1
252
B. Roy et al. / Tetrahedron Letters 52 (2011) 1250–1252
Scheme 2. Synthesis of 3TCMP 1 starting from H-phosphonate 3 (ROH = 3TC). Reagents and conditions: (a) BSA/DMF, 10 min, rt; (b) DCSO, 10 min, rt; (c) methanol/DBU 97/3
v/v 30 min, rt. Measured 31P NMR chemical shifts are indicated above each compound.
product could be converted to compound 2 by treatment in dilute
alkali medium (data not shown). Purification of lamivudine 5 -
diphosphate was performed by anion exchange chromatography
followed by chromatography on reverse phase. The yield of the
diphosphorylation step was 56%.
14. Brzezinski, B.; Grech, E.; Malarski, Z.; Sobczyk, L. J. Chem. Soc., Perkin Trans. 2
1991, 857.
0
1
1
5. Kraszewski, A.; Stawinski, J. J. Pure Appl. Chem. 2007, 79, 2217–2227.
6. Jankowska, J.; Sobkowski, M.; Stawinski, J.; Kraszewski, A. Tetrahedron Lett.
1994, 35, 3355–3358.
17. Excess of phosphonylating agent was also rapidly hydrolyzed to phenyl H-
phosphonate monoester. Compound 3 was separated from the phenyl H-
In conclusion, the present method is highly effective for the
31
phosphonate monoester on a silica gel column. Compound 3 (3TCHP): P NMR
0
1
synthesis of lamivudine 5 -diphosphate. The best synthetic route
(300 MHz, D
2
O): d 6.5 ( JP–H = 642.4 Hz); HPLC: t
R
= 19.3 min (kmax 272 nm).
0
18. Wada, T.; Mochizuki, A.; Sato, Y.; Sekine, M. Tetrahedron Lett. 1998, 39, 7123–
126.
to lamivudine 5 -monophosphate was through H-phosphonate oxi-
7
dation by DCSO. Follow-up of the reaction course by HPLC was
found to be more convenient than 31P NMR analysis since it is a
much lower time- and material-consuming technique.
1
9. The mixture was diluted with water–pyridine (1:1, v/v) and washed with
dichloromethane. The aqueous layers were concentrated to dryness to give
compound 1 as a colorless foam. The nucleotide was purified by Sephadex
DEAE A-25 resin ion exchange column chromatography with
a linear
gradient (0–0.5 M) of 0.5 M ammonium bicarbonate. Compound
1
31
3
Acknowledgments
(3TCMP):
calcd for
t = 13.9 min (kmax 272 nm).
R
P
NMR (300 MHz, D O):
d
3.5 ( J
P–H
= 5.5 Hz). HRMS (m/z)
2
ꢀ
C
8
H
N
11 3
O
6
PS (MꢀNa) , 308.0130; found 308.0106. HPLC:
We thank Sophie Moureu, Matthieu Bergès, Juliette Langlais,
Aboudou Haidari and Flaurence Lormeau for their technical assis-
tance. This work was financially supported by the Scientific council
of University Montpellier 2 and also by the Agence Nationale de
Recherches sur le Sida (contract number 2008-090).
2
0. Calleri, E.; Ceruti, S.; Cristalli, G.; Martini, C.; Temporini, C.; Parravicini, C.;
Volpini, R.; Daniele, S.; Caccialanza, G.; Lecca, D.; Lambertucci, C.; Trincavelli,
M. L.; Marucci, G.; Wainer, I. W.; Ranghino, G.; Fantucci, P.; Abbracchio, M. P.;
Massolini, G. J. Med. Chem. 2010, 53, 3489–3501.
0
2
1. To
a
solution of the tri-n-butylammonium salt of lamivudine 5 -
monophosphate (0.15 mmol) dissolved in 1 mL of dry DMF was added 36
lL
of tri-n-butylamine (0.15 mmol). The solution was stirred for 20 min at room
temperature. After evaporation of the solvent under anhydrous conditions, the
residue was suspended in 1.4 mL of dry DMF, then N,N -carbonyldiimidazole
References and notes
0
(
122 mg, 0.75 mmol) was added and the mixture was stirred for 3 h at room
temperature. Excess of CDI was eliminated by addition of methanol (49 L,
.2 mmol) and the mixture was stirred for 30 min. Then 6 mL (3 mmol) of a
.5 M solution of tri-n-butylammonium phosphate in dry DMF were added.
1
2
.
.
Mathe, C.; Gosselin, G. Antiviral Res. 2006, 71, 276–281.
l
(a) Alexandre, J. A. C.; Roy, B.; Topalis, D.; Pochet, S.; Perigaud, C.; Deville-
Bonne, D. Nucleic Acids Res. 2007, 35, 4895–4904; (b) Gondeau, C.; Chaloin, L.;
Lallemand, P.; Roy, B.; Périgaud, C.; Barman, T.; Varga, A.; Vas, M.; Lionne, C.;
Arold, S. T. Nucleic Acids Res. 2008, 36, 3620–3629.
Krishnan, P. G. E.; Lam, W.; Dutschman, G. E.; Grill, S. P.; Cheng, Y. C. J. Biol.
Chem. 2003, 278, 36726–36732.
Coates, J.; Cammack, N.; Jenkison, H.; Mutton, I.; Pearson, B.; Storer, R.;
Cameron, J.; Penn, C. Antimicrob. Agents Chemother. 1992, 36, 733–739.
Romeo, G.; Chiacchio, U.; Corsaro, A.; Merino, P. Chem. Rev. 2010, 110, 3337–
1
0
The mixture was stirred for another 17 h. The solvent was removed under high
vacuum. P NMR analysis of the crude reaction product in D O showed a
2
31
3.
4.
5.
characteristic doublet (d ꢁꢀ10.5 and ꢀ11.9 ppm, JP–P = 17 Hz) assigned to the
diphosphate product along with a singulet signal at ꢁ0 ppm corresponding to
excess tri-n-butylammonium phosphate. The progress of the reaction was also
monitored by HPLC-UV. The mixture dissolved in water was purified by
Sephadex DEAE A-25 resin ion exchange column chromatography with a linear
gradient 0–0.5 M of ammonium bicarbonate, followed by chromatography on
RP-18. The triethylammonium counter ions were exchanged to sodium by
passing the nucleotide solution through a Dowex-AG 50WX2-400 column.
Compound 2 (3TCDP) was obtained as a white solid after lyophilization (56%
3
370.
6
7
.
.
Bedse, G.; Kumar, V.; Vingh, S. J. Pharm. Biomed. Anal. 2009, 49, 55–63.
Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter, C. D. J. Org. Chem. 1987, 52,
1
794–1801.
8
9
.
.
Hoard, D. E.; Ott, D. G. J. Am. Chem. Soc. 1965, 87, 1785–1788.
Yoshikawa, M.; Kato, T.; Takenishi, T. Tetrahedron Lett. 1967, 50, 5065–5068.
= 18.4 min (kmax 272 nm). 1H NMR (300 MHz, D
= 5.4 Hz, J = 4.4 Hz, 1H, H-1’),
= 3.5 Hz, 1H, H-4’), 4.35 (m, 1H, H
= 5.4 Hz, J = 12.2 Hz, 1H, H -2’), 3.20
-2’). C NMR (D O): d 166.1 (C-4),
57.0 (C-2), 141.9 (C-6), 95.9 (C-5), 87.5 (C-1’), 84.3 (C-4’), 66.1 (C-5’), 36.7 (C-
yield). HPLC: t
6,5 = 7.6 Hz, 1H, H-6), 6.30 (pseudo, t, J
R
2
O): d 8.04 (d,
J
5
5
1
0
,2
0
a
0 0
1 ,2 b
1
1
1
0. Jansen, R. S.; Rosing, H.; Schellens, J. H. M.; Beijnen, J. H. Nucleosides Nucleotides
.73 (d, J5,6 = 7.6 Hz, 1H, H-5), 5.45 (t, J
’), 4.22 (m, 1H, H -5’), 3.52 (dd, J
= 4.4 Hz, J = 12.2 Hz, 1H, H
4
0
,5
0
a
-
Nucleic Acids 2010, 29, 14–26.
0
0
0
0
b
1
,2
a
2
a,2
b
a
1. Risbood, P. A.; Kane, C. T.; Hossain, M. T.; Vadapalli, S.; Chadda, S. K. Bioorg.
Med. Chem. Lett. 2008, 18, 2957–2958.
2. Crauste, C.; Lefebvre, I.; Hovaneissian, M.; Puy, J.-Y.; Roy, B.; Peyrottes, S.;
Cohen, S.; Guitton, J.; Dumontet, C.; Perigaud, C. J. Chromatogr., B 2009, 877,
13
(
dd, J
1
0
,2
0
b
2
0
a,2
0
b
b
2
1
2
(
31
’). P NMR (D
2
O): d ꢀ8.8 (d, JP–P = 20.7 Hz), ꢀ11.1 (d, JP–P = 20.7 Hz). HRMS
ꢀ
m/z) calcd for C
8
H
11
N
3
O
9
P
2
SNa (MꢀNa) , 409.9589; found 409.9588.
1
417–1425. Under the HPLC conditions described in this paper, the retention
4
2
2. In HPLC-UV, the retention time of N -(methylcarbamoyl)lamivudine is
6.3 min (kmax 292 nm). Mass spectroscopy was carried out on triple
time (min) are: cytosine, 8.2; 3TC, 20.1; 3TCMP, 13.9. kmax was 272 nm except
for cytosine (266 nm).
2
a
quadrupole mass spectrometer TSQ Quantum Ultra (Thermo Fisher Scientific
Inc). The molecular formula of N -(methylcarbamoyl)lamivudine was
1
3. 0.5 mmol (50 mg) of 3TC was dissolved in 1 mL triethylphosphate under argon.
4
The solution was cooled at 0 °C before addition of freshly distilled POCl
3
and
ꢀ
determined to be C10
46).
3. Sigmund, H.; Pfleiderer, W. Helv. Chim. Acta 1994, 77, 1267–1280.
H
15
N
3
O
11
P
2
S by negative-ion ESI-MS ([MꢀH] at m/z
allowed to proceed at the same temperature. At time intervals, an aliquot of
the reaction media was submitted to hydrolysis with triethylammonium
bicarbonate 1 M pH 7.5 prior to analysis by HPLC-UV.
4
2