Synthesis of nucleoside 3′,5′-cyclic boranophosphorothioate, a new type of cyclic nucleotide

Article abstract of DOI:10.1039/b207350a

The first examples of a borane-containing doubly P-modified chiral cyclic nucleoside monophosphate (cNMP), e.g., thymidine and 5-fluoro-2′-deoxyuridine 3′,5′-cyclic boranophosphorothioates, have been synthesized; these cNMP analogues with increased lipoph

Full text of DOI:10.1039/b207350a

Synthesis of nucleoside 3A,5A-cyclic boranophosphorothioate, a new type
of cyclic nucleotide†
Ping Li and Barbara Ramsay Shaw*
Paul M.Gross Chemical Laboratory, Department of Chemistry, Duke University, Durham, North Carolina,
27708-0346, USA. E-mail: brs@chem.duke.edu; Fax: 1-919-6601618; Tel: 1-919-6601551
Received (in Cambridge, UK) 26th July 2002, Accepted 3rd August 2002
First published as an Advance Article on the web 4th November 2002
The first examples of a borane-containing doubly P-
modified chiral cyclic nucleoside monophosphate (cNMP),
e.g., thymidine and 5-fluoro-2A-deoxyuridine 3A,5A-cyclic
boranophosphorothioates, have been synthesized; these
cNMP analogues with increased lipophilicity could be
potential anticancer prodrugs and useful probes for mecha-
nistic studies.
example, treatment of nucleoside 5A-monophosphate with
dicyclohexylcarbodiimide (DCC) provides a convenient ap-
proach to synthesize normal cNMP.8a–c However, in our case,
this method failed to give the desired compound 5a from
thymidine 5A- or 3A-boranophosphorothioate (7 or 9)4c (Scheme
1). Reaction of thymidine 5A-boranophosphorothioate P-diiso-
propylamidate 6 with two equiv. of 5-ethylthio-1H-tetrazole9
was also unsuccessful. A similar intramolecular reaction of
thymidine 3A-boranophosphorothioate P-diisopropylamidate
810 gave compound 5a, but in less than 10% yield.11 The steric
hinderance of a primary and secondary alcohol may account for
the reactivity difference between compounds 6 and 8.12
Nucleoside 3A,5A-cyclic monophosphates (cNMPs) 1 (Fig. 1),
such as cAMP and cGMP, are important regulators of
biochemical processes.1 For example, cAMP acts as a mediator
of hormone action and modulator of enzymatic activity. To
better understand these processes, various modified cNMPs
have been prepared to probe the stereochemical and mechanistic
aspects of phosphoryl- and nucleotidyl transfer reactions and
their inhibition.2 However, to the best of our knowledge, only
the 3A,5A-cyclic nucleoside phosphorothioate (cNMPS) 2 and
phosphorodithioate (cNMPS2) 3 (Fig. 1) have been well studied
Fig. 1 cNMP, its analogues and their abbreviations.
as P-modified analogues of the cNMPs.2a–c,3 More analogues
could be useful in order to fully understand processes involving
cNMPs.2b By structurally combining the phosphorothioate and
boranophosphate, our group recently reported the synthesis of
dithymidine boranophosphorothioate and thymidine 5A-[a-P-
borano, a-P-thio]triphosphate,4 wherein the two nonbridging
phosphoryl oxygen atoms were replaced with a sulfur atom and
borane group (BH3). The excellent lipophilicity for dithymidine
boranophosphorothioate relative to natural dithymidine phos-
phate makes this modification a convenient basis for creation of
possible anticancer prodrugs.4a For example, 5-fluoro-2A-
deoxyuridine monophosphate (FdUMP) is a well-known cancer
inhibitor that inhibits thymidylate synthase, a target enzyme for
the control of cell growth. However, the high polarity of
FdUMP makes it difficult to penetrate the cell membrane.
Several prodrugs based on the delivery of FdUMP have been
synthesized and studied in vitro, but few appear to be efficient.5
By virtue of replacing two non-bridging phosphoryl oxygens in
cNMP with a more lipophilic sulfur atom and borane group, we
propose to synthesize the first class of doubly-modified
nucleoside 3A,5A-cyclic boranophosphorothioate (cNMPBS)
compounds 5. Based on the studies of other cyclic monop-
hosphate analogues6 and boranophosphates,7 the cNMPBS may
more readily penetrate into cells and be hydrolyzed to the
corresponding nucleoside 5A-monophosphate.
Scheme 1 Possible approaches for synthesizing cTMPBS 5a.
Since the direct cyclization of boranophosphorothioates was
unsuitable, we decided to explore the possibility of introducing
the modification into a nucleoside 3A,5A-cyclophosphite. We
developed the procedure here for the one-pot synthesis of
cNMPBS 5 (Scheme 2) based on a modification of the methods
reported by our group for the synthesis of 3A,5A-cyclic
boranomonophosphate (cNMPB) 44,13a and by Eckstein for the
synthesis of dithiotriphosphates.13b One crucial step in the
synthesis of cNMPBS 5 is the formation of phosphoramidite 11
by the reaction of nucleoside 10 with a bis-functional phosphor-
amide, such as hexamethylphosphoramide (HMPA),14 bis(dii-
sopropylamino)chlorophosphine, and diisopropylphosphorami-
dous dichloride. By using HMPA with thorough deoxygenation
of the solvent (acetonitrile) and slowly increasing the reaction
temperature to 65 °C, we were able to obtain a relatively high
yield ( > 95%) of compound 11. Phosphoramidite 11 was then
transformed to phosphite triester 12 by reaction with 4-ni-
trophenol in the presence of 5-ethylthio-1H-tetrazole. Upon in
situ boronation, compound 1315 was formed and exhibited a
broad 31P NMR peak centered at 107 ppm, which is character-
Typically, nucleoside 3A,5A-cyclic monophosphates can be
prepared from the nucleoside monophosphates or their deriva-
tives via intramolecular dehydration and/or cyclization.8 For
† Electronic supplementary information (ESI) available: Experimental
details and spectral data. See http://www.rsc.org/suppdata/cc/b2/b207350a/
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CHEM. COMMUN., 2002, 2890–2891
This journal is © The Royal Society of Chemistry 2002

We thank Drs Zinaida Sergueeva, Dmitri Sergueev and
Mikhail Dobrikov for helpful discussion and critical comments.
This work was supported by NIH grant RO1-AI-52061 to B. R.
S.
Notes and references
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Scheme 2 Synthesis of cNMPBS 5.
istic of boranophosphite.16 Of several boronating reagents tried,
borane–dimethyl sulfide gave the best yield after a 4 h
treatment. Introduction of a sulfur atom at this stage is another
key point in our synthesis. After evaporation of the solvent,
boranophosphite 13 could be directly and quantitatively
converted to cNMPBS 5 via Li2S substitution in DMF/dioxane
without the addition of 18-crown-6 used in the synthesis of
thymidine 5A-[a-P-borano, a-P-thio]triphosphate.4a
Nucleoside 3A,5A-cyclic boranophosphorothioate 5a and 5b
are the only known P-disubstituted chiral cyclic monop-
hosphate with a negative charge. The borano-, thio-disubstitu-
tion will change the electron distribution over the phosphate, as
well as the interactions between cyclic monophosphate and
metal ions.7b,17 These properties could make cNMPBS 5 useful
in elucidating the stereochemical course and role of metal ions
in phosphoryl and nucleotidyl transfer reactions.
In partitioning experiments, the partition coefficients, de-
fined as the ratio of concentration in octan-1-ol to that in water,
were 1.7 3 1022, 2.2 3 1022, 5.1 3 1025 and 7.6 3 1025 for
cTMPBS 5a, cFdUMPBS 5b, cTMP and cFdUMP, re-
spectively. Thus, 3A,5A-cyclic boranophosphorothioates
cTMPBS 5a and cFdUMPBS 5b were 340- and 290-fold more
lipophilic than parental cTMP and cFdUMP, accordingly. A
good lipophilicity in combination with the negative charge and
good solubility in water could make 3A,5A-cyclic nucleoside
boranophosphorothioates promising as prodrug reagents.
In summary, we have synthesized a new type of doubly P-
modified chiral cyclic NMP, e.g., cNMPBS 5a,b,† by a one-pot
reaction in 65–70% overall yield after isolation. The two P-
diastereoisomers of cNMPBS were separated by RP-HPLC.
The increased lipophilicity and the putatively different substrate
properties imparted by the borane group7 make the cNMPBS a
potential anticancer prodrug and a new probe for mechanistic
studies on cNMP.
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=
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