Chemical and enzymatic synthesis of 4′-thio-β-D- arabinofuranosylcytosine monophosphate and triphosphate

Article abstract of DOI:10.1081/NCN-200061797

N⁴-Acetyl-1-(2, 3-di-O-acetyl-4-thio-β-D-arabinofuranosyl) cytosine (2) was synthesized in three steps from 1-(4-thio-β-D- arabinofuranosyl)cytosine (1). The reaction of this partially blocked 4′-thio-ara-C derivative 2 with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4- one gave the 5′-phosphitylate derivative 3, which on reaction with pyrophosphate gave the 5′-nucleosidylcyclotriphosphite 4. Product 4 was then oxidized with iodine/pyridine/water and deblocked with concentrated ammonium hydroxide to provide the desired 4′-thio-ara-C-5′- triphosphate 5. This triphosphate 5 was converted to 4-thio-ara-C-5′- monophosphate 6 by treatment with snake venom phosphodiesterase I. The details of the synthesis, purification, and characterization of both nucleotides are described. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200061797

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CHEMICAL AND ENZYMATIC SYNTHESIS OF 4′-THIO-β-D-
ARABINOFURANOSYLCYTOSINE MONOPHOSPHATE AND
TRIPHOSPHATE
A. S. Fowler a , K. N. Tiwari a , S. R. Campbell a & J. A. Secrist a
a Southern Research Institute , Birmingham, Alabama, USA
Published online: 15 Nov 2011.
To cite this article: A. S. Fowler , K. N. Tiwari , S. R. Campbell & J. A. Secrist (2005) CHEMICAL AND ENZYMATIC SYNTHESIS
OF 4′-THIO-β-D-ARABINOFURANOSYLCYTOSINE MONOPHOSPHATE AND TRIPHOSPHATE, Nucleosides, Nucleotides and Nucleic
Acids, 24:5-7, 533-537, DOI: 10.1081/NCN-200061797
To link to this article: http://dx.doi.org/10.1081/NCN-200061797
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):533–537, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200061797
CHEMICAL AND ENZYMATIC SYNTHESIS OF
4’-THIO-b-D-ARABINOFURANOSYLCYTOSINE MONOPHOSPHATE
AND TRIPHOSPHATE
5
A. S. Fowler, K. N. Tiwari, S. R. Campbell, and J. A. Secrist III
Southern
Research Institute, Birmingham, Alabama, USA
N4-Acetyl-1-(2, 3-di-O-acetyl-4-thio-b-D-arabinofuranosyl)cytosine (2) was synthesized in three
5
steps from 1-(4-thio-b-D-arabinofuranosyl)cytosine (1). The reaction of this partially blocked 4’-thio-
ara-C derivative 2 with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one gave the 5’-phosphitylate
derivative 3, which on reaction with pyrophosphate gave the 5’-nucleosidylcyclotriphosphite 4. Product 4
was then oxidized with iodine/pyridine/water and deblocked with concentrated ammonium hydroxide to
provide the desired 4’-thio-ara-C-5’-triphosphate 5. This triphosphate 5 was converted to 4’-thio-ara-
C -5’-monophosphate 6 by treatment with snake venom phosphodiesterase I. The details of the synthesis,
purification, and characterization of both nucleotides are described.
INTRODUCTION
In an ongoing search for new nucleoside analogues with selective anticancer
activity, a series of 4’-thionucleosides has been synthesized in our laboratories.[1]
One of these compounds, 4’-thio-ara-C, which was originally synthesized by
Whistler et al.[2,3] in the early seventies, was subsequently resynthesized by us in a
more efficient manner.[4] This nucleoside has shown very good anticancer activity
in a broad spectrum of human tumor xenografts including colon, non-small cell
lung, prostate, renal, breast, and pancreatic cancers.[4] This compound is currently
being evaluated in phase I clinical trials. In order for us to study its mechanism of
action and other pharmacological properties, we needed to synthesize the
monophosphate and the triphosphate of 4’-thio-ara-C. Because of the inherent
This investigation was supported by the National Cancer Institute, National Institute of Health (POl-
CA34200). The authors are indebted to Dr. James M. Riordan, Mr. Mark Richardson, and Ms. Joan Bearden for
spectral data and to Ms. Stephanie Brannon for help in preparation of this poster.
Address correspondence to A. S. Fowler, Southern Research Institute, Birmingham, AL, USA.
533
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A. S. Fowler et al.

A. S. Fowler et al.
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SCHEME 1
difficulty associated with the stability of dichloridates of this class of compounds,[5]
we were unable to synthesize the monophosphate or the triphosphate of 4’-thio-ara-
C by traditional methods.[6,7] Previously, Ludwig and Eckstein reported a new
method for the synthesis of 5’-O-(1-thiotriphosphates) and 5’-triphosphates using 2-
chloro-4H-1,3,2-benzodioxaphosphorin-4-one.[8] The use of this multifunctional
reagent was found to give good yields of 5’-O-(1-thiotriphosphates) and 5’-
triphosphates. Since this reagent was commercially available, we decided to use
this approach in the synthesis of 4’-thionucleotides. The details of these syntheses
are reported below.
FIGURE 1 HPLC Assays: (A) purified 4’-S-araCMP; (B) purified 4’-S-araCTP; (C) crude 4’-S-araCTP. Elution
conditions: column, Waters mBondapak C18, 10 mm; eluent A, 0.01 M NH4H2PO4 and .005 M PIC A
(tetrabutylammonium phosphate) ion-pairing reagent (pH 5.0); eluent B, MeOH; elution, 20-min linear gradient
from 10 to 40% eluent B (chromatogram A) or 20–50% eluent B (chromatograms B & C) at a flow rate of 1 mL/min.

536
A. S. Fowler et al.
CHEMISTRY
The synthesis of N4-acetyl-2’, 3’-di-O-acetyl-4’-thio-ara-C 2 was carried out from
4’-thio-ara-C 1 in a one-flask sequence of blocking 5’ with dimethoxytrityl followed
by acetylation with acetic anhydride in pyridine and subsequent detritylation with
trifluoroacetic acid. After silica gel column chromatography of the evaporated
crude mixture, a portion of this appropriately blocked nucleoside 2 (0.1 mmol) was
dissolved in anhydrous pyridine (0.1 mL) and anhydrous dioxane (0.3 mL). A
freshly prepared 1 M solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in
anhydrous dioxane (0.1 mL) was then added to the well stirred solution of 2. After
10 min, a well-mixed solution of 0.5 M bis(tri-n-butylammonium)pyrophosphate in
anhydrous DMF (0.3 mL) and tri-n-butylamine (0.1 mL) was quickly injected, and
the reaction mixture was stirred at room temperature for 10 min. A solution of 1%
iodine in pyridine/water (98/2, v/v) (2 mL) was then added, and the mixture was
stirred for 15 min. Excess iodine was destroyed by adding a few drops of a 5%
aqueous solution of sodium bisulfite, and the reaction solution was evaporated to
dryness. The resulting residue was dissolved in water (10 mL) and allowed to stand
at room temperature for 30 min before concentrated ammonium hydroxide (20 mL)
was added. After 3 h, the solution was evaporated to dryness, and the residue was
dissolved in water. This solution was applied to an ion-exchange column
(16 cm  2 cm i.d.) containing 5 grams of DEAE Sephadex A-25 in the
bicarbonate form. The column was eluted with a linear gradient of 0.01–1.0 M
triethylammonium bicarbonate buffer (TEAB) at 4°C with UV monitoring at
254 nm. Product containing fractions were pooled, frozen, and lyophilized to give 5
(41 mg) as the triethylammonium salt hydrate. A portion of 5 (9 mg) was treated
with snake venom phosphodiesterase I (22 units) for 46 h at room temperature. The
resulting monophosphate 6 was purified as for 5 except using 750 mg of DEAE
Sephadex (6 cm  1 cm i.d. column) and a linear gradient of 0.01–0.35 M TEAB.
Monophosphate 6 (4 mg) was also isolated as the triethylammonium salt hydrate.
The structures and purities of nucleotides 5 and 6 were verified by HRMS (ESI-
1
TOF), H NMR, 31P NMR, and HPLC.
REFERENCES
1. Secrist, J.A., III; Parker, W.B.; Tiwari, K.N.; Messini, L.; Shaddix, S.C.; Rose, L.M.; Bennett, L.L., Jr.;
Montgomery, J.A. The synthesis and biological activity of certain 4’-thionucleosides. Nucleosides Nucleotides
1995, 14, 675–686.
2. Whistler, R.L.; Doner, L.W.; Nayak, U.G. 4-Thio-D-arabinofuranosylpyrimidine nucleosides. J. Org. Chem.
1971, 36, 108–110.
3. Ototani, N.; Whistler, R.L. Preparation and antitumor activity of 4’-thio analogs of 2,2’-anhydro-1-b-D-
arabinofuranosylcytosine. J. Med. Chem. 1974, 17, 535–537.
4. Tiwari, K.N.; Shortnacy-Fowler, A.T.; Cappellacci, L.; Parker, W.B.; Waud, W.R.; Montgomery, J.A.; Secrist,
J.A., III. Synthesis of 4’-thio-b-D-arabinofuranosylcytosine (4’-thio-Ara-C) and comparison of its anticancer
activity with that of Ara-C. Nucleosides Nucleotides Nucleic Acids 1999, 19, 329–340.
5. Hancox, E.; Walker, R.T. Some reactions of 4’-thionucleosides and their sulfones. Nucleosides Nucleotides
1996, 15, 135–148.

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7. Ludwig, J. A new route to nucleoside 5’-triphosphates. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16,
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