Synthesis of Novel Phosphoramidate Prodrugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4317
5-F lu or o-2′-d eoxy-5′-u r id yl 1-ben zotr ia zolyl bis(2-br o-
m oeth yl)p h osp h or a m id a te (1b) was prepared as described
for 3a from 7b in 58% yield. 5-FUdR (1.6 mmol) was used
instead of thymidine: Rf ) 0.38 (10% MeOH:CHCl3); 1H NMR
(acetone-d6) δ 8.08-7.41 (5H, m), 6.30 (1H, m), 4.61 (3H, m),
4.23 (1H, m), 3.62 (8H. m), 2.31 (2H, m); 31P NMR (acetone) δ
-13.60, -14.13; HRMS (Cl9H22Br2FN6O7P) calcd (M + H)+
656.9696, found 656.9703.
5-F lu or o-2′-d eoxy-5′-u r id yl 1-ben zotr ia zolyl p ip er id yl-
p h osp h or a m id a te (1c) was prepared as described for 3a from
7c in 57% yield. 5-FUdR (1 mmol) was used instead of
thymidine: Rf ) 0.33 (10% MeOH:CHCl3); 1H NMR (CDCl3) δ
8.05-7.35 (5H, m), 6.34 (1H, m), 4.60-4.13 (4H, m), 3.36 (4H,
br), 2.47 (1H, br), 2.20 (1H, m), 1.65 (6H, br); 31P NMR (THF,
ref ) H3PO4) δ -14.88, -15.30; HRMS (C20H24FN6O7P) calcd
(M + H)+ 511.1506, found 511.1506.
5′-Th ym id yl 1-Ben zotr ia zolyl P ip er id ylp h osp h or a m i-
d a te (3c). Phosphoramidate 3c was prepared from piperidyl
dichloride (7c) (4.13 mmol) and thymidine as described for 3a
in 43% yield: Rf ) 0.47 (9:1 CHCl3:EtOH); 1H NMR (CDCl3) δ
9.03 and 8.96 (1H, s, 1:1.3 mixture), 8.05 (1H, m), 7.70 (1H,
m), 7.56 (1H, m), 7.46-7.28 (2H, m), 6.37 and 6.26 (1H, t, 1.3:1
mixture), 4.48 (3H, m), 4.21-4.00 (1H, m), 3.76 (1H, s), 3.27
(4H, m), 2.45-1.99 (2H, m), 1.94 and 1.80 (3H, s, 1.3:1
mixture), 1.62 (6H, m); 31P NMR (CDCl3) δ -14.83, -15.05
(1:1.3 mixture); HRMS (C21H26N6O7P) calcd 506.1679 (M +
H)+, found 506.1670.
concentrated to a volume of 20 mL. Toluene (10 mL) was
added, and the mixture was concentrated again to a volume
of 20 mL. The coevaporation with toluene was repeated (×3)
to remove pyridine. After the final coevaporation with toluene,
the crude mixture was evaporated to dryness and the residue
was passed through a short plug of silica gel (10:90 MeOH:
CHCl3) to remove any remaining amine hydrochloride salts
and then purified by chromatography on silica gel (5:95 MeOH:
CHCl3) to afford compound 4a as a white foam (1.48 g, 68%).
This product was identical in all respects to that obtained from
method 1.
5′-Th ym id yl 5-n itr o-2-fu r ylm eth yl N-m eth yl-N-(2-br o-
m oeth yl)p h osp h or a m id a te (13) was prepared as described
for compound 4a (method 2) on a 4.13-mmol scale in 77%
yield: Rf ) 0.34 (5:95 MeOH:CHCl3); 1H NMR (CDCl3) δ 8.94
(1H, bs), 7.35 (1H, d, J ) 1 Hz), 7.29 (1H, d, J ) 4 Hz), 6.89
(1H, d, J ) 4 Hz), 6.25 (1H, q, J ) 6 Hz), 5.10 and 5.00 (2H,
d, J ) 5 Hz, 1:1 mixture), 4.54 (1 H, m), 4.26 (2H, m), 4.06
(1H, m), 3.48 (4H, m), 2.73 (3H, d, J ) 10 Hz), 2.41 (1H, m),
2.22 (1H, m), 1.92 and 1.79 (3H, 1:1 mixture); 31P NMR (CDCl3)
δ 14.65, -14.75 (1:1 mixture of diastereomers); MS (C18H24
-
BrN4O10P) calcd (M + H)+ 567.0492, found 567.0481.
5-F lu or o-2′-d eoxy-5′-u r id yl 5-n it r o-2-fu r ylm et h yl N-
m eth yl-N-(2-br om oeth yl)p h osp h or a m id a te (2a ) was pre-
pared from 1a on a 0.9-mmol scale as described for 4a in 75%
yield: Rf ) 0.29 (10% MeOH:CHCl3); 1H NMR (CDCl3) δ 7.80
(1H, m), 7.36 (1H, m), 6.74 (1H, m), 6.24 (1H, m), 5.15 (2H, d,
J ) 9.0 Hz), 4.58 (1H, m), 4.31 (2H, m), 4.09 (1H, m), 3.52
(4H, m), 2.77 (3H, d, J ) 9.3 Hz), 2.48 (12H, m), 2.23 (1H, m);
31P NMR (acetone) δ -14.64; HRMS (C17H2lBrFN4O10P) calcd
(M + Na)+ 593.0060, found 593.0063.
5-F lu or o-2′-d eoxy-5′-u r id yl 5-n itr o-2-fu r ylm eth yl bis-
(2-br om oeth yl)p h osp h or a m id a te (2b) was prepared from
7b on a 0.4-mmol scale as described for 4a in 70% yield: Rf )
0.32 (10% MeOH:CHCl3); 1H NMR (acetone-d6) δ 7.90 (1H, m),
7.49 (lH, d, J ) 3.6 Hz), 6.95 (1H, d, J ) 3.5 Hz), 6.23 (1H,
rn), 5.21 (2H, m), 4.42 (1H, m), 4.28 (2H, m), 4.11 (1H, m),
3.42 (8H, m), 2.32 (2H, m); 31P NMR (MeOH) δ -15.13. Anal.
Calcd for C18H22Br2FN4O10P: C 32.55, H 3.34, N 8.84. Found:
C 32.64, H 3.34, N 8.46.
5-F lu or o-2′-d eoxy-5′-u r id yl 5-n itr o-2-fu r ylm eth yl p ip -
er id ylp h osp h or a m id a te (2c) was prepared on a 0.5-mmol
scale from 1c as described for 4a in 71% yield: Rf ) 0.19 (10%
MeOH:CHCl3); 1H NMR (CDCl3) δ 7.89 (1H, m), 7.30 (1H, m),
6.72 (1H, m), 6.25 (1H, m), 5.05 (2H, m), 4.58 (1H, m), 4.35
(2H, m), 4.16 (1H, m), 3.08 (4H, br), 2.48 (lH, m), 2.19 (lH, m),
1.54 (6H, br); 31P NMR (CH2Cl2) δ -15.50, -15.71; HRMS
(C19H24FN4PO10) calcd (M + Na)+ 541.1112, found 541.1118.
5′-Th ym id yl b en zyl p ip er id ylp h osp h or a m id a t e (4c)
was prepared from 3c (210 mg, 0.42 mmol) as described above
in 71% yield, and the product was isolated by silica gel
chromatography (1:3:3 hexanes:EtOAc:acetone f 10% MeOH:
CHCl3) as a 2:1 mixture of diastereomers (as determined by
1H NMR): Rf ) 0.25 (1:3:3 hexanes:EtOAc:acetone); 1H NMR
(CDCl3) δ 9.51 and 9.42 (1H, s, 2:1 mixture), 7.50 (1H, s), 7.36
(5H, m), 6.33 (1H, t), 5.01 (2H, m), 4.50 (1H, m), 4.20 (2H, m),
4.09 (1H, m), 3.29 (1H, s), 3.06 (4H, m), 2.41(1H, m), 2.07 (1H,
m), 1.85 and 1.79 (3H, s, 2:1 mixture), 1.50 (6H, m); 31P NMR
(CDCl3) δ -17.89; HRMS (C22H30N3O7P) calcd 480.1900 (M +
H)+, found 480.1922.
5′-Th ym id yl Ben zyl N-Meth yl-N-(2-br om oeth yl)p h os-
p h or a m id a te (4a ). Meth od 1: Benzyl alcohol (0.056 mL, 0.54
mmol) was dissolved in THF (1 mL) under an atmosphere of
argon, and a few crystals of 2,2′-dipyridyl indicator were added.
Ethylmagnesium bromide (0.72 mL of ca. 0.75 M solution in
THF, 0.54 mmol) was added dropwise to the mixture at room
temperature until a pale pink color persisted. The resulting
thick mixture containing the alkoxide was cooled to -20 °C.
Phosphoramidate 3a (100 mg, 0.18 mmol) was dissolved in
THF (1 mL) and added dropwise to the alkoxide at -20 °C. A
color change from pink to colorless was observed after the
addition of 3a . The reaction mixture was warmed to 0 °C over
1 h and then diluted with CHCl3 (10 mL). The organic mixture
was washed with saturated NaHCO3 (1 × 25 mL), dried over
Na2SO4 and concentrated. The residue was purified by silica
gel chromatography (5 f 10% MeOH:CHCl3) to yield 4a (67
mg, 70%, white foam) as a 1:2 mixture of diastereomers (as
determined by 1H and 31P NMR): Rf ) 0.42 (10% MeOH:
CHCl3); 1H NMR (CDCl3) δ 10.05 and 9.92 (1H, s, 2:1 mixture),
7.37 (5H, s), 6.28 (1H, t, J ) 10.4 Hz), 5.04 (2 H, dd, J HCNP
)
9.0 Hz), 4.50 (1H, m), 4.23 (2H, dd, J HCNP ) 7.5 Hz), 4.08 (1H,
m), 3.41 (4H, m), 3.05 (1H, s), 2.65 (3H, d, J ) 9.6 Hz), 2.42
(1H, m), 2.12 (1H, m), 1.88 and 1.82 (3H, s, 2:1 mixture); 31P
NMR (CDCl3) δ -14.47, -14.57 (1:2 mixture); HRMS (C20H27
-
N3O7BrP) calcd 532.0848 (M + H)+, found 532.0847.
Meth od 2: Benzyl alcohol (0.85 mL, 8.26 mmol) was
dissolved in CH3CN/CH2Cl2 (5:20 mL) and cooled to -78 °C.
Phosphorus trichloride (4.13 mL, 2.0 M in CH2Cl2) was added
slowly followed by the dropwise addition of diisopropylethyl-
amine (2.16 mL, 12.4 mmol). The reaction mixture was allowed
to stir at -78 °C for 15 min. N-Methyl-N-(2-bromoethyl)amine
hydrobromide (1.81 g, 8.26 mmol) was dissolved in anhydrous
CH3CN (20 mL) and added to the reaction mixture dropwise.
Diisopropylethylamine (4.32 mL, 24.8 mmol) was added drop-
wise and the reaction mixture was warmed to -60 °C and
stirred for 20 min. Thymidine (1.0 g, 4.13 mmol) was coevapo-
rated several times with anhydrous pyridine (6 × 30 mL) and
then dissolved in pyridine (30 mL) and cooled to -45 °C. The
mixture of thymidine in pyridine was then titrated with the
reaction mixture containing intermediate 11a until thymidine
disappeared. The disappearance of thymidine was monitored
by TLC (90:10 CHCl3:MeOH). The reaction mixture was
oxidized by the dropwise addition of tert-butyl hydroperoxide
(1.65 mL, 5.0-6.0 M in decane) at -45 °C and warmed to 0
°C over 30 min. Saturated aqueous NH4Cl (50 mL) was added,
and the aqueous layer was extracted with CH2Cl2 (3 × 50 mL).
The combined organic layers were dried over Na2SO4 and
5′-Th ym id yl N-Meth yl-N-(2-br om oeth yl)p h osp h or a m -
id ic Acid , Tr ieth yla m m on iu m Sa lt (5a ). Phosphoramidate
4a (10.0 mg, 0.019 mmol) was dissolved in THF (1 mL). Pd/C
(10%, 5 mg) was suspended in THF (1 mL) and transferred to
the flask containing phosphoramidate 4a . The flask was
equipped with a balloon filled with hydrogen, and the reaction
mixture was stirred for 10 min at room temperature. Triethyl-
amine (2.8 µL, 0.020 mmol) was added and stirring was
continued for 1-2 min. The catalyst was filtered, and the
filtrate was concentrated to 0.50 mL and transferred to an
NMR tube. Complete conversion to phosphoramidate anion 5a
was confirmed by 31P NMR. The remaining THF was removed
by rotary evaporation and the unstable product 5a was
immediately characterized by mass spectrometry or used in a