Activation mechanisms of nucleoside phosphoramidate prodrugs

Article abstract of DOI:10.1021/jm000302b

A series of thymidine and tetrahydrofurfuryl phosphoramidates bearing haloethyl or piperidyl substituents was synthesized and used to investigate the activation mechanisms of nucleoside phosphoramidate prodrugs. Structure assignments for the tetrahydrofurfuryl reaction products were confirmed by comparison to authentic samples. Structural assignments for thymidine phosphoramidate reaction products were made by analogy to the tetrahydrofurfuryl products. Generation of the phosphoramidate anion leads to cyclization and subsequent nucleophilic attack at carbon and phosphorus of the resulting aziridinium ion intermediate to give the observed products. Nucleophilic attack by water at carbon and phosphorus occurs without selectivity, supporting a mechanism of action of haloethylamine nucleoside prodrugs involving intracellular release of the nucleotide. Activation of the benzotriazolyl piperidyl phosphoramidates is followed by P-N bond hydrolysis; this reaction is subject to specific acid catalysis and to nucleophilic catalysis by 1-hydroxybenzotriazole. These results suggest that the mechanism of action of the piperidyl nucleoside phosphoramidates involves the intracellular release of the active nucleotide following P-N bond cleavage, presumably by the action of an endogenous phosphoramidase.

Full text of DOI:10.1021/jm000302b

J . Med. Chem. 2000, 43, 4319-4327
4319
Activa tion Mech a n ism s of Nu cleosid e P h osp h or a m id a te P r od r u gs
Caren L. Freel Meyers and Richard F. Borch*
Department of Chemistry, 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 thymidine and tetrahydrofurfuryl phosphoramidates bearing haloethyl or piperidyl
substituents was synthesized and used to investigate the activation mechanisms of nucleoside
phosphoramidate prodrugs. Structure assignments for the tetrahydrofurfuryl reaction products
were confirmed by comparison to authentic samples. Structural assignments for thymidine
phosphoramidate reaction products were made by analogy to the tetrahydrofurfuryl products.
Generation of the phosphoramidate anion leads to cyclization and subsequent nucleophilic
attack at carbon and phosphorus of the resulting aziridinium ion intermediate to give the
observed products. Nucleophilic attack by water at carbon and phosphorus occurs without
selectivity, supporting a mechanism of action of haloethylamine nucleoside prodrugs involving
intracellular release of the nucleotide. Activation of the benzotriazolyl piperidyl phosphora-
midates is followed by P-N bond hydrolysis; this reaction is subject to specific acid catalysis
and to nucleophilic catalysis by 1-hydroxybenzotriazole. These results suggest that the
mechanism of action of the piperidyl nucleoside phosphoramidates involves the intracellular
release of the active nucleotide following P-N bond cleavage, presumably by the action of an
endogenous phosphoramidase.
In tr od u ction
5′-phosphate.2 However, little is known about the
reactivity of haloethyl phosphoramidate anions bearing
ester substituents.
The development of nucleoside prodrugs capable of
undergoing intracellular activation to the corresponding
nucleotide has become an area of intense interest.1 Our
interest in developing new methods for the intracellular
delivery of nucleotides has resulted in the synthesis of
several biologically active nucleoside phosphoramidates
that act as prodrugs for the known thymidylate syn-
thase inhibitor, 5-fluoro-2′-deoxyuridine 5′-monophos-
phate (FdUMP).2 These nucleotide prodrug analogues
exploit the reactivity of a haloethyl 5′-phosphoramidate
moiety that undergoes intracellular activation to the
haloethyl phosphoramidate anion followed by rapid
P-N bond hydrolysis to liberate the corresponding
nucleotide.
The haloethyl nucleoside phosphoramidates prepared
in this laboratory bear structural resemblance to the
known anticancer agent phosphoramide mustard.3 The
use of haloethyl phosphoramidates as alkylating agents
for the treatment of cancer is well-documented.4 Lude-
man and others5 have carried out extensive investiga-
tions of the activation mechanisms of phosphoramide
mustard and related phosphorodiamidates that are
capable of undergoing cyclization to the corresponding
aziridinium ion intermediate. In these cases, the desired
reaction at carbon of the aziridinium ion by nucleophiles
present in DNA results in alkylation of DNA, although
significant hydrolysis of the P-N bond is known to occur
as well.5 It was anticipated that the haloethyl moiety
of our nucleoside prodrug analogues would undergo
P-N bond hydrolysis following activation and, as a
consequence, serve as a masking group for the desired
Thus, activation studies were carried out in this
laboratory on model haloethyl and piperidyl phospho-
ramidates for which the parent nucleoside phosphora-
midate prodrugs are known to have growth inhibitory
activity.2 The purpose of these studies was to provide
convincing evidence that the activation process involves
hydrolysis of the P-N bond, supporting a mechanism
of action involving intracellular release of the desired
nucleotide. The complexity of the results obtained in
studies carried out on model haloethyl phosphorami-
dates raised numerous questions about the proposed
activation mechanisms of the parent nucleotide phos-
phoramidate prodrugs.2 Furthermore, the piperidyl
phosphoramidate analogues, which were assumed to be
stable analogues, displayed surprising growth inhibitory
activity in L1210 cells. These perplexing results prompted
a closer investigation of the activation mechanisms of
nucleotide phosphoramidate prodrugs. We report herein
the synthesis and activation mechanisms of model
thymidine and tetrahydrofurfuryl phosphoramidates.
Resu lts a n d Discu ssion
Syn th esis of Ha loeth yl P h osp h or a m id a tes. The
reaction profiles observed for the nucleoside phospho-
ramidate analogues were complex. Therefore, tetrahy-
drofurfuryl phosphoramidates were synthesized and
used as models for the investigation of the activation
processes. The facile synthesis and characterization of
authentic tetrahydrofurfuryl phosphoramidates have
allowed us to confirm the structures of reaction products
in this system. Confirmation of the structurally related
but more complex nucleoside phosphoramidate reaction
products was then accomplished by analogy to the
tetrahydrofurfuryl model system.
* 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/jm000302b CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/05/2000

4320 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Freel Meyers and Borch
Ch a r t 1
Sch em e 2a
a
Reagents and conditions: (a) i. tetrahydrofurfuryl alcohol,
TEA, CH2Cl2, -10 °C to rt, 20 min, ii. benzyl alcohol, TEA, CH2Cl2,
0 °C, 1 h; (b) CH3NHCH2CH2X, TEA, THF or CH3CN, rt, 1 h; (c)
i. H2, Pd/C, THF, rt, 5 min, ii. TEA.
Sch em e 1a
a
Reagents and conditions: (a) tetrahydrofurfuryl alcohol, LiH-
MDS, THF, -78 °C to rt, 45 min; (b) HOBT, TEA, THF, rt, 1 h 15
min.
F igu r e 1. Reaction of phosphoramidate anion 1a in cacody-
late buffer (ca. 100 mM, pH 7.4, 37 °C): A, solvolysis product
10; B, amine-trapped product 11; C, buffer product 12; TMP,
thymidine 5′-monophosphate. Chemical shifts are reported
relative to triphenylphosphine oxide reference. See Results and
Discussion section for details.
Thymidyl phosphoramidate anion 1a and benzotria-
zolyl thymidyl phosphoramidate 2a (Chart 1) were
prepared as described in the accompanying paper.2
Benzotriazolyl tetrahydrofurfuryl N-methyl-N-(2-bro-
moethyl)phosphoramidate (3) was prepared as a 1:1
mixture of diastereomers as shown in Scheme 1. Treat-
ment of phosphoryl dichloride 46 with the lithium
alkoxide of tetrahydrofurfuryl alcohol in THF afforded
the tetrahydrofurfuryl phosphoryl monochloride 5 in
87% yield. Reaction of 5 with 1-hydroxybenzotriazole
(HOBT) in the presence of triethylamine resulted in the
formation of the desired benzotriazolyl phosphoramidate
3 in 67% yield.
Preparation of haloethyl phosphoramidate anion 6a
was accomplished as shown in Scheme 2. Phosphorus
oxychloride was reacted with tetrahydrofurfuryl alcohol
in the presence of triethylamine, and the resulting
intermediate was treated with benzyl alcohol in triethy-
lamine to form the phosphodiester monochloride 7 in
55% yield as a 1:1 mixture of diastereomers. Compound
7 was treated with N-methyl-N-(2-bromoethyl)amine
hydrobromide in the presence of triethylamine in THF
to give benzyl ester 8a in 70% yield. Catalytic hydro-
genolysis in THF resulted in quantitative conversion of
benzyl ester 8a to phosphoramidate anion 6a (as
determined by 31P NMR). Haloethyl phosphoramidate
anion 6a is unstable and was therefore used im-
mediately in the 31P NMR kinetics experiments de-
scribed below.
31P NMR Kin etics of N-Meth yl-N-(2-br om oeth yl)-
p h osp h or a m id a tes. The reactions of haloethyl phos-
phoramidates (Chart 1) were studied by using 31P NMR
under model physiologic conditions (4.5:1 0.4 M cacody-
late buffer/CH3CN, 37 °C, pH 7.4). 31P Chemical shifts
are reported in ppm using 1% triphenylphosphine oxide
in benzene-d6 as the coaxial reference. A kinetic analysis
of the reaction was performed using the Quattro Pro
optimization routine as described in the Experimental
Section. The results for compound 1a are shown in
Figure 1. Thymidine phosphoramidate 1a (-16.12 ppm)
disappeared with a rate constant k ) 0.19 ( 0.02 min-1
(t1/2 ) 3.7 min). Disappearance of 1a resulted in the
formation of four products (Scheme 3): solvolysis prod-
uct 10 (A, -14.82 ppm), amine-trapped product 11 (B,
-15.94 ppm), transient unknown product 12 (C, -16.52
ppm), and thymidine monophosphate (TMP, -21.77
ppm). The steady-state concentrations of products 10
and TMP were ca. 43% and 43%, respectively, of the

Mechanisms of Nucleoside Phosphoramidate Prodrugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4321
Sch em e 3a
a
Reagents and conditions: (a) 4.5:1 0.4 M cacodylate buffer/acetonitrile, pH 7.4, 37 °C.
consumed. Slow conversion of unknown product 12,
presumably to TMP, was also observed.
The complex mixture of phosphate and phosphora-
midate anion products observed in the reaction of
phosphoramidate 1a made product isolation and char-
acterization difficult as a method for structure verifica-
tion. An alternative strategy for product characteriza-
tion was sought using 31P NMR. Characterization of the
peak corresponding to TMP (Figure 1) was accomplished
by 31P NMR comparison to authentic TMP. Addition of
commercially available TMP to the NMR tube contain-
ing the reaction mixture of compound 1a resulted in an
increase in the resonance at -21.77 ppm, confirming
the structure assignment for the reaction product TMP.
Reaction products 10-12 were subsequently character-
ized by comparison to reaction products formed in the
structurally analogous tetrahydrofurfuryl model system.
F igu r e 2. Reaction of phosphoramidate anion 1a in cacody-
late buffer (ca. 100 mM, pH 7.4, 37 °C). Data points were
measured from 31P NMR peak areas; the solid lines represent
the best-fit values calculated from least-squares minimization
using the Quattro Pro optimization routine: (9) phosphora-
midate anion 1a ; (b) solvolysis product 10; (4) hydrolysis
product TMP; (0) amine-trapped product 11; (O) buffer-trapped
product 12.
Tetrahydrofurfuryl phosphoramidate 6a was synthe-
sized (Scheme 2) and monitored under model physiologic
conditions by 31P NMR. The experiment carried out on
compound 6a revealed a reaction profile nearly identical
to that observed for thymidine phosphoramidate 1a
(Scheme 3). The resonance for tetrahydrofurfuryl phos-
phoramidate anion 6a disappeared (k ) 0.34 ( 0.02
min-1, t1/2 ) 2.0 min) to give four products (Scheme 4):
solvolysis product 13 (-14.63 ppm), a small amount of
amine-trapped product 14 ( -15.69 ppm), transient
unknown product 15 (-16.17 ppm), and hydrolysis
product, tetrahydrofurfuryl monophosphate 16 (-22.37
ppm). The reaction products were characterized by 31P
NMR comparison to authentic phosphate and phospho-
ramidates as described below.
total mixture. Minor products 11 and 12 made up <13%
of the total product composition.
The kinetic analysis of the data obtained in the
reaction of thymidine phosphoramidate anion 1a is
shown in Figure 2. The analysis revealed that the rates
of formation for all reaction products were identical,
suggesting that the rate-limiting step for the conversion
of phosphoramidate anion to trapped and P-N bond-
cleaved products is the cyclization of anion 1a to the
aziridinium ion intermediate. The concentrations of
solvolysis product 10 and amine-trapped product 11
remained constant after phosphoramidate anion 1a was
Syn t h esis of Au t h en t ic P r od u ct s. Hydroxyethyl
phosphoramidate anion 6b was prepared as described

4322 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Freel Meyers and Borch
Sch em e 4
Sch em e 5a
a
Reagents and conditions: (a) tetrahydrofurfuryl alcohol, TEA,
CH2Cl2, -40 °C to rt, 20 min; (b) 3-hydroxypropionitrile, TEA,
THF, -10 °C to rt, overnight; (c) concd NH4OH, rt, 24 h.
pearance of phosphoramidate anion 6a resulted in the
formation of solvolysis product 13 and monophosphate
16 (Scheme 4) as expected. In addition, the formation
of a new product 9 (-15.61 ppm) was observed. Confir-
mation of the structure assignment for this reaction
product was accomplished by 31P NMR comparison to
authentic N-methyl-N-(2-dimethylaminoethyl)phospho-
ramidate anion 9. Authentic compound 9 was prepared
from phosphodiester 7 as shown in Scheme 2. Addition
of authentic phosphoramidate anion 9 to the NMR tube
containing the reaction mixture resulted in an increase
in the 31P NMR resonance at -15.61 ppm. This obser-
vation was sufficient to confirm the occurrence of amine
attack on the carbon of the aziridinium ion in the
reaction of tetrahydrofurfuryl phosphoramidate anion
6a and thymidine phosphoramidate anion 1a .
The 31P chemical shift of 15 (Scheme 4) indicates that
it is most likely a phosphoramidate ester. Disappear-
ance of this 31P NMR resonance, ostensibly to mono-
phosphate 16, was also observed in the reaction of
compound 6a at 37 °C. It was conceivable that a
component of the buffer might participate in the reac-
tion of phosphoramidate anion 6a .5 Thus, the reaction
of phosphoramidate 6a was carried out in distilled water
over 30 min at room temperature. A 31P NMR spectrum
of the sample revealed the presence of unreacted phos-
phoramidate anion 6a , solvolysis product 13, amine-
trapped product 14, and monophosphate 16 as before
(Scheme 4). However, the unknown product 15 was not
observed in the absence of buffer. This result implicated
the participation of buffer in the formation of 15, and
efforts toward further characterization of this peak were
abandoned. It should be noted that amine-trapped
product 14 and buffer product 15 are simply artifacts
of the closed-system environment in these 31P NMR
experiments and contribute <15% to the total product
composition.
for phosphoramidate anion 6a in Scheme 2. Following
catalytic hydrogenolysis, phosphoramidate anion 6b was
added to the NMR tube containing the reaction mixture
of haloethyl phosphoramidate anion 6a . The resulting
increase in the 31P resonance at -14.63 ppm (13,
Scheme 4) verified the peak assignment for solvolysis
product 13 in the reaction of compound 6a . By analogy,
it was concluded that the structure assignment for
solvolysis product 10 in the reaction of thymidine
phosphoramidate 1a (Scheme 3) was correct.
Authentic tetrahydrofurfuryl monophosphate 17 was
synthesized as shown in Scheme 5. Phosphorus oxy-
chloride was treated with tetrahydrofurfuryl alcohol in
the presence of triethylamine to give phosphoryl dichlo-
ride 18 in 94% yield. Subsequent treatment with 3-hy-
droxypropionitrile in the presence of triethylamine
afforded phosphotriester 19 in 54% yield. Removal of
the cyanoethyl protecting groups in aqueous ammonium
hydroxide followed by lyophilization of the resulting
aqueous mixture gave authentic monophosphate 17 in
97% yield. Addition of authentic tetrahydrofurfuryl
monophosphate 17 to the NMR tube containing the
reaction mixture of phosphoramidate 6a resulted in an
increase in the resonance at -22.37 ppm (16, Scheme
4). This result confirmed the peak assignment for
hydrolysis product 16.
Kin etics of Ben zotr ia zolyl Ha loeth yl P h osp h o-
r a m id a t e E st er s. The reaction profiles exhibited by
phosphoramidate anions 1a and 6a provided a founda-
tion for additional mechanistic studies carried out on
the benzotriazolyl phosphoramidate esters 2a and 3
(Chart 1). It was anticipated that activation of the
benzotriazolyl phosphoramidate esters to the corre-
sponding phosphoramidate anions would occur via hy-
drolysis of the benzotriazolyl group.
It was hypothesized that ring opening of N-methy-
laziridine released by nucleophilic displacement at
phosphorus could result in the formation of a variety of
secondary amines capable of trapping the aziridinium
ion present in the reaction mixture to form the phos-
phoramidate product 14 (Scheme 4). The plausibility of
amine trapping at carbon of the aziridinium ion was
tested in an experiment in which the reaction of
compound 6a was carried out in the presence of excess
dimethylamine (5 equiv) at room temperature. Disap-
A
31P NMR experiment was carried out on benzo-
triazolyl thymidine phosphoramidate 2a under model
physiologic conditions. Phosphoramidate ester 2a ap-

Mechanisms of Nucleoside Phosphoramidate Prodrugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4323
Sch em e 6
F igu r e 3. Activation of benzotriazolyl phosphoramidate 2a
in 4.5:1 cacodylate buffer/CH3CN (ca. 100 mM, pH 7.4, 37
°C): D, phosphoramidate anion 20; E, solvolysis product 10;
F , amine-trapped product 11; G, OBT-trapped product 21; H,
OBT phosphate 22; TMP, thymidine 5′-monophosphate.
peared as two resonances (-12.50, -12.78 ppm) char-
acteristic of a mixture of diastereomers (Figure 3).
Apparent first-order hydrolysis of 2a (k ) 0.15 min-1
(
0.02, t1/2 ) 4.7 min) resulted in the formation of
phosphoramidate anion 20 (D, -15.99 ppm) which
appeared as a single resonance (Scheme 6). The ob-
served conversion of two resonances to a single phos-
phoramidate anion resonance indicated a collapse of the
diastereomeric mixture to a single isomer. The short
half-life of the OBT ester suggests that nucleoside
phosphoramidate prodrugs containing this delivery
group are unlikely to be clinically useful.
As shown in Scheme 6, phosphoramidate anion 20
was converted with a rate constant k ) 0.20 ( 0.01
min-1 (t1/2 ) 3.4 min) to solvolysis product 10 (E), amine-
trapped product 11 (F ), and TMP as observed in the
reaction of phosphoramidate anion 1a (Scheme 3).
However, little or no buffer product 12 was observed,
and two new resonances appeared: a new product 21
(G, -15.59 ppm) and P-N bond-cleaved product 22 (H,
-25.73 ppm). A kinetic analysis of the data obtained
indicated that the concentrations of solvolysis product
10, new product 21, and amine-trapped product 11
remained constant after phosphoramidate anion 20 was
consumed, and product 22 underwent slow conversion
to TMP (k ) 0.0042 min-1 ( 0.001 min-1, t1/2 ) 164
min).
) 1.6 min) to solvolysis product 13, amine-trapped
product 14, and tetrahydrofurfuryl monophosphate 16
as observed in the reaction of phosphoramidate anion
6a (Scheme 4). In addition, a new product 24 (-15.54
ppm) and P-N bond-cleaved product 25 (-25.25 ppm)
were observed. As expected, the concentrations of sol-
volysis product 13, OBT-trapped product 24, and amine-
trapped product 14 remained constant after phospho-
ramidate anion was consumed, and product 25 under-
went slow conversion to tetrahydrofurfuryl monophos-
phate 16 (k ) 0.011 min-1, t1/2 ) 61 min).
The structure assignments for products 24 and 25 are
supported by data obtained in an experiment of phos-
phoramidate anion 6a carried out in the presence of
excess HOBT (5 equiv). In addition to the formation of
reaction products 13-16 (Scheme 4), the resonances
corresponding to products 24 and 25 were observed as
the major products. The resonance at -15.54 ppm is
consistent with trapping at carbon of the aziridinium
ion by the nucleophile HOBT. More interestingly, the
resonance at -25.25 ppm is thought to arise from
nucleophilic attack by HOBT at phosphorus; cleavage
of the P-N bond by nucleophiles other than water has
not been observed previously in these systems. Ideally,
confirmation of the structure of product 25 might be
accomplished by 31P NMR comparison to authentic
compound. However, the inherent reactivity of this
intermediate made isolation and characterization very
difficult. Numerous attempts to prepare authentic OBT
The 31P NMR resonances corresponding to reaction
products 21 (-15.59 ppm) and 22 (-25.73 ppm) ap-
peared only in the reaction of benzotriazolyl phospho-
ramidate 2a (Figure 3) and not in the reaction of the
phosphoramidate anion itself (1a , Figure 1). On the
basis of this observation, it was hypothesized that
products 21 and 22 arise from nucleophilic attack by
HOBT at carbon and phosphorus of the aziridinium ion,
respectively. Confirmation of these structure assign-
ments was sought by comparison to the structurally
analogous reaction products formed in the reaction of
tetrahydrofurfuryl phosphoramidate ester 3 (Scheme 1).
A 31P NMR experiment carried out on benzotriazolyl
tetrahydrofurfuryl phosphoramidate 3 (Scheme 6) re-
vealed a reaction profile nearly identical to that ob-
served for thymidine phosphoramidate 2a (Scheme 6).
Apparent first-order hydrolysis of 3 (k ) 0.094 min-1
,
t1/2 ) 7.4 min) resulted in the formation of the corre-
sponding phosphoramidate anion (-15.89 ppm) which
was converted with a rate constant k ) 0.43 min-1 (t1/2

4324 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Freel Meyers and Borch
Sch em e 7a
the reduction of the nitro group.4b However, in this case
the phosphoramidate anion was converted exclusively
to a single trapped product; no P-N bond cleavage
occurred. Bisulfite liberated during the reaction pre-
sumably traps the aziridinium ion more effectively than
water and predominates over hydrolysis of the P-N
bond. This hypothesis was confirmed in an experiment
carried out on compound 6a in which excess bisulfite
was added. In this case, the resonance for phosphora-
midate anion 6a disappeared to give a single resonance
(-15.81 ppm) corresponding to the bisulfite-trapped
product.
a
Reagents and conditions: (a) HOBT, pyridine, THF, 0 °C to
This result prompted the consideration of glutathione
(GSH), the most abundant intracellular thiol (intracel-
lular concentrations 1-10 mM), in this reaction. An
experiment was carried out on phosphoramidate 6a in
which excess GSH (5 equiv, ca. 400 mM) was added.
Phosphoramidate anion 6a disappeared to form solvoly-
sis and hydrolysis products 13 and 16 in addition to the
presumed GSH-trapped phosphoramidate. No P-N
bond cleavage by GSH was observed, and the overall
contribution of GSH-trapped phosphoramidate was
<50% of the total composition. Furthermore, the con-
version of phosphoramidate anion to hydrolysis product
was reduced by <50% in the presence of 400 mM GSH.
In general, polarizable nucleophiles (i.e. nitrogen and
sulfur nucleophiles) show a preference for reaction at
the more polarizable electrophilic center, in this case,
carbon of the aziridinium ion. These data are consistent
with experimental data obtained in studies of the
reactivities of various nucleophiles on a series of phos-
phoryl substrates.7 In general, sulfur nucleophiles are
not reactive at phosphorus while oxygen nucleophiles
are known to react at both phosphorus and saturated
carbon centers. Reactions of amine nucleophiles at
phosphorus are highly dependent upon nitrogen basicity
and steric effects.
rt; (b) 4.5:1 0.4 M cacodylate buffer/THF, pH 7.4, 37 °C.
phosphate were unsuccessful. Thus, an alternative
strategy for the structural verification of OBT phosphate
25 was sought.
The characterization of OBT phosphate 25 was ulti-
mately accomplished in a 31P NMR experiment in which
the conversion of OBT phosphate 25 to tetrahydrofur-
furyl monophosphate 16 was reproduced. This strategy
(Scheme 7) involved the hydrolysis of the highly reactive
bis(benzotriazolyl) intermediate 26 generated in situ
from phosphoryl dichloride 18. Intermediate 26 was
prepared by reaction of 18 with HOBT (2 equiv) in the
presence of pyridine (Scheme 7). Following removal of
pyridine hydrochloride, the reaction mixture was used
immediately in a 31P NMR hydrolysis experiment.
Complete conversion of the presumed bis(benzotriazolyl)
intermediate 26 to OBT phosphate 25 (-25.17 ppm) was
essentially instantaneous. OBT phosphate 25 under-
went conversion to tetrahydrofurfuryl phosphate 16
(-22.39 ppm) with a rate constant k ) 0.011 ( 0.01
min-1 (t1/2 ) 64 min), a value in excellent agreement
with that obtained in the reaction of benzotriazolyl
tetrahydrofurfuryl phosphoramidate 3 (k ) 0.011 min-1).
The structure assignment is also supported by a peak
at m/z 300 corresponding to 25 in the mass spectrum of
a sample taken early in the hydrolysis of 26.
Syn th esis a n d Kin etics of P ip er id yl P h osp h or a -
m id a te An a logu es. The piperidyl nucleoside phospho-
ramidate prodrugs prepared in this laboratory2 exhib-
ited significant growth inhibitory activity, although they
were expected to be “stable” analogues. Thus, the
activation mechanism of these compounds was explored
using the model thymidine phosphoramidates 1b and
2b shown in Chart 1. Phosphoramidates 1b and 2b were
prepared as described in the accompanying paper.2 The
behavior of the piperidyl phosphoramidate anion was
studied in a 31P NMR kinetics experiment carried out
on compound 1b under model physiologic conditions.
Phosphoramidate 1a (-15.55 ppm) hydrolyzed slowly
(t1/2 ∼ 11 days) at pH 7.4 resulting in the formation of
TMP. Additional studies were performed in which the
rate of P-N bond hydrolysis was measured as a function
of both pH and buffer concentration. On the basis of
data obtained in these studies, it was concluded that
the P-N bond hydrolysis reaction is specific acid-
catalyzed but not subject to general acid catalysis. The
mechanistic data obtained in these studies suggested
that, although the biological activity of piperidyl phos-
phoramidate prodrug analogues2 is consistent with
intracellular liberation of the nucleotide, this most likely
occurs by the action of endogenous phosphoramidase.
Protonation of the piperidyl nitrogen at physiologic pH
Nu cleop h ilic Tr a p p in g of th e Azir id in iu m Ion .
The product distributions in the reactions of phospho-
ramidates 1a , 2a , 3, and 6a (Chart 1) are governed
largely by the reactions of the aziridinium ion involving
water as the nucleophile. In all cases, water reacts
without selectivity at carbon and phosphorus. Trapping
of the aziridinium ion at carbon and cleavage of the P-N
bond by nucleophiles other than water was observed as
well. Reaction of HOBT at carbon and phosphorus also
occurred without selectivity. On the contrary, amines
were found to react only at carbon of the aziridinium
ion intermediate. No P-N bond aminolysis was ob-
served in the reactions of phosphoramidates 1a , 2a , 3,
and 6a .
This evidence of exclusive nucleophilic attack at
carbon of the aziridinium ion prompted a closer look at
some puzzling results obtained in activation studies
performed on nucleoside phosphoramidate prodrugs
containing a nitrofuryl delivery moiety.2 Chemical
activation of nucleoside phosphoramidates bearing a
nitrofuryl group can be carried out using sodium
dithionite as a chemical reducing agent. As expected,
treatment of the nitrofuryl nucleoside phosphoramidate
with aqueous sodium dithionite results in immediate
expulsion of the phosphoramidate anion, presumably via

Mechanisms of Nucleoside Phosphoramidate Prodrugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4325
Sch em e 8
uents. We conclude that the mechanism of action of
nucleoside phosphoramidates containing the N-methyl-
N-(2-bromoethyl) substituent proceeds through an aziri-
dinium ion intermediate followed by hydrolysis of the
P-N bond to release the active nucleotide intracellu-
larly. The mechanism of action of the piperidyl nucleo-
side phosphoramidates is most likely the intracellular
release of the active nucleotide. However, simple pro-
tonation on nitrogen followed by hydrolysis of the P-N
bond does not account for the observed growth inhibition
at physiologic pH. Cleavage of the P-N bond, in this
case, presumably takes place by the action of an
endogenous phosphoramidase.
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 a 250 MHz Bruker instrument. 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 refer-
ence (triphenylphosphine oxide/toluene-d8 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 temperature 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 µm). Visualization
of the plates was accomplished 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 diisopropylethylamine were distilled prior to use. Tet-
rahydrofurfuryl alcohol and benzyl alcohol were distilled from
sodium prior to use. Piperidine, N,N,N′-trimethylethylenedi-
amine and 3-hydroxypropionitrile were distilled from calcium
hydride prior to use. HOBT was dried in a 90 °C oven.
N-Methyl-N-(2-bromoethyl)amine hydrobromide and 2-(me-
thylamino)ethanol were dried by coevaporation with acetoni-
trile prior to use.
31P NMR Kin etics Exp er im en ts. 31P NMR kinetics ex-
periments were carried out at 37 °C (or ambient temperature,
24-26 °C) on a 250 MHz Bruker NMR using the Bruker
variable-temperature unit to control the probe temperature.
The actual sample temperature was taken manually before
and after each experiment using a Fluke digital thermometer
and thermocouple.
Typically, the benzotriazolyl phosphoramidate or phospho-
ramidate anion (0.015-0.076 mmol) was dissolved in a small
amount of organic solvent (ca. 90 µL CH3CN or THF) and
diluted to 0.50 mL with buffer (0.1-0.4 M cacodylate or acetate
buffer, ca. 490 µL). The reaction start time was noted at the
time of dilution. The homogeneous reaction mixture was
adjusted to the appropriate pH with dilute NaOH or HCl and
transferred to a 5-mm NMR tube at room temperature. The
sample was inserted into the AC 250 Bruker NMR probe and
allowed to equilibrate for about 5 min prior to the start of
acquisition. The elapsed time was noted at the start of
acquisition. Data were collected for 1 or 2 h (longer experi-
ments were followed manually by 31P NMR). Spectra were
acquired every 2.5 min for 30 min, then every 5 min for 30
min. For 2-h experiments, spectra were acquired every 10 min
for an additional hour. The pH of the reaction mixture was
recorded at the end of the experiment. In general, a pH range
followed by hydrolysis of the P-N bond cannot account
for the biological activity observed for these analogues.
A
31P NMR kinetics experiment carried out on ben-
zotriazolyl phosphoramidate 2b under model physiologic
conditions revealed a slightly different reaction profile.
As expected, the resonances for ester 2b were replaced
by a resonance corresponding to the piperidyl phospho-
ramidate anion 27 (t1/2 ) 9.7 min, Scheme 8). Apparent
nucleophilic displacement of the piperidyl group by
HOBT resulted in the formation of covalent intermedi-
ate 28, which then hydrolyzed to give the nucleotide,
TMP. Additional studies showed a strong dependence
upon HOBT concentration for the rate of conversion of
piperidyl phosphoramidate anion 1b to TMP; a 3-fold
increase in the rate of conversion to TMP was observed
when the reaction was carried out in the presence of 2
equiv of HOBT. These data suggest that P-N bond
hydrolysis of piperidyl phosphoramidates is subject to
nucleophilic catalysis by HOBT.
Su m m a r y
A series of thymidine and tetrahydrofurfuryl phos-
phoramidates was synthesized and used as chemical
models for the analogous nucleoside phosphoramidate
prodrugs.2 These analogues were studied using 31P
NMR for the purpose of increasing our understanding
of the reactivity of phosphoramidate anions and for
clarifying the proposed mechanism of action for the
5-fluoro-2′-deoxyuridine phosphoramidate prodrugs re-
ported in the accompanying paper.2
The structure assignments of phosphate and phos-
phoramidate products in complex phosphoramidate
reaction mixtures were accomplished by comparison of
the products to synthesized authentic materials. A
knowledge of the reactivity of the aziridinium ion
intermediate at carbon and phosphorus toward different
nucleophiles was obtained, providing insight into the
inherent problems associated with certain types of
chemical activation of phosphoramidate prodrugs con-
taining a haloethylamine substituent. Studies carried
out on the P-N bond hydrolysis reactions of piperidine
phosphoramidates were instrumental for the resolution
of the mechanism of activation of phosphoramidates
incapable of forming the aziridinium ion intermediate.
The mechanistic results obtained have provided a
foundation on which to draw conclusions regarding the
mechanism of action of nucleoside phosphoramidates
containing either haloethylamine or piperidine substit-

4326 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Freel Meyers and Borch
of 7.0-8.0 was maintained for experiments whose target pH
was 7.4. The relative concentrations of reaction intermediates
were determined by measuring the peak areas. The relative
concentration for each reactant or product at any given time
is represented as a percent of the total.
Reactions involving the addition of a nucleophile (i.e. HOBT,
DDTC or dimethylamine) were carried out in the following
manner. The nucleophile (5-15 equiv) was added to a small
amount of buffer (0.4 M cacodylate or acetate buffer, ca. 490
µL) and the resulting solution was adjusted to the appropriate
pH using dilute NaOH or HCl. The mixture was added to a
vial containing the phosphoramidate in CH3CN (ca. 90 µL).
The experiment proceeded as described above.
Kin etic An a lyses. The kinetic analyses for the reactions
described were carried out using the optimization routine in
the Quattro Pro Program. The 31P NMR peak areas were
measured, and the product composition was determined at
each time point as a percentage of the total material. Rate
expressions were derived for each reaction, and the rate
constants were determined by minimization of the least-
squares difference between observed and calculated product
composition at each time point. The rate of disappearance of
phosphoramidate anion (kdis, min-1) was also determined by
performing a linear regression of the 31P NMR peak areas.
Na2SO4 and concentrated. Purification of the crude product
by silica gel chromatography (3:1 hexanes:EtOAc) afforded
benzyl ester 7 as a clear oil (819 mg, 55%). The product was
isolated as a 1:1 mixture of diastereomers (as determined by
31P NMR): Rf ) 0.20 (3:1 hexanes:EtOAc); 1H NMR (CDCl3) δ
7.39 (s, 5H), 5.22 (dd, 2H, J HCNP ) 8.9 Hz), 4.15 (m, 3H), 3.83
(m, 2H), 1.99 (m, 1H), 1.90 (m, 2H), 1.69 (m, 1H); 31P NMR
(CDCl3) δ -21.25, -21.33 (1:1 mixture).
Tetr a h yd r ofu r fu r yl Ben zyl N-Meth yl-N-(2-br om oeth -
yl)p h osp h or a m id a te (8a ). N-Methyl-N-(2-bromoethyl)amine
hydrobromide (159 mg, 0.73 mmol), which was prepared
according to Fries,6 was suspended in THF (3 mL) under an
atmosphere of argon. Phosphoryl monochloride 7 (211 mg, 0.73
mmol) was dissolved in THF (2 mL) and added to the reaction
mixture in one portion. Triethylamine (0.12 mL, 0.87 mmol)
was added dropwise at room temperature, and the reaction
mixture was stirred for 45 min. Triethylamine hydrochloride
was removed by filtration and the filtrate was concentrated.
The residue was purified by silica gel chromatography (1:1
EtOAc:hexanes f 100% EtOAc) to give 8a (204 mg, 70%) as a
1
clear oil: Rf ) 0.18 (1:1 EtOAc:hexanes); H NMR (CDCl3) δ
7.37 (m, 5H), 5.03 (d, 2H, J ) 7.7 Hz), 4.12 (m, 1H), 3.96 (m,
2H), 3.82 (m, 2H), 3.41 (m, 4H), 2.69 (d, 3H, J ) 9.6 Hz), 1.98
(m, 1H), 1.89 (m, 2H), 1.66 (m, 1H); 31P NMR (CDCl3) δ -16.09;
HRMS (C15H23NO4BrP) calcd 392.0626 (M + H)+, found
392.0636.
Tetr a h yd r ofu r fu r yl N-Meth yl-N-(2-br om oeth yl)p h os-
p h or a m id och lor id a te (5). A solution of tetrahydrofurfuryl
alcohol (0.81 g, 7.80 mmol) in THF (20 mL) was cooled to -78
°C under an atmosphere of argon. LiHMDS (8.60 mL of 1.0 M
solution in THF, 8.60 mmol) was added dropwise. The reaction
mixture was stirred at -78 °C for 5 min. Phosphoramidic
dichloride 4 (2.00 g, 7.80 mmol) was dissolved in THF (5 mL)
and added in one portion to the alkoxide at -78 °C. The
reaction mixture was warmed to room temperature over 45
min and quenched with saturated NH4Cl (10 mL). The reaction
mixture was added to EtOAc (25 mL) in a separatory funnel.
The aqueous layer was extracted with EtOAc, and the com-
bined organic layers were dried over Na2SO4 and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (100:10:0.5 CHCl3:EtOAc:MeOH) to yield 5
(2.19 g, 87%) as a clear oil: Rf ) 0.44 (100:10:0.5 CHCl3:EtOAc:
Tetr a h yd r ofu r fu r yl Ben zyl N-Meth yl-N-(2-h yd r oxy-
eth yl)ph osph or am idate (8b). 2-(Methylamino)ethanol (0.041
mL, 0.52 mmol) was dissolved in THF (1.5 mL) under an
atmosphere of argon. Triethylamine (0.082 mL, 0.58 mmol)
was added in one portion followed by the addition of molecular
sieves (4 Å, 0.5 mL). Phosphoryl monochloride 7 was dissolved
in THF (1 mL) and added dropwise at room temperature. The
reaction mixture was stirred at room temperature for 1 h and
then decanted from the molecular sieves. The sieves were
washed with CHCl3 (4 × 2 mL). The combined organic
solutions were concentrated and purified by silica gel chro-
matography (95:5 CHCl3:MeOH) to afford 8b (88 mg, 78%) as
a clear oil. The product was isolated as a 1:1 mixture of
diastereomers (as determined by 1H and 31P NMR): Rf ) 0.41
(95:5 CHCl3:MeOH); 1H NMR (CDCl3) δ 7.36 (m, 5H), 5.02 and
5.03 (d, 2H, J ) 7.8 Hz, 1:1 mixture), 4.10 (m, 1H), 3.97 (m,
2H), 3.82 (m, 2H), 3.68 (t, 2H, J ) 5.0 Hz), 3.16 (m, 2H), 2.67
and 2.68 (d, 3H, J ) 9.8 Hz, 1:1 mixture), 2.57 (d, 1H), 1.97
(m, 1H), 1.89 (m, 2H), 1.64 (m, 1H); 31P NMR (CDCl3) δ -15.06,
-15.09 (1:1 mixture); HRMS (C15H24NO5P) calcd 330.1470 (M
+ H)+, found 330.1469.
1
MeOH); H NMR (CDCl3) δ 4.14 (m, 3H), 3.85 (m, 2H), 3.49
(m, 4H), 2.82 (d, 3H, J ) 12.9 Hz), 2.02 (m, 1H), 1.93 (m, 2H),
1.69 (m, 1H); 31P NMR (CDCl3) δ -8.39.
Tet r a h yd r ofu r fu r yl 1-Ben zot r ia zolyl N-Met h yl-N-(2-
br om oeth yl)p h osp h or a m id a te (3). HOBT (65.4 mg, 0.50
mmol) and triethylamine (0.074 mL, 0.53 mmol) were dissolved
in THF (1 mL) and added dropwise to phosphoramidic chloride
5 (141 mg, 0.44 mmol) in THF (4 mL) at room temperature.
The reaction mixture was stirred at room temperature under
argon for 1 h 15 min. Triethylamine hydrochloride was
removed by filtration and the filtrate was concentrated. The
residue was purified by silica gel chromatography (4:1 CHCl3:
EtOAc) to give benzotriazolyl phosphoramidate 3 (124 mg,
67%) as a clear oil that was a 1:1 mixture of diastereomers
(as determined by 31P NMR): Rf ) 0.52 (4:1 CHCl3:EtOAc);
1H NMR (CDCl3) δ 7.99 (d, 1H, J ) 8.4 Hz), 7.73 (d, 1H, J )
8.2 Hz), 7.53 (t, 1H, J ) 8.2 Hz), 7.39 (t, 1H, J ) 7.3 Hz), 4.24
(m, 3H), 3.83 (m, 2H), 3.41 (m, 4H), 2.92 (d, 3H, J ) 10.2 Hz),
1.99 (m, 1H), 1.91 (m, 2H), 1.69 (m, 1H); 31P NMR (CDCl3) δ
-15.84, -16.02 (1:1 mixture); HRMS (C14H20N4O4BrP) calcd
419.0484 (M + H)+, found 419.0486.
Tetr a h yd r ofu r fu r yl Ben zyl P h osp h or och lor id a te (7).
Tetrahydrofurfuryl alcohol (0.50 mL, 5.16 mmol) was dissolved
in CH2Cl2 (20 mL) and cooled to -10 °C under argon.
Phosphorus oxychloride (0.48 mL, 5.16 mmol) was added in
one portion followed by the dropwise addition of triethylamine
(0.79 mL, 5.68 mmol), and the reaction was warmed to room
temperature over 20 min. The reaction mixture was then
cooled to 0 °C and benzyl alcohol (0.53 mL, 5.16 mmol) was
added dropwise followed by the dropwise addition of triethy-
lamine (0.79 mL, 5.68 mmol). Stirring was continued at 0 °C
for 1 h. The viscous reaction mixture was poured into brine
(25 mL), and the aqueous layer was extracted with CHCl3 (2
× 20 mL). The combined organic layers were dried over
Tetr a h yd r ofu r fu r yl Ben zyl N-Meth yl-N-(2-d im eth yl-
a m in oeth yl)p h osp h or a m id a te (8c). Benzyl ester 7 (210 mg,
0.72 mmol) was dissolved in anhydrous CH3CN (6 mL) under
argon. Freshly distilled N,N,N′-trimethylethylenediamine (0.080
mL, 1.08 mmol) was dissolved in CH3CN (3 mL) and added
dropwise over 5 min. The homogeneous reaction mixture was
stirred at room temperature for 1 h and then concentrated
under reduced pressure. The residue was dissolved in water
(25 mL) and the aqueous solution was adjusted to pH 12 with
1 M NaOH. The solution was extracted with CHCl3 (8 × 10
mL). The combined organic layers were dried over Na2SO4 and
concentrated. The residue was chromatographed on silica gel
(EtOH) to afford 8c (193 mg, 75%) as a clear oil. It should be
noted that attempts to purify the crude product by reverse-
phase chromatography using CH3CN/H2O (0.1% TFA) resulted
1
in decomposition of the product: Rf ) 0.14 (EtOH); H NMR
(CDCl3) δ 7.37 (m, 5H), 5.02 (d, 2H, J ) 7.4 Hz), 4.12 (m, 1H),
3.95 (m, 2H), 3.81 (m, 2H), 3.12 (m, 2H), 2.66 (d, 3H, J ) 9.8
Hz), 2.41 (t, 2H, J ) 7.2 Hz), 2.23 (s, 6H), 1.99 (m, 1H), 1.88
(m, 2H), 1.68 (m, 1H); 31P NMR (CDCl3) δ -16.08; HRMS
(C17H29N2O4P) calcd 357.1943 (M + H)+, found 357.1925.
Tetr a h yd r ofu r fu r yl N-Meth yl-N-(2-br om oeth yl)p h os-
p h or a m id a te Tr ieth yla m m on iu m Sa lt (6a ). Phosphora-
midate 8a (7.0 mg, 0.018 mmol) was dissolved in THF (1 mL).
Pd/C (10%, 5 mg) was suspended in THF (1 mL) and trans-
ferred to the flask containing phosphoramidate 8a . The flask
was equipped with a balloon filled with hydrogen, and the

Mechanisms of Nucleoside Phosphoramidate Prodrugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4327
reaction mixture was stirred for 5 min at room temperature.
Triethylamine (2.6 µL, 0.019 mmol) was added and stirring
was continued for 1-2 min. The catalyst was filtered and the
filtrate was concentrated to 0.50 mL. Complete conversion to
phosphoramidate anion 8a was observed by 31P NMR. The
remaining THF was removed by rotary evaporation and the
unstable product 6a was immediately analyzed by mass
spectrometry or used for a kinetics experiment: 31P NMR
(THF) δ -17.41; HRMS (C8H17NO4BrP, free acid) calcd 302.0157
(M + H)+, found 302.0157.
Tetr ah ydr ofu r fu r yl N-Meth yl-N-(2-h ydr oxyeth yl)ph os-
p h or a m id a te Tr ieth yla m m on iu m Sa lt (6b). Phosphora-
midate anion 6b was prepared from benzyl ester 8b (5.7 mg,
0.017 mmol) as described for the preparation of compound 6a .
Quantitative conversion of benzyl ester 8b to phosphoramidate
anion 6b was observed by 31P NMR. Phosphoramidate anion
6b was concentrated for mass spectral analysis and used
without further purification: 31P NMR (0.4 M cacodylate
buffer/CH3CN, pH 7.4, 37 °C) δ -14.63; HRMS (C8H18NO5P,
free acid) calcd 240.1001 (M + H)+, found 240.1007.
Tetr a h yd r ofu r fu r yl N-Meth yl-N-(2-d im eth yla m in oeth -
yl)p h osp h or a m id a te Tr ieth yla m m on iu m Sa lt (9). Au-
thentic phosphoramidate 9 was prepared as described for
compound 6a from benzyl ester 8c (6.6 mg, 0.018 mmol).
Quantitative conversion of benzyl ester 8c to phosphoramidate
anion 9 was observed by 31P NMR. Phosphoramidate anion 9
was concentrated for mass spectral analysis and used without
further purification: 31P NMR (0.4 M cacodylate buffer/
CH3CN, pH 7.4, 37 °C) δ -15.55; HRMS (C10H23N2O4P, free
acid) calcd 267.1474 (M + H)+, found 267.1476.
phate 17 (7.3 mg, 97%) as a waxy white solid. Authentic
compound 17 was used without further purification: 31P NMR
(0.4 M cacodylate buffer/CH3CN, pH 7.4, 37 °C) δ -22.37;
HRMS (C5H9O5P) calcd 183.0422 (M + H)+, found 183.0425.
Tetr a h yd r ofu r fu r yl 1-Ben zotr ia zolyl P h osp h a te (25).
HOBT (324 mg, 2.40 mmol) and pyridine (0.21 mL, 2.60 mmol)
were dissolved in THF (3.5 mL) and cooled to -10 °C under
an atmosphere of argon. Phosphoryl dichloride 18 (250 mg,
1.14 mmol) was dissolved in THF (1.5 mL) and added dropwise.
The reaction mixture was warmed to room temperature and
stirred for 2 h. Pyridine hydrochloride was removed by passing
the reaction mixture through a plug of cotton. A portion of the
filtered reaction mixture (90 µL) was diluted with distilled
water (500 µL) and immediately analyzed by electrospray
ionization mass spectrometry. Mass spectral data confirmed
the presence of OBT phosphate 25.31P NMR data obtained on
an analogous mixture in 0.4 M cacodylate buffer/THF (4.5:1)
confirmed the quantitative conversion of bis-benzotriazolyl
phosphate 26 to OBT phosphate 25. OBT phosphate 25 was
used without further purification in a kinetics experiment
carried out on a buffered mixture (4.5:1 0.4 M cacodylate
buffer/ THF) containing 25: 31P NMR (4.5:1 0.4 M cacodylate
buffer/THF, pH 7.4, 37 °C) δ -25.25; HRMS (C11H14N3O5P,
free acid) calcd 300.0749 (M + H)+, found 300.0734.
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) (a) 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. (b) 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.
(2) Freel Meyers, C. L.; Borch, R. F.; Hong, L.; J oswig, C. Synthesis
and biological activity of novel 5-fluoro-2′-deoxyuridine phos-
phoramidate prodrugs. J . Med. Chem. 2000, 43, 4313-4318
(accompanying manuscript).
(3) (a) Colvin, M.; Padgett, C. A.; Fenselau, C. A biologically active
metabolite of cyclophosphamide. Cancer Res. 1973, 33, 915-918.
(b) Takamizawa, A.; Rochino, Y.; Hamashima, T.; Iwata, T.
Studies of cyclophosphamide metabolites and their related
compounds. Chem. Pharm. Bull. 1972, 20, 1612-1616. (c)
Struck, R. F.; Kirk, M. C.; Mellett, L. B.; El Dareer, S.; Hill, D.
L. Urinary metabolites of the antitumor agent cyclophospha-
mide. Mol. Pharmacol. 1971, 7, 519-529.
(4) (a) Flader, C.; Liu, J .; Borch, R. F. Development of novel quinone
phosphorodiamidate prodrugs targeted to DT-diaphorase. J .
Med. Chem. 2000, 43, 3157-3167. (b) 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 phosphora-
midates as hypoxia-selective alkylating agents. J . Med. Chem.
2000, 43, 2258-2265. (c) Colvin, M.; Brundrett, R. B.; Kan, M.-
N. N.; J ardine, I.; Fenselau, C. Alkylating properties of phos-
phoramide mustard. Cancer Res. 1976, 36, 1121-1126. (d)
Brookes, P.; Lawley, P. D. The reaction of mono- and di-
functional alkylating agents with nucleic acids. Biochem. J .
1961, 80, 496-503.
(5) Shulman-Roskes, E.; Noe, D. A.; Gamcsik, M. P.; Marlow, A. L.;
Hilton, J .; Hausheer, F. H.; Colvin, O. M.; Ludeman, S. M. The
partitioning of phosphoramide mustard and its aziridinium ions
among alkylation and P-N bond hydrolysis reactions. J . Med.
Chem. 1998, 41, 515-529.
(6) Fries, K. M.; J oswig, C.; Borch, R. F. Synthesis and evaluation
of 5-fluoro-2′-deoxyuridine phosphoramidate pnalogs. J . Med.
Chem. 1995, 38, 2672-2680.
(7) (a) Hudson, R. F. Structure and mechanism in organo-phospho-
rus chemistry; Academic Press: New York, 1965; Chapter 4. (b)
Edwards, J . O.; Pearson, R. G. The factors determining nucleo-
philic reactivities J . Am. Chem. Soc. 1962, 84, 16-24. (c) Clark,
V. M.; Todd, A. R. Studies on phosphorylation. Part VI. The
reaction between organic bases and esters of the oxy-acids on
phosphorus. An interpretation based on a comparison of certain
aspects of the chemistry of sulphur and phosphorus J . Chem.
Soc. 1950, 2023-2029.
Tetr a h yd r ofu r fu r yl P h osp h or od ich lor id a te (18). Phos-
phorus oxychloride (0.91 mL, 9.79 mmol) was added in one
portion to tetrahydrofurfuryl alcohol (1.00 g, 9.79 mmol)
dissolved in CH2Cl2 (25 mL) at -40 °C under an atmosphere
of argon. Triethylamine (1.50 mL, 10.77 mmol) was added
dropwise and the reaction mixture was warmed to room
temperature over 20 min. The viscous mixture was poured over
ice and the water layer was extracted with CH2Cl2 (2 × 25
mL). Combined organic layers were dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified
by silica gel chromatography (3:1 hexanes:EtOAc) to give 18
(2.01 g, 94%) as a clear oil: Rf ) 0.30 (3:1 hexanes:EtOAc); 1H
NMR (CDCl3) δ 4.27 (m, 3H), 3.88 (m, 2H), 2.07 (m, 1H), 1.96
(m, 2H), 1.75 (m, 1H); 31P NMR (CDCl3) δ -17.14. HRMS
(C5H9O3Cl2P) calcd 218.9745 (M + H)+, found 218.9756.
Tetr a h yd r ofu r fu r yl Bis(2-cya n oeth yl) P h osp h a te (19).
Phosphoryl dichloride 18 (100 mg, 0.46 mmol) was dissolved
in THF (3 mL) and cooled to -10 °C under an atmosphere of
argon. 3-Hydroxypropionitrile (0.062 mL, 0.91 mmol) and
triethylamine (0.130 mL, 0.96 mmol) were dissolved in THF
(2 mL) and added dropwise at -10 °C. The reaction mixture
was warmed to room temperature and stirred overnight.
Triethylamine hydrochloride was removed by filtration, and
the filtrate was concentrated under reduced pressure. Purifi-
cation of the crude product by silica gel chromatography (1:1
EtOAc:CHCl3) afforded 19 (71 mg, 54%) as a colorless oil: Rf
1
) 0.15 (EtOAc:CHCl3); H NMR (CDCl3) δ 4.31 (m, 4H), 4.10
(m, 3H), 3.85 (m, 2H), 2.80 (t, 4H, J ) 6.2 Hz), 2.02 (m, 1H),
1.93 (m, 2H), 1.65 (m, 1H); 31P NMR (CDCl3) δ -29.80; HRMS
(C11H17N2O5P) calcd 289.0953 (M + H)+, found 289.0939.
Tetr ah ydr ofu r fu r yl P h osph ate Diam m on iu m Salt (17).
Phosphotriester 19 (10.0 mg, 0.035 mmol) was dissolved in
CH3CN (90 µL) and added to concentrated ammonium hy-
droxide (500 µL) at room temperature. The mixture was
transferred to an NMR tube, and the reaction was monitored
by 31P NMR for 30 min. Instantaneous removal of the first
cyanoethyl group followed by slow removal of the second
cyanoethyl group was observed. The half-life for removal of
the second cyanoethyl group was calculated (t1/2 ) 194 min),
and the reaction mixture was transferred to a vial and allowed
to stir for 24 h (>7 half-lives). The aqueous mixture was frozen
and lyophilized without further purification to give monophos-
J M000302B