Aryl nucleoside H-phosphonates. Part 15: Synthesis, properties and, anti-HIV activity of aryl nucleoside 5′-α-hydroxyphosphonates

Article abstract of DOI:10.1016/j.bmc.2005.10.048

Aryl nucleoside 5′-H-phosphonates 4 bearing AZT or 2′,3′-dideoxyuridine moieties were subjected to reaction with various aromatic aldehydes to produce nucleoside 5′-α- hydroxyphosphonate derivatives 2 as potential anti-HIV agents. Stability of the title c

Full text of DOI:10.1016/j.bmc.2005.10.048

Bioorganic & Medicinal Chemistry 14 (2006) 1924–1934
Aryl nucleoside H-phosphonates. Part 15: Synthesis, properties
and, anti-HIV activity of aryl nucleoside 50-a-hydroxyphosphonates
a
a
a
a
´
´
Agnieszka Szymanska, Marzena Szymczak, Jerzy Boryski, Jacek Stawinski,
Adam Kraszewski,a,* Gabriella Collu,b Giseppina Sanna,b Gabriele Giliberti,b
Roberta Loddob and Paolo La Collab,*
aInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan´, Poland
`
Dipartimento di Scienze e Tecnologie Biomediche, Universita di Cagliari, Cittadella Universitaria, 09042 Monserrato, Cagliari, Italy
b
Received 19 July 2005; revised 19 October 2005; accepted 25 October 2005
Available online 14 November 2005
Abstract—Aryl nucleoside 50-H-phosphonates 4 bearing AZT or 20,30-dideoxyuridine moieties were subjected to reaction with var-
ious aromatic aldehydes to produce nucleoside 50-a-hydroxyphosphonate derivatives 2 as potential anti-HIV agents. Stability of the
title compounds in cell culture media was investigated and three distinct decomposition pathways were identified. The anti-HIV
activity of hydroxyphosphonates 2 correlates well with the type and extent of their chemical or enzymatic degradation in culture
medium (RPMI 1640 containing 10% FBS), suggesting that aryl nucleoside 50-hydroxyphosphonates 2 act as depot forms of the
parent antiviral nucleosides.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
two nucleosides, for example, 20,30-dideoxyinosine
(ddI) and 30-azido-30-deoxythymidine (AZT).12
Nucleoside analogues currently used in AIDS therapy
caused by HIV infections are pro-drugs that, following
uptake by the cells, are stepwise phosphorylated into
the corresponding 50-triphosphates. As such, they are
substrates for the HIV reverse transcriptase (RT) and,
upon incorporation into the growing DNA chain, termi-
nate the synthesis of the viral DNA.1,2 Since phosphor-
ylation of nucleoside analogues is indispensable for their
antiviral activity, efforts have been focused to prepare
derivatives that might efficiently enter the cell and be
converted into bioactive forms.3–11
Based on these promising results, we recently devel-
oped dinucleoside phosphonate-phosphates26 of type
1
(Scheme 1) as potential anti-HIV agents. In
O H
O
AZT O P C O P O AZT
PhO Ar
OPh
1
0.1 M PB, pH 7.4
or RPMI/FBS
The approach of using precursors, which could deliver
into the cell two identical or different phosphorus-linked
antiviral nucleosides, has received considerable atten-
tion.12–25 Ex vivo studies have shown that, in many
instances, such precursors are endowed with consider-
able anti-HIV activity and that synergistic potentiation
may be obtained following simultaneous delivery of
O
O
-O P O AZT
PhO P O AZT
HO C H
+
OPh
Ar
3
2c
AZT = 3'-deoxy-3'-azidothymidin-5'-yl
Ph = phenyl
Ar = 4-methoxyphenyl
PB = 0.1 M phosphate buffer pH 7.4
FBS = fetal bovine serum
RPMI = RPMI 1640
Keywords: Antiviral; Pro-drugs; Nucleotides; Nucleoside analogues;
HIV; a-Hydroxyphosphonates.
Corresponding authors. Tel.: +48 61 852 85 03; fax: +48 61 852 05 32
(A.K.); tel.: +39 070 675 4147; fax: +39 070 675 4210 (P.L.C.); e-mail
addresses: akad@ibch.poznan.pl; placolla@unica.it
*
Scheme 1. Decomposition of 1 in PB, pH 7.4, and RPMI/FBS 10% (v/v)
37 ꢁC.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.048

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A. Szymanska et al. / Bioorg. Med. Chem. 14 (2006) 1924–1934
1925
HIV-1-infected MT-4 cells, 30-azido-30-deoxythymidin-
50-yl phenyl (30-azido-30-deoxythymidin-50-yl phenyl
HIV-infected cells.16,18,19,28 In this context, it was inter-
esting to investigate the antiviral properties of hydrox-
yphosphonates of type 2 (Scheme 2) as their structural
features should permit a precise modulation of their
chemical and pharmacokinetic properties by a proper
choice of two aromatic units Ar1 and Ar2.
phosphinyl)-4-methoxybenzyl phosphate
1
showed
anti-HIV potency (EC50 = 0.01 lM) equal to that of
AZT (EC50 = 0.01 lM). The decomposition pathway
of compound 1 in cell culture medium (RPMI 1640
containing 10% heat-inactivated foetal bovine serum
FBS) indicated the rapid formation (HPLC, t1/
2 < 5 min) of 30-azido-30-deoxythymidin-50-yl phenyl
hydroxy(4-methoxyphenyl)methanephosphonate 2 and
30-azido-30-deoxythymidin-50-yl phenyl phosphodiester
3 in equal amounts (Scheme 1).27
Dinucleoside hydroxyphosphonates can be prepared by
the addition of an aldehyde to the corresponding
H-phosphonate diesters in the presence of base, but this
usually requires high temperature, large excess of base
and prolonged reaction times.28 Analogous synthesis
of aryl nucleoside hydroxyphosphonates 2 would call
for the corresponding aryl nucleoside H-phosphonates
4 as intermediates, but these are much more labile than
dinucleoside H-phosphonates and show high propensity
towards disproportionation under basic conditions.29
Indeed, even under relatively mild reaction conditions,
for example, addition of an aldehyde to H-phosphonate
diesters in THF in the presence of triethylamine,28
formation of significant amount of side products was
observed in the case of aryl H-phosphonate diesters
(31P NMR spectroscopy). For this reason, it was
necessary to develop a new synthetic protocol that
would permit efficient synthesis of aryl nucleoside
hydroxyphosphonates 2.
Phosphodiester 3 can be metabolized to provide the cor-
responding nucleoside monophosphate. However, since
the anionic structure of the latter one may prevent effi-
cient crossing of the cell membrane, it is likely that the
observed anti-HIV activity of 1 was due to the genera-
tion of uncharged AZT phenyl hydroxyphosphonate
2c that might undergo further enzymatic transformation
into biologically active species. To verify this assump-
tion, we synthesised a series of nucleoside aryl a-hydrox-
yphosphonates of type 2 (Scheme 2) and investigated
their chemical and anti-HIV properties.
2. Results and discussion
To this end, AZT phenyl H-phosphonate 4a [generated
in situ by coupling of AZT H-phosphonate and phenol
in methylene chloride in the presence of pyridine (10 mo-
lar equiv) and diphenyl chlorophosphate (DPCP)]30 was
allowed to react with benzaldehyde 5a (5 molar equiv) in
the presence of a slight excess of diisopropyl ethylamine
(DIPEA, 2.5 molar equiv in respect to 4a), instead of
2.1. Synthesis of a-hydroxyphosphonates of type 2 via
aryl H-phosphonate four intermediates
Recent studies by Meier et al. have shown that dinucle-
oside analogues of hydroxybenzylphosphonates bearing
two AZT moieties exhibit potent antiviral activity in
O
O
Ar1O P O
B
Ar1O P O
HO CH
Ar2
B
DIPEA or
2,6-lutidine
O
O
+
Ar2CHO
H
5
X
X
2
4
4a; B = thymin-1-yl, X = N3, Ar1 = phenyl
2a; B = thymin-1-yl, X = N3,
Ar1 = Ar2 = phenyl
4b; B = thymin-1-yl, X = N3, Ar1 = 4-methoxyphenyl
4c; B = thymin-1-yl, X = N3, Ar1 = 2,6-dimethylphenyl
4d; B = uracil-1-yl, X = H, Ar1 = phenyl
2b; B = thymin-1-yl, X = N3
Ar1 = phenyl, Ar2 = 4- methylphenyl
2c; B = thymin-1-yl, X = N3
5a; Ar2 = phenyl
Ar1 = phenyl, Ar2 = 4-methoxyphenyl
2d; B = thymin-1-yl, X = N3
5b; Ar2 = 4-methylphenyl
5c; Ar2 =4-methoxyphenyl
5d; Ar2 = 4-chlorophenyl
5e; Ar2 = 4-nitrophenyl
5f; Ar2 = pyridin-3-yl
Ar1 = phenyl, Ar2 = 4-chlorophenyl
2e; B = thymin-1-yl, X = N3
Ar1 = enyl, Ar2 = 4-nitrophenyl
ph
2f; B = thymin-1-yl, X = N3
Ar1 = phenyl, Ar2 = pyridin-3-yl
2g; B = thymin-1-yl, X = N3
DIPEA = diisopropylethylamine
Ar1 = 4-methoxyphenyl, Ar2 = pyridin-3-yl
2h; B = thymin-1-yl, X = N3
Ar1 = 2,6-dimethylphenyl, Ar2 = pyridin-3-yl
2i; B = uracil-1-yl, X = H
Ar1 = phenyl, Ar2 = 4-methoxyphenyl
Scheme 2. Scheme of synthesis of aryl nucleoside a-hydroxy(aryl)methanephosphonates of type 2.

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A. Szymanska et al. / Bioorg. Med. Chem. 14 (2006) 1924–1934
1926
Table 1. Octanol/water partition coefficient (logP), 31P NMR and HPLC data of hydroxyphosphonates 2 and their decomposition products
b,c
tR [min]
Compound
dPa,c(ppm)
LogP
2a
2b
2c
2d
2e
2f
18.72, 18.76, 18.97, 19,05
18.88, 18.96, 19.11, 19.24
18.82, 18.85, 18.98, 19,09
17,96, 18.05, 18,08, 18,26
17.10, 17.14, 17.31, 17.54
18.05, 18.22, 18.43, 18.59
17.80, 17.98, 18.08, 18.36
17.54, 17.68, 17.81, 18.12
27.2, 27.7
28.2, 28.3, 28.8
27.8, 28.1, 28,31, 28.46
29.5, 29.9
28.9, 29.1, 29.2
23.4, 23.7
23.4, 23,6
25.4, 26.0
26.4, 26.7, 26.9
13.9
1.49
1.88
1.48
1.53
0.99
ꢀ0.14
ꢀ0.23
0.56
2g
2h
2I
6
18.96, 19.12
1
5.41d of t, JHP = 631 Hz, JHP = 5.5 Hz
1.27
3
8a
8b
8c
9
16.08, 16.27
15.09, 15.31
15.81, 16.05
ꢀ6.26, ꢀ6.29
ꢀ6.00
17.4
14.6
—
—
—
—
—
—
—
0.06
14.5
32.0, 32.2
18.1
12.1
3
10
11
AZT
0.44
3
52.95, 53.45 (2t, JHP = 6.4 Hz)
—
—
—
a Chemical shifts in spectra recorded with heteronuclear decoupling.
b tR, retention time; Hypersil ODS, 4.6 · 250 mm (for details, see Section 4).
c Multiplicity of signals and peaks is due to the presence of diastereoisomers.
triethylamine (Scheme 2). 31P NMR spectroscopy re-
vealed that the reaction was completed within 10 min
and the corresponding a-hydroxyphosphonate 2a was
the only nucleotidic product observed. In the 31P NMR
spectra, a-hydroxyphosphonates 2 gave rise to a group
of four signals centred at ca. 18 ppm, due to the presence
of four diastereomers (two chiral centres: at the phospho-
rus atom and at the phosphonate carbon centre) (Table
1). These conditions were also suitable for the reactions
of other aldehydes with electron-donating (5a–c) or
weakly electron-withdrawing substituents (5d), and affor-
ded the corresponding hydroxyphosphonates 2a–d in
70–80% yields after silica gel column chromatography.
compound of type 2 in the above culture medium con-
taining 5% DMSO were incubated at 37 ꢁC and the for-
mation of metabolites was monitored by HPLC. Since
hydroxyphosphonates 2 were stable in acidic media
(vide infra), 0.01 M triethylammonium trifluoroacetate,
pH 3.2, was used as a polar solvent for HPLC analyses.
The products formed upon decomposition of 2 were
unambiguously identified by co-elution with authentic
samples obtained by other methods (vide infra and
Section 4).
All aryl nucleoside a-hydroxyphosphonates 2 appeared
to be rather unstable (t1/2 10–30 min) in the cell culture
medium and decomposed in different ways depending
on the aryl moiety Ar2 in the hydroxy(aryl)methylene
part of the molecule, and on the aryl group Ar1 bound
to the phosphorus centre (Scheme 3). Three decomposi-
tions pathways (A, B and C), primarily determined by
the electron density in Ar2, were identified: (i) path A,
which is likely to be initiated by a spontaneous dissoci-
ation of hydroxyphosphonates 2 into the parent
H-phosphonate diesters 4 and the corresponding alde-
hydes 5; (ii) path B, which results from hydrolysis of
the phosphoester aryl moiety and represents, under
these reaction conditions, a dead-end product; (iii) path
C, which involves as an initial step a phosphonate-phos-
phate rearrangement to produce the corresponding
phosphotriesters 9. The relative contribution of path-
ways A, B and C for a particular hydroxyphosphonate
2 is determined by electronic nature combination of
Ar1 and Ar2 groups present in the compound.
Aldehydes with strong electron-withdrawing substitu-
ents (5e and 5f) in the presence of DIPEA reacted faster
with H-phosphonates 4 than with aldehydes 5a–c, but
produced several side products resonating at dP 27–
32 ppm (ca. 20%). To suppress these side reactions, we
replaced DIPEA (pKa > 11) with a weaker base 2,6-luti-
dine (pKa 6.6).31 This caused the condensation reactions
to proceed slowly (overnight), however, the correspond-
ing hydroxyphosphonates 2e–h were formed without
noticeable contamination of side products (31P NMR
spectroscopy).
Structure and purity of all synthesised AZT (or ddU)
aryl hydroxyphosphonates 2 were established by 1H,
31P and HRMS spectroscopy, and also by chromato-
graphic methods (TLC, HPLC).
2.2. Stability of hydroxyphosphonates 2 in cell culture
medium
Decomposition paths of phenyl esters 2a–f bearing dif-
ferent Ar2 substituents were investigated. a-Hydrox-
Stability of all hydroxyphosphonates 2 was investigated
in the same cell culture medium (RPMI 1640 containing
10% FBS, pH 7.6), in which cultivation of T-lympho-
cytes was carried out during in vitro cytotoxicity and
anti-HIV assays. To this end, 2 mM solutions of each
yphosphonates
2a–c
bearing
electron-donating
substituents on Ar2 decomposed exclusively according
to pathway A (Scheme 3, Table 2) and rapidly pro-
duced AZT H-phosphonate 6, which then slowly
underwent dephosphonylation to AZT most likely by

´
A. Szymanska et al. / Bioorg. Med. Chem. 14 (2006) 1924–1934
1927
O
O
P
H
O
Ar1O P O AZT
-O
O AZT
Ar1OH
+
Ar2
C
+
H
5
H
7
A
6
4
O
P
O
P
B
Ar1OH
-O
O AZT +
Ar1O
O AZT
AZT
HO CH
Ar2
HO CH
8
Ar2
C
2
O
O
O
Ar1O
O AZT
-O P O AZT + Ar1OH
O-
P
Ar1O P O AZT
O
O-
7
10
CH2Ar2
3
9
7a; Ar1 = phenyl
AZT
7b; Ar1 = 4-methoxyphenyl
7c; Ar1 = 2,6-dimethylphenyl
8a; Ar2 = 4-chlorophenyl
8b; Ar2 = 4-nitrophenyl
8c; Ar2 = pirydin-3-yl
9; Ar1 = phenyl, Ar2= 4-nitrophenyl
3; Ar1 = phenyl
Scheme 3. Decomposition paths of AZT aryl hydroxyphosphonates of type 2 in cell culture media.
Table 2. Stability and decomposition paths of hydroxyphosphonates of type 2 in cell culture mediaa and their anti-HIV potencyb
Compound
Ar1
Ar2
RPMI/FBS t1/2 (min)
Path
CC50 (lM)
EC50 (lM)
SI
A (%)
B (%)
C (%)
2a
2b
2c
27.2
19.4
13.1
100
—
—
—
—
49
42
49
0.06
0.05
0.04
817
100
100
—
—
840
CH3
1225
1000
OCH3
2d
13.1
92
8
—
50
0.05
Cl
2e
2f
9.0
15.9
26.3
57.6
25.8
24
55
47
45
42
—
—
29
—
—
—
—
37
>100
27
0.05
0.04
740
>2500
2700
4900
5
NO2
N
2g
2h
2I
58
0.01
N
N
OCH3
H3C
CH3
100
100
49
0.01
>100
>20
OCH3
6
1175
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
55
ndc
>100
27
0.01
ndc
2.4
5500
—
42
8b
8c
9
Stable 6 days
Stable 6 days
9901
0.01
0.01
2700
5000
AZT
—
50
a RPMI 1640/FBS 9:1 (v/v), 37 ꢁC.
b MT-4 cells, HIV IIIB strain.
c Not determined.

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A. Szymanska et al. / Bioorg. Med. Chem. 14 (2006) 1924–1934
1928
the 50-nucleotidase-like but not phosphatase-like32
activity of FBS. Since 31P NMR spectroscopy proved
formation of phosphonic rather than phosphoric acid
at this step, it seems that AZT H-phosphonate 6 does
not need to be oxidized to the phosphate form to be
substrate of FBS 50-nucleotidase-like enzymatic activity
as it has been suggested previously.33 This lent support
to the opinion that AZT H-phosphonate 6 is rather a
nucleoside vehicle,15 although some data indicated that
6 to some extent can also act as a pronucleotide.34
2.3. Chemistry underlying the A, B and C decomposition
pathways
In 0.1 M phosphate buffer (pH 7.4), the decomposition
of compounds 2a–i followed the same initial steps ob-
served in culture medium, although at 5- and 6-fold low-
er rates (data not shown). These findings are consistent
with data by Meier et al. concerning the decomposition
of dinucleoside hydroxybenzylphosphonates.28
Factors influencing decomposition of dinucleoside
hydroxyphosphonates according to path C have been dis-
cussed in detail by Meier et al.16 and seem to apply also to
the decomposition of the aryl nucleoside a-hydrox-
yphosphonates investigated in this study. Formation of
phosphotriesters 9 occurs most likely via a phospho-
nate-phosphate rearrangement35,37 and involves an intra-
molecular nucleophilic attack of the hydroxyl function on
the phosphorus centre of hydroxyphosphonates 2, fol-
lowed by the P–C bond scission. Since the latter process
occurs in a rate-determining step and consists of move-
ment of electrons towards the benzylic carbon of the
hydroxyphosphonate, the carbanion-stabilizing substitu-
ents in Ar2 should facilitate this process. Consistent with
this mechanism, the pathway C was observed only for
hydroxyphosphonate 2e bearing the 4-nitrophenyl group
(Ar2).
In the case of hydroxyphosphonates 2d–f, in which the
Ar2 group is electron deficient due to the presence of
electron-withdrawing substituents, two additional
decomposition routes, B and C, became available. a-
Hydroxyphosphonate 2d predominantly (92%) pro-
duced AZT H-phosphonate 6 via path A, although the
formation of a-hydroxyphosphonate monoester 8a
(8%) through path B became evident. The contribution
of path B to the decomposition of 2f bearing pyridin-
3-yl as an Ar2 group was higher (45%), approaching that
of path A (55%). In the case of a-hydroxyphosphonate
diester 2e with the most electron-withdrawing 4-nitro-
phenyl group as Ar2, the third decomposition path C,
triggered by the phosphonate-phosphate rearrange-
ment,35–37 could be observed (29% of 9). The produced
AZT 4-nitrobenzyl phenyl phosphotriester (9) was slow-
ly (Table 2) but chemoselectively hydrolysed to AZT
phenyl phosphodiester 3, followed by transformation
to AZT monophosphate 10 and, ultimately, to AZT
(Table 2). The last two transformations, that is, forma-
tion of AZT monophosphate 10 and its dephosphoryla-
tion to AZT, are apparently enzymatic reactions
catalysed by FBS phosphodiesterases and phosphatases,
respectively, because compounds 3 and 10 were stable
for at least 6 days in RPMI lacking FBS.
The path B, a SN2(P) reaction, is characteristic for
hydroxyphosphonates bearing a good leaving group at
the phosphorus and for this reason it was observed for
aryl nucleoside a-hydroxyphosphonates 2, but not for
dinucleoside derivatives.16 For the hydroxyphosphonates
investigated, the decomposition pathway B became
apparent only for compounds 2d–g. All of them have elec-
tron-deficient Ar2 groups (4-chlorophenyl, 4-nitrophenyl
and 3-pyridyl) which apparently facilitate the nucleophil-
ic attack at the phosphorus centre with expulsion of the
phenoxy (for 2d–f) or the p-methoxyphenoxy group (for
2g). As expected, phenyl esters of a-hydroxyphospho-
nates 2 with electron-rich Ar2 groups (e.g., 2a–c,
Ar2 = phenyl, 4-methylphenyl and 4-methoxyphenyl,
respectively) did not undergo this kind of reaction. A
decreasing contribution of the hydrolytic path B within
the series 2f > 2g > 2h is consistent with the pKa values
of the leaving groups involved (phenyl, 4-methoxyphenyl,
2,6-dimethylphenyl, respectively; vide supra) and, in the
case of the 2,4-dimethylphenolyl ester 2h, with steric
factors which may interfere with solvation of the 2,6-
dimethylphenoxy moiety and thus lower its leaving group
ability.
It was also interesting to know to what extent electronic
properties of an aryl ester group Ar1 can modify a relative
contribution of the decomposition pathways of hydrox-
yphosphonates 2 in cell culture media. To this end, the sta-
bility in cell culture medium of a-hydroxyphosphonates
2f–h with Ar2 = pyridin-3-yl and Ar1 = phenyl
(pKa = 9.98) (2f), 4-methoxyphenyl (pKa = 10.20) (2g)
or 2,6-dimethylphenyl (pKa = 10.59)31 (2h) was
investigated.
The replacement of a phenyl (compound 2f) by a p-meth-
oxyphenyl group (compound 2g) had only a marginal ef-
fect on the decomposition mode (Table 2); however, the
introduction of a 2,6-dimethylphenyl group (hydrox-
yphosphonate 2h) completely prevented degradation
through the hydrolytic pathway B. The fact that 2h
decomposes exclusively according to path A might ex-
plain its high antiviral potency. From the point of view
of tuning chemical and biological properties of a-hydrox-
yphosphonates of type 2, a potentially important
feature is that the introduction of an electron rich aryl
ester group (Ar1) increases the stability of these com-
pounds, as it is apparent from the corresponding t1/2
values: 2f, Ar1 = phenyl, 15.9 min; 2g, Ar1 = 4-methoxy-
phenyl, 26.3 min; 2h, Ar1 = 2,6-dimethylphenyl 57.6 min
(Table 2).
From the point of view ofthe potential antiviral activityof
a-hydroxyphosphonates, the most important decomposi-
tion pathway is, apart from C, path A which, ultimately,
generates nucleoside H-phosphonate monoesters and
the corresponding nucleosides. This seems to be a general
pathway for decomposition of hydroxyphosphonates
under hydrolytic conditions and is observed for both
dinucleoside and aryl nucleoside hydroxyphosphonates.
Spontaneous generation of an aldehyde and the corre-
sponding H-phosphonate diester, postulated by Meier

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A. Szymanska et al. / Bioorg. Med. Chem. 14 (2006) 1924–1934
1929
et al. for dinucleoside hydroxyphosphonates, was also ob-
O
P
served with the aryl nucleosides 2 (Scheme 3, path A). The
most likely reason for this is that a-hydroxyphosphonates
are adducts of H-phosphonate diesters and aldehydes,
and this reaction is apparently reversible with the equilib-
rium shifted to the left. Under mildly basic hydrolytic
conditions, an equilibrium concentration of an H-phos-
phonate diester is rapidly depleted due to fast hydrolysis
of H-phosphonate diesters, and the equilibrium shifts to
the right. This results in decomposition of a-hydrox-
yphosphonate derivatives according to pathway A. For
hydroxyphosphonates 2 (e.g., for compounds 2a–c, 2h
and 2i) for which this route was the only reaction pathway
available, 31P NMR spectroscopy revealed the presence of
only starting material 2 and H-phosphonate monoester 6.
Ar1O
HO C Ar2
O AZT
H
2c
O
O
Ar1O
O AZT
Ar2
5c
P
H
+
H
4a
To provide evidence for the existence of a true equilibrium
between a-hydroxyphosphonate and the corresponding
substrates, a trapping experiment was designed to show
intermediacy of the H-phosphonate diester 4, generated
from a hydroxyphosphonate 2 under anhydrous condi-
tions. For this purpose, a-hydroxyphosphonate 2c was
dissolved in methylene chloride containing elemental sul-
fur and left overnight. The 31P NMR spectra of the reac-
tion mixture showed that the starting material 2c (four
signals at ca. 19 ppm) disappeared and two new resonanc-
es in a region typical for aryl nucleoside phosphorothioate
diesters38 (dP 52.8 and 53.5 ppm; two diastereoisomers)
appeared. After work-up and column chromatography,
two compounds were isolated from the reaction mixture:
the aldehyde 5c and the AZT phenyl phosphorothioate 11
(1H, 31P NMR, TLC, comparison with authentic sam-
ples). In another experiment, a-hydroxyphosphonate 2c
was treated in acetonitrile with ethanol (10 molar equiv)
and iodine (2 molar equiv) in the presence of triethyl-
amine. After an overnight standing, the 31P NMR spec-
troscopy revealed that 2c was completely converted into
AZT ethyl phenyl phosphate 12. These results are consis-
tent with the generation of H-phosphonate diester 4a
which, in the presence of elemental sulfur, underwent: sul-
furization to the corresponding phosphorothioate 11, or
in the presence of iodine underwent oxidative coupling
with ethanol to produce the phosphotriester 12. These re-
sults support the postulated equilibrium between a-
hydroxyphosphonate 2 and its substrate 5 + 4 (Scheme
4) involved in the decomposition of a-hydroxyphospho-
nates of type 2 according to path A.
I2/EtOH
S8
O
O
Ar1O
O AZT
P
Ar1O P O AZT
S-
11
OEt
12
Ar1 = phenyl
Ar2 = 4-methoxyphenyl
Et = ethyl
Scheme 4. Reactions of AZT phenyl hydroxy(4-methoxyphenyl)meth-
anephosphonate with elemental sulfur under anhydrous conditions
and with ethanol in the presence of iodine.
wild type HIV-1, strain IIIB using AZT as reference
compound.
Taking into account the observed decomposition path-
ways of a-hydroxyphosphonates 2 in RPMI/FBS and
assuming that both AZT H-phosphonate 6 and AZT
monophosphate 10 probably act as depots for AZT,15,39
it was expected that all a-hydroxyphosphonates 2 that
decompose according to paths A and C should show sim-
ilar EC50 indices. Data in Table 2 are in agreement with
this general consideration. Small variation in antiviral
activity (EC50 0.01–0.06 lM) might be attributed to dif-
ferent rates and decomposition pathways of a-hydrox-
yphosphonates
2 to AZT H-phosphonate 6 (or
phosphate 10) in cell culture medium. The latter com-
pound, being dephosphonylated by phosphatase-like
activity of FBS, released (t1/2 = 1175 min) the active
pro-drug AZT. AZT phenyl a-hydroxyphosphonate 2e
(Ar2 = 4-nitrophenyl), which decomposed via A, B, and
C paths, did not show an improved selectivity index. Both
activity and cytotoxicity for this compound may come
from the produced AZT H-phosphonate 6 as well as from
AZT 4-nitrobenzyl phosphotriester 9, which itself was
highly active but also very cytotoxic.
Although the above equilibria for all hydroxyphospho-
nates 2 investigated are completely to the left (equilibri-
um concentrations of the corresponding H-phosphonate
diesters are usually below the detection level of 31P
NMR spectroscopy), it is apparent that the electronic
nature of the aryl groups Ar1 and Ar2 may influence
both the position of the equilibria and the rates with
which these are established, and ultimately may deter-
mine the decomposition pathways of these compounds.
The family of a-hydroxyphosphonates 2f–h bearing an
electron-deficient Ar2 group (pyridin-3-yl) and different
aryl ester groups Ar1 (phenyl, 4-methoxyphenyl and
2,6-dimethylphenyl, respectively) deserves some com-
ments. a-Hydroxyphosphonate 2f (Ar1 = phenyl), which
decomposed according to paths A and B, showed high
2.4. Antiviral potency of type 2 hydroxyphosphonates
a-Hydroxyphosphonates 2 were evaluated for their in vi-
tro cytotoxicity and anti-HIV-1 activity in MT-4-based
assays. The antiviral activity was determined against

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A. Szymanska et al. / Bioorg. Med. Chem. 14 (2006) 1924–1934
1930
anti-HIV potency (EC50 = 0.04 lM) and the lowest
cytotoxicity (CC50 > 100 lM) among the compounds
tested. These results were somewhat surprising in the
light of the very low antiviral activity of 8 and also of
the low antiviral potency but high cytotoxicity of nico-
tine aldehyde that was generated during the decomposi-
tion of 2f (Table 2). The a-hydroxyphosphonate 2g
(Ar1 = 4-methoxyphenyl) (EC50 = 0.01 lM) was more
potent than 2f (EC50 = 0.04 lM) but, at the same time,
highly cytotoxic (CC50 = 27 lM), probably due to the
release of 4-methoxyphenol (CC50 = 39 lM). When 4-
methoxyphenyl was replaced by 2,6-dimethylphenyl as
an Ar1 group, compound 2h underwent decomposition
exclusively according to path A (vide supra) and, due
to the low cytotoxicity of the generated 2,6-dimethyl-
phenol (CC50 > 100 M), CC50 and EC50 indices for 2h
were identical to those of AZT alone. For this reason,
compound 2h may be interesting as a potential fragment
of a more complex molecular framework because of its
low rate of decomposition in cell culture medium.
investigations, a-hydroxyphosphonates 2f–h bearing a
3-pyridyl moiety as Ar2 and different aryl ester groups
Ar1 are promising candidates for incorporation into a
more complex molecular framework to be evaluated for
their antiviral activity.
Finally, it is worth noting that all a-hydroxyphospho-
nates 2 studied herein are very stable under acidic condi-
tions (e.g., phosphate buffer, pH 2.0, >6 days; HPLC
analysis). This property, together with high lipophilicity
of a-hydroxyphosphonates 2 (Table 1), makes these
types of compounds attractive candidates for oral
administration in AIDS therapy.
4. Experimental
1H and 31P NMR spectra were recorded on a 300
MHz spectrometer. The 31P NMR experiments were car-
ried out in 5 mm tubes using 0.1 mol/mL concentration of
phosphorus-containing compounds and using H3PO4 as
external reference (inner tube). For column chromatogra-
phy, Kieselgel 60 Merck was used. The amount of water in
solvents was measured with Karl Fisher coulometric titra-
tion. Methylene dichloride was dried over P2O5, distilled
Finally, we examined the anti-HIV potency of a-hydrox-
yphosphonate 2i bearing 20,30-dideoxyuridine (ddU).
Since ddU is not phosphorylated by cellular kinases,40
it shows antiviral activity only when ddUMP is external-
ly delivered.41–43 The low anti-HIV potency observed for
compound 2i (Table 2) indicates that the a-hydrox-
yphosphonates 2 investigated herein act probably as
nucleoside vehicles and not as pro-nucleotides.
˚
and kept over molecular sieves 4 A until the amount of
water was less than 10 ppm. Pyridine and 2,6-lutidine
˚
were stored over molecular sieves 4 A until the amount
of water was below 20 ppm. Triethylamine and diisopro-
pylethylamine were distilled and stored over CaH2. Alde-
hydes 5a–f were of commercial grade from Aldrich.
RPMI-1640 cell culture medium and foetal bovine serum
(FBS) used for studies of stability of compounds were
from Sigma. HPLC analysis was carried out on a Hypersil
ODS column (4.6 · 250 mm, 5 lm); flow rate 1.5 mL/
min; solvent A—0.01 M triethylammonium trifluoroace-
tate, pH 3.02; solvent B—A/acetonitrile 4:1 (v/v); events:
5 min A 100%, linear gradient of B 0–100% in 40 min, A
100%—15 min wash. For quantification of peaks LKB
2221 integrator was used. Octanol/water partition coeffi-
cients were determined using the published procedure.44
3. Conclusions
We have developed efficient synthetic protocols for the
preparation of aryl nucleoside a-hydroxyphosphonates
2 which allow the introduction of various combinations
of aryl ester (Ar1) and a-arylmethylphosphonate (Ar2)
groups. The method makes use of easily available nucle-
oside H-phosphonate monoesters which, upon in situ
conversion into the corresponding aryl nucleoside H-
phosphonate diesters 4, are subjected to the reaction
with aromatic aldehydes 5 to produce a-hydrox-
yphosphonates 2 with high yields.
The reference compounds used for the identification of
some reaction products or intermediates were obtained
as follows: AZT H-phosphonate 6 and ddU H-phospho-
nate in the reaction of 30-azido-30-deoxythymidine or
20,30-dideoxyuridine with pyrophosphonic acid in pyri-
dine;45 AZT phenyl phosphorothioate 11 was obtained
following the earlier described procedure;38 phosphotriest-
ers 9 and 12 were obtained by condensation of phosphate
diester 3 with 4-nitrobenzyl alcohol or ethanol aided with
2,4,6-triisopropylbenzenesulfonyl chloride in methylene
chloride in the presence of N-methylimidazole.46
Aryl nucleoside a-hydroxyphosphonates 2 can be consid-
ered as potential antiviral drugs, whose reactivity can be
modulated by the proper choice of Ar1 and Ar2 groups.
Stability studies in cell culture medium showed that,
depending on the electronic structure of the aryl groups,
a-hydroxyphosphonates 2 may undergo decomposition
via three reaction pathways, A, B and C, to produce the
corresponding nucleosides, nucleotides, phenols, and
aldehydes.
Biological studies showed that the antiviral activity of a-
hydroxyphosphonates 2 mostly correlated with the
released AZT H-phosphonate monoester, and AZT as a
final metabolite. This is in line with the observed chemical
decomposition pathways for the investigated com-
pounds. All a-hydroxyphosphonates 2 showed high anti-
viral activity and it seems that their cytotoxicity can be
controlled (and probably improved) by a proper combi-
nation of Ar1 and Ar2 groups. At the present stage of
4.1. Antiviral assay procedures
4.1.1. Compounds. Compounds subjected to biological
assays were solubilized in DMSO at 200 mM and then
diluted in culture medium.
4.1.2. Cells and viruses. MT-4, C8166, and H9/IIIB cells
were grown at 37 ꢁC in a 5% CO2 atmosphere in RPMI
1640 medium, supplemented with 10% foetal calf serum

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A. Szymanska et al. / Bioorg. Med. Chem. 14 (2006) 1924–1934
1931
(FCS), 100 IU/mL penicillin G and 100 lg/mL strepto-
mycin. Cell cultures were checked periodically for the
absence of mycoplasma contamination with a MycoTect
Kit (Gibco). Human immunodeficiency viruses type-1
(HIV-1, IIIB strain) were obtained from supernatants
of persistently infected H9/IIIB cells. The HIV-1 stock
solutions had titres of 4.5 · 106 50% cell culture infec-
tious dose (CCID50)/mL.
nates 4, a respective aldehyde 5 was added (5 molar
equiv) and the reaction mixture was left standing over-
night at rt. After completion of the reaction (31P
NMR), the mixture was diluted with methylene chlo-
ride, washed with aqueous 1 M KH2PO4, pH 4.0, and
worked-up as described above.
a-Hydroxyphosphonates 2 were isolated by a silica gel 60
column chromatography using a stepwise gradient (0–
10%) of isopropanol in methylene chloride. Fractions
containing pure products were collected and evaporated
yielding non-hygroscopic foams. After freeze–drying
from benzene, compounds 2 were obtained usually
as white amorphous solids (yellow for p-nitrobenzyl
derivatives).
4.1.3. HIV titration. Titration of HIV was performed in
C8166 cells by the standard limiting dilution method
(dilution 1:2, four replica wells per dilution) in 96-well
plates. The infectious virus titre was determined by light
microscope scoring of syncytia after 4 days of incubation.
Virus titres were expressed as CCID50/mL.
4.3. 30-Azido-30-deoxythymidin-50-yl phenyl
hydroxy(phenyl)methanephosphonate (2a)
4.1.4. Anti-HIV assays. The activity of test compounds
against multiplication of wt HIV-1, in acutely infected
cells, was based on the inhibition of virus-induced cyto-
pathicity in MT-4 cells. Briefly, 50 lL of culture medium
containing 1 · 104 cells was added to each well of flat-bot-
tom microtiter trays containing 50 lL culture medium
with or without various concentrations of test com-
pounds. Then 20 lL of HIV suspensions (containing
the appropriate amount of CCID50 to cause complete
cytopathicity at day 4) was added. After incubation at
37 ꢁC, cell viability was determined by the 3-(4,5-dimeth-
ylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT)
method. Alternatively, p24 levels were determined by
an immunoenzymatic kit (Abbott). The cytotoxicity of
test compounds was evaluated in parallel with their anti-
viral activity and was based on the viability of mock-in-
fected cells, as monitored by the MTT method.
Obtained from H-phosphonate diester 4a and benzalde-
hyde 5a. The addition reaction in the presence of DI-
PEA was completed in 10 min. Yield 0.18 g (70%). H
NMR (CDCl3) d 7.54–7.05 (m, 11H), 6.13–6.03 (m,
1H), 5.54, 5.26, 5.22 (3d, 1H, J = 3.3, 2.7, and 2.7 Hz),
4.34–3.89 (m, 4H), 2.36–1.90 (m, 2H), 1.83, 1.81, 1.78,
1.77 (4d, 3H, J = 1.2 Hz); HRMS [MH]+: 514.1486,
calcd for C23H25O7N5P: 514.1491.
1
4.4. 30-Azido-30-deoxythymidin-50-yl phenyl a-hydroxy
(4-methylphenyl)methanephosphonate (2b)
Obtained from H-phosphonate diester 4a and 4-methyl-
benzaldehyde 5b. The addition reaction in the presence
of DIPEA was completed in 40 min. Yield 0.38 g (77%).
1H NMR (CDCl3) d 7.41–7.06 (m, 10H), 6.15–6.04 (m,
1H), 5.24–5.20 (m, 1H), 4.32–3.89 (m, 4H), 2.33 (s, 3H),
2.29–1.85 (m, 2H), 1.80, 1.78, 1.75 (3s, 3H); HRMS
[MH]+: 528.1650, calcd for C24H27O7N5P: 528.1648.
4.2. General procedure for the synthesis of aryl nucleoside
a-hydroxyphosphonates of type 2
Nucleosid-50-yl H-phosphonate (1 molar equiv) and the
respective phenol (1.5 molar equiv) were rendered anhy-
drous by co-evaporation of added pyridine and then
were dissolved in methylene chloride containing pyridine
[10% (v/v)] (0.1 mmol/1 mL). The coupling was effected
by the addition of diphenyl chlorophosphate (DPCP, 3
molar equiv) to the reaction mixture. When the forma-
tion of aryl nucleosid-50-yl H-phosphonate 4 was com-
pleted (ca. 20 min., 31P NMR), aldehyde 5 (5 molar
equiv) and diisopropylethylamine (DIPEA, 2.5 molar
equiv; for aldehydes 5a–d) were added. After the reac-
tions were completed (10–80 min, 31P NMR) the reac-
tion mixture was diluted with methylene chloride
(three times the initial volume) and acetic acid (1 molar
equiv) was added to neutralize excess amine. The result-
ing mixture was washed with water, organic layer sepa-
rated, and after drying (Na2SO4 anhyd) it was
evaporated under vacuum.
4.5. 30-Azido-30-deoxythymidin-50-yl phenyl a-hydroxy
(4-methoxyphenyl)methanephosphonate (2c)
Obtained from H-phosphonate diester 4a and 4-meth-
oxybenzaldehyde 5c. The addition reaction in the pres-
ence of DIPEA was completed in 80 min. Yield 0.46 g
1
(73%). H NMR (CDCl3) d 7.44–6.88 (m, 10 H), 6.16–
6.05 (m, 1H), 5.19–5.14 (m, 1H), 4.36–3.90 (m, 4H),
3.82, 3.80, 3.79 (3s, 3H), 2.39–1.90 (m, 2H), 1.85, 1.83,
1.80, 1.79 (3d, 3H, J = 1.2, 1.2, and 1.2 Hz); HRMS
[MH]+: 544.1584, calcd for C24H27O8N5P: 544.1597.
4.6. 30-Azido-30-deoxythymidin-50-yl phenyl hydroxy
(4-chlorophenyl)methanephosphonate (2d)
Obtained from H-phosphonate diester 4a and 4-chloro-
benzaldehyde 5d. The addition reaction in the presence
of DIPEA was completed in 10 min. Yield 0.42 g
When instead of DIPA, 2,6-lutidine [10% (v/v)] was used
as a base in condensation of nucleosid-50-yl H-phospho-
nates and phenols the reactions were carried out in
methylene chloride containing 2,6-lutidine [10% (v/v)]
(0.1 mmol/1 mL) using the same ratios of reagents
(condensations were usually completed in 20 min, 31P
NMR). To produce aryl nucleosid-50-yl H-phospho-
1
(77%). H NMR (CDCl3) d 7.46–7.03 (m, 10H), 6.08–
5.98 (m, 1H), 5.27, 5.24, 5.23, 5.19 (4s, 1H), 4.40–4.10
(m, 3H), 3.99–3.90 (m, 1H), 2.43–2.05 (m, 2H), 1.83,
1.80, 1.79 (s, 2d, 3H, J = 1.2 and 0.9 Hz). HRMS
[MH]+ 548.1106, calcd for C23H24O7N5 35ClP: 548.1102.

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A. Szymanska et al. / Bioorg. Med. Chem. 14 (2006) 1924–1934
1932
4.7. 30-Azido-30-deoxythymidin-50-yl phenyl hydroxy(4-
nitrophenyl)methanephosphonate (2e)
4.12. Synthesis of reference nucleoside a-hydrox-
yphosphonates 8
Obtained from H-phosphonate diester 4a and 4-nitro-
benzaldehyde 5e. The addition reaction in the presence
of 2,6-lutidine was completed in 45 min. Yield 0.92 g
Aryl nucleoside a-hydroxyphosphonate 2 (0.5 mmol)
was dissolved in a mixture of acetonitrile/water/triethyl-
amine 2:1:1 (v/v) (5 mL). After the reaction was com-
pleted (31P NMR), the solvents were evaporated and
the residue dissolved in methylene chloride was applied
into the silica gel column prepared in isopropanol. Reac-
tion mixtures consist of variable amounts of 8 (8a 47%,
8b 27%, and 8c 82%) and other decomposition products.
Compounds 8a–c were isolated using a stepwise gradient
of water [0–6% (v/v)] in isopropanol containing triethyl-
amine [3% (v/v)]. Fractions containing pure products 8
were collected and evaporated yielding a white foam.
After freeze–drying from benzene/methanol, compounds
8a–c were obtained as non-hygroscopic white amor-
phous solids.
1
(80%). H NMR (CDCl3) d 8.22–8.19, 7.75–7.68 (2m,
4H), 7.26–7.04 (m, 6H), 5.98–5.88 (m, 1H), 5.45–5.35
(m, 1H), 4.47–4.23 (m, 3H), 4.01–3.89 (m, 1H), 2.54–
2.27 (m, 2H), 1.85, 1.84, 1.82 (3d, 3H, J = 1.2, 0.9, and
1.2 Hz); HRMS [MH]+: 559.1337, calcd for
C23H24O9N6P: 559.1342.
4.8. 30-Azido-30-deoxythymidin-50-yl phenyl
hydroxy(pyridin-3-yl)methanephosphonate (2f)
Obtained from H-phosphonate diester 4a and pyridin-3-
aldehyde 5f. The addition reaction in the presence of
2,6-lutidine was completed in 24 h. Yield 1.16 g (55%).
1H NMR (CDCl3) d 8.70–8.67 (m, 1H), 8.52–8.50 (m,
1H), 7.90–7.84 (m, 1H), 7.30–7.02 (m, 7H), 5.92 (m,
1H), 5.30–5.25 (m, 1H), 4.44–4.16 (m, 3H), 3.91–3.90
(m, 1H), 2.30–2.24 (m, 2H), 1.82–1.77 (m, 3H); HRMS
[MH]+: 515.1427, calcd for C22H24O7N6P: 515.1444.
4.13. 30-Azido-30-deoxythymidin-50-yl hydroxy(4-chloro-
phenyl)methanephosphonate triethylammonium salt
(8a)
1H NMR (CDCl3) d 7.69,7.63 (2d, 1H, J = 0.9 and
1.2 Hz), 7.52–7.47 (m, 2H), 7.28–7.26 (m, 2H) 6.19–
6.12 (m, 1H) 4.92, 4.90, 4.88, 4.85 (4s, 1H), 4.41–4.37
(m, 1H), 4.30–3.94 (m, 3H), 2.92 (q, J = 7.4 Hz, 6H)
2.38–2.26 (m, 2H),1.93–1.92 (m, 3H), 1.18 (t,
J = 7.4 Hz, 9H). HRMS [M]ꢀ: 470.0646, calcd for
C17H18O7N5P: 470.0632.
4.9. 30-Azido-30-deoxythymidin-50-yl 4-methoxyphenyl
hydroxy(pyridin-3-yl)methanephosphonate (2g)
Obtained from H-phosphonate diester 4b and pyridin-3-
aldehyde 5f. The addition reaction in the presence of
2,6-lutidine was completed in 24 h. Yield 0.19 g (52%).
1H NMR (CDCl3) d 8.68 (br m, 1H), 8.50 (br m, 1H),
7.90–7.82 (m, 1H), 7.23–6.67 (m, 6H), 5.90 (br m, 1H),
5.26–5.23 (m,1H), 4.37–4.15 (m, 3H), 3.93–3.88 (m,
1H), 3.70, 3.69 (2s, 3H), 2.26 (br m, 2H), 1.77 (s, 3H).
HRMS [MH]+: 545.1552, calcd for C23H26O8N6P:
545.1550.
4.14. 30-Azido-30-deoxythymidin-50-yl hydroxy(4-nitro-
phenyl)methanephosphonate (8b)
1H NMR (CDCl3) d 8.18–8.15 (m, 2H), 7.78–7.73 (m,
2H), 7.66, 7.62 (2d, J = 1.2 and 0.9 Hz, 1H), 6.15–6.08
(m, 1H), 5.08, 5.06, 5.03, 5.00 (4s, 1H), 4.40–4.37 (m,
1H), 4.30–3.91 (m, 3H), 2.94 (q, J = 7.4 Hz, 6H), 2.41–
2.28 (m, 2H), 1.93 (s, 3H), 1.17 (t, J = 7.4 Hz, 9H);
HRMS [M]ꢀ: 481.0888, calcd for C17H18O9N6P:
481.0873.
4.10. 30-Azido-30-deoxythymidin-50-yl 2,6-dimethylphenyl
hydroxy(pyridin-3-yl)methanephosphonate (2h)
Obtained from H-phosphonate diester 4c and pyridin-3-
aldehyde 5f. The addition reaction in the presence of
2,6-lutidine was completed in 48 h. Yield 0.15 g (60%).
1H NMR (CDCl3) d 8.78–8.75 (m, 1H), 8.52 (s, 1H),
8.06–7.7 (m, 1H), 7.38–6.93 (m, 7H), 6.02–5.79 (m,
1H), 5.44–5.30 (m, 1H), 4.45–4.31 (m, 1H), 4.21–4.09
(m, 2H), 3.97–3.83 (m, 1H), 2.43–2.28 (m, 2H), 2.14,
2.13 (2s, 6H), 1.90, 1.86, 1.85 (3d, 3H, J = 0.6, 0.9,
and 1.2 Hz). HRMS [MH]+: 543.1748, calcd for
C24H28O7N6P: 543.1757.
4.15. 30-Azido-30-deoxythymidin-50-yl hydroxy(pyridin-3-
yl)methanephosphonate (8c)
1H NMR (CDCl3) d 8.76–8.75 (m, 1H), 8.46–8.44 (m,
1H), 7.95–7.89 (m, 1H), 7.69, 7.65, (2d, J = 1.2 and
1.2 Hz), 7.27–7.23 (m, 1H), 6.18–6.11 (m, 1H), 4.98,
4.96, 4.94, 4.91 (4s, 1H), 4.44–4.40 (m, 1H), 4.30–
4.02 (m, 3H), 2.87 (q, J = 7.4 Hz, 6H), 2.44–2.24
(m, 2H), 1.92–1.91 (m, 3H), 1.13 (t, J = 7.4 Hz,
9H); HRMS [M]ꢀ: 437.0983, calcd for C16H18O7N6P:
437.0975.
4.11. 20,30-Dideoxy-20,30-didehydrouridin-50-yl phenyl
a-hydroxy(4-methoxyphenyl)methanephosphonate (2i)
4.16. 30-Azido-30-deoxythymidin-50-yl 4-nitrobenzyl
phenyl phosphate (9)
Obtained from H-phosphonate diester 4d and 4-meth-
oxybenzaldehyde 5c. The addition reaction in the pres-
ence of DIPEA was completed in 2 h. Yield 0.22 g
1
Yield 0.185 g (73%). H NMR (CDCl3) d 8.78–8.76 (m,
1
(63%). H NMR (CDCl3) d 7.46–6.87 (m, 10H), 6.06–
1H), 8.23–8.17 (m, 2H), 7.51–7.15 (m, 8H), 6.19–6.15
(m, 1H), 5.30,5.28, 5.27, 5.25 (4s, 2H), 4.48–4.24 (m,
3H), 4.03–4.01 (m, 1H), 2.48–2.24 (m, 2H), 1.86, 1.82
(2d, J = 1.2 and 1.2 Hz); HRMS [MH]+: 559.1322, calcd
for C23H24O9N6P: 559.1342.
5.91 (m, 1H), 5.66, 5.62, 5.54, 5.51 (4d, 1H, J = 8.1,
7.5, and 8.7 Hz), 4.32–4.15 (m, 3H), 3.81, 3.80, 3.80,
3.79 (4s, 3H), 2.40–1.64 (m, 4H). HRMS [MH]+:
488.1342, calcd for C23H25N2O8P: 488.1349.

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A. Szymanska et al. / Bioorg. Med. Chem. 14 (2006) 1924–1934
1933
4.17. 30-Azido-30-deoxythymidin-50-yl phenyl phosphoro-
thioate triethylammonium salt (11)
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1
Yield 0.069 g (64%). H NMR (CDCl3) d 8.16 (s, 1H),
7.83, 7.75 (2d, J = 1.2 and 1.2 Hz), 7.15–7.09 (m, 4H),
6.95–6.93 (m, 1H), 6.33–6.27 (m, 1H), 4.43–4.28 (m,
3H), 4.09–4.08 (m, 1H)3.15 (q, J = 7.4 Hz, 6H), 2.37–
2.25 (m, 2H), 1.93, 1.90 (2d, J = 1.2 and 1.2 Hz, 3H),
1.28 (t, J = 7.4 Hz, 9H); HRMS [M]ꢀ: 438.0625, calcd
for C16H17O6N5P: 438.0637.
4.18. 30-Azido-30-deoxythymidin-50-yl ethyl phenyl phos-
phate (12)
1
Yield 0.195 g (86%). H NMR (CDCl3) d 8.59 (s, 1H),
7.40–7.19 (m, 6H), 6.26–6.20 (m, 1H), 4.67–4.21 (m,
5H), 4.06–4.02 (m, 1H), 2.45–2.19 (m, 2H), 1.88, 1.86
(2d, J = 0.9 and 1.2 Hz, 3H), 1.40–1.33 (m, 3H); HRMS
[MH]+: 451.1263, calcd for C18H22N5O7P: 451.1257.
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Acknowledgments
Financial support from the State Committee for Scien-
tific Research, Republic of Poland (PBZ-KBN 059/
T09/19 grant to A.K.), and Regione Autonoma Sarde-
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gna (Assessorato Sanita) is gratefully acknowledged.
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