Synthesis of a phosphoramidate pro-drug of 6-thio-7-deaza-2′-deoxyguanosine (TDG): A regioselective phosphorylation

Article abstract of DOI:10.1016/S0040-4039(00)02045-1

6-Thio-7-deaza-2′-deoxyguanosine-5′-triphosphate (TDG-TP) is a potent inhibitor of human telomerase. Regioselective synthesis of the 5′-phenyl methoxyalaninyl phosphate pro-drug of 6-thio-7-deaza-2′-deoxyguanosine (TDG) has been achieved in good yields by the reaction of TDG with phenyl methoxyalaninyl phosphochloridate in the presence of N-methylimidazole at -70°C. This pro-drug of TDG is effective in producing measurable levels of TDG-TP in A549 cells.

Full text of DOI:10.1016/S0040-4039(00)02045-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 565–567
Synthesis of a phosphoramidate pro-drug of
6
-thio-7-deaza-2%-deoxyguanosine (TDG): a regioselective
phosphorylation
†
Devinder Kumar, Brian Kanz, Blain M. Mamiya, Jonathan T. Kern and Sean M. Kerwin*
Division of Medicinal Chemistry, College of Pharmacy, Institute for Cellular and Molecular Biology,
University of Texas at Austin, Austin, TX 78712, USA
Received 11 November 2000; accepted 14 November 2000
Abstract—6-Thio-7-deaza-2%-deoxyguanosine-5%-triphosphate (TDG-TP) is a potent inhibitor of human telomerase. Regioselective
synthesis of the 5%-phenyl methoxyalaninyl phosphate pro-drug of 6-thio-7-deaza-2%-deoxyguanosine (TDG) has been achieved in
good yields by the reaction of TDG with phenyl methoxyalaninyl phosphochloridate in the presence of N-methylimidazole at
−
70°C. This pro-drug of TDG is effective in producing measurable levels of TDG-TP in A549 cells. © 2001 Elsevier Science Ltd.
All rights reserved.
6
,7
There has been continued interest in the synthesis and
biological evaluation of nucleoside analogs capable o1f
delivering the corresponding nucleotides inside cells.
Of particular interest are the (aryloxy)phosphoramidate
nucleotide monophosphate pro-drugs pioneered by
TDG (3)
(2-amino-9-(2%-deoxy-b-D-erythro-pento-
furanosyl)-7H-pyrrolo[2,3-d]pyrimidin-6-thione; 6-thio-
7-deaza-2%-deoxyguanosine) was prepared from 3%,5%-
ditoluyl protected intermediate as previously reported.
Phenyl methoxyalaninyl phosphochloridate (4) as a 1:1
diastereoisomeric mixture was prepared as reported by
4
2
2
McGuigan and co-workers. The (aryloxy)phosphora-
midates of AZT (1) and d4T (2) bypass the dependence
of the parent nucleosides on thymidine kinase-mediated
activation, displaying full activity in thymidine kinase-
the reaction of L-alanine methyl ester hydrochloride
with phenylphosphorodichloridate in the presence of
triethylamine in dry dichloromethane. While previous
workers have employed this material after precipitation
2
,3
deficient cells. We have recently shown that 6-thio-7-
deaza-2%-deoxyguanosine-5%-triphosphate (TDG-TP) is
a very potent inhibitor of human telomerase, with
2
of the triethylamine hydrochloride with ether, we have
found that the regioselective coupling described here
proceeds much better when the phenyl methoxyalaninyl
phosphochloridate prepared in this fashion is further
purified by rapid flash silica gel column chromatogra-
phy using dichloromethane as the eluant. The purified
4
an IC50 of 60 nM (Fig. 1). Here we report the regio-
selective synthesis of the 5%-(phenyl methoxyalaninyl)-
phosphate derivative of 6-thio-7-deaza-2%-deoxyguano-
sine (TDG), and the ability of this TDG pro-drug,
when incubated with A549 lung cancer cells, to afford
detectable levels of intracellular TDG-TP.
1
13
31
material was characterized by H, C, P NMR and
HRMS and was stable for weeks when stored under an
8
inert atmosphere in the freezer.
*
Corresponding author. Fax: +1-512-232-2606; e-mail: skerwin@mail.utexas.edu
On leave from Guru Jambheshwar University, Hisar-125001, Haryana, India.
†
0
040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)02045-1

5
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D. Kumar et al. / Tetrahedron Letters 42 (2001) 565–567
5
Fig. 1. Inhibition of human telomerase by TDG-TP. A. PAGE of telomerase reactions employing 5%-biotinylated d(TTAGGG)
3
32
primer, dATP, dTTP, [a- P]dGTP, and crude telomerase extract from HeLa cells in the presence of RNase or increasing
concentrations of TDG-TP. B. Plot of telomerase inhibition data from A.
To a solution of 50 mg of 3 in 10 ml THF and 4 equiv.
of N-methylimidazole (NMI) at −70°C was slowly
added a solution of 1.2 equiv. of 4 in 10 ml THF.
Immediate evaporation of the reaction mixture fol-
lowed by column chromatography of the residue
afforded the phosphoramidate derivative 5 in 60%
yield. The phosphoramidate 5 was isolated as an insep-
arable 2:1 mixture of diastereoisomers due to the phos-
room temperature, followed by the addition of 1 equiv.
of 4; however, this procedure only led to very low (ca.
9%) and variable yields of the desired phosphoramidate
5
.
6
We incubated 2×10 A549 human lung cancer cells with
various concentrations (20–100 mM) of TDG (3) for
4
–24 h to determine the amount of TDG-TP formed
9
phate stereocenter. The direct conversion of 3 to 5 is
intracellularly. After incubation, the cells were rinsed
PBS), trypsinized, collected, and lysed in HClO fol-
noteworthy in that the selective coupling of the 5%-
hydroxyl group proceeds in better yields than for previ-
ously reported 5%-selective couplings involving
(
4
lowed by centrifugation. HLPC analysis (Partisil-10
SAX anion exchange, ammonium phosphate buffer
gradient pH 2.8–3.7, UV detection 344 nm) of the
neutralized supernatant showed no measurable intracel-
lular TDG-TP. In contrast, A549 cells treated with
1
0,11
3%-unprotected pyrimidine nucleosides,
despite the
presence of other potentially nucleophilic sites on the
purine base in the case of 3 (e.g. 6-thio and 2-amino
groups). The clean conversion of 3 to 5 could only be
achieved at −70°C; reactions performed at room tem-
perature or 0°C resulted in the formation complex
mixtures of products including the 3%,5%-bis(phosphor-
amidate) along with small amounts of 5. We also
attempted the preparation of 5 by treating a THF/pyri-
dine solution of 3 with 2 equiv. of tBuMgCl in THF at
2
0–100 mM of 5 showed measurable levels of TDG-TP
after 4–24 h of incubation (Fig. 2).
We are currently investigating the biological effects of
the TDG pro-drug 5 in vitro and in vivo, and will
report on this work in due course.
Fig. 2. HPLC chromatogram of the supernatant from A549 cells incubated with 80 mM 5 for 4 h demonstrating the presence of
TDG-TP (peak at 53.38 min, confirmed by MS and co-elution with authentic material).

D. Kumar et al. / Tetrahedron Letters 42 (2001) 565–567
567
Acknowledgements
MHz, CDCl ): l 8.74, 8.50. CIMS m/z: 278/280 (3:1,
3
+
MH ); CIHRMS m/z calc%d for C H ClO P: 278.0348,
10
14
4
found 278.0344.
9. Compound 5 was isolated as a colorless glass: H NMR
500 MHz, DMSO-d ) [the resonances for the minor
1
This work was supported by a grant from the National
Institutes of Health (CA-67769).
(
6
diastereomer, when resolved, are indicated by an aster-
isk]: l 7.38–7.32 (m, 2H, C H (m)), 7.21–7.13 (m, 3H,
6
5
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(
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2
(
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13
%-H ), 1.21, 1.18* (d X 2, 3H, J=7 Hz, CH ). C NMR
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6
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(
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1
6
1
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3
31
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3
+
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21 27
5
7
1
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yield: H NMR (300 MHz, CDCl ): l 7.37–7.32 (m, 2H),
3
7
1
.25–7.18 (m, 3H), 4.44–4.36 (m, 1H), 4.23–4.10 (m,
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13
J=7.1, 0.8 Hz). C NMR (75.5 MHz, CDCl ): l 171.08–
3
1
5
71.20, 149.66, 129.88, 125.96, 120.53–120.47, 52.75–
31
2.82, 50.32–50.62, 20.47–20.54.
P
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.
.