Convenient and regioselective synthesis of nucleoside phosphoramidate monoesters

Article abstract of DOI:10.1055/s-2005-871582

We have developed a convenient and efficient method for the synthesis of nucleoside phosphoramidate monoesters. This step-wise synthesis, consisting of an ester exchange, reaction of the 5′-OH of nucleosides with fluorenylmethyl aryl phosphite, and an Ath

Full text of DOI:10.1055/s-2005-871582

LETTER
1927
Convenient and Regioselective Synthesis of Nucleoside Phosphoramidate
Monoesters
Synthesis
i
of
N
uc
g
a
phoramidat
n
e
Monoester
g
Zhu,a Hua Fu,*a Yuyang Jiang,a,b Yufen Zhaoa
a
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education, Department of Chemistry,
Tsinghua University, Beijing 100084, P. R. China
b
Key Laboratory of Chemical Biology, Guangdong Province, Graduate School of Shenzhen, Tsinghua University,
Shenzhen 518057, P. R. China
Fax +86(10)62781695; E-mail: fuhua@mail.tsinghua.edu.cn
Received 14 May 2005
through a P–N bond cleavage by phosphoramidases to
yield the corresponding nucleoside monophosphates.2d,3
These studies have provided valuable insight to the design
of phosphoramidate-based pronucleotides.1a,b Chloro-
Abstract: We have developed a convenient and efficient method
for the synthesis of nucleoside phosphoramidate monoesters. This
step-wise synthesis, consisting of an ester exchange, reaction of the
5¢-OH of nucleosides with fluorenylmethyl aryl phosphite, and an
Atherton–Todd reaction, gave fluorenylmethyl nucleoside 5¢-phos- phosphate3a,4 and DCC-mediated5 coupling methods are
phoramidates; final removal of the fluorenylmethyl group yielded
the desired target products in good yields.
usually applied to the synthesis of this kind of phosphor-
amidates, however, most of these methods required the
Key words: phosphoramidate, ester-exchange, diphenyl phosphite,
nucleoside, Atherton–Todd reaction
2¢,3¢-OH and NH2 to be protected prior to phosphorylation
of 5¢-OH. Wagner et al.3b used direct phosphorylation of
nucleosides – treatment of 5¢-OH phosphorus oxychloride
in triethyl phosphate, hydrolysis to nucleoside monophos-
In general, the biological activity of antiviral and anti- phate,5 and finally coupling of the nucleoside monophos-
cancer nucleosides is dependent on the host cell kinase phate with the amino acid methyl esters in tert-butanol
activity producing their active triphosphorylated forms. and water using DCC, afforded the corresponding phos-
However, the phosphorylating yields of these nucleosides phoramidates. The method usually needs a long reaction
are very low because they are unnatural substrates for the time, the overall yields are low, and purification of the
corresponding kinases. The development of nucleoside nucleoside monophosphate over an ion-exchange column
prodrugs capable of undergoing intracellular activation to (BioRad AG1-X8) is troublesome, so it is necessary to
the corresponding nucleotides has become an area of in- develop a new general and efficient approach to nucleo-
tense interest.1 Nucleoside amino acid phosphoramidates side phosphoramidate monoesters.
show promise as potential pronucleotides.1a,b Moreover,
The Atherton–Todd reaction is considered to be an effec-
nucleoside phosphoramidate monoesters are potent anti-
tive approach for the construction of the P–N bond from
viral and/or anticancer agents with enhanced activity and
H-phosphonates; the synthetic route is summarized in
reduced cytotoxicity.2 These nucleoside phosphoramidate
Scheme 1.
monoesters are thought to exert their biological functions
HO
B
O
R = OH, B = A (a), U (b)
R = H, B = T (c)
O
P
O
P
H
OH
R
Py
OPh
O
PhO
Fm
OPh
Fm
O
4
+
Fm OH
2
Py
H
1
3
O
O
O
AAOMe
CCl4 / Et3N
N
Fm
P
O
B
–
H
O
P
H
O
B
O
P
O
B
O
O
O
HN
HC R'
HN
OH
HC R'
R
5
OH
R
OH
R
COOCH3
COOCH3
6
7
Scheme 1 Synthetic route of nucleoside phosphoramidate monoesters
SYNLETT 2005, No. 12, pp 1927–1929
0
1
.0
8
.2
0
0
5
Advanced online publication: 07.07.2005
DOI: 10.1055/s-2005-871582; Art ID: U15005ST
© Georg Thieme Verlag Stuttgart · New York

1928
J. Zhu et al.
LETTER
The ester-exchange reaction with fluorenylmethanol (Fm- result of the coupling with 4¢-CH, however, the 1H NMR
OH) and 1.2 equivalents of diphenyl phosphite (31P NMR, spectra of nucleoside phosphoramidates (6 and 7) showed
1.40 ppm) in anhydrous pyridine at –5 °C under a nitrogen multiple peaks due the coupling of the H in P-5¢-OCH2
atmosphere led to 3 with a minor amount of difluorenyl- with P and H in 4¢-CH. The regioselective phosphoryl-
methyl phosphite as side product (ca. 6%, 31P NMR deter- ation of 5¢-OH is attributed to the high reactivity of 5¢-OH
mination, 31P NMR, d = 9.00 ppm); the reaction was compared with 2¢,3¢-OH, and NH2.
completed within ten minutes. The corresponding nucleo-
Compared with the previous approaches to nucleoside
side (1.4 equiv) in pyridine was added dropwise to the
phosphoramidates, this method has its advantages. The
resulting solution at –5 °C, the solution was warmed to
reactions are operationally simple and fast, the total yields
40 °C, and the reaction was complete within 1 h. 31P NMR
are high, and the desired target products are easily puri-
spectroscopy showed that the major product fluorenyl-
fied.
methyl nucleoside 5¢-H-phosphonate (31P NMR, d 9.60–
In conclusion, we have developed a new method for the
10.00ppm) along with side products difluorenylmethyl
synthesis of nucleoside phosporamidate monoesters,
regioselective ester-exchange, reaction of the 5¢-OH of
nucleosides with fluorenylmethyl aryl phosphite, and
Atherton–Todd reaction gave fluorenylmethyl nucleoside
5¢-phosporamidates; removal of the fluorenylmethyl
yielded the desired target products in good yields.
phosphite and dinucleoside phosphite (31P NMR, d 10.50
ppm) were present in the solution. Pyridine was removed
from the solution under reduced pressure; subsequent
Atherton–Todd reaction of the residue 5 (the crude prod-
uct fluorenylmethyl nucleoside 5¢-H-phosphonate) with
1.2 equivalents of amino acid methyl ester in THF in the
presence of CCl4 and Et3N provided fluorenylmethyl
nucleoside 5¢-phosphoramidate (6) in 73–82% isolated
overall yields. Treatment of 6 with piperidine in CH2Cl2
led to the target products 7. A total isolated yield of 65–
75% was achieved (1→7, Table 1).
Fluorenylmethyl phenyl H-phosphonate (3): Fm-OH (196 mg, 1
mmol) in anhydrous pyridine (3 mL) was added dropwise to diphe-
nyl phosphite (281 mg, 1.2 mmol) in anhydrous pyridine (3 mL),
and the solution was stirred at –5 °C. The reaction was complete
within 10 min. 31P NMR (pyridine): d = 5.30 ppm.
Table 1 31P NMR and Total Yields of the Synthesized Compounds
Fluorenylmethyl nucleoside 5¢-H-phosphonates (5): Nucleoside
(1.4 mmol) in anhydrous pyridine (3 mL) was added to a solution of
the above. The resulting solution was stirred for about 1 h at 40 °C,
and concentrated under reduced pressure to provide the crude prod-
uct fluorenylmethyl nucleoside 5¢-H-phosphonate 5 (mixture of
diastereoisomers, ratio 1:1), 31P NMR (pyridine): d = 9.8 ppm (two
peaks, 1JP-H = 692 Hz).
Compound NuOH R¢
31P NMR
(d/ppm)
Yield
(%)
7aa
7ab
7ac
7ad
7ae
7ba
7bb6
7bc
7bd
7be
7ca
7cb
7cc
A
A
A
A
A
U
U
U
U
U
T
CH3
7.29
6.83
6.82
6.65
6.77
6.58
7.01
6.85
7.02
6.80
6.83
6.86
6.57
7.00
8.70, 8.50
67
73
73
66
65
70
73
73
71
66
68
74
75
70
82
CH(CH3)2
CH2Ph
Fluorenylmethyl nucleoside 5¢-phosphoramidates (6): Amino
acid methyl ester hydrogen chloride (1.2 mmol), Et3N (2 mmol),
and CCl4 (0.5 mL) were mixed together in anhydrous THF (5 mL);
the crude product fluorenylmethyl nucleoside 5¢-H-phosphonate (5)
in anhydrous THF (2 mL) was added dropwise to the solution at
–5 °C, and the solution was stirred for 30 min at r.t. The Atherton–
Todd reaction produced fluorenylmethyl nucleoside 5¢-phosphora-
midate 6 in almost quantitative yield (31P NMR). The solvent was
removed by rotary evaporation, the residue was dissolved in CH2Cl2
(5 mL), and then the solution was washed with 0.1 M HCl (3 ꢀ 3
mL). Purification by silica gel column chromatography (CH2Cl2–
MeOH, 10:1) gave the fluorenylmethyl nucleoside 5¢-phosphorami-
date 6 (mixture of diastereoisomers, ratio 1:1). 31P NMR: d = 8.60
(2 peaks). Overall yield from 1 to 6: 73–82%.
CH2COOCH3
CH2OH
CH3
CH(CH3)2
CH2Ph
CH2COOCH3
CH2OH
Nucleoside 5¢-phosphoramidate monoesters (7): Piperidine (1
mL) was added to 6 in CH2Cl2 (5 mL) and the solution was stirred
for 5 min. The solvent and piperidine were removed on a rotary
evaporator. Purification by silica gel column chromatography
(PrOH–H2O–NH3ꢀH2O, 7:1:2) gave the desired target product nu-
cleoside phosphoramidates 7 in 65–75% (total isolated yield from 1
to 7). These compounds were identified by 31P, 1H, and 13C NMR
spectroscopy and ESI-MS.
CH3
T
CH(CH3)2
CH2Ph
T
7cd
6bb6
T
CH2COOCH3
CH(CH3)2
U
Acknowledgment
Regioselective reaction of fluorenylmethyl aryl phosphite
with the 5¢-OH of nucleosides was confirmed by 1H NMR
spectroscopy. The 5¢-CH21of the free nucleosides should
appear as doublets in the H NMR spectrum, which is a
This work was supported by the Excellent Dissertation Foundation
of the Chinese Ministry of Education (No. 200222), the Excellent
Young Teacher Program of MOE, P. R. C., and the National Natural
Science Foundation of China (Grant No. 20472042).
Synlett 2005, No. 12, 1927–1929 © Thieme Stuttgart · New York

LETTER
Synthesis of Nucleoside Phosphoramidate Monoesters
1929
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(6) Spectroscopic data of selected compounds: Fm-U-P-
LeuOCH3 (6bb). Overall yield from 1 to 6: 82%. 1H NMR
(300 MHz, CDCl3): d = 0.77–0.82 (d, 6 H, CH3, 3J = 6.00
Hz), 1.26 (m, 2 H, b-CH2), 1.40 (m, 1 H, g-CH), 3.49–3.62
(m, 4 H, OCH3, a-CH), 3.85 (m, 5¢-CH2), 3.99 (Fm-CH),
4.02 (m, 1 H, 4¢-CH), 4.14 (t, 1 H, 3¢-CH), 4.25 (t, 1 H, 2¢-
CH), 5.21 (2 H, Fm-CH2), 5.81 (1 H, 1¢-CH), 5.62 (d, 1 H, 3-
CH), 7.74 (d, 1 H, 2-CH), 7.20–7.55 (8 H, Fm-Ar-). 13
C
NMR (75 MHz, CDCl3): d = 21.67 (CH3), 22.73 (CH3),
24.54 (g-CH), 43.84 (b-CH2), 48.09 (Fm-CH), 52.34
(OCH3), 52.86 (a-CH), 64.80 (5¢-CH2), 68.46 (2¢-CH), 69.84
(3¢-CH), 72.40 (Fm-CH2), 83.10 (4¢-CH), 89.81 (1¢-CH),
102.70 (3-CH), 120.12, 124.98, 127.26, 127.97, 140.34,
141.42 (Fm-Ar), 143.17 (2-CH), 151.21 (6-C), 163.98 (3-C),
174.68 (CO). 31P NMR (121 MHz, CDCl3): d = 8.70, 8.50.
ESI-MS (Positive): m/z = 630 [M + H]+. U-P-LeuOCH3
(7bb). Total yield from 1 to 7: 73%. 1H NMR (300 MHz,
D2O): d = 0.73–0.75 (d, 6 H, CH3, 3J = 6.00 Hz), 1.38 (m, 2
H, b-CH2), 1.54 (m, 1 H, g-CH), 3.60–3.62 (4 H, m, OCH3,
a-CH), 3.83–3.94 (m, 5¢-CH2), 4.12 (m, 1 H, 4¢-CH), 4.18 (t,
1 H, 3¢-CH), 4.23 (t, 1 H, 2¢-CH), 5.84 (1 H, 1¢-CH), 5.83 (d,
1 H, 3-CH), 7.85 (d, 1 H, 2-CH). 13C NMR (75 MHz,
CDCl3): d = 21.45 (CH3), 21.98 (CH3), 24.26 (g-CH), 43.09
(d, b-CH2, 3JP-C = 7.16 Hz), 52.61 (OCH3), 53.48 (a-CH),
63.60 (5¢-CH2), 69.91 (2¢-CH), 73.87 (3¢-CH), 83.53 (d, 4¢-
CH, 3JP-C = 8.61 Hz), 88.65 (1¢-CH), 102.69 (3-CH), 141.86
(2-CH), 151.81 (6-C), 166.19 (3-C), 178.27 (CO). 31P NMR
(121 MHz, D2O): d = 7.01. ESI-MS (Negative): m/z = 450
[M – H]–.
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Synlett 2005, No. 12, 1927–1929 © Thieme Stuttgart · New York