Acyclic nucleoside phosphonates with a branched 2-(2-phosphonoethoxy)ethyl chain: Efficient synthesis and antiviral activity

Article abstract of DOI:10.1016/j.bmc.2011.06.045

Series of novel acyclic nucleoside phosphonates (ANPs) with various nucleobases and 2-(2-phosphonoethoxy)ethyl (PEE) chain bearing various substituents in β-position to the phosphonate moiety were prepared. The influence of structural alternations on anti

Full text of DOI:10.1016/j.bmc.2011.06.045

Bioorganic & Medicinal Chemistry 19 (2011) 4445–4453
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Acyclic nucleoside phosphonates with a branched 2-(2-phosphonoethoxy)ethyl
chain: Efficient synthesis and antiviral activity
Dana Hocková a, , Antonín Holy , Graciela Andrei , Robert Snoeck , Jan Balzarini
⇑
a
b
b
b
´
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, CZ-16610 Prague 6, Czech Republic
b Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of novel acyclic nucleoside phosphonates (ANPs) with various nucleobases and 2-(2-phosphono-
ethoxy)ethyl (PEE) chain bearing various substituents in b-position to the phosphonate moiety were pre-
pared. The influence of structural alternations on antiviral activity was studied. Several derivatives
exhibit antiviral activity against HIV and vaccinia virus (middle micromolar range), HSV-1 and HSV-2
(lower micromolar range) and VZV and CMV (nanomolar range), although the parent unbranched
PEE–ANPs are inactive. Adenine as a nucleobase and the methyl group attached to the PEE chain proved
to be a prerequisite to afford pronounced antiviral activity.
Received 21 April 2011
Revised 14 June 2011
Accepted 15 June 2011
Available online 22 June 2011
Keywords:
Acyclic nucleoside phosphonates
Drug design
Ó 2011 Elsevier Ltd. All rights reserved.
Antiviral activity
Purines
Pyrimidines
1. Introduction
Further structure-activity relationship studies in the series of
PMEA congeners included modification of the parent molecule
Acyclic nucleoside phosphonates (ANPs) possess significant anti-
viral and cytostatic activity.1 These nucleotide analogs contain an
isopolar phosphonomethyl moiety instead of the nucleotide phos-
phate ester group which excludes their enzymatic degradation
and/or eliminates problems linked to intracellular phosphorylation
necessary for nucleoside activation. They are excellent templates for
drug design because of the absence of the labile glycosidic bond and
the flexibility of the acyclic chain which enables the compounds to
adopt a suitable conformation in their target enzyme. Among ANPs,
2-(phosphonomethoxy)alkyl derivatives of purine and pyrimidine
bases are particularly potent. 9-[2-(Phosphonomethoxy)ethyl]ade-
nine (PMEA, Fig. 1) is active against DNA viruses and retroviruses2;
its prodrug, adefovir dipivoxil3 has been approved for hepatitis B
virus therapy (Hepsera™).4 Attachment of the hydroxymethyl
group to the 2-(phosphonomethoxy)ethyl chain leads to another ac-
tive compound, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]
adenine ((S)-HPMPA, Fig. 1), with potent anti-DNA-viral activity.5
Substitution of the aliphatic PMEA side chain by the methyl group
results in (R)-9-[2-(phosphonomethoxy)propyl] adenine (PMPA,
Fig. 1) with high potency and selectivity against HIV-1 and
HIV-26; its oral prodrug tenofovir disoproxil fumarate has been
approved for treatment of HIV infection (Viread™) and recently also
HBV infection.6c
both in the side chain (e.g., unsaturated or aryl ANPs)7 and in the
heterocyclic moiety. They demonstrated that the margins of struc-
tural alteration are very narrow. Except for the antiviral activity of
the cytosine derivative (S)-HPMPC (Vistide™),6 the choice of the
base is limited mostly to adenine, guanine and 2,6-diaminopurine,
and to their 8-aza and 3-deaza congeners. The specificity of antivi-
ral action is predominantly determined by the structure of the side
chain.
ANPs with the chain elongated by CH2 group – 2-(phosphono-
ethoxy)ethyl (PEE) compounds8 – do not exhibit any significant
antiviral activity, but their 6-oxopurine derivatives (PEEG and
PEEHx, Fig. 1) posses anti-malarial activity. They inhibit hypoxan-
thine-guanine-xanthine phosphoribosyltransferase (HGXPRT), the
NH2
NH2
N
N
N
N
N
N
N
N
OH
R
O P OH
O
P
OH
OH
α
O
O
β
PMEA:
(R)-PMPA: R = CH3
(S)-HPMPA: R = CH2OH
R = H
PEEG: Z=NH2
PEHx: Z=H2
⇑
Corresponding author.
E-mail address: lasice@uochb.cas.cz (D. Hocková).
Figure 1.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.045

4446
D. Hocková et al. / Bioorg. Med. Chem. 19 (2011) 4445–4453
key enzyme of the purine salvage pathway essential for the repli-
The resulting intermediate 1 was directly alkylated by ethyl bro-
moacetate under mild conditions to avoid elimination reaction.
The ethyl (alkoxy)acetate moiety in compound 2 can serve as a
latent hydroxyethyl group. These esters 2 were reduced by bor-
ane in THF to obtain desired hydroxy derivatives 3 with the al-
ready built-in ether bond. The overall yield of the three step
sequence was 69% for 3a and 58% for 3d.
cation and survival of the parasite Plasmodium falciparum, with Ki
values of 0.1 (PEEG) and 0.3 (PEEHx) l
M, respectively.9 To investi-
gate the effect of branching of the phosphonoethoxyethyl chain on
selectivity and ability to inhibit PfHGXPRT or human HGPRT, sub-
stituents were added either to the carbon attached directly to the
phosphorus atom (
a-branched derivatives) or to the adjacent car-
bon (b-branched derivatives). While the
a
-branched PEE-6-oxopu-
The resulting alcohols 3a–3c10 and 3d were introduced under
Mitsunobu conditions into the 9-position of the purine or the 1-po-
sition of pyrimidine bases (Scheme 1). Thus 6-chloropurine was
alkylated by alcohols 3a–3d and 2-amino-6-chloropurine by 3a
and 3d to form ANP diethyl esters 4a–4d and 5a,d, respectively
(details of the synthesis of 4a–4c and 5a were given in our previous
paper9). The same procedure followed by base-deprotection was
used for alkylation of 3-benzoylthymine and 3-benzoyluracil to
form the pyrimidine derivatives 6a,d and 7a,d, respectively. As ex-
pected from our previous experience, the yields of the Mitsunobu
reaction were moderate (45–70%).
Further procedures in the purine series (Scheme 2) were based
on a modification of the 6-position in heterocyclic moiety followed
by cleavage of the esters groups under the standard conditions (1.
Me3SiBr/acetonitrile, 2. hydrolysis).
Thus heating of 6-chloropurine derivatives 4a–4d in methanolic
ammonia in autoclave afforded intermediates 8a–8d. After subse-
quent cleavage of the ethyl groups, adenine b-branched PEE-com-
pounds 9a–9d were obtained. During this reaction the benzyl
group was partially cleaved from 8d and, in addition to 9d, hydroxy
derivative 9e was isolated as the second product.
A routine procedure was applied to hydrolyze halogen in the
6-position of the purine derivatives 4d and 5d to obtain hypoxan-
thine and guanine ANPs. Reaction with 75% aqueous trifluoroacetic
acid gave 6-oxopurine compounds 10d and 11d, respectively, and
sufficient amounts of these products were debenzylated by
hydrogenolysis (Pd/C) to form hydroxymethyl PEE-derivatives
10e and 11e. Finally, diethyl esters 10d,e and 11d,e were trans-
formed to the target free phosphonic acids 12d,e and 13d,e.
The same procedure as for the above mentioned adenine ANPs
was applied for the synthesis of diaminopurine compound 14, in
this case starting from 2-amino-6-chloro derivative 5a.
rines were only very weak inhibitors, some of the b-branched
compounds bind tightly to the enzymes.10,11
Herein, we report on the synthesis and antiviral activity of a ser-
ies of ANPs with a b-branched PEE moiety. To further investigate
the structure–activity relationship in this group of compounds,
the influence of the nucleobase and of various substituents at the
PEE-chain was studied. The prepared ANPs can be divided into
three groups: (A) derivatives 9a–9e with adenine as a base and var-
iant b-substituents; (B) derivatives 12a,10 13a,10 14, 15a, 16a and
18a with 2-(1-phosphonopropan-2-yloxy)ethyl chain and various
purine or pyrimidine bases as analogs of PMP-compounds; (C)
derivatives 12e, 13e, 15e and 16e (and their benzyl-protected
congeners 12d, 13d, 15d and 16d) with various nucleobases and
2-(3-hydroxy-1-phosphonopropan-2-yloxy)ethyl chain as analogs
with a structure motif derived from HPMP-ANPs.
2. Chemistry
Previously published syntheses of branched phosphonates suf-
fered mainly from the elimination reactions. For this extensive
series of b-branched PEE–ANPs we decided to improve our
previously published method10 for the preparation of diethyl
2-(2-hydroxyethoxy)alkylphosphonates 3a–3c. The procedure
was significantly simplified and can provide convenient and rapid
access to a broad spectrum of phosphonate derivatives. The main
improvement compared to our previously published step-vise
procedure10 was the one-pot sequence without the isolation of
the branched alcohol after the first step. Starting from commer-
cially available diethyl methylphosphonate (Scheme 1), diethyl
lithiomethylphosphonate preformed in situ in dry THF under ar-
gon atmosphere was reacted with the corresponding aldehydes.
O
R
O
P
O
P
R
O
P
O
O
O
O
nBuLi/THF
O
O
Br
O
O
O
H3C
LiO
O
O
R
H
2
BH3-THF
O
N
X
R
HN
R
O
P
a: R = Me
PPh3/DIAD/THF
O
O
O
b: R = Et
HO
O
c: R = CH2Ph
d: R = CH2OCH2Ph
for 6: 1-benzoyl thymine
for 7: 1-benzoyl uracil
6a, 6d: X = Me
7a, 7d: X = H
O
O
3
P OEt
OEt
PPh3/DIAD/THF
2-amino-6-chloropurine
PPh3/DIAD/THF
6-chloropurine
Cl
Cl
N
N
N
N
N
N
N
H2N
N
O
O
P OEt
OEt
P OEt
OEt
R
O
5
O
4
R
Scheme 1.

D. Hocková et al. / Bioorg. Med. Chem. 19 (2011) 4445–4453
4447
NH2
Cl
N
N
N
N
N
NH3/MeOH
N
N
N
O
P OY
OY
O
8: Y = Et
9: Y = H
Me3SiBr/CH3CN
P OEt
OEt
O
4
O
R
R
O
N
O
from 4d
TFA/H2O
a: R = Me
b: R = Et
N
N
N
HN
HN
Me3SiBr/CH3CN
c: R = CH2Ph
N
N
Z
d: R = CH2OCH2Ph
e: R = CH2OH
Z
O
O
P OEt
OEt
P OH
OH
O
O
from 5d
TFA/H2O
12d,12e: Z = H
10d, 10e: Z = H
R
R
13d,13e: Z = NH2
11d, 11e: Z = NH2
H2/Pd/C
Cl
NH2
N
N
N
N
N
from 5a
1. NH3/MeOH
N
H2N
N
H2N
N
O
P OEt
OEt
O
P OH
OH
2. Me3SiBr/CH3CN
14
5
O
O
R
CH3
Scheme 2.
O
NH2
X
R
HN
N
1. TPSCl/CH3CN/Et3N
2. NH4OH
O
N
O
N
O
O
O
P OY
OY
O
a: R = Me
P OY
OY
d: R = CH2OCH2Ph
e: R = CH2OH
CH3
6: X = Me, Y = Et
15: X = Me, Y = H
17: Y = Et
Me3SiBr/CH3CN
Me3SiBr/CH3CN
Me3SiBr/CH3CN
18: Y = H
7: X = H, Y = Et
16: X = H, Y = H
Scheme 3.
In the pyrimidine series (Scheme 3) the benzyl derivatives 6d
and 7d, prepared by the Mitsunobu approach, were debenzylated
by hydrogenolysis (Pd/C) to form hydroxymethyl compounds 6e
and 7e, respectively. The uracil methyl-PEE diester 7a was con-
verted into the cytosine derivative 17 by the treatment with
2,4,6-triisopropylbenzenesulfonyl chloride followed by amination
with ammonium hydroxide.12 The diethyl esters derived from
cytosine (17), thymine (6a,e) and uracil (7a,e) were treated with
bromotrimethylsilane and after subsequent hydrolysis the free
phosphonic acids 18, 15a,e and 16a,e were isolated, respectively.
compounds were also evaluated against retroviruses [(i.e., human
immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2)]. All
compounds were also examined against several RNA viruses,
including vesicular stomatitis virus (VSV), Coxsackie B4 virus,
respiratory syncytial virus (RSV), parainfluenza virus type 3, reovi-
rus-1, Sindbis virus and Punta Toro virus. None of the compounds
showed activity against the different RNA viruses, except for HIV-1
and HIV-2.
Compounds 9d,e, 12a–12d, 13a–13c, 14, 15e and 16e proved to
be inactive against herpes- and poxviruses. Compounds, that in
contrast to the parent unbranched PEE–ANPs,8 exhibited at least
moderate anti-herpes and antipoxvirus activities are summarized
in Table 1. The antiviral activity was observed mainly for an ade-
nine as a nucleobase and for a methyl group attached to the PEE
chain. Thus, compound 9a that possesses both features emerged
as the most active b-branched 9-(phosphonoethoxyethyl) ANPs
3. Biological activity
The entitled novel b-branched 9-(phosphonoethoxyethyl) ANPs
9a–9e, 12d,e, 13d,e, 14, 15a,e, 16a,e and 18, as well as previously
published10 guanine (13a–13c) and hypoxanthine (12a–12c)
derivatives, were evaluated against various DNA viruses, including
poxviruses [i.e., vaccinia virus (VACV)], herpesviruses [i.e., herpes
simplex virus type 1 (HSV-1)] and type 2 (HSV-2), thymidine ki-
nase-deficient HSV-1 (acyclovir-resistant, ACVr), varicella-zoster
virus (VZV), and human cytomegalovirus (HCMV) (Table 1). The
with EC50 values in the range of 0.07–2.3
man herpesviruses and of 27 M against vaccinia virus. Although
this compound only altered cell morphology at a concentration
of 322 M, it appeared to be cystostatic for HEL (human embryonic
lung) fibroblasts with a CC50 (concentration inhibiting cell growth
lM against different hu-
l
l

4448
D. Hocková et al. / Bioorg. Med. Chem. 19 (2011) 4445–4453
Table 1
Antiviral and cytotoxic/static activity of the compounds against herpes- and poxviruses in cell culture
Compounds Base R
Antiviral activity: EC50
(
l
M)a
Cytotoxic/static activity
VZV
HSV-1
(KOS)
HSV-2
(G)
HSV-1
Cytomegalovirus Vaccinia Cell
Cell
TK–
virus
Morphology growth
(KOS ACV)
TK+ strains
OKA
TK– strains
07/1 YS/R
AD-169
strain
Davis
strain
Lederle MCC
strain
M)b
CC50
(l
M)c
(
l
YS
9a
9b
9c
12e
13d
13e
15a
16a
A
A
A
Me
Et
CH2Ph
0.10 0.13 0.07 0.04 0.11 0.06 0.07 0.09 1.3
42 19 12 22 18 43 238 111 286 111 251 95 159
2.9 3.4 2.9 2.6 4.7 3.2 2.5 3.1 53
0
2.3 1.40 1.7
0
0.93 0.23 0.90 0.6 27 4.7 322
13 10
>317
P247 27
>315
>240
300
>342
291 76
307
254 200
1,338 484
543 492
243
>317
>265
>315
>240
>300
>342
>360
>361
>317
>265
>315
>240
>300
>342
>360
>361
0
86 47
315
53
0
59 50
>315
>240
165
68 22
>315
>240
165
248
34 1.8
>361
Hx CH2OH
N.D.
88
197
94
>300
48
N.D.
N.D.
N.D.
N.D.
12
208
>240
156
171
43
>315
>240
261
>342
97 36
>361
G
G
T
U
C
CH2OCH2Ph N.D.
CH2OH
Me
Me
Me
109
>300
47
6.3 2.7 13 4.6
130 168
>240
156
154
34 13
>361
N.D.
N.D.
9.9
N.D.
N.D.
188
36 4.1
>361
0
18
N.D.
>361
1.3 0.6
Cidofovir
Acyclovir
Ganciclovir
0.29 0.25 0.10 0.06 N.D.
0.9 0.1
1.0 0.1 0.79 0.51 0.70 0.29 7.3 4.6 >317
3.64 1.24 1.56 0.84 71.6 31.6 76.9 41.3 0.30 0.17 0.23 0.15 7.3 4.6 N.D.
N.D. N.D. N.D. N.D. 0.017 0.01 0.023 0.01 0.12 0.07 5.9 3.1 5.1 2.0 >100
N.D.
>250
>444
>394
N.D.: not determined.
a
Concentration required to reduce virus-induced cytopathicity or viral plaque formation by 50%.
Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology (human embryonic lung (HEL) fibroblasts; for details see Section
b
5).
c
Cytotoxic concentration required to reduce cell growth by 50% (human embryonic lung (HEL) fibroblasts; for details see Section 5).
R
O
P
Nucleobase
OH
OH
O
by 50%) value of 13
l
M. Hence, selectivity indices (ratio CC50:EC50
)
compounds 9b and 9c. As observed for their cytostatic activity
against human fibroblast HEL cell proliferation, when evaluated
against murine leukemia cells (L1210), murine mammary carci-
noma cells (FM3A) and human T-lymphocyte cells (CEM), 9a was
far more cytostatic than 9b and 9c (Table 2).
for compound 9a were of 120–190 for different VZV strains, 14–15
for HCMV, 6–10 for HSV, and <1 for vaccinia virus compared to,
respectively, 880–2540, 195–282, 322–363, and 35 for cidofovir.
Replacement of the Me group by an Et (9b) or CH2Ph (9c) group
in 9a resulted in decreased cytostatic activities (CC50 values of,
respectively, >317 and 247 lM) but also in diminished antiviral
4. Conclusions
activities against herpes- and poxviruses. The benzyl-substituted
derivative showed antiviral activity in between the methyl (most
active) and ethyl (least active) derivatives. Also, replacement of
the methyl by a hydroxymethyl (9e) annihilated the anti-DNA virus
activity. Substitution of the adenine nucleobase in 9a by uracil
(16a), thymine (15a) or cytosine (18) resulted in lower cystostatic
effects but also in a virtually complete loss of antiviral potency.
Among the different b-branched 9-(phosphonoethoxyethyl)
ANPs, only 9a was able to inhibit the replication of HIV-1
The methodology for the synthesis of b-branched acyclic nucle-
oside phosphonates with PEE moiety was significantly improved
and a series of new purine and pyrimidine derivatives were
synthesized. Although the parent unsubstituted PEE–ANPs were
antivirally inactive,8 attachment of a methyl, ethyl or benzyl sub-
stituent to the acyclic aliphatic chain lead to antivirally active com-
pounds, especially in the adenine series. These new results
contribute to the extensive SAR project aimed at the development
of new selective antiviral drugs.
(EC50 = 11 4.0
cultures. Its antiretroviral activity was in the same concentration
range as PMEA (adefovir) (EC50: 7.4 1.7 for HIV-1 and
7.0 1.1 M for HIV-2). However, the cystostatic activity of 9a in
CEM cells (CC50 = 20 6.3 M) may account for the anti-HIV activ-
lM) and HIV-2 (EC50 = 9.0 6.0 lM) in CEM cell
lM
5. Experimental
l
l
Unless otherwise stated, solvents were evaporated at 40 °C/2 kPa,
and the compounds were driedover P2O5 at 2 kPa. NMR spectra were
recorded on Bruker Avance 400 (1H at 400 MHz, 13C at 100.6 MHz)
spectrometers with TMS as internal standard or referenced to the
residual solvent signal. Mass spectra were measured on a ZAB-EQ
(VG Analytical) spectrometer. The chemicals were obtained from
commercial sources (Sigma–Aldrich) or prepared according to the
published procedures. Dimethylformamide and acetonitrile were
distilled from P2O5 and stored over molecular sieves (4 Å). THF was
distilled from sodium benzophenone ketyl under argon. Deionisa-
tion was performed on Dowex 50 ꢀ 8 (H+-form) columns by the fol-
lowing procedure: after application of crude product the column
was washed with wateruntil the UV absorption dropped. Thereafter,
the column was eluted with 2.5% aqueous NH3. Chromatography on
Dowex 1 ꢀ 2 (acetate form) was as follows: after application of the
ity observed and thus, the observed anti-HIV activity might not be
due to a direct inhibition of virus replication, but rather an indirect
cellular antimetabolic activity. Compound 9a also demonstrated a
higher cystostatic activity against the cell cultures compared to
Table 2
Inhibitory effect of the adenine ANP derivatives on the proliferation of murine
leukemia cells (L1210), murine mammary carcinoma cells (FM3A) and human
T-lymphocyte cells (CEM)
Compound
R
CC50
FM3A
2.7 1.3
432
90 37
(
l
M)
L1210
CEM
9a
9b
9c
Me
Et
CH2Ph
10 0.0
>660
273 88
20 6.3
>660
530
0

D. Hocková et al. / Bioorg. Med. Chem. 19 (2011) 4445–4453
4449
aqueous solution of the crude product onto the column, it was
washed with water until the UV absorption dropped. The column
was then eluted with a gradient of dilute acetic or formic acid (0–
1 M). All tested ANPs were characterized by 1H NMR, 13C NMR and
mass spectrometry. The purity of the target compounds was deter-
mined by combustion elemental analysis (C, H, N).
from 3d and 6-chloropurine). 1H NMR (DMSO-d6): 8.77 s, 1H and
8.68 s, 1H (H-2 and H-8); 7.29 m, 3H and 7.18 m, 2H (Ar); 4.45
dd, 2H, J(10,20) = 5.1, Jg = 10.0 (H-10); 4.35 d, 2H, J = 3.5 (CH2Ph);
3.96 t, 2H, J(20,10) = 5.3 (H-20); 3.90 m, 4H (Et); 3.76 m, 1H (H-30);
3.47 dd, 1H, J(50a,30) = 3.5, Jg = 10.6 (H-50a); 3.39 dd, H,
J(50b,30) = 6.1, Jg = 10.6 (H-50b); 1.95 td, H, J(40,30) = 6.1,
J(40,P) = 18.1 (H-40); 1.15 t, H, J = 7.1 (Et). 13C NMR (DMSO-d6):
151.83 (C-4); 151.22 (C-2); 148.73 (C-6); 147.70 (C-8); 138.01
(Ar); 130.59 (C-5); 128.02, 2C (Ar); 127.22 (Ar); 127.10, 2C (Ar);
74.05 (C-30); 72.03 (CH2Ph); 71.76 d, J(P,C) = 10.9 (C-50); 66.69
(C-20); 60.79 m, 2C (Et); 43.95 (C-10); 27.50 d, J(P,C) = 138.3
(C-40); 16.01 m (Et). MS (ESI): m/z = 483 [M+H]+.
5.1. Diethyl 3-(benzyloxy)-2-(2-hydroxyethoxy)
propylphosphonate (3d)
A solution of n-butyllithium in hexane (2.5 M, 16 ml) was added
slowly to a stirred solution of diethyl methanephosphonate (5 g,
33.3 mmol) in dry THF (50 ml) cooled to ꢁ78 °C under argon atmo-
sphere. After 15 min of vigorous stirring benzyloxyacetaldehyde
(5 g, 33.3 mmol) was added. The reaction mixture was allowed to
rise to ꢁ30 °C and stirred for 5 h. Then ethyl bromoacetate
(4.5 ml, 40 mmol) was added and the temperature of the reaction
mixture was allowed to rise slowly to room temperature. After
overnight stirring diethyl ether (250 ml) was added and the
mixture was washed with saturated NaCl solution. The organic
layer was dried over anhydrous magnesium sulfate and solvent
evaporated. The crude intermediate 2d (ethyl 2-(1-(benzyloxy)-
3-(diethoxyphosphoryl)propan-2-yloxy)acetate) was purified by
chromatography on silica gel (hexane-CHCl3–MeOH) and used in
the following step.
5.4. Diethyl 9-[2-(3-benzyloxy-1-phosphonopropan-2-yloxy)
ethyl]-2-amino-6-chloropurine (5d)
Water (10 ml) was added and the reaction mixture was heated
at 70 °C for 24 h. Diethyl ether (250 ml) was added and the mixture
was washed with saturated NaCl solution. The organic layer was
dried over anhydrous magnesium sulfate and solvent evaporated.
The pure product was obtained after chromatography on silica
gel (chloroform–MeOH), yield 59% (starting from 3d and 2-ami-
no-6-chloropurine). 1H NMR (DMSO-d6): 8.10 s, H (H-8); 7.30 m,
H and 7.23 m, H (Ar); 6.91 s, H (NH2); 4.38 d, H, J = 2.7 (CH2Ph);
4.18 dd, H, J(10,20) = 4.8, Jg = 8.3 (H-10); 3.86 t, 2H, J(20,10) = 5.3
(H-20); 3.90 m, 4H (Et); 3.75 m, 1H (H-30); 3.50 dd, 1H,
J(50a,30) = 3.3, Jg = 10.5 (H-50a); 3.41 dd, 1H, J(50b,30) = 6.1, Jg = 10.5
(H-50b); 1.96 m, 2H (H-40); 1.16 t, 6H, J = 7.0 (Et). 13C NMR
(DMSO-d6): 159.58 (C-2); 153.94 (C-4); 149.09 (C-6); 143.45
(C-8); 138.06 (Ar); 128.05, 2C (Ar); 127.24 (Ar); 127.19, 2C (Ar);
123.11 (C-5); 70.06 (C-30); 72.14 (CH2Ph); 71.76 d, J(P,C) = 10.3
(C-50); 66.73 (C-20); 60.77 m, 2C (Et); 43.18 (C-10); 27.55 d,
J(P,C) = 138.55 (C-40); 16.02 d, J(P,C) = 5.7 (Et). MS (ESI): m/z = 498
[M+H]+.
1H NMR (DMSO-d6): 7.33 m, 5H (Ar); 4.49 s, 2H (CH2Ph); 4.22 s,
2H (CH2COOEt); 4.08 dd, 2H, J = 14.2 and 7.0 (Et); 3.98 m, 4H (Et);
3.84 m, 1H (CH); 3.61 dd, 1H, J = 10.5 and 3.3 (CH2O-a); 3.54 dd,
1H, J = 10.5 and 6.0 (CH2O-b); 2.06 m, 2H (CH2P); 1.17 m, 9H
(CH3). 13C NMR (DMSO-d6): 169.82 (CO); 138.16 (Ar); 128.07, 2C
(Ar); 127.26 (Ar); 127.24, 2C (Ar); 74.33 (CH); 72.16 (CH2); 71.96
d, J(P,C) = 8.7 (CH2); 66.85 (CH2); 71.96 dd, J(P,C) = 6.2 and 13.58
(CH2OP); 59.98 (CH2); 27.57 d, J(P,C) = 137.8 (CH2P); 16.04 d,
J(P,C) = 6.0 (CH3); 13.89 (CH3).
Intermediate 2d was cooled to ꢁ20 °C and a solution of borane in
THF (1.0 M, 55 ml) was added under argon atmosphere. The reac-
tion mixture was stirred at room temperature for 3 days. Methanol
(20 ml) was added at ꢁ20 °C and the solution was concentrated
after the evolution of hydrogen had stopped. The residue was puri-
fied by chromatography on silica gel (hexane-CHCl3–MeOH) and
product 3d (6.7 g) was obtained in 58% yield in three steps.
1H NMR (DMSO-d6): 7.34 m, 5H (Ar); 4.50 s, 2H (CH2Ph); 43.97
m, 4H (Et); 3.84 m, 1H (CH); 3.58 m, 2H; 3.51 m, 2H and 3.47 m, 2H
(CH2); 2.00 m, 2H (CH2P); 1.31 t, 6H (CH3). MS (ESI): m/z = 347
[M+H]+.
5.5. Diethyl 1-[2-(1-phosphonopropan-2-yloxy)ethyl]thymine
(6a)
The solvent was evaporated and methanol (30 ml) and 1 M
NaOMe in methanol (6 ml) were added. The solution was stirred
overnight, neutralized by 0.5 M aqueous HCl and solvent evapo-
rated. The crude mixture was purified by chromatography on silica
gel (chloroform–MeOH), yield 65% (starting from 3a and 3-ben-
zoylthymine). 1H NMR (DMSO-d6): 11.23 br s, 1H (NH); 7.48 s,
1H (H-6); 3.96 m, 4H (Et); 3.75 m, 2H (H-10); 3.66 m, 1H (H-30);
3.56 t, 2H, J(20,10) = 5.4 (H-20); 2.04 m, 1H (H-40a); 1.87 m, 1H (H-
40b); 1.74 s, 3H (CH3); 1.21 t, 6H, J = 7.0 (Et); 1.16d, 3H,
J(30,50) = 6.1 (H-50). 13C NMR (DMSO-d6): 164.12 (C-4); 150.72 (C-
2); 141.95 (C-6); 107.68 (C-5); 70.66 (C-30); 65.06 (C-20); 60.73
dd, J(P,C) = 6.3 and 11.9 (Et); 47.05 (C-10); 32.22 d, J(P,C) = 136.0
(C-40); 20.58 d, J(P,C) = 7.9 (C-50); 16.08 d, J(P,C) = 5.9 (Et); 11.68
(CH3-5). MS (ESI): m/z = 349 [M+H]+.
5.2. Synthesis of compounds4d, 5d, 6a,d, and 7a,d by Mitsunobu
reaction – general procedure
To a solution of triphenylphosphine (3.5 g, 13.4 mmol) in dry
THF (45 ml) cooled to ꢁ30 °C under argon atmosphere diisopropy-
lazadicarboxylate (DIAD, 2.4 ml, 12.6 mmol) was added slowly. The
mixture was stirred for 30 min and this preformed complex was
added to the reaction mixture containing corresponding heterocy-
clic base (6-chloropurine, 2-amino-6-chloropurine, 3-benzoylthy-
mine or 3-benzoyluracil; 6.5 mmol), dry THF (30 ml) and diethyl
phosphonate 3a10 or 3d (4.3 mmol) at ꢁ30 °C under argon. The
resulting mixture was slowly warmed to room temperature and
stirred for 48 h. Further procedure differs for corresponding
derivatives:
5.6. Diethyl 1-[2-(3-benzyloxy-1-phosphonopropan-2-yloxy)
ethyl]thymine (6d)
The same procedure as for thymine derivative 6a was applied,
yield 70% (starting from 3d and 3-benzoylthymine). 1H NMR
(DMSO-d6): 11.24 br s, 1H (NH); 7.46 s, 1H (H-6); 7.30 m, 5H
(Ar); 4.47 s, 2H (CH2Ph); 3.95 m, 4H (Et); 3.76 m, 3H (H-10 and
H-30); 3.69 t, 2H, J(20,10) = 5.0 (H-20); 3.53 dd, 1H, J(50a,30) = 3.5,
Jg = 10.5 (H-50a); 3.45 dd, 1H, J(50b,30) = 6.0, Jg = 10.5 (H-50b); 1.98
m, 2H (H-40); 1.20 t, 6H, J = 7.1 (Et).13C NMR (DMSO-d6): 164.12
(C-2); 150.74 (C-4); 141.95 (C-6); 138.15 (Ar); 128.09, 2C (Ar);
127.26 (Ar); 127.19, 2C (Ar); 107.64 (C-5); 74.07 (C-30); 72.20
5.3. Diethyl 9-[2-(3-benzyloxy-1-phosphonopropan-2-yloxy)
ethyl]-6-chloropurine (4d)
The solvent was evaporated and the crude mixture was purified
by chromatography on silica gel (MeOH–CHCl3), yield 53% (starting

4450
D. Hocková et al. / Bioorg. Med. Chem. 19 (2011) 4445–4453
(CH2Ph); 71.75 d, J(P,C) = 10.1 (C-50); 66.72 (C-20); 60.82 m, 2C (Et);
47.15 (C-10); 27.55 d, 2C, J(P,C) = 138.2 (C-40); 16.05 d, J(P,C) = 5.8
(Et); 11.68 (CH3). MS (ESI): m/z = 455 [M+H]+.
5.11. 9-[2-(1-Phosphonobutan-2-yloxy)ethyl]adenine (9b)
Starting from 6-chloropurine derivative 4b, yield 54% in two
steps. 1H NMR (DMSO-d6): 8.14 s, 1H and 8.11 s, 1H (H-2 and
H-8); 7.28 br s, 2H (NH2); 4.27 t, 2H, J(10,20) = 5.2 (H-10); 3.82 dt,
1H, J(20a,10) = 4.8, Jg = 9.9 (H-20a); 3.68 dt, 1H, J(20b,10) = 5.7,
Jg = 10.7 (H-20b); 3.46 m, 1H (H-30); 1.80 ddd, 1H, J(40a,30) = 4.6,
Jg = 14.9, J(40a,P) = 19.6 (H-40a); 1.61 m, 2H (H-40b and H-50a);
1.36 td, 1H, J(50b,30) = J(50b,60) = 7.1, Jg = 14.2 (H-50b); 0.63 t, 3H,
J(60,50) = 7.3 (H-60). 13C NMR (DMSO-d6): 155.59 (C-6); 151.88
(C-2); 149.32 (C-4); 141.24 (C-8); 118.44 (C-5); 76.12 (C-30);
65.75 (C-20); 43.17 (C-10); 32.45 d, J(P,C) = 134.1 (C-40); 26.96 d,
J(P,C) = 5.7 (C-50); 8.71 (C-60). Anal. Calcd for C11H18N5O4P.1/
2H2O: C, 40.74; H, 5.91; N, 21.60. Found: C, 40.44; H, 5.85; N,
21.31. MS (ESIꢁ): m/z = 314 [MꢁH]ꢁ.
5.7. Diethyl 1-[2-(1-phosphonopropan-2-yloxy)ethyl]uracil (7a)
The same procedure as for thymine derivative 6a was applied,
yield 45% (starting from 3a and 3-benzoyluracil). 1H NMR
(DMSO-d6): 11.24 br s, 1H (NH); 7.57 d, 1H, J(6,5) = 7.8 (H-6);
5.51 d, 1H, J(5,6) = 7.8 (H-5); 3.96 m, 4H (Et); 3.78 dd, 2H,
J(10,20) = 9.8 and 5.2 (H-10); 3.67 m, 1H (H-30); 3.57 dd, 2H,
J(20,10) = 8.8 and 5.3 (H-20); 2.04 m, 1H (H-40a); 1.86 m, 1H (H-
40b); 1.21 t, 6H (Et); 1.16 d, 3H, J(30,50) = 6.1 (H-50).13C NMR
(DMSO-d6): 163.55 (C-4); 150.76 (C-2); 146.19 (C-6); 100.13 (C-
5); 70.71 (C-30); 65.025 (C-20); 60.75 dd, J(P,C) = 6.3 and 11.1 (Et);
47.36 (C-10); 32.23 d, J(P,C) = 136.0 (C-40); 20.59 d, J(P,C) = 7.8 (C-
50); 16.08 d, J(P,C) = 5.8 (Et). MS (ESI): m/z = 335 [M+H]+.
5.12. 9-[2-(3-Phenyl-1-phosphonopropan-2-yloxy)ethyl]
adenine (9c)
5.8. Diethyl 9-[2-(3-benzyloxy-1-phosphonopropan-2-yloxy)
ethyl]uracil (7d)
Starting from 6-chloropurine derivative 4c, yield 48% in two
steps. 1H NMR (DMSO-d6): 8.12 s, 1H and 7.95 s, 1H (H-2 and H-
8); 7.31 br s, 2H (NH2); 7.10 m, 3H and 7.00 m, 2H (Ar); 4.20 m,
2H (H-10); 3.78 m, 2H and 3.63 ddd, 1H, J = 4.5, 6.9 and 10.6. (H-
20 and H-30); 2.91 dd, 1H, J(50a,30) = 3.8, Jg = 13.5 (H-50a); 2.65 dd,
1H, J(50b,30) = 7.0, Jg = 13.9 (H-50b); 1.79 ddd, 1H, J(40a,30) = 5.2,
Jg = 15.0, J(40a,P) = 19.3 (H-40a); 1.66 ddd, 1H, J(40b,30) = 7.4,
Jg = 15.0, J(40b,P) = 17.4 (H-40b). 13C NMR (DMSO-d6): 155.60 (C-
6); 151.80 (C-2); 149.24 (C-4); 141.13 (C-8); 138.23 (Ar); 129.20,
2C (Ar); 127.70, 2C (Ar); 125.70 (Ar); 118.49 (C-5); 76.24 (C-30);
66.22 (C-20); 43.12 (C-10); 40.31 d, J(P,C) = 7.0 (C-50); 32.53 d,
J(P,C) = 132.0 (C-40). Anal. Calcd for C16H20N5O4P.4/5H2O: C,
49.05; H, 5.56; N, 17.88. Found: C, 48.98; H, 5.65; N, 17.87. MS
(ESIꢁ): m/z = 376 [MꢁH]ꢁ.
The same procedure as for thymine derivative 6a was applied,
yield 70% (starting from 3d and 3-benzoyluracil). 1H NMR (DMSO-
d6): 11.25 br s, 1H (NH); 7.56 d, J = 7.9, 1H (H-6); 7.30 m, 5H (Ar);
5.46 d, H, J = 7.9 (H-5); 4.47 s, H (CH2Ph); 3.95 m, H (Et); 3.79 m, H
(H-10 and H-30); 3.69 t, 2H, J(20,10) = 5.1 (H-20); 3.53 dd, 1H,
J(50a,30) = 3.4, Jg = 10.5 (H-50a); 3.44 dd, 1H, J(50b,30) = 6.0, Jg = 10.5
(H-50b); 1.99 m, 2H (H-40); 1.20 t, 6H, J = 7.1 (Et). 13C NMR (DMSO-
d6): 163.55 (C-2); 150.77 (C-4); 146.16 (C-6); 138.13 (Ar); 128.09,
2C (Ar); 126.24, 3C (Ar); 100.08 (C-5); 74.06 (C-30); 72.19 (CH2Ph);
71.75 d, J(P,C) = 10.3 (C-50); 66.68 (C-20); 60.83 m, 2C (Et); 47.42
(C-10); 27.58 d, 2C, J(P,C) = 138.1 (C-40); 16.05 m, J(P,C) = 5.9 (Et).
MS (ESI): m/z = 441 [M+H]+.
5.9. Synthesis of 9-[2-(1-phosphonoalkan-2-yloxy)ethyl]-ade-
nines 9a–9e – general procedure
5.13. Diethyl 9-[2-(3-benzyloxy-1-phosphonopropan-2-yloxy)
ethyl]adenine (8d)
A solution of 6-chloropurine derivatives 4a-4c10 or 4d(1 mmol)
in methanolic ammonia (60 ml) was stirred in an autoclave at
70 °C for 30 h, solvent was evaporated and the residue co-distilled
with acetonitrile. A mixture of this crude diethyl ester adenine
intermediate 8a–8d, acetonitrile (20 ml) and BrSiMe3 (2 ml) was
stirred overnight at room temperature. After evaporation and co-
distillation with acetonitrile, the residue was treated with aqueous
ammonia (2.5%) and evaporated to dryness. The residue was ap-
plied onto a column of Dowex 50 ꢀ 8 (H+ form, 40 ml) and washed
with water. Elution with 2.5% aqueous ammonia and evaporation
afforded crude product as ammonium salt. This residue was ap-
plied onto a column of Dowex 1 ꢀ 2 (acetate, 50 ml), washed with
water followed by a gradient of acetic acid (0–0.5 M) and formic
acid (0.5 M). The main UV-absorbing fraction was evaporated and
co-distilled with water to obtain product as a white solid.
Starting from 6-chloropurine derivative 4d, intermediate, yield
67%. 1H NMR (DMSO-d6): 8.13 s, 1H and 8.11 s, 1H (H-2 and H-
8); 7.28 m, 5H, 2H (Ar); 7.19 s, 2H (NH2); 4.39 d, 2H, J = 3.7
(CH2Ph); 4.27 dd, 2H, J(10,20) = 5.1, Jg = 8.3 (H-10); 3.97 m, 2H (H-
20); 3.88 m, 6H (H-20 and Et); 3.76 m, 1H (H-30); 3.49 dd, 1H,
J(50a,30) = 3.4, Jg = 10.5 (H-50a); 3.41 dd, 1H, J(50b,30) = 6.0, Jg = 10.5
(H-50b); 1.96 m, 2H (H-40); 1.16 t, 6H, J = 7.1 (Et). 13C NMR
(DMSO-d6): 155.78 (C-6); 152.14 (C-2); 149.37 (C-4); 141.02 (C-
8); 138.10 (Ar); 128.05, 2C (Ar); 127.20, 3C (Ar); 118.48 (C-5);
74.05 (C-30); 72.13 (CH2Ph); 71.73 d, J(P,C) = 10.7 (C-50); 67.08
(C-20); 60.80 m, 2C (Et); 43.07 (C-10); 27.57 d, J(P,C) = 138.2 (C-
40); 16.02 m (Et). MS (ESI): m/z = 464 [M+H]+.
5.14. 9-[2-(3-Benzyloxy-1-phosphonopropan-2-yloxy)ethyl]
adenine (9d) and 9-[2-(3-Hydroxy-1-phosphonopropan-2-yl-
oxy)ethyl]adenine (9e)
5.10. 9-[2-(1-Phosphonopropan-2-yloxy)ethyl]adenine (9a)
Starting from intermediate 8d, yield 45% of 9d and 27% of deb-
enzylated product 9e.
Starting from 6-chloropurine derivative 4a, yield 61% in two
steps. 1H NMR (DMSO-d6): 8.16 s, 1H and 8.13 s, 1H (H-2 and H-
8); 7.36 br s, 2H (NH2); 4.27 t, 2H, J(10,20) = 5.3 (H-10); 3.73 t, 2H,
J(20,10) = 5.3 (H-20); 3.65 m, 1H (H-30); 1.87 ddd, 1H, J(40a,30) = 4.6,
Jg = 14.7, J(40a,P) = 19.4 (H-40a); 1.58 ddd, 1H, J(40b,30) = 8.7,
Jg = 14.7, J(40b,P) = 17.4 (H-40b); 1.12 d, 3H, J(30,50) = 6.1 (H-50).
13C NMR (DMSO-d6): 155.31 (C-6); 151.53 (C-2); 149.24 (C-4);
141.34 (C-8); 118.37 (C-5); 71.31 (C-30); 65.30 (C-20); 43.08
(C-10); 35.22 d, J(P,C) = 132.0 (C-40); 20.74 d, J(P,C) = 5.0 (C-50). Anal.
Calcd for C10H16N5O4P.1/2H2O: C, 38.71; H, 5.52; N, 22.57. Found:
C, 38.67; H, 5.53; N, 22.45. MS (ESI): m/z = 302 [M+H]+.
9d: 1H NMR (DMSO-d6): 8.14 s, 1H and 8.13 s, 1H (H-2 and H-
8); 7.28 m, 5H, 2H (Ar); 7.22 s, 2H (NH2); 4.37 d, 2H, J = 3.7
(CH2Ph); 4.28 t, 2H, J(10,20) = 5.2 (H-10); 3.86 m, 2H (H-20); 3.79
m, 1H (H-30); 3.57 dd, 1H, J(50a,30) = 3.0, Jg = 10.6 (H-50a); 3.40 dd,
1H, J(50b,30) = 6.0, Jg = 10.6 (H-50b); 1.77 m, 2H (H-40). 13C NMR
(DMSO-d6): 155.58 (C-6); 151.82 (C-2); 149.27 (C-4); 141.22
(C-8); 138.26 (Ar); 128.03, 2C (Ar); 127.11, 3C (Ar); 118.41 (C-5);
74.82 (C-30); 72.32 (CH2Ph); 72.15 d, J(P,C) = 10.6 (C-50); 66.96
(C-20); 43.14 (C-10); 30.22 d, J(P,C) = 134.1 (C-40). Anal. Calcd for

D. Hocková et al. / Bioorg. Med. Chem. 19 (2011) 4445–4453
4451
C17H22N5O5P.3/2H2O: C, 47.00; H, 5.80; N, 16.12. Found: C, 47.20;
H, 5.86; N, 16.26. MS (ESIꢁ): m/z = 406 [MꢁH]ꢁ.
(C-50); 67.01 (C-20); 60.83 m, 2C (Et); 43.07 (C-10); 27.55 d, 2C,
J(P,C) = 137.9 (C-40); 16.40 m, J(P,C) = 5.9 (Et). MS (ESI): m/z = 480
[M+H]+.
9e: 1H NMR (DMSO-d6): 8.19 s, 1H and 8.13 s, 1H (H-2 and H-8);
7.28 s, 2H (NH2); 4.27 t, 2H, J(10,20) = 5.2 (H-10); 3.83 dd, 2H,
J(20,10) = 5.4, Jg = 8.7 (H-20); 3.61 m, 1H (H-30); 3.49 dd, 1H,
J(50a,30) = 3.8, Jg = 11.5 (H-50a); 3.33 dd, 1H, J(50b,30) = 6.1, Jg = 11.5
(H-50b); 1.71 m, 2H (H-40). 13C NMR (DMSO-d6): 155.61 (C-6);
151.84 (C-2); 149.26 (C-4); 141.32 (C-8); 118.38 (C-5); 76.80
(C-30); 67.05 (C-20); 63.60 d, J(P,C) = 8.2 (C-50); 43.17 (C-10); 30.44
d, J(P,C) = 133.9 (C-40). Anal. Calcd for C10H16N5O5P.3/4H2O: C,
35.20; H, 5.51; N, 20.52. Found: C, 35.49; H, 5.35; N, 20.39. MS
(ESIꢁ): m/z = 316 [MꢁH]ꢁ.
5.19. Debenzylation of 6d, 7d, 10d and 11d – general procedure
Benzyl derivative (1.5 mmol) was hydrogenated at atmospheric
pressure in methanol (20 ml, 0.1 ml of 4.5 M HCl in DMF added)
over 10% palladium on charcoal (0.2 g) under stirring at room tem-
perature for 20 h. The mixture was filtered through a pad of Celite,
the catalyst was washed with hot water and hot methanol (100 ml
each) and the solvent was evaporated to obtain the crude product
in quantitative yield.
5.15. 9-[2-(1-Phosphonopropan-2-yloxy)ethyl]diaminopurine
(14)
5.20. Diethyl 9-[2-(3-hydroxy-1-phosphonopropan-2-yloxy)
ethyl]thymine (6e)
Starting from 2-amino-6-chloropurine derivative 5a10 the same
procedure was used as above; yield 54% in two steps. 1H NMR
(DMSO-d6): 7.76 s, 1H and 7.19 s, 1H (H-2 and H-8); 6.32 br s,
2H (NH2); 4.10 t, 2H, J(10,20) = 5.1 (H-10); 3.66 t, 2H, J(20,10) = 5.1
(H-20); 3.65 m, 1H (H-30); 1.86 ddd, 1H, J(40a,30) = 4.5, Jg = 14.8,
J(40a,P) = 19.2 (H-40a); 1.57 ddd, 1H, J(40b,30) = 8.7, Jg = 14.8,
J(40b,P) = 17.4 (H-40b); 1.14 d, 3H, J(30,50) = 6.0 (H-50). 13C NMR
(DMSO-d6): 159.01 (C-2); 155.63 (C-6); 150.70 (C-4); 138.03
(C-8); 112.71 (C-5); 71.60 (C-30); 65.67 (C-20); 42.76 (C-10); 35.56
d, J(P,C) = 131.7 (C-40); 20.85 d, J(P,C) = 5.8 (C-50). Anal. Calcd for
Starting from 6d. 1H NMR (DMSO-d6): 11.22 br s, 1H (NH); 7.52
s, 1H (H-6); 3.95 m, 4H (Et); 3.76 t, 2H, J(10,20) = 5.2 (H-10); 3.66 t,
2H, J(20,10) = 5.2 (H-20); 3.55 m, 1H (H-30); 3.44 dd, 1H,
J(50a,30) = 4.3, Jg = 11.4 (H-50a); 3.37 dd, 1H, J(50b,30) = 5.6, Jg = 11.4
(H-50b); 1.92 m, 2H (H-40); 1.21 t, 6H, J = 7.1 (Et). 13C NMR
(DMSO-d6): 164.15 (C-2); 150.77 (C-4); 142.03 (C-6); 107.65
(C-5); 75.94 (C-30); 66.69 (C-20); 63.02 d, J(P,C) = 11.3 (C-50);
60.76 m, 2C (Et); 47.15 (C-10); 27.50 d, 2C, J(P,C) = 138.3 (C-40);
16.07 d, J(P,C) = 5.7 (Et); 11.70 (CH3). MS (ESI): m/z = 365 [M+H]+.
C
10H17N6O4PꢂH2O: C, 35.93; H, 5.73; N, 25.14. Found: C, 35.89; H,
5.55; N, 24.96. MS (ESIꢁ): m/z = 315 [MꢁH]ꢁ.
5.21. Diethyl 9-[2-(3-hydroxy-1-phosphonopropan-2-yloxy)
ethyl]uracil (7e)
5.16. Transformation of 6-chloropurine and 2-amino-6-chloro-
purine derivatives 4d and 5d to hypoxanthine and guanine
derivatives 10d and 11d – general procedure
Starting from 7d. 1H NMR (DMSO-d6): 11.23 br s, 1H (NH); 7.60
d, J = 7.9, 1H (H-6); 5.50 d, 1H, J = 7.9 (H-5); 4.75 t, 1H, J = 5.6 (OH);
3.96 m, 4H (Et); 3.79 t, 2H, J(10,20) = 5.1 (H-10); 3.67 t, 2H,
J(20,10) = 5.1 (H-20); 3.55 m, 1H (H-50a); 3.44 m, 1H (H-50b); 1.92
m, 2H (H-40); 1.21 t, 6H, J = 7.0 (Et). 13C NMR (DMSO-d6): 163.60
(C-2); 150.81 (C-4); 146.26 (C-6); 100.12 (C-5); 76.04 (C-30);
66.69 (C-20); 63.06 d, J(P,C) = 11.5 (C-50); 60.78 m, 2C (Et); 47.50
(C-10); 27.52 d, 2C, J(P,C) = 138.4 (C-40); 16.08 m, J(P,C) = 5.9 (Et).
MS (ESI): m/z = 351 [M+H]+.
The 6-chloro derivative 4d or 5d (2 mmol) was dissolved in tri-
fluoroacetic acid (75%, 20 ml) and stirred overnight. The solvent
was evaporated and the residue co-distilled with water (3ꢀ) and
ethanol. The crude mixture was purified by chromatography on sil-
ica gel (chloroform–MeOH).
5.17. Diethyl 9-[2-(3-benzyloxy-1-phosphonopropan-2-yloxy)
ethyl]hypoxanthine (10d)
5.22. Diethyl 9-[2-(3-hydroxy-1-phosphonopropan-2-yloxy)
ethyl]hypoxanthine (10e)
Starting from 4d, yield 67%. 1H NMR (DMSO-d6): 12.30 br s, 1H
(NH); 8.11 s, 1H and 8.04 s, 1H (H-2 and H-8); 7.28 m, 5H (Ar); 4.40
d, 2H, J = 3.5 (CH2Ph); 4.28 dd, 2H, J(10,20) = 4.9, Jg = 8.8 (H-10); 3.91
m, 6H (H-20 and Et); 3.76 m, 1H (H-30); 3.49 dd, 1H, J(50a,30) = 3.4,
Jg = 10.5 (H-50a); 3.41 dd, 1H, J(50b,30) = 5.9, Jg = 10.5 (H-50b); 1.96
m, 2H (H-40); 1.17 t, 6H, J = 7.0 (Et). 13C NMR (DMSO-d6): 156.36
(C-6); 148.14 (C-4); 145.30 (C-2); 140.46 (C-8); 138.08 (Ar);
128.05, 2C (Ar); 127.23 (Ar); 127.19, 2C (Ar); 123.40 (C-5); 74.07
(C-30); 72.13 (CH2Ph); 71.78 d, J(P,C) = 10.5 (C-50); 67.16 (C-20);
60.80 m, 2C (Et); 43.53 (C-10); 27.55 d, J(P,C) = 138.3 (C-40); 16.00
m (Et). MS (ESI): m/z = 465 [M+H]+.
Starting from 10d. 1H NMR (DMSO-d6): 8.11 s, 1H and 8.03 s, 1H
(H-2 and H-8); 4.83 t, 1H, J = 5.2 (OH); 4.26 dd, 2H, J(10,20) = 5.2,
Jg = 8.8 (H-10); 3.91 m, 4H (Et); 3.86 t, 2H, J = 4.5 (H-2v); 3.58 m,
1H (H-3v); 3.40 m, 2H (H-5); 1.89 m, 2H (H-40); 1.17 t, 6H, J = 7.0
(Et).13C NMR (DMSO-d6): 156.46 (C-6); 148.17 (C-4); 145.21 (C-
2); 140.56 (C-8); 123.62 (C-5); 75.98 (C-30); 67.14 (C-20); 63.00 d,
J(P,C) = 11.6 (C-50); 60.75 m, 2C (Et); 43.50 (C-10); 27.48 d,
J(P,C) = 138.4 (C-40); 16.02 m (Et). MS (ESI): m/z = 375 [M+H]+.
5.23. Diethyl 9-[2-(3-hydroxy-1-phosphonopropan-2-yloxy)
ethyl]guanine (11e)
5.18. Diethyl 9-[2-(3-benzyloxy-1-phosphonopropan-2-yloxy)
ethyl]guanine (11d)
Starting from 11d. 1H NMR (DMSO-d6): 11.69 br s, 1H (NH);
8.97 s, 1H (H-8); 7.28 s, 2H (NH2); 4.21 t, 2H, J(10,20) = 3.5 (H-10);
3.91 m, 6H (H-20 and Et); 3.59 m, 1H (H-30); 3.45 dd, 1H,
J(50a,30) = 3.6, Jg = 11.5 (H-5va); 3.35 dd, 1H, J(50b,30) = 5.6,
Jg = 11.5 (H-50b); 1.93 m, 2H (H-40); 1.18 t, 6H, J = 7.0 (Et).13C
NMR (DMSO-d6): 155.13 (C-6); 153.64 (C-2); 149.67 (C-4);
137.62 (C-8); 116.29 (C-5); 75.99 (C-30); 65.83 (C-20); 62.93 d,
2C, J(P,C) = 12.3 (Et); 60.77 d, J(P,C) = 13.4 (C-50); 44.30 (C-10);
27.32 d, J(P,C) = 138.8 (C-40); 16.02 d, 2C, J(P,C) = 5.8 (Et). MS
(ESIꢁ): m/z = 390 [MꢁH]ꢁ.
Starting from 5d, yield 61%. 1H NMR (DMSO-d6): 10.70 br s, 1H
(NH); 7.88 s, 1H (H-8); 7.26 m, 5H (Ar); 6.54 s, 2H (NH2); 4.42 d,
2H, J = 2.8 (CH2Ph); 4.08 dd, 2H, J(10,20) = 5.0, Jg = 8.0 (H-10); 3.93
m, 4H (Et); 3.82 t, 2H, J(20,10) = 5.2 (H-20); 3.75 m, 1H (H-30); 3.51
dd, 1H, J(50a,30) = 3.4, Jg = 10.5 (H-50a); 3.42 dd, 1H, J(50b,30) = 6.0,
Jg = 10.5 (H-50b); 1.97 m, 2H (H-40); 1.18 t, 6H, J = 7.0 (Et). 13C
NMR (DMSO-d6): 156.18 (C-6); 153.60 (C-2); 150.77 (C-4);
138.09 (Ar); 137.62 (C-8); 128.07, 2C (Ar); 127.24, 3C (Ar);
118.29 (C-5); 74.07 (C-30); 72.17 (CH2Ph); 71.76 d, J(P,C) = 10.6

4452
D. Hocková et al. / Bioorg. Med. Chem. 19 (2011) 4445–4453
5.24. Diethyl 1-[2-(1-phosphonopropan-2-yloxy)ethyl]cytosine
(17)
5.28. 9-[2-(3-Benzyloxy-1-phosphonopropan-2-yloxy)ethyl]
guanine (13d)
Compound 7a (470 mg, 1.4 mmol), Et3N (0.6 mL), and 2,4,6-tri-
isopropylbenzenesulfonyl chloride (1.56 g, 5.08 mmol) were stir-
red in CH3CN (20 mL) at room temp. for 3 days. NH4OH (25%,
5 mL) was added, the mixture was stirred for 24 h, and the solvents
were evaporated. The residue in ethyl acetate was washed with
brine, the aqueous fraction was than washed with five portions
of CHCl3, and the organic fractions were dried with MgSO4 and
concentrated under reduced pressure. The crude product was puri-
fied by flash chromatography to give 350 mg (75%). 1H NMR
(DMSO-d6): 7.50 d, 1H, J(6,5) = 7.2 (H-6); 5.61 d, 1H, J(5,6) = 7.2
(H-5); 3.96 m, 4H (Et); 3.74 dd, 2H, J(10,20) = 11.7 and 5.7 (H-10);
3.63 m, 1H (H-30); 3.54 t, 2H, J(20,10) = 5.2 (H-20); 2.01, 1H (H-
40a); 1.85 m, 1H (H-40b); 1.20 t, 6H (Et); 1.15 d, 3H, J(30,50) = 6.1
(H-50). 13C NMR (DMSO-d6): 165.76 (C-4); 155.48 (C-2); 146.99
(C-6); 92.53 (C-5); 70.63 (C-30); 65.29 (C-20); 60.75 dd, J(P,C) = 6.3
and 12.3 (Et); 48.63 (C-10); 32.25 d, J(P,C) = 135.7 (C-40); 20.64 d,
J(P,C) = 7.3 (C-50); 16.09 d, J(P,C) = 5.4 (Et). MS (ESI): m/z = 334
[M+H]+.
Starting from 11d, yield 92%. 1H NMR (DMSO-d6): 10.55 br s, 1H
(NH); 7.71 s, 1H (H-8); 7.30 m, 5H (Ar); 6.45 s, 2H (NH2); 4.41 q,
2H, J = 12.10 (CH2Ph); 4.06 t, 2H, J(10,20) = 5.1 (H-10); 3.78 m, 3H
(H-20 and H-30); 3.59 dd, 1H, J(50a,30) = 2.7, Jg = 10.5 (H-50a); 3.42
dd, 1H, J(50b,30) = 6.5, Jg = 10.5 (H-50b); 1.78 m, 2H (H-40). 13C
NMR (DMSO-d6): 156.54 (C-6); 153.35 (C-2); 150.89 (C-4);
138.27 (Ar); 137.68 (C-8); 128.08, 2C (Ar); 127.16, 3C (Ar);
116.00 (C-5); 74.75 (C-30); 72.29 d, J(P,C) = 7.1 (C-50); 72.13
(CH2Ph); 66.97 (C-20); 42.83 (C-10); 30.30 d, J(P,C) = 134.5 (C-40).
Anal. Calcd for C17H22N5O6P.1/2H2O: C, 47.22; H, 5.36; N, 16.20.
Found: C, 47.20; H, 5.16; N, 16.26. MS (ESIꢁ): MS (ESIꢁ): m/
z = 422 [MꢁH]ꢁ.
5.29. 9-[2-(3-Hydroxy-1-phosphonopropan-2-yloxy)ethyl]
guanine (13e)
Starting from 11e, yield 81%. 1H NMR (DMSO-d6): 10.76 br s, 1H
(NH); 8.02 s, 1H (H-8); 6.59 s, 2H (NH2); 4.09 t, 2H, J(10,20) = 5.0 (H-
10); 3.77 t, 2H, J(20,10) = 5.0 (H-20); 3.59 m, 1H (H-30); 3.50 dd, 1H,
J(50a,30) = 3.8, Jg = 11.4 (H-50a); 3.35 dd, 1H, J(50b,30) = 6.0, Jg = 11.4
(H-50b); 1.73 m, 2H (H-40). 13C NMR (DMSO-d6): 156.02 (C-6);
153.72 (C-2); 150.69 (C-4); 137.82 (C-8); 116.09 (C-5); 76.65
(C-30); 66.69 (C-20); 63.55 m, (C-50); 43.19 (C-10); 30.38 d,
J(P,C) = 128.8 (C-40). Anal. Calcd for C14H23N4O6PꢂH2O: C, 34.19;
H, 5.17; N, 19.94. Found: C, 34.36; H, 4.96; N, 19.85. MS (ESIꢁ):
m/z = 332 [MꢁH]ꢁ.
5.25. Synthesis of phosphonic acids 12d,e, 13d,e, 15a,e, 16a,e
and 18 – general procedure
A mixture of diethyl ester (1 mmol), acetonitrile (20 ml) and
BrSiMe3 (1 ml) was stirred overnight at room temperature. After
evaporation and co-distillation with acetonitrile, the residue was
treated with aqueous ammonia (2.5%) and evaporated to dryness.
This residue was applied onto a column of Dowex 1 ꢀ 2 (acetate,
50 ml), washed with water followed by a gradient of acetic acid
(0–1 M) and formic acid (0.25–1 M). The main UV-absorbing frac-
tion was evaporated and co-distilled with water to obtain product
as a white solid.
5.30. 1-[2-(1-Phosphonopropan-2-yloxy)ethyl]thymine (15a)
Starting from 6a, yield 90%. 1H NMR (DMSO-d6): 11.22 br s, 1H
(NH); 7.47 s, 1H (H-6); 3.75 m, 2H (H-10); 3.65 m, 1H (H-30); 3.53 t,
2H, J(20,10) = 3.3 (H-20); 1.90 m, 1H (H-40a); 1.74 s, 3H (CH3); 1.60
m, 1H (H-40b); 1.16 d, 3H, J(30,50) = 6.1 (H-50).13C NMR (DMSO-
d6): 164.16 (C-4); 150.74 (C-2); 142.08 (C-6); 107.62 (C-5); 71.36
(C-30); 64.89 (C-20); 47.11 (C-10); 35.25 d, J(P,C) = 132.4 (C-40);
20.77 d, J(P,C) = 4.9 (C-50); 11.69 (CH3-5). Anal. Calcd for
5.26. 9-[2-(3-Benzyloxy-1-phosphonopropan-2-yloxy)ethyl]
hypoxanthine (12d)
Starting from 10d, yield 55%. 1H NMR (DMSO-d6): 12.56 br s,
1H (NH); 8.50 s, 1H and 8.12 s, 1H (H-2 and H-8); 7.27 m, 5H
(Ar); 4.39 d, 2H, J = 3.6 (CH2Ph); 4.32 t, 2H, J = 5.1 (H-10); 3.87
m, 2H (H-20); 3.78 m, 1H (H-30); 3.56 dd, 1H, J(50a,30) = 3.0,
Jg = 10.5 (H-50a); 3.41 dd, 1H, J(50b,30) = 6.4, Jg = 10.5 (H-50b);
1.76 m, 2H (H-40). 13C NMR (DMSO-d6): 155.59 (C-6); 147.80
(C-4); 146.11 (C-2); 140.46 (C-8); 138.21 (Ar); 128.03, 2C (Ar);
127.17 (Ar); 127.11, 2C (Ar); 121.53 (C-5); 74.73 (C-30); 71.30 d,
J(P,C) = 10.4 (C-50); 72.10 (CH2Ph); 66.72 (C-20); 43.97 (C-10);
30.33 d, J(P,C) = 137.8 (C-40). Anal. Calcd for C17H21N4O6P.1/
4H2O: C, 49.46; H, 5.25; N, 13.57. Found: C, 49.12; H, 5.07; N,
13.91. MS (ESIꢁ): m/z = 407 [MꢁH]ꢁ As a side product 30% of
12e was isolated.
C
10H17N2O6P: C, 41.10; H, 5.86; N, 9.59. Found: C, 39.85; H, 5.93;
N, 9.69. MS (ESIꢁ): m/z = 291 [MꢁH]ꢁ.
5.31. 9-[2-(3-Hydroxy-1-phosphonopropan-2-yloxy)ethyl]
thymine (15e)
Starting from 6e, yield 73%. 1H NMR (DMSO-d6): 11.20 br s, 1H
(NH); 7.54 s, 1H (H-6); 3.75 t, 2H, J(10,20) = 5.2 (H-10); 3.64 t, 2H,
J(20,10) = 5.2 (H-20); 3.57 m, 1H (H-30); 3.50 dd, 1H, J(50a,30) = 3.9,
Jg = 11.4 (H-50a); 3.35 dd, 1H, J(50b,30) = 5.9, Jg = 11.4 (H-50b); 1.71
m, 2H (H-40). 13C NMR (DMSO-d6): 164.17 (C-2); 150.80 (C-4);
142.17 (C-6); 107.60 (C-5); 76.61 (C-30); 66.52 (C-20); 63.52 d,
J(P,C) = 10.0 (C-50); 47.15 (C-10); 30.37 d, 2C, J(P,C) = 134.3 (C-40);
11.70 (CH3). Anal. Calcd for C10H17N2O7P.1/2H2O: C, 37.86; H,
5.72; N, 8.83. Found: C, 37.62; H, 5.61; N, 8.57. MS (ESIꢁ): m/
z = 307 [MꢁH]ꢁ.
5.27. 9-[2-(3-Hydroxy-1-phosphonopropan-2-yloxy)ethyl]
hypoxanthine (12e)
5.32. 1-[2-(1-Phosphonopropan-2-yloxy)ethyl]uracil (16a)
Starting from 10e, yield 76%. 1H NMR (DMSO-d6): 12.46 br s, 1H
(NH); 8.40 s, 1H and 8.09 s, 1H (H-2 and H-8); 4.30t, 2H, J = 5.1 (H-
10); 3.84 t, 2H, J = 4.3 (H-20); 3.59 m, 1H (H-30); 3.49 dd, 1H,
J(50a,30) = 3.9, Jg = 11.5 (H-50a); 3.33 dd, 1H, J(50b,30) = 6.1, Jg = 11.5
(H-50b); 1.71 m, 2H (H-40). 13C NMR (DMSO-d6): 155.89 (C-6);
147.90 (C-4); 145.81 (C-2); 140.58 (C-8); 122.17 (C-5); 76.62 (C-
30); 66.71 (C-20); 63.54 d, J(P,C) = 8.7 (C-50); 43.81 (C-10); 30.31 d,
J(P,C) = 134.4 (C-40). Anal. Calcd for C10H15N4O6P.1/2MeOH: C,
37.73; H, 5.13; N, 16.76. Found: C, 37.42; H, 5.41; N, 16.68. MS
(ESIꢁ): m/z = 317 [MꢁH]ꢁ.
Starting from 7a, yield 87%. 1H NMR (DMSO-d6): 7.60 d, 1H,
J(6,5) = 7.8 (H-6); 5.50 d, 1H, J(5,6) = 7.8 (H-5); 3.78 dd, 2H,
J(10,20) = 8.9 and 4.8 (H-10); 3.60 m, 1H (H-30); 3.51 dd, 2H,
J(20,10) = 8.9 and 4.8 (H-20); 1.75 m, 1H (H-40a); 1.37 m, 1H (H-40b);
1.15 d, 3H, J(30,50) = 6.0 (H-50). 13C NMR (DMSO-d6): 163.60 (C-4);
150.77 (C-2); 146.42 (C-6); 99.99 (C-5); 72.42 (C-30); 64.45 (C-20);
47.41 (C-10); 36.68 d, J(P,C) = 129.5 (C-40); 20.96 d, J(P,C) = 3.1 (C-
50). Anal. Calcd for C9H15N2O6PꢂNH3: C, 36.61; H, 6.15; N, 14.23.
Found: C, 36.47; H, 6.08; N, 14.03. MS (ESIꢁ): m/z = 277 [MꢁH]ꢁ.

D. Hocková et al. / Bioorg. Med. Chem. 19 (2011) 4445–4453
4453
5.33. 9-[2-(3-Hydroxy-1-phosphonopropan-2-yloxy)ethyl]uracil
(16e)
into 96-well microtiter plates and allowed to proliferate for 24 h.
Then, medium containing different concentrations of the test com-
pounds was added. After 3 days of incubation at 37 °C, the cell
number was determined with a Coulter counter. The cytostatic
concentration was calculated as the CC50, or the compound con-
centration required to reduce cell proliferation by 50% relative to
the number of cells in the untreated controls. CC50 values were
estimated from graphic plots of the number of cells (percentage
of control) as a function of the concentration of the test com-
pounds. Alternatively, cytotoxicity of the test compounds was ex-
pressed as the minimum cytotoxic concentration (MCC) or the
compound concentration that caused a microscopically detectable
alteration of HEL cell morphology.
Starting from 7e, yield 66%. 1H NMR (DMSO-d6): 11.33 br s, 1H
(NH); 7.74 d, J = 7.9, 1H (H-6); 5.59 d, 1H, J = 7.8 (H-5); 3.90 t, 2H,
J(10,20) = 5.1 (H-10); 3.75 t, 2H, J(20,10) = 5.1 (H-20); 3.61 dd, 1H,
J(50a,30) = 3.9, Jg = 11.4 (H-50a); 3.46 dd, 1H, J(50b,30) = 5.9, Jg = 11.4
(H-50b); 1.83 m, 2H (H-40). 13C NMR (DMSO-d6): 163.60 (C-2);
150.81 (C-4); 146.26 (C-6); 100.12 (C-5); 76.04 (C-30); 66.69 (C-
20); 63.06 d, J(P,C) = 11.5 (C-50); 60.78 m, 2C (Et); 47.50 (C-10);
27.52 d, 2C, J(P,C) = 138.4 (C-40); 16.08 m, J(P,C) = 5.9 (Et). Anal.
Calcd for C9H15N2O7P.1/2H2O: C, 35.65; H, 5.32; N, 9.24. Found:
C, 35.24; H, 5.20; N, 8.97. MS (ESI): m/z = 351 [M+H]+.
Cytostatic activities against L1210 (murine leukemia), FM3A
(murine mammary carcinoma) and CEM (human T-lymphoblast)
cell lines were measured essentially as originally described for
the mouse leukemia L1210 cell assays.13
5.34. 1-[2-(1-Phosphonopropan-2-yloxy)ethyl]cytosine (18)
Starting from 17, yield 38%. 1H NMR (DMSO-d6): 7.58 d, 1H,
J(6,5) = 7.2 (H-6); 7.48 br s, 2H (NH2); 5.67 d, 1H, J(5,6) = 7.2 (H-
5); 3.76 dd, 2H, J(10,20) = 10.8 and 5.3 (H-10); 3.62 m, 1H (H-30);
3.52 t, 2H, J(20,10) = 5.2 (H-20); 1.86 m, 1H (H-40a); 1.55 m, 1H (H-
40b); 1.15 d, 3H, J(30,50) = 6.1 (H-50). 13C NMR (DMSO-d6): 164.84
(C-4); 154.39 (C-2); 147.28 (C-6); 92.55 (C-5); 71.47 (C-30); 64.91
(C-20); 48.62 (C-10); 35.52 d, J(P,C) = 132.1 (C-40); 20.81 d,
J(P,C) = 4.5 (C-50). Anal. Calcd for C9H16N3O5P.1/2H2O.1/2MeOH:
C, 38.35; H, 6.09; N, 14.50. Found: C, 38.20; H, 5.88; N, 14.22. MS
(ESIꢁ): m/z = 276 [MꢁH]ꢁ.
Acknowledgments
This work is a part of the research project AVOZ40550506 of the
Institute of Organic Chemistry and Biochemistry and was sup-
ported by the Grant Agency of the Czech Republic (Grant No.
P207/11/0108) and Centre for New Antivirals and Antineoplastics
(1M0508, Ministry of Education, Youth and Sports of the Czech
Republic). This study was also supported by Gilead Sciences, Inc.
(Foster City, CA, USA) and the ‘‘Geconcerteerde Onderzoeksacties’’
(GOA), Krediet nr. 10/014’’ of the KU Leuven. We also thank Anita
Camps, Steven Carmans, Frieda De Meyer, Leentje Persoons, Leen
Ingels, Wim Van Dam, Lies Van den Heurck and Lizette van Berc-
kelaer for excellent technical assistance with the antiviral/cyto-
static assays.
5.35. Antiviral activity assays
The compounds were evaluated against the following viruses:
herpes simplex virus type
1 (HSV-1) strain KOS, thymidine
kinase-deficient (TK-) HSV-1 KOS strain resistant to ACV (ACVr),
herpes simplex virus type 2 (HSV-2) strains Lyons and G, vari-
cella-zoster virus (VZV) strain Oka, TK-VZV strain 07-1, human
cytomegalovirus (HCMV) strains AD-169 and Davis, vaccinia virus
Lederle strain, human immunodeficiency virus (HIV) type 1 (IIIB)
and type 2 (ROD), respiratory syncytial virus (RSV) strain Long,
vesicular stomatitis virus (VSV), Coxsackie B4, Parainfluenza 3,
Reovirus-1, Sindbis, Punta Toro, feline coronavirus (FIPV), influenza
A virus subtypes H1N1 and H3N2, and influenza B virus. The anti-
viral assays, other than HIV, were based on inhibition of virus-in-
duced cytopathicity or plaque formation in human embryonic
lung (HEL) fibroblasts, African green monkey kidney cells (Vero),
human epithelial cervix carcinoma cells (HeLa), Crandell-Rees fe-
line kidney cells (CRFK), or Madin Darby canine kidney cells
(MDCK). Confluent cell cultures in microtiter 96-well plates were
inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose
to infect 50% of the cell cultures) or with 20 plaque forming units
(PFU) and the cell cultures were incubated in the presence of vary-
ing concentrations of the test compounds. Viral cytopathicity or
plaque formation (VZV) was recorded as soon as it reached com-
pletion in the control virus-infected cell cultures that were not
treated with the test compounds. Antiviral activity was expressed
as the EC50 or compound concentration required to reduce virus-
induced cytopathicity or viral plaque formation by 50%. The meth-
odology of the anti-HIV assays was as follows: human CEM
(ꢃ3 ꢀ 105 cells/ml) were infected with 100 CCID50 of HIV-1(IIIB)
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Cytostatic activity measurements were based on the inhibition
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