Design and synthesis of phosphotyrosine peptidomimetic prodrugs

Article abstract of DOI:10.1021/jm060142p

A novel approach to the intracellular delivery of aryl phosphates has been developed that utilizes a phosphoramidate-based prodrug approach. The prodrugs contain an ester group that undergoes reductive activation intracellularly with concomitant expulsion

Full text of DOI:10.1021/jm060142p

3368
J. Med. Chem. 2006, 49, 3368-3376
Design and Synthesis of Phosphotyrosine Peptidomimetic Prodrugs
Hugo Garrido-Hernandez, Kyung D. Moon, Robert L. Geahlen, and Richard F. Borch*
Department of Medicinal Chemistry and Molecular Pharmacology, and the Cancer Center, Purdue UniVersity, West Lafayette, Indiana 47907
ReceiVed February 8, 2006
A novel approach to the intracellular delivery of aryl phosphates has been developed that utilizes a
phosphoramidate-based prodrug approach. The prodrugs contain an ester group that undergoes reductive
activation intracellularly with concomitant expulsion of a phosphoramidate anion. This anion undergoes
intramolecular cyclization and hydrolysis to generate aryl phosphate exclusively with a t1/2 ) ∼20 min.
Phosphoramidate prodrugs (8-10) of phosphate-containing peptidomimetics that target the SH2 domain
were synthesized. Evaluation of these peptidomimetic prodrugs in a growth inhibition assay and in a cell-
based transcriptional assay demonstrated that the prodrugs had IC50 values in the low micromolar range.
Synthesis of phosphorodiamidate analogues containing a P-NH-Ar linker (16-18) was also carried out in
the hope that the phosphoramidates released might be phosphatase-resistant. Comparable activation rates
and cell-based activities were observed for these prodrugs, but the intermediate phosphoramidate dianion
underwent spontaneous hydrolysis with a t1/2 ) ∼30 min.
Scheme 1a
Introduction
Protein tyrosine phosphorylation is a common intracellular
signaling event that controls numerous signaling pathways.
Specific sequences, contained within a protein that undergoes
phosphorylation on tyrosine, can serve as recognition sites for
other proteins. Src homology 2 (SH2) domains mediate these
protein-protein interactions. SH2 domains are noncatalytic
motifs of about 100 amino acids that are contained in a variety
of proteins.1,2 The ability of these SH2 domains to bind to
phosphotyrosine-containing sequences is governed by the im-
mediately adjacent C-terminal sequence to the phosphotyrosine
residue. In particular, it has been found that the SH2 domains
of the cytoplasmic tyrosine kinases Src and Lck bind with
micromolar affinity to the peptide sequence pY-E-E-I.3,4 Src
and Lck have been implicated in a variety of disorders that
include breast cancer, colon cancer, autoimmune diseases, and
osteoporosis, and therefore they constitute good targets to
develop new therapeutic agents.5-8
a Reagents: (a) p-cresol, Et3N, CH2Cl2, -10° to 0 °C, 30 min; (b) benzyl
alcohol, Et3N, 0 °C, 1 h; (c) N-methyl-N-(2-bromo-ethyl)amine hydrobro-
mide, Et3N, THF, 20 min, rt; (d) N-methyl-N-(4-bromobutyl) amine
hydrobromide, i-Pr2NEt, CH2Cl2, -20 °C to rt, 4 h; (e) p-cresol, LiHMDS,
THF; (f) nitrofurfuryl alcohol, LiHMDS, THF.
generation of cell membrane permeable phosphotyrosine mi-
metic prodrugs.
Several peptidomimetics that target the SH2 domain of Lck
have been developed.9-11 However, the presence of a highly
ionizable phosphate group in these compounds compromises
their cell membrane permeability. To overcome this problem,
several less anionic phosphotyrosine isosteres have been de-
veloped and included in the design of SH2 domain inhibitors.12-14
Another approach that has been used is the generation of
phosphotyrosine prodrugs in the form of phosphoesters that are
simply activated by enzymatic hydrolysis.15 However, due to
the ubiquitous presence of esterases, this type of approach cannot
be considered selective. We have developed previously a series
of cell membrane permeable nucleoside phosphoramidate pro-
drugs that deliver nucleotides intracellularly.16-18 These phos-
phoramidates are composed of a phosphoester delivery group
and a masking phosphoramide group. The release of the active
nucleotide is triggered by the enzymatic transformation and
elimination of the delivery group, which is followed by a
spontaneous intramolecular cyclization and hydrolysis. Herein
we report the application of this prodrug technology to the
development of aryl phosphoramidates and apply it to the
Results and Discussion
(a) Model Compounds. In an effort to simplify the reaction
and experimental conditions, initial studies were carried out on
model p-cresol phosphoramidates. Two different phosphorami-
dates were synthesized (Scheme 1). Phosphoramidate 1 con-
tained a 2-bromoethyl masking group19 and a benzyl ester as a
conditional activating group.20 Phosphoramidate 2 contained an
extended 4-bromobutyl masking group and a nitrofurfuryl ester
as an activating group.17 The syntheses of phosphoramidates 1
and 2 are illustrated in Scheme 1. Phosphorus oxychloride
(POCl3) was consecutively reacted with p-cresol and benzyl
alcohol in the presence of triethylamine followed by reaction
with N-methyl-N-(2-bromoethylamine) hydrobromide to gener-
ate phosphoramidate 1. For the synthesis of phosphoramidate
2, POCl3 was reacted with N-methyl-N-(4-bromobutylamine)
hydrobromide in the presence of diisopropylethylamine to
generate the phosphoramidic chloride which was then consecu-
tively reacted with the lithium alkoxides of p-cresol and
nitrofurfuryl alcohol.
On the basis of the results obtained from the study of
nucleotide phosphoramidate prodrugs, it was expected that the
aryl phosphoramidates would behave in a similar manner, i.e.,
* To whom correspondence should be addressed. Tel: (765) 494-1403.
Fax: (765) 494-1414. E-mail: borch@purdue.edu.
10.1021/jm060142p CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/11/2006

Synthesis of Phosphotyrosine Peptidomimetic Prodrugs
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3369
Scheme 3a
Scheme 2
a Reagents: (a) N-methyl-4-chlorobutylamine hydrochloride, i-Pr2NEt,
CH2Cl2, 0 °C to rt, 4 h; (b) 6a-c, i-Pr2NEt, CH2Cl2; (c) LiHMDS,
nitrofurfuryl alcohol, THF, -30 °C, 5 h; (d) Pd(PPh3)4, TolSO2Na, THF/
H2O.
following delivery group activation and elimination, a phos-
phoramidate anion A would be formed (Scheme 2). The
inductive effect of the oxygen anion would drive the intramo-
lecular cyclization to yield the formation of the reactive
intermediate B. This intermediate could react with water
following pathway a and/or b to generate solvolysis product C
and/or the hydrolysis product D, respectively.
Scheme 4
To test this hypothesis, phosphoramidate 1 was hydrogeno-
lyzed and its subsequent transformation was monitored by 31P
NMR. The analysis showed that when n ) 2 the phosphora-
midate anion A disappeared with a half-life of 60 min (0.4 M
cacodylate buffer, pH ∼ 7.4, 22 °C) and that the desired aryl
phosphate D represented approximately 25% of the product.
Attack of water at the carbon of the aziridinium ring clearly
predominates in this system. In contrast, phosphoramidate A
(n ) 4) (generated by reduction of 2 with sodium dithionite)
was converted to the aryl monophosphate D quantitatively with
a half-life of <3 min (0.4 M cacodylate buffer, pH ∼ 7.4, 22
°C). It is interesting to note that cyclization to the five-membered
ring is much faster than to the three-membered ring in this
phosphoramidate.
(b) Peptidomimetic Prodrugs. The results obtained from the
model compounds prompted the application of this strategy to
the synthesis of phosphotyrosine peptidomimetic prodrugs.
Peptidomimetics targeted to the SH2 domain were selected to
demonstrate the feasibility of the approach. However, it was
discovered subsequently that the bromobutyl compound 2 was
unstable during storage and appeared to decompose via a
reaction initiated by intramolecular cyclization. In an attempt
to enhance the stability of the phosphoramidate esters while
retaining facile cyclization of the phosphoramidate anion
intermediate, the bromine in the masking group was replaced
with chlorine. Compound 11 (Scheme 4), which has low
micromolar binding affinity to pp60src,21 was selected as the
initial peptidomimetic for the prodrug approach (e.g., 8). By
analogy, two additional phosphoramidate prodrugs 9 and 10 that
differ in the spacer between the P and P+3 sites were
synthesized (Scheme 4). To accomplish the synthesis of
phosphoramidates 8-10, a convergent approach was used
involving preparation of two building blocks: (1) a carboxy-
containing aryl phosphoramidate and (2) a benzylamine. The
synthesis of the carboxy-containing building blocks is illustrated
in Scheme 3. Reaction of POCl3 and N-methyl-N-(4-chlorobu-
tylamine) hydrochloride produced phosphoramidic dichloride
3, which could be isolated and purified. We have shown
previously that allyl esters can be used to protect carboxylic
acids in the presence of phosphoramidates,22 so allyl esters 6a-c
were prepared by refluxing the respective free acids in allyl
bromide in the presence of diisopropylethylamine.23 These allyl
esters were then reacted with 3 in the presence of diisopropyl-
ethylamine to generate phosphoramidic monochlorides 4a-c,
respectively. Addition of preformed nitrofurfuryl lithium alkox-
ide to 4a-c generated the corresponding phosphoramidates
which were deallylated to give the carboxylic acids 5a-c. These
aryl phosphoramidates were then coupled to benzylamine 7
(synthesized according to the published procedure)21 in the
presence of PyBOP and diisopropylethylamine to generate
phosphoramidate prodrugs 8-10 (Scheme 4).
To verify that the free phosphates could be released from
their corresponding prodrugs, phosphoramidate 8 was subjected
to reductive activation using sodium dithionite, the residue was
dissolved in 0.4 M cacodylate buffer, final pH 7.2, and its
transformation studied by 31P NMR kinetics. The phosphora-
midate anion derived from 8 generated phosphate 11 with a
half-life of 20 min at 37 °C and 2.5 h at room temperature
(Scheme 4).
(c) Phosphorodiamidates. Because both phosphate ionization
and stability to phosphatase degradation can be modulated by
replacement of the linking oxygen in phosphotyrosine mimet-
ics,24,25 the replacement of oxygen by NH was explored in these
peptidomimetics. p-Toluidine phosphate 12b (Scheme 5) was

3370 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Garrido-Hernandez et al.
Scheme 5a
Scheme 6a
a Reagents: (a) POCl3, i-Pr2NEt, CH2Cl2, 0 °C to rt; (b) Na2CO3 or
NaOH, CH3CN/H2O; (c) allyl bromide, DMF; (d) allyl alcohol, cat. H2SO4,
reflux; (e) Ac2O, NaOH, H2O/acetone; (f) H2, Pd/C, NH.
a Reagents: (a) 3, i-Pr2NEt, (ClCH2)2, reflux; (b) POCl3, i-Pr2NEt,
CH2Cl2, -5 °C; (c) N-methyl-4-chlorobutylamine hydrochloride, i-Pr2NEt,
CH2Cl2, -5 °C; (d) LiHMDS, nitrofuryl alcohol, THF, -20 °C; (e)
Pd(PPh3)4, TolSO2Na, THF/H2O; (f) PyBOP, i-Pr2NEt, CH2Cl2.
Figure 1. Chemical shift titration curve of p-toluidine phosphoramidate
12b.
generated in situ from basic hydrolysis of phosphoramidic
dichloride 12a and explored as a simple model. The pKa2 for
this compound was determined using the pH-dependent chemical
shift change of the 31P NMR resonance and found to be 6.9
(Figure 1). The overall 31P NMR chemical shift change of about
1.25 ppm suggests that protonation of 12b occurs on oxygen
rather than nitrogen.26,27
Figure 2. Formation and hydrolysis of NH-phosphate from phospho-
ramidate 19 at 37 °C, pH ) 7.2. (b), phosphoramidate 19; (9), NH-
phosphate 20; (2), inorganic phosphate.
Synthesis of the analogous phosphorodiamidate peptidomi-
metic prodrugs 16-18 is outlined in Schemes 5 and 6. Allyl
ester 13a was prepared by reaction of p-aminobenzoic acid
potassium salt with allyl bromide in DMF. Attempts to prepare
13b and 13c28 under similar conditions, however, resulted in
allylation of the amino group. These compounds were prepared
successfully using Fischer esterification. Phosphorylation of 13a
was slow and generated 14a in poor yield even after reflux with
phosphoramidic dichloride 3 for 10 days. Intermediates 14b and
14c were prepared in modest yield using a two-step procedure
in which 13b or 13c was reacted with POCl3 followed by
reaction with N-methyl-N-(4-chlorobutylamine) hydrochloride.
Addition of 5-nitrofurfuryl lithium alkoxide to the three different
phosphorodiamidic chlorides 14a-c generated the correspond-
ing phosphorodiamidates which were cleaved to the correspond-
ing acids 15a-c by treatment with p-TolSo2Na and Pd(PPh3).
Finally, the peptidomimetics 16-18 were prepared by coupling
15a-c with benzylamine 7 in the presence of PyBOP and
diisopropylethylamine.
Scheme 7
To demonstrate that the phosphorodiamidates would undergo
activation and release of the analogous phosphoramidate dianion,
prodrug 16 was activated by sodium dithionite reduction and
the subsequent reactions (Scheme 7) studied by 31P NMR; the
kinetic profile is shown in Figure 2. Although conversion of
the phosphorodiamidate 19 (round symbols) to the phosphate
analogue 20 (square symbols) is clearly evident at early time
points, this product is unstable and proceeds to hydrolyze to
the corresponding aniline 21 and inorganic phosphate (triangle
symbols). Kinetic analysis (0.4 M cacodylate buffer, pH 7.2,
37 °C) gives rate constants k1 ) 0.052 and k2 ) 0.022 min-1
,

Synthesis of Phosphotyrosine Peptidomimetic Prodrugs
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3371
binding affinity (6 µM) for the free phosphate 11,21 consistent
with intracellular prodrug activation and phosphate release.
These results are also consistent with the targeting of phosphate
11 to the Lck SH2 domain, but it is also possible that binding
of this phosphate to other sites could contribute to the reduced
levels of luciferase expression. Although it was anticipated that
the difference in length of the spacer between the P and P+3
groups would affect the activity of the prodrugs, it appears that
all three structures can be accommodated at the binding site. It
is surprising that the activities of prodrugs 16-18 are compa-
rable to those of 8-10, considering that the phosphorodiami-
dates released from the former compounds are hydrolytically
unstable. However, it is likely that the phosphoramidates
released from 8-10 will be substrates for intracellular phos-
phatases, so the intracellular lifetimes of the enzymatically labile
phosphoramidates and hydrolytically labile phosphorodiamidates
may in fact be comparable.
Figure 3. pH-rate profile for the hydrolysis of phosphoramidate 12b,
37 °C.
Table 1. Response of Jurkat Cells and the NCI Tumor Cell Panel to
Prodrugsa
Conclusions
cpd
NFAT IC50, µMb
NCI panel GI50, µMc
A novel approach for the intracellular delivery of aryl
phosphate peptidomimetics has been developed. This approach
consists of cell membrane permeable phosphoramidate prodrugs
that undergo intracellular enzymatic activation followed by
intramolecular cyclization and hydrolysis to generate the dian-
ionic phosphate. Although the half-life for activation (cycliza-
tion) of the phosphoramidate is slower than that for aliphatic
phosphoramidate prodrugs (20 min vs < 5 min, respectively),
the slower activation rate is still adequate for the generation of
a biological response. Attempts to utilize a phosphorodiamidate
(by replacing the P(O)-O-Ar bond with P(O)-NH-Ar) that
generates a presumed phosphatase-resistant ligand resulted in
a prodrug of comparable activity, although NMR studies showed
that the phosphoramide dianion undergoes spontaneous hy-
drolysis with a half-life of ∼30 min. Efforts to extend this work
to phosphatase-resistant difluoromethylphosphonate analogues
are underway and will be reported in due course.
8
9
10
16
17
18
31 ( 3
9 ( 5
21 ( 4
16 ( 5
20 ( 4
28 ( 3
12 ( 7
3 ( 1
6 ( 5
18 ( 10
NDd
ND
a NFAT assay, cells exposed to drug for 6 h; NCI panel, cells exposed
to drug for 48 h. See Experimental Section for details. b Mean ( SE, n )
4. c Mean ( SD for 62 cell lines in the panel. d Not determined.
corresponding to half-lives of 13 and 32 min, respectively. The
pH dependence of the phosphoramide hydrolysis reaction was
investigated using p-toluidine phosphate 12b; the pH-rate
profile (Figure 3) clearly demonstrates that this is an acid-
catalyzed reaction and that the rate of hydrolysis is near half-
maximal at physiologic pH.
Biological Evaluation
Although the chemical activation and 31P analysis had
provided insight into the ability of these prodrugs to generate
aryl phosphates, there was the need to evaluate the feasibility
of this approach in cell-based systems. Therefore, the phospho-
ramidate prodrugs 8-10 and 16-18 were evaluated in a cell-
based assay that consists of a human leukemia Jurkat J77 T
cell line transiently transfected with a luciferase reporter gene
driven by four adjacent binding sites for the NFAT (nuclear
factor of activated T cells) transcription factor. The activation
of NFAT occurs in response to the mobilization of calcium
following cross-linking of the T cell antigen receptor and is
dependent on the expression of Lck with a functional SH2
domain.8,29 Thus, inhibitors directed against the SH2 domain
of Lck block both the mobilization of calcium and the
subsequent activation of gene transcription.12,30
Transiently transfected Jurkat cells were incubated with
phosphoramidates 8-10 and 16-18 for 1 h at concentrations
from 0.1 to 1000 µM and then activated with a combination of
anti-CD3 antibody and PMA. At the end of the 6 h incubation
period the cells were lysed, supernatant was collected, and the
luciferase activity was measured. The Src family selective
tyrosine kinase inhibitor PP231 was also studied as a positive
control; it showed an IC50 ) 0.9 µM. The growth inhibitory
activity of the compounds was also evaluated in the NCI tumor
cell panel. The results from the cell-based assays are shown in
Table 1. The prodrugs showed modest and similar activity in
both the luciferase and growth inhibition assays. The cell-based
IC50 values for prodrug 8 are in the same range as the published
Experimental Section
Materials and Methods. NMR spectra were recorded using a
250 MHz Bruker spectrometer equipped with a 5 mm multinuclear
1
probe. H chemical shifts are reported in parts per million using
tetramethylsilane as an internal reference. 31P NMR spectra were
1
obtained using broadband H decoupling, and chemical shifts are
reported in parts per million using 1% triphenylphosphine oxide
in benzene-d6 as a coaxial insert. 31P NMR kinetics carried out at
37 °C were conducted using a Bruker variable temperature unit.
Silica gel grade 60 was used to carry out all chromatographic
purifications. HPLC analysis was done using a Beckman System
Gold equipped with a 168 detector set to 250 nm, a 126 solvent
module, and an econosphere C18 column (5 µM, 4 × 250 mm)
from Alltech Associates. Mass spectral analyses were obtained from
the Mass Spectrometry Laboratory at Purdue University, West
Lafayette, IN. All anhydrous reactions were carried out under argon,
using flamed dried flasks, and all organic solvents were distilled
prior to use.
31P NMR Studies. Kinetics experiments were carried out as
described previously.17 Briefly, the compound (∼20 mg) was
dissolved in acetonitrile (80 µL); a solution of the activating agent
(sodium dithionite; 3 equiv) in cacodylate buffer (500 µL, 0.4 M,
pH ) 7.4) was added. The reaction mixture was transferred to a 5
mm NMR tube, and data acquisition was started with the probe
maintained at 37 °C. Spectra were acquired every 2.5 min for 30
min and when necessary every 10 min for additional time. Time
points were assigned to each data acquisition from the start of the
reaction. The integration of the peak areas was used to determine
the relative concentration of the intermediates and products.

3372 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Garrido-Hernandez et al.
Kinetic Analysis. The methodology has been described.20 The
31P peak areas were measured, and the product composition was
determined at each time point as a percentage of the total material.
For simple first-order reactions, the natural log of the peak area vs
time was analyzed by linear regression and the rate constant
obtained from the slope of the line. For multistep reactions, 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 using the Quattro Pro optimization routine.
Luciferase Assay. J77 Jurkat cells (2 × 107 cells in 500 µL of
FBS-free RPMI media) were transiently transfected with 15 µg of
NF-AT-luciferase plasmid by electroporation (300 V, 800 µF),
transferred to a flask with 10 mL of FBS-containing RPMI media,
and incubated for 24 h. Cells were then harvested, resuspended in
12 mL of FBS-containing RPMI media, and divided into a 6-well
plate (2 mL/well). Drug stock solutions were prepared in DMSO,
and each well was treated with a different concentration and
incubated for 1 h. Each well was then treated with 10 µL of a
solution of 1.25 µg of PMA (Calbiochem) in 115 µL of serum-
free RPMI media, followed by the addition of 2 µg/well of anti-
CD3 antibody (Pharmingen). The cells were incubated for 6 h, spun
down, washed with PBS, lysed (1× Promega lysis buffer) for 15
min, and centrifuged. The supernatant was collected and stored at
-78 °C overnight. Luciferase activity was measured, using a Lumat
LB 9501 luminometer, from a mixture of 50 µL of luciferase
substrate (Promega) and 10 µL of supernatant. The data obtained
were analyzed by sigmoidal curve fit of relative luciferase units
(RLU) vs log of drug concentration. The results are expressed as
the drug concentration that generates a 50% decrease in RLU (IC50)
of the control value (DMSO).
Triethylamine (0.090 mL, 0.78 mmol) was added dropwise to a
solution of N-Methyl-N-2-bromoethylamine hydrobromide (0.070
g, 0.34 mmol) and the phosphoryl monochloride prepared above
(0.10 g, 0.34 mmol) in dry THF (1.2 mL) at room temperature.
The reaction mixture was stirred for 80 min and filtered through a
plug of cotton, and the filtrate was evaporated to give an oil. The
crude product was purified using column chromatography (10:1
CHCl3/EtOAc) to yield 1 (60 mg, 47%) as an oil. Rf ) 0.56 (10:1
1
CHCl3/EtOAc). H NMR (CDCl3): δ 7.35 (s, 5H), 7.10 (m, 4H),
5.10 (d, 2H), 4.48-3.21 (m, 4H), 2.70 (d, 3H), 2.29 (s, 3H). 31P
NMR (CDCl3): δ -19.7. MS (ESI) m/z 398/400 (M + H)+.
N-Methyl-N-(4-bromobutyl) O-(5-Nitrofuryl-2-methyl) O-(4-
Methyl)phenyl Phosphoramidate (2). Methyl bromobutyl phos-
phoramidic dichloride (1.0 g, 3.6 mmol) (prepared as described
for the chlorobutyl compound 3) was dissolved in dry THF (5 mL)
and cannulated to a pre-cooled solution of p-cresol (0.38 g, 3.6
mmol) and LiHMDS (0.1 M solution in THF, 3.9 mL, 3.9 mmol)
in dry THF (18 mL) at -78 °C. The reaction mixture was stirred
and warmed to room temperature over 3 h. This mixture was then
cannulated to a pre-cooled solution of nitrofurfuryl alcohol (0.51
g, 3.6 mmol) and LiHMDS (3.9 mL, 3.9 mmol) in dry THF (18
mL) at -78 °C. The reaction mixture was then warmed to -15 °C
and stirred for 2.5 h. The reaction mixture was quenched with
saturated ammonium chloride and extracted with EtOAc (2 × 10
mL). The organic extracts were combined, washed with brine, dried
over MgSO4, filtered, and evaporated to yield a brown oil. The
residue was purified using column chromatography to yield 2 (0.75
g, 46%) as an orange oil. Rf ) 0.5 (100:10:0.5, CHCl3/EtOAc/
1
MeOH). H NMR (CDCl3): δ 7.26 (d, 1H), 7.09 (m, 4H,), 6.621
(d, 1H), 5.06 (d, 2H), 3.38 (t, 2H), 3.09 (m, 2H), 2.71 (d, 3H),
2.31 (s, 3H), 1.76 (m, 2H), 1.63 (m, 2H). 31P NMR (CDCl3): δ
-19.0. MS (ESI) m/z 461/463 (M + H)+.
NCI Human Tumor Cell Line Screen. Details of the methodol-
ogy are described at http://dtp.nci.nih.gov/branches/btb/ivclsp.html.
Briefly, cells are grown in supplemented RPMI 1640 medium for
24 h, then incubated with drug for 48 h at five concentrations from
10-8 to 10-4 M. The assay is terminated by addition of cold
trichloroacetic acid, and the cells are fixed and stained with
sulforhodamine B. Bound stain is solubilized, and the absorbance
is read on an automated plate reader. Percentage growth inhibition
is calculated from time zero, control growth, and the five concentra-
tion level absorbances.
N-Methyl-N-(4-chlorobutyl) Phosphoramidic Dichloride (3).
POCl3 (2.4 mL, 25 mmol) was added to a pre-cooled solution of
N-methyl-N-(4-chlorobutyl)amine hydrochloride (4.0 g, 25 mmol)
in dry CH2CL2 (40 mL) at 0 °C, followed by the addition of
triethylamine (7.1 mL, 51 mmol) diluted in dry CH2CL2 (20 mL).
The reaction mixture was removed from the cooling bath, stirred
overnight at room temperature, quenched with saturated ammonium
chloride, and extracted with CH2CL2 (3 × 10 mL). The organic
extracts were combined, washed with brine, dried over MgSO4,
filtered, and evaporated to dryness to yield an oil. The crude product
was purified using column chromatography (3:1 hexanes/EtOAc)
to yield 12 (4.8 g, 80%) as a clear oil. Rf ) 0.45 (3:1 Hex/EtOAc).
1H NMR (CDCl3): δ 3.58 (t, 2H), 3.29 (m, 2H), 2.84 (d, 3H),
1.79 (m, 4H). 31P NMR (CDCl3): δ -6.6.
Allyl 4-Hydroxybenzoate (6a). Diisopropylethylamine (6.3 mL,
36 mmol) was added to a suspension of 4-hydroxybenzoic acid
(5.0 g, 36 mmol) in allyl bromide (86 mL). The reaction mixture
was refluxed for 2 h and cooled to room temperature. Excess allyl
bromide was evaporated under reduced pressure, and the remaining
oil was dissolved in EtOAc (50 mL) and washed with water (3 ×
10 mL). The organic extract was washed with brine, dried over
MgSO4, filtered, and evaporated to yield a yellow oil. The crude
product was purified using column chromatography (10:1 CHCl3/
EtOAc) to yield 6a (5.0 g, 78%) as a white solid, mp 100-102
°C. Rf ) 0.44 (10:1 CHCl3/EtOAc). 1H NMR (CDCl3): δ 7.99 (d,
2H), 6.86 (d, 2H), 6.02 (m, 1H), 5.30 (m, 2H), 4.80 (d, 2H). MS
(ESI) m/z 179 (M + H)+.
N-Methyl-N-(4-bromobutyl)amine Hydrobromide. Hydrobro-
mic acid (20 mL, 48% by wt) was added slowly with stirring to
N-methyl-N-4-hydroxybutylamine (4.0 g, 0.39 mmol) at 0 °C, and
the reaction mixture was heated to reflux for 2 h. A distillation
apparatus was attached, 10 mL of distillate was collected, and an
additional 10 mL of 48% HBr was added. After the mixture was
refluxed for 4 h, the procedure was repeated, and the reaction
mixture was refluxed overnight. Approximately 20 mL of distillate
was removed from the reaction mixture, and the still pot residue
was poured into acetone that was cooled to -78 °C. A white
precipitate formed that was collected by filtration (4.24 g, 42%),
1
mp 86-88 °C. H NMR (D2O): δ 2.84 (t, 2H), 2.39 (t, 2H), 2.04
(s, 3H), 1.23 (m, 4H).
N-Methyl-N-(2-bromoethyl) O-Benzyl O-(4-Methyl)phenyl
Phosphoramidate (1). Triethylamine (0.37 mL, 2.6 mmol) was
diluted in dry CH2CL2 (1 mL) and added slowly to a pre-cooled
solution of p-cresol (0.25 mL, 2.4 mmol) and POCl3 (0.22 mL, 2.4
mmol) in dry CH2CL2 (6 mL) at -10 °C. The reaction mixture
was stirred and warmed to 0 °C over 30 min. Benzyl alcohol (0.25
mL, 2.4 mmol) was added, followed by triethylamine (0.37 mL,
2.6 mmol) diluted in dry CH2CL2 (1 mL). The reaction was stirred
for 1 h at 0 °C, quenched with saturated ammonium chloride, and
extracted with CH2CL2 (3 × 5 mL). The organic extracts were
combined, washed with brine, dried over MgSO4, filtered, and
evaporated to yield an oil. The crude product was subjected to
column chromatography (3:1 hexanes/EtOAc) to give the phos-
phoryl monochloride (0.35 g, 50%) as an oil, which was used
directly in the next step. Rf ) 0.49 (3:1 hexanes/EtOAc). 1H NMR
(CDCl3): δ 7.38 (s, 5H), 7.10 (m, 4H), 5.29 (m, 2H), 2.32 (s, 3H).
31P NMR (CDCl3): δ -24.0.
Allyl 4-Hydroxyphenylacetate (6b). Allyl ester 6b was syn-
thesized from phenylacetic acid (2.0 g, 13 mmol) as described for
6a and obtained as an oil (2.3 g, 89%). Rf ) 0.31 (10:1 CHCl3/
EtOAc). 1H NMR (CDCl3): δ 7.16 (d, 2H), 6.76 (d, 2H), 5.92 (m,
1H), 5.28 (m, 2H), 4.60 (d, 2H), 3.64 (s, 2H).
N-Acetyl-L-tyrosine Allyl Ester (6c). Allyl ester 6c was
synthesized from Ac-Tyr-OH (10 g, 45 mmol) as described for 6a
and obtained as an oil (9.2 g, 80%). 1H NMR (CDCl3): δ 7.00 (d,
2H), 6.77 (d, 2H), 5.97 (m, 2H), 5.31 (m, 2H), 4.89 (m, 1H), 4.61
(d, 2H,), 3.13 (m, 2H), 2.00 (s, 3H).
N-Methyl-N-(4-chlorobutyl) O-4-Carboallyloxyphenyl Phos-
phoramidic Chloride (4a). Diisopropylethylamine (0.73 mL, 4.2

Synthesis of Phosphotyrosine Peptidomimetic Prodrugs
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3373
mmol) was added neat to a pre-cooled solution of phosphoramidic
dichloride 3 (0.50 g, 2.1 mmol) and allyl ester 6a (0.37 g, 2.1 mmol)
in dry CH2CL2 (8 mL) at -15 °C. The reaction mixture was stirred
for 1 h at -15 °C and 3 h at -5 °C, then quenched with saturated
ammonium chloride, and extracted with CH2CL2 (2 × 10 mL). The
organic extracts were combined, washed with brine, dried over
MgSO4, filtered, and evaporated to give an oil. The crude product
was purified using column chromatography (10:1 CHCl3/EtOAc)
to yield 4a (0.50 g, 63%) as a clear oil. Rf ) 0.60 (10:1 CHCl3/
(5c). Phosphoramidate 5c (0.15 g, 27%) was synthesized in two
steps from 4c (0.48 g, 1.0 mmol) as described for 5a. Rf ) 0.41
1
(10:1 CHCl3/EtOAc). H NMR (CDCl3): δ 7.30-7.25 (m, 1H),
7.23-6.83 (m, 4H), 6.80-6.47 (m, 1H), 5.09 (d, 2H), 5.01-4.59
(m, 2H), 3.76-3.33 (m, 2H), 3.36-2.98 (m, 4H), 2.98-2.55 (m,
3H), 2.14-1.77 (d, 3H), 1.89-1.55 (m, 4H). 31P NMR (CDCl3):
δ -19.3, -19.4 (1:1 mixture of diastereomers). MS (ESI) m/z 532/
534 (M + H)+.
5-(1-Aminoethyl)-2-cyclohexylmethoxybenzamide (7). Com-
pound 7 was prepared from 5-acetylsalicylamide according to a
1
EtOAc). H NMR (CDCl3): δ 8.10 (d, 2H,), 7.34 (d, 2H), 6.09-
21
5.98 (m, 1H), 5.45-5.28 (m, 2H), 4.84-4.81 (m, 2H), 3.57 (t, 2H),
3.35-3.17 (m, 2H), 2.84 (d, 3H), 1.82-1.79 (m, 4H). 31P NMR
(CDCl3): δ -13.1. MS (ESI) m/z 380/382 (M + H)+.
three-step procedure described by Lunney et al.
N-Methyl-N-(4-chlorobutyl) O-(5-Nitrofuryl-2-methyl) O-(4-
(1-(3-Carbamoyl-4-cyclohexylmethoxyphenyl)ethylcarbamoyl))-
phenyl Phosphoramidate (8). Diisopropylethylamine (0.11 mL,
0.62 mmol) was added neat to a pre-cooled solution of phospho-
ramidate 5a (0.13 g, 0.28 mmol), amine 7 (0.090 g, 0.31 mmol),
and PyBOP (0.16 g, 0.31 mmol) in dry CH2CL2 (2 mL) at 0 °C.
The reaction was stirred for 10 min at 0 °C and 30 min at room
temperature, then quenched with saturated ammonium chloride, and
extracted with CH2CL2 (3 × 5 mL). The organic extracts were
combined, washed with brine, dried over MgSO4, filtered, and
evaporated to yield an oil. The crude product was purified using
column chromatography (10% MeOH in EtOAc) to yield 8 (13
N-Methyl-N-(4-chlorobutyl) O-(4-Carboallyloxymethyl)phe-
nyl Phosphoramidic Chloride (4b). Phosphoramidic chloride 4b
was synthesized from allyl ester 6b (0.20 g, 10 mmol) as described
for 4a. The crude product was purified using column chromatog-
raphy (10:1 CHCl3/EtOAc) to yield 4b a clear oil (0.13 g, 32%).
1
Rf ) 0.71 (10:1 CHCl3/EtOAc). H NMR (CDCl3): δ 7.32-7.19
(m, 4H), 5.98-5.82 (m, 1H), 5.31-5.20 (m, 2H), 4.60 (d, 2H),
3.64 (s, 2H), 3.58 (t, 2H), 3.25-3.18 (m, 2H), 2.83 (d, 3H), 1.80-
1.78 (m, 4H). 31P NMR (CDCl3): δ -12.7. MS (ESI) m/z 394/396
(M + H)+.
1
N-Methyl-N-(4-chlorobutyl) O-(4-((S)-2-Acetylamino-2-car-
boallyloxyethyl))phenyl Phosphoramidic Chloride (4c). Phos-
phoramidic chloride 4c was synthesized from 6c (0.50 g, 1.9 mmol)
as described for 4a, with the exception that the reaction mixture
was stirred overnight. The crude product was purified using column
chromatography (5% MeOH in CH2CL2) to yield 4c (0.49 g, 55%)
as an oil. Rf ) 0.57 (5% MeOH in CH2CL2). 1H NMR (CDCl3:) δ
7.20-7.09 (m, 4H), 5.99-5.78 (m, 2H), 5.35-5.25 (m, 2H), 4.93-
4.85 (m, 1H), 4.61 (d, 2H), 3.58 (t, 2H), 3.26-3.13 (m, 4H), 2.83
(d, 3H), 2.00 (s, 3H), 1.96-1.75 (m, 4H). 31P NMR (CDCl3): δ
-12.7. MS (ESI) m/z 465/467 (M + H)+.
mg, 67%) as a white foam. H NMR (CDCl3): δ 8.25 (d, 1H),
7.72 (d, 2H), 7.50 (d, 1H), 7.24 (m, 3H), 6.95 (d, 2H), 6.63 (d,
1H), 5.28 (t, 1H), 5.07 (d, 2H), 3.93 (d, 2H), 3.52 (t, 2H), 3.09 (m,
2H), 2.70 (d, 3H), 1.87-1.58 (m, 14H), 1.48-0.85 (m, 4H). 31P
NMR (CDCl3): δ -21.2. HRMS (ESI) C33H42ClN4O9P calculated
705.2456 (M + H)+, found 705.2432. Anal. (C33H42ClN4O9P‚H2O)
C, H, N.
N-Methyl-N-(4-chlorobutyl) O-(5-Nitrofuryl-2-methyl) O-(1-
(3-Carbamoyl-4-cyclohexylmethoxyphenyl)methyl)phenyl Phos-
phoramidate (9). Compound 9 was obtained from 5b (0.12 g, 0.25
mmol) as described for the preparation of 8. The crude product
was purified using column chromatography (5% MeOH in EtOAc)
to yield 9 (0.10 g, 55%) as a brown foam. Rf ) 0.44 (5% MeOH
N-Methyl-N-(4-chlorobutyl) O-(5-Nitrofuryl-2-methyl) O-(4-
Carboxy)phenyl Phosphoramidate (5a). Phosphoramidic monochlo-
ride 4a (0.63 g, 1.2 mmol) was dissolved in dry THF (2 mL) and
cannulated to a pre-cooled solution of nitrofurfuryl alcohol (0.19
g, 1.3 mmol) and LiHMDS (1.0 M solution in THF, 1.5 mL, 1.5
mmol) in dry THF (2 mL) at -78 °C. The reaction was brought to
-40 °C and stirred for 5.5 h. Saturated ammonium chloride was
added, and the mixture was extracted with ethyl acetate (3 × 5
mL). The organic extracts were combined, washed with brine, dried
over MgSO4, filtered, and evaporated to yield a dark oil. The crude
product was purified by column chromatography (10:1 CHCl3/
EtOAc) to yield the allyl ester of phosphoramidate 5a (0.30 g, 52%)
as an orange oil. Rf ) 0.25 (10:1 CHCl3/EtOAc). 1H NMR
(CDCl3): δ 8.05 (m, 2H), 7.26 (m, 3H), 6.64 (d, 1H), 6.05 (m,
1H), 5.31 (m, 2H), 5.09 (d, 2H), 4.82 (d, 2H), 3.53 (t, 2H), 3.1 (m,
2H), 2.72 (d, 3H), 1.67 (m, 4H). 31P NMR (CDCl3): δ -21.7.
Sodium p-toluenesulfinate (0.29 g, 1.6 mmol) was dissolved in
water (2.5 mL) and added to a solution of the allyl ester (0.71 g,
1.5 mmol) and Pd(PPh3)4 (80 mg, 73 µmol) in THF (6 mL). The
reaction mixture was stirred for 30 min at room temperature, diluted
with diethyl ether, and washed with water (3 × 5 mL). The aqueous
extracts were combined, washed with ether (1 × 5 mL), acidified
to pH 3 with 2% HCl, and extracted with EtOAc. The organic
extracts were combined, washed with brine, dried over MgSO4,
filtered, and evaporated to yield phosphoramidate 5a (0.51 g, 78%)
1
in EtOAc). H NMR (CDCl3): δ 8.10-8.00 (m, 1H), 7.99-7.86
(m, 1H), 7.45-7.09 (m, 7H), 6.98-6.85 (m, 1H), 6.69-6.56 (m,
1H), 6.05-5.89 (m, 1H), 5.87-5.69 (m, 1H), 5.19-4.95 (m, 3H),
4.02-3.81 (m, 2H), 3.64-3.29 (m, 4H), 3.26-2.92 (m, 2H), 2.87-
2.52 (m, 3H), 2.02-1.57 (m, 11H), 1.54-0.89 (m, 6H). 31P NMR
(CDCl3): δ -18.9, -19.0 (1:1 mixture of diastereomers). HRMS
(ESI) C34H44ClN4O9P calculated 719.2613 (M + H)+, found
719.2600. Anal. (C34H44ClN4O9 P‚2H2O) C, H, N. H: calcd, 6.41;
found, 5.89.
N-Methyl-N-(4-chlorobutyl) O-(5-Nitrofuryl-2-methyl) O-(4-
((S)-2-Acetylamino-2-(1-(3-carbamoyl-4-cyclohexylmethoxyphe-
nyl)ethylcarbamoyl)ethyl))phenyl Phosphoramidate (10). Com-
pound 10 was obtained from 5c (0.15 g, 0.27 mmol) as described
for the preparation of 8. The crude product was purified using
column chromatography (10% MeOH in EtOAc) to yield 10 (0.13
1
g, 58%) as a brown foam. Rf ) 0.34 (10% MeOH in EtOAc). H
NMR (CDCl3): δ 8.26-8.11 (m, 1H), 8.09-7.99 (m, 1H), 7.95-
7.76 (m, 1H), 7.24-7.09 (m, 2H), 7.08-6.99 (m, 2H), 6.96-6.82
(m, 1H), 6.72-6.55 (m, 1H), 6.50-6.20 (m, 1H), 6.18-6.54 (m,
1H), 5.16-4.82 (m, 3H), 4.79-4.46 (m, 1H), 4.03-3.78 (m, 2H),
3.62-3.43 (m, 2H), 3.15-2.81 (m, 5H), 2.80-2.51 (m, 3H), 2.04-
1.89 (m, 3H), 1.87-1.56 (m, 10H), 1.49-0.90 (m, 8H). 31P NMR
(CDCl3): δ -18.9. HRMS (ESI) C37H49ClN5O10P calculated
790.2984 (M + H)+, found 790.2981. Anal. (C37H49ClN5O10P‚H2O)
C, H, N.
1
as an orange foam. H NMR (CDCl3): δ 8.08 (d, 2H), 7.31 (m,
3H), 6.66 (d, 1H), 5.11 (d, 2H), 3.53 (t, 2H), 3.13 (m, 2H), 2.74
(d, 3H), 1.71 (m, 4H). 31P NMR (CDCl3): δ -20.4.
N-(4-Methylphenyl)phosphoramidic Dichloride (12a). A solu-
tion of diisopropylethylamine (0.81 mL, 4.7 mmol) in CH2CL2 (3
mL) was added slowly to a pre-cooled solution of p-toluidine (0.50
g, 4.7 mmol) and POCl3 (0.43 mL, 4.7 mmol) in CH2CL2 (30 mL)
at -10 °C. The cooling bath was removed, and the reaction was
stirred overnight at room temperature. Saturated ammonium chloride
was added, the layers were separated, and the aqueous layer was
extracted with CH2CL2. The combined organic layers were washed
with brine, dried over MgSO4, filtered, and evaporated. The crude
product was purified by column chromatography (10:1 CHCl3:
N-Methyl-N-(4-chlorobutyl) O-(5-Nitrofuryl-2-methyl) O-(4-
Carboxymethyl)phenyl Phosphoramidate (5b). Phosphoramidate
5b (0.12 g, 36%) was synthesized in two steps from 4b (0.20 g,
1
0.51 mmol) as described for 5a. H NMR (CDCl3): δ 7.26-7.04
(m, 5H), 6.60 (d, 1H), 5.43-5.19 (m, 2H), 5.06 (d, 2H), 3.62 (s,
2H), 3.51 (t, 2H), 3.18-2.96 (m, 2H), 2.71 (d, 3H), 1.77-1.55
(m, 4H). 31P NMR (CDCl3): δ -19.0.
N-Methyl-N-(4-chlorobutyl) O-(5-Nitrofuryl-2-methyl) O-(4-
((S)-2-Acetylamino-2-carboxyethyl))phenyl Phosphoramidate

3374 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Garrido-Hernandez et al.
EtOAc) to yield 12a (0.72 g, 69%) as an oil. NMR (CDCl3): δ
7.22 (d, 2H), 7.16 (d, 2H), 2.30 (s, 3H). 31P NMR (CDCl3): δ
-16.6.
Kinetics. A solution of 12a (20 mg) in acetonitrile (300 µL)
was added to a solution of Na2CO3 (20 mg) in water (300 µL).
The solution was mixed for 5 min, passed through a short plug of
Celite, and diluted in cacodylate buffer (400 µL, 0.4 M). The pH
was adjusted to 7.4, and the resulting solution was monitored by
31P NMR at 37 °C.
N-(4-Methylphenyl)phosphoramidic Acid (12b). A solution of
12a (0.50 g, 2.2 mmol) in acetonitrile (5 mL) was added slowly to
a solution of NaOH (0.18 g, 4.5 mmol) in H2O (10 mL) at 0 °C.
The pH was monitored near the end of the addition to ensure that
the pH remained >11. The solution was then stirred for 30 min,
warmed to room temperature, and filtered through Celite. This
solution of 12b as its disodium salt was suitable for all further
experiments. 31P NMR (D2O): δ -24.8.
Allyl 4-Aminobenzoate (13a). Allyl bromide (0.99 mL, 11
mmol) was added to a suspension of potassium p-aminobenzoate
(2.0 g, 11 mmol) in DMF (40 mL). The reaction mixture was stirred
overnight, diluted with water, and extracted with EtOAc (3 × 15
mL). The organic extracts were combined, washed with brine, dried
over MgSO4, filtered, and evaporated to yield an oil. The crude
product was purified by column chromatography (10:1 CHCl3/
EtOAc) to yield 13a (2.0 g, 70%) as a foam. 1H NMR (CDCl3): δ
7.99 (d, 2H), 7.08 (d, 2H), 6.05-5.97 (m, 1H), 5.51-5.30 (m, 2H),
4.82-4.78 (m, 2H).
were separated, the organic layer was washed with brine, dried over
MgSO4, filtered, and evaporated to yield an oil. The crude product
was purified by column chromatography (10:1 CHCl3/EtOAc) to
1
yield 14a (0.22 g, 20%) as an oil. H NMR (CDCl3): δ 7.99 (d,
2H), 7.08 (d, 2H), 6.05-5.97 (m, 1H), 5.57 (d, 1H), 5.51-5.30
(m, 2H), 4.82-4.78 (m, 2H), 3.54 (t, 2H), 3.36-3.13 (m, 2H), 2.74
(d, 3H), 1.87-1.64 (m, 4H). 31P NMR (CDCl3): δ -11.3. MS (ESI)
m/z 379/381 (M + H)+.
N-Methyl-N-(4-chlorobutyl) N-(4-Carboallyloxymethyl)phe-
nyl Phosphorodiamidic Chloride (14b). Diisopropylethylamine
(0.27 mL, 1.6 mmol) was added to a pre-cooled solution of 13b
(0.3 g, 1.6 mmol) and POCl3 (0.2 mL, 1.6 mmol) in dry CH2CL2
(3 mL) at -5 °C. The reaction mixture was stirred for 10 min.
N-Methyl-N-(4-chlorobutyl)amine hydrochloride (0.37 g, 2.4 mmol)
was dissolved in dry CH2CL2 (2 mL) and added to the reaction
mixture at -5 °C. Diisopropylethylamine (0.82 mL, 4.7 mmol) in
dry CH2CL2 (1 mL) was then added slowly to the mixture. The
reaction mixture was stirred and warmed to room temperature over
3 h, saturated ammonium chloride was added, and the aqueous layer
was extracted with CH2CL2 (3 × 10 mL). The organic extracts
were combined, washed with brine, dried over MgSO4, filtered,
and evaporated to yield an oil. The crude product was purified by
column chromatography (10:1 CHCl3/EtOAc) to yield 14b (0.62
1
g, 34%) as a yellow oil. H NMR (CDCl3): δ 7.19 (d, 2H), 7.01
(d, 2H), 6.00-5.79 (m, 1H), 5.41-5.17 (m, 3H), 4.59 (d, 2H), 3.60
(s, 2H), 3.49 (bs, 2H), 3.27-3.09 (m, 2H), 2.71 (d, 3H), 1.84-
1.59 (m, 4H). 31P NMR (CDCl3): δ -10.6. MS (ESI) m/z 393/395
(M + H)+.
Allyl 4-Aminophenylacetate (13b). A mixture of 4-aminophe-
nylacetic acid (0.50 g, 3.3 mmol), allyl alcohol (20 mL), and
concentrated H2SO4 (1 mL) was refluxed for 2 h and then allowed
to cool to room temperature. The reaction mixture was poured over
ice, basified (pH ∼ 10) with Na2CO3, and extracted with EtOAc
(3 × 10 mL). The organic extracts were combined, washed with
brine, dried over MgSO4, filtered, and evaporated to dryness to
yield an oil. The crude product was purified using column
chromatography (10:1 CHCl3/EtOAc) to yield 13b (0.63 g, 51%)
N-Methyl-N-(4-chlorobutyl) N-(4-(2-((S)-Acetylamino-2-car-
boallyloxy)ethyl))phenyl Phosphorodiamidic Chloride (14c).
Phosphorodiamidic chloride 14c was obtained from 13c (0.50 g,
1.9 mmol) as described for the preparation of 14b. The crude
product was purified by column chromatography to yield 14c (0.30
1
g, 33%) as a white foam. H NMR (CDCl3): δ 7.19-6.91 (m,
4H), 6.00-5.79 (m, 2H), 5.41-5.22 (m, 2H), 4.97-4.78 (m, 1H),
4.60 (d, 2H), 3.59-3.46 (m, 2H), 3.28-2.97 (m, 4H), 2.71 (d, 3H),
1.99 (s, 3H), 1.81-1.57 (m, 4H). 31P NMR (CDCl3): δ -10.5.
MS (ESI) m/z 464/466 (M + H)+.
1
as an oil. Rf ) 0.34 (10:1 CHCl3/EtOAc). H NMR (CDCl3): δ
7.06 (d, 2H), 6.61 (d, 2H), 6.00-5.80 (m, 1H), 5.32-5.11 (m, 2H),
4.57 (d, 2H), 3.50 (s, 2H).
N-Methyl-N-(4-chlorobutyl) O-(5-Nitrofuryl-2-methyl) O-(4-
Carboxy)phenyl Phosphorodiamidate (15a). LiHMDS (1.0 M
solution in THF, 0.65 mL, 0.65 mmol) was added slowly to a pre-
cooled solution of nitrofurfuryl alcohol (0.08 g, 0.59 mmol) in dry
THF (1 mL) at -78 °C. The mixture was then cannulated to a
pre-cooled solution of 14a (0.22 g, 0.59 mmol) in dry THF (1 mL)
at -20 °C. The reaction was stirred for 4 h at -20 °C and then
quenched with saturated ammonium chloride. The reaction mixture
was extracted with ethyl acetate (3 × 5 mL). The organic extracts
were combined, washed with brine, dried over MgSO4, filtered,
and evaporated to yield an oil. The crude product was purified by
column chromatography (10% MeOH in EtOAc) to give the allyl
ester (0.11 g, 40%) as a brown foam. 1H NMR (CDCl3): δ 7.96 9
(d, 2H), 7.26 (d, 1H), 6.99 (d, 2H), 6.65 (d, 1H), 6.16-5.84 (m,
1H), 5.51-5.19 (m, 3H), 5.09 (d, 2H), 4.92-4.73 (m, 2H), 3.52
N-Acetyl-4-aminophenylalanine Allyl Ester (13c). Ac2O was
added slowly to a solution of 4-nitrophenylalanine (3.0 g, 13 mmol)
and NaOH (1.1 g, 26 mmol) in a 3:1 mixture of H2O/acetone (120
mL). After the mixture was stirred for 1 h, acetone was removed
under reduced pressure. The remaining aqueous solution was
basified (pH ∼10) with saturated sodium bicarbonate and extracted
with EtOAc (3 × 10 mL). The organic extracts were discarded.
The aqueous layer was acidified (pH ∼ 3) with dilute HCl and
extracted with EtOAc. The organic extracts were combined, washed
with brine, dried over MgSO4, filtered, and evaporated to give
1
N-acetyl-4-nitrophenylalanine (3.4 g, 74%) as a white solid. H
NMR (DMSO-d6): δ 8.16 (d, 1H), 8.11 (d, 2H), 7.48 (d, 2H), 4.45
(m, 1H), 3.20-2.87 (m, 2H), 1.71 (s, 3H). A portion of this product
(2.5 g, 9.6 mmol) was dissolved in ethanol (100 mL), 10% Pd/C
(0.5 g) was added, and the mixture was agitated in a hydrogen
atmosphere (60 psi) for 1 h. The reaction mixture was filtered and
evaporated to give N-acetyl-4-aminophenylalanine (2.13 g, 100%)
(t, 2H), 3.22-2.99 (m, 2H), 2.69 (d, 3H), 1.85-1.61 (m, 4H). 31
P
NMR (CDCl3): δ -15.6. MS (ESI) m/z 486/488 (M + H)+.
1
Sodium p-toluenesulfinate (45 mg, 0.25 mmol) was dissolved
in water (0.6 mL) and added to a solution of the allyl ester (0.11
g, 0.23 mmol) and Pd(PPh3)4 (13 mg, 11 µmol) in THF (1.2 mL).
The reaction mixture was stirred for 45 min at room temperature,
and then diluted with diethyl ether and washed with water (3 × 2
mL). The aqueous extracts were combined, washed with ether (1
× 4 mL), acidified to pH 3 with 2% HCl, and extracted with EtOAc
(3 × 3 mL). The organic extracts were combined, washed with
brine, dried over MgSO4, filtered, and evaporated to yield 15a (80
as a white solid. H NMR (DMSO-d6): δ 8.01 (d, 1H), 6.81 (d,
2H), 6.40 (d, 2H), 4.23 (m, 1H), 2.90-2.40 (m, 2H), 1.72 (s, 3H).
This compound (5.19 g, 23.4 mmol) was converted to the allyl
ester 13c as described for the preparation of 13b. The crude product
was purified using column chromatography (5% MeOH in CH2-
1
CL2) to yield 13c (3.6 g, 60%) as an oil. H NMR (CDCl3): δ
6.86 (d, 2H), 6.60 (d, 2H), 6.00-5.79 (m, 1H), 5.40-5.21 (m, 2H),
4.82 (m, 1H), 4.60 (d, 2H), 3.02 (m, 2H), 1.98 (s, 3H).
N-Methyl-N-(4-chlorobutyl) N′-(4-Carboallyloxy)phenyl Phos-
phorodiamidic Chloride (14a). Diisopropylethylamine (0.51 mL,
2.9 mmol) was added to a mixture of phosphoramidic dichloride 3
(0.70 g, 2.9 mmol) and allyl ester 13a (0.52 g, 2.9 mmol) in dry
dichloroethane (10 mL). The reaction mixture was refluxed for 10
days, allowed to cool room temperature, diluted with CH2CL2 (10
mL), and quenched with saturated ammonium chloride. The layers
1
mg, 76%) as a yellow foam. H NMR (CDCl3, TMS) δ: 7.96 (d,
2H), 7.26 (d, 1H), 6.99 (d, 2H), 6.65 (d, 1H), 6.45-6.25 (m, 1H),
5.09 (d, 2H), 3.52 (t, 2H), 3.22-2.99 (m, 2H), 2.69 (d, 3H), 1.85-
1.61 (m, 4H). 31P NMR (CDCl3) δ: -15.4. HPLC (gradient 30%
to 100% CH3CN/H2O [0.1% TFA] over 35 min): 9.37 min, 100%.
MS (ESI) m/z 444/446 (M - H)-.

Synthesis of Phosphotyrosine Peptidomimetic Prodrugs
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3375
N-Methyl-N-(4-chlorobutyl) O-(5-Nitrofuryl-2-methyl) O-(4-
Carboxymethyl)phenyl Phosphorodiamidate (15b). Phospho-
rodiamidate 15b was obtained as a yellow foam (0.10 g, 40%) from
14b (0.21 g, 0.54 mmol) by the two step sequence described for
(gradient 30% to 100% CH3CN/H2O [0.1% TFA] over 35 min):
18.25 min, 50%; 18.42 min, 50%. HRMS (ESI) m/z C37H50ClN6O9P
calculated 789.3147 (M + H)+, found 789.3144. Anal. (C37H50-
ClN6O9P‚3H2O) C, H, N. H: calcd, 6.69; found, 6.13. N: calcd,
9.97; found, 9.36.
1
the preparation of 15a. H NMR (CDCl3): δ 7.19-7.06 (m, 3H),
7.88 (d, 2H), 6.51 (d, 1H), 5.82 (d, 1H), 5.01 (d, 2H), 3.51 (s, 2H),
3.48 (t, 2H), 3.11-2.97 (m, 2H), 2.61 (d, 3H), 1.79-1.51 (m, 4H).
31P NMR (CDCl3): δ -14.4. HPLC (gradient 30% to 100% CH3-
CN/H2O [0.1% TFA] over 35 min): 11.48 min, 100%. MS (ESI)
m/z 456/458 (M - H)-.
Acknowledgment. Financial support from the National
Cancer Institute (Grant R01 CA34619) is gratefully acknowl-
edged. Support from the Purdue Cancer Center Support Grant
P30 CA23168 for services provided by the NMR and Mass
Spectrometry Shared Resources is appreciated.
N-Methyl-N-(4-chlorobutyl) O-(5-Nitrofuryl-2-methyl) O-(4-
(2-((S)-Acetylamino-2-carboxy)ethyl))phenyl Phosphorodiami-
date (15c). Phosphorodiamidate 15c was obtained as a foam (0.12
g, 38%) from 14c (0.29 g, 0.63 mmol) by the two-step sequence
Supporting Information Available: Elemental analysis data
of 8-10 and 16-18. This material is available free of charge via
the Internet at http://pubs.acs.org.
1
described for the preparation of 15a. H NMR (CDCl3): δ 7.00-
6.81 (m, 3H), 6.75-6.62 (d, 3H), 6.28-6.15 9 (m, 2H), 5.21-
5.00 (m, 2H), 4.92-4.61 (bs, 1H), 3.51 (bs, 2H), 3.19-2.92 (m,
4H), 2.62 (m, 3H), 1.99 (bs, 3H), 1.69-1.52 (m, 4H). 31P NMR
(CDCl3): δ -15.1. HPLC (gradient 30% to 100% CH3CN/H2O
[0.1% TFA] over 35 min): 10.12 min, 100%. MS (ESI) m/z 531/
533 (M + H)+.
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N-Methyl-N-(4-chlorobutyl) O-(5-Nitrofuryl-2-methyl) O-(4-
(1-(3-Carbamoyl-4-cyclohexylmethoxyphenyl)ethylcarbamoyl))-
phenyl Phosphorodiamidate (16). Diisopropylethylamine (0.07
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1
as a yellow foam. H NMR (CDCl3): δ 8.08 (d, 1H), 7.87 (bs,
1H), 7.34 (dd, 1H), 7.20 (d, 1H), 7.11 (d, 2H), 6.99-6.81 (m, 4H),
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