Synthesis and biological activity of novel 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs

Article abstract of DOI:10.1021/jm000301j

A series of novel haloethyl and piperidyl phosphoramidate FdUMP prodrug analogues has been synthesized, and the growth inhibitory activity of these compounds has been evaluated against L1210 mouse leukemia cells. All compounds exhibited potent inhibition

Full text of DOI:10.1021/jm000301j

J . Med. Chem. 2000, 43, 4313-4318
4313
Syn th esis a n d Biologica l Activity of Novel 5-F lu or o-2′-d eoxyu r id in e
P h osp h or a m id a te P r od r u gs
Caren L. Freel Meyers, Liping Hong, Carolyn J oswig, and Richard F. Borch*
Departments of Chemistry and Pharmacology, University of Rochester, Rochester, New York 14642, and Department of
Medicinal Chemistry and Molecular Pharmacology and the Cancer Center, Purdue University, West Lafayette, Indiana 47907
Received J uly 18, 2000
A series of novel haloethyl and piperidyl phosphoramidate FdUMP prodrug analogues has been
synthesized, and the growth inhibitory activity of these compounds has been evaluated against
L1210 mouse leukemia cells. All compounds exhibited potent inhibition of L1210 cell
proliferation with IC50 values in the nanomolar range. Growth inhibition was reversed by the
addition of 5 µM thymidine, suggesting a mechanism of action involving the intracellular release
of FdUMP. 31P NMR studies carried out on model haloethyl phosphoramidates confirm the
release of nucleotide via cyclization of the phosphoramidate anion to the aziridinium ion
intermediate followed by hydrolysis of the P-N bond. The data suggests that <50% of the
prodrug is converted to FdUMP intracellularly by this pathway. Piperidyl phosphoramidate
analogues are also converted to nucleotide intracellularly, presumably by the action of an
endogenous phosphoramidase.
In tr od u ction
benzotriazolyl moiety would undergo direct hydrolysis
while the nitrofuryl moiety would undergo bioreductive
activation11 to generate the same phosphoramidate
anion (B) intracellularly as shown in Scheme 2. Three
amide groups were investigated; the methyl bromoethyl
and bis(bromoethyl) groups were selected on the basis
of previous findings10 that, following generation of the
phosphoramidate anion (B), they would cyclize to the
aziridinium ion (C) and undergo P-N bond hydrolysis
to liberate the corresponding nucleotide (D; Scheme 2).
The piperidine analogues 1c and 2c (Scheme 1) were
synthesized because, although the corresponding phos-
phoramidate anion is chemically stable, it presumably
undergoes bioactivation resulting in significant growth
inhibitory activity.10 Finally, the 5′-amino analogue 10
(Scheme 3) was prepared to determine whether the
corresponding nucleotide analogue might also show
growth inhibitory properties; presumably this compound
would not be a substrate for 5′-nucleotidase. We report
herein the synthesis of nitrofuryl and benzotriazolyl
nucleotide prodrugs and the growth inhibitory activity
of these compounds.
Inhibition of DNA biosynthesis has become a key
objective in the design of anticancer chemotherapeutic
agents.1,2 To this end, several modified nucleoside and
pyrimidine antimetabolites requiring intracellular phos-
phorylation have been developed as inhibitors of the
enzyme thymidylate synthase (TS), a key enzyme for
the endogenous production of thymidine 5′-monophos-
phate (TMP).3,4 The emergence of resistance to such
analogues, likely as a result of reduced enzymatic
activation by the tumor cell,5 has prompted efforts to
develop prodrugs that release nucleotides intracellularly
and circumvent the requirement for intracellular
phosphorylation.6-8 For example, the amino acid phos-
phoramidate derivatives of antiviral and antitumor
nucleosides7 have demonstrated potential as nucleotide
prodrugs. However, the initial step in the activation of
these compounds can potentially occur extracellularly,
because it relies on the function of nonspecific esterases.
Furthermore, the final activation step requires cleavage
of the P-N bond, presumably catalyzed by an endog-
enous phosphoramidase;8 this process may be suf-
ficiently slow that little intracellular accumulation of
the nucleotide occurs.9
Resu lts a n d Discu ssion
Ch em istr y. Syn th esis of Nu cleosid e P h osp h or -
a m id a tes. Target compounds 1 and 2 were synthesized
according to the procedure outlined in Scheme 1. Phos-
phoryl dichlorides 7a -c were prepared by the reaction
of phosphorus oxychloride with the appropriate amine
(6a -c) in the presence of triethylamine.10 Reaction of
7a -c with 2 equiv of 1-hydroxybenzotriazole in the
presence of pyridine afforded the highly reactive bis-
(benzotriazolyl) phosphoramidate intermediates 8a -c.
Reaction of 8a -c in situ with 5-FdU in the presence of
1-methylimidazole as a catalyst afforded diastereomeric
mixtures of compounds 1a -c in 43-60% yield. Nitro-
furyl and benzyl nucleoside phosphoramidates 2a -c
were prepared in 70-75% yield as diastereomeric
mixtures from the corresponding benzotriazolyl phos-
Our interest in the development of phosphoramidate
nucleotide prodrugs10 has led to the design and synthe-
sis of a new series of haloethyl nucleoside phosphor-
amidates, 1a ,b and 2a ,b (Scheme 1), that undergo facile
P-N bond hydrolysis following intracellular activation
to liberate the known nucleotide TS inhibitor, 5-fluoro-
2′-deoxyuridine 5′-monophosphate (FdUMP). The benzo-
triazolyl and nitrofuryl moieties were introduced as
delivery groups for the corresponding 5-fluorodeoxy-
uridyl phosphoramidates. It was anticipated that the
* To whom correspondence should be addressed at: Department of
Medicinal Chemistry and Molecular Pharmacology, Purdue University,
West Lafayette, IN 47907. Tel: (765) 494-1403. Fax: (765) 494-1414.
E-mail: rickb@pharmacy.purdue.edu.
10.1021/jm000301j CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/10/2000

4314 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Freel Meyers et al.
Sch em e 1a
a
Reagents and conditions: (a) triethylamine, CH2Cl2, -40 to 0 °C, 1 h; (b) HOBT, pyridine, 0 °C to rt, 2 h; (c) 5-fluorodeoxyuridine (or
thymidine), N-methylimidazole, rt, overnight; (d) nBuLi (or RMgBr) and benzyl alcohol (or 1-hydroxymethyl-5-nitrofuran), THF, rt, then
1 (or 3), -20 to 0 °C, 1 h; (e) H2, 10% Pd/C, THF, 10 min, rt; (f) triethylamine, rt, 2 min.
Sch em e 2
Sch em e 3a
a
Reagents and conditons: (a) LiHMDS, 2-hydroxymethyl-5-
nitrofuran, THF, -7.8 to -10 °C, 3 h; (b) 5-fluoro-5′-amino-2′-
phoramidates 1a -c and either 2-hydroxymethyl-5-
nitrofuran or benzyl alcohol in the presence of base.
5′-Amino nucleoside phosphoramidate analogue 10
was prepared as shown in Scheme 3. Treatment of 7a
with 2-hydroxymethyl-5-nitrofuran in the presence of
LiHMDS afforded phosphoryl monochloride 9 in 81%
yield. Subsequent reaction of 9 with 5-fluoro-5′-amino-
2′-deoxyuridine (prepared according to Garnett et al.)12
in the presence of poly(4-vinylpyridine) (PVP) provided
the 5′-amino nucleoside phosphoramidate 10 in 61%
yield.
Thymidine phosphoramidates 3 and 4 were synthe-
sized to use as model compounds for 31P NMR experi-
ments; it was assumed that the 5-substituent on the
pyrimidine ring would have little effect on the reaction
rates of the phosphoramidate anion. Compound 13 was
prepared for the purpose of conducting studies to
ascertain the stability of the nitrofuryl analogues in
serum-containing medium. Thymidine phosphorami-
dates 3 and 4 were prepared as described above (Scheme
1). Quantitative conversion of the benzyl nucleoside
phosphoramidates 4a ,c to the corresponding phosphor-
deoxyuridine, PVP, EtOH, 0 °C to rt, 6 h.
amidate anions 5a ,c was accomplished by catalytic
hydrogenolysis followed by treatment with triethyl-
amine. The phosphoramidate anion prepared by this
method was used without further purification in the 31P
NMR experiments discussed below. More recently, the
development of a one-pot strategy for the synthesis of
nucleoside phosphoramidate analogues was undertaken
in an attempt to maximize the conversion of nucleoside.
This alternative approach for the synthesis of phosphor-
amidates such as 4a (Scheme 4) employs the in situ
generation of a highly reactive phosphorus(III) chloride
intermediate for the phosphorylation of the nucleoside.
Phosphorus trichloride was reacted with benzyl alcohol
in the presence of diisopropylethylamine followed by
reaction with N-methyl-N-(2-bromoethyl)amine hydro-
bromide to generate monochloro intermediate 11. Thy-
midine in pyridine was then titrated slowly with 11; the
rapid disappearance of nucleoside made it possible to
monitor the reaction by TLC. It should be noted that
fast addition of 11 resulted in the formation of 3′-

Synthesis of Novel Phosphoramidate Prodrugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4315
Sch em e 4a
Sch em e 5
were not expected to exhibit significant in vitro activity.
However, potencies comparable to the corresponding
haloethyl phosphoramidates were observed for 1a ,c at
the longest drug exposure time (48 h). It was initially
hypothesized that the activity observed in the piperidyl
phosphoramidate series was consistent with a mecha-
nism of action involving intracellular liberation of the
phosphoramidate anion followed by protonation on the
piperidyl nitrogen at physiologic pH and hydrolysis of
the P-N bond to generate FdUMP. However, mecha-
nistic studies carried out on model piperidyl phosphor-
amidates13 indicate that hydrolysis is too slow to account
for the observed inhibition of cell proliferation. Presum-
ably, the piperidyl phosphoramidate anion undergoes
enzymatic activation to FdUMP.
a
Reagents and conditions: (a) benzyl alcohol or 1-hydroxy-
methyl-5-nitrofuran, diisopropylethylamine, CH3CN/CH2Cl2 (1:3),
-78 °C, 15 min; (b) N-methyl-N-(2-bromoethyl)amine hydrobro-
mide, CH3CN/CH2Cl2 (1:1.5), -78 to -60 °C, 20 min; (c) thymidine,
pyridine, -50 °C; (d) tert-butyl hydroperoxide, -50 to 0 °C, 30 min.
Ta ble 1. Growth Inhibition in L1210 Mouse Leukemia Cells
IC50, nM
treatment time, h
compd
R1
CH2CH2Br
CH2CH2Br
CH2CH2CH2CH2CH2
CH2CH2Br
R2
2
8
24
429 178 22
CH2CH2Br 276 96 11
843 733 77 13
1027 347 56 16
48
3.3
2.5
1a
1b
1c
2a
2b
2c
10
5-FU
CH3
CH3
CH2CH2Br
CH2CH2CH2CH2CH2
CH2CH2Br
CH2CH2Br 2074 519 103 18
1592 292 100 36
CH3
1101 504 108 34
2200 630 220 125
To determine whether the nitrofuryl phosphoramidate
analogues are stable under standard L1210 cell assay
conditions (10% horse serum in Fischer’s medium, pH
7.4, 37 °C), an experiment was performed in which
HPLC was used to monitor the stability of thymidyl
phosphoramidate 13. Under these conditions, >95% of
compound 13 remained after 8 h; 13 subsequently
disappeared over a longer time period (t1/2 ) 18 h).
phosphorylated and 3′,5′-bisphosphorylated byproducts.
Oxidation of phosphoramidite 12 with tert-butyl hydro-
peroxide afforded phosphoramidate 4a in 68% yield.
Phosphoramidate 6a was prepared in 77% yield (Scheme
4) using this strategy. Phosphorylation of nucleosides
using this approach has resulted in higher overall yields
and is the method of choice for the preparation of
nucleoside phosphoramidates.
B. 31P NMR Stu d ies. A 31P NMR study was carried
out for the purpose of elucidating the chemical activa-
tion of this series of nucleoside phosphoramidates and
to provide convincing evidence of P-N bond hydrolysis.
The first NMR experiment was designed to provide
information about the reactions of the phosphoramidate
anion, a key precursor in this prodrug strategy. Thymidyl
phosphoramidate anion 5a was synthesized as described
above, and its reactions were monitored by 31P NMR
under model physiologic conditions (ca. 100 mM in 0.4
M cacodylate buffer, pH 7.4, 37 °C). The resonance for
5a (A, -16.12 ppm) disappeared (t1/2 ) 3.7 min) to give
two major products (Scheme 5). The product of primary
biological interest is the nucleotide (B, -21.77 ppm),
arising from P-N bond hydrolysis of the aziridinium
ion. A second product (C, -14.82 ppm) is also formed
as a result of ring opening of the aziridinium ion. Two
minor byproducts were also formed and accounted for
<15% of the total product. Integration of the resonances
corresponding to trapping of the aziridinium ion by
water (solvolysis product, C) and the desired hydrolysis
of the P-N bond (nucleotide, B) reveals a solvolysis-to-
hydrolysis product ratio of 1:1. These data confirm the
occurrence of P-N bond hydrolysis to liberate nucleotide
in the activation of N-methyl-N-(2-bromoethyl) nucleo-
side phosphoramidate anion, but they also suggest that
Biologica l Activity. A. In Vitr o Activity. Com-
pounds 1a -c, 2a -c, and 10 were evaluated for growth
inhibitory activity against L1210 mouse leukemia cells;
the results are summarized in Table 1. All analogues
exhibited potent inhibition of cell proliferation that was
reversed by the addition of 5 µM thymidine, indicating
that these compounds act via inhibition of thymidylate
synthase. Compounds 1a ,b exhibited comparable poten-
cies and were significantly more potent than 5-FU.
Nitrofuryl analogues 2a ,b were less potent than benzo-
triazolyl analogues 1a ,b at all drug treatment times,
presumably as a result of slower enzymatic activation
of the nitrofuryl phosphoramidates. A similar trend was
observed for the 5′-amino nucleoside phosphoramidate
10. The data obtained for the haloethyl phosphorami-
date series are consistent with a proposed mechanism
of action involving intracellular generation of the phos-
phoramidate anion followed by cyclization to the aziri-
dinium ion and subsequent hydrolysis of the P-N bond
to liberate FdUMP.
Piperidyl phosphoramidates 1c and 2c are not capable
of undergoing the same type of activation as the
haloethyl nucleoside phosphoramidates and, as a result,

4316 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Freel Meyers et al.
using UV and/or the following stains: 1% 4-(p-nitrobenzyl)-
pyridine in acetone followed by heating and subsequent
treatment with 3% KOH in methanol (for detection of haloethyl
functionality), 3% phosphomolybdic acid in methanol followed
by heating, or p-anisaldehyde dip (1.85% p-anisaldehyde,
20.5% sulfuric acid, 0.75% acetic acid in 95% EtOH) followed
by heating.
All reactions were carried out under an atmosphere of
nitrogen or argon unless otherwise specified or reagents
containing water were used. All organic solvents were distilled
prior to use unless otherwise specified. Pyridine, triethylamine
and diisopropylamine were distilled prior to use. 1-Hydroxy-
benzotriazole and all nucleosides were dried by coevaporation
with pyridine prior to use.
In Vitr o Gr ow th In h ibition . Stock solutions of drugs were
prepared in 95% ethanol, and serial dilutions of drug were
prepared in ethanol such that 50 µL of drug solution added to
10 mL of cell suspension gave the desired final concentration.
L1210 cells in exponential growth were suspended in Fischer’s
medium supplemented with 10% horse serum, 1% glutamine,
and 1% antibiotic-antimycotic solution to give 10-mL volumes
e50% of the prodrug is ultimately converted to the
nucleotide.
A second 31P NMR experiment was carried out on
benzotriazolyl phosphoramidate 3a to confirm that
initial activation of the phosphoramidate prodrugs 1a -c
leads to the corresponding phosphoramidate anions. A
solution of phosphoramidate 3a was prepared (ca. 100
mM in 4.5:1 0.4 M cacodylate buffer/acetonitrile), and
the mixture was monitored by 31P NMR under physi-
ologic conditions (pH 7.4, 37 °C). As expected, the
resonances corresponding to the diastereomeric mixture
of compound 3a (-12.50, -12.78 ppm) were replaced
(t1/2 ) 4.7 min) by a single resonance corresponding to
the phosphoramidate anion (-15.99 ppm). The subse-
quent reactions of phosphoramidate anion resulted in
the formation of a variety of products arising both from
cleavage of the P-N bond and ring opening of the
aziridinium ion.13 Again, solvolysis product and the
desired nucleotide were formed in a 1:1 ratio.
of cell suspension at
a
final density of 3-6 × 104/mL.
A similar experiment was carried out (ca. 100 mM,
0.4 M cacodylate buffer, pH 7.4, 37 °C) on the piperidyl
phosphoramidate anion 5c.13 The resonance for pip-
eridyl nucleoside phosphoramidate anion 5c (-16.11
ppm) disappeared slowly (t1/2 ∼ 11 days) to give the
corresponding nucleotide. The exceptionally slow con-
version of piperidyl phosphoramidate anion to nucleo-
tide under physiologic conditions suggests that pro-
tonation on nitrogen followed by P-N bond hydrolysis
does not account for the observed growth inhibitory
activity of phosphoramidate prodrugs 1c and 2c. Recent
studies8 suggest that endogenous phosphoramidase
activity may account for the observed P-N bond hy-
drolysis in related phosphoramidate systems. Presum-
ably 5c undergoes a similar enzymatic conversion.
Appropriate volumes of the drug solution were transferred to
the cell suspensions, and incubation was continued for 2, 8,
24, or 48 h. The cells were spun down, resuspended in fresh
drug-free medium, and returned to the incubator. Cell counts
were determined 48 h after initiation of drug treatment. The
procedure was repeated for a range of drug concentrations and
the cell counts (reported as percent of control values) were
plotted versus concentration for each drug treatment time (the
control cell densities were in the range 5-7 × 105/mL). The
data are reported as IC50 values in nM.
5′-Th ym id yl 1-Ben zot r ia zolyl N-Met h yl-N-(2-b r om o-
eth yl)p h osp h or a m id a te (3a ). Phosphoramidic dichloride
(7a ) (1.05 g, 4.13 mmol) was dissolved in THF (3 mL) and
added dropwise to a stirred solution of 1-hydroxybenzotriazole
(1.11 g, 8.24 mmol) and pyridine (0.67 mL, 8.24 mmol) in THF
(20 mL) at 0 °C under an atmosphere of argon. The reaction
mixture was removed from the 0 °C bath and stirred at room
temperature for 2 h. The mixture was transferred to two
Wheaton tubes, and the pyridine hydrochloride was removed
by centrifugation under an atmosphere of argon. The super-
natant containing the reactive bis(benzotriazolyl) intermediate
8a was added in one portion to a stirred solution of thymidine
(0.50 g, 2.06 mmol) in pyridine (2 mL) at room temperature.
1-Methylimidazole (0.20 mL, 2.47 mmol) was added dropwise,
and stirring was continued at room temperature for 12 h. THF
was removed under reduced pressure. The residue was diluted
with CHCl3 (50 mL) and washed with saturated NaHCO3 (1
× 25 mL) to remove excess HOBT. The organic layer was
washed with saturated NH4Cl (2 × 25 mL) to remove 1-meth-
ylimidazole. The aqueous layers were combined and extracted
with CHCl3 (25 mL). The combined organic layers were dried
over Na2SO4 and concentrated under reduced pressure. The
residue was purified by silica gel chromatography (75:115:10
EtOAc:CHCl3:EtOH) to give 3a (649 mg, 56%) as a white foam
that was a 1:1.4 mixture of diastereomers (as determined by
1H and 31P NMR): Rf ) 0.17 (75:115:10 EtOAc:CHCl3:EtOH);
1H NMR (CDCl3) δ 9.18 and 9.10 (1H, s, 1:1.4 mixture), 8.00
(1H, t) 7.71 (1H, m) 7.60-7.29 (3H, m), 6.34 and 6.27 (1 H, t,
1.4:1 mixture), 4.98 (1H, s), 4.53 (3H, m), 4.25-4.05 (1H, m),
3.48 (4H, m), 2.94 (3H, d, J ) 10.3), 2.52-2.15 (2H, m), 1.92
and 1.82 (3H, s, 1.4:1 mixture); 31P NMR (CDCl3) δ -14.56,
-14.98 (1:1.4); HRMS (C19H24N6O7BrP) calcd 559.0706 (M +
H)+, found 559.0707
Su m m a r y
The synthesis of a new series of nucleoside phosphor-
amidate prodrugs bearing benzotriazolyl and nitrofuryl
esters was accomplished. Potent inhibition of L1210 cell
proliferation was observed for the 5-fluorodeoxyuridyl
analogues that was reversed by the addition of thymi-
dine. 31P NMR activation studies in model systems
confirm the release of nucleotide via P-N bond hydroly-
sis of the aziridinium ion intermediate. However, the
data suggests that e50% of the prodrug is converted to
FdUMP intracellularly. Finally, the piperidyl phosphor-
amidate anion is stable under model physiologic condi-
tions; the activity of this compound presumably results
from enzymatic conversion to FdUMP.
Exp er im en ta l Section
1
Ma ter ia ls a n d Meth od s. All 31P and H NMR spectra were
recorded on either a 250 or 270 MHz Bruker instrument unless
specified otherwise. All 31P NMR spectra were acquired using
broadband gated decoupling. 31P chemical shifts are reported
in parts per million using 1% triphenylphosphine oxide in
benzene-d6 as the coaxial reference (triphenylphosphine oxide/
benzene-d6 has a chemical shift of +24.7 ppm relative to 85%
phosphoric acid). Variable-temperature 31P NMR kinetics
experiments were controlled using the Bruker variable tem-
perature unit. 1H chemical shifts are reported in parts per
million from tetramethylsilane. Flash chromatography using
silica gel grade 60 (230-400 mesh) was carried out for all
chromatographic separations. Thin-layer chromatography was
performed using Analtech glass plates precoated with silica
gel (250 microns). Visualization of the plates was accomplished
5-F lu or o-2′-d eoxy-5′-u r id yl 1-ben zotr ia zolyl N-m eth yl-
N-(2-br om oeth yl)p h osp h or a m id a te (1a ) was prepared from
7a as described for 3a in 60% yield. 5-FUdR (2 mmol) was
1
used instead of thymidine: Rf ) 0.41 (10% MeOH:CHCl3); H
NMR (CDCl3) δ 8.03-7.34 (5H, m), 6.23 (1H, m), 4.43 (3H,
m), 4.12 (1H, m), 3.51 (4H, m), 3.01 (3H, d, J ) 4.3 Hz), 2.49
(1H, m), 2.23 (1H, m); 31P NMR (THF) δ -13.25, -13.71;
HRMS (C18H21BrFN6O7P) calcd 563.0455 (M + H)+, found
563.0444.

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

4318 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Freel Meyers et al.
(2) Danenberg, P. V. Thymidylate synthetase-A target enzyme in
cancer chemotherapy. Biochim. Biophys. Acta 1977, 473, 73-
92.
kinetics experiment. Further attempts to isolate the reaction
product resulted in decomposition of the phosphoramidate
anion: 31P NMR (4.5:1 0.4 M cacodylate buffer:CH3CN, pH
7.4, 37 °C) δ -15.99; HRMS (C13H21N3O7BrP) calcd 442.0379
(M + H)+, found 442.0372.
(3) (a) Heidelberg, C.; Danenberg, P. V.; Langenbach, R. J . Thymidy-
late synthetase: Mechanism of inhibition by 5-fluoro-2′-deoxy-
uridylate. Biochem. Biophys. Res. Commun. 1972, 48, 1565-
1571. (b) Santi, D. V.; McHenry, C. S.; Sommer, H. Mechanism
of interaction of thymidylate synthetase with 5-fluorodeoxy-
uridylate. Biochemistry 1974, 13, 471-481. (c) Heidelberger, C.;
Hartmann, K.-U. Studies on fluorinated pyrimidines XIII.
Inhibition of thymidylate synthase. J . Biol. Chem. 1961, 236,
3006-3013. (d) Cohen, S. S.; Flaks, J . G.; Barner, H. D.; Loeb,
M. R.; Lichtenstein, J . The mode of action of 5-fluorouracil and
its derivatives. Proc. Natl. Acad. Sci. U.S.A. 1958, 44, 1004-
1012.
(4) (a) Santi, D. V. Perspectives on the design and biochemical
pharmacology of inhibitors of thymidylate synthetase. J . Med.
Chem. 1980, 23, 103-111. (b) Maggiora, L.; Chang, C. C. T.-C.;
Torrence, P. F.; Mertes, M. P. 5-Nitro-2′-deoxyuridine 5′-
phosphate. A mechanism-based inhibitor of thymidylate syn-
thetase. J . Am. Chem. Soc. 1981, 103, 3192-3198.
(5) Inaba, M.; Naoe, Y.; Mitsuhashi, J . Mechanisms for 5-fluorouracil
resistance in human colon cancer DLD-1 cells. Biol. Pharm. Bull.
1998, 21, 569-573.
(6) Farquhar, D.; Chen, R.; Khan, S. 5′-[4-(Pivaloyloxy)-1,3,2-
dioxaphosphorinan-2-yl]-2′-deoxy-5-fluorouridine: A membrane-
permeating prodrug of 5-fluoro-2′-deoxyuridylic acid (FdUMP).
J . Med. Chem. 1995, 38, 488-495.
(7) (a) Winter, H.; Maeda, Y.; Uchida, H.; Mitsuya, H.; Zemlicka, J .
Phosphodiester amidates of unsaturated nucleoside analogues:
Synthesis and anti-HIV activity. J . Med. Chem. 1997, 40, 2191-
2195. (b) Valette, G.; Pompon, A.; Girardet, J .-L.; Cappellacci,
L.; Franchetti, P.; Grifantini, M.; La Colla, P.; ′Loi, A. G.;
Pe´rigaud, C.; Gosselin, G.; Imbach, J .-L. Decomposition path-
ways and in vitro HIV inhibitory effects of IsoddA pronucleo-
tides: Toward a rational approach for intracellular delivery of
nucleoside 5′- monophosphates. J . Med. Chem. 1996, 39, 1981-
1990. (c) McGuigan, C.; Cahard, D.; Sheeka, H. M.; De Clercq,
E.; Balzarini, J . Aryl phosphoramidate derivatives of d4T have
improved anti-HIV efficacy in tissue culture and may act by the
generation of a novel intracellular metabolite. J . Med. Chem.
1996, 39, 1748-1753. (d) McGuigan, C.; Pathirana, R. N.;
Balzarini, J .; De Clerq, E. Intracellular delivery of bioactive AZT
nucleotides by aryl phosphate derivatives of AZT. J . Med. Chem.
1993, 36, 1048-1052.
5′-Th ym id yl P ip er id ylp h osp h or a m id ic Acid , Tr ieth yl-
a m m on iu m Sa lt (5c). Phosphoramidate 4c (7.0 mg, 0.015
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 4c. The flask was equipped with
a balloon filled with hydrogen, and the reaction was stirred
for 10 min at room temperature. Triethylamine (2.2 µL, 0.016
mmol) was added and stirring was continued for 1-2 min. The
catalyst was filtered and the filtrate was concentrated by
rotary evaporation. Complete conversion to phosphoramidate
anion 5c was confirmed by 31P NMR. Phosphoramidate anion
5c was used without further purification for a kinetics experi-
ment: 31P NMR (4.5:1 0.4 M cacodylate buffer:CH3CN, pH 7.4,
37 °C) δ -16.11; HRMS (C15H24N3O7P) calcd 390.1430 (M +
H)+, found 390.1428.
5-Nitr o-2-fu r ylm eth yl N-Meth yl-N-(2-br om oeth yl)ph os-
p h or a m id och lor id a te (9). To a solution of 2-hydroxymethyl-
5-nitrofuran (1.08 g, 7.55 mmol) in THF (5 mL) at -78 °C was
added LiHMDS (7.60 mL of 1 M solution in THF, 7.6 mmol)
dropwise. The reaction was continued at -78 °C for 5 min.
The resulting alkoxide was added to a solution of phosphoryl
dichloride 7a (2.02 g, 7.92 mmol) in THF (3 mL) at -78 °C.
The reaction mixture was slowly warmed to -12 °C over 3 h,
quenched with saturated aqueous NH4CI, and extracted with
EtOAc. The organic layer was dried and concentrated. The
residue was purified by chromatography (0 to 1% MeOH:
CHCl3) to afford 9 (2.21 g, 81%) as a pale yellow oil: Rf 0.43
1
(0.7% MeOH:CHCl3); H NMR (CDCl3) δ 7.32 (IH, d, J ) 4.3
Hz), 6.77 (1H, d, J ) 4.2 Hz), 5.20 (2H, m), 3.50 (4H, m), 2.84
(3H, d, J ) 11.3 Hz); 31P NMR (CH2Cl2) δ -7.71.
5-F lu or o-5′-a m in o-2′-d eoxy-5′-u r id yl 5-Nit r o-2-fu r yl-
m eth yl N-Meth yl-N-(2-br om oeth yl)ph osph or am idate (10).
To a suspension of 5-fluoro-5′-amino-2′-deoxyuridine12 (119 mg,
0.485 mmol) in anhydrous EtOH (2 mL) at 0 °C was added
the phosphoramidic chloride 9 (184 mg, 0.509 mmol) in THF
(1 mL) Cross-linked poly(4-vinylpyridine) (PVP; 500 mg, 25%
cross-linked) was then added. The mixture was allowed to stir
at room temperature for 6 h then filtered through a small pad
of Celite to remove the PVP. The filtrate was concentrated and
chromatographed (10% MeOH:CHC13) to yield 10 (168 mg,
61%) as a glassy solid: Rf ) 0.35 (MeOH:CHC13 1:5); 1H NMR
(300 MHz, CD3OD) δ 7.92 (1H, dd, J ) 6.8, 1.3 Hz), 7.43 (1H,
d, J ) 3.7 Hz), 6.80 (1H, d, J ) 3.7 Hz), 6.16 (1H, m), 5.05
(2H, m), 4.35 (1H, m), 3.89 (1H, m), 3.54 (2H, m), 3.42 (2H,
m), 3.19 (2H, m), 2.75 (3H, m), 2.23 (2H, m); 31P NMR (EtOH)
δ -6.85; HRMS (C17H22BrFN5O9P) calcd 574.0314 (M + Na)+,
found 574.0302.
(8) Abraham, T. W.; Kalman, T. I.; McIntee, E. J .; Wagner, C. R.
Synthesis and biological activity of aromatic amino acid phos-
phoramidates of 5-fluoro-2′-deoxyuridine and 1-Β-arabinofur-
anosylcytosine: Evidence of phosphoramidase activity. J . Med.
Chem. 1996, 39, 4569-4575.
(9) McIntee, E. J .; Remmel, R. P.; Schinazi, R. F.; Abraham, T. W.;
Wagner, C. R. Probing the mechanism of action and decomposi-
tion of amino acid phosphomonoester amidates of antiviral
nucleoside prodrugs. J . Med. Chem. 1997, 40, 3323-3331.
(10) Fries, K. M.; J oswig, C.; Borch, R. F. Synthesis and evaluation
of 5-fluoro- 2′-deoxyuridine phosphoramidate analogues. J . Med.
Chem. 1995, 38, 2672-2680.
(11) Borch, R. F.; Liu, J .; Schmidt, J . P.; Marakovits, J . T.; J oswig,
C.; Gipp, J . J .; Mulcahy, R. T. Synthesis and evaluation of
nitroheterocyclic phosphoramidates as hypoxia-selective alkyl-
ating agents. J . Med. Chem. 2000, 43, 2258-2265.
(12) Henn, T. F. G.; Garnett, M. C.; Chabra, S. R.; Bycroft, B. W.;
Baldwin, R. W. Synthesis of 2′-deoxyuridine and 5-fluoro-2′-
deoxyuridine derivatives and evaluation in antibody targeting
studies J . Med. Chem. 1993, 36, 1570.
(13) Freel Meyers, C. L.; Borch, R. F. Activation mechanisms of
nucleoside phosphoramidate prodrugs. J . Med. Chem. 2000, 43,
4319-4327 (accompanying manuscript).
Ack n ow led gm en t. Financial support from Grants
R01 CA34619 and T32 CA09634, provided by the
National Cancer Institute, is gratefully acknowledged.
Refer en ces
(1) Cancer Chemotherapeutic Agents; Foye, W. D., Ed.; American
Chemical Society: Washington, DC, 1995; Chapter 3.
J M000301J