Synthesis and antiviral activity evaluation of acyclic 2′-azanucleosides bearing a phosphonomethoxy function in the side chain

Article abstract of DOI:10.1016/j.bmc.2009.04.054

Acyclic 2′-azanucleosides with a phosphonomethoxy function in the side chain were obtained by coupling of diethyl {2-[N-(pivaloyloxymethyl)-N-(p-toluenesulfonyl)amino]ethoxymethyl}phosph onate with the pyrimidine nucleobases via the Vorbrueggen-type proto

Full text of DOI:10.1016/j.bmc.2009.04.054

Bioorganic & Medicinal Chemistry 17 (2009) 3756–3762
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Bioorganic & Medicinal Chemistry
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Synthesis and antiviral activity evaluation of acyclic 20-azanucleosides bearing
a phosphonomethoxy function in the side chain
a,
b
b
*
´
Mariola Koszytkowska-Stawinska , Erik De Clercq , Jan Balzarini
a Faculty of Chemistry, Warsaw University of Technology, ul. Noakowskiego 3, 00-664 Warszawa, Poland
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Acyclic 20-azanucleosides with a phosphonomethoxy function in the side chain were obtained by coupling
of diethyl {2-[N-(pivaloyloxymethyl)-N-(p-toluenesulfonyl)amino]ethoxymethyl}phosphonate with the
pyrimidine nucleobases via the Vorbrüggen-type protocol. The compounds were evaluated in vitro for
activity against a broad variety of RNA and DNA viruses.
Article history:
Received 28 January 2009
Revised 23 April 2009
Accepted 25 April 2009
Available online 3 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Azanucleosides
Acyclic nucleoside phosphonates
Nucleoside analogs
Antiviral agents
Sulfonamides
1. Introduction
moiety (Fig. 1, analogues 1, 2 and 3, respectively)5 as well as N-
(phosphonomethyl)prolinol derivatives 46 (Fig. 1) have also been
Nucleoside analogues need to be in vivo phosphorylated to their
triphosphates to display antiviral activity.1 The first step of this
three-step phosphorylation sequence, leading to the corresponding
monophosphates, is essential for their activity. Low activity of
nucleoside analogues may often result from poor in vivo monopho-
sphorylation. To circumvent this problem, acyclic nucleoside phos-
phonates (ANPs), that is, derivatives with a phosphonate P–C–O-
linkage instead of a phosphoester P–O–C one, were developed.2
Since ANPs are stable analogues of nucleoside monophosphates,
in contrast to the ‘classical’ acyclic nucleoside analogues (such as
acyclovir, ganciclovir or penciclovir),1 they do not require activa-
tion by the first phosphorylation.3 Moreover, the presence of the
phosphonate group makes ANPs resistant towards phosphomo-
noesterases and nucleotidases, enzymes involved in nucleotide
catabolism. Owing to the specific mode of action, ANPs enhance
a broad spectrum of antiviral activity.3 Several of them (i.e., cidofo-
vir, adefovir, and tenofovir, Fig. 1) have been approved for the
treatment of viral infections.3 However the therapeutic use of the
ANP drugs is limited by poor oral bioavailability, potential nephro-
toxicity or development of resistant viral strains.3 In attempts to
overcome these problems, a variety of structural analogues were
synthesized and biologically evaluated.4 Among them, compounds
with the aza function at the 10-, 30-, or 40-position of the acyclic
developed. Among the synthesized derivatives, compounds 2
(B = Gua or Cyt) were endowed with a weak anti-herpes virus
activity.5a
These findings inspired us to evaluate the antiviral activity of
compounds 8 (Scheme 1) being phosphonomethyl ethers of the
previously reported, antivirally inactive N-(2-hydroxyethyl) azanu-
cleosides.7c Considering a unique mode of action of ANPs, we
decided to examine the effect of the O-phosphonomethyl modifica-
tion of the parent hydroxy derivatives on their antiviral activity.
Compounds 8 can be regarded as aza-congeners of cidofovir. Tak-
ing into account the location of the aza function at the 20-position
of the side chain, they can be also considered as complementary to
the abovementioned 10-, 30-, and 40-aza analogues of the acyclic
nucleoside phosphonates.
2. Results and discussion
Generally, the synthetic approaches reported for the aforemen-
tioned acyclic aza-analogues 1–3 (Fig. 1) involved two key steps:
(a) transformation of a nucleobase into the corresponding N-ami-
no- or N-aminoalkyl derivative; and (b) alkylation (or acylation)
of the resultant precursor at the exocyclic amino group with a spe-
cific synthon bearing a dialkylphosphoryl function, followed by
deprotection of the intermediate phosphonate esters.5 Our strategy
for the synthesis of the target 20-azanucleosides 8 is outlined in
Scheme 1. Based on our previous experience in synthesizing
* Corresponding author. Tel.: +48 22 234 5442; fax: +48 22 628 2741.
E-mail address: mkoszyt@ch.pw.edu.pl (M. Koszytkowska-Stawin´ ska).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.054

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M. Koszytkowska-Stawinska et al. / Bioorg. Med. Chem. 17 (2009) 3756–3762
1'
B
Ade
O
B
3'
N
H
H
N
X
(*)
R
HO
O
P(O)(OH)2
P(O)(OH)2
P(O)(OH)2
R
B
1
: X = CH2 or C=O
2: B = Ade, Gua, Ura, Cyt
Cidofovir
Adefovir
Tenofovir
(S)-CH2OH Cyt
H
)-CH
Ade
Ade
OH
B
H
B
N
(
R
3
N
P(O)(OH)2
HO
4'
P(O)(OH)2
3
: B = Ade, Gua, Ura, Cyt
4: B = Ade, Gua, Thy, Ura, Cyt
Figure 1. The ANP drugs and their aza analogues.
R1
R2
N
N
Pyrimidine
nucleobase
OPiv
O
N
N
X
O
P(OR)2
O
Ts
O
P(OR)2
O
7
Ts
O
P(OH)2
O
Vorbrüggen's
conditions
5
(X = CH2CH2OMs)
8
or 6 (X = H)
R1 = H, CH3, F
R2= OH, NH2
R = alkyl
Scheme 1. Synthetic strategy for the target 2’-azanucleosides 8.
nucleoside aza-analogues,7 we decided to achieve compounds 8 via
simultaneous introduction of the aza and dialkylphosphoryl func-
tion to the nucleobase in one reaction step, that is, by N-alkylation
of the nucleobase with the phosphonylated N-(pivaloyloxymeth-
yl)sulfonamide 7 under Vorbrüggen’s conditions.
(thymine, 5-fluorouracil or uracil) was performed according to
the one-pot base silylation/nucleoside coupling procedure using
N,O-bis(trimethylsilyl)acetamide (BSA) as the silylating agent and
tin(IV) chloride as the Lewis acid [Scheme 3, conditions (i)]. The
thymine derivative 13a and the 5-fluorouracil derivative 13b were
Synthesis of N-(pivaloyloxymethyl)sulfonamide 7 from diethyl
(2-mesyloxyethoxy)methylphosphonate8 5 or diethyl hydroxy-
methylphosphonate 6 is shown in Scheme 2. Starting from diethyl
(2-mesyloxyethoxy)methylphosphonate 5, the p-toluenesulfonyla-
mino function of 7 was introduced by two methods [Scheme 2,
conditions (i)–(iii) or (iv)]. The first one involved the following
steps: (i) transformation of 5 into azide 9 using sodium azide at
room temperature;9 (ii) reduction of 9 to amine 10 under Stauding-
er’s conditions;9 and (iii) N-sulfonation of 10 with p-toluenesulfo-
nyl chloride; the overall yield of 11 was 59%. The second method
[conditions (iv)], that is, direct reaction of 5 with p-toluenesulfon-
amide in the presence of anhydrous potassium carbonate, afforded
11 in 68% yield. We also tried to prepare 11 by reaction of diethyl
obtained as the only products in a yield of 83% and 55%,
respectively. The only exception was the reaction of 7 with uracil;
the desired azanucleoside 13c (the major product, 73%) and 1,3-
disubstituted uracil 14 (7%) were obtained. The minor product 14
was readily separated by column chromatography. Coupling of 7
with N4-benzoylcytosine under the same conditions, followed by
treatment of the crude product (not shown) with ammonium
hydroxide gave the cytosine derivative 13d in 80% yield [Scheme
3, conditions (ii)].
The N-1 substitution pattern of the aza analogues 13 was con-
firmed by the interactions observed in the 2D NMR spectra of
13a and 13b (Fig. 2). In the 1H–1H ROESY spectrum of 13a, the
1H–1H interaction between H-6 of thymine and the H-10 protons
was seen. In the 1H–13C HMBC spectrum of 13b, in turn, the
1H–13C couplings of the H-10 protons with both C-6 and C-2 of 5-
fluorouracil, as well as the coupling between H-6 and C-10 were
observed.
hydroxymethylphosphonate
6 with N-(p-toluenesulfonyl)aziri-
dine10 12 in the presence of sodium hydride [conditions (v)]. How-
ever, this approach was not satisfactory; 11 was obtained in a low
yield of 23%. In the next step [conditions (vi)], N-alkylation of 11
with chloromethyl pivalate in the presence of anhydrous potas-
sium carbonate gave N-(pivaloyloxymethyl)sulfonamide 7 in a
high yield of 96%. Reaction of 7 with the pyrimidine nucleobases
The final step [Scheme 3, conditions (iii)], dealkylation of 13
with bromotrimethylsilane (TMSBr) followed by treatment of the
reaction mixture with a water/acetone mixture, afforded the
iv: TsNH2
(68%)
H
OPiv
iii: TsCl
(79%)
vi:PivO Cl
(96%)
N
N
R
i
O
P(OEt)2
O
Ts
O
P(OEt)2
O
11
Ts
O
P(OEt)2
O
7
5
,
R = OMs
9
,
R = N3 (93%)
, R = NH2 (80%)
v
ii
10
(23%)
+
HO P(OEt)2
N
Ts
12
Ts = 4-CH3-C6H4-SO2-
Ms = CH3SO2-
O
Piv = (CH3)3CC(O)-
6
Scheme 2. Reagents and conditions: (i) NaN3, DMF, rt, 3 d; (ii) (1) Ph3P, toluene, rt, 1 h; (2) H2O; (iii) NEt3, DCM, rt, 20 h; (iv) K2CO3, DMF, 50 °C, 3 d; (v) NaH, DMF, rt, 10 h;
(vi) K2CO3, DMF, rt, 3 d.

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M. Koszytkowska-Stawinska et al. / Bioorg. Med. Chem. 17 (2009) 3756–3762
CH3
NH2
O
N
N
N
NH
OPiv
O
O
4-Bz-Cytosine
i: Thymine
ii: N
N
N
N
O
P(OR)2
O
O
P(OEt)2
O
O P(OR)2
Ts
Ts
Ts
O
13d
8d
, R = Et (80%)
, R = H (82%)
13a
8a
, R = Et (83%)
, R = H (78%)
7
iii
iii
i: Uracil
i: 5-F-Uracil
F
O
NH
O
NH
O
N
N
N
N
+
O
O
O
O
O
N
N
N
N
(EtO)2P
P(OR)2
O
P(OEt)2
O
O
Ts
P(OR)2
O
O
Ts
Ts
Ts
O
14
13c
8c
(7%)
, R = Et (73%)
13b, R = Et (55%)
8b, R = H (63%)
iii
iii
, R = H (80%)
Scheme 3. Reagents and conditions: (i) (1) nucleobase (thymine, 5-F-uracil or uracil), BSA, CH3CN, rt, 1 h; (2) 7, SnCl4/CH2Cl2, CH3CN, rt, 2 d; (ii) (1) N4-Bz-cytosine, BSA,
CH3CN, rt, 1 h; (2) 7, SnCl4/CH2Cl2, CH3CN, rt, 2 d; (3) NH4OH, MeOH, rt, 20 h; (iii) (1) TMSBr, CH3CN, rt, 20 h; (2) acetone, H2O, rt, 2 h.
reovirus-1, Sindbis virus, Coxsackie B4 virus, or Punta Toro virus;
(d) HEL cells infected with cytomegalovirus (AD-169 or Davis
strain); (e) HEL cells infected with varicella-zoster virus (TK+ strain
OKA or TKꢀ strain 07/1); and (f) Crandell-Rees Feline Kidney
(CRFK) cells infected with feline corona virus or feline herpes virus.
The antiviral activity was expressed as (i) the effective concentra-
tion (EC50) required to reduce virus-induced cytopathic effect by
50%, as determined by visual scoring of the CPE [assays (a)–(c)]
or by measuring the cell viability with the colorimetric forma-
zan-based MTS assay [assays (f)]; or (ii) the effective concentration
(EC50) required to reduce virus plaque formation by 50% [assays (d)
and (e)]. None of the evaluated compounds, at the highest (subtox-
F
CH3
H-1', 5.23 ppm
C-1, 61.08 ppm
6
H6
H
O
NH
O
H-1', 5.24 ppm
H-6, 7.37 ppm
2
N
N
H
H
H
H
NH
O
O
1'
1'
H-6, 7.73 ppm
C-6, 127.67 ppm
O
O
N
N
P(OEt)2
O
P(OEt)2
O
Ts
Ts
C-2, 150.08 ppm
13a
13b
Figure 2. The principal 1H–1H ROESY correlation and the 1H–13C HMBC couplings
observed for 13a and 13b, respectively.
desired phosphonic acids 8 in 63–82% yield. Structure and purity of
derivatives 8a–c was verified by standard NMR and IR techniques
as well as by elemental analysis. In contrast to 8a–c, solubility of
the cytosine derivative 8d in water (5 mg/ca. 100 ml of water)
and in conventional organic solvents, including dimethylformam-
ide and dimethylsulfoxide, was very low. For this reason, only IR
spectrum and elemental analysis was performed for 8d. Therefore,
in an attempt to transform 8d into the corresponding sodium salt,
a suspension of 8d in a water/DMSO mixture was shaken with
DOWEXÒ 88 ion-exchange resin (Na-form). Contrary to our expec-
tation that 8d would gradually dissolve upon the treatment with
the ion-exchange resin, the suspension (which was then identified
by elemental analysis as the unchanged 8d) was still observed.
Therefore, the time of shaking was prolonged for six months. Nev-
ertheless, the conversion of 8d to its sodium salt was not satisfac-
tory. Filtration of the unchanged 8d and evaporation of the
solvents from the filtrate afforded a small amount of a solid (8d–
s, 15 mg from 50 mg of 8d) which was identified by elemental
analysis as an equimolar mixture of 8d and its monosodium salt
(not shown). The 1H NMR spectrum of the mixture allowed us to
confirm the structure of 8d. Unfortunately, solubility of 8d–s in
DMSO or in water was too low to measure the 13C NMR spectrum.
ic) concentrations used (i.e., 10–100
viruses examined.
lg/mL), was active against the
4. Conclusion
A series of 20-azanucleosides 8a–d containing the phosphono-
methoxy function in the side chain were obtained by coupling of
diethyl
{2-[N-(pivaloyloxymethyl)-N-(p-toluenesulfonyl)amino]
ethoxymethyl}phosphonate 7 with the pyrimidine nucleobases
under the Vorbrüggen’s conditions followed by dealkylation
of the intermediate phosphonate esters. The biological results
demonstrate that replacement of the 3-hydroxy-2-(phosphono-
methoxy)-propyl moiety in cidofovir with the N-(2-phosphono-
methoxyethyl)-N-(p-toluenesulfonyl)aminoethyl function causes
annihilation of the antiviral activity at subtoxic concentrations.
This lack of antiviral activity may be due to the poor cell membrane
permeability of the free phosphonic acids 8, resulting from the
polarity of the phosphono group.2 Considering the fact that mask-
ing of the phosphono group with a lipophilic function decreases
the polarity of ANPs,2,11 we have undertaken research on such
modification of 8. This study as well as antiviral activity evaluation
of masked derivatives of 8, including diesters 13, will be a subject
of a forthcoming publication.
3. Antiviral activity evaluation
5. Experimental
Compounds 8a–d and the 8d–s mixture were evaluated in vitro
for activity against a variety of RNA and DNA viruses, using the fol-
lowing cell-based assays: (a) HEL cells infected with herpes sim-
5.1. Materials and methods
plex virus type
1 (KOS), herpes simplex virus type 2 (G),
acyclovir-resistant herpes simplex virus type 1 (TKꢀ KOS ACVr),
vaccinia virus, or vesicular stomatitis virus; (b) HeLa cells infected
with vesicular stomatitis virus, Coxsackie B4 virus, or respiratory
syncytial virus; (c) Vero cells infected with parainfluenza-3 virus,
Pre-coated Merck Silica Gel 60 F-254 plates were used for thin-
layer chromatography (TLC, 0.2 mm), spots were detected under
UV light (254 nm). Column chromatography was performed using
silica gel (200–400 mesh, Merck). High Resolution Mass Spectra

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M. Koszytkowska-Stawinska et al. / Bioorg. Med. Chem. 17 (2009) 3756–3762
(Electrospray Ionisation, ESI) were performed on a MarinerÒ spec-
trometer in positive ionization mode. The IR spectra were recorded
on a Perkin–Elmer System 2000 spectrometer in KBr disc; resolu-
dropwise within 15 min. The mixture was stirred at room tempera-
ture overnight, diluted with dichloromethane (100 mL), washed with
brine (2 ꢁ 20 mL), and dried. The solvent was distilled off. The resi-
due was purified by column chromatography (chloroform/acetone,
98:2?80:20, v/v) to give 11 as an oil (2.85 g, 79%). dH (CDCl3,
tion was 2 cmꢀ1; absorption maxima ( max) are given in cmꢀ1
m
and quoted as ‘s’ strong, ‘m’ moderate, ‘w’ weak, ‘br’ broad. The
1H NMR spectra were measured on a Varian Gemini-200BB
400 MHz) 1.34 (t, JH–H 7.0, 6H), 2.43 (s, 3H), 3.12–3.16 (m, 2H),
3
2
(200 MHz) or on
a
Varian Mercury-400BB spectrometer
3.62–3.65 (triplet-like multiplet, 2H), 3.73 (d, JH–P 8.0, 2H), 4.16
(400 MHz). The 13C NMR spectra were recorded on a Varian Gem-
ini-200BB spectrometer at 50 MHz. 1H and 13C chemical shifts (d)
are reported in parts per million (ppm) relative to the solvent sig-
nals (CDCl3, dH (residual CHCl3) 7.26 ppm, dC 77.16 ppm; or DMSO-
d6, dH (residual DMSO-d5) 2.50 ppm, dC 39.52 ppm), or relative to
2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS), which was used
as an internal standard for the spectra taken in D2O; signals are
quoted as ‘s’ (singlet), ‘d’ (doublet), ‘t’ (triplet), ‘q’ (quartet), ‘m’
(multiplet), and ‘br s’ (broad singlet). Coupling constants (J) are
reported in Hertz. The 1H–1H ROESY (Rotating frame Overhauser
Effect Spectroscopy) for 13a was measured on a Varian Mercury-
400BB spectrometer in CDCl3. The 1H–13C HMBC (Heteronuclear
Multiple Bond Correlation) for 13b was measured on a Varian
VNMRS 500 spectrometer in CDCl3. Elemental analyses were
performed in a Perkin Elmer 2400 apparatus. Anhydrous MgSO4
was employed as a drying agent. Solvents were distilled off under
reduced pressure in a rotating evaporator.
(dq, JH–P 8.4, JH–H 7.0, 4H), 5.29 (triplet-like multiplet, 1H, NH),
7.29–7.31 (m, 2H), 7.73–7.76 (m, 2H). dC (CDCl3, 50 MHz) 16.55 (d,
3
3
2
1
3JC–P 5.7), 21.52, 42.87, 62.54 (d, JC–P 6.5), 65.33 (d, JC–P 166.2),
3
71.98 (d, JC–P 9.9), 127.09, 129.70, 137.08, 143.40. mmax (NaCl)
1162s (S@O), 1330s (S@O), 3151 m (N–H). HRMS m/z C14H24NO6-
NaPS (M+Na)+ 388.0954, found 388.0951.
5.2.2. Method B [Scheme 2, conditions (iv)]
A mixture of 5 (1.3 g, 4.5 mmol), p-toluenesulfonamide (7.67 g,
45.0 mmol), anhydrous potassium carbonate (0.62 g, 4.5 mmol),
and anhydrous DMF (15 mL) was stirred at 50 °C (an oil bath) for
3 days and then evaporated to dryness under vacuum at 80 °C
(0.05 mmHg). The residue was diluted with ethyl ether (100 mL)
and filtered through a Celite pad. The filtrate was washed with
water (3 ꢁ 20 mL), dried, filtered, and evaporated to dryness under
reduced pressure. The residue was purified by column chromatog-
raphy (chloroform/acetone, 98:2?80:20, v/v) to afford 11 as an oil
(1.12 g, 68%).
5.2. Preparation of diethyl [2-(p-toluenesulfonylamino)ethoxy]-
methylphosphonate (11)
5.2.3. Method C [Scheme 2, conditions (v)]
A mixture of diethyl hydroxymethylphosphonate 6 (0.57 g,
3.4 mmol), sodium hydride (60% suspension in mineral oil,
0.14 g, 3.4 mmol) and anhydrous DMF (10 mL) was stirred in an
ice-water bath for 1 h under argon atmosphere. N-(p-Toluenesulfo-
nyl)aziridine 12 (0.34 g, 1.7 mmol) was added in one portion. After
1 day of stirring at room temperature, the mixture was poured into
water (75 mL) and extracted with ethyl acetate (3 ꢁ 20 mL). The
combined extracts were washed with water (2 ꢁ 10 mL) and dried.
The low boiling solvents were distilled off under reduced pressure
and residual DMF was removed under vacuum at 80 °C
(0.05 mmHg). The residue was purified by column chromatogra-
phy (chloroform) to yield 11 as an oil (0.14 g, 23%).
5.2.1. Method A [Scheme 2, conditions (i)–(iii)]
5.2.1.1. Diethyl (2-azidoethoxy)methylphosphonate (9)9. A mix-
ture of diethyl (2-mesyloxyethoxy)methylphosphonate 5 (3.0 g,
10.3 mmol), sodium azide (6.72 g, 103.0 mmol) and anhydrous
DMF (60 mL) was stirred at room temperature for 3 days and then
evaporated to dryness under vacuum at 80 °C (0.05 mmHg). The res-
idue was diluted with water (50 mL) and extracted with dichloro-
methane (3 ꢁ 20 mL). The combined extracts were dried, filtered,
and evaporated to dryness under reduced pressure to give 9 as an
oil (2.28 g, 93%). An analytical sample was obtained by column chro-
3
matography (chloroform). dH (CDCl3, 200 MHz) 1.34 (td, JH–H 7.0,
4JH–P 0.4, 6H), 3.37–3.42 (triplet-like multiplet, 2H), 3.73–3.78 (trip-
2
3
let-like multiplet, 2H), 3.84 (d, JH–P 8.2, 2H), 4.17 (dq, JH–P 8.2, 3J
5.3. Diethyl {2-[N-(pivaloyloxymethyl)-N-(p-toluenesulfonyl)-
amino]ethoxymethyl}phosphonate (7)
3
7.0, 4H). dC (CDCl3, 50 MHz) 16.61 (d, JC–P 5.7), 50.77, 62.71 (d,
H–H
1
3
2JC–P 6.5), 65.57 (d, JC–P 165.0), 72.20 (d, JC–P 10.2).
Chloromethylpivalate(6.39 g, 42.4 mmol, 6.1 mL)was addedtoa
stirred mixture of 11 (3.1 g, 8.5 mmol) and anhydrous potassium
carbonate (11.7 g, 85.0 mmol) in dry DMF (50 mL) at room temper-
ature. The mixture was stirred for 5 days at room temperature and
then poured into ice-cold water (250 mL). The organic phase was ex-
tracted with dichloromethane (3 ꢁ 50 mL). The extracts were com-
bined and washed with water (2 ꢁ 20 mL), dried and filtered. The
solvent was distilled off. The residue was purified by column chro-
matography (chloroform) to give 7 as an oil (3.92 g, 96%). dH (CDCl3,
400 MHz) 0.99 (s, 9H), 1.35 (t, 3JH–H 7.2, 6H), 2.42 (s, 3H), 3.36–3.42
(triplet-like multiplet, 2H), 3.78–3.81 (m, 4H), 4.17 (dq, 3JH–P 8.0, 3J
5.2.1.2. Diethyl (2-aminoethoxy)methylphosphonate (10)9.
A
mixture of triphenylphosphine (4.88 g, 18.6 mmol) and dry toluene
(20 mL) was cooled on an ice-water bath and a solution of 9
(2.94 g, 12.4 mmol) in dry toluene (2.5 mL) was added dropwise
within 30 min. The mixture was stirred at room temperature for
1 h. Water was then added and stirring was continued for
15 min. The aqueous layer was separated, extracted with diethyl
ether (3 ꢁ 20 mL), and evaporated to dryness under vacuum at
80 °C (0.05 mmHg) to afford 10 as an oil (2.09 g, 80%). An analytical
sample was obtained by column chromatography (chloroform/
3
4
methanol, 95:5, v/v). dH (CDCl3, 200 MHz) 1.34 (td, JH–H 7.0, JH–P
0.4, 6H), 1.55 (br s, 2H, NH2), 2.84–2.89 (triplet-like multiplet,
7.2, 4H), 5.53 (s, 2H), 7.28–7.30 (m, 2H), 7.73–7.76 (m, 2H). dC
H–H
3
(CDCl3, 50 MHz) 16.63 (d, JC–P 6.1), 21.64, 26.93, 45.91, 62.68 (d,
2
1
3
2H), 3.57–3.63 (triplet-like multiplet, 2H), 3.80 (d, JH–P 8.4, 2H),
2JC–P 6.5), 65.36 (d, JC–P 165.8), 71.86 (d, JC–P 9.4), 73.16, 127.73,
3
3
4.17 (dq, JH–P 8.0, JH–H 7.0, 4H). dC (CDCl3, 50 MHz) 16.63 (d,
129.98, 137.08, 143.99, 177.43. mmax (NaCl) 1729s (C@O). HRMS m/
3JC–P 5.3), 41.77, 62.53 (d, JC–P 6.8), 62.30 (d, JC–P 165.8), 75.96
(d, JC–P 10.7).
z C20H34NO8NaPS (M+Na)+ 502.1635, found 502.1653.
2
1
3
5.4. General procedure for coupling of 7 with thymine or 5-
fluorouracil
5.2.1.3. Diethyl [2-(p-toluenesulfonylamino)ethoxy]methylphos-
phonate (11). A mixture of 10 (2.09 g, 9.86 mmol), triethylamine
(2.0 g, 19.7 mmol, 2.8 mL) and dichloromethane (10 mL) was cooled
in an ice-water bath and a solution of p-toluenesulfonyl chloride
(2.8 g, 14.8 mmol) in dichloromethane (10 mL) was then added
A
mixture of a nucleobase (thymine or 5-fluorouracil)
(2.0 mmol) and N,O-bis(trimethylsilyl)acetamide (BSA, 815 mg,
4.0 mmol, 1.0 mL) in dry acetonitrile (10 mL) was stirred at room

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M. Koszytkowska-Stawinska et al. / Bioorg. Med. Chem. 17 (2009) 3756–3762
3
3
4
temperature for 1 h under argon atmosphere. A solution of 7
(1.0 mmol) in dry acetonitrile (1 mL), and subsequently 1 M solu-
tion of tin(IV) chloride in dichloromethane (3 mL) was added.
The reaction mixture was kept at room temperature for 2 days.
Ethyl acetate (50 mL) and then a saturated sodium bicarbonate
solution (1 mL) was added. The mixture was stirred for 1 h and fil-
tered through a Celite pad. The organic phase was separated,
washed with brine (3 ꢁ 10 mL), dried and filtered. The volatiles
were distilled off. The residue was purified by column chromatog-
raphy (chloroform/methanol, 98:2, v/v) to yield azanucleoside 13a
or 13b.
8.2, JH–H 7.2, 4H), 5.26 (s, 2H), 5.71 (dd, JH–H 8.2, JH–H 2.2, 1H),
7.29–7.31 (m, 2H), 7.61–7.64 (m, 3H), 9.14 (br s, 1H, NH). dC (CDCl3,
50 MHz) 16.63 (d, 3JC–P 5.3), 21.66, 48.43, 61.35, 62.58 (d, 2JC–P 6.5),
1
3
65.27 (d, JC–P 165.4), 72.33 (d, JC–P 10.6), 102.64, 126.99, 130.10,
136.68, 144.25, 144.46, 151.24, 163.47. mmax (KBr) 1694br s
(C@O). HRMS m/z C19H28N3O8NaPS (M+Na)+ 512.1227, found
512.1240.
5.5.2. 1,3-Bis{N-(p-toluenesulfonyl)-N-[2-(diethylphosphoryl-
methoxy)ethyl]aminomethyl}-1H,3H-pyrimidin-2,4-dione (14)
dH (CDCl3, 200 MHz) 1.19 (m, 12H), 2.39 (s, 6H), 3.54–3.74 (m,
12H), 4.06–4.20 (m, 8H), 5.24 (s, 2H), 5.33 (s, 2H), 5.64 (d, JH–H
8.0, 1H), 7.28–7.37 (m, 4H), 7.52 (d, JH–H 8.0, 1H), 7.60–7.76 (m,
3
3
5.4.1. Diethyl {2-[N-(5-methyl-1H,3H-pyrimidin-2,4-dion-1-
ylmethyl)-N-(p-toluenesulfonyl)amino]ethoxymethyl}phos-
phonate (13a)
3
4H). dC (CDCl3, 50 MHz) 16.60 (d, JC–P 5.3), 21.63, 47.97, 49.48,
2
1
54.57, 61.85, 62.56 (d, JC–P 6.5), 65.17 (d, JC–P 165.4), 72.17 (d,
3
According to the general procedure, 13a was obtained from 7
(960 mg, 2.0 mmol) and thymine. Chromatographic purification
gave 13a (834 mg, 83%) as an oil. dH (CDCl3, 400 MHz) 1.32 (t,
3JC–P 10.4), 72.31 (d, JC–P 10.8), 102.00, 127.02, 127.54, 129.70,
130.08, 136.67, 136.92, 142.58, 143.69, 144.39, 151.67, 162.54.
mmax (KBr) 1699br
s (C@O). HRMS m/z C34H52N4NaO14P2S2
3JH–H 7.2, 6H), 1.90 (d, JH–H 0.8, 3H), 2.41 (s, 3H), 3.55–3.58 (trip-
(M+Na)+ 889.2289, found 889.2311.
4
3
let-like multiplet, 2H), 3.68–3.72 (m, 4H), 4.14 (dq, JH–P 8.2,
3JH–H 7.2, 4H), 5.24 (s, 2H), 7.26–7.31 (m, 2H), 7.60–7.65 (m, 2H),
5.6. Diethyl {2-[N-(4-amino-1H-pyrimidin-2-on-1-ylmethyl)-N-
(p-toluenesulfonyl)amino]ethoxymethyl}phosphonate (13d)
4
7.37 (q, JH–H 0.8, 1H), 8.98 (br s, 1H, NH). dC (CDCl3, 50 MHz)
3
2
12.40, 16.62 (d, JC–P 5.7), 21.65, 48.26, 60.99, 62.58 (d, JC–P 6.5),
1
3
65.25 (d, JC–P 165.8), 72.20 (d, JC–P 10.2), 111.16, 126.98, 130.03,
136.93, 139.85, 144.39, 151.37, 164.05. mmax (KBr) 1689br s
(C@O). HRMS m/z C20H30N3O8NaPS (M+Na)+ 526.1389, found
526.1390.
According to the procedure used for the coupling of 7 with thy-
mine or 5-fluorouracil, a solution of the silylated N4-benzoylcyto-
sine in dry acetonitrile (10 mL) was prepared from N4-
benzoylcytosine (435 mg, 2.0 mmol) and BSA (815 mg, 4.0 mmol,
1.0 mL) and then treated with a solution of 7 (480 mg, 1.0 mmol)
in dry acetonitrile (1 mL) and 1 M solution of tin(IV) chloride in
dichloromethane (3 mL). After workup, the reaction mixture was
passed through a short chromatographic column (chloroform/
methanol, 98:2, v/v) in order to separate the unreacted N4-ben-
zoylcytosine. Fractions with RF value of 0.25 were combined and
evaporated to dryness. The residue was dissolved in methanol
(10 mL) and aqueous ammonium hydroxide (25 mL) was added.
The mixture was kept at room temperature overnight. The volatiles
were distilled off, and the residue was purified by column chroma-
tography (chloroform/methanol, 95:5, v/v) to afford 13d (392 mg,
80%) as a white solid; mp 135–137 °C. dH (CDCl3, 400 MHz) 1.32
(t, JH–H 7.2, 6H), 2.40 (s, 3H), 3.59–3.61 (triplet-like multiplet,
2H), 3.67–3.73 (m, 4H), 4.14 (dq, JH–P 8.0, JH–H 7.2, 4H), 5.29 (s,
2H), 5.86 (d, JH–H 7.2, 1H), 6.38 (br s, 2H, NH2), 7.25–7.28 (m,
2H), 7.60–7.63 (m, 3H). dC (CDCl3, 50 MHz) 16.70, 21.66, 47.75,
61.64, 62.72, 65.12 (d, JC–P 166.2), 72.14 (d, JC–P 10.4), 95.55,
127.00, 130.02, 136.88, 144.11, 145.00, 156.98, 166.53. mmax (KBr)
1656s (C@O), 3118 m (N–H), 3363 m (N–H). HRMS m/z
C19H29N4O7NaPS (M+Na)+ 511.1387, found 511.1412.
5.4.2. Diethyl {2-[N-(5-fluoro-1H,3H-pyrimidin-2,4-dion-1-yl-
methyl)-N-(p-toluenesulfonyl)amino]ethoxymethyl}phospho-
nate (13b)
According to the general procedure, 13b was obtained from 7
(487 mg, 1.0 mmol) and 5-fluorouracil. Chromatographic purifica-
tion gave 13b (285 mg, 55%) as an oil. dH (CDCl3, 400 MHz) 1.32
3
[t, JH–H 7.2, 6H, (CH3–CH2–O)2P(O)–], 2.43 (s, 3H, CH3–C6H4–SO2–
), 3.52–3.54 (triplet-like multiplet, 2H, –O–CH2–CH2–N–), 3.68–
3
3.72 [m, 4H, –O–CH2–CH2–N–, –P(O)–CH2–O–], 4.14 [dq, JH–P 8.4,
3JH–H 7.2, 4H, (CH3–CH2–O)2P(O)–], 5.23 (s, 2H, –N–CH2–N–),
7.31–7.34 (m, 2H, Ar-H-ortho to CH3), 7.65–7.68 (m, 2H, Ar-H-
3
3
ortho to SO2), 7.73 (d, JH–F 5.2, 1H, –N–CH@CF–), 9.31 (br s, 1H,
3
3
3
NH). dC (CDCl3, 50 MHz) 16.23 [d, JC–P 5.3, (CH3–CH2–O)2P(O)–],
3
21.31 (CH3–C6H4–SO2–), 48.14 (–O–CH2–CH2–N–), 61.08 (–N–
2
1
CH2–N–), 62.45 [d, JC–P 6.5, (CH3–CH2–O)2P(O)–], 64.74 [d, JC–P
3
1
3
165.4, –P(O)–CH2–O–], 71.74 (d, JC–P 11.0, –O–CH2–CH2–N–),
126.77 (Ar-C-ortho to SO2), 127.67 (d, JC–F 33.4, –N–CH@CF–),
2
129.84 (Ar-C-ortho to CH3), 136.09 (Ar-C-ipso to SO2), 140.17 [d,
1JC–F 236.0, –CH@CF–C(O)–], 144.23 (Ar-C-ipso to CH3), 150.08 [–
2
N–C(O)–N–], 157.39 [d, JC–F 26.2, @CF–C(O)–N–]. mmax (KBr)
1706br
s
(C@O), 1672s (C@O). HRMS m/z C19H27N3O8FNaPS
5.7. General procedure for the dealkylation of 13
(M+Na)+ 530.1133, found 530.1125.
A solution of 13 (1.0 mmol) in dry acetonitrile (10 mL) was
cooled in an ice-water bath under argon atmosphere and trimeth-
ylsilyl bromide (TMSBr, 10 mmol) was added dropwise. The mix-
ture was left at room temperature overnight and evaporated to
dryness under reduced pressure. The residue was diluted with an
acetone/water mixture (1:9, v/v, 10 mL). The resulting solution
was stirred at room temperature until a white solid precipitated
out (ca. 2 h). The suspension was cooled in the refrigerator (5 °C)
overnight. The solid was filtered off, washed with the acetone/
water mixture (1:9, v/v), and purified by crystallization (acetone/
water, 1:9, v/v) or boiling with the same solvent to yield 8a–d.
5.5. Coupling of 7 with uracil
According to the procedure used for the coupling of 7 with thy-
mine or 5-fluorouracil, a solution of the silylated uracil in dry ace-
tonitrile (15 mL) was prepared from uracil (361 mg, 3.2 mmol) and
BSA (1.3 g, 6.4 mmol, 1.6 mL) and then treated with a solution of 7
(746 mg, 1.6 mmol) in dry acetonitrile (1.5 mL) and 1 M solution of
tin(IV) chloride in dichloromethane (4.8 mL). Column chromatog-
raphy (chloroform/methanol, 98:2, v/v) provided azanucleoside
13c (558 mg, 73%) as an oil and 14 (49 mg, 7%) as an oil.
5.5.1. Diethyl {2-[N-(1H,3H-pyrimidin-2,4-dion-1-ylmethyl)-N-
(p-toluenesulfonyl)amino]ethoxymethyl}phosphonate (13c)
5.7.1. {2-[N-(5-Methyl-1H,3H-pyrimidin-2,4-dion-1-ylmethyl)-N-
(p-toluenesulfonyl)amino]ethoxymethyl}phosphonic acid (8a)
According to the general procedure, 8a was obtained from 13a
(582 mg, 1.2 mmol). Crystallization gave 8a (405 mg, 78%) as a
3
dH (CDCl3, 400 MHz) 1.32 (t, JH–H 7.2, 6H), 2.41 (s, 3H), 3.54–
3
3.56 (triplet-like multiplet, 2H), 3.69–3.72 (m, 4H), 4.14 (dq, JH–P

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M. Koszytkowska-Stawinska et al. / Bioorg. Med. Chem. 17 (2009) 3756–3762
white powder; mp 119–123 °C. dH (DMSO-d6, 400 MHz) 1.74 (d,
5.8. Antiviral activity assays
4JH–H 0.8, 3H), 2.37 (s, 3H), 3.47–3.51 (m, 4H), 3.58–3.60 (triplet-
like multiplet, 2H), 3.84 (br s, 2H, OH), 5.15 (s, 2H), 7.31–7.33 (m,
The compounds were evaluated against the following viruses:
herpes simplex virus type 1 (HSV-1) strain KOS, thymidine ki-
nase-deficient (TKꢀ) HSV-1 strain KOS resistant to ACV (ACVr), her-
pes simplex virus type 2 (HSV-2) strain G, varicella zoster virus
(VZV) strain Oka, TK- VZV strain 07-1, human cytomegalovirus
(HCMV) strains AD-169 and Davis, vaccinia virus Lederle strain,
respiratory syncitial virus (RSV) strain Long, vesicular stomatitis
virus (VSV), Coxsackie B4, Parainfluenza 3, Reovirus-1, Sindbis,
Punta Toro, feline coronavirus and feline herpes virus. The antiviral
assays were based on inhibition of virus-induced cytopathicity or
plaque formation in human embryonic lung (HEL) fibroblasts, Afri-
can green monkey cells (Vero), human epithelial cells (HeLa) or
MTT dye staining of virus-infected feline kidney (CRFK) cell cul-
tures. Confluent cell cultures in microtiter 96-well plates were
inoculated with 100 CCID50 of virus (1CCID50 being the virus dose
to infect 50% of the cell cultures), or with 20 plaque forming units
(PFU) (for VZV) or with 100 PFU (for HCMV). After a 1–2 h adsorp-
tion period, residual virus was removed, and the cell cultures were
incubated in the presence of varying concentrations of the test
compounds. Viral cytopathicity or plaque formation (VZV) was re-
corded as soon as it reached completion in the control virus-in-
fected cell cultures that were not treated with the test
compounds. Antiviral activity was expressed as the EC50 or com-
pound concentration required to reduce virus-induced cytopathic-
ity or viral plaque formation by 50%.
4
2H), 7.35 (q, JH–H 0.8, 1H), 7.65–7.67 (m, 2H), 11.22 (br s, 1H,
NH). dC (DMSO-d6, 50 MHz) 12.08, 21.15, 48.09, 60.87, 66.55 (d,
3
1JC–P 159.4), 71.48 (d, JC–P 10.6), 108.95, 126.92, 129.96, 136.83,
140.54, 143.91, 151.26, 164.21. mmax (KBr) 2771br w (PO–H). Anal.
Calcd for C16H22N3O8PSꢂH2O: C, 41.29; H, 5.20; N, 9.03; S, 6.89.
Found: C, 41.36; H, 5.35; N, 9.08; S, 6.46.
5.7.2. {2-[N-(5-Fluoro-1H,3H-pyrimidin-2,4-dion-1-ylmethyl)-
N-(p-toluenesulfonyl)amino]ethoxymethyl}phosphonic acid
(8b)
According to the general procedure, 8b was obtained from 13b
(277 mg, 0.5 mmol). Crystallization gave 8b (156 mg, 63%) as a
white powder; mp 166–168 °C. dH (DMSO-d6, 400 MHz) 2.38 (s,
2
3H), 3.46 (d, JH–P 8.8, 2H), 3.51–3.59 (m, 4H), 5.14 (s, 2H), 7.38–
3
7.40 (m, 2H), 7.71–7.73 (m, 2H), 7.91 (d, JH–F 6.4, 1H), 11.83 (d,
4JH–F 4.4, 1H, NH). dC (DMSO-d6, 50 MHz) 21.03, 48.15, 61.31,
1
3
2
66.48 (d, JC–P 159.4), 71.20 (d, JC–P 10.6), 126.87, 129.10 (d, JC–F
1
33.4), 129.89, 136.54, 139.25 (d, JC–F 229.2), 143.85, 149.74,
157.24 (d, JC–F 25.8). mmax (KBr) 1662s, 1688s, 1723s, 2695br w
2
(PO–H). Anal. Calcd for C15H19FN3O8PS: C, 39.91; H, 4.24; N, 9.31;
S, 7.10. Found: C, 39.86; H, 4.24; N, 9.29; S, 7.21.
5.7.3. {2-[N-(1H,3H-Pyrimidin-2,4-dion-1-ylmethyl)-N-(p-
toluenesulfonyl)amino]ethoxymethyl}phosphonic acid (8c)
According to the general procedure, 8c was obtained from 13c
(465 mg, 0.9 mmol). Crystallization gave 8c (331 mg, 80%) as a
white powder; mp 184–186 °C. dH (DMSO-d6, 400 MHz) 2.37 (s,
3H), 3.46–3.50 (m, 4H), 3.56–3.59 (triplet-like multiplet, 2H),
5.9. Cytotoxicity assays
Cytostatic measurements were based on the inhibition of cell
growth. HEL cells were seeded at 5 ꢁ 103 cells/well into 96-well
microtiter plates and allowed to proliferate for 24 h. Then, medium
containing different concentrations of the test compounds 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 concentration re-
quired 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 compounds. Alternatively,
cytotoxicity for cell morphology was expressed as the minimum
cytotoxic concentration (MCC) or the compound concentration
that caused a microscopically detectable alteration of cell (i.e.,
HEL, Vero, HeLa) morphology.
3
4
4.19 (br s, 2H, OH), 5.15 (s, 2H), 5.59 (dd, JH–H 8.0, JH–H 1.6, 1H),
3
7.37–7.39 (m, 2H), 7.49 (d, JH–H 8.0, 1H), 7.68–7.70 (m, 2H),
11.28 (br s, 1H, NH). dC (DMSO-d6, 50 MHz) 21.07, 48.13, 61.10,
1
3
66.55 (d, JC–P 159.4), 71.26 (d, JC–P 10.5), 101.34, 126.88, 129.95,
136.47, 143.86, 145.04, 151.18, 163.53. mmax (KBr) 2823br w (PO–
H). Anal. Calcd for 2C15H20N3O8PSꢂH2O: C, 40.73; H, 4.78; N, 9.50;
S, 7.25. Found: C, 40.76; H, 4.55; N, 9.56; S, 7.35.
5.7.4. {2-[N-(4-Amino-1H-pyrimidin-2-on-1-ylmethyl)-N-(p-
toluenesulfonyl)amino]ethoxymethyl}phosphonic acid (8d)
According to the general procedure, 8d was obtained from 13d
(374 mg, 0.8 mmol). Boiling of the crude product with a mixture of
acetone and water (2 ꢁ 5 mL, 1:9, v/v) gave 8d (272 mg, 82%) as a
white powder; mp >218 °C (dec). Its solubility in DMSO or in water
(5 mg/ca. 100 ml of water) was too low to measure the 1H and 13
C
Acknowledgments
NMR spectra. mmax (KBr) 1688s, 1725s, 2737br w (PO–H). Anal.
Calcd for C15H21N4O7PS: C, 41.67; H, 4.90; N, 12.96; S, 7.42. Found:
C, 41.58; H, 4.81; N, 12.74; S, 7.40.
The synthetic part of this work was financially supported by
Ministry of Science and Higher Education, Poland, Project No. 1
T09B 013 30. The virological part of the work was supported by
the Concerted Research Actions (GOA), Project No. 05/19 of the K.
U. Leuven. The technical assistance of Mrs. Leentje Persoons, Frieda
Demeyer, Vicky Broeckx, Anita Camps, Lies Van den Heurck and
Steven Carmans was highly appreciated.
A suspension of 8d (50 mg) in the DMSO (50 mL)/water (50 mL)
mixture was shaken with DowexÒ 88 ion-exchange resin (Na-form,
500 mg) for six months. The suspension was decanted from ion-ex-
change resin. The unchanged 8d (identified by elemental analysis,
23 mg) was filtered off, was washed with the DMSO/water mixture
and dried under high vacuum. The filtrate was evaporated to dry-
ness under high vacuum. The residue was twice crystallized from
water to give a white solid 8d–s (15 mg) which was identified by
elemental analysis as an equimolar mixture of 8d and its monoso-
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2
´
2. Holy, A. Curr. Pharm. Des. 2003, 9, 2567.
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3
´
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