Aryl nucleoside H-phosphonates. Part 16: Synthesis and anti-HIV-1 activity of di-aryl nucleoside phosphotriesters

Article abstract of DOI:10.1016/j.bmc.2009.02.033

Di-aryl nucleoside phosphotriesters have been explored as a new type of pronucleotides for the purpose of anti-HIV-1 therapy and efficient synthetic protocols, based on H-phosphonate chemistry, have been developed for the preparation of this class of comp

Full text of DOI:10.1016/j.bmc.2009.02.033

Bioorganic & Medicinal Chemistry 17 (2009) 3489–3498
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Aryl nucleoside H-phosphonates. Part 16: Synthesis and anti-HIV-1 activity
of di-aryl nucleoside phosphotriesters
´
´
Joanna Romanowska, Agnieszka Szymanska-Michalak, Jerzy Boryski, Jacek Stawinski,
Adam Kraszewski*
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan´, Poland
Roberta Loddo, Giuseppina Sanna, Gabriella Collu, Barbara Secci, Paolo La Colla*
Department of Biomedical Sciences and Technologies University of Cagliari, 09042 Monserrato (CA), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Di-aryl nucleoside phosphotriesters have been explored as a new type of pronucleotides for the purpose
of anti-HIV-1 therapy and efficient synthetic protocols, based on H-phosphonate chemistry, have been
developed for the preparation of this class of compounds. It was found that anti-HIV-1 activity of the
phosphotriesters bearing an antiviral nucleoside moiety (AZT, ddA) and also ddU was due, at least par-
tially, to intracellular conversion into the corresponding nucleoside 50-monophosphates, and their effi-
ciency correlated well with the pKa values of the aryloxy groups present.
Received 9 October 2008
Revised 11 February 2009
Accepted 15 February 2009
Available online 23 February 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Nucleoside analogues
Pro-nucleotides
Phosphotriesters
Anti-HIV-1 activity
Pro-drugs
1. Introduction
properly masked and became neutral, this should facilitate cell
membrane penetration and increase concentration of drug inside
To disclose their antiviral activity, nucleoside analogues have to
be converted with the help of cellular enzymes, such as nucleoside
and nucleotidyl kinases, into the respective nucleoside 50-triphos-
phates (NTP).1 The formation of triphosphates occurs in a stepwise
manner and usually the first phosphorylation step, that is, the syn-
thesis of nucleoside 50-monophosphates, is the crucial step for NTP
formation. Since phosphorylation is indispensable for biological
activity, nucleoside analogues that are poor substrate for phos-
phorylating enzymes (e.g., 20,30-dideoxyuridine), are usually inac-
tive.2 Due to this, nucleoside analogues lose their antiviral
potency in nucleoside kinase deficient cells.3
To by-pass this enzymatic monophosphorylation step, efforts
were focused on delivery into the cell 50-monophosphates of nucle-
oside analogues4 (for review5–8). Unfortunately, under physiologi-
cal conditions, nucleoside 50-monophosphates exist as dianions
and cannot cross negatively charged cell membranes.9 Hence, it
was assumed that if a phosphate moiety of mononucleotide is
the cell. This idea, called pronucleotide approach, triggered studies
on various types of nucleotide derivatives, whose intracellular con-
version to the desired nucleoside 50-monophosphates would occur
via chemical and/or enzymatic hydrolysis of the phosphate mask-
ing groups. Typical examples include cyclic phosphate derivatives
(e.g., nucleoside saligenyl phosphates, cyclo-Sal),10 or nucleoside
phosphoramidates.11,12 Another class of pronucleotides is repre-
sented by pivaloyloxymethyl (POM7), dithioethyl (DTE13) and
S-acyl-2-thioethyl (SATE13–15) nucleoside phosphotriesters, whose
bio-activation is triggered by enzymatic cleavage of a carboxylic
acid ester group (POM and SATE), or a disulfide bond of the side
chain (DTE), followed by a spontaneous intramolecular conversion
into the corresponding nucleoside monophosphates. Synthesis and
biological activity of these compounds were widely studied and
were subjects of several reviews.5,6
Somehow surprisingly, simple alkyl16 or aryl17 nucleoside phos-
photriesters have not received much attention, most likely due to
mediocre anti-HIV-1 activity (exception, bis-4-nitrophenyl AZT
phosphate) observed in the early experiments.18 However, since
there is a huge choice of alkyl and particularly aryl groups, with
different structural and electronic features, finding a good masking
group should be a feasible task.
* Corresponding authors. Tel.: +39 070 675 4147; fax: +39 070 675 4210 (P.L.C.);
tel.: +48 61 852 85 03; fax: +48 61 852 05 32 (A.K.).
E-mail addresses: placolla@unica.it (P.L. Colla), Adam.Kraszewski@ibch.poznan.
pl (A. Kraszewski).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.033

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R. Loddo et al. / Bioorg. Med. Chem. 17 (2009) 3489–3498
In practice, the problem is more complex because an ideal
pling of respective aryl nucleoside phosphodiesters of type 4 and
phenols 2, using TPS-Cl/NMeIm reagent system.22 This approach
was of our interest because of an easy synthetic access in our lab-
oratory to a variety of aryl nucleoside phosphodiesters23 and a
wide commercial offer of phenol or pyridinol derivatives. Prelimin-
ary experiments showed (31P NMR analysis) that condensation of
30-azido-30-deoxythymidine (AZT) pyridin-3-yl phosphodiester
4af with phenol 2a produced rapidly (ca. 15 min) the expected
AZT phenyl pyridin-3-yl phosphotriester 5aa almost quantita-
tively. Unfortunately, in contrast to this, under the same reaction
conditions, condensations of phenyl phosphodiesters 4aa with
pyridin-3-ol 2f were very slow and produced a complex mixture
of products (31P NMR analysis). Although this approach worked
well in some cases, it cannot be considered as a general synthetic
method for the preparation of nucleoside phosphotriesters.
The second approach studied, namely, an iodine promoted oxi-
dative coupling of aryl nucleoside H-phosphonate diesters of type
3 with 5 M excess of a phenol or pyridinol of type 2, provided
nearly quantitatively (31P NMR analysis) the desired phosphotri-
esters 5 (Scheme 1). The starting aryl nucleoside H-phosphonates
3, were easily accessible via a condensation of nucleoside H-phos-
phonates 1 with various phenols or pyridinols (OHAr1 2) (1.2 M -
equiv), promoted by diphenyl chlorophosphate (DPCP) or pivaloyl
chloride (PvCl), in methylene chloride (or other neutral solvent)
in the presence of a limited amount of pyridine.24 Due to their high
pronucleotide has to fulfil several, sometimes contradictory, crite-
ria. Specifically, (i) a pronucleotide should have proper stability in
physiological media and be converted inside the cell into biologi-
cally active form with optimal kinetics, (ii) it should be well soluble
in water, (iii) it should be neutral and/or sufficiently lipophilic to
be able to cross cell membrane and enter the cell, (iv) the pronucle-
otides and their metabolites should not be toxic.
During our studies on anti-HIV-1 activity of uncharged nucleo-
side a
-hydroxyphosphonates,19 we noticed that compounds, bear-
ing a pyridyl residue in the C-phosphonate fragment of the
molecule, were well soluble in water, had significant antiviral
activity and were relatively non-toxic. Favourable physicochemical
properties prompted us to investigate phosphotriesters bearing a
pyridyl protecting group (e.g., AZT, ddA and ddU derivatives), as
potential pronucleotides.
2. Results and discussion
2.1. Synthesis of aryl nucleoside phosphodiesters of type 4 and
di-aryl nucleoside phosphotriesters of type 5
For the synthesis of nucleoside phosphotriesters of type 5 we
investigated two approaches. The first one, a ‘step by step’ conden-
sation (classical phosphotriester approach20,21), consisted of cou-
Scheme 1. General scheme for the synthesis of di-aryl nucleoside phosphotriesters of type 5. Reagents and conditions: (i) diphenyl chlorophosphate (DPCP ꢀ1.2 M equiv) in
CH2Cl2/pyridine 9:1 (v/v); (ii) I2 (1.5 M equiv) in pyridine (0.3 M solution); (iii) I2 (1.5 M equiv) in pyridine/water (4:1, v/v); (iv) 2,4,6-tri-isopropylbenzenesulfonyl chloride
(TPS-Cl, 3 M equiv) in methylene chloride containing N-methylimidazole (NMeIm, 10% by volume).

R. Loddo et al. / Bioorg. Med. Chem. 17 (2009) 3489–3498
3491
reactivity,24 H-phosphonate diesters of type 3 were not isolated,
but subjected in situ to the reaction with a second phenol or pyrid-
inol OHAr2 2 (5 M equiv), in the presence of iodine (1.5–2 equiv).
This produced rapidly (<3 min) and cleanly (31P NMR analysis)
the desired di-aryl nucleoside phosphotriesters 5.
chemical hydrolysis (experiments in RPMI), and how good sub-
strates they were to phosphoesterases (experiments in RPMI/FBS
media). Although enzymatic activities present in the cell culture
media (from FBS) are ca. 10–50 times weaker than those found in
cells,29,30 it is usually assumed that data from the experiments in
RPMI and RPMI/FBS can be extrapolated to the analogous metabolic
events in the living cell. It was found that all aryl nucleoside phos-
In these reactions, 5 M excess of OHAr2 2 was necessary to
ensure rapid formation of unsymmetrical (Ar1–Ar2) di-aryl phos-
photriester 5. Under such reaction conditions, symmetrical phos-
photriesters (Ar1–Ar1) were not formed as judged from the 31P
NMR spectra and chromatographic analysis (HRTLC).
phodiesters 4aa–cb (2 lM solutions) were stable within 6 days in
RPMI at 37 °C (HPLC analysis). Their stability under analogous con-
ditions but in RPMI/FBS varied and depended on a phosphoester
aryl group, type of modification in the sugar ring, and a nucleobase
in the nucleoside moiety (Table 1).
These reactions are believed to proceed via iodophosphate
intermediates,25–27 that in the presence of pyridine, reacted rapidly
with hydroxylic components to produce di-aryl phosphotriesters
of type 5. Since the whole reaction sequence, from 1 to 5, pro-
ceeded cleanly and nearly quantitatively (31P NMR analysis), one
could consider it a simple and efficient one-pot approach to the
synthesis of diaryl nucleoside phosphotriesters. After purification
by a silica gel short column chromatography28 and freeze-drying
from benzene, compounds 5 were obtained as amorphous solids
in satisfactory yields (50–70%). The correctness of their structures
and high purity were proven by spectral (1H, 31P NMR, and HRMS)
and chromatographic (HPLC) analyses.
As it is apparent from data in Table 1, the rates of hydrolyses do
not vary much along the AZT nucleotide aryl esters 4aa–ak series,
although in the case of phosphodiester 4ae, it seems that the
inductive effect of two chlorine atoms (o- and p-) made 2,4-dichlo-
rophenol the best leaving group and the presence of chlorine atoms
do not affect the interaction with an enzyme active centre during
hydrolysis. Considering only the t1/2 values within the AZT pyridi-
nyl phosphoesters 4af–ak series, it seems that best substrates for
hydrolytic enzymes are nucleotides, bearing the pyridin-3-yl, pyri-
din-4-yl and 5-chloropyridin-3-yl groups (4af, 4ag and 4ak,
respectively) and these were nearly equivalent in respect to enzy-
matic hydrolysis to phenol derivatives 4aa–ad.
Aryl nucleoside phosphodiesters derived from ddU (4ba–bc)
and ddA (4ca) appeared to be distinctly poor substrates compared
to AZT nucleotides, most likely due to weaker interaction of the
20,30 dideoxyribose moieties of ddU and ddA with the enzyme ac-
tive centre. Nevertheless, phosphodiesters 4ba, 4bc and 4c were
still good substrates for hydrolytic enzymes of FBS and certainly
can be considered as a potentially useful metabolite in the anabolic
path to the desired nucleoside 50-triphosphate in the cell.
It can be tentatively concluded that all of the investigated aryl
nucleoside phosphodiesters 4 were substrates for enzymatic activ-
ities present in FBS and the observed differences in t1/2 values of
their hydrolysis can be useful in tuning pronucleotide properties
of phosphotriester of type 5 (vide infra).
2.2. Decomposition of aryl nucleoside phosphodiesters 4 in
RPMI and cell culture media [RPMI/FBS 9:1 (v/v)]
For phosphotriesters of type 5 to act as true pronucleotides, both
steps in their intracellular conversion into the corresponding nucle-
oside 50-monophosphates, that is, chemical hydrolysis to produce
nucleoside phosphodiester, and the subsequent enzymatic hydro-
lysis to nucleoside 50-phosphate (Scheme 2), are equally important.
In the first step, chemical properties of a phosphotriester masking
groups permit usually prediction of its hydrolytic stability. Rates
of the removal of protecting groups from phosphodiesters are often
difficult to predict, due to enzymatic catalysis usually involved in
this step. Considering that the leaving groups are often structurally
unrelated to natural substrates of enzymatic reaction (nucleoside or
nucleotide), it was necessary to check experimentally susceptibility
of nucleoside phosphodiesters of type 4 (Scheme 2) to enzymatic
hydrolysis. To this end, we prepared a set of aryl nucleoside phos-
phodiesters 4aa–cb, which were the expected intermediates during
conversion of pronucleotides 5 into the corresponding nucleotide
50-monophosphates 6, and examined their stability in RPMI and
in RPMI/FBS 9:1 (v/v) media. These experiments should provide
information to what extent these compounds were prone to simple
2.3. Decomposition of di-aryl nucleoside phosphotriesters 5 in RPMI
Next, we examined stability of di-aryl nucleoside phosphotri-
esters 5 in RPMI. Since no enzymatic activity is present in this med-
ium, the observed decomposition of 5 had to be due to chemical
hydrolysis. This was particularly useful in the case of unsymmetri-
cal phosphotriesters of type 5 (Ar1 – Ar2), since it provided infor-
Scheme 2. Decomposition of di-aryl nucleoside phosphotriesters 5 in RPMI and aryl nucleoside phosphodiesters 4 in RPMI/FBS 37 °C.

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R. Loddo et al. / Bioorg. Med. Chem. 17 (2009) 3489–3498
Table 1
Stability of aryl nucleoside phosphodiester 4 in the cell culture mediaa
Compound
Ar
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
Cl
H3C
N
N
N
Br
CN
Cl
Cl
t1/2 (min)b
568
330
478
425
182
233
4bc
484
949
Compound
4ai
4aj
4ak
4ba
4bb
4ca
4cb
Cl
Ar
N
N
N
N
N
Cl
N
CH3
1732
t1/2 (min)b
976
436
1506
3013
1414
2166
3850
a
RPMI 1640/FBS 9:1 (v/v), 37 °C.
b
In each case enzymatic hydrolysis of 4 produced the respective nucleoside (AZT, ddU or ddA)-50-monophosphate 6. For all experiments one lot of RPMI and FBS was used.
mation which phosphoester bond, and to what extent, was weaker
and in consequence, which phosphodiester 4 would be produced as
a major product. In Table 2, nucleotide phosphodiesters, produced
during decomposition of nucleoside phosphotriesters 5 in RPMI,
are shown. The ratio of different phosphodiesters 4 formed, indi-
cated relative stability of the phosphoester bonds in the studied
phosphotriesters 5.
Data from Table 2 show that the half-lives (t1/2) of phosphotriest-
ers 5 in RPMI medium depended on acidity of the phenol moieties in
5, as it can be seen, for example, along the series of phosphotriesters
5aa, 5ac–5af. The influence of an aryl group on the stability of the
phosphoester bonds was as expected from electronic effects of the
substituents in the aromatic ring. For pyridin-3-yl phosphotriesters
5ak–5an, the same rules seemed to operate, although compounds,
Table 2
Stability of di-aryl nucleoside phosphotriester 5 in RPMIa, their cytotoxicityb (CC50) against MT-4 cells and antiviral activityc (EC50) against HIV-1IIIB
Compounds
AZT
Ar1
Ar2
RPMI t1/2 (min)
Produced 4 and their ratiod
CC50
60
(lM)
EC50
0.01
(
lM)
SI
—
—
—
—
>6000
5aa
9.9831
8.6031
3013
770
4aa > 4af ꢁ3:1
>100
0.05
>2000
N
5ab
7.74e,f32
4aa
>100
0.01
>10000
N
5ac
5ad
5ae
9.3831
10.031
3300
2660
173
4ab > 4af ꢁ2:1
4ac > 4af ꢁ2:1
4af
P100
94
0.03
0.03
0.02
>3333
3133
2900
N
N
N
Cl
Br
7.9531
58
CN
Cl
5af
7.8531
396
4af
>100
0.01
>10,000
N
N
Cl
N
5ag
5ah
295
513
4af
4af
>100
>100
0.01
0.02
>10,000
>5000
N
N
H3C
H3C
5ai
5aj
9.5033
1034
1283
4ah
4ai
>100
>100
0.02
0.04
>5000
>2500
N
N
8.9033
CH3
N
N
CH3

R. Loddo et al. / Bioorg. Med. Chem. 17 (2009) 3489–3498
3493
Table 2 (continued)
Compounds
Ar1
Ar2
RPMI t1/2 (min)
Produced 4 and their ratiod
4aa > 4ah ꢁ2:1
CC50
(l
M)
EC50
0.1
(
lM)
SI
H3C
5ak
5al
4951
>100
>1000
>1111
N
2665
4aa > 4ai ꢁ2:1
>100
0.09
N
CH3
N
Cl
5am
5an
963
4aa
4aa
P100
0.03
0.04
>3333
2500
1506
100
N
Cl
ddU
5ba
—
—
—
—
>100
>100
>100
38
1
3850
4ba > 4bb ꢁ3:1
>2
N
5bb
1610
420
4ba
4bb
>100
>100
3.0
14
>33
>7
N
5bc
N
N
ddA
—
—
—
—
>100
>100
5.2
23
>19
>4
5c
5331
4ca > 4cb ꢁ3:1
N
a
RPMI 1640, 37 °C. Compounds 5 exhibited nearly the same stability also in RPMI/FBS (9:1, v/v: data not shown).
b
c
Compound concentration (lM) required to reduce the viability of mock-infected MT-4 cells by 50%, as determined by the MTT method.
Compound concentration (lM) required to achieve 50% protection from virus-induced cytopathogenicity, as determined by the MTT method. Data represent mean values
(+SD) for three independent determinations. Variation among triplicate samples was less than 15%.
d
Calculated on the basis of area of peaks corresponding to particular compounds—not corrected.
e
pKa of OH group in respective ArOH.
f
Corrected value with regard to hydroxypyridine-pyridone tautomerism.32
with lipophilic substituent at the ortho position (e.g., Cl or CH3 as in
5ak and 5am), were about twice as stable as those bearing the same
substituents at the para or meta position (5al and 5an, respectively).
These differences can be explained, at least in part, by less efficient
hydration of the leaving aryloxyl group, analogously to what we ob-
5aa–an series, the potency, comparable or higher to that of the
parent nucleoside (AZT), disclosed the least stable in the RPMI
medium compounds, that is, 5ab, 5af, 5ag and also 5ae, 5ah and
5ai (t1/2 300–1000 min). All of them bear at least one aryl group
with electron-withdrawing substituents that facilitates chemical
hydrolysis to the corresponding nucleoside phosphodiesters of
type 4. Since enzymatic hydrolysis of the resulting phosphodiest-
ers of type 4 proceeded faster than the chemical hydrolysis step,
it could be suggested that for the compounds investigated, the
antiviral potency depended on stability of phosphotriesters of type
5. This rule seems to be held also for phosphotriesters 5ba–bc in
the 20,30-dideoxyuridine (ddU) series and for 20,30-dideoxyadeno-
sine (ddA) pronucleotide 5c.
served earlier during hydrolysis of a
-hydroxyphosphonates.19 Phos-
photriesters 5, bearing 4-pyridyl group (e.g., 5ab or 5bb), were
distinctly less stable than the analogous 3-pyridyl protected nucle-
otides (5aa and 5bb, respectively), which can be attributed to a
stronger inductive effect (electron-withdrawing) of an endocyclic
nitrogen atom in the position 4 of the pyridine ring.
Another important observation was that ddU and ddA phospho-
triesters (5ba, 5bb and 5c) were apparently more hydrolytically
stable than the analogous AZT derivatives (5aa and 5ab, respec-
tively). It is likely that, the inductive effect of the 3-azido function
in AZT, although distant, still enhances electrophilicity of the phos-
phorus center in phosphotriesters 5a. In the case of phosphotriest-
ers 5ba–bc and 5c, the 30-methylene group may have an electron-
donating character and lowers electrophilicity of the phosphorus
center. Thus, detail structural features of all the groups attached
to the phosphorus center have to be taken into consideration when
designing a new type of pronucleotides.
Antiviral potency of diphenyl AZT phosphotriester is known to
be rather poor (EC50 0.3 lM) most likely due to its high stability
in RPMI (t1/2 > 6 days). Taking this into account, it can be postu-
lated that at least one of the aryloxy groups of a potential pronucle-
otide of type 5 should have a pKa value lower than that of
unsubstituted phenol (pKa 9.98), in order to secure efficient gener-
ation of phosphodiesters 4, the first intermediate in anabolic multi-
step path of enzymatic formation of AZT 50-triphosphate. It is
worth stressing that the above correlation is also valid for ddU-de-
rived phosphotriesters 5ba–bc, although these compounds were
found to be generally more stable (higher the t1/2 values, Table 2)
than the analogous AZT derivatives.
2.4. Cytotoxicity and antiviral activity
All di-aryl nucleoside phosphotriesters investigated disclosed
significant anti-HIV-1 potency. Along the AZT phosphotriester
Since anti-HIV-1 potency of the investigated phosphotriesters 5
correlated mainly with their susceptibility to chemical hydrolysis,

3494
R. Loddo et al. / Bioorg. Med. Chem. 17 (2009) 3489–3498
an important question arose, namely, what was the mode of action
of these compound? Are they true pronucleotides or are they vehi-
cles for antiviral nucleosides, only?
4. Experimental
1H and 31P NMR spectra were recorded on 300 MHz or
400 MHz machines. The 31P NMR (121 MHz) experiments were
carried out in 5 mm tubes using 0.1 M solutions of the phospho-
rus-containing compound. 31P NMR chemical shifts are reported
in ppm relative to 85% H3PO4 in water, used as an external stan-
dard. Mass spectra of phosphodiesters of type 4 were recorded
with liquid secondary ion mass technique (LSIMS) using Cs+
(12 keV) for ionisation. FAB technique was used for measuring
mass spectra of phosphotriesters of type 5. The amount of water
in solvents was measured with Karl Fisher coulometric titration.
Methylene dichloride was dried over P2O5, distilled, and kept over
molecular sieves 4 Å until the amount of water was less than
10 ppm. Pyridine was stored over molecular sieves 4 Å until the
amount of water was below 20 ppm. Triethylamine was distilled
and stored over CaH2. For column chromatography Silica gel 60
(Merck) was used. For TLC analysis, the precoated plates (Merck
Silica gel 60 F254) were used. Phenols and pyridinols were com-
mercial grade from Aldrich. RPMI-1640 cell culture medium and
heat non inactivated foetal bovine serum (FBS) used for studies
of stability of compounds were from Sigma (R7256 and F7524,
respectively). HPLC analysis was carried out on a Hypersil ODS
To address this question, along with AZT nucleotides, the anal-
ogous phosphotriesters, bearing the ddU nucleoside moiety, were
prepared. It is known that ddU nucleoside is a very poor substrate
for thymidine kinase and it cannot be converted into its 50-mono-
phosphate, and in consequence, into anti-HIV-1 active tri-phos-
phate.2 However, when ddU-derived nucleotides with properly
masked 50-phosphate groups were used, they were found to be
highly potent anti-HIV-1 agents. This suggested that these com-
pounds could act as pronucleotides and by-pass a thymidine kinase
phosphorylation process in the cell.34
In these studies we found a significant anti-HIV-1 activity of
ddU phosphotriesters 5ba–bc, and this is a strong evidence that
these compounds can act, at least partially, as true pronucleotides,
delivering into the cell a notable proportion of nucleoside 50-mono-
phosphates. The same is most likely true for the AZT phosphotri-
esters (for instance 5ab and 5ag) that under similar conditions
also exhibited high anti-HIV-1 potency.
Although, most of the compounds investigated were not
toxic (CC50 >100
lM), some of them (5ac, 5ad, 5ae, 5am and
5an) showed noticeable (CC50 6 100
l
M) cytotoxicity, which
probably came from the known toxicity of halophenols,35
formed as hydrolytic metabolites (with exception of 4-cyano-
phenyl phosphotriester 5ae). In this context rather surprising
was the lack of cytotoxicity of phosphotriester 5af, bearing
2,4-dichlorophenyl phosphoester group. It is also important to
note that 2-, 3- and 4-pyridinols, examined separately, were
column (4.6 ꢂ 250 mm, 5
lm); flow rate 1.5 mL/min; solvent
A—0.01 M triethylammonium acetate, pH 7.2; solvent B—A/aceto-
nitrile 4:1 (v/v); events: 5 min A 100%, linear gradient of B 0–
100% in 30 min, A 100%—10 min wash. For quantification of peaks
Waters Breeze Software was used.
Nucleoside H-phosphonates of type 1 and aryl nucleoside phos-
phodiesters of type 4 were obtained following procedures de-
scribed earlier.36,23 All synthesized compounds were of purity
better than 97% as judged from 1H NMR spectroscopy.
not cytotoxic (CC50 >100 lM). These results should be taken
into account while designing of new anti-HIV-1 pronucleotides
of type 5.
We have also examined antiviral activity of all di-aryl nucleo-
side phosphotriesters 5 against HIV-1 variants containing the
mutations 181C (N119), 103N+181C (A17), 103R+179D+225H
(EFVR), 67N+70R+215F+219Q (AZTR) and 41L+74V+106A+215Y
(MDR-1). The details of these experiments, which are consistent
with those discussed above, are given in Supplementary data.
4.1. Biological assays
4.1.1. Compounds
Compounds were dissolved in DMSO at 100 mM and then di-
luted in culture medium.
3. Conclusions
4.1.2. Cells and viruses
Cell lines were purchased from American Type Culture Collec-
tion (ATCC). The absence of mycoplasma contamination was
checked periodically by the Hoechst staining method. CD4+ human
T-cells containing an integrated HTLV-1 genome (MT-4) were used
to support the multiplication of HIV-1.
We have prepared a series of aryl nucleoside phosphates 4 and
found that all of them are good substrates for a phosphoesterase-
type of activity present in FBS, and probably also for similar enzy-
matic activities in the living cell. These results kindle the hope that,
if phosphodiesters of type 4 will be delivered or in situ produced in
the cell, they should be converted into nucleoside 50-monophos-
phates 6 and further into the corresponding triphosphates, that
are true anti-HIV-1 agents. On this basis, we designed di-aryl
nucleoside phosphotriesters 5, bearing AZT, ddU or ddA moiety,
as potential anti-HIV-1 pronucleotides. For the preparation of these
compounds we developed a highly efficient one-pot synthesis,
which consisted of a condensation of nucleoside 50-H-phospho-
nates with the appropriate phenol or pyridinol, followed by
oxidative coupling of a pyridinol or another phenol, promoted by
iodine.
All the compounds investigated showed anti-HIV-1 activity and
since it was observed for both AZT- and ddU-derived phosphotri-
esters 5, we can assume that these compounds act, at least
partially, as true pronucleotides, delivering inside the cells notable
amounts of the nucleoside 50-monophosphates, rather than corre-
sponding nucleosides.
Chemical stability of phosphotriesters 5 determines how easy
the corresponding phosphodiesters of type 4 are formed and this
may serve for a tentative prediction of anti-HIV-1 activity of these
compounds.
4.1.3. Cytotoxicity assays
For cytotoxicity evaluations, exponentially growing cells derived
from human haematological tumors [CD4+ human T-cells contain-
ing an integrated HTLV-1 genome (MT-4)] were seeded at an initial
density of 1 ꢂ 105 cells/ml in 96 well plates in RPMI-1640 medium
supplemented with 10% foetal calf serum (FCS), 100 units/mL pen-
icillin G and 100 lg/mL streptomycin. Cell cultures were then incu-
bated at 37 °C in a humidified, 5% CO2 atmosphere in the absence or
presence of serial dilutions of test compounds. Cell viability was
determined after 96 h at 37 °C by the MTT method.37
4.1.4. Antiviral assay
Activity of compounds against Human Immunodeficiency virus
type-1 (HIV-1) was based on inhibition of virus-induced cytopath-
ogenicity in MT-4 cells acutely infected with a multiplicity of infec-
tion (m.o.i.) of 0.01. Briefly, 50
were added to each well of flat-bottom microtitre trays containing
l
L of RPMI containing 1 ꢂ 104 MT-4
50
Then, 20
l
L of RPMI, without or with serial dilutions of test compounds.
L of an HIV-1 suspension containing 100 CCID50 were
l

R. Loddo et al. / Bioorg. Med. Chem. 17 (2009) 3489–3498
3495
added. After a 4-day incubation, cell viability was determined by
the MTT method.
J = 7.2 Hz, 6H), 2.38–2.22 (m, 2H), 1.89 (d, J = 0.9 Hz, 3H), 1.28 (t,
3
J = 7.2 Hz, 9H); 31P NMR (CDCl3) d ꢀ6.27 (t, JHP = 5.5 Hz); HRMS
[MꢀEt3NH+]ꢀ: 423.0815, calcd for C15H16N6O7P: 423.0818.
4.1.5. Aryl nucleoside phosphodiesters of type 4
4.1.5.1. 30-Azido-30-deoxythymidyn-50-yl phenyl phosphate tri-
ethylammonium salt (4aaY).ield 0.49 g (94%). 1H NMR (CDCl3) d
12.24 (br s, 1H, exch. D2O), 8.00 (br s, exch. D2O), 7.73 (q,
J = 0.9 Hz, 1H), 7.28–7.24 (m, 5H), 6.27 (t, J = 6.7 Hz, 1H), 4.33–
4.28 (m, 1H), 4.22–4.18 (m, 2H), 4.03–4.01 (m, 1H), 3.04 (q,
J = 7.2 Hz, 6H), 2.33–2.23 (m, 2H), 1.89 (d, J = 0.9 Hz, 3H), 1.32 (t,
4.1.5.8. 30-Azido-30-deoxythymidyn-50-yl 2-methylpyridin-3-yl
phosphate triethylammonium salt (4ahY).ield 0.09 g (54%). 1H
NMR (CDCl3) d 9.35 (br s, 1H, exch. D2O), 8.17 (d, J = 4.8 Hz, 1H),
7.74 (d, J = 8.1 Hz, 1H), 7.66 (q, J = 0.9 Hz, 1H), 7.00 (dd, J = 8.1 Hz,
J = 4.2 Hz, 1H), 6.25 (t, J = 6.6 Hz, 1H), 4.38 (m, 1H), 4.20 (m, 2H),
4.02 (m, 1H), 3.01 (q, J = 7.2 Hz, 6H), 2.53 (s, 3H), 2.33 (m, 2H),
1.86 (d, J = 0.9 Hz, 3H), 1.28 (t, J = 7.2 Hz, 9H); 31P NMR (CDCl3) d
3
J = 7.2 Hz, 9H); 31P NMR (CDCl3) d ꢀ5.83 (t, JHP = 6.4 Hz); HRMS
[MꢀEt3NH+]ꢀ: 422.0846, calcd for C16H17N5O7P: 422.0865.
ꢀ5.23 (t, JHP = 6.4 Hz); HRMS [MꢀEt3NH+]ꢀ: 437.0983, calcd for
3
C16H18N6O7P: 437.0975.
4.1.5.2. 30-Azido-30-deoxythymidyn-50-yl 4-chlorophenyl phos-
phate triethylammonium salt (4abY).ield 0.14 g (81%). 1H NMR
(CDCl3) d 12.02 (br s, 1H, exch. D2O), 8.76 (br s, exch. D2O), 7.67
(br s, 1H), 7.19 (s, 4H), 6.26 (t, J = 6.6 Hz, 1H), 4.36–4.31 (m, 1H),
4.24–4.11 (m, 2H), 4.00 (br m, 1H), 3.05 (q, J = 7.2 Hz, 6H), 2.38–
2.21 (m, 2H), 1.88 (br s, 3H), 1.33 (t, J = 7.2 Hz, 9H). 31P NMR
4.1.5.9. 30-Azido-30-deoxythymidyn-50-yl 6-methylpyridin-3-yl
phosphate triethylammonium salt (4aiY).ield 0.12 g (73%). 1H
NMR (CDCl3) d 11.89 (br s, 1H, exch. D2O), 9.17 (br s, 1H, exch.
D2O), 8.40 (s, 1H), 7.66 (q, J = 0.9 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H),
7.05 (d, J = 8.4 Hz, 1H), 6.26 (t, J = 6.6 Hz, 1H), 4.38–4.33 (m, 1H),
4.27–4.13 (m, 2H), 4.01 (br m, 1H), 3.05 (q, J = 7.2 Hz, 6H), 2.47
(s, 3H), 2.39–2.24 (m, 2H), 1.85 (d, J = 0.9 Hz, 3H), 1.31 (t,
3
(CDCl3) d ꢀ5.38 (t, JHP = 6.4 Hz); HRMS [MꢀEt3NH+]ꢀ: 456.0500,
calcd for C16H16N5O7PCl: 456.0476.
3
J = 7.2 Hz, 9H); 31P NMR (CDCl3) d ꢀ5.46 (t, JHP = 6.4 Hz); HRMS
4.1.5.3. 30-Azido-30-deoxythymidyn-5’-yl 4-bromophenyl phos-
phate triethylammonium salt (4acY).ield 0.16 g (87%). 1H NMR
(CDCl3) d 11.91 (br s, 1H, exch. D2O), 9.24 (br s, exch. D2O), 7.64
(br s, 1H), 7.32 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 6.24 (t,
J = 6.6 Hz, 1H), 4.35–4.30 (m, 1H), 4.22–4.10 (m, 2H), 3.98 (br m,
1H), 3.03 (q, J = 7.2 Hz, 6H), 2.35–2.20 (m, 2H), 1.85 (br s, 3H),
[MꢀEt3NH+]ꢀ: 437.0993, calcd for C16H18N6O7P: 437.0975.
4.1.5.10. 30-Azido-3’-deoxythymidyn-50-yl 2-chloropyridin-3-yl
phosphate triethylammonium salt (4ajY).ield 0.14 g (71%). 1H
NMR (CDCl3) d 11.87 (br s, 1H, exch. D2O), 9.00 (br s, 1H, exch.
D2O), 8.07 (dd, J = 4.8 Hz, J = 1.5 Hz, 1H), 7.97 (dd, J = 8.4 Hz,
J = 1.5 Hz, 1H), 7.66 (q, J = 0.9 Hz, 1H), 7.16 (dd, J = 8.4 Hz,
J = 4.8 Hz, 1H), 6.26 (t, J = 6.6 Hz, 1H), 4.46–4.37 (m, 1H), 4.28–
4.17 (m, 2H), 4.04–4.01 (m, 1H), 3.06 (q, J = 7.2 Hz, 6H), 2.40–
2.25 (m, 2H), 1.89 (d, J = 0.9 Hz, 3H), 1.32 (t, J = 7.2 Hz, 9H); 31P
3
1.31 (t, J = 7.2 Hz, 9H); 31P NMR (CDCl3) d ꢀ5.53 (t, JHP = 6.4 Hz);
HRMS [MꢀEt3NH+]ꢀ: 499.9960, calcd for C16H16N5O7PBr:
499.9971.
3
4.1.5.4. 30-Azido-30-deoxythymidyn-50-yl 4-cyanophenyl phos-
phate triethylammonium salt (4adY).ield 0.15 g (81%). 1H NMR
(CDCl3) d 11.86 (br s, 1H, exch. D2O), 8.90 (br s, 1H, exch. D2O),
7.63 (q, J = 0.9 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz,
2H), 6.22 (t, J = 6.6 Hz, 1H), 4.33–4.31 (m, 1H), 4.20–4.08 (m, 2H),
3.99 (br m, 1H), 3.06 (q, J = 7.2 Hz, 6H), 2.32–2.21 (m, 2H), 1.88
(d, J = 0.9 Hz, 3H), 1.35 (t, J = 7.2 Hz, 9H); 31P NMR (CDCl3) d
NMR (CDCl3) d ꢀ5.99 (t, JHP = 6.4 Hz); HRMS [MꢀEt3NH+]ꢀ:
457.0450, calcd for C15H15N6O7PCl: 457.0429.
4.1.5.11. 30-Azido-30-deoxythymidyn-5’-yl 5-chloropyridin-3-yl
phosphate triethylammonium salt (4akY).ield 0.15 g (79%). 1H
NMR (CDCl3) d 11.86 (br s, 1H, exch. D2O), 8.82 (br s, 1H, exch.
D2O), 8.45 (d, J = 1.5 Hz, 1H), 8.26 (d, J = 1.8 Hz, 1H), 7.71 (dd,
J = 1.8 Hz, J = 1.5 Hz, 1H), 7.62 (q, J = 0.9 Hz, 1H), 6.26 (t,
J = 6.3 Hz, 1H), 4.40–4.34 (m, 1H), 4.28–4.14 (m, 2H), 4.01 (br m,
1H), 3.06 (q, J = 7.2 Hz, 6H), 2.42–2.25 (m, 2H), 1.88 (d, J = 0.9 Hz,
3
ꢀ6.15 (t, JHP = 6.4 Hz); HRMS [MꢀEt3NH+]ꢀ: 447.0800, calcd for
C17H16N6O7P: 447.0818.
4.1.5.5. 30-Azido-30-deoxythymidyn-50-yl 2,4-dichlorophenyl
phosphate triethylammonium salt (4aeY).ield 0.17 g (82%). 1H
NMR (CDCl3) d 11.95 (br s, 1H, exch. D2O), 8.56 (br s, 1H, exch.
D2O), 7.68 (br s, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.33 (s, 1H), 7.10 (d,
J = 8.7 Hz, 1H), 6.26 (t, J = 6.7 Hz, 1H), 4.40–4.35 (m, 1H), 4.24–
4.16 (m, 2H), 4.02–3.98 (m, 1H), 3.07 (q, J = 7.2 Hz, 6H), 2.36–
2.24 (m, 2H), 1.88 (br s, 3H), 1.36 (t, J = 7.2 Hz, 9H); 31P NMR
3H), 1.34 (t, J = 7.2 Hz, 9H). 31P NMR (CDCl3)
d -5.63 (t,
3JHP = 6.4 Hz); HRMS [MꢀEt3NH+]ꢀ: 457.0420, calcd for
C15H15N6O7PCl: 457.0429.
4.1.5.12. 20,30-Dideoxyuridin-5’-yl phenyl phosphate triethyl-
ammonium salt (4ba). Yield 0.07 g (76%). 1H NMR (CDCl3) d
11.94 (br s, 1H, exch. D2O), 9.83 (br s, 1H, exch. D2O), 7.84 (d,
J = 8.1 Hz, 1H), 7.22–7.15 (m, 4H), 6.98–6.93 (m, 1H), 6.00–5.96
(m, 1H), 5.51 (d, J = 8.1 Hz, 1H), 4.24–4.18 (m, 2H), 4.07–4.00 (m,
1H), 2.97 (q, J = 7.2 Hz, 6H), 2.29–2.22 (m, 1H), 1.97–1.87 (m,
3
(CDCl3) d ꢀ5.88 (t, JHP = 6.4 Hz) HRMS [MꢀEt3NH+]ꢀ: 490.0065,
calcd for C16H15N5O7P: 490.0086.
4.1.5.6. 30-Azido-30-deoxythymidyn-50-yl pyridin-3-yl phos-
phate triethylammonium salt (4afY).ield 0.14 g (86%). 1H NMR
(CDCl3) d 11.67 (br s, 1H, exch. D2O), 9.87 (br s, 1H, exch. D2O),
8.50 (s, 1H), 8.24 (br s, 1H), 7.65 (m, 2H), 7.18 (m, 1H), 6.20 (t,
J = 6.6 Hz, 1H), 4.41–4.32 (m, 1H), 4.26–4.10 (m, 2H), 4.03–3.94
(m, 1H), 3.01 (q, J = 7.2 Hz, 6H), 2.32–2.23 (m, 2H), 1.80 (s, 3H),
3H), 1.24 (t,, J = 7.2 Hz, 9H). 31P NMR (CDCl3)
d
ꢀ5.83 (t,
3JHP = 5.5 Hz); HRMS [MꢀEt3NH+]ꢀ: 367.0682, calcd for
C15H16N2O7P: 367.0700.
4.1.5.13. 20,30-Dideoxyuridin-50-yl pyridin-3-yl phosphate tri-
ethylammonium salt (4bbY).ield 0.07 g (72%). 1H NMR (CDCl3) d
11.73 (br s, 1H, exch. D2O), 10.16 (br s, 1H, exch. D2O), 8.45 (br s,
1H), 8.2 (d, J = 4.4 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.62 (d,
J = 8.8 Hz, 1H), 7.17 (dd, J = 8.8 and 4.4 Hz, 1H), 5.97–5.95 (m,
1H), 5.53 (d, J = 8.4 Hz, 1H), 4.21–4.17 (m, 2H), 4.05–4.00 (m,
1H), 2.99 (q, J = 7.2 Hz, 6H), 2.29–2.24 (m, 1H), 1.97–1.87 (m,
3
1.28 (t, J = 7.2 Hz, 9H); 31P NMR (CDCl3) d ꢀ6.09 (t, JHP = 6.4 Hz);
HRMS [M-Et3NH+]ꢀ: 423.0797, calcd for C15H16N6O7P: 423.0818.
4.1.5.7. 30-Azido-30-deoxythymidyn-50-yl pyridin-4-yl phos-
phate triethylammonium salt (4agY).ield 0.22 g (84%). 1H NMR
(CDCl3) d 9.76 (br s, 1H, exch. D2O), 8.44 (d, J = 5.7 Hz, 2H), 7.63
(q, J = 0.9 Hz, 1H), 7.20 (d, J = 5.7 Hz, 2H), 6.23 (t, J = 6.6 Hz, 1H),
4.38–4.32 (m, 1H), 4.24–4.12 (m, 2H), 4.03–3.97 (m, 1H), 3.02 (q,
3H), 1.24 (t, J = 7.2 Hz, 9H). 31P NMR (CDCl3)
d
ꢀ5.88 (t,
3JHP = 5.5 Hz); HRMS [MꢀEt3NH+]ꢀ: 368.0642, calcd for
C14H15N3O7P: 368.0648.

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R. Loddo et al. / Bioorg. Med. Chem. 17 (2009) 3489–3498
3
4.1.5.14. 20,30-Dideoxyuridin-50-yl pyridin-4-yl phosphate tri-
ethylammonium salt (4bcY).ield 0.08 g (80%). 1H NMR (CDCl3) d
11.5 (br s, 1H, exch. D2O), 9.95 (br s, 1H, exch. D2O), 7.99 (d,
J = 8.4 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 6.59 (d, J = 7.5 Hz, 2H),
6.10–6.04 (m, 1H), 5.86 (d, J = 8.4 Hz, 1H), 4.40–4.22 (m, 2H),
4.17–4.05 (m, 1H), 3.17 (q, J = 7.2 Hz, 6H), 2.51–2.38 (m, 1H),
2.18–1.93 (m, 3H), 1.26 (t, J = 7.2 Hz, 9H); 31P NMR (CDCl3) d
t (partially overlapping), JHP = 6.4 Hz). HRMS [MH]+ 501.1311,
calcd for C21H22N6O7P: 501.1288.
4.1.6.2. 30-Azido-30-deoxythymidyn-50-yl phenyl pyridin-4-yl
phosphate (5ab). Yield 0.09 g (40%). 1H NMR (DMSO-d6) d
11.37 (br s, 1H, exch. D2O), 8.59 (two br, s 1H), 8.28 (m, 1H),
7.71 (br s, 1H), 7.44–7.39 (m, 2H), 7.32–7.14 (m, 4H), 7.04–6.92
(m, 1H), 6.17–6.09 (m, 1H), 4.59–4.37 (m, 2H), 4.06–3.95 (m,
2H), 2.51–2.21 (m, 2H), 1.74 and 1.66 (two s, 3H); 31P NMR (CDCl3)
d ꢀ12.19 (m); HRMS [MH]+ 501.1319, calcd for C21H22N6O7P:
501.1288.
3
ꢀ6.03 (t, JHP = 5.5 Hz); HRMS [MꢀEt3NH+]ꢀ: 368.0615, calcd for
C14H15N3O7P: 368.0648.
4.1.5.15. 20,30-Dideoxyadenosin-50-yl phenyl phosphate trieth-
ylammonium salt (4caY).ield 0.08 g (84%). 1H NMR (CDCl3) d 8.55
(s, 1H), 7.29 (d, J = 5.7 Hz, 2H), 7.21 (t, J = 5.7 Hz, 2H), 6.98 (t,
J = 5.7 Hz, 1H), 6.34–6.32 (m, 1H), 4.38–4.30 (m, 2H), 4.16–4.10
(m, 1H), 3.04 (q, J = 7.2 Hz, 6H), 2.50–2.41 (m, 1H), 2.37–2.26 (m,
1H), 2.18–2.06 (m, 1H), 2.03–1.96 (m, 1H), 1.32 (t, J = 7.2 Hz, 9H).
4.1.6.3. 30-Azido-30-deoxythymidyn-50-yl 4-chlorophenyl pyri-
din-3-yl phosphate (5ac). Yield 0.18 g (65%). 1H NMR (DMSO-
d6) d 11.35 (br s, 1H, exch. D2O), 8.52 (m, 1H), 8.47 (m, 1H), 7.73
and 7.69 (two m, 1H), 7.50–7.46 (m, 3H), 7.43–7.42 (m, 1H),
7.37–29 (m, 1H), 6.15 (t, J = 6.6 Hz, 1H), 4.62–4.46 (m, 3H), 4.08–
4.04 (m, 1H), 2.45–2.30 (m, 2H), 1.65 (s, 3H); 31P NMR (CDCl3) d
ꢀ11.12 and ꢀ11.14 (m); HRMS [MH]+ 535.0891, calcd for
C21H21N6O7P: 535.0898.
3
31P NMR (CDCl3) d ꢀ6.12 (t, JHP = 5.5 Hz); HRMS [MꢀEt3NH+]ꢀ:
390.0975, calcd for C16H17N5O5P: 390.0967.
4.1.5.16. 20,30-Dideoxyadenosin-50-yl pyridin-3-yl phosphate tri-
ethylammonium salt (4cbY).ield 0.08 g (81%). 1H NMR (CDCl3) d
8.58 (s, 1H), 8.53 (d, J = 2.7 Hz, 1H), 8.22 (s, 1H), 8.18 (d,
J = 4.8 Hz, 1H), 7.18 (dm, J = 8.7 Hz, 1H), 7.13 (dd, J = 8.7 and
4.8 Hz, 1H), 6.33 (dd, J = 6.6 and 3.6 Hz, 1H), 4.41–4.29 (m, 2H),
4.16–4.10 (m, 1H), 3.06 (q, J = 7.2 Hz, 6H), 2.52–2.40 (m, 1H),
2.38–2.28 (m, 1H), 2.2–2.08 (m, 1H), 1.99–1.92 (m, 1H), 1.24 (t,
4.1.6.4. 30-Azido-30-deoxythymidyn-50-yl 4-bromophenyl pyri-
din-3-yl phosphate (5ad). Yield 0.15 g (52%). 1H NMR (CDCl3) d
8.7 (br s, 1H, exch. D2O) 8.58–8.48 (m, 2H), 7.60 and 7.55 (two m,
1H), 7.50–7.43 (m, 2H), 7.35–7.23 (m, 1H), 7.21–20 (m, 1H), 7.13–
7.08 (m, 2H), 6.15 (t, J = 6.6 Hz, 1H), 4.61–4.44 (m, 2H), 4.35–4.28
(m, 1H), 4.08–4.03 (m, 1H), 2.51–2.32 (m, 2H), 1.64 (s, 3H); 31P
NMR (CDCl3) d ꢀ11.86 and ꢀ11.91 [two t (partially overlapping),
3
J = 7.2 Hz, 9H); 31P NMR (CDCl3) d ꢀ6.09 (t, JHP = 5.5 Hz); HRMS
[MꢀEt3NH+]ꢀ: 391.0914, calcd for C15H16N6O5P: 391.0920.
3JHP = 6.4 Hz);
HRMS
[MH]+
581.0376,
calcd
for
C21H21N6O7PBr(81): 581.0372.
4.1.6. A typical procedure for the synthesis of bis-aryl
nucleoside phosphate triesters 5
4.1.6.5. 30-Azido-30-deoxythymidyn-50-yl 4-cyanophenyl pyri-
din-3-yl phosphate (5ae). Yield 0.12 g (75%). 1H NMR (CDCl3)
d 9.3 (br s, 1H, exch. D2O), 8.55 (br s, 1H), 7.71–7.65 (m, 2H),
7.61–7.57 (m, 1H), 7.37–7.33 (m, 4H), 7.17 and 7.16 (two q,
JJ = 1.2 Hz), 6.08 and 6.07 (two t, JJ = 6.4 Hz), 4.61–4.56 (m, 1H),
4.54–4.49 (m, 1H), 4.37–4.30 (m, 1H), 4.07–4.04 (m, 1H), 2.49–
2.45 (m, 2H), 1.83 and 1.82 (two d, JJ = 1.2 Hz, 3H); 31P NMR
(CDCl3) d ꢀ12.52 and ꢀ12.59 [two t (partially overlapping),
3JHP = 6.4 Hz). HRMS [MH]+ 526.1226, calcd for C22H21N7O7P:
526.1240.
Nucleoside-50-yl H-phosphonate 1 (1 M equiv) and a phenol
or pyridinol HOAr1 2 (1.2–1.5 M equiv) were rendered anhy-
drous by co-evaporation of added pyridine, and then were dis-
solved in methylene chloride containing pyridine [10% (v/v)]
(0.1 mmol/1 mL). The condensation was effected by the addition
of diphenyl chlorophosphate (1.2 M equiv) or pivaloyl chloride
(1.5 M equiv) to the reaction mixture. When the formation of
aryl nucleosid-50-yl H-phosphonate
3 was completed (ca.
20 min, 31P NMR), the reaction mixture was added to a solution
of a pyridinol or another phenol HOAr2 2 (5 equiv) to be cou-
pled with, and then iodine (1.5–3 M equiv) in pyridine (1 mL)
was added. After the reaction was complete (ca. 5 min, 31P
NMR), the excess of iodine was decomposed with ethanethiol,
and the solvents were removed by evaporation. The oily residue
was dissolved in methylene chloride (3 mL), washed with aque-
ous 0.1 M KH2PO4 buffer (pH 6) and the organic phase was
dried over Na2SO4. Phosphotriesters 5 were then purified by re-
versed phase silica gel chromatography using a stepwise gradi-
ent of acetone (0–40%) in water, or by a Silica gel 60 column
using a stepwise gradient (0–6%) of isopropanol in methylene
chloride. Fractions containing pure products 5 were collected
and evaporated, yielding non-hygroscopic foams. After freeze-
drying from benzene, products were obtained as white amor-
phous solids.
4.1.6.6. 30-Azido-30-deoxythymidyn-50-yl 2,4-dichlorophenyl
pyridin-3-yl phosphate (5af). Yield 0.13 g (77%). 1H NMR
(CDCl3) d 9.0 (br s, 1H, exch. D2O), 8.60–8.52 (m, 2H), 7.63–7.59
(m, 1H), 7.46–7.44 (m, 1H), 7.36 (s, 1H), 7.34–7.33 (m, 1H), 7.26–
7.21 (m, 2H), 6.17–6.13 (m, 1H), 4.66–4.61 (m, 1H), 4.58–4.50
(m, 1H), 4.36–4.31 (m, 1H), 4.08–4.07 (m, 1H), 2.50–2.35 (m,
2H), 1.84 and 1.82 (two s, 3H); 31P NMR (CDCl3) d ꢀ12.20 and
ꢀ12.29 (m); HRMS [MH]+ 569.0489, calcd for C21H20N6O7P:
569.0508.
4.1.6.7. 30-Azido-30-deoxythymidyn-50-yl bis-pyridin-3-yl phos-
phate (5ag). Yield 0.14 g (68%). 1H NMR (DMSO-d6) d 11.34 (br s,
1H, exch. D2O), 8.55 (m, 2H), 8.47 (m, 2H), 7.76 and 7.74 (two m,
2H), 7.51–7.41 (m, 3H), 6.15 (t, J = 6.6 Hz, 1H), 4.67–4.48 (m, 3H),
Mutplicities of signals in 1H and 31P NMR spectra of phosphotri-
4.09–4.05 (m, 1H), 2.45–2.30 (m, 2H), 1.65 (s, 3H); 31P NMR (CDCl3)
3
ꢀ11.63 (t, JHP = 6.4 Hz). HRMS [MH]+ 502.1253, calcd for
esters of type
5 were often complex due to a mixture of
d
diastereoisomers.
C20H21N7O7P: 502.1240.
4.1.6.1. 30-Azido-30-deoxythymidyn-50-yl phenyl pyridin-3-yl
phosphate (5aa). Yield 0.22 g (89%). 1H NMR (DMSO-d6) d
11.35 (br s, 1H, exch. D2O), 8.51 (br s, 1H), 8.47 (m, 1H), 7.72 and
7.69 (two br s, 1H), 7.49–7.36 (m, 4H), 7.30–7.25 (m, 3H), 6.26 (t,
J = 6.6 Hz, 1H), 4.70–4.47 (m, 3H), 4.10–4.02 (m, 1H), 2.45–2.30
(m, 2H), 1.65 (s, 3H). 31P NMR (CDCl3) d ꢀ10.98 and ꢀ11.03 [two
4.1.6.8. 30-Azido-30-deoxythymidyn-50-yl pyridin-3-yl pyridin-4-
yl phosphate (5ah). Yield 0.07 g (30%). 1H NMR (CDCl3) d 9.0 (br
s, 1H, exch. D2O), 8.62 (m, 2H), 8.56 and 8.51 (two m, 1H), 7.62–
7.56 (m, 1H), 7.36–7.29 (m, 1H), 7.22–7.17 (m, 3H), 6.15 (m, 1H),
4.63–4.47 (m, 2H), 4.37–4.29 (m, 1H), 4.09–4.02 (m, 1H), 2.47–
2.42 (m, 2H), 1.84 and 1.83 (two s, 3H); 31P NMR (CDCl3) d

R. Loddo et al. / Bioorg. Med. Chem. 17 (2009) 3489–3498
3497
ꢀ12.72 and 12.75 (m); HRMS [MH]+ 502.1248, calcd for
4.1.6.16. 20,30-Didehydrouridin-50-yl phenyl pyridin-4-yl phos-
phate (5bb). Yield 0.07 g (49%). 1H NMR (CDCl3) d 9.51 (br s, 1H,
exch. D2O), 8.56 (br s, 2H), 7.51 and 7.48 (two d, JJ = 8.1 Hz, 1H),
7.40–7.32 (m, 2H), 7.26–7.16 (m, 5H), 6.07–6.03 (m, 1H), 5.58
and 5.57 (two d, JJ = 8.1 Hz, 1H), 4.58–4.50 (m, 1H), 4.43–4.28
(m, 2H), 2.47–2.34 (m, 1H), 2.13–1.87 (m, 3H); 31P NMR (CDCl3)
d ꢀ12.59 and ꢀ12.61 (m). HRMS [MH]+ 446.1106, calcd for
C20H21N3O7P: 446.1117.
C20H21N7O7P: 502.1240.
4.1.6.9. 30-Azido-30-deoxythymidyn-50-yl bis-2-methylpyridin-
3-yl phosphate (5ai). Yield 0.17 g (62%). 1H NMR (CDCl3) d 8.4
(br s, 1H, exch. D2O), 8.38 (m, 2H), 7.62–7.58 (m, 2H), 7.19 (br s,
1H), 7.17–7.10 (m, 2H), 6.09 (t, t, J = 6.6 Hz, 1H), 4.60–4.46 (m,
2H), 4.33–4.27 (m, 1H), 4.08–4.03 (m, 1H), 2.52 (s, 3H), 2.50 (s,
3H), 2.47–2.40 (m, 2H), 1.83 (s, 3H); 31P NMR (CDCl3) d -10.86 (t,
3JHP = 6.4 Hz); HRMS [MH]+ 530.1571, calcd for C22H25N7O7P:
530.1553.
4.1.6.17. 20,30-Didehydrouridin-50-yl pyridin-3-yl pyridin-4-yl
phosphate (5bc). Yield 0.07 g (32%). 1H NMR (CDCl3) d 11.3 (br
s, 1H, exch. D2O), 8.54 (br s, 1H), 8.22–8.11 (m, 3H), 7.72 and
7.71 (two d, JJ = 6.0, 1H), 7.53 and 7.52 (two d, JJ = 6.0 Hz), 7.49
(d, J = 8.4 Hz, 1H), 7.20–7.14 (m, 2H), 6.04 and 6.02 (two d,
JJ = 6.8 Hz), 5.61 (d, J = 8.4 Hz, 1H), 4.57–4.52 (m, 1H), 4.45–4.39
(m, 1H), 4.24–4.19 (m, 1H), 2.49–2.40 (m, 1H), 2.36–2.27 (m,
1H), 2.11–1.99 (m, 2H); 31P NMR (CDCl3) d ꢀ12.47 (m). HRMS
[MH]+ 447.1059, calcd for C19H20N4O7P: 447.1070.
4.1.6.10. 30-Azido-30-deoxythymidyn-50-yl bis-6-methylpyridin-
3-yl phosphate (5aj). Yield 0.21 g (80%). 1H NMR (DMSO-d6) d
11.34 (br s, 1H, exch. D2O), 8.34 (br s, 2H), 7.63–7.57 (m, 2H),
7.37 (s, 1H), 7.30 and 7.27 (two d, J = 8.9 Hz, 2H), 6.14 (t,
J = 6.6 Hz, 1H), 4.62–4.46 (m, 3H), 4.08–4.04 (m, 1H), 2.45 (s, 3H),
2.44 (s, 3H), 2.42–2.31 (m, 2H), 1.63 (s, 3H); 31P NMR (CDCl3) d
3
ꢀ10.28 (t, JHP = 7.2 Hz); HRMS [MH]+ 530.1534, calcd for
C22H25N7O7P: 530.1553.
4.1.6.18. 20,30-Didehydroadenosin-50-yl phenyl pyridin-3-yl
phosphate (5ca). Yield 0.22 g (94%). 1H NMR (DMSO-d6) d 8.44
(br s, 1H), 8.43 (m, 1H), 8.27 (two s, 1H), 8.13 (two s, 1H), 7.65–
7.60 (m, 1H), 7.41–7.13 (m, 6H), 6.29–6.25 (m, 1H), 4.52–4.45
(m, 2H), 4.44–4.36 (m, 1H), 2.55–2.42 (m, 2H), 2.19–2.12 (m,
2H); 31P NMR (CDCl3) d ꢀ11.09 and ꢀ11.14 [two t (partially over-
lapping), 3JHP = 6.4 Hz). HRMS [MH]+ 469.1372, calcd for
C21H22N6O5P: 469.1390.
4.1.6.11. 30-Azido-30-deoxythymidyn-50-yl 2-methylpyridin-3-yl
phenyl phosphate (5ak). Yield 0.16 g (64%). 1H NMR (DMSO-d6)
d 11.36 (br s, 1H, exch. D2O), 8.32 and 8.31 (two d, J = 4.5 Hz, 1H),
7.66 and 7.63 (two s, 1H), 7.45–7.38 (m, 3H), 7.30–7.23 (m, 4H),
6.15 and 6.14 (two t, JJ = 6.6 Hz, 1H), 4.61–4.45 (m, 3H), 4.10–
4.04 (m, 1H), 2.44–2.30 (m, 2H), 2.36 (s, 3H), 1.65 (s, 3H); 31P
NMR (CDCl3) d ꢀ10.83 and ꢀ10.85 (t, JHP = 7.2 Hz); HRMS [MH]+
515.1455, calcd for C22H24N6O7P: 515.1444.
Acknowledgements
4.1.6.12. 30-Azido-30-deoxythymidyn-50-yl 6-methylpyridin-3-yl
phosphate phenyl (5al). Yield 0.18 g (71%). 1H NMR (DMSO-d6)
d 11.33 (br s, 1H, exch. D2O), 8.35 (br s, 1H), 7.60–7.54 (m, 1H),
7.45–7.39 (m, 3H), 7.30–7.24 (m, 4H), 6.15 (t, J = 6.6 Hz), 4.60–
4.46 (m, 3H), 4.06 (m, 1H), 2.44 and 2.43 (two s, 3H), 1.65 (s,
3H); 31P NMR (CDCl3) d ꢀ10.75 and ꢀ10.84 (m); HRMS [MH]+
515.1454, calcd for C22H24N6O7P: 515.1444.
We are grateful to Dr. Y. Hagiwara and Dr. K. Izawa from Aji-
nomoto Co., Inc., Kawasaki, Japan for a gift of ddA. Financial sup-
port from the government of the Polish Republic (Project No.
PBZ-MNiSW—07/I/2007; 2008–2010) is gratefully acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.02.033.
4.1.6.13. 30-Azido-30-deoxythymidyn-50-yl 2-chloropyridin-3-yl
phenyl phosphate (5am). Yield 0.09 g (54%). 1H NMR (DMSO-
d6) d 11.36 (br s, 1H, exch. D2O), 8.32–8.30 (m, 1H), 7.90 and
7.87 (two br s, 1H), 7.52–7.39 (m, 4H), 7.30–7.25 (m, 3H), 6.15
and 6.14 (two t, JJ = 6.6 Hz, 1H), 4.67–4.47 (m, 3H), 4.11–4.05 (m,
1H), 2.48–2.30 (m, 2H), 1.66 (s, 3H); 31P NMR (CDCl3) d ꢀ12.18
and ꢀ12.33 (two t, JHP = 7.2 Hz); HRMS [MH]+ 535.0906, calcd for
C21H22N6O7PCl (35): 535.0898.
References and notes
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exch. D2O), 8.54 (br s, 1H), 8.49–8.47 (m, 1H), 7.61–7.56 (m, 1H),
7.51 and 7.49 (two d, J = 8.1 Hz, 1H), 7.40–7.18 (m, 6H), 6.07–
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