Synthesis and resolution of dinucleotide(TpAZT) phosphoramidates

Article abstract of DOI:10.1081/SCC-120021847

Dinucleotide (TpAZT) phosphoramidates were synthesized through Atherton-Todd reaction of dinucleoside H-phosphonates and amino acid methyl esters, and their diastereomers (R_p and S_pu) were separated by crystallization. It was showed that the cheap methyl esters of natural alanine and phenylalanine could act as new chiral auxiliaries for large-scale synthesis of dinucleotide analogs.

Full text of DOI:10.1081/SCC-120021847

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Synthesis and Resolution of Dinucleotide(TpAZT)
Phosphoramidates
Changxue Lin , Hua Fu , Guangzhong Tu & Yufen Zhao
To cite this article: Changxue Lin , Hua Fu , Guangzhong Tu & Yufen Zhao (2003) Synthesis
and Resolution of Dinucleotide(TpAZT) Phosphoramidates, Synthetic Communications, 33:14,
2553-2562, DOI: 10.1081/SCC-120021847
To link to this article: http://dx.doi.org/10.1081/SCC-120021847
Published online: 20 Aug 2006.
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SYNTHETIC COMMUNICATIONSꢀ
Vol. 33, No. 14, pp. 2553–2562, 2003
Synthesis and Resolution of
Dinucleotide(TpAZT) Phosphoramidates
2
Changxue Lin,1 Hua Fu,1, Guangzhong Tu,
*
and Yufen Zhao1,
*
1The Key Laboratory of Bioorganic Phosphorus
Chemistry, Ministry of Education, Department of
Chemistry, School of Life Sciences and Engineering,
Tsinghua University, Beijing, P.R. China
2Beijing Institute of Microchemistry, Beijing, P.R. China
ABSTRACT
Dinucleotide (TpAZT) phosphoramidates were synthesized through
Atherton-Todd reaction of dinucleoside H-phosphonates and amino
acid methyl esters, and their diastereomers (Rp and Sp) were sepa-
rated by crystallization. It was showed that the cheap methyl esters of
natural alanine and phenylalanine could act as new chiral auxiliaries
for large-scale synthesis of dinucleotide analogs.
*Correspondence: Hua Fu and Yufen Zhao, The Key Laboratory of Bioorganic
Phosphorus Chemistry, Ministry of Education, Department of Chemistry,
School of Life Sciences and Engineering, Tsinghua University, Tsinghua East
Road, Beijing 100084, P.R. China; E-mail: fuhua@tsinghua.edu.cn and
yfzhao@tsinghua.edu.cn.
2553
DOI: 10.1081/SCC-120021847
Copyright & 2003 by Marcel Dekker, Inc.
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com

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2554
Lin et al.
Key Words: Dinucleotide; Phosphoramidate; anti-HIV; Chiral
separation.
INTRODUCTION
Despite the recent introduction of HIV protease and integratase
inhibitors, reverse transcriptase represents an attractive target for the
chemotherapy of human immunodeficiency virus (HIV).[1–3] Nucleoside
analogues are widely used as antiviral agents in the treatments of AIDS
and the AIDS related complex. 30-Azido-50-deoxythymidine (AZT),
initially tested as anticancer agent, is an inhibitor of HIV-1 reverse
transcriptase(RT) and the first drug clinically used for the treatment of
AIDS.[4,5]
However, it has been proved that AZT must be phosphorylated
intracellularly to their active triphosphate form before acting as compet-
itive inhibitor or alternate substrate (chain terminators) of HIV RT.[6]
Because the cellular kinases involved in activiting the nucleoside prodrugs
are usually specific,[7] it is thought the replacement of ddNs with natural
nucleoside, such as thymidine, could improve the rate of phosphorylation
and inhibit the HIV-RT. Dinucleoside phosphate derivatives have
attracted great attentions as anti-HIV drugs. Various homo- and heter-
dinucleoside, such as AZT-P-AZT, AZT-P-ddI, AZT-P-ddA, have been
synthesized and tested for HIV-infected MT-2 cells.[8] It was found that
dinucleside analogues showed enhanced anti-HIV potency relative to
monomers. In addition, AZT-P-ddI was 10 times less toxic than AZT
to human granulocytemacrophage progenitor cells.[9]
By far, most of the dinucleoside prodrugs were constructed by two
unnatural nucleosides, i.e., 20,30-dideoxynucleside (ddNs). Hakimelahi
et al. also found that dinucleotide phosphoramidates conjugated with
methyl ester of alanine were completely resistant to snake venom and
spleen enzyme, and these phosphoramidates showed superior bioavail-
ability and profound antiviral activity.[10] On the other hand, a pair of
diastereomers are usually formed for the synthesis of dinucleotide ana-
logs due to phosphorus chiral center, however, these diastereomers show
great difference in biological activity and cellular toxity for specificity of
enzymes. For example, Meier et al. observed the difference of anitiviral
activity between two diastereomers of cyclosal pronuclesides is 3–80
folds.[11] Dinucleotide phosphonamidates bearing with a D-amino acid
ester moiety exhibited lower activity and less cellular toxity than the
corresponding phosphoramidates bearing with a L-amino acid ester
moiety.[10]

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Dinucleotide(TpAZT) Phosphoramidates
2555
In this article, a series of dinucleotide phosphoramidates were
synthesized by Atherton-Todd reaction, the single diastereomers
containing alanine and phenylalanine methyl esters were separated by
crystallization.
RESULTS AND DISCUSSION
Dinucleotide phosphoramidates were prepared as shown in Sch. 1.
Reaction of 50-DMTr-thymidine and diphenyl phosphite in dry pyridine
at room temperature under nitrogen atmosphere and following hydro-
lysis in triethylamine and water led to 2, dinucleoside H-phosphate 3 was
obtained after coupling of 2 and AZT by pivaloyl chloride. Atherton-
Todd reaction of 3 and amino acid methyl ester in CCl4/NEt3/H2O/
CH3CN solution at room temperature gave product 4. Product 5 was
obtained as white foam after 50-deprotection of 4 in formic acid and
purification on silica gel column chromatography.
Compounds 5 were obtained as mixtures of diastereoisomers as it is
1
difficult to isolate and their structures were confirmed by 31P, H, and
13C NMR. When these compounds containing methyl ester of alanine or
phenylalanine were dissolved in methanol and kept in refrigerator (À5ꢀC)
Scheme 1. Synthetic route of dinucleotide phosphoramidates.

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2556
Lin et al.
Figure 1. 31P NMR spectra of 5b (a), the mother solution (b), and the precipitate
redissolved in methanol (c).
for about one week, these diastereoisomers can be completely separated
by crystallization. For example, Fig. 1 shows 31P NMR spectra of dia-
stereoisomers 5b, the mother solution and the precipitate redissolved in
methanol. 5b in methanol shows two peaks at 31P NMR 9.23 and
9.04 ppm, the precipitated white solid was filtered and redissolved in
methanol, its 31P NMR showed a single peak at 9.23 ppm, the remained
mother solution exhibited another single peak at 9.04 ppm. 1H and
13C NMR spectroscopies also confirmed each structures of separated
diastereomers. In addition dinucleotide phosphoramidates containing
phenylalanine methyl ester showed the same results.
It is worthwhile noting that the above results only occurred for
dinucleotide phosphoramidate containing methyl esters of alanine and
phenylalanine. For dinucleotide phosphoramidates containing glycine
methyl ester, structural difference between Rp and Sp diastereomers is
minor because of absence of chiral carbon in glycine, it is difficult to
isolate diastereomers by crystallization. For dinucleotide phosphorami-
dates containing valine, leucine and iso-leucine methyl esters, they could
not be precipitated from the solution because of highly hydrophobic side
chains of these amino acids. So methyl esters of alanine and phenyl-
alanine can be better chiral auxiliaries for separation of the diastereo-
meric dinucleotide phosphoramidates (RP and SP).
In conclusion, we synthesized some dinucleotide (TpAZT) phosphor-
amidates. The introduction of alanine or phenylalanine methyl etser
improved structural difference of diastereomers (Rp and Sp) that made
a single diastereomer firstly precipitate from solution, and another one
remained in mother solution. The attractive and cheap method could be
used for chiral separation of large-scale PS-dinucleotides. Activity for

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Dinucleotide(TpAZT) Phosphoramidates
2557
separated diastereomers (Rp and Sp) of dinucleotide phosphoramidates
containing methyl esters of alanine and phenylalanine is being tested,
chiral separation of PS-dinucleotide phosphoramidates is in progress.
EXPERIMENTAL
General Procedure
All reactions were monitored by TLC on silica gel GF254, Column
chromatography was performed using silica gel 230–400 mesh. Pyridine
1
were dried over CaH2 by reflux 4ꢁ5 h. H NMR and 13C NMR spectra
were recorded (internal standard tetramethylsilane) on Bruker AM 500
type spectrometer using CD3 OD as the solvent. 31P NMR spectra were
1
taken on Bruker AC 200 spectrometer at 81 MHz under H decoupled
conditions. 31P NMR chemical shift are reported in ppm downfield (þ) or
upfield (À) from external 85% H3PO4 as reference. Mass spectra were
obtained using a Bruker Esquire ion-trap mass spectrometer in positive
ion mode.
Synthesis of 50-dimethoxytritylthymidine 1. The starting material
DMTrT was prepared according to the published procedure.[15]
Synthesis of 50-dimethoxytritylthymidine-30-H-phosphonate 2. Accord-
ing to the Lit.[16] 50-Dimethoxytritylthymidine-30-H-phosphonate was
synthesized by adding 50-dimethoxytritylthymidine to the solution of
diphenyl phosphite in dry pyridine at room temperature, and reacted
for 20 min. Ten milliliters of triethylamine and water (1:1) was added
to the reaction solution, the reaction lasted another 20 min. After
evaporation of the solvent, 2 was obtained as brittle yellow foam after
column chromatography. Yield: 84%.
Synthesis of O-(50-dimethoxytrityl-20-deoxythmidin-30-yl)-O0-(30-azido-
20-deoxythymidin-50-yl)H-phosphonate 3. Zero point eight gram
(1.1 mmol) 50-O-dimethoxytritylthymidine-30-phosphonate 2 and 0.267 g
(1 mmol) AZT were dissolved in anhydrous pyridine and coevaporated
twice. The residue was then dissolved in 10 mLanhydrous pyridine,
406 mLPivCl (3.06 mmol) was added dropwise, the reaction mixture
was stired at room temperature for 10 min; after addition of a few
drops of water, the solvent was removed under reduced pressure. The
residue was dissolved in 25 mLEtOAc and washed with saturated NaCl
solution; the organic phase was separated and dried with anhydrous
Na2SO4. The crude product was purified with column chromatography
(CHCl3/MeOH 40:1). After concentration, the product was obtained as

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2558
Lin et al.
white foam. Yield: 72.7%. 31P NMR (methanol): ꢀ 8.88, 8.04 ppm, JP–H
721 Hz (mixture of diastereoisomers).
Synthesis of O-(20-deoxythmidin-30-yl) -O0-(30-azido-20- deoxythymi-
din-50-yl)N-amino acid methyl ester phosphoramidate 5. Two millimolars
compound 3 in 2 mLCH 3CN was added dropwise to 0.22 mmol L-amino
acid methyl ester hydrochloride in solution (100 mL H2O, 70 mLNEt ,
3
100 mLCCl 4, and 5 mLCH CN) and stirred at room temperature until 3
3
disappeared (31P NMR determination). The crude product 4 was
obtained after the solvents were removed by rotary evaporation, and
then treated with HCOOH and CH2Cl2 until the deprotection wad
completed (TLC dertermination). The solution was neutralized with a
few drops of saturated NaHCO3, and then concentrated under reduced
pressure. The product 5 was purified by column chromatography
(CHCl3:MeOH from 40:1 to 20:1).
Spectral Data for Compounds 5a–e
T-p-AZT-GlyOCH3 5a. Yield: 53.4%. 31P NMR (CD3OD, 81 MHz):
1
ꢀ 10.55œ10.35. H NMR (CD3OD, 500 MHz): ꢀ 7.82, 7.80 (d, 1H, H-6),
7.55–7.54 (d, 1H, H-6), 6.31–6.28 (m, 1H, H-10), 6.17–6.14 (m, 1H, H-10),
5.14–5.11 (m, 1H, H-30), 4.46–4.45 (m, 1H, H-30), 4.33–4.18 (m, 4H,
2 ꢂ H-50), 3.81, 3.79 (d, 2H, CH2), 3.77, 3.74 (d, 2H, 2 ꢂ H-40), 3.71 (d,
3H, OCH3), 2.53–2.32 (m, 4H, 2 ꢂ H-20), 1.89–1.87 (dd, 6H, 2 ꢂ C5-
CH3). 13C NMR (CD3OD, 125 MHz): ꢀ 173.28 (COOMe), 166.30
(2 ꢂ C-4), 152.35, 150.18 (2 ꢂ C-2), 137.88 (2 ꢂ C-6), 11.97œ111.85,
111.81 (2 ꢂ C-5), 87.39, 87.36, 87.22, 87.17, 86.58, 86.50, 83.74, 83.67
(2 ꢂ C-40, 2 ꢂ C-10), 79.24–78.98 (q, C-30), 67.15, 66.79 (C-30), 62.67–
61.60 (q, 2 ꢂ C-50), 52.68 (CH2), 51.35 (OMe), 39.78, 37.65, 39.62
(2 ꢂ C-20), 12.55, 12.46 (2 ꢂ C5-CH3). ESI-MS [M þ H]þ m/z: 643,
[M þ Na]þ m/z: 665.
T-p-AZT-AlaOCH3 5b. Yield: 55.8%. 31P NMR (CD3OD, 81 MHz):
1
ꢀ 9.03, 9.24. H NMR (CD3OD, 500 MHz): ꢀ 7.83, 7.80 (d, 1H, H-6),
7.55, 7.52 (d, 1H, H-6), 6.31–6.29 (m, 1H, H-10), 6.16–6.13 (m, 1H, H-
10), 5.16–5.02 (m, 1H, H-30), 4.50–4.41 (m, 1H, H-30), 4.32–4.04 (m, 4H,
2 ꢂ H-50), 3.95–3.88 (m, 1H, CH), 3.80–3.78 (2s, 2H, 2 ꢂ H-40), 3.72, 3.71
(d, 3H, OCH3), 2.56–2.30 (m, 4H, 2 ꢂ H-20), 1.90–1.87 (m, 6H, 2 ꢂ C5-
CH3), 1.41, 1.39, 1.37 (t, 3H, CH3). 13C NMR (CD3OD, 125 MHz): ꢀ
175.73 (COOMe), 166.29 (2 ꢂ C-4), 152.36, 150.17 (2 ꢂ C-2), 137.99,
137.92, 137.89 (137.82) (2 ꢂ C-6), 112.04, 111.90, 111.80 (2 ꢂ C-5),
87.36, 87.25, 87.20, 86.62, 86.07, 83.61 (2 ꢂ C-40, 2 ꢂ C-10), 79.46, 79.36,
79.02 (C-30), 67.04, 66.64, 66.60 (C-30), 62.71, 62.57, 61.75, 61.48

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Dinucleotide(TpAZT) Phosphoramidates
2559
(2 ꢂ C-50), 52.85 (CH), 51.35 (OMe), 39.74, 37.61 (2 ꢂ C-20), 20.42, 20.36
(CH3), 12.56, 12.47 (2 ꢂ C5-CH3). ESI-MS [M þ H]þ m/z: 657,
[M þ Na]þ m/z: 679.
T-p-AZT-PheOCH3 5c. Yield: 57.2%. 31P NMR (CD3OD, 81 MHz):
1
ꢀ 9.12, 8.65. H NMR (CD3OD, 500 MHz): ꢀ 7.79, 7.77 (d, 1H, H-6),
7.47, 7.42 (d, 1H, H-6), 7.30–7.19 (m, 5H, Ph), 6.23–6.21 (m, 1H, H-10),
6.13–6.07 (m, 1H, H-10), 4.96–4.93 (m, 1H, H-30), 4.33–4.22 (m, 1H, H-
30), 4.11–3.93 (m, 4H, 2 ꢂ H-50), 3.85–3.84 (m, 1H, CH), 3.74, 3.73 (2s,
2H, 2 ꢂ H-40), 3.72, 3.71 (d, 3H, OCH3), 3.19–2.81 (m, 2H, CH2Ph),
2.40–2.18 (m, 4H, 2 ꢂ H-20), 1.88–1.86 (m, 6H, 2 ꢂ C5-CH3). 13C NMR
(CD3OD, 125 MHz): ꢀ 174.89, 174.80 (COOMe), 166.29 (2 ꢂ C-4), 152.30,
150.13 (2 ꢂ C-2), 138.64, 138.48, 137.87, 137.78 (2 ꢂ C-6), 130.60–127.97
(Ph), 111.99, 111.94, 111.82, 111.77 (2 ꢂ C-5), 87.28, 87.25, 87.15, 86.55,
86.46, 85.97, 83.51 (2 ꢂ C-40, 2 ꢂ C-10), 79.61, 79.57, 78.84 (C-30), 66.80,
66.75, 66.47 (C-30), 62.70, 62.47, 61.83, 61.50 (2 ꢂ C-50), 57.87, 57.65
(CH), 52.86, 52.83 (OMe), 40.76, 39.67 (2 ꢂ C-20), 37.74, 37.64
(CH2Ph), 12.62, 12.47 (2 ꢂ C5-CH3). ESI-MS [M þ H]þ m/z: 733,
[M þ Na]þ m/z: 755.
T-p-AZT-ValOCH3 5d. Yield: 51.9%. 31P NMR (CD3OD, 81 MHz):
ꢀ 9.93. 1H NMR (CD3OD, 500 MHz): ꢀ 7.83, 7.82 (d, 1H, H-6), 7.55, 7.52
(d, 1H, H-6), 6.32–6.29 (m, 1H, H-10), 6.17–6.14 (m, 1H, H-10), 5.11–5.05
(m, 1H, H-30), 4.50–4.41 (m, 1H, H-30), 4.32–4.06 (m, 4H, 2H-50), 3.80–
3.78 (2s, 2H, 2 ꢂ H-40), 3.74, 3.73 (d, 3H, OCH3), 3.65–3.59 (m, 1H, CH),
2.58–2.33 (m, 4H, 2 ꢂ H-20), 2.10–2.05 (CH(CH3)2) 1.91–1.88 (m, 6H,
2 ꢂ C5-CH3), 0.98–0.94 (m, 6H, 2 ꢂ CH3). 13C NMR (CD3OD,
125 MHz): ꢀ 174.98 (COOMe), 166.27 (2 ꢂ C-4), 152.36, 152.15 (2 ꢂ C-
2), 138.04, 137.98, 137.89, 137.83 (2 ꢂ C-6), 112.06, 112.03, 111.90, 111.81
(2 ꢂ C-5), 87.35, 87.25, 87.20, 86.72, 86.09, 83.64, 83.53 (2 ꢂ C-40, 2 ꢂ C-
10), 79.57, 79.53, 79.06 (C-30), 67.33, 67.29, 66.75, 66.71 (C-30), 62.73,
62.60, 61.80, 61.56 (2 ꢂ C-50), 53.03 (CH), 52.67 (OMe), 39.76, 37.58
(2 ꢂ C-20), 32.02, 32.96 (CH(CH3)2), 19.71, 18.60, 18.30, 18.23
(2 ꢂ CH3), 12.63, 12.54 (2 ꢂ C5-CH3). ESI-MS [M þ H]þ m/z: 685,
[M þ Na]þ m/z: 707.
T-p-AZT-LeuOCH3 5e. Yield: 54.3%. 31P NMR (CD3OD, 81 MHz):
1
ꢀ 9.43, 9.24. H NMR (CD3OD, 500 MHz): ꢀ 7.83, 7.81 (d, 1H, H-6),
7.55, 7.51 (d, 1H, H-6), 6.31–6.27 (m, 1H, H-10), 6.17–6.12 (m, 1H, H-
10), 5.12–5.02 (m, 1H, H-30), 4.41–4.40 (m, 1H, H-30), 4.30–4.04 (m, 4H,
2 ꢂ H-50), 3.89–3.84 (m, 1H, CHCOO), 3.79–3.78 (2s, 2H, 2 ꢂ H-40), 3.72,
3.71 (d, 3H, OCH3), 2.55–2.33 (m, 4H, 2 ꢂ H-20), 1.90–1.86 (m, 6H,
2 ꢂ C5-CH3), 1.79–1.76 (m, 1H, CH(CH3)2), 1.58–1.54 (m, 2H, CH2),
0.93–0.92 (m, 6H, 2 ꢂ CH3). 13C NMR (CD3OD, 125 MHz): ꢀ 175.92
(COOMe), 166.28 (2 ꢂ C-4), 152.33, 152.15 (2 ꢂ C-2), 137.93, 137.98,

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2560
Lin et al.
137.83 (2 ꢂ C-6), 112.02, 111.85, 111.81 (2 ꢂ C-5), 87.40, 87.36, 87.20,
86.69, 86.61, 83.64, 83.60 (2 ꢂ C-40, 2 ꢂ C-10), 79.43, 79.08, 79.05 (C-30),
66.97, 66.93 (C-30), 62.69, 62.59, 61.82, 61.72 (2 ꢂ C-50), 54.41, 54.30
(CHNH), 52.79 (OMe), 43.82, 43.76 (CH(CH3)2), 39.83, 37.71 (2 ꢂ C-
20), 32.02, 32.96 (CH(CH3)2), 25.73, 25.70 (CH2), 23.28, 21.87, 21.83
(2 ꢂ CH3), 12.63, 12.53 (2 ꢂ C5-CH3). ESI-MS [M þ H]þ m/z: 699,
[M þ Na]þ m/z: 721.
Diastereomer in mother solution of 5b. 31P NMR (CD3OD, 81 MHz):
ꢀ 9.03. 1H NMR (CD3OD, 500 MHz): ꢀ 7.81 (s, 1H, H-6), 7.56 (s, 1H, H-
6), 6.32–6.29 (m, 1H, H-10), 6.18–6.15 (m, 1H, H-10), 5.06–5.03 (m, 1H,
H-30), 4.51–4.48 (m, 1H, H-30), 4.32–4.05 (m, 4H, H-50), 3.92–3.90 (m,
1H, CH), 3.79, 3.78 (2s, 2H, H-40), 3.72 (s, 3H, CO2CH3), 2.57–2.34 (m,
4H, H-20), 1.89, 1.88 (2s, 6H, CH3), 1.39, 1.38 (2s, 3H, CH3). 13C NMR
(CD3OD, 125 MHz): ꢀ 175.73 (COOMe), 166.29 (C-2), 152.35, 150.17 (C-
4), 137.89, 137.92 (C-6), 111.81, 111.80 (C-5), 87.25, 87.20 (C-40), 86.62,
86.07 (C-10), 83.61, 83.55 (C-40), 79.47, 79.37 (C-30), 66.74, 66.60 (C-30),
62.71, 61.48 (C-50), 52.85 (CH), 51.35 (OMe), 39.74, 37.61 (C-20), 20.42,
20.36 (CH3), 12.56, 12.47 (CH3). ESI-MS [M þ Na]þ m/z: 695.
Diastereomer of precipitation of 5b. 31P NMR (CD3OD, 81 Hz): ꢀ
1
9.24. H NMR (CD3OD, 500 Hz): ꢀ 7.84 (s, 1H, H-6), 7.53 (s, 1H, H-
6), 6.30–6.29 (m, 1H, H-10), 6.16–6.14 (m, 1H, H-10), 5.15–5.12 (m, 1H,
H-30), 4.44–4.41 (m, 1H, H-30), 4.27–4.05 (m, 4H, H-50), 3.97–3.94 (m,
1H, CH), 3.81, 3.80 (2s, 2H, H-40), 3.73 (s, 3H, CO2CH3), 2.57–2.36 (m,
4H, H-20), 1.91, 1.89 (2s, 6H, CH3), 1.42, 1.40 (2s, 3H, CH3). 13C NMR
(CD3OD, 125 Hz): ꢀ 175.75 (COOMe), 166.31 (C-2), 152.39, 152.18 (C-4),
137.95, 137.90 (C-6), 112.02, 111.82 (C-5), 87.42, 87.38 (C-40), 86.72,
86.13 (C-10), 83.70, 83.64 (C-40), 79.06, 79.02 (C-30), 67.11, 67.07 (C-30),
62.59, 61.78 (C-50), 52.85 (CH), 51.51 (OMe), 39.74, 37.61 (C-20), 20.41,
20.36 (CH3), 12.53, 12.44 (CH3). ESI-MS [M þ Na]þ m/z: 695.
Diastereomer in mother solution of 5c. 31P NMR (CD3OD, 81 MHz):
ꢀ 9.12. 1H NMR (CD3OD, 500 MHz): ꢀ 7.80 (s, 1H, H-6), 7.48 (s, 1H, H-
6), 7.31–7.21 (m, 5H, Ph), 6.24–6.21 (m, 1H, H-10), 6.14–6.10 (m, 1H,
H-10), 4.96–4.94 (m, 1H, H-30), 4.33–4.24 (m, 1H, H-30), 4.12–3.95 (m, 4H,
2 ꢂ H-50), 3.85–3.84 (m, 1H, CH), 3.74, 3.73 (2s, 2H, 2 ꢂ H-40), 3.71 (s,
3H, OCH3), 3.20–2.81(m, 2H, CH2Ph), 2.42–2.19 (m, 4H, 2 ꢂ H-20),
1.89–1.87 (m, 6H, 2 ꢂ C5-CH3). 13C NMR (CD3OD, 125 MHz): ꢀ
174.89 (COOMe), 166.27 (2 ꢂ C-4), 152.30 (2 ꢂ C-2), 137.89, 137.80
(2 ꢂ C-6), 130.60–127.95 (Ph), 111.82, 111.77 (2 ꢂ C-5), 87.28, 87.21,
87.15, 86.55, 86.46, 83.53 (2 ꢂ C-40, 2 ꢂ C-10), 79.61, 79.57 (C-30), 66.81,
66.75 (C-30), 62.70, 62.48, 61.83, 61.50 (2 ꢂ C-50), 57.87, 57.65 (CH),
52.86, 52.84 (OMe), 40.73, 39.67 (2ꢂC-20), 37.75, 37.67 (CH2Ph), 12.61,
12.47 (2 ꢂ C5-CH3). ESI-MS [M þ H]þ m/z: 733, [M þ Na]þ m/z: 755.

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Dinucleotide(TpAZT) Phosphoramidates
2561
Diastereomer of precipitation of 5c. 31P NMR (CD3OD, 81 MHz): ꢀ
8.65. 1H NMR (CD3OD, 500 MHz): ꢀ 7.78 (s, 1H, H-6), 7.43 (s, 1H, H-6),
7.32–7.20 (m, 5H, Ph), 6.24–6.21 (m, 1H, H-10), 6.14–6.11 (m, 1H, H-10),
4.97–4.95 (m, 1H, H-30), 4.33–4.26 (m, 1H, H-30), 4.12–3.97 (m, 4H,
2 ꢂ H-50), 3.86–3.84 (m, 1H, CH), 3.74, 3.73 (2s, 2H, 2 ꢂ H-40), 3.72 (s,
3H, OCH3), 3.19–2.80 (m, 2H, CH2Ph), 2.39–2.17 (m, 4H, 2 ꢂ H-20),
1.89–1.88 (m, 6H, 2 ꢂ C5-CH3). 13C NMR (CD3OD, 125 MHz): ꢀ
174.80 (COOMe), 166.24 (2 ꢂ C-4), 150.13 (2 ꢂ C-2), 138.68, 138.49
(2 ꢂ C-6), 130.62–127.97 (Ph), 111.99, 111.94 (2 ꢂ C-5), 87.29, 87.15,
86.57, 86.46, 85.93, 83.51 (2 ꢂ C-40, 2 ꢂ C-10), 79.58, 78.84 (C-30), 66.77,
66.42 (C-30), 62.74, 62.47, 61.83, 61.50 (2 ꢂ C-50), 57.87, 57.65 (CH),
52.85, 52.82 (OMe), 40.76, 39.65 (2 ꢂ C-20), 37.72, 37.63 (CH2Ph), 12.62,
12.48 (2 ꢂ C5-CH3). ESI-MS [M þ H]þ m/z: 733, [M þ Na]þ m/z: 755.
ACKNOWLEDGMENT
The authors would like to thank the financial supports from the
National Natural Science Foundation of China (No. 29902003,
20132020 and 20175026), the Ministry of Science and Technology, the
Chinese Ministry of Education and Tsinghua University.
REFERENCES
1. Vlieghe, P.; Clerc, T.; Pannecouque, C.; Witvrouw, M.; De Clercq, E.;
Salles, J.P.; Kraus, J.L. Synthesis of new covalently bound kappa-
carrageenan-AZT conjugates with improved anti-HIV activities. J.
Med. Chem. 2002, 45, 1275.
2. Wong, A.; Toth, I. Lipid, sugar and liposaccharide based delivery
systems. Curr. Med. Chem. 2001, 8, 1123.
3. Gallo, J.M. Delivery of anti-HIV nucleosides to the central-nervous
system. Adv. Drug Deliver Rev. 1994, 14, 199.
4. Mitsuya, H.; Weinhold, K.J.; Furman, P.A.; Clair, M.H. 30-Azido-30-
deoxythymidine(BWA509U): an agent that inhibits the infectivity
and cytopathic effect of human T-cell lymphotropic virus type III/
lympadenopathy-associated virus in vitro. Proc. Natl. Acad. Sci.
1985, 82, 7096.
5. Fischl, M.A.; Richman, D.D.; Grieco, M.H.; Gottlieb, M.S. The
efficacy of azidothymidine(AZT) in the treatment of patients with
AIDs and AIDs-related complex. A double blind, placebo-controlled
trial. N. Engl. J. Med. 1987, 317, 185.

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2562
Lin et al.
6. Wanger, C.R.; Iyer, V.V.; McIntee, E. J. Pronucleotides: toward the
in vivo delivery of antiviral and anticancer nucleotides. Med. Res.
Rev. 2000, 20, 417.
7. De Clerq, E. Nucleosides Nucleotides 1994, 13, 1271.
8. Busso, M.; Mian, A.M.; Hahn, E.F.; Resnick, L. AIDS Res. Hum.
Retroviruses 1988, 4, 449.
9. Shinazi, R.F.; Sommadossi, J.P.; Saalmann, V.; Cannon, D.L.; Xie,
M.Y.; Hart, G.C.; Smith, G.A.; Hahn, E.F. Antimicrob. Agents
Chemother. 1990, 34, 1061.
10. Hakimelahi, G.H.; Movahedi, A.A.M.; Sadeghi, M.M.; Tsay, S.C.;
Hwu, J.R. Design, synthesis and structure-activity relationship of
novel dinucleotide analogs as agents against herpes and human
immunodeficiency viruses. J. Med. Chem. 1995, 38, 4648.
11. Meier, C.; Lorey, M.; De Clercq, E.; Balzarini, J. CycloSal-20,30-
dideoxy-20,30-didehydrothymidine
monophosphate
(cycloSal-
d4TMP): synthesis and antiviral evaluation of a new d4TMP
delivery system. J. Med. Chem. 1998, 41, 1417.
12. Lu, Y.; Just, G. Stereoselective synthesis of R-P- and S-P-dithymi-
dine phosphorothioates via chiral indolooxazaphosphorine inter-
mediates derived from tryptophan. Angew. Chem. Int. Ed. 2000,
39, 4521.
13. Kers, A.; Stawinski, J. A new type of nucleotide analogue with 4-
pyridylphosphonate internucleotide linkage. Tetrahedron Lett.
1999, 40, 4263.
14. Garegg, P.J.; Regberg, T.; Stawinkski, J.; Stromberg, R. Formation
of internucleotidic bonds via phosphonate intermediates. Chemica
Scipta 1985, 25, 280.
15. Peyser, J.R.; Ferris, J.P. The rates of hydrolysis of thymidyl-30,50-
thymidine-H-phosphonate: the possible role of nucleic acids linked
by diesters of phosphorous acid in the origins of life. Origins Life
Evol. B. 2001, 31 (4), 363.
16. Jankowska, J.; Sobkowski, M.; Stawinski, J.; Kraszewski, A.
Studies on aryl H-phosphonates. 1. An efficient method for the
preparation of deoxyribonucleoside and ribonucleoside 30-H-
phosphonate monoesters by transesterification of diphenyl H-phos-
phonate. Tetrahedron Lett. 1994, 35 (20), 3355.
Received in the Netherlands November 11, 2002