Aryl H-phosphonates 17: (N-aryl)phosphoramidates of pyrimidine nucleoside analogues and their synthesis, selected properties, and anti-HIV activity

Article abstract of DOI:10.1021/jm2001103

New synthetic protocol for the preparation of nucleoside 5′-(N-aryl)phosphoramidate monoesters 4 was developed. It consisted of a condensation of the corresponding nucleoside 5′-H-phosphonates with aromatic- or heteroaromatic amines promoted by diphenyl phosphorochloridate, followed by oxidation of the produced H-phosphonamidates with iodine/water. 5′-(N-Aryl)phosphoramidate monoesters derived from 3′-azido- 3′-deoxythymidine (AZT) or 2′,3′-dideoxyuridine (ddU) nucleosides and various aromatic and heteroaromatic amines were evaluated as potential anti-HIV drugs. It was found that these compounds act most likely as pronucleotides and that some of them have therapeutic indices superior to those of the reference AZT.

Full text of DOI:10.1021/jm2001103

ARTICLE
pubs.acs.org/jmc
Aryl H-Phosphonates 17: (N-Aryl)phosphoramidates of Pyrimidine
Nucleoside Analogues and Their Synthesis, Selected Properties, and
Anti-HIV Activity
Joanna Romanowska,† Michaz Sobkowski,† Agnieszka Szymaꢀnska-Michalak,† Krystian Kozodziej,†
Aleksandra Dabrowska,‡ Andrzej Lipniacki,‡ Andrzej Piasek,‡ Zofia M. Pietrusiewicz,† Marek Figlerowicz,†,§
)
Andrzej Guranowski,|| Jerzy Boryski,† Jacek Stawiꢀnski,† and Adam Kraszewski*,†
†Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznaꢀn, Poland
‡National Institute of Medicines, Chezmska 30/34, 00-725 Warsaw, Poland
§Institute of Computing Science, Poznaꢀn University of Technology, Piotrowo 2, 60-965 Poznaꢀn, Poland
Faculty of Biochemistry and Biotechnology, Life Science University, Wozyꢀnska 35, 60-637 Poznaꢀn, Poland
S Supporting Information
b
ABSTRACT: New synthetic protocol for the preparation of
nucleoside 50-(N-aryl)phosphoramidate monoesters 4 was de-
veloped. It consisted of a condensation of the corresponding
nucleoside 50-H-phosphonates with aromatic- or heteroaro-
matic amines promoted by diphenyl phosphorochloridate,
followed by oxidation of the produced H-phosphonamidates
with iodine/water. 50-(N-Aryl)phosphoramidate monoesters derived from 30-azido-30-deoxythymidine (AZT) or 20,30-dideoxyur-
idine (ddU) nucleosides and various aromatic and heteroaromatic amines were evaluated as potential anti-HIV drugs. It was found
that these compounds act most likely as pronucleotides and that some of them have therapeutic indices superior to those of the
reference AZT.
’ INTRODUCTION
enzymes into the respective nucleotide and subsequently into
their 50-triphosphates that inhibit proliferation of viruses.
Although some of the above requirements for pro-nucleotides
are contradictory (e.g., lipophilicity vs solubility in water or
stability vs susceptibility to metabolism), several types of pro-
nucleotides fulfill to some extent these criteria and play an
important role in drugs used for treating of viral infections or
cancer. Among these compounds, nucleoside pivaloyloxymethyl
phosphotriesters (POM),24 nucleoside S-acetyl-thioethyl phos-
photriesters (SATE),25 nucleoside cyclosaligenyl phosphotrie-
sters (cycloSal nucleotides),26 and phosphoramidate diesters
have received the most attention,27,28 which hopefully will
expand the range of antiviral drugs. It is worth noting that until
now there was only one pro-nucleotide (tenofovir disoproxil
fumarate, VIREAD)29 that was used in AIDS and chronic
hepatitis B therapy.
The findings by C. R. Wagner et al., that nucleoside amino acid
derived phosphoramidate monoesters are exceptionally highly
active against HIV,6 caused some confusion in the pro-nucleotide
concept and criteria since being pronucleotides, they were
inactive in HIV infected TKꢀ cells. Later, it was found that
phosphoramidic pro-nucleotides derived from simple amines are
also very active against HIV.30 These findings suggested that
Modifications at the phosphorus center of natural products
was proved to be an exceptionally valuable tool in studies of
reaction mechanisms particularly those catalyzed by enzymes.1ꢀ3
In most cases, P-modified analogues of natural compounds
exhibit new, sometimes unique chemical and biological proper-
ties. This was particularly well demonstrated for compounds
bearing phosphoramidic bonds, which found many applications
in molecular biology,4ꢀ8 medicinal chemistry,9ꢀ11 and in cancer
therapy (e.g., cyclophosphamide).12 In nucleotide and oligonu-
cleotide chemistry, phosphoramidites and phosphoramidates
occupy a well established position. While nucleoside phospho-
ramidites13 are commonly used as basic building blocks in
modern chemical synthesis of oligonucleotides, modified oligo-
nucleotides bearing either30-50 PꢀN bonds14,15 orinternucleotide
aminoalkyl phosphoramidate moieties (cationic oligonucleotides)16
are well established as potential antisense or antigene agents.
Phosphor- and phosphonamidates derived from nucleoside
analogues were subjects of intensive studies due to their
promising pro-drug activity against various viruses (particularly
HIV)9,17ꢀ19 and in cancer therapy.20ꢀ23 These compounds
fulfill the criteria of so-called pro-nucleotides, i.e., they are
(i) uncharged, (ii) highly lipophilic, (iii) reasonably soluble in
water, (iv) stable in physiological environment, (v) enter the
target cells without affecting the nucleotide skeleton, and after
this (vi) can be converted chemically or by means of cellular
Received: January 31, 2011
Published: August 11, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Nucleoside (N-Aryl)phosphoramidates 4a
a Reagents and conditions: (i) DPCP (1.5 molar equiv with respect to 1) in DCM/pyridine 2: 1 (v/v);. (ii) I2 (2.0 molar equiv) in pyridine containing
water (50 molar equiv).
Table 1. 31P NMR Data and Times of Oxidation of Nucleo-
’ RESULTS AND DISCUSSION
side H-Phosphonamidates 3 to the Respective
Nucleoside Phosphoramidates 4
Chemistry. In our previous studies on the synthesis and
properties of nucleoside (N-alkyl)-H-phosphonamidates, we
observed that formation of the PꢀN bond strongly depended
on the condensing agent used and the basicity of the reaction
media.31 It was also found that a competing reaction of
N-alkylamines with a condensing agent significantly affected yields
of the desired nucleoside (N-alkyl)-H-phosphonamidates.
In contrast to these, we found that N-arylamines 2 used in this
study [except, 4-aminopyridine (2f), vide infra] reacted smoothly
with nucleoside H-phosphonate in the presence of DPCP as
condensing agent, producing rapidly (<5 min) and nearly
quantitatively (31P NMR) putative (N-aryl)-H-phosphonami-
dates 3 (Scheme 1), whose structures were tentatively assigned
on the basis of their chemical shifts, splitting pattern, and
coupling constancies recorded in 31P NMR spectra (Table 1).
Unfortunately, isolation of the above species was impossible due
to their partial instability during workup and/or silica gel
chromatography. Coupling reactions were performed under mild
conditions, i.e., at room temperature, in DCM containing 10%
(v/v) of pyridine using a slight excess of an N-arylamine 2 and
DPCP (both 1.5 molar equiv). As was mentioned above, the
reaction worked well for all aromatic amines investigated except
for 4-aminopyridine, for which a complex mixture of products
was formed (31P NMR spectroscopy).
These differences between N-alkylamines and N-arylamines
investigated herein, can be attributed, at least partly, to the
disparity in their pKa values. For N-alkylamines (pKa >10), high
basicity was responsible for the side reactions observed and high
nucleophilicity for a partial loss of chemoselectivity during the
aminolysis of the pivalic-H-phosphonic mixed anhydride. Con-
sequently, for N-alkylamines specific synthetic protocols had to
be developed depending on the reactivity of the amine used.
In contrast to these, N-arylamines 2aꢀe are much weaker
bases (pKa for 2a ꢀ 4.6; 2b ꢀ 4.58; 2c ꢀ 3.99; 2d ꢀ 6.71; 2e ꢀ
6.03),32 and thus, undesired side-reactions with activated H-
phosphonate31 (vide supra) or subsequent disproportionation33
were significantly suppressed. However, their relatively low
nucleophilicity was apparently sufficient to allow rapid and
quantitative formation of the desired nucleoside (N-aryl)-H-
phosphonamidates 3.
cpd
δP[ppm]
1JHP[Hz]
2JHP[Hz]
3JHP[Hz]
ta[min]
3aa
3ab
3ac
3ad
3ae
3bd
3be
4aa
4ab
4ac
4ad
4ae
4af
7.68, 7.99b,c
7.91, 8.23b,c
7.43, 7.73b,c
7.32, 8.42b,d
7.43, 7.89b,d
7.52, 7.88b,d
6.62, 7.05b,d
ꢀ1.73 dtf
654.9, 656.7
655.7, 654.0
655.1, 653.4
664.0, 670,4
656.0, 653.0
671.6, 673.4
656.1, 656.9
7.3
7.3
7.4
7.9
8.3
8.3
8.2
8.2
7.3
6.4
7.3
6.4
7.0
6.4
5.4
6.4
6.4
<5
30
<5
<5
<5
<5
<5
7.4
7.9
n. o.e
n. o.e
n. o.e
n. o.e
7.3
ꢀ1.38 dtf
6.4
ꢀ2.13 dtf
7.3
ꢀ4.47 dtf
6.4
ꢀ2.17 dtf
7.0
ꢀ4.43 dtf
6.4
4bd
4be
4bf
ꢀ3.82 dtf
5.4
ꢀ2.50 dtf
6.4
ꢀ4.29 dtf
6.4
a Time required for the completion of the oxidation of 3 into 4. b Two
c
diastereomers. In DCM/pyridine 1: 9 (v/v), doublet of doublets of
triplets. d In DCM/pyridine 1: 9 (v/v), doublet of triplets. n.o.: not
e
observed. f In DMSO-d6. Upon the addition of D2O, due to the exchange
of the hydrogen atom of the bridging phosphoramidate group, a triplet
was observed in the 31P NMR spectra.
nucleoside phosphoramidates may constitute a unique group of
nucleotide derivatives with high antiviral or anticancer potency.
Some efforts were devoted to recognize this problem, but they were
focused on rather narrow groups of compounds, especially amino
acids or some simple N-alkylamine derived phosphoramidates.10,17
In the course of our studies on nucleoside H-phosphonates,
we have investigated the synthesis and properties of nucleoside
(N-alkyl)phosphonamidates.31 In this article, we present our
studies on nucleoside (N-aryl)phosphon- and (N-aryl)phosphor-
amidates, which have properties distinguishing them from other
phosphoramidates and which can make them attractive anti-HIV
agents. Among aryl amines investigated, the main emphasis was
placed on 2-, 3-, and 4-pyridynylamines due to their electronic
and physicochemical properties, as well as their favorable lipo-
philic/hydrophilic character.
The basicity of 4-aminopyridine (2f) (pKa 9.13) markedly
differs from those of 2- and 3-aminopyridines (2d, 2e respectively)
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as well as of aniline (2a) and its derivatives (2bꢀc) (vide supra).
This was probably a critical factor responsible for the low
efficiency of synthesis of H-phosphonamidate of type 3 via a
direct coupling of 4-aminopyridine and nucleoside H-phospho-
nate 1, similar to that observed for highly basic N-alkylamines
(pKa > 9.0).32 Thus, to obtain (N-pirydin-4-yl)phosphoramidate
4af required for this study, another approach, which secured
higher yield, was developed.
Since the attempted isolation of the intermediate species of
type 3 failed (vide supra), the putative nucleoside (N-aryl)-H-
phosphonamidates 3 were subjected in situ to oxidation. In
contrast to nucleoside (N-alkyl)-H-phosphonamidates,31 which
were resistant to oxidation with iodine under the standard
conditions commonly used for oxidation of H-phosphonate
diesters,34,35 we assumed that this reaction might work well in
the case of analogous N-aryl phosphonamidate analogues 3.
Considering that oxidation of H-phosphonates with iodine
proceeds via their tricoordinated tautomers (λ3σ3), we expected
that the presence of an electron-withdrawing aryl group should
make the (N-aryl)-H-phosphonamidates more prone to oxida-
tion. Indeed, when these compounds were treated with an
iodineꢀwater system, all H-phosphonamidates 3 investigated
yielded the respective (N-aryl)phosphoramidates 4 quantita-
tively. The influence of an aryl group on the oxidation of 3 could
be observed along the series of 3aꢀc, in which H-phosphona-
midate 3b bearing the least electron-withdrawing aryl group
(4-methylphenyl one) required the longest time (30 min), while
the other H-phosphonamidates 3 were oxidized much faster
(<5 min, Table 1). All products 4 were isolated by silica gel flash
column chromatography and after freeze-drying were obtained as
white solids with high yields (>60%). Their purity and structure
were confirmed with chromatography (TLC and HPLC) and
spectral (1H, 13C and 31P NMR spectroscopy) analyses.
Since both steps in the synthesis of nucleoside aryl phosphor-
amidates 4 (Scheme 1) proceeded smoothly and nearly quanti-
tatively (31P NMR), the above approach could be recommended
as a convenient method for the synthesis of N-arylphosphor-
amidates derived from amines of pKa < 8 (for synthesis of
4-aminopyridine derivative 4af and 4bf, vide infra).
During the purification and isolation of phosphoramidates 4,
we noticed an interesting phenomenon, namely, that triethylam-
monium or pyridinium salts of these compounds upon evapora-
tion from polar solvents (particularly water) were rapidly loosing
the cationic parts forming the corresponding acids (1H NMR
spectroscopy). Such a phenomenon was not observed for the
triethylammonium salt of 2-, 3- or 4-pyridyl phosphodiesters
(AZT derivatives)36 that did not show a detectable (1H NMR)
loss of triethylammonium cation even upon repeated (several
times) evaporation from aqueous solutions.
This behavior of phosphoramidates can be related to the
presence of the PꢀN bond, which, due to back-donation of
electrons from the nitrogen to phosphorus center,37 should make
these compounds less acidic than the corresponding phosphate
derivatives. Unfortunately, pKa values of phosphoramidate mono-
esters are usually unavailable,38 but the acidity of N-(n-butyl)
phosphoramidic acid (pKa 2.92 and 9.88)39 vs n-butyl
phosphoric acid (pKa 1.80 and 6.84)32 seems to support this
assumption.
Figure 1. Some possible ionic structures of phosphoramidates 4 in
solution.
Scheme 2. Synthesis of Di(1H-1,2,4-Triazol-1-yl)-
[N-(pyridin-4-yl)]phosphoramidate (6)
various N-site protonation is unknown. Structural and electronic
features of these species may have important biological bearing
for chemical decomposition of (N-aryl)phosphoramidates in vivo
as well as for their cellular up-take. Under physiological condi-
tions, a fraction of neutral (II) and zwitter-ionic (III and IV)
forms is probably not very high, but this may be sufficient to
enable their uptake via passive diffusion (neutral molecule II) or
the flipꢀflop mechanism40 (zwitter-ion III and IV). One should
note that although various phosphoramidates have been pro-
posed as drugs or prodrugs, the importance of their pKa values
and sites of protonation for the potential biological activity has
never been discussed.
In contrast to amines 2aꢀe, coupling the 4-aminopyridine 2f
with nucleosideH-phosphonates 1 appeared to be ineffective in any
of the several variants of this method examined. Thus, it was
necessary to find another method for the synthesis of nucleoside
[N-(pyridin-4-yl)]phosphoramidates 4f. For this purpose, we
turned our attention to phosphoryl tris-triazole41 5, which in
reaction with an equimolar amount of 4-aminopyridine (2f) in
dioxane at room temperature produced di(1H-1,2,4-triazol-1-yl)-
[N-(pyridin-4-yl)]phosphoramidate (6), which can be considered
as a new phosphorylating agent for the introduction of basic
aromatic amines into 50-monophosphate nucleosides (Scheme 2).
The reaction proceeded smoothly, and it was complete within
ca. 15 min (31P NMR spectroscopy). Product 6 precipitated from
the reaction mixture as a white solid, and after filtering off,
washing, and drying, pure triamide 6 was obtained in ca. 90%
yield. Its structure and purity were determined by spectral
methods (1H, 13C and 31P NMR, HRMS).
The low acidity of phosphoramidate monoesters 4 can make
these compounds under physiological conditions exist as an
equilibrium mixture of different charged and uncharged forms
(e.g., IꢀIV in Figure 1), although the exact preference for O- vs
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Scheme 3. Synthesis of Nucleosid-50-yl-[N-(pyridin-4-yl)]phosphoramidates 4af and 4bfa
a Reagents and conditions: (i) 6 (2 M equiv) and 7 (1 M equiv) in pyridine, 75 °C, 10 min. (ii) H2O excess, 1 h.
This new phosphorylating reagent was applied for the intro-
duction of the (N-pyridin-4-yl)phosphoramidate moiety into the
50-position of the nucleoside analogues used in this study
(Scheme 3).
cellular kinases, the observed anti-HIV activity of compound 4bf
has to be attributed to its internalization and its further conver-
sion into the respective 20,30-dideoxyuridin-50-yl phosphate.43
Because the acute cytotoxicity of AZT is due to the accumulation
of AZT 50-monophosphate (AZTMP) in cells, one can speculate
that nucleoside phosphoramidates 4 have favorable pharmaco-
kinetic characteristics (slower release of the corresponding
nucleoside monophosphate than its conversion into di- and
triphosphates) that maximize the therapeutic effect. Finally, a
very low cytotoxicity of all aminopyridines 2dꢀf (Table 2) (the
expected metabolites of (N-pyridinyl)phosphoramidates 4) can
be certainly the added value when considering phosphoramidates
4 as potential candidates for anti-HIV drugs.
Unfortunately, a pronucleotide mode of action of nucleoside
(N-pyridinyl)phosphoramidates 4 was not verified in the experi-
ments with CEM TKꢀ HIV-1 infected cells, in which none of the
examined compounds (of AZT or ddU series, 4a and 4b,
respectively) showed detectable HIV inhibitory activity. It is
symptomatic that similar observations were reported by C. R.
Wagner et al., and J.-L. Imbach et al. during studies on another
type of nucleoside phosphoramidates, namely, nucleoside ami-
no-acid derived phosphoramidates44 and nucleoside SATE [N-
alkyl(or N-aryl)]phosphoramidates,45 respectively. They found
that these compounds in normal cells acted as pronucleotides
whereas in TKꢀ cells were inactive against HIV. It was suggested,
that the lack of anti-HIV activity in TKꢀ cells was due to the
reduced substrate affinity of certain nucleoside phosphorami-
dates bearing an aromatic N-substituent (for instance phenyl
group) for phosphoramidases; thus, the conversion of this type of
nucleoside phosphoramidates to the respective nucleoside 50-
phosphate derivatives was prevented. It is possible that for the
same reasons phosphoramidates 4adꢀaf bearing heteroaromatic
pyridinyl group studied herein also did not show anti-HIV
activity in thymidine kinase deficient cells, while they were highly
active in TK+ HIV infected cells.
To this end, to a suspension of di(1H-1,2,4-triazole)-
[N-(pyridin-4-yl)]phosphoramidate (6) (2 molar equiv) in
pyridine at 75 °C, nucleoside 7 (1 molar equiv) dissolved in
pyridine was added stepwise with vigorous stirring. After 10 min,
the reaction was quenched with the added excess of water to
hydrolyze all reactive species present in the reaction mixture
(excess of phosphorylating reagent and the intermediate nucleo-
side (N-pyridyn-4-yl)phosphotriazolide generated). This furn-
ished nucleoside [N-(pyridin-4-yl)]phosphoramidate 4af and
4bf that after workup and isolation by silica gel column chroma-
tography were obtained in 60%ꢀ70% yields. This simple proto-
col can be considered as a convenient approach for a chemo-
selective introduction of the [N-(pyridin-4-yl)]phosphoramidate
moiety into nucleosides, even those that are thermally rather
fragile (for instance ddU).
It is worth noticing that [N-(pyridin-4-yl)]phosphoramidates
4af and 4bf after chromatography were obtained as free acids,
similar to the N-pyridin-2-yl and N-pyridin-3-yl regio isomers
4adꢀae and 4bdꢀbe discussed above.
Biological Evaluation. Anti-HIV activity of nucleoside (N-aryl)
phosphoramidates 4 and their decomposition in cell culture media
were evaluated (Table 2).
These data indicate the high anti-HIV potency of AZT (N-aryl)
phosphoramidates 4aaꢀaf but among them aminopyridinyl
derivatives 4adꢀaf stood out clearly. A striking feature of all
compounds 4adꢀf is their very low cytotoxicity, which was not
observed even when the cells were grown in cell culture media
enriched with the tested compounds up to a concentration of 200
μM. High selectivity indices, SI50, significantly exceeding a value
of 80000 for all AZT (N-pyridinyl)phosphoramidates 4adꢀf,
make these compounds exceptionally good candidates for pro-
drugs in AIDS therapy. It is worth noticing that compounds
4adꢀf retained their high anti-HIV potency during several days
of the experiments and during this time their cytotoxicity was
below a measurable level (data not shown). The concentration-
dependent anti-HIV activity of ddU [N-(pyridin-4-yl)]pho-
sphoramidate 4bf can be considered as proof (at least partially)
that this type of nucleotide analogues are pronucleotides, i.e.,
they enter the infected cell and release the corresponding
nucleotides, which are subsequently converted by cellular kinases
into nucleoside triphosphates. Since ddU is not a substrate for
Stability of Nucleoside Phosphoramidates 4 in Cell Cul-
ture Media. In order to have a deeper insight into the mode of
action of compounds 4 as potential pro-nucleotides, first we
checked their stability and decomposition pathways in cell
culture media. To find out the chemical factors involved in a
possible metabolism, nucleoside (N-aryl)phosphoramidates 4aaꢀf
and 4bdꢀf were incubated in RPMI at 37 °C, and progress of the
decomposition was monitored by HPLC analysis. Under these
conditions, all investigated compounds remained unchanged for
at least 6 days, proving their stability in RPMI. In contrast, in cell
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Table 2. Stability and Anti-HIV Potency of Nucleoside (N-Aryl)phosphoramidates 4
c
a Retention time in HPLC analysis. For details, see the Experimental Section. b In RPMI/FBS 9:1 (v/v), 37 °C; concn 2 mM. Cmax: maximal
concentration used in the assay. d Concentration (μM) required to reduce the viability of mock-infected CEM-T4 cells by 50% or 90%, respectively, as
determined by the MTT method. e Concentration (μM) required to achieve 50% and 90% (respectively) protection from virus-induced
cytopathogenicity. f SI50: selectivity index CC50/EC50. SI values were rounded off respectively. g n. o.: in the tested range of concentrations cytotoxicity
was not observed. hSee ref 42.
culture media [RPMI/FBS 9:1 (v/v), 37 °C], which contained
enzymatic activities (including those of phosphoesterase type),36
the examined compounds 4 underwent slow decomposition with
half-life times (t1/2) varying from 22 h (4bf) to several days (4af)
(Table 2). The observed decomposition can be attributed to
enzymatic activities introduced into cell culture media with FBS,
and the differences in t1/2 are probably due to different substrate
affinities of the AZT (4a series) and ddU (4b series) derivatives.
The main products observed (HPLC) during the hydrolysis of
all the investigated nucleoside (N-pyridinyl)phosphoramidates
4 were the respective nucleosides (AZT or ddU) and their
50-monophosphate derivatives (NMPs) as minor products. This
might suggest that compounds 4 decomposed by hydrolysis of
the phosphoramidic bond to produce NMP and/or by hydrolysis
of phosphoester bond to produce the respective nucleoside and
(N-pyridinyl)phosphoramidate. To clarify this issue, in a separate
experiment AZTMP was incubated in RPMI/FBS, and it was
found (HPLC) that it was readily (t1/2 3.6 h, Table 2) depho-
sphorylated with phosphatase-like activity present in FBS. This
may suggest that decomposition of the examined compounds 4
proceeded via the respective NMPs, which were subsequently
dephosphorylated and produced the observed AZT or ddU.
Because in cell culture media compounds 4 decomposed very
slowly (Table 2), the suggestion that they enter the cell un-
changed and are metabolized into biologically active nucleotide
(most likely nucleosid-50-yl phosphate) seems plausible.
In order to recognize a potential involvement of cell surface
components or other factors of cellular origin, which potentially
may influence the metabolism of the examined compounds,
additional experiments were carried out, in which the stability
of nucleoside (N-pyridinyl)phosphoramidates 4adꢀaf in the cell
culture media in the presence of MT-4 cells was investigated.
Under such conditions, no essential differences in the degrada-
tion rates and decomposition paths of nucleoside (N-pyridinyl)-
phosphoramidates 4adꢀaf as compared to the neat cell culture
media were observed (results are not shown). The above results
indicated that in in vitro experiments these compounds preserved
their structure and that HIV infected cells are exposed for a long
period of time to unchanged compounds 4ad-af.
Trying to find possible degradation paths of the studied
compounds, we looked for enzymes that potentially may be
involved in the conversion of (N-pyridinyl)phosphoramidates
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Figure 2. Influence of nucleoside (N-pyridinyl)phosphoramidates 4adꢀaf on the HIV-1 RT polymerase activity.
4adꢀaf into AZTMP. To this end, the studied compounds were
treated with different HIT-proteins, some of which exhibited the
nucleoside phosphoramidase activity (Fhit proteins46 and some
Hint proteins47). Unfortunately, we found that none of the above
nucleoside (N-pyridinyl)phosphoramidates was hydrolyzed by
these enzymes (results not shown). To find out a possible origin
of exceptionally high anti-HIV activity of the investigated com-
pounds, their HIV-1 RT inhibitory potency was also investigated
(vide infra).
(e.g., DCM) containing a controlled amount of pyridine and then
can be oxidized with an iodine/water system into the respective
nucleoside (N-aryl)phosphoramidates 4. The rate of the latter
reaction depended on electronic features of the aryl group present,
and for more electron-withdrawing aryls, oxidation was faster. In this
respect, nucleoside (N-aryl)phosphoramidates 4differ from their N-
alkyl counterparts that are very resistant to oxidation.31 Hence, the
procedures described herein provide a versatile and efficient ap-
proach for the synthesis of (N-aryl)phosphoramidates. The
exceptions were 4-aminopyridine derivatives, which were synthe-
sized using the new phosphorylating agent, di(1H-1,2,4-ditriazol-1-yl)-
[N-(pyridin-4-yl)]phosphoramidate 6. This reagent can be re-
commended for the introduction of [N-(pyridin-4-yl)phosphor-
amidate functionality into nucleosides and probably also into
other biologically important compounds.
All AZT derived phosphoramidates revealed high anti-HIV
potency, and among them, [N-(pyridinyl)]phosphoramidates
(4adꢀaf) stood out with their very low cytotoxicity and very
high antiviral activity. These features make phosphoramidates 4
attractive candidates for new anti-HIV pro-drugs. Although their
mode of action is still unclear, the observed concentration-
dependent anti-HIV activity of the ddU derivate (4bf) strongly
points out that these compounds act as pronucleotides. Unfortu-
nately, this was not confirmed in experiments with TK-deficient
cells, in which none of examined phosphoramidates 4 was active
against HIV. The case seems to be not simple and certainly
requires careful conceptual and experimental verification and this
is the subject of intensive studies in our laboratories. Finally, we
would like to conclude that nucleoside (N-aryl)phosphorami-
dates 4 (particularly 4aeꢀaf) irrespective of their mode of action,
might be considered as new attractive anti-HIV pro-drugs due to
their exceptionally convenient pharmacokinetic parameters and
very low cytotoxicity.
Nucleoside (N-Pyridinyl)phosphoramidates 4 As Poten-
tial HIV-1 RT Inhibitors. Theoretically, nucleoside (N-pyridi-
nyl)phosphoramidates 4 can inhibit a polymerase activity of HIV
RT by mechanical blocking its active site or via allosteric
interaction(s). Considering the high stability of compounds 4
in cell culture media (and possibly also in the cell), one might
suspect that they could inhibit viral polymerase without conver-
sion into the respective nucleoside triphosphates. To verify the
above hypothesis, the inhibitory capacity of AZT (N-pyridinyl)-
phosphoramidates 4adꢀf was tested in the standard in vitro
reverse transcription reaction involving recombinant HIV-1 RT.
The reaction mixtures contained DNA template, 50(32P)-radi-
olabeled primer, all four natural nucleosid-50-yl triphosphates
and variable amounts of the examined compounds 4adꢀf. After
incubation for 1 h at 45 °C, the reaction mixtures were analyzed
by electrophoresis in a 12% denaturating polyacrylamide gel. The
efficacy of DNA synthesis was determined by autoradiography.
As a positive control, an analogous series of the reactions with
efavirenz (well established non-nucleoside HIV RT inhibitor)
was carried out. A negative control was a standard reaction
mixture without any inhibitor. The obtained data clearly showed
(Figure 2) that none of the investigated compounds demon-
strated inhibitory activity in concentrations e3 μM. The above
results might strongly suggest that it is not nucleoside (N-
pyridinyl)phosphoramidates 4 but their metabolites [e.g., AZT
50-triphosphate (AZTTP)]48 that inhibit viral RT in the cell
culture experiments.
’ EXPERIMENTAL SECTION
General Methods and Materials. 1H, 13C, and 31P NMR spectra
were recorded on Varian Unity BB VT 300 MHz or Bruker Avance II
400 MHz machines. The 31P NMR experiments were carried out in 5
mm tubes using 0.1 M solutions of the phosphorus-containing com-
pounds. 31P NMR chemical shifts are reported in ppm relative to 85%
H3PO4 in water (external standard) and with the exception of com-
pound 6 are listed in Table 1. Mass spectra were recorded with the ESI
technique with negative ionization with accuracy below 5 ppm. HPLC
analyses were performed on a C18 (5.0 μm) 4.6 mm ꢁ 150 mm column
The tested compounds are indicated above each set of
analytical runs. Lanes 1ꢀ10 correspond to the increasing con-
centration of each compound: 0.03; 0.06; 0.15; 0.3; 0.45; 0.6; 1.2;
1.5; 2.1 and 3 μM, respectively. Lane C, control, reaction without
inhibitor; 21nt, primer, 21 nucleotide long; 21ntC, primer
control lane; PP, polymerization product; Efv, efavirenz.
Conclusions. In conclusion, we found that nucleoside (N-aryl)-
H-phosphonamidates 3 can be effectively generated (the only
product observed in the 31P NMR spectra) in neutral solvents
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Journal of Medicinal Chemistry
ARTICLE
using solvent system A, 0.1 M triethylammonium acetate, and B, A/
acetonitrile 1: 4 (v/v), and gradient B in A 0%ꢀ50% within 20 min with
a flow rate of 1.5 mL/min, at 35 °C. For TLC analysis, the precoated
plates (Merck silica gel F254) were used, and for column chromatogra-
phy silica gel Si 60, 35ꢀ70 mesh (Merck) was used.
kinase and γ-[32P]ATP). HIV-1 RT was preincubated in a reaction
buffer (50 mM Tris-HCl at pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM
DTT) with increasing amounts of each compound (5 min at 45 °C), and
after the DNA primer and template were added, the polymerizations
were initiated by the addition of dNTPs to the final concentration of 50
μM. The above reaction mixtures were incubated for 60 min at 45 °C,
and after that, the products were separated in a 12% denaturing
polyacrylamide gel and visualized by autoradiography with Phosphor-
Imager Typhoon (Molecular Dynamics). To determine the efficiency of
polymerization, the amount of the fully extended product (FP) in each
sample was quantified using the MultiGage program.
Syntheses. General Procedure for the Synthesis of Nucleosid-50-
yl (N-Aryl)phosphoramidates 4 except for 4af and 4bf. Nucleoside H-
phosphonate 3 (1 mmol) and 2- or 3-aminopyridine (1.5 mmol) were
rendered anhydrous by the evaporation of the added pyridine (3 ꢁ
10 mL) and then dissolved in 10 mL of DCM containing 10% (v/v) of
pyridine. To this, DPCP (1.5 mmol) was added, and the reaction
mixture was left for 5 min. To oxidize the produced nucleoside, H-
phosphonate 3 iodine (2 mmol), dissolved in pyridine containing water
(50 mmol), was added, and after 5ꢀ30 min (see Table 1), excess of
iodine was decomposed with the added ethanethiol and the solvent
evaporated. The remaining residue dissolved in water (10 mL/1 mmol
of nucleoside) was washed with DCM (3 ꢁ 25 mL/1 mmol of
nucleoside). The aqueous layer was evaporated, and the desired product
was isolated with method A or B (vide infra).
DCM before use for reactions was dried over P2O5, distilled, and kept
over molecular sieves 4 Å until the amount of water was less than 10
ppm. Commercial grade anhydrous pyridine was stored over molecular
sieves of 4 Å until the amount of water was below 20 ppm. The amount
of water in solvents was measured with Karl Fisher coulometric titration.
Each evaporation of solvents was performed with a rotary evaporator
under reduced pressure using water bath temperature not exceeding
40 °C. Nucleosid-50-yl H-phosphonates were obtained as described
earlier.49
As judged from 1H, 13C, and 31P NMR spectra and HPLC analyses
(Waters 1525; Waters 2487 UV detector, λ254), the purity of the
obtained compounds was as least 97% except for compounds 4bdꢀbf
whose purity was ca. 90%.
RPMI-1640 cell culture medium and heat non-inactivated fetal
bovine serum (FBS) used for studies of stability of compounds were
from Sigma (R7256 and F7524 respectively).
Cytotoxicity and Anti-HIV Activity. CC50 and CC90 Para-
meters. For the determination of compound cytotoxicity, CEM-T4
and CEM-A cells were cultured in standard conditions (37 °C, 5% CO2)
on 96 wells culture plates in media RPMI/FCS 10% (v/v). The
experiments were carried out in media containing tested compounds
in concentrations of the appropriate range. Cultures in neat medium
(RPMI, 10% FCS) were used as a control. Viability of cells was
determined after 7 days using the MTT test50 in which to each well of
a culture plate was added 10 μL of MTT solution (5 mg/mL), and
cultures were incubated for 3 h at a temperature of 37 °C. After
centrifugation, the supernatant was removed, and DMSO was added
for lysis of the cells and to dissolve crystals of farmazan. Color intensity
was measured with a plate reader (λ560 nm).
Anti-HIV Activity (EC50 and EC90). To determine EC50 and EC90,
CEM-T4 cells were preincubated (96 flat bottom wells culture plates)
for 24 h in standard conditions (37 °C, 5% CO2) and in standard
medium [RPMI, FCS 10% (v/v)] enriched with tested compounds in
concentrations 0,001ꢀ20 μM. In each well, 20000 cells were suspended
in the solution of the tested compound (200 μL). For each concentra-
tion, cultures were run in triplicate. As a reference, media containing
AZT in concentrations of 1.25 μM, 2.5 μM, 5.0 μM, 10.0 μM, and 20
μM were used. As a reference of maximal replication of HIV-1, culture in
neat standard medium [RPMI/FCS 10% (v/v)] was used. After 24 h of
incubation in medium enriched with a tested compound and AZT, cells
were inoculated with a known amount of HIV, and after 8 days, the
amount of viral protein p24 was measured with the MTT method.51 For
each tested compound and for each concentration, the measurements of
EC50 and EC90 were done in triplicate.
Inhibition of HIV RT. Recombinant HIV-1 reverse transcriptase
(HIV-1 RT) was obtained with HIV-1 RT bacterial expression vector
generously provided by Stephen H. Hughes from the National Cancer
Institute, Frederick, MD, USA. A 50 nt DNA template (length of 50
nucleotide units) and primer (length of 21 nucleotide units) were
purchased from IBA. T4 polynucleotide kinase was purchased from
Promega and γ-[32P]ATP from Amersham. All chemicals were from
Sigma. Sequences of DNA molecules used in the experiments are presented
below, and the complementary fragments are underlined. Template,
50-TTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAA-
CAGGGACTTG-30; primer, 50-CAAGTCCCTGTTCGGGCGCCA-30.
The inhibition HIV-1 RT potency with nucleoside (N-pyridyl)pho-
sphoramidates 4adꢀf was tested in vitro in a primer extension reaction,
involving a 50 nt DNA template a cDNA primer (length 21 nucleotide
units, 32P labeled at the 50 end in a reaction involving T4 polynucleotide
Purification by Method A (for Compounds 4aaꢀac). The crude
oily residue dissolved in a minimum volume of methanol was applied to a
silica gel column (4 ꢁ 7 cm, equilibrated with 2-propanol) and washed
with 2-propanol. Separation was carried out with a flash chromatography
system using a gradient of water (0ꢀ20%) in 2-propanol containing 5%
(v/v) of triethylamine. Fractions containing a pure compound were
collected and evaporated, and the triethylammonium salt of nucleotide 4
was solidified by freeze-drying from benzeneꢀmethanol.
Purification by Method B (for Compounds 4adꢀaf and 4bdꢀbf).
The crude residue was dissolved in a minimum volume of toluene/
methanol 1:1 (v/v) and loaded on a silica gel column (loaded and
equilibrated with toluene/methanol 5:1, v/v). A separation was run
using gradient (20ꢀ50%, v/v) of methanol in toluene. Fractions
containing pure compound 4 were collected and evaporated. After
freeze-drying (from benzeneꢀmethanol), pure products 4adꢀaf and
4bdꢀbf were obtained as white dry powder.
3-Azido-30-deoxythymidyn-50-yl (N-Phenyl)phosphoramidate,
1
Triethylammonium Salt (4aa). Yield, 0.22 g (71%). H NMR (400
MHz) δH (D2O) 1.28 (9H, t, J = 7.3 Hz), 1.87 (3H, s), 2.42 (2H, m),
3.18 (6H, q, J = 7.3 Hz), 4.01 (1H, m), 4.08 (2H, m), 4.32 (1H, m), 6.14
(1H, t, J = 6.6 Hz), 6.86 (1H, t, J = 7.8 Hz), 7.00 (2H, d, J = 7.8 Hz), 7.19
(2H, t, J = 7.8 Hz), 7.58 (1H, s). 13C NMR (75 MHz) δC (D2O) 7.8,
11.2, 35.6, 46.2, 60.0, 63.9 (d, J = 7.0 Hz), 82.6 (d, J = 10 Hz), 84.6, 110.9,
116.4 (d, J = 7.0 Hz), 119.8, 128.6, 137.0, 141.5, 151.0, 165.9. HRMS
m/z 421.1045, calcd for [C16H18N6O6P]ꢀ 421.1031.
3-Azido-30-deoxythymidyn-50-yl (N-4-Methylphenyl)phosphorami-
1
date, Triethylammonium Salt (4ab). Yield, 0.09 g (53%). H NMR
(400 MHz) δH (D2O) 1.30 (9H, t, J = 7.6 Hz), 1.85 (3H, d, J = 0.8 Hz),
2.19 (3H, s), 3.19 (6H, q, J = 7.6 Hz), 3.97ꢀ4.01 (1H, br. m), 4.04ꢀ4.11
(2H, br. m), 4.30 (1H, m), 6.16 (1H, t, J = 6.8 Hz), 6.90 (2H, d, J = 8.4
Hz), 6.98 (2H, d, J = 8.4 Hz), 7.54 (1H, q, J = 0.8 Hz). 13C NMR (100
MHz) δC (D2O) 8.2, 11.6, 19.5, 35.9, 46.6, 52.1, 60.4, 64.2 (d, J = 4.7
Hz), 83.0 (d, J = 9.0 Hz), 84.9, 111.2, 117.1 (d, J = 7.0 Hz), 129.2, 129.7,
137.3, 139.2, 151.4, 166.1. HRMS m/z 435.1293, calcd for [C17H20-
N6O6P]ꢀ 435.1287.
3-Azido-30-deoxythymidyn-50-yl (N-4-Chlorophenyl)phosphorami-
1
date, Triethylammonium Salt (4ac). Yield, 0.23 g (73%). H NMR
(400 MHz) δH (D2O) 1.31 (9H, t, J = 7.2 Hz), 1.84 (3H, s), 2.48 (2H, t,
J = 6.8 Hz), 3.21 (6H, q, J = 7.2 Hz), 4.01 (1H, m), 4.13 (2H, m), 4.36
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Journal of Medicinal Chemistry
ARTICLE
(1H, m), 6.17 (1H, t, J = 6.8 Hz), 6.94 (2H, d, J = 8.8 Hz), 7.13 (2H, d, J =
8.8 Hz), 7.45 (1H, s). 13C NMR (75 MHz) δC (D2O), 7.8, 11.2, 35.4,
46.2, 59.8, 64.0 (d, J = 5.0 Hz), 82.5 (d, J = 10 Hz), 84.7, 110.9, 117.9 (d,
J = 7.0 Hz), 123.8, 128.0, 137.0, 140.3, 151.0, 165.7. HRMS m/z
455.0631, calcd for [C16H17ClN6O6P]ꢀ 455.0641.
oily residuedissolved in water (10 mL/1 mmol of nucleoside) was washed
with methylene chloride (3 ꢁ 25 mL/1 mmol of nucleoside), and the
aqueous layer was evaporated. Compounds 4af and 4bf were isolated with
purification procedure B (see above).
3-Azido-30-deoxythymidin-50-yl [N-(Pyridin-4-yl)]phosphoramidate
(4af). Yield, 0.50 g, 59%, 1H NMR (300 MHz) δH (D2O) 1.85 (3H, s),
2,51 (2H, m), 4.10 (3H, m), 4.41 (1H, dt, J = 6.0 Hz), 6.13 (1H, t,
J = 6.6 Hz), 7.22 (2H, d, J = 6.3 Hz), 7.52 (1H, s), 8.19 (1H, d, J =
6.3 Hz). 13C NMR (75 MHz) δC (D2O), 11.1, 35.4, 59.6, 64.2 (d, J = 5.1
Hz), 82.6 (d, J = 9.1 Hz), 85.2, 111.2, 112.2 (d, J = 7.0 Hz), 137.5, 142.4,
151.3, 155.9, 166.1. HRMS m/z 422.1006, calcd for [C15H17N7O6P]ꢀ
422.0983.
3-Azido-30-deoxythymidin-50-yl [N-(Pyridin-2-yl)]phosphorami-
1
date (4ad). Yield, 0.13 g (84%). H NMR (400 MHz) δH (D2O)
1.84 (3H, s), 2.50 (2H, m), 4.04 (1H, m), 4.14 (2H, m), 4.40 (1H, m, J =
6.4 Hz), 6.16 (1H, t, J = 6.4 Hz), 6.87 (1H, m, J = 8.4 Hz, 6.4 Hz), 7.03
(1H, d, J = 8.4 Hz), 7.51 (1H, s), 7.60 (1H, m, J = 6.4 Hz, 1.6 Hz), 8.30
(1H, d, J = 6.4 Hz). 13C NMR (100 Hz) δC (D2O) 11.5, 35.8, 60.1, 64.6
(d, J = 4.0 Hz), 82.8 (d, J = 9.1 Hz), 85.0, 110.9, 111.2, 116.4, 137.3,
138.6, 147.0, 151.5, 154.0 (d, J = 5.0 Hz), 166.4. HRMS m/z 422.0912,
calcd for [C15H17N7O6P]ꢀ 422.0983.
2,30-Dideoxyuridin-50-yl [N-(Pyridin-4-yl)]phosphoramidate (4bf).
Yield, 0.46 g, 62%. 1H NMR (400 MHz) δH (D2O) 1.90 (1H, m), 2.10
(2H, m), 2.45 (1H, m), 3.92 (1H, dt, J = 5.6 and 11.6 Hz), 4.13 (1H, dt,
J = 3.2 and 11.6 Hz), 4.36 (1H, m), 5.68 (1H, d, J = 8.0 Hz), 6.05 (1H,
dd, J = 2.8 and 7.2 Hz), 7.05 (2H, d, J = 6.4), 7.70 (1H, d, J = 8.0 Hz),
8.17 (2H, d, J = 6.4). 13C NMR (75 MHz) δC (D2O), 24.8, 30.7, 66.1
(d, J = 5.0 Hz), 80.1 (d, J = 9.1 Hz), 86.4, 101.5, 112.1 (d, J = 7.5 Hz),
141.9, 146.6, 151.8, 152.2, 166.6. HRMS m/z 367.0813, calcd for
[C14H16N4O6P]ꢀ 367.0870.
3-Azido-30-deoxythymidin-50-yl [N-(Pyridin-3-yl)]phosphorami-
1
date (4ae). Yield, 0.14 g (92%), H NMR (400 MHz) δH (D2O),
1.82 (3H, s), 2.50 (2H, m), 4.04 (1H, m), 4.13 (2H, m), 4.34 (1H, dt, J =
6.4 Hz), 6.17 (1H, t, J = 6.4 Hz), 7. Twenty-two (1H, dd, J = 8.4, 4.8 Hz),
7.44 (1H, m), 7.46 (1H, s), 7.96 (1H, d, J = 4.8 Hz), 8.18 (1H, m). 13C
NMR (100 Hz) δC (D2O) 11.4, 35.7, 60.0, 64.6 (d, J = 5.0 Hz), 82.6 (d,
J = 9.1 Hz), 85.2, 111.1, 125.9, 129.5 (d, J = 8.0 Hz), 132.1 (d, J = 8.0 Hz),
134.9, 137.5, 141.5, 151.3, 166.1. HRMS m/z 422.0910, calcd for
[C15H17N7O6P]ꢀ 422.0983.
’ ASSOCIATED CONTENT
2,30-Dideoxyuridin-50-yl [N-(Pyridin-2-yl)]phosphoramidate (4bd).
Yield, 0.11 g (88%). 1H NMR (400 MHz) δH (D2O), 1.93 (1H, m), 2.13
(2H, m), 2.46 (1H, m), 4.00 (1H, m), 4.20 (1H, m), 4.36 (1H, m), 5.73
(1H, d, J = 8.0 Hz), 6.05 (1H, m), 7.10 (1H, dd, J = 6.6 and 7.2 Hz), 7.20
(1H, d, J = 8.8 Hz), 7.72 (1H, d, J = 8.0 Hz), 7.98 (1H, dd, J = 6.6 and 8.8
Hz), 8.06 (1H, d, J = 7.2 Hz). 13C NMR (100 Hz) δC (D2O) 24.4, 30.1,
66.1 (d, J = 5.6 Hz), 79.7 (d, J = 8.4 Hz), 85.8, 101.2, 113.6 (d, J = 5.3
Hz), 115.6, 139.0, 141.6 (d, J = 2.1 Hz), 143.2, 151.0, 151.7 (d, J =
2.6 Hz), 165.6. HRMS m/z 367.0776, calcd for [C14H16N4O6P]ꢀ
367.0813.
Supporting Information. 1H, 13C, and 31P NMR spectra
S
b
of all new compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Institute of Bioorganic Chemistry, Polish Academy o Sciences,
Noskowskiego 12/14, 61-704 Poznaꢀn, Poland. Tel: +48-61 852
85 03. Fax: +48-61-852 05 32. E-mail: adam.kraszewski@ibch.
poznan.pl; akad@ibch.poznan.pl.
2,30-Dideoxyuridin-50-yl [N-(Pyridin-3-yl)]phosphoramidate (4be).
Yield, 0.12 g (93%). 1H NMR (400 MHz) δH (D2O) 1.90 (1H, m), 2.12
(2H, m), 2.43 (1H, m), 3.93 (1H, m), 4.13 (1H, m), 4.36 (1H, m), 5.64
(1H, d, J = 8.0 Hz), 6.05 (1H, m), 7.37 (1H, dd, J = 5.1 and 8.4 Hz), 7.60
(1H, ddd, J = 1.2, 2.7, and 8.5 Hz), 7.68 (1H, d, J = 8.0 Hz), 8.03 (1H, d,
J = 4.6 Hz), 8.22 (1H, d, J = 2.2 Hz). 13C NMR (100 Hz) δC (D2O) 24.5,
30.3, 65.7 (d, J = 5.2 Hz), 79.8 (d, J = 9.4 Hz), 86.0, 101.1, 124.4, 126.2
(d, J = 6.2 Hz), 135.2 (d, J = 8.1 Hz), 137.7, 139.7, 141.5, 151.0, 165.7.
HRMS m/z 367.0778, calcd for [C14H16N4O6P]ꢀ 367.0813
’ ACKNOWLEDGMENT
Financial support from the Government of the Polish Repub-
lic (project No. PBZ-MNiSW-07/I/2007; 2008ꢀ2010) is grate-
fully acknowledged.
Synthesis of Di(1H-1,2,4-triazol-1-yl) [N-(Pyridin-4-yl)]phosphora-
midate 6. To a solution of phosphoryl-tris-(1H-1,2,4-triazole) 5 in
dioxane (1 mmol/5 mL), 4-aminopyridine (equimolar amount) dis-
solved in pyridine (1 mmol/1.25 mL) was added. After 10ꢀ20 min,
di(1H-1,2,4-triazol-1-yl) [N-(pyridin-4-yl)]phosphoramidate 6 started
to precipitate as a white solid. After 1 h, the solvent was decanted, and the
remaining solid was washed with 1,4-dioxane (3ꢁ 5 mL), diethyl ether
(1ꢁ 10 mL), and dried under vacuum. Yield, 91%. Mp 167ꢀ170 °C (not
corrected). 1H NMR (400 MHz) δH (DMSO-d6), 6.95 (2H, d, J = 6.8
Hz), 8.13 (4H, m), 9.03 (2H, s). 13C NMR (100 MHz) δC (DMSO-d6),
158.0, 154.4 (d, J = 16.3 Hz), 149.8 (d, J = 8.2 Hz), 139.6, 118.5 (d, J =
23.0 Hz). 13P NMR (121 MHz) δP (DMSO, ppm), ꢀ14.5 (s); HRMS
(ꢀ) m/z 275.0786, calcd for [C9H8N8OP]ꢀ 275.0559
’ ABBREVIATIONS USED
AZT, 30-azido-30-deoxythymidine; AZTMP, 30-azido-30-deox-
ythymidin-50-yl monophosphate; AZTTP, 30-azido-30-deoxythy-
midin-50-yl triphosphate; DCM, dichloromethane; ddU, 20,30-
dideoxyuridine; DMSO, dimethyl sulfoxide; DPCP, diphenyl
chlorophosphate; DTT, dithiothreitol; ESI, electron spray ioni-
zation; FBS, fetal bovine serum; HRMS, high-resolution mass
spectrometry; NMP, nucleosid-50-yl monophosphate; RPMI, Roswell
Park Memorial Institute medium; SATE, S-acetyl-thioethyl;
TKꢀ, thymidine kinase-deficient
Procedure for the Synthesis of Nucleoside [N-(Pyridin-4-yl)]pho-
sphoramidates 4af and 4bf. To a suspensionofdi(1H-1,2,4-triazol-1-yl)
[N-(pyridin-4-yl)]phosphoramidate 6 (2 molar equiv) in pyridine
(1 mmol/12.5 mL) at 75 °C (oil bath), AZT or ddU (1 molar equiv)
dissolved in pyridine (1 mmol/10 mL) was added in five equal portions in
1 min intervals with stirring (magnetic bar). The reaction was continued
for ca. 10 min at 75 °C and cooled down to room temperature, and after a
large excess of water was added (10% v/v), the whole mixture was left for
1 h. The solvent was evaporated (rotary evaporator), and the remaining
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