Conjugation of amino acid O-methyl esters with AZT-5'-O-phosphorothioate and phosphorodithioate

Article abstract of DOI:10.1016/S0040-4039(00)01635-X

Based upon 1,3,2-oxathia(-dithia)phospholane chemistry, 5'-O-derivatisation of AZT with the O-methyl esters of L-phenylalanine and L-tryptophan was performed and the corresponding 5'-aminoacidophosphoramidothioates or phosphoramidodithioates of AZT were obtained in satisfactory yield. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01635-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 9139–9142
Conjugation of amino acid O-methyl esters with
AZT-5%-O-phosphorothioate and phosphorodithioate
Janina Baraniak, Renata Kaczmarek and Wojciech J. Stec*
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies PAS, Sienkiewicza 112,
90-363 Ło´dz´, Poland
Received 20 July 2000; revised 5 September 2000; accepted 20 September 2000
Abstract
Based upon 1,3,2-oxathia(-dithia)phospholane chemistry, 5%-O-derivatisation of AZT with the O-methyl
esters of -phenylalanine and -tryptophan was performed and the corresponding 5%-aminoacidophospho-
L
L
ramidothioates or phosphoramidodithioates of AZT were obtained in satisfactory yield. © 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: AZT; N-phosphorothioylated amino acid; oxathia(-dithia)phospholane ring-opening condensation.
Since the discovery of the therapeutic efficacy of 3%-azido-3%-deoxythymidine (AZT) against
human immunodeficiency virus (HIV),1 extensive research towards AZT-derivatisation enhanc-
ing antiviral activity has been carried out in numerous laboratories.2 It has been reported
recently that 5%-O-conjugation of 3%-azido-3%-deoxythymidine (AZT) with N-phosphorylated
aromatic amino acids provides the corresponding 3%-azido-3%-deoxythymidine-5%-aminoacido-
phosphoramidates (1) which exhibit many-fold higher activity against HIV-1 replication in
PBMCs cells than the parent AZT.3a It has also been shown, that due to their negative charge,
compounds 1 are considerably more water soluble and stable in human blood plasma. As an
amino acid, -tryptophan, was found to be the best choice: in vitro, compound 1b was 8-fold
L
more active when compared to free AZT, without any symptoms of toxicity.3b
* Corresponding author. Tel: +4842 681 97 44; fax: +4842 681 54 83; e-mail: wjstec@bio.cbmm.lodz.pl
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01635-X

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In these studies, all compounds of type 1 described were obtained using phosphoramidite
methodology.4 Their low toxicity and enhanced anti-HIV activity prompted us to search for
more efficient synthetic routes providing compounds of type 1 and also their phosphorothio- or
phosphorodithioate analogues. To reach this goal we adapted methodology that is based upon
the oxathiaphospholane chemistry developed in this laboratory.5
Although that approach had been designed for the stereocontrolled synthesis of oligo(nu-
cleoside phosphorothioate)s, it has been independently applied to the synthesis of N3%“P5%-
dinucleotides6 and O-alkyl-N-alkyl phosphoramidothioates.7 The synthetic strategy presented in
this communication consisted of two steps: (i) the simple derivatisation of the corresponding
amino acid-O-methyl esters (2) either with 2-chloro-1,3,2-oxathiaphospholane (3) or 2-chloro-
1,3,2-dithiaphospholane (4) (Scheme 1) and (ii) the reaction of resulting N-phosphorothioylated
amino acid esters 5 or 6 with AZT (Scheme 2).
Scheme 1.
Scheme 2.
Accordingly the O-methyl esters of
L
-phenylalanine (2a) or -tryptophan (2b) were treated
L
with an equimolar amount of 2-chloro-1,3,2-oxathia(dithia-)phospholane (3 or 4) in pyridine
solution in the presence of elemental sulphur. These reactions were nearly quantitative and
resulted in the formation of 2-N-(carbomethoxyaminoacido)-[2-thiono-1,3,2-oxathia(1,3,2-
dithia)phospholanes] (5 or 6). Compounds 5–6 were isolated from the reaction mixture by silica

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1
gel column chromatography† and characterised by H NMR, 31P NMR and FAB-MS analysis
(Table 1).‡ Condensation of 5 or 6 with an equimolar amount of AZT in the presence of DBU
occurred smoothly providing compounds 7–8 in high yield.§
Table 1
Physicochemical characteristics of compounds 1 and 5–8
Comp. no.
31P NMR [ppm] Chemical shifts (H3PO4)
FAB-MS (M−1) m/z
Yield [%]
5a
5b
6a
6b
7a
7b
8a
8b
1a
1b
96.50, 95.59a
96.54, 95.52b
101.02a
316
355
332
371
523
562
539
578
507
546
89
75
84
72
87
92
95
93
80
82
101.04a
58.51, 58.24b
58.50, 58.43a
103.21a
103.48a
7.01c
7.20c
a NMR spectra were determined in CDCl3.
b NMR spectra were determined in CD3CN.
c NMR spectra were determined in CD3OD.
Due to the asymmetry of the phosphorus atom, compounds 7 consisted of diastereomeric
mixtures and can be separated into diastereomerically pure species by means of HPLC.
Compounds 8 are P-achiral. Their physicochemical characteristics are also presented in Table 1.
Moreover, compounds 7 dissolved in a solution of OXONE® (potassium peroxymonosulfate,
buffered solution, pH 6.5) underwent conversion into compounds 1, described earlier, in high
yield. It should be emphasised that the present methodology allowed us to obtain not only the
known compounds 1, but also a new class of phosphorothio- and phosphorodithio-analogues
(compounds 7 and 8). Since the chromatographic resolution of diastereomeric mixtures of
compounds 7 is feasible, antiviral activity of the pure species and compounds 8 is under
investigation. The synthetic strategy presented in this paper, in comparison with the previously
described one based upon phosphoramidite methodology, is more efficient and does not require
the use of an excess of amino acid.
† As an eluent chloroform:hexane (7:3) was used.
‡ 1H NMR data were in accordance with literature2 and are not included in this communication.
§ The synthesis of compound 7b: compound 5b (0.178 g, 0.5 mmol) was dissolved in dry acetonitrile (2 ml) and to
this solution was added a solution of 3%-azido-3%-deoxythymidine (0.134 g, 0.5 mmol) and DBU (0.083 g, 0.55 mmol)
in dry acetonitrile (3 ml). After 3 h the reaction mixture was concentrated under reduced pressure, and the crude
product was purified by silica gel column chromatography (0“15% methanol in chloroform) to provide 7b in 92%
yield. Separation into diastereomerically pure 7 was performed by HPLC.

9142
Acknowledgements
This project was financially assisted by the State Committee for Scientific Research (KBN),
grant no. 4 P05F 006 17 (to W.J.S.).
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