Nucleoside 3′,5′-cyclic H-phosphonates, new useful precursors for the synthesis of nucleoside 3′,5′-cyclic phosphates and their analogues

Article abstract of DOI:10.1021/ol4016404

Nucleoside H-phosphonates activated with a condensing agent spontaneously formed nucleoside 3′,5′-cyclic H-phosphonates. The cyclization was stereoselective and produced one of the P-diastereomers in preponderance (de ca. 80%). Nucleoside 3′,5′-cyclic H-phosphonates were stereochemically unstable and underwent epimerization affording the thermodynamically more stable diastereomer as a major product (de ca. 80%). They were susceptible to hydrolysis, transesterification, and oxidation and by changing oxidation protocols nucleoside 3′,5′-cyclic phosphate analogues, e.g. phosphodiesters, phosphorothioate diesters, and phosphotriesters, were obtained.

Full text of DOI:10.1021/ol4016404

ORGANIC
LETTERS
Nucleoside 30,50-Cyclic H‑Phosphonates,
New Useful Precursors for the Synthesis
of Nucleoside 30,50-Cyclic Phosphates and
Their Analogues
XXXX
Vol. XX, No. XX
000–000
Malgorzata Rozniewska, Jacek Stawinski, and Adam Kraszewski*
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14,
ꢀ
61-704 Poznan, Poland
adam.kraszewski@ibch.poznan.pl
Received June 12, 2013
ABSTRACT
Nucleoside H-phosphonates activated with a condensing agent spontaneously formed nucleoside 30,50-cyclic H-phosphonates. The cyclization
was stereoselective and produced one of the P-diastereomers in preponderance (de ca. 80%). Nucleoside 30,50-cyclic H-phosphonates were
stereochemically unstable and underwent epimerization affording the thermodynamically more stable diastereomer as a major product (de ca. 80%).
They were susceptible to hydrolysis, transesterification, and oxidation and by changing oxidation protocols nucleoside 30,50-cyclic phosphate
analogues, e.g. phosphodiesters, phosphorothioate diesters, and phosphotriesters, were obtained.
Nucleoside 30,50-cyclic phosphates (e.g., cAMP, cGMP)
play an important role in living organisms acting as second
messengers in numerous cellular events such as activation
of protein kinases, cyclic nucleotide binding proteins or
cAMP receptor proteins, regulation of ion channels, etc.1,2
Recently, some disorder in cAMP pathways and an aber-
rant activation of cAMP-controlled genes have been im-
plicated in tumor progression.3,4 This diverse biological
activity of these rather simple molecules has stirred interest
among chemists for the preparation of various analogues
of nucleoside 30,50-cyclic phosphates as molecules for
potential biomedical intervention. Typical examples here
are nucleoside 30,50-cyclic phosphorothioates,5,6 the corre-
sponding phosphoramidates derivatives7ꢀ9 (especially
those with defined stereochemistry at the phosphorus
center), and stereospecifically isotope labeled cAMP
derivatives.8,10 Many of these analogues were found to
be very useful tools for elucidation of the mechanistic
aspects of various enzymatic reactions.11
From a synthetic point of view, different chemistries
were applied for the preparation of nucleoside cyclic
phosphotriesters. The most common approach involves
condensation of the corresponding cyclic phosphodies-
ters with an alcohol aided by a condensing agent, or an
(6) Zielinski, W. S.; Stec, W. J. J. Am. Chem. Soc. 1977, 99, 8365–
8366.
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P. J.; Barret, K. E.; Tsien, R. Y. J. Biol. Chem. 1993, 268, 6316–6322.
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r
10.1021/ol4016404
XXXX American Chemical Society

alkylation reaction with a suitable alkylating agent.12ꢀ14
Other methods make use of bifunctional phosphorylating
reagents which, after phosphorylation of one hydroxyl
group of a suitably protected nucleoside, form a 30,50 six-
membered ring in a subsequent intramolecular cyclization
reaction.15ꢀ17 All these methods are multistep and afford
final nucleoside 30,50-cyclic phosphotriesters in moderate
or poor yield.16,17 This can be a serious drawback when
working with starting materials that are not readily acces-
sible (e.g., antiviral nucleoside or nucleotide analogues) or
with compounds for which diverse modifications at the
phosphorus center are required.
The chemistry of phosphorus compounds with
a phosphorus atom embedded in a six-membered
H-phosphonate ring fused to a ribose moiety has been
rather poorly explored in nucleotide chemistry, and
nucleoside 30,50-cyclic H-phosphonates have not been
prepared yet. There are few examples of the synthesis of
H-phosphonate phosphinane derivatives of simple
diols18ꢀ22 described in literature, and these compounds
have unique properties that distinguish them from
tricoordinated phosphite and tetracoordinated phos-
phate analogues.
As a model reaction for the formation of nucleoside
30,50-cyclic H-phosphonate diesters we chose activation
of thymidine 30-H-phosphonate monoester 1a with piva-
loyl chloride. To this end, we added pivaloyl chloride
(1.2 equiv) to a 0.1 mM solution of H-phosphonate 1a in
dichloromethane/pyridine (95:5, v/v). The first 31P NMR
spectrum recorded (after ca. 3 min) revealed that the
starting material 1a had completely disappeared and two
main resonances (ratio 9:1, ca. 95% of the phosphorus
containing compounds), centered at δP = 1.4 and 3.4 ppm
1
1
(2 m, JHP = 741.8 Hz, JHP = 699.7 Hz, respectively),
appeared.24 The chemical shifts and multiplicities of these
signals were quite different from those expected for the
putative intermediate mixed anhydride 2a (δP = 0.8 ppm
1
3
and 0.9 ppm, 2 dd, JHP = 745.2 and 746.2 Hz, JHP
=
8.1 Hz)25 and indicated formation of the desired thymidine
cyclic H-phosphonate 3a. In agreement with this, in situ
oxidation of a diastereomeric mixture of the putative cyclic
H-phosphonate 3a with iodine (vide infra) produced thy-
midine 30,50-cyclic phosphate (cTMP, 6a in Scheme 2) that
was identical to an original sample of this compound
prepared in another way.
A similar course of the reaction was also observed for
other nucleoside 30-H-phosphonates investigated (1bꢀd),
and thus, the described above synthetic protocol was used
for generation of nucleoside 30,50-cyclic H-phosphonates
of type 3 discussed in this paper.
In this paper we would like to describe our studies on the
synthesis and properties of nucleoside 30,50-cyclic H-phos-
phonates as new synthetic intermediates for the prepara-
tion of various analogues of nucleoside 30,50-cyclic
phosphates.
Attempted synthesis of nucleoside 30,50-cyclic H-phos-
phonates using a bifunctional phosphonylating reagent,
diphenyl H-phosphonate, and unprotected thymidine as a
model deoxyribonucleoside gave complex reaction mix-
tures, in contrast to the reaction with 20,30-unprotected
ribonucleosides which smoothly afforded the correspond-
ing nucleosides 20,30-cyclic H-phosphonates.23 In this sit-
uation we explored another approach that was based on a
condensing agent activation of nucleoside H-phosphonate
monoesters bearing an unprotected hydroxyl function,
followed by intramolecular cyclization (Scheme 1). Since
comparable results were obtained for isomeric nucleoside
30- or 50-H-phosphonate monoesters and for different
condensing agents, we confined our discussion below to
the reactions of nucleoside 30-H-phophonates and pivaloyl
chloride used as an activator.
Scheme 1. Formation of Nucleoside 30,50-Cyclic H-Phosphonates
and Their Sulfurization
We also assigned the absolute configuration at the
phosphorus center in 3a using stereochemical correlation
analysis. To this end, to a diastereomeric mixture of
thymidine cyclic H-phosphonate 3a (ratio 9:1) preformed
as described above, elemental sulfur was added (3 equiv).
The reaction was fast, and after ca. 5 min the 31P NMR
spectrum indicated the presence of two compounds
resonating at 55.94 and 54.69 ppm (ratio ca. 8:1). These
were identified as the corresponding thymidine 30,5-cyclic
phosphorothioate diesters by comparison with original
samples.5 Intensities of the signals suggested that the
(12) Engels, J. W.; Schlaeger, E.-J. J. Med. Chem. 1977, 20, 907–911.
(13) Kruppa, J.; Keely, S.; Schwede, F.; Schultz, C.; Barret, K. E.;
Jastorff, B. Bioorg. Med. Chem. Lett. 1997, 7, 945–948.
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Soc. 1990, 112, 7475–7482.
(17) Nelson, K. A.; Bentrude, W. G.; Stezer, W. N.; Hutchinson, J. P.
J. Am. Chem. Soc. 1987, 109, 4058–4064.
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(19) Cullis, P. M.; Lee, M. J. Chem. Soc., Chem. Commun. 1992,
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Kraszewski, A. Tetrahedron Lett. 2001, 42, 8055–8058.
(24) Under these reaction conditions we also observed formation of
variable amounts of linear condensation products (ca. 5%, signals at
6ꢀ7 ppm). However, when we decreased the concentration of 1a to
0.01 mM, the formation of linear products was practically completely
eliminated.
(25) Cieslak, J.; Sobkowski, M.; Kraszewski, A.; Stawinski, J. Tetra-
hedron Lett. 1996, 37, 4561–4564.
B
Org. Lett., Vol. XX, No. XX, XXXX

downfield signal (δP = 55.99 ppm, RP-thymidin-30,50-diyl
phosphorothioate, 4a5) was formed from the major dia-
stereomer 3a resonating at δP = 1.35 ppm, and the upfield
signal (δP = 54.69 ppm, SP-thymidin-30,50-diyl phosphor-
othioate, 4a5) arose from the minor diastereomer 3a (δP =
1.35 ppm). In an analogous experiment in which the ratio
of diastereomers of cyclic H-phosphonate 3a was opposite
(ca. 1:7, vide infra), a major product of the reaction was the
upfield isomer SP-thymidin-30,50-diyl phosphorothioate 4a
(δP = 54.69 ppm), and the ratio of the SP/RP diastereomers
4a was inverted compared to the previous experiment
(8:1 vs 6.7:1 in this experiment). The produced thymidine
30,50-cyclic phosphorothioates 4 were purified by silica gel
column chromatography, and their structures were unam-
biguously determined by spectroscopic methods (1H, 13C,
and 31P NMR, HRMS).
the final equilibrium ratio of ca. 9:1. This meant that the
kinetic product of the reaction in Scheme 1, SP-thymidine
cyclic H-phosphonate 3a, upon standing, was converted
into the thermodynamic product, RP-3a.33 Mechanistic
aspects of this phenomenon are under investigation.
To demonstrate the synthetic utility of the nucleoside
30,50-cylic H-phosphonates 3 and to gain additional insight
into their reactivity, we subjected these compounds to
various oxidation protocols. Since, during the oxidation
of H-phosphonate diesters with iodine, reactive iodopho-
sphates are formed, that are highly susceptible to nucleo-
philic substitution at the phosphorus center,34ꢀ38 we
attempted to develop this as a new method for the pre-
paration of nucleoside 30,50-cyclic phosphodiesters of type
6 andalsoforthesynthesisof lessexplored, until now, alkyl
or aryl nucleoside 30,50-cyclic phosphotriesters of type 7
and 8, respectively (Scheme 2).
Since the stereochemical correlation analysis above in-
volved one stereospecific reaction (sulfurization) that pro-
ceeds with retention of configuration, it implies that the
high-field resonating diastereomers of cyclic H-phospho-
nate 3a (δP = 1.35 ppm) has an SP configuration (or DP
using DP/LP notation26,27), and the low-field resonating
diastereomer of 3a (δP = 3.54 ppm), an RP configuration
(or LP using DP/LP notation26,27) at the phosphorus center
(Scheme 1).28 Since a similar pattern of signals was ob-
served also for the reactions involving other nucleosides
1bꢀd, we assumed that the stereochemical assignments
shown in Scheme 1 can be extended to other nucleoside
30,50-cyclic H-phosphonates investigated in this paper.
H-Phosphonate diesters derived from nucleosides, sug-
ars, lipids, etc. are usually stable enough to be purified
by silica gel chromatography and then stereospecifically
converted into various P(V)-derivatives.29ꢀ32 By way of
contrast, attempted isolation of nucleoside 30,50-cyclic
H-phosphonates of type 3 failed, due to instability of these
compounds during aqueous workup. This susceptibility
to hydrolysis and transesterification with alcohols (data
not shown) may result from some strain or distortion of
a 1,3,2-dioxaphosphinane ring imparted by the fused
deoxyribose moiety. This increased reactivity of nucleo-
side 30,50-cyclic H-phosphonates 3 compared to acyclic
H-phosphonate diesters is also manifested in the config-
urational instability of these compounds. For example, we
observed that when a reaction mixture containing a mix-
ture of RP /SP-diastereomers 3a was left at room tempera-
ture for 5 h, a gradual change in the ratio of the
diastereomers occurred from an initial value of ca. 1:9 to
Scheme 2. Synthesis of Nucleoside 30,50-Cyclic Phosphates and
Phosphotriesters
The efficacy of such an approach was checked by
generating thymidine 30,50-cyclic H-phosphonate 3a (vide
supra) and adding this to the reaction mixture of I2
(1.2 equiv, dissolved in anhydrous pyridine), followed by
an excess of water (after 30 s) (Scheme 2). We chose a
separate addition of iodine and water, instead of a stan-
dard oxidation protocol (treatment of H-phosphonate
diesters with an aqueous pyridine solution of iodine36),
to minimize the risk of hydrolysis of 3a under the reaction
(33) In 1969 Mikozajczyk21 postulated epimerization at the phos-
phorus center of 4-methyl-2-oxo-1,3,2-dioxaphosphinane to explain the
stereoselectivity observed during hydrolysis of the corresponding phos-
phorochloridite. More recently, Cullis and Lee19 reported that during
distillation at elevated temperature equilibration of the RP and SP
diastereomers of 5-tert-butyl-2-oxo-1,3,2-dioxaphosphinane occurred.
No mechanistic aspect of this phenomenon was discussed.
(26) Sobkowski, M.; Stawinski, J.; Kraszewski, A. Nucleosides,
Nucleotides Nucleic Acids 2006, 25, 1363–1.
(27) Sobkowski, M.; Stawinski, J.; Kraszewski, A. Nucleosides,
Nucleotides Nucleic Acids 2006, 25, 1377–1389.
(34) von Nylen, P. Z. Anorg. Allg. Chem. 1938, 235, 161–183.
(35) Saunders, B. C.; Stark, B. P. Tetrahedron 1958, 4, 169–177.
(36) Garegg, P. J.; Regberg, T.; Stawinski, J.; Stromberg, R. J. Chem.
Soc., Perkin Trans. 1 1987, 1269–1273.
(28) Change from the RP to SP or SP to RP configuration in Scheme 1
for the stereoretentive sulfurization step is due to the priority order of
substituents in the ICP convention.39 Since a sense of chirality at the
phosphorus center is preserved in this reaction, the DP/LP stereochemi-
cal descriptors remain invariant.26,27
ꢀ
(37) Romanowska, J.;Szymanska-Michalak, A.; Boryski, J.; Stawinski,
J.; Kraszewski, A.; Loddo, R.; Sanna, G.; Collu, G.; Secci, B.; La Colla, P.
(29) Stawinski, J.; Kraszewski, A. Acc. Chem. Res. 2002, 35, 952–960.
(30) Bollmark, M.; Stawinski, J. Tetrahedron Lett. 1996, 37, 5739–
5742.
(31) Zain, R.; Stawinski, J. J. Org. Chem. 1996, 61, 6617–6622.
(32) Stawinski, J.; Thelin, M. J. Org. Chem. 1991, 56, 5169–5175.
Bioorg. Med. Chem. 2009, 17, 3489–3498.
(38) Romanowska, J.; Sobkowski, M.; Szymanska-Michalak, A.;
Kozodziej, K.; Dabrowska, A.; Lipniacki, A.; Piasek, A.; Pietrusiewicz,
)
Z. M.; Figlerowicz, M.; Guranowski, A.; Boryski, J.; Stawinski, J.;
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Kraszewski, A. J. Med. Chem. 2011, 54, 6482–6491.
Org. Lett., Vol. XX, No. XX, XXXX
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conditions. Both steps of the reaction, oxidation with
iodine and hydrolysis, were rapid, and after evaporation
of the solvents and aqueous workup, the desired thymidine
30,50-cyclic phosphate 6a was isolated in high yield (82%)
by silica gel column chromatography.
Analogous reactions for other cyclic H-phosphonates 3
containing standard 20-deoxyribonucleosides (20-deoxy-
adenosine 3b, 20-deoxycytidine 3c, and 20-deoxyguanosine
3d derivatives, generated from the corresponding nucleo-
side 30-H-phosphonates 1bꢀd) worked equally well afford-
ing after deprotection and silica gel chromatography the
corresponding nucleoside cyclic phosphates 6bꢀd in con-
sistently high yield (74%ꢀ82%, calculated on the starting
nucleoside H-phosphonates 1).
The synthetic potential of the putative cyclic iodopho-
sphate intermediates 5 (Scheme 2) was also exploited in
the preparation of alkyl and aryl nucleoside 30,50-cyclic
phosphotriesters of type 7 or 8, respectively. To this end,
cyclic iodophosphate intermediates 5 (generated in situ
from 3 as described above) were treated with a moderate
excess (3.0 molar equiv) of the respective alcohol
(methanol, ethanol, 2-isopropanol) dissolved in pyridine.
The reactions were rapid (<3 min) and stereoselective,
affording practically quantitatively two diastereomers of
triesters 7 in a ratio of ca. 3:1 (downfield/upfield resonating
diastereomer in the 31P NMR spectra).
A similar course of the reaction was also observed for
oxidative condensations with phenols. For example, un-
substituted phenol or 3-hydroxypyridine reacted smoothly
under the analogous conditions with in situ generated
5a affording the corresponding cyclic triesters 8aa and
8ab that, after standard workup, were purified by reversed-
phase column chromatography and isolated in 66%
and 54% yield, respectively. The lower yields observed
for the aryl phosphotriesters were, most likely, due to the
incomplete stability of these compounds during chro-
matography.
the infected cell. We were delighted to find that cyclic
triester 7ad indeed revealed high anti-HIV potency (EC50
0.01 μM, MT-4) along with very low cytotoxicity (CC50
.
350 μM; SI . 35000), which makes it a very promising
anti-HIV pro-drug candidate. This type of compound
most likely acts as an anti-HIV pro-nucleotide and poten-
tially can be useful in AIDS therapy. Further studies are in
progress in our laboratory.
To summarize, condensing agent-activated nucleoside
30- or 50-H-phosphonate monoesters undergo a stereose-
lective, intramolecular cyclization to produce SP-nucleo-
side 30,50-cyclic H-phosphonates 3 as the major products
(de 80%). It was found that this type of cyclic H-phospho-
nate was significantly more susceptible to hydrolysis and
transesterification than its acyclic counterpart and, upon
standing, underwent epimerization at the phosphorus
center to afford the thermodynamically more stable
RP-diastereomers of 3 (de 80%). Absolute P-configura-
tions of thymidine 30,50-cyclic H-phosphonate 3a were
established using stereochemical correlation analysis. To
demonstrate the synthetic utility of this new type of inter-
mediates, we converted cyclic H-phosphonates of type
3 under mild conditions into the corresponding cyclic
phosphorothioates, phosphodiesters, and diverse cyclic
phosphotriester derivatives. The procedures developed
are simple, efficient, high yielding, and thus provide
new entries to biologically important nucleoside 30,50-cylic
phosphate analogues of type 6, 7, 8, and others.
We also found that a nucleoside 1,3,2-dioxaphosphi-
nane scaffold may exert a beneficial effect on the pharma-
cological activity of a potential drug as exemplified by
the high anti-HIV potency and very low cytotoxicity of
the 30-azido-30-deoxythymidine (AZT) derivative 7ad.
Acknowledgment. Financial support from the National
Science Centre of Poland (Project No. 2011/01/B/ST5/
06414) is gratefully acknowledged.
We also applied the above oxidative coupling procedure
for the reaction involving thymidine cyclic H-phosphonate
3a and 30-azido-30-deoxythymidine (AZT) to produce the
AZT thymidine 30,50-cyclic phosphotriester 7ad, as a po-
tential vehicle for delivery of an anti-HIV agent (AZT) into
Supporting Information Available. Synthetic protocols,
1H, 13C, 31P NMR spectra, HRMS data, and HPLC
(purity criteria). This material is available free of charge
via the Internet at http://pubs.acs.org.
(39) Prelog, V.; Helmchen, G. Angew. Chem., Int. Ed. Engl. 1982,
21, 567–583.
The authors declare no competing financial interest.
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