Synthesis and antiviral properties of novel analogues of monophosphate and diphosphate bioactive forms of acyclovir

Article abstract of DOI:10.1016/S0014-827X(00)00046-X

New analogues (compounds 6, 7 and 9) of the mono- (8) and diphosphate (10) bioactive forms of the antiherpes drug acyclovir are described. In compound 6, the monophosphate moiety of 8 was replaced by an aminosulfonyloxy group, while in compounds 7 and 9,

Full text of DOI:10.1016/S0014-827X(00)00046-X

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 322–327
New Compounds
Synthesis and antiviral properties of novel analogues of
monophosphate and diphosphate bioactive forms of acyclovir
Marco Macchia a,*, Guido Antonelli b, Simone Bertini a, Federico Calvani a,
Valeria Di Bussolo c, Filippo Minutolo a, Ramon Tesoro d, Imperio Tonetti a,
Ombretta Turriziani d
a Dipartimento di Scienze Farmaceutiche, Uni6ersita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy
b Dipartimento di Biomedicina, Uni6ersita` di Pisa, Via San Zeno 39, 56127 Pisa, Italy
c Dipartimento di Chimica Bioorganica e Biofarmacia, Uni6ersita` di Pisa, Via Bonanno 33, 56126 Pisa, Italy
d Istituto di Virologia, Uni6ersita` La Sapienza, Viale di Porta Tiburtina 28, 00185 Rome, Italy
Received 15 December 1999; accepted 8 March 2000
Abstract
New analogues (compounds 6, 7 and 9) of the mono- (8) and diphosphate (10) bioactive forms of the antiherpes drug acyclovir
are described. In compound 6, the monophosphate moiety of 8 was replaced by an aminosulfonyloxy group, while in compounds
7 and 9, a phosphonoacetamidoxy and an O-ethyl phosphonoacetamidoxy moiety are, respectively present instead of the
diphosphate one of 10. None of the compounds synthesized proved to possess an appreciable activity on herpes simplex virus
(HSV) or human immunodeficiency virus (HIV). © 2000 Elsevier Science S.A. All rights reserved.
Keywords: Antiviral; HSV-1; HIV-1; Acyclovir-monophosphate; Acyclovir-diphosphate
cially against the resting viral cells which possess only
one low level of kinase [7,8]; nucleotide mono- di- and
1. Introduction
triphosphate themselves would partially or totally by-
The nucleoside analogues currently used in therapy
pass this requirement for intracellular activation, but
against herpes simplex virus (HSV) or human im-
they are poor drug candidates as they are metabolically
munodeficiency virus (HIV) infections, such as acy-
unstable and become degraded rapidly to the parent
clovir (ACV) [1], ganciclovir (DHPG) [2] or Zidovudine
nucleosides.
(AZT) [3], Lamivudine (3TC) [4] and Stavudine (D4T)
[5] are not active per se: their antiviral activity is due to
For this reason, over the last few years various
chemically and metabolically stable analogues of nucle-
their transformation to the corresponding triphosphate
otide and acyclonucleotide mono-, di- and triphosphate
bioactive forms, which are formed through three dis-
have been studied as a good approach for antiviral
tinct intracellular phosphorylation steps by the action
therapy against HSV and HIV. Examples of this class
of cellular kinases. The resulting triphosphate nucle-
include monophosphate acyclonucleotide analogues
otide analogues then block viral replication through the
such as 9-[(3-hydroxy-2-(phosphonylmethoxy)propyl]-
inhibition of enzymes such as DNA polymerases or
adenine (HPMPA) [9], and 9-[2-(phosphonylmethoxy)-
reverse transcriptases [1,6].
ethyl]adenine (PMEA) [10], in which the monophos-
The process of activation to triphosphates represents
phate moiety of the metabolite of acyclonucleotides is
the limiting step for the efficacy of such drugs, espe-
replaced by a stable phosphonomethyloxy group.
HPMPA and PMEA proved to possess a broad-spec-
trum anti-HSV activity and a potent anti-HIV activity,
respectively (Fig. 1).
* Corresponding author. Tel.: +39-050-500209; fax: +39-050-
40517.
E-mail address: nmacchia@farm,unipi.it (M. Macchia).
0014-827X/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(00)00046-X

M. Macchia et al. / Il Farmaco 55 (2000) 322–327
323
In previous papers, we described the synthesis and
the antiviral properties of compounds 1, 2 and 4
[11,12], designed as stable analogues of the mono- (3)
and diphosphate (5) metabolites of 5-iodo-2%-de-
oxyuridine (IDU), a drug actually used in therapy
against HSV whose activity is due to its bioactive
triphosphate form.
In particular, while compound 4, in which a phos-
phonoacetamido moiety is present instead of the
diphosphate one of 5, was practically devoid of any
appreciable antiviral activity, compounds 1 and 2, in
which the mono- and diphosphate moiety of 3 and 5
were replaced, respectively by an aminosulfonyloxy and
aminosulfonylaminocarbonyloxy moiety, proved to
possess an appreciable activity against herpes virus type
1 (HSV-1), even if slightly lower than that of IDU. The
latter results indicated that the aminosulfonyloxy and
aminosulfonylaminocarbonyloxy moieties of 1 and 2
might be considered, at least for HSV-1, as partial
bioisosters, respectively of the mono- and diphosphate
portions of the metabolite of IDU 3 and 5 (Fig. 2).
As a part of our search for new chemically and
metabolically stable analogues of nucleotide and acy-
clonucleotide mono-, di- and triphosphate, we here
report the synthesis and the antiviral properties of new
analogues (compounds 6, 7 and 9) of the mono- (8) and
diphosphate (10) bioactive forms of the antiherpes drug
acyclovir. In particular, compound 6, the monophos-
phate moiety of 8 was replaced by an aminosulfonyloxy
group, as in the case of the previously reported stable
analogue 1 of IDU-monophosphate 3, while com-
pounds 7 and 9 contain, respectively a phosphonoac-
etamidoxy and an O-ethyl phosphonoacetamidoxy
moiety instead of the diphosphate one of 10. The choice
to use a phosphonoacetamidoxy and an O-ethyl phos-
phonoacetamidoxy portion as potential bioisosters of
the diphosphate moiety of 10, was based on our previ-
ous studies in the field of antitumour agents [13–15] in
which we found that, in the case of a series of ana-
logues of geranylgeranyldiphosphate, an intermediate
of the cholesterol biosynthetic pathway involved in the
isoprenylation of RAS superfamily GTP-binding
proteins such as Rho, Rac and CdC42, these moieties
were able to act as stable isosters of the diphosphate
group (Fig. 3).
Fig. 1.
2. Chemistry
The aminosulfonyl derivative 6 was synthesized as
reported in Scheme 1. Reaction of 2-benzoylamino-9-
(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one (11)
[16] with sodium hydride and aminosulfonyl chloride in
anhydrous DMF afforded the intermediate 12, which
was then treated with a saturated methanolic solution
of ammonia, to obtain the N-debenzoylated final
product 6.
Phosphonic derivatives 7 and 9 were prepared using
the synthetic procedure shown in Scheme 2. 2-Benzoy-
lamino-9-[2-(phthalimidoxy)ethoxymethyl]-1,9-dihydro-
purin-6-one (13) [16] was subjected to hydrazinolysis to
give the aminoxy derivative 14, which was then treated
with diethylphosphonoacetic acid in the pres-ence of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hy-
drochloride (EDC) and 1-hydroxybenzotriazole
Fig. 2.
(HOBt), affording the phosphonated intermediate 15.

324
M. Macchia et al. / Il Farmaco 55 (2000) 322–327
The O-ethylphosphonoacetamidoxy derivative 9 was
directly obtained from intermediate 15 by alkaline hy-
drolysis with NaOH in dioxane.
3. Biological results and conclusions
Compounds 6, 7 and 9 were tested for their antiviral
activity against herpes virus type 1 (HSV-1), using
VERO cells infected with the HSV-1 strain HF and for
their HIV-1 inhibitory activity, using lymphoblastoid
CD4+ cells (C8166), infected with the HIV strain
HTLV-IIIB derived from chronically infected H9 cells.
At a concentration of 50 mM all the compounds were
found to be practically devoid of any appreciable an-
tiviral activity on both HSV-1 and HIV-1.
These data indicate that the aminosulfonyloxy moi-
ety of compound 6, which was used as a potential
bioisoster of the monophosphate portion of the bioac-
tive form of ACV 8, is not able to confer any antiviral
activity to ACV, contrary to previous findings in the
case of IDU-monophosphate analogue 1, for which
such type of modification led to an appreciable activity
against herpes virus type 1 (HSV-1), even if slightly
lower than that of IDU.
Furthermore, also the phosphonoacetamidoxy and
the O-ethyl phosphonoacetamidoxy moieties of com-
pounds 7 and 9 were not able to act as bioisosters of
the diphosphate portion of the bioactive form of ACV
10; however the possibility cannot be excluded that the
inactivity found for compounds 7 and 9 might be
ascribed to distribution problems.
4. Experimental
Fig. 3.
4.1. Chemistry
Reaction of 15 with trimethylsilyl bromide (TMSBr)
and collidine followed by treatment with aqueous
sodium hydroxide, led to the removal of the ethyl
groups from the phosphonate moiety of 15 to yield
compound 16. The N-benzoyl group in 16 was removed
by alkaline hydrolysis with sodium hydroxide in diox-
ane, to give the final phosphonoacetamidoxy-substi-
tuted compound 7.
Melting points were determined on a Kofler hot-
stage apparatus and are uncorrected. IR spectra for
comparison of compounds were taken as paraffin oil
mulls or as liquid films on a Mattson 1000 FTIR
1
spectrometer. H NMR spectra of all compounds were
obtained with a Varian Gemini-200 instrument operat-
1
ing at 200 MHz. For H NMR spectra, only the most
Scheme 1. Reagents and conditions. (i) NH2SO2Cl, NaH, DMF, r.t., 48 h; (ii) sat. NH3/MeOH, 0°C, 72 h.

M. Macchia et al. / Il Farmaco 55 (2000) 322–327
325
Scheme 2. Reagents and conditions. (i) NH2NH2, MeOH, r.t., 1 h; (ii) diethylphosphonoacetic acid, EDC, HOBt, DMF, r.t., 24 h; (iii) (a) TMSBr,
collidine, CH2Cl2, r.t., 12 h; (b) aqueous NaOH; (iv) NaOH, dioxane, 30°C, 24 h.
significant details are reported. Mass spectra were
recorded on a VG 70-250S mass spectrometer. Analyti-
cal TLCs were carried out on 0.25-mm layer silica gel
plates containing a fluorescent indicator; spots were
detected under UV light (254 nm). Column chro-
matographies were performed using 230–400 mesh sil-
ica gel (Macherey-Nagel Silica Gel 60 Art. no. 815381).
Sodium sulfate was always used as the drying agent.
Evaporations were performed in vacuo (rotating
evaporator).
(s, 1H), 11.96 (s, 1H), 8.21 (s, 1H), 8.14–7.50 (m, 7H),
5.52 (s, 2H), 4.55–4.38 (m, 2H), 3.85–3.70 (m, 2H). MS
(FAB): m/z 409 [M+H]+.
4.1.2. Synthesis of 2-amino-9-(2-aminosulfonyloxy-
ethoxymethyl)-1,9-dihydropurin-6-one (6)
A suspension of compound 12 (0.30 g, 0.73 mmol) in
a saturated solution of ammonia in methanol (6 ml)
was stirred at 0°C for 72 h. The solvent was then
removed under vacuum and the residue was recrystal-
lized from water to give compound 6 as a white solid
1
4.1.1. Synthesis of 2-benzoylamino-9-(2-amino-
(0.085 g, 0.28 mmol, 38% yield). H NMR (200 MHz,
sulfonyloxyethoxymethyl)-1,9-dihydropurin-6-one (12)
A solution of compound 11 [16] (1.40 g, 4.25 mmol)
in anhydrous DMF (85 ml) was treated with 0.51 g
(12.8 mmol) of NaH under nitrogen and stirred at 0°C
for 15 min. Then, a solution of aminosulfonyl chloride
(1.22 g, 10.6 mmol) in anhydrous THF (15 ml) was
added and stirring was continued for 48 h at room
temperature. The excess of NaH was quenched with
EtOH (2 ml) and the resulting suspension was concen-
trated to give a solid which was recrystallized from
MeOH, yielding 1.18 g (2.89 mmol, 68% yield) of 12 as
Py-d5): l 9.58 (s, 2H), 8.16 (s, 1H), 7.98-7.89 (br, 2H),
5.67 (s, 2H), 4.74–4.68 (m, 2H), 4.10–4.05 (m, 2H). MS
(FAB): m/z 305 [M+H]+.
4.1.3. Synthesis of 2-benzoylamino-9-(2-aminoxy-
ethoxymethyl)-1,9-dihydropurin-6-one (14)
A solution of compound 13 [16] (5.19 g, 10.9 mmol)
in methanol (500 ml) was treated with hydrazine mono-
hydrate (0.53 ml, 11 mmol), and the resulting mixture
was stirred for 1 h at room temperature. The solvent
was removed under vacuum and the residue was
1
a white solid. H NMR (200 MHz, DMSO-d6): l 12.35

326
M. Macchia et al. / Il Farmaco 55 (2000) 322–327
1
purified by column chromatography on silica gel, using
a 30:10:1 mixture of CHCl3/MeOH/NH3 as the eluant,
yielding 14 as a white solid (1.69 g, 4.91 mmol, 45%
yield). 1H NMR (200 MHz, DMSO-d6): l 12.32 (s, 1H),
11.92 (s, 1H), 8.20–7.51 (m, 6H), 5.62 (s, 2H), 4.03 (br,
2H), 3.57 (s, 4H). MS (FAB): m/z 345 [M+H]+.
disodic salt (0.015 g, 0.037 mmol, 77% yield). H NMR
(200 MHz, DMSO-d6): l 11.98 (s, 1H), 8.13–7.78 (m,
4H), 5.55 (s, 2H), 3.61–3.44 (m, 4H), 2.46 (d, JHP=20
Hz, 2H). MS (FAB, anionic): m/z 383 [M−Na]−, 180
[M−2Na]2−
.
4.1.7. Synthesis of the monosodium salt of 2-amino-
9-[2-(O-ethylphosphonoacetamidoxy)ethoxymethyl]-1,9-
dihydropurin-6-one (9)
4.1.4. Synthesis of 2-benzoylamino-9-[2-(O,O%-
diethylphosphonoacetamidoxy)ethoxymethyl]-1,9-
dihydropurin-6-one (15)
A solution of 15 (0.200 g, 0.383 mmol) in dioxane (10
ml) was treated with a 1 N aqueous solution of NaOH
(2.0 ml, 2.0 mmol) and the resulting reaction mixture
was stirred at 30°C for 24 h. The solvent was then
removed under vacuum and the crude product was
purified by reverse-phase column chromatography on
Rp18-coated silica gel, using water as the eluant, to
obtain 9 as the monosodic salt (0.099 g, 0.24 mmol,
62% yield). 1H NMR (200 MHz, DMSO-d6): l 11.91 (s,
1H), 8.15–7.81 (m, 4H), 5.54 (s, 2H), 3.93 (q, J=7.2
Hz, 2H), 3.60–3.45 (m, 4H), 2.19 (d, JHP=20 Hz, 2H),
1.24 (t, J=7.2 Hz, 3H). MS (FAB, anionic): m/z 389
[M−Na]−.
A solution of diethylphosphonoacetic acid (0.063 g,
0.33 mmol) and HOBt (0.043 g, 0.32 mmol) in anhy-
drous DMF (13 ml) was stirred at 0°C for 30 min.
Compound 14 (0.10 g, 0.29 mmol) and EDC hy-
drochloride (0.061 g, 0.32 mmol) were then added to
this solution. The reaction mixture was then stirred at
room temperature for 24 h. The solvent was removed
under vacuum and the oily residue was purified by
column chromatography on silica gel (CHCl3/MeOH
9:1), yielding product 15 as a white solid (0.14 g, 0.26
1
mmol, 90% yield). H NMR (200 MHz, DMSO-d6): l
12.36 (s, 1H), 11.91 (s, 1H), 8.21–7.48 (m, 7H), 5.54 (s,
2H), 4.27–3.87 (m, 4H), 3.58–3.43 (m, 4H), 2.90 (d,
J
HP=22 Hz, 2H), 1.24 (t, J=6.8 Hz, 6H). MS (FAB):
4.2. Assay of anti-HSV-1 acti6ity
m/z 523 [M+H]+.
The HSV-1 strain HF, obtained from ATCC (Man-
assas VA), was used to infect VERO cells. These cells
were maintained in MEM supplemented with 10% fetal
calf serum, glutamine and gentamicin. RS cells, used to
backtitrate the virus, were maintained in MEM supple-
mented with 10% fetal calf serum and sodium pyruvate.
VERO and RS cells were originally purchased from
Istituto Zooprofilattico Sperimentale della Lombardia e
dell’Emilia (Brescia, Italy).
The antiviral activity of compounds was evaluated in
terms of inhibition of viral yield in the presence of the
drugs. Briefly VERO cells were infected with HSVHF at
a multiplicity of infection (MOI) of 0.05, and treated
with different dilutions of compounds. After 48 h, the
virus released in the supernatant was harvested and
then titrated on RS cells by the standard limiting
dilution method (0.5 log ratio, three replicates per
dilution) on 96-well microtitre plates. The infectious
titre was calculated by the method of Reed and
Muench [17].
4.1.5. Synthesis of the disodium salt of
2-benzoylamino-9-[2-(phosphonoacetamidoxy)-
ethoxymethyl]-1,9-dihydropurin-6-one (16)
A solution of 15 (0.090 g, 0.17 mmol) in anhydrous
dichloromethane (1 ml) was treated under nitrogen with
trimethylsilyl bromide (0.14 ml, 1.0 mmol) and 2,4,6-
collidine (0.05 ml, 0.3 mmol). The resulting solution
was stirred for 12 h at room temperature. The solvent
was removed under vacuum and the residue was treated
with a solution of NaOH (0.052 g, 1.3 mmol) in water
(3 ml) for 30 min under stirring. Final removal of water
under vacuum afforded a crude product which was
purified by reverse-phase column chromatography on
Rp18-coated silica gel, using H2O/MeOH 95:5 as the
eluant, to give 16 as the disodic salt (0.077 g, 0.15
1
mmol, 91% yield). H NMR (200 MHz, DMSO-d6): l
12.38 (s, 1H), 11.95 (s, 1H), 8.24–7.50 (m, 7H), 5.57 (s,
2H), 3.61–3.45 (m, 4H), 2.48 (d, JHP=20 Hz, 2H). MS
(FAB, anionic): m/z 487 [M−Na]−, 232 [M−2Na]2−
.
4.1.6. Synthesis of the disodium salt of 2-amino-9-
[2-(phosphonoacetamidoxy)ethoxymethyl]-1,9-
dihydropurin-6-one (7)
4.3. Assay of anti-HIV-1 acti6ity
A solution of 16 (0.026 g, 0.048 mmol) in dioxane
(1.3 ml) was treated with a 1 N aqueous solution of
NaOH (0.3 ml, 0.3 mmol) and the resulting mixture
was stirred at 30°C for 24 h. The solvent was removed
under vacuum and the crude product was purified by
reverse-phase column chromatography on Rp18-coated
silica gel using water as the eluant, to give 7 as the
The HTLV-IIIB strain of HIV-1 was derived from
chronically infected H9 cells. Acute infection with HIV-
1 was carried out in the CD4+ lymphoblastoid cell
line C8166, containing the HTLV-I genome and ex-
pressing only the tax gene [18]. These cells were main-
tained in RPMI supplemented with 10% fetal calf
serum and gentamicin.

M. Macchia et al. / Il Farmaco 55 (2000) 322–327
327
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cells by the standard limiting dilution method (0.5 log
ratio, three replicates per dilution) on 96-well microtitre
plates. The infectious titre, expressed as tissue culture
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