. Moreover, this
assignment is also in accordance with the empirical rule
based on chemical shift differences between H-40 and H-
50 (Ád), which should be smaller for the b anomer,
compared to the a one.29 In the case of a-anomer 3, Ád
was 0.49 ppm, whereas in the case of the b anomer 4 Ád
was 0 ppm.
Some of the compounds, in particular those with an
intact and unsubstituted adenine moiety( 24 and 26), are
substrate for adenosine deaminase (ADA), enzyme that
converts adenine nucleosides into 6-hydroxypurine
nucleosides by hydrolytic deamination. Those com-
pounds have been tested in all experiments both in
presence and in absence of erythro-hydroxynonyl-
adenine (EHNA), a reference ADA inhibitor, to assess
if and how ADA activitycan modifytheir biological
actions.
Tables 1 and 2 report on biological results on BHV-1
and MVV assays, respectively.
BHV-1 inhibition. On BHV-1 all compounds showed an
EC50 equal to or higher than 50 mM, whereas some of
them were able to reduce the viral titre of (or close to) 3
log units (i.e., 6a, 8, 9, 10, 25). This result is worth of
Scheme 2. Synthesis of (2-chloro-)6-cycloalkylamino-9-(2,3-dideoxy-
b-d-glyceropentofuranosyl)-9H-purines: (i) R-NH2; (ii) NH3/CH3OH;
(iii) Pd/C, H2, OHꢀ.

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D. Salvatori et al. / Bioorg. Med. Chem. 10 (2002) 2973–2980
notice, given the fact that the same efficacyis shown by
the reference compound Ganciclovir, an antiviral drug
used in the therapyof human herpesvirus infections.
differences in biological activityonlyin the cytotoxicity
test. In fact, compound 24 showed a lower toxicitywhen
EHNA was present, whereas compound 26 had an
opposite behavior. In antireplicative assays, the two
compounds were almost inactive in both tests.
Almost all compounds showed to be cytotoxic against
cells used for virus replication; onlyfew of them had
little or no toxicity. One of them is compound 6a which,
with a CD50 >400 mM, along with its previously
underlined abilityof reducing virus titre, is an interest-
ing starting point for further developments of anti-
BHV-1 drugs. From cytotoxicity data, the presence of a
chlorine atom in the 2-position clearlyraises activity
against cell line, whereas the opposite effect can be seen,
in manycases, for viral titre reduction (see, e.g., 5a vs 6a
and 7 vs 8).
MVV inhibition. Table 2 shows reduction of virus titre
and EC50 on MVV. It shows also CD50 on sheep chon-
drocytes, used for virus replication, and CD50/EC50
ratio, which accounts for safetyof the compounds when
used as antivirals in vitro.
All compounds show a moderate to good abilityof
reducing virus titre, with the highest efficacydemon-
strated
by
N6-cycloheptyl-2-chloro-20,30-dideoxy-
adenosine (6a), with a reduction of 4 log units compared
to the control. Manyother nucleosides were able to
reduce virus titre by3 log units, demonstrating that
MVV is overall more sensitive to tested compounds
than BHV-1.
The most active against the replication of BHV-1 of the
tested nucleosides possess some structural analogies: an
intact purine ring, and the lack of the hydroxyl group in
position 20. These observations can be useful in the
optimization of anti-BHV-1 drugs.
From anti-replicative and cytotoxicity data some inter-
esting structure–activityrelationships can be drawn.
Assayed nucleosides possess high activity against MVV,
Testing of compounds 24 and 26 both in presence and
in absence of the inhibitor of ADA EHNA showed
Table 1. Effect of compounds on bovine herpes virus (BHV-1, strain
IBR-LA), grown on fetal kidneycalf cell line MDBK
Table 2. Effect of compounds on sheep maedi/visna lentivirus (MVV,
strain KV1772) replicating on sheep chondrocytes G81092
Viral titreb
EC50c (mM)
a
a
Compd
CD50 (mM) Viral titreb EC50c (mM) CD50/EC50
Compd
CD50 (mM)
5a
5b
6a
6b
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
24d
25
26
26d
180ꢁ26
315ꢁ46
>400
6.50
7.50
5.50
7.50
7.50
5.74
5.50
5.50
7.50
7.50
7.50
7.50
7.50
7.50
7.50
7.50
7.50
7.50
7.50
7.50
7.50
7.50
7.50
5.50
7.50
7.50
7.50
7.50
7.50
8.50
7.50
5.50
8.50
50
>50
50
>50
>50
50
5a
5b
6a
6b
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
24d
25
26
26d
180ꢁ30
125ꢁ25
280ꢁ25
308ꢁ10
234ꢁ50
312ꢁ30
98ꢁ25
108ꢁ33
225ꢁ25
105ꢁ20
100ꢁ20
126ꢁ30
236ꢁ45
230ꢁ55
260ꢁ26
205ꢁ25
145ꢁ45
210ꢁ45
270ꢁ40
>400
4.50
3.50
2.50
4.50
3.75
4.50
3.75
5.50
4.75
6.50
6.50
5.50
4.50
4.75
4.75
4.75
3.50
3.75
3.50
5.50
4.50
3.75
3.50
4.50
4.75
4.50
3.75
5.50
3.50
4.00
5.50
<1.00
6.50
0.41
0.027
0.15
8.6
0.15
1.5
439
4630
1867
36
1560
208
363
72
15
7
667
8
87
163
520
137
967
244ꢁ22
<50
>400
130ꢁ11
320ꢁ33
359ꢁ50
>400
50
50
0.27
1.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
15
15
0.15
15
2.7
1.41
0.5
1.5
0.15
0.5
1.5
1.6
0.15
0.41
0.027
1.5
1.5
1.5
131ꢁ11
>400
185ꢁ35
120ꢁ20
180ꢁ30
228ꢁ22
191ꢁ28
293ꢁ37
255ꢁ41
>400
420
180
>250
1300
>976
6667
<33
>267
>267
<3
>27
>2667
>98
>27
>60
<50
<50
195ꢁ45
>400
110ꢁ15
50
50
180ꢁ50
<50
>400
>400
<50
>400
>400
>400
>400
>300
<50
250ꢁ30
<50
<50
105ꢁ15
140ꢁ10
260ꢁ20
<50
>50
>50
>50
>50
>50
>50
>50
50
27
28
29
30
27
28
29
30
15
15
0.15
4.1
15
31
Ganciclovir
Virus control
31
3TC
Virus control
<50
5
aConcentration of compound able to give 50% of the maximal effect.
bMaximal dilution (negative log of concentration) at which the virus is
still able to give cytopathic effects.
cConcentration of compound able to lower virus effects by50%.
dCompound tested in presence of EHNA, inhibitor of adenosine
deaminase.
aConcentration of compound able to give 50% of the maximal effect.
bMaximal dilution (negative log of concentration) at which the virus is
still able to give cytopathic effects.
cConcentration of compound able to lower virus effects by50%.
dCompound tested in presence of EHNA, inhibitor of adenosine
deaminase.

D. Salvatori et al. / Bioorg. Med. Chem. 10 (2002) 2973–2980
2977
with EC50 ranging from 15 mM to 27 nM. These data
are definitelyof interest, speciallywhen compared to the
5 mM EC50 of the reference compound 20,30-dideoxy-30-
thiacytidine (3TC).
well above 1000. These data can help in identifying a
new class of anti-HIV compounds, and in finding
therapeutics for viral infections of animals.
With the exception of 20,30-dideoxyadenosine (24), N6-
cycloalkyl derivatives 5–22 showed an average abilityto
inhibit virus replication higher than that of N6-unsub-
stituted nucleosides 23–31, suggesting that a substituent
in this position is able to improve compound’s activity.
On the other hand, the same average difference can be
seen in cytotoxicity assays. In fact, with few exceptions,
N6-unsubstituted compounds showed a CD50 higher
than 400 mM, maximal assayconcentration, whereas
CD50 of N6-cycloalkyl derivatives is ranging between 98
(9) and 312 (8) mM.
Experimental
Chemistry
Compounds 24–27 were purchased from Sigma-Aldrich
s.r.l. (Milan, Italy); 1H NMR spectra were obtained
with a Varian VX 300 MHz spectrometer. UV spectra
were recorded on a Varian Cary1E UV–Visible spec-
trophotometer. Thin-layer chromatography (TLC) was
carried out on precoated TLC plates with silica gel 60
F-254 (Merck). For column chromatography, silica gel
60 (Merck) was used. Elemental analysis were per-
formed on a Fisons Instruments EA 1108 apparatus and
results are within ꢁ0.4% of theoretical values.
More in detail, purine derivatives (5a–14, 23–27) data
clearlyshow that a chlorine atom in the 2 position
improves antireplicative activity, without a parallel
increase in cytotoxicity (5b vs 6b, 7 vs 8, 9 vs 10, 13 vs
14). Also the size of the cycloalkyl substituent in N6
modulates activity, with larger (cycloheptyl) ring bring-
ing about in most cases higher efficacythan smaller
(cyclopropyl) one (5b vs 5a, 13 vs 11, 16 vs 15).
Dideoxyribose clearly showed to be the sugar of choice
for inhibiting viral replication (5a, 5b, 6a, 6b, vs 7–10,
and vs 11–14, 23 vs 25, 24 vs 26).
2,6-Dichloro-9-(2,3-dideoxy-ꢀ-D-glyceropentofuranosyl)-
9H-purine (3)and 2,6-dichloro-3-(2,3-dideoxy- ꢁ-D-gly-
ceropentofuranosyl)-9H-purine (4). To a suspension of
1.00 g (5.29 mmol) of 2,6-dichloropurine (1) in 20 mL of
hexamethyldisilazane (HMDS) was added a catalytic
amount of (NH4)2SO4 and the mixture was heated at
reflux for 5 h. The excess solvent was evaporated, the
residue coevaporated three times with 15 mL of dry
toluene, and then dissolved in 20 mL of drymethylene
Looking at deazapurine derivatives (15–22, 28–31), 1-
deazanucleosides possess higher antireplicative activity
than 3-deaza analogues (17 vs 18, 19 vs 21, 29 vs 31).
chloride.
2,3-Dideoxy-1-O-methyl-5-O-(4-methylben-
zoyl)-d-glycero-pentofuranoside (2)26 (1.524 g, 5.29
mmol) was added, the solution cooled on ice, and then
1.0 mL of TMS–triflate was added. The clear solution
was stirred at rt overnight, added of 10 mL of a cold
saturated solution of Na2CO3, then extracted with
CH2Cl2 (3ꢂ20 mL). Organic layers were reunited,
washed with cold water, dried (Na2SO4), filtered, and
evaporated. The residue was purified on a flash chro-
matographycolumn, eluting with a gradient of ethly
acetate in cyclohexane (15–85 to 20–80, v/v). Evapora-
tion of the fractions containing the products afforded
0.785 g (1.93 mmol, 36%) of 3 (a anomer) and 0.523 g
(1.28 mmols, 24%) of 4 (b anomer) as chromato-
graphicallypure thick oils.
0
Preferred sugar moietyis the 3 -deoxyribose (19 vs 16,
29 vs 28). As alreadyshown for purine derivatives, both
N6-cycloalkyl substitution (16 vs 28, 20 vs 30, 21 vs 31)
and a chlorine atom in the 2 position (19 vs 20, 29 vs 30)
are able to increase activityof nucleosides.
The most active compounds proved to be 5b (2-chloro-
N6-cycloheptyl-20,30-dideoxyadenosine) and 24 (20,30-
dideoxyadenosine, when tested in presence of the ADA
inhibitor EHNA), with a Ki for both of 27 nM. Those
nucleosides show also the highest CD50/ED50 ratio, of
4630 and 6667, respectively. Furthermore, compound
24, when tested without ADA inhibitor, is about 15
times less active as anti-replicative drug (EC50=410
nM), demonstrating that the inosine derivative formed
byaction of ADA possess a lower abilityin inhibiting
virus replication.
(3): UV lmax 274 (EtOH, e 5500), 273 (pH=1, e 5850),
1
273 (pH=12, e 6300); H NMR (DMSO-d6): d 2.01 (m,
1H, CH2), 2,21 (m, 1H, CH2), 2.36 (s, 3H, CH3), 2.48–
2.70 (m, 2H, CH2), 4.17–4.40 (m, 2H, CH2-50), 4.78 (m,
1H, H-40), 6.49 (dd, 1H, J1=5.8 Hz, J2=4.2 Hz, H-10),
7.37 (d, 2H, J=8.1 Hz, 2 Ar–H), 7.91 (d, 2H, J=8.1
Hz, 2 Ar–H), 8.92 (s, 1H, H-8). Anal. calcd C 53.09, H
3.96, N 13.76; found C 52.73, H 3.81, N 13.88.
Conclusion
We have synthesized new N6-cycloalkyl-20,30-dideoxy-
derivatives of adenosine and tested them, together with
other numerous purine nucleoside analogues, on BHV-1
(bovine herpes virus, verysimilar to human herpes
viruses) and MVV (maedi-visna virus, sheep retrovirus,
animal model for HIV) replication. Biological data
demonstrated that tested compounds are veryactive in
inhibiting MVV replication and possess a moderate
cytotoxicity, resulting in antiviral nucleosides with
cytotoxicity/antireplicative activity ratio in some cases
(4): UV lmax 274 (EtOH, e 5600), 274 (pH=1, e 5800),
273 (pH=12, d 6200); 1H NMR (DMSO-d6): d 2.20 (m,
2H, CH2), 2.35 (s, 3H, CH3), 2.60 (m, 2H, CH2), 4.30–
4.50 (m, 3H, H-40 and CH2-50), 6.32 (dd, 1H, J1=6.9
Hz, J2=2.4 Hz, H-10), 7.24 (d, 2H, J=8.1 Hz, 2 Ar–H),
7.65 (d, 2H, J=8.1 Hz, 2 Ar–H), 8.83 (s, 1H, H-8).
Anal. calcd C 53.09, H 3.96, N 13.76; found C 52.86, H
4.18, N 13.42.

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D. Salvatori et al. / Bioorg. Med. Chem. 10 (2002) 2973–2980
2-Chloro-6-cyclopropylamino-9-(2,3-dideoxy-ꢁ-D-glycero-
pentofuranosyl)-9H-purine (5a). Compound 4 (282 mg,
0.69 mmol) was added of 5 mL of cyclopropylamine.
The solution was stirred at room temperature for 3 h,
and then the excess of amine was evaporated. The resi-
due was added of 20 mL of methanol saturated at 0 ꢃC
with ammonia, and stirred at rt in a tightlyclosed flask
for 3 days. The solvent was then evaporated in vacuo,
and the residue chromatographed on a silica gel col-
umn, eluting with CHCl3–CH3OH–CH3CN (95:2.5:2.5,
v/v). Evaporation of the fractions containing the pro-
duct afforded 180 mg (0.58 mmol, 84%) of 5a as chro-
matographicallypure vitreous solid.
C 56.71, H 6.22, N 25.44; found C 56.59, H 6.18, N
25.16.
6-Cycloheptylamino-9-(2,3-dideoxy-ꢁ-D-glyceropentofur-
anosyl)-9H-purine (6b). Compound 5b (150 mg, 0.41
mmol) was dissolved in 20 mL of ethanol and added of
1.5 mL of NaOH 2 N and a catalytic amount of Pd/C
10%; the suspension was stirred in a Parr apparatus
under an atmosphere of H2 (50 psi) for 7 h. The catalyst
was filtered off, the solvent removed in vacuo, and the
residue chromatographed on a silica gel column, eluting
with CHCl3-CH3OH–CH3CN (95:2.5:2.5, v/v). Evap-
oration of the fractions containing the product afforded
85 mg (0.26 mmol, 63%) of 6b as chromatographically
pure vitreous solid.
1H NMR (DMSO-d6): d 0.58–0.83 (m, 4H, CH2–CH2
cycloprop.), 2.04 (m, 2H, CH2 rib.), 2.37 (m, 2H, CH2
rib.), 2.99 (m, 1H, CH cycloprop.), 3.42–3.69 (m, 2H,
CH2-50), 4.12 (m, 1H, H-40), 4.96 (t, 1H, J=5.5 Hz,
OH), 6.18 (dd, 1H, J1=6.5 Hz, J2=3.7 Hz, H-10), 8.40
(s, 1H, H-8), 8.45 (br s, 1H, NH). Anal. calcd C 50.41,
H 5.21, N 22.61; found C 50.08, H 5.18, N 22.33.
1H NMR (DMSO-d6): d 1.40–2.08 (m, 14H, 12H cyclo-
hept. and CH2 rib.), 2.42 (m, 2H, CH2 rib.), 3.42–3.65
(m, 2H, CH2-50), 4.11 (m, 1H, H-40), 4.26 (m, 1H, CH-
cyclohept.), 5.06 (t, 1H, J=6.0 Hz, OH), 6.22 (t, 1H,
J=5.0 Hz, H-10), 7.56 (d, 1H, J=8.4 Hz, NH), 8.18 (s,
1H, H-2), 8.34 (s, 1H, H-8). Anal. calcd C 61.61, H 7.60,
N 21.13; found C 61.30, H 7.91, N 20.87.
2-Chloro-6-cycloheptylamino-9-(2,3-dideoxy-ꢁ-D-glycero-
pentofuranosyl)-9H-purine (5b). Compound 4 (310 mg,
0.76 mmol) was added of 5 mL of cycloheptylamine.
The solution was stirred at room temperature for 3 h,
then the excess of amine was evaporated. The residue
was added of 20 mL of methanol saturated at 0 ꢃC with
ammonia, and stirred at 40 ꢃC in a tightlyclosed flask
for 24 h. The solvent was then evaporated in vacuo, and
the residue chromatographed on a silica gel column,
eluting with CHCl3–CH3OH–CH3CN (97:1.5:1.5, v/v).
Evaporation of the fractions containing the product
afforded 188 mg (0.51 mmol, 67%) of 5b as a
chromatographicallypure vitreous solid.
Biology
Chemicals. All the chemicals used were reagent’s
grade or better. Eagle’s minimum essential medium
(EMEM) and Dulbecco’s minimum essential medium
(DMEM) were from Biospa Italia; antibiotics were from
Sigma.
Foetal calf serum was from BioWhittaker (USA); Try-
pan blue and MTT were from Sigma (USA); microtiter
plates from Nunc (Denmark).
1H NMR (DMSO-d6): d 1.35–2.13 (m, 14H, 12H cylo-
hept. + CH2 rib.), 2.36 (m, 2H, CH2 rib.), 3.44–3.70 (m,
2H, CH2-50), 4.11 (m, 1H, H-40), 4.82 (m, 1H, CH cylo-
hept.), 4.96 (t, 1H, J=5.5 Hz, OH), 6.16 (dd, 1H,
J1=6.3 Hz, J2=3.7 Hz, H-10), 8.20 (d, 1H, J=8.5 Hz,
NH), 8.38 (s, 1H, H-8). Anal. calcd C 55.81, H 6.61, N
19.14; found C 55.45, H 6.83, N 18.77.
Virus. MVV: The biological clone of increased neuro-
virulence KV 177231 was used. Stock viruses were pre-
pared in cell line G81092 of sheep chondrocytes. The
viral strain had a titre of 106,50 TCID50/0.025 mL
(TCID50=virus dilution causing 50% of cytopathic
effects).
The sheep chondrocytes cell line G81092 was grown in
DMEM with antibiotics and 5% foetal calf serum.
6-Cyclopropylamino-9-(2,3-dideoxy-ꢁ-D-glyceropentofur-
anosyl)-9H-purine (6a). Compound 5a (140 mg, 0.45
mmol) was dissolved in 20 mL of ethanol and added of
1 mL of NaOH 2 N and a catalytic amount of Pd/C
10%; the suspension was stirred in a Parr apparatus
under an atmosphere of H2 (50 psi) for 5 h. The catalyst
was filtered off, the solvent removed in vacuo, and the
residue chromatographed on a silica gel column, eluting
with CHCl3–CH3OH–CH3CN (90:5:5, v/v). Evapora-
tion of the fractions containing the product afforded 90
mg (0.33 mmol, 73%) of 6a as chromatographically
pure vitreous solid.
BHV-1: Strain IBR-LA32 of BHV-1 at its 52nd passage on
33
Madin and Darbybovine cell line (MDBK) was used.
The viral strain had a titre of 108,50 TCID50/0.025 mL.
MDBK cells were grown in EMEM with antibiotics
enriched with 5% foetal calf serum. The concentration
of serum was reduced to 2% in both culture systems
when cultures were subjected to virus inoculation.
Evaluation of cytotoxicity of tested compounds
1H NMR (DMSO-d6): d 0.58–0.80 (m, 4H, CH2–CH2
cycloprop.), 2.06 (m, 2H, CH2 rib.), 2.41 (m, 2H, CH2
rib.), 3.05 (m, 1H, CH cycloprop.), 3.45–3.70 (m, 2H,
CH2-50), 4.12 (m, 1H, H-40), 5.07 (t, 1H, J=5.6 Hz,
OH), 6.24 (t, 1H, J=5.0 Hz, H-10), 7.94 (br s, 1H,
NH), 8.25 (s, 1H, H-2), 8.37 (s, 1H, H-8). Anal. calcd
Logarithmically growing chondrocyte cells were trypsi-
nized and adjusted to 2ꢂ105 cells/mL in MEM supple-
mented with 2% foetal calf serum. Logarithmically
growing MDBK cells were trypsinized and adjusted to
2ꢂ103 cells/mL in MEM supplemented with 10% foetal
calf serum.

D. Salvatori et al. / Bioorg. Med. Chem. 10 (2002) 2973–2980
2979
Aliquots of 0.1 mL were used to seed each well of 96-
well plates; the plates were then incubated at 37 ꢃC. 24
h later, the culture medium was removed, and fresh
medium containing serial dilutions from 0.5 to 400
mM of each test compound was inoculated using three
wells of the plate for each dilution and 100 mL of
inoculum per well. The plates were incubated at 37 ꢃC
for 7 days.
Cytopathic effects (CPE)inhibition assay.. For this test,
24 h monolayers grown in 96-well plates were also used.
The wells of the plate were inoculated with 1ꢂ104
TCID50 or with 3ꢂ105 TCID50 of BHV-1 or MVV,
respectively, in an inoculum of 0.025 mL per well.
After one h incubation at 37 ꢃC, six wells of the plates
were first inoculated with each of the following con-
centrations of the compounds: 0.005, 0.05, 0.5, 5, 50 mM
in an inoculum of 0.025 mL per well.
To evaluate the cytotoxicity of the compounds under
studythe following methods were applied.
Then, 0.05 mL of the culture medium was added to each
well and the plates were incubated at 37 ꢃC. The reading
were made when the CPE were complete in the controls,
that is after 5 (BHV-1) or 7 (MVV) days of incubation.
Light microscopy. Monolayers were prepared as above
and incubated for 7 days with different concentrations
of the compounds prepared as mentioned. Then the
cells were fixed with ethanol and stained with the
Giemsa method to facilitate the visualisation of cell
morphology.
The antiviral activities of the compounds were expressed
as the compound concentration that caused a 50%
reduction of cytopathology (50% effective concentration,
EC50).
MTT method. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT; Thiazol blue) is a water-
soluble tetrazolium salt yielding a yellowish solution
when prepared in media or salt solutions lacking phenol
red. Dissolved MTT is converted to an insoluble purple
formazan bycleavage of the tetrazolium ring bydehy-
drogenases. This water insoluble formazan was solubi-
lized using isopropanol and the dissolved material was
characterised spectrophotometricallyieylding absor-
bance as a function of concentration of converted dye.
In contrast to dead cells, active mitochondrial dehy-
drogenases of living cells will cause this conversion.34
The cells were prepared and compounds included as
described above. A solution was prepared bydissolving
5 mg of MTT in 1 mL of medium without phenol red.
The solution was filtered through a 0.2 mm filter and
stored at 2–8 ꢃC. The MTT solution was added to each
culture in a volume of 100 mL per well and the plate was
incubated at 37 ꢃC for 3–4 h. At the end of the incu-
bation period, the medium was removed and the con-
verted dye was solubilized in acidic isopropanol (0.04–
0.1 N HCl in absolute isopropanol). Absorbance of
converted dye was measured at wavelength of 540 nm
with background subtraction at 690 nm.
Acknowledgements
The authors wish to thank Prof. G. Castrucci (Labora-
toryof virology‘V. Cilli’, Universityof Perugia, Italy)
for providing continuous stimulating discussion and
exchange of reagents and ideas. Authors wish also to
thank mrs Natalina Cammertoni for excellent technical
assistance. Financial support byUniversityof Camerino
(Fondo di Ricerca d’Ateneo) and European Community
(COST action no. D13/0009/00) is gratefullyacknowl-
edged.
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