Phosphate prodrugs for amines utilizing a fast intramolecular hydroxy amide lactonization

Article abstract of DOI:10.1021/jo961477p

A novel phosphate prodrug system for amines, amino acids, peptides, and peptide mimetics, which utilizes a fast hydroxy amide lactonization of a 3-(2'-hydroxy-4',6'-dimethylphenyl)-3,3-dimethylpropionic amide system, was developed. Prodrugs of five model amine/amino acids, including p-anisidine, GlyOMe, PheOMe, LysOMe, and Asp-α-OMe, were synthesized. The syntheses of these model phosphate prodrugs were accomplished by coupling the amine or the protected amino acids with 3-[2'-(dibenzylphosphono)oxy-4',6'-dimethylphenyl]-3,3-dimethylpropion ic acid using coupling agents such as bis(2-oxo-3-oxazolidinyl)phosphinic chloride and 1-(3-dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride, followed by hydrogenolysis. These phosphate prodrugs were evaluated as substrates for the human placental alkaline phosphatase (AP). The structural features of the amine/amino acids attached to the carboxylic acid group of the promoiety were not found to significantly affect the substrate activity for AP, as evidenced by the small variations observed in the Michaelis-Menten parameters (K(m) and V(max)) of the phosphate prodrugs. Results obtained from this study suggest that such a phosphate prodrug system may be applied to a variety of structurally diverse amine-containing drugs.

Full text of DOI:10.1021/jo961477p

8636
J . Org. Chem. 1996, 61, 8636-8641
P h osp h a te P r od r u gs for Am in es Utilizin g a F a st In tr a m olecu la r
Hyd r oxy Am id e La cton iza tion
Michalis G. Nicolaou, Chong-Sheng Yuan, and Ronald T. Borchardt*
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047
Received August 1, 1996X
A novel phosphate prodrug system for amines, amino acids, peptides, and peptide mimetics, which
utilizes a fast hydroxy amide lactonization of a 3-(2′-hydroxy-4′,6′-dimethylphenyl)-3,3-dimethyl-
propionic amide system, was developed. Prodrugs of five model amine/amino acids, including
p-anisidine, GlyOMe, PheOMe, LysOMe, and Asp-R-OMe, were synthesized. The syntheses of these
model phosphate prodrugs were accomplished by coupling the amine or the protected amino acids
with 3-[2′-(dibenzylphosphono)oxy-4′,6′-dimethylphenyl]-3,3-dimethylpropionic acid using coupling
agents such as bis(2-oxo-3-oxazolidinyl)phosphinic chloride and 1-(3-dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride, followed by hydrogenolysis. These phosphate prodrugs were
evaluated as substrates for the human placental alkaline phosphatase (AP). The structural features
of the amine/amino acids attached to the carboxylic acid group of the promoiety were not found to
significantly affect the substrate activity for AP, as evidenced by the small variations observed in
the Michaelis-Menten parameters (Km and Vmax) of the phosphate prodrugs. Results obtained
from this study suggest that such a phosphate prodrug system may be applied to a variety of
structurally diverse amine-containing drugs.
In tr od u ction
ing anticancer agents with a variety of structural features
may be used for cellular targeting.12,13
Phosphate prodrugs have been successfully utilized to
overcome a variety of drug delivery problems that might
otherwise have compromised the therapeutic utilities of
the parent drugs.1-8 Structurally, they consist of a
phosphate group directly attached to the drug molecule
in the form of phosphomonoesters or phosphoramidates
or indirectly attached with a chemical linker known as
a promoiety. The ionic nature of the phosphate group
in these prodrugs may significantly improve the solubility
and dissolution rate of poorly soluble drugs. In the
presence of alkaline phosphatase (AP), an enzyme widely
distributed in a variety of tissues such as liver, kidney
tubules, intestinal epithelium, etc., phosphomonoesters
and phosphoramidates were shown to undergo hydrolysis
to release the parent alcohol or amine and inorganic
phosphate.9-11 In addition, certain tumors could be
artificially enriched in AP with the aid of monoclonal
antibodies; thus, phosphate derivatives of amine-contain-
For example, a phosphate prodrug of the antiseizure
drug phenytoin (recently approved by the FDA) was
shown to enhance the aqueous solubility and dissolution
rate as well as the oral bioavailability of this drug due
to the presence of AP in the small intestine, especially
in the jejunum.14-16
The major prerequisites for the success of a phosphate
prodrug approach are adequate aqueous chemical stabil-
ity and the complete in vivo enzymatic conversion of the
prodrug to the active drug species. Unfortunately, not
all phosphomonoesters and phosphoramidates are both
chemically stable and good substrates for AP. Phospho-
monoesters of sterically hindered secondary and tertiary
alcohols suffer from slow rates of bioconversion,17,18 and
many phosphoramidates suffer from poor chemical sta-
bility.19 For example, phosphorylation at the secondary
hydroxyl group of the poorly soluble anticancer agent
Taxol significantly improved its solubility. However, AP-
mediated bioconversion of this prodrug was extremely
slow.20
* To whom correspondence should be addressed. Tel: (913)-864-
3427. Fax: (913)-864-5736. E-Mail: Borchardt@Smissman.hbc.ukans.
edu.
Recently, through either rational drug design or com-
binatorial chemistry approaches, peptide and peptide
mimetics have emerged as a new generation of thera-
peutic agents. However, the therapeutic utility of these
X Abstract published in Advance ACS Abstracts, November 1, 1996.
(1) Sinkula, A. A.; Yalkowsky, S. H. J . Pharm. Sci. 1975, 64, 181-
210.
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29, 455-473.
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L.; Brown, J . P.; Hellstrom, I.; Hellstrom, K. E. Proc. Natl. Acad. Sci.
U.S.A. 1988, 85, 4842-4846.
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Sci. 1984, 73, 1068-1073.
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(19) Bruice, T. C.; Bencovic, S. Bioorganic Mechanisms; W. A.
Benjamin, Inc: New York, 1966; Vol. 2, pp 71-84.
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J . O.; Mamber, S. W.; Doyle, T. W. Bioorg. Med. Chem. Lett 1993, 3,
1357.
S0022-3263(96)01477-6 CCC: $12.00 © 1996 American Chemical Society

Phosphate Prodrugs for Amines
J . Org. Chem., Vol. 61, No. 24, 1996 8637
Sch em e 1
esters, LysOMe and AspOMe (Scheme 2). We report here
that all five phosphate prodrugs were converted equimo-
larly to the corresponding amine or amino acid by AP,
and the structure of the amides did not significantly
affect the rate of bioconversion from the phosphate
prodrugs to the parent amine/amino acids. These results
suggest that this promoiety may be used for a variety of
amine-containing drugs with different structural fea-
tures.
Resu lts a n d Discu ssion
Syn th esis. The synthesis of the model phosphate
prodrugs 8a -e was accomplished as shown in Scheme
2. Starting from the commercially available 3,5-dimeth-
ylphenol (1) and methyl 3,3-dimethylacrylate in meth-
anesulfonic acid the lactone 2 was prepared. The use of
methanesulfonic acid as both the solvent and the catalyst
was reported earlier by Carpino et al.25 for the synthesis
of a similar lactone. It was found to afford a higher yield
and easier product isolation compared to the method
published earlier by our laboratory.22 Efforts to open the
lactone 2 to its corresponding hydroxy acid, followed by
subsequent phosphorylation of the phenolic hydroxyl
group and attachment of the amine/amino acids to the
carboxylic acid, were met with little success. Thus, the
syntheses of the model prodrugs were accomplished by
reduction of lactone 2 to the diol 3 with lithium alumi-
num hydride (LAH) using a modification of a procedure
reported earlier by our laboratory.23 The use of a
saturated aqueous solution of NH4Cl to quench the
unreacted LAH simplified the product isolation by en-
abling removal of the lithium aluminum salts by simple
filtration over the filter agent Celite. Selective protection
of the alkylhydroxyl group of the diol 3 as a tert-
butyldimethyl silyl ether with tert-butyldimethylsilyl
chloride (TBDMSCl) in CH2Cl2 under basic conditions
yielded the intermediate compound 4. Phosphorylation
of the phenolic hydroxyl group of 4 was accomplished
using tetrabenzyl pyrophosphate. Efforts to synthesize
5 with other reagents, such as bis(2,2,2-trichloroethyl)
phosphorochloridate or phosphorus oxychloride, were
unsuccessful. Complete reviews on phosphorylation have
appeared elsewhere.29,30 Difficulty in phosphorylating
the phenolic hydroxyl group of 4 with diphenyl phos-
phorochloridate was also observed by Ueda et al.28 The
carboxylic acid 6 was synthesized after in situ deprotec-
tion of the silyl ether and subsequent oxidation using
potassium fluoride and J ones reagent in acetone. Potas-
sium fluoride in conjunction with the J ones reagent has
been used previously to synthesize ketones from their
corresponding silyl-protected secondary alcohols.31 This
synthetic procedure for obtaining the carboxylic acid 6
was simpler and more efficient than the previous syn-
thetic procedures used to form other derivatives of the
phenolic propionic acids from their corresponding alco-
hols.23 The use of pyridinium chlorochromate to oxidize
the alcohol to the aldehyde followed by further oxidation
with KMnO4 to the desirable final product is no longer
necessary;23 thus, three steps are reduced to one with
relatively high yield (72%). Ueda et al.28 also reported
the use of J ones reagent for the synthesis of carboxylic
acid 6 after deprotection and isolation of the correspond-
ing alcohol of 5 followed by subsequent oxidation with
the J ones reagent.
compounds is often limited by poor solubility or drug
delivery problems.21 Thus, new approaches to accom-
modate the phosphate group into peptide and peptide
mimetics in order to enhance their solubility and improve
their delivery are needed.
Recently, Amsberry and co-workers22-24 and Carpino
et al.25 have reported the use of the phenolic propionic
acids 9 (R′ ) H or OH) shown in Scheme 1 as promoieties
for amines and alcohols. Derivatization of the phenolic
group of 9 (R′ ) H) with a carboxylic acid yields 10, which
is an esterase sensitive prodrug.23 Oxidation of 9 (R′ )
OH) to the quinone 11 yields a redox-sensitive pro-
drug.24,25
Upon hydrolysis of the carboxylic acid in 10 or reduc-
tion of the quinone in 11, the hydroxyamides 9 (R′ ) H)
and 9 (R′ ) OH), respectively, are generated. These
hydroxyamides rapidly lactonize (t1/2 ) 1 min at pH )
7.4) to release equimolar amounts of the amine-contain-
ing drug and the inactive lactone 2.22 The reason for the
fast lactonization is the trimethyl substitution, “trimethyl
lock”, which for steric reasons leads to a productive
“cissoid” conformation favorable to lactonization.26,27
In a recent communication, Ueda et al.28 reported the
use of a phosphate ester of 3-(2′-hydroxy-4′,6′-dimeth-
ylphenyl)-3,3-dimethylpropionic acid to derivatize a sec-
ondary alcohol on Taxol. This Taxol prodrug exhibited
substantially better solubility than Taxol itself, and it
was hydrolyzed to the parent drug by AP. In order to
extend the utility of this promoiety to improve the
solubility of amine-containing drugs and/or target amine-
containing drugs to tissues rich in AP, we synthesized a
series of model phosphate derivatives 8a -e (Scheme 2)
and evaluated their substrate activity for human pla-
cental AP. The model amine-containing compounds used
in this study included anisidine, a neutral aromatic
amine, two neutral amino acid methyl esters, GlyOMe
and PheOMe, and two charge-bearing amino acid methyl
(21) Oliyai, R. Adv. Drug. Delivery Rev. 1996, 19, 275-286.
(22) Amsberry, K. L.; Borchardt, R. T. J . Org. Chem. 1990, 55, 5867-
5877.
(23) Amsberry, K. L.; Gerstenberger, A. E.; Borchardt, R. T. Pharm.
Res. 1991, 8, 455-461.
(24) Amsberry, K. L.; Borchardt, R. T. Pharm. Res. 1991, 8, 323-
330.
(25) Carpino, L. A.; Triolo, S. A.; Berglund, R. A. J . Org. Chem. 1989,
54, 3303-3310.
(26) Milstien, S.; Cohen, L. A. Proc. Natl. Acad. Sci. U.S.A. 1970,
67, 1143-1147.
(27) Wang, B.; Nicolaou, M.; Liu, S.; Borchardt, R. T. Bioorg. Chem.
1996, 24, 39-49.
(29) Slotin, L. A. Synthesis 1977, 737-752.
(30) Lemmen, P.; Richter, W.; Werner, B.; Karl, R.; Stumpf, R.; Ugi,
I. Synthesis 1993, 1-10.
(28) Ueda, Y.; Mikkilineni, A. B.; Knipe, J . O.; Rose, W. C.; Casazza,
A. M.; Vyas, D. M. Bioorg. Med. Chem. Lett 1993, 3, 1761-1766.
(31) Muzart, J . Synthesis 1993, 11-26.

8638 J . Org. Chem., Vol. 61, No. 24, 1996
Nicolaou et al.
Sch em e 2
Coupling the amine/amino acids (a-e, Scheme 2) with
the carboxylic acid 6 was accomplished using either 1-(3-
dimethylamino)propyl)-3-ethylcarbodiimide hydrochlo-
ride (EDC) plus 4-(dimethylamino)pyridine (4-DMAP) or
bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOPCl) as
coupling reagents. In both cases, the byproducts, 1-(3-
dimethylamino)propyl)-3-ethyl urea (EDU) and the bis-
(2-oxo-3-oxazolidinyl)phosphoric acid formed after the
coupling, can be removed by washing the reaction milieu
with H2O. The desired model prodrugs 8a -e were
obtained after hydrogenolytic debenzylation of 7a -e in
either THF or CH3OH. The amino acid methyl esters
(b-e, Scheme 2) were carefully chosen so that the
functional groups were benzyl protected and hydro-
genolysis afforded the final products in one step. Model
prodrugs 8d -e were obtained initially as colorless oils;
however, after addition of H2O, freezing, and subsequent
lyophilization, the final products were obtained as amor-
phous solids.
Biologica l Eva lu a tion . The phosphate monoesters
of the phenolic propionic amides 8a -e synthesized in this
study were examined for their ability to release the model
amine/amino acids in the presence of human placenta AP.
This conversion process includes two steps: a rate-
determining dephosphorylation step catalyzed by AP to
generate the intermediate hydroxy amide 9a -e (R′ ) H)
(Schemes 1 and 2) followed by a fast intramolecular
lactonization step to release the model amine/amino acids
and the lactone 2 (R ) H). As shown in Scheme 1, the
amount of lactone formed upon hydrolysis of the prodrug
should be equal to the original amount of the prodrug.
Figure 1 shows a representative time course for the
bioconversion from prodrug 8a to lactone 2 (R′ ) H). As
expected, an equimolar amount of lactone 2 (R′ ) H) was
formed from 8a . This bioconversion process was followed
by HPLC as shown in Figure 2.
F igu r e 1. Time profile for the enzymatic degradation of the
prodrug 8a (b) and the appearance of the lactone 2 (9)
(Scheme 2): pH ) 7.4, 100 mM MOPS buffer, 37 °C, 50 µM of
8a , and 32.9 µg or AP. One millimolar ZnCl2 and 1 mM MgCl2
were added to the buffer solutions. The samples were analyzed
by HPLC.
This may be attributed to the basicity of the ω-amine
nitrogen. It has been suggested that as the acidity of
the substrate increases the Km value decreases, and in
this case, the basic amino group of lysine may contribute
to such an increase in Km.9,32
One reason for choosing the phosphate esters of the
phenolic propionic acid as a prodrug strategy for amines
is that phenolic phosphates are regarded as good sub-
strates for AP. Previous studies suggested that the rate
of the AP-mediated release of the parent alcohol or phenol
may depend on the pKa value of the hydroxyl leaving
group.33,34 The relatively lower pKa values of phenols
compared to other aliphatic alcohols may be one reason
that aromatic phosphates were found to be good sub-
strates for AP.33,34 An important criterion for a prodrug
system that aims to deliver a variety of amine-containing
drugs of wide structural variation, including functional
To assess the effects of the structural features of the
amides on the AP-mediated conversion of prodrugs to the
model amine/amino acids, the five model prodrugs (8a -
e) were evaluated as substrates for human placenta AP.
Their Michaelis-Menten kinetic parameters (Km and
Vmax) are provided in Table 1. For compounds 8a -d
(32) Walker, P. G.; King, E. J . Biochem. J . 1950, 47, 93-95.
(33) Varia, S. A.; Schuller, S.; Stella, V. J . J . Pharm. Sci. 1984, 73,
1074-1080.
there were no significant differences in either Km or Vmax
values. In contrast, compound 8e showed slightly larger
Km and Vmax values than the other model compounds.
(34) Hall, A. H.; Williams, A. Biochemistry 1986, 25, 4784-4790.

Phosphate Prodrugs for Amines
J . Org. Chem., Vol. 61, No. 24, 1996 8639
through biological membranes.35 A phosphate ester of
this prodrug system was recently reported to improve the
solubility of Taxol and to exhibit phosphatase-mediated
bioconversion to the anticancer agent Taxol.28
Con clu sion s
The applicability of the phosphate phenolic propionic
amide system shown in Scheme 1 was examined. Five
model compounds were synthesized and shown to be
substrates for human placental AP. The small variations
in the Michaelis-Menten parameters (Km and Vmax
)
among the prodrugs for AP suggest that this system may
be used for amine drugs with a wide variety of structural
features. Future studies will be focused on in vivo
bioconversion of model prodrugs of this phenolic propionic
amide system.
F igu r e 2. HPLC chromatograms of the lactone 2 (A) and the
model prodrug 8a (B): (A) column, 15 cm C18 Hypersil; mobile
phase, 60% aqueous acetonitrile; UV detection, λ ) 225 nm;
flow rate, 1 mL/min; (B) column, 15 cm C18 Hypersil; mobile
phase, 40% acetonitrile in 20 mM aqueous phosphate buffer
pH ) 3, 20 mM TBA phosphate; UV detection, λ ) 240 nm;
flow rate 1 mL/min.
Exp er im en ta l Section
Gen er a l. 1H NMR spectra were recorded on a Varian XL
300 (300 MHz). Chemical shifts are expressed in parts per
million (δ) relative to residual solvents as internal standards.
Mass spectral analyses were conducted by The University of
Kansas Mass Spectral Laboratory, and elemental analyses
were determined by The University of Kansas Elemental
Analysis Laboratory, Lawrence, KS. HPLC analyses were
conducted using a Shimadzu 10A system equipped with a UV
spectrophotometric detector. All materials used for the syn-
thesis of the described compounds were purchased from
Aldrich Chemical Co., Milwaukee, WI, except bis(2-oxo-3-
oxazolidinyl)phosphinic chloride (BOPCl) which was purchased
from TCI America, Portland, OR, and the protected amino
acids were purchased from Bachem California, Torrance, CA.
HPLC solvents and 3-(N-morpholino)propanesulfonic acid
(MOPS) buffer were purchased from Fisher Scientific, Pitts-
burgh, PA, and alkaline phosphatase (P 3895, type XXIV) was
purchased from Sigma Chemical Co., St. Louis, MO. All
materials were used as received.
Gen er a l Meth od s. 4,4,5,7-Tetr a m eth yl-3,4,-d ih yd r o-
cou m a r in (2). 3,5-Dimethylphenol (1) (5.00 g, 41.00 mmol)
was added to methanesulfonic acid (5 mL) followed by addition
of methyl 3,3-dimethylacrylate (5.14 g, 45.03 mmol). The
mixture was brought to 70 °C using a thermostated oil bath
and was allowed to react for 12 h with constant stirring. The
reaction mixture was then diluted with H2O (1 L) and
extracted three times with 200 mL portions of EtOAc. The
extracts were combined and washed three times with 5%
aqueous NaHCO3 (200 mL × 3) and saturated aqueous NaCl
solution (100 mL) and dried over anhydrous MgSO4. Solvent
removal by rotary evaporation and recrystallization from
EtOAc-hexane afforded the pure lactone 2 in 87% yield.
Structural data for this compound are described elsewhere.22
3-(2′-Hyd r oxy-4′,6′-d im et h ylp h en yl)-3,3-d im et h ylp r o-
p a n ol (3). The synthesis of 3-(2′-hydroxy-4′,6′-dimethylphen-
yl)-3,3-dimethylpropanol (3) was performed by LAH reduction
of 2, as previously described,23 with a minor modification.
Instead of using aqueous HCl to quench and dissolve the
lithium/aluminum salts, a saturated solution of NH4Cl was
used. The insoluble white precipitate (lithium/aluminum
salts) was removed by filtration over Celite and the resulting
solution was mixed with H2O (100 mL) and extracted with
Et2O (200 mL × 3). The ethereal extracts were combined and
dried over MgSO4. Solvent removal by rotary evaporation and
recrystallization in EtOAc/hexane afforded the analytically
pure, white crystalline product 3 in 90% yield.
Ta ble 1. Mich a elis-Men ten P a r a m eter s for Com p ou n d s
8a -8e a n d P h en ol P h osp h a te
a
Vmax
a
model prodrug
8a
Km (mM)
((nmol/min)/mg of protein)
16.5
15.6
22.3
21.3
74.3
4.6
175.7
183.3
170.9
156.7
264.2
202.5
8b
8c
8d b
8e
phenol phosphate
a
Km and Vmax values were obtained after curve fitting the initial
rate data for 8a -8e and phenol phosphate to the Michaelis-
b
Menten equation. Performed in 50 mM MOPS and quenched
with 2 M acetic acid in acetonitrile.
groups, charge, size, and steric hindrance, is to be equally
cleavable by AP regardless of the structural features. On
the basis of data generated in this study, prodrugs 8a -e
have similar Michaelis-Menten parameters when tested
as substrates for human placental AP (Table 1). This
system was found particularly useful for amines since
their release does not depend on direct hydrolysis of the
amide bond but, rather, on the enzymatic lability of the
phenolic ester bond.
To further probe the structural contribution to the AP
kinetic parameters (Km and Vmax), the Michaelis-Menten
parameters for phenol phosphate were also determined.
As shown in Table 1, the Km value (4 µM) of phenol
phosphate was lower than the Km values (15.6-74.3 µM)
observed for prodrugs 8a -e, suggesting that the high
degree of methylation and the restricted conformation of
the phenolic propionic amide system may slightly restrict
binding to the active site of the enzyme. However, phenol
phosphate has a Vmax value (202.5 (nmol/min)/mg of
protein) similar to the Vmax values (156.7-264.2 (nmol/
min)/mg of protein) for the prodrugs 8a -e, suggesting
that the enzyme may catalyze the dephosphorylation
reaction equally well, regardless of the structural features
in the molecule.
A major advantage of the phenolic propionic acid
promoiety is that by applying different derivatization
methods to the phenolic hydroxyl group, one may cir-
cumvent different barriers to drug delivery, including
solubility, permeability, targeting, etc. For example, we
have shown previously that ester prodrugs of the phenolic
propionic amide type could significantly reduce the
hydrophilicity of amines with high pKa values, including
peptides,23 and could possibly enhance their transport
1-O-ter t-Bu tyld im eth ylsilyl-3-(2′-h yd r oxy-4′,6′-d im eth -
ylp h en yl)-3,3-d im eth ylp r op a n ol (4). The synthesis and
structural data for 1-O-tert-butyldimethylsilyl-3-(2′-hydroxy-
4′,6′-dimethylphenyl)-3,3-dimethylpropanol (4) have been pre-
viously reported by this laboratory and appeared elsewhere.23
(35) Pauletti, G. M.; Gangwar, S.; Wang, B.; Siahaan, T. J .; Vander
Velde, D. G.; Borchardt, R. T. Pharm. Res. 1995, 12, S-208.

8640 J . Org. Chem., Vol. 61, No. 24, 1996
Nicolaou et al.
1-O-ter t-Bu tyld im eth ylsilyl-3-[2′-(d iben zylp h osp h on o)-
oxy-4′,6′-d im eth ylp h en yl]-3,3-d im eth ylp r op a n ol (5). 1-O-
tert-Butyldimethylsilyl-3-(2′-hydroxy-4′,6′-dimethylphenyl)-3,3-
dimethylpropanol (4) (2.74 g, 8.5 mmol) was added to 200 mL
of dry THF followed by addition of solid potassium tert-
butoxide (1.04 g, 9.30 mmol). The mixture was heated to 60
°C in an oil bath for approximately 5 min, becoming pale yellow
in color. To this warm reaction mixture was added the solid
tetrabenzyl pyrophosphate, which was synthesized based on
the method of Khorana and Todd36 and recrystallized from
benzene/hexane. Approximately 10 min after the addition of
the tetrabenzyl pyrophosphate, an insoluble white precipitate
formed. The reaction mixture was kept under continuous
stirring at 60 °C for 45 min and then allowed to cool to room
temperature. To the reaction milieu was added hexane (200
mL), and the insoluble white precipitate was removed by
filtration. After removal of all solvents by rotary evaporation,
a viscous residue was obtained. The desired product was
eluted from a silica gel column with 20% EtOAc in hexane and
isolated as a green-yellow oil (4.29 g, 87%): 1H NMR (CDCl3)
δ 7.32 (10 H, s), 7.10 (1 H, s), 6.71 (1 H, s), 5.11 (4 H, d, J )
8.13 Hz), 3.48 (2 H, t, J ) 7.26 Hz), 2.50 (3 H, s), 2.17 (3 H, s),
2.09 (2 H, t, J ) 7.26 Hz), 1.53 (6 H, s), 0.83 (9 H, s), 0.05 (6
H, s); MS (FAB) m/ z 583 (M+ + 1). Anal. Calcd for C33H47O5-
PSi: C, 68.01; H, 8.13. Found: C, 68.00; H, 8.50.
3-[2′-(Dib en zylp h osp h on o)oxy-4′,6′-d im et h ylp h en yl]-
3,3-d im eth ylp r op ion ic Acid (6). 1-O-(tert-Butyldimethyl-
silyl)-3-[2′-(dibenzylphosphono)oxy-4′,6′-dimethylphenyl]-3,3-
dimethylpropanol (5) (4.3g 7.39 mmol) was dissolved in 60 mL
of acetone followed by addition of solid KF (0.43 g, 29.6 mmol).
The above mixture was placed in an ice bath, and J ones
reagent (6.1 mL), whose preparation was previously de-
scribed,37 was added dropwise over a period of 20 min under
continuous stirring. The reaction mixture was stirred for 2
h; after that time, 2-propanol (2.7 mL) was added to quench
the residual J ones reagent, and the reaction was allowed to
continue for an additional 20 min. The reaction mixture was
further concentrated under reduced pressure rotary evapora-
tion, and EtOAc (100 mL) and H2O (100 mL) were added. The
organic layer was separated, and the aqueous layer was
extracted with EtOAc (100 mL × 3). The organic extracts were
then combined and washed with saturated NaCl aqueous
solution (100 mL × 2) and dried over anhydrous MgSO4.
Removal of the solvents by rotary evaporation afforded a
yellow-green oil which, upon recrystallization from Et2O and
hexane, afforded the desired white, solid product (2.56 g, 72%
yield): 1H NMR (CDCl3) δ 7.33 (10 H, m), 6.99 (1 H, s), 6.73
(1 H, s), 5.07-5.13 (4 H, m), 2.84 (2 H, s), 2.50 (3 H, s), 2.13 (3
H, s), 1.61 (6 H, s); MS (FAB) m/ z 483 (M+ + 1). Anal. Calcd
for C27H31O6P: C, 67.22; H, 6.43. Found: C, 67.30; H,6.38.
Gen er a l Meth od I for P r ep a r a tion of Am id es. All
glassware was flame dried, and the reaction was kept under
argon atmosphere at all times. To anhydrous CH2Cl2 (20 mL)
were added solid 3-[2-(dibenzylphosphono)oxy-4′,6′-dimeth-
ylphenyl]-3,3-dimethylpropionic acid (6) (200 mg, 0.415 mmol),
triethylamine (TEA) (126 mg, 1.25 mmol) and the amine or
the protected amino acid. This was mixed and cooled to 0 °C
in an ice bath. BOPCl (159 mg, 0.62 mmol) was added all at
once to the solid form. The reaction mixture was kept under
continuous stirring and an argon atmosphere for 12 h while
it was gradually allowed to reach ambient temperature.
Solvent was removed under reduced pressure rotary evapora-
tion, and the obtained residue was mixed with EtOAc (50 mL)
and 5% aqueous citric acid solution (50 mL). The organic layer
was separated and the aqueous layer was washed with EtOAc
(50 mL × 3). The EtOAc extracts were combined and
concentrated under reduced pressure rotary evaporation. The
desired product was eluted from a silica gel column.
mixture was dissolved and cooled to 0 °C. To the cooled
solution were added 4-DMAP (2 mg, 0.016 mmol) and EDC
(119 mg, 0.62 mmol), and the reaction mixture was kept under
continuous stirring for 15 min. The amine or the protected
amino acid in the solid form was then added at once, and the
reaction mixture was stirred for 24 h while it was gradually
allowed to reach ambient temperature. The solvent was then
removed under reduced pressure rotary evaporation, and the
desired product was eluted from a silica gel column.
p -An isid in e -3-[2′-(d ib e n zylp h osp h on o)oxy-4′,6′-d i-
m eth ylp h en yl]-3,3-d im eth ylp r op ion ic Am id e (7a ). Com-
pound 7a was prepared and purified according to the general
method II for the preparation of amides as described above.
To prepare compound 7a the amine used was p-anisidine (56
mg, 0.456 mmol). The desired product was eluted from a silica
gel column with EtOAc/hexane (80:20) and recrystallized from
EtOAC/hexane (165 mg, 67%): 1H NMR (CDCl3) δ 8.30 (1 H,
s), 7.36 (10 H, s), 7.18 (2 H, d, J ) 8.88 Hz), 6.94 (1 H, s), 6.73
(2 H, d, J ) 9.06 Hz), 6.68 (1 H, s), 5.18-5.12 (4 H, m), 3.74
(3 H, s), 2.68 (2 H, s), 2.45 (3 H, s), 2.11 (3 H, s), 1.67 (6 H, s);
MS (FAB) m/ z 588 (M+ + 1). Anal. Calcd for C34H38NO6P:
C, 69.50; H, 6.47; N, 2.39. Found: C, 69.19; H, 6.80; N, 2.10.
GlyOMe-3-[2′-(d ib en zylp h osp h on o)oxy-4′,6′-d im et h -
ylp h en yl]-3,3-d im eth ylp r op ion ic Am id e (7b). Compound
7b was prepared and purified according to the general method
I for the preparation of amides as described above. To prepare
compound 7b the amino acid used was GlyOMe (58 mg, 0.46
mmol). The desired white, crystalline solid product was
obtained after elution from a silica gel column with EtOAc/
hexane (80:20) (85 mg, 37%): 1H NMR (CDCl3) δ 7.34 (10 H,
m), 7.03 (1 H, s), 6.72 (1 H, s), 6.54 (1 H, t, J ) 5.58 Hz), 5.20-
5.08 (4 H, m), 3.67 (2 H, d, J ) 5.46 Hz), 3.62 (3 H, s), 2.67 (2
H, s), 2.49 (3 H, s), 2.15 (3 H, s), 1.62 (6 H, s); MS (FAB) m/ z
554 (M+ + 1). Anal. Calcd for C30H36NO7P: C, 65.10; H, 6.51;
N, 2.53. Found: C, 65.21; H, 6.89; N, 2.40.
L-P h eOMe-3-[2′-(d iben zylp h osp h on o)oxy-4′,6′-d im eth -
ylp h en yl]-3,3-d im eth ylp r op ion ic Am id e (7c). Compound
7c was prepared and purified according to the general method
I for the preparation of amides as described above. To prepare
compound 7c the amino acid used was L-PheOMe (99 mg, 0.46
mmol). The desired product, a yellow oil, was obtained after
elution from a silica gel column with EtOAc/hexane (80:20)
(222 mg, 83%): 1H NMR (CDCl3) δ 7.36-7.25 (10 H, m), 7.19-
7.17 (3 H, m), 7.07 (1 H, s), 6.93-6.90 (2 H, m), 6.70 (1 H, s),
6.53 (1 H, d, J ) 7.71 Hz), 5.13-5.00 (4 H, m), 4.63 (1 H, q, J
) 6.84 Hz), 3.56 (3 H, s), 2.87-2.70 (2 H, m), 2.61 (2 H, s),
2.47 (3 H, s), 2.15 (3 H, s), 1.59 (3 H, s), 1.58 (3 H, s); MS
(FAB) m/ z 644 (M+ + 1). Anal. Calcd for C37H42NO7P: C,
69.05; H, 6.53; N, 2.18. Found: C, 69.00; H, 6.80; N, 2.40.
Lys-E-CBZ-r-OMe-3-[2′-(d iben zylp h osp h on o)oxy-4′,6′-
dim eth ylph en yl]-3,3-dim eth ylpr opion ic Am ide (7d). Com-
pound 7d was prepared and purified according to the general
method I for the preparation of amides as described above. To
prepare compound 7d the amino acid used was N-ꢀ-CBZ-L-
LysOMe (153 mg, 0.46 mmol). The desired product was
obtained after elution from a silica gel column with EtOAc/
hexane (50:50) (251 mg, 81%): 1H NMR (CDCl3) δ 7.41-7.28
(15 H, m), 6.98 (1 H, s), 6.77 (1 H, d, J ) 7.32 Hz), 6.71 (1 H,
s), 5.29 (1 H, m), 5.18-5.02 (6 H, m), 4.25-4.21 (1 H, m), 3.61
(3 H, s), 3.14-3.03 (2 H, m), 2.73 (1 H, d, J ) 13.23 Hz), 2.47
(3 H, s), 2.46 (1 H, d, J ) 13.14 Hz), 2.11 (3 H, s), 1.67 (3 H,
s), 1.56 (3 H, s), 1.30-1.47 (4 H, m), 1.10-0.90 (2 H, m); MS
(FAB) m/ z 759 (M+ + 1). Anal. Calcd for C42H51N2O9P: C,
66.49; H, 6.72; N, 3.69. Found: C, 66.09; H, 7.10; N, 3.98.
Asp-r-OMe-â-ben zyl-3-[2′-(diben zylph osph on o)oxy-4′,6′-
dim eth ylph en yl]-3,3-dim eth ylpr opion ic Am ide (7e). Com-
pound 7e was prepared and purified according to the general
method I for the preparation of amides as described above. To
prepare compound 7e the amino acid used was Asp-R-OMe-
â-Obzl (164 mg, 0.50 mmol). The synthesis of this protected
amino acid is described later in this section. The desired
product was eluted from a silica gel column with EtOAc/hexane
gradient from 30-40% (218 mg, 75%): 1H NMR (CDCl3) δ
7.37-7.28 (15 H, m), 7.03 (1 H, S), 6.88 (1 H, d, J ) 7.83 Hz),
6.69 (1 H, s), 5.20-5.06 (4 H, m), 5.05 (2 H, s), 4.72-4.66 (1
H, m), 3.55 (3 H, s), 2.72-2.51 (4 H, m), 2.48 (3 H, s), 2.11 (3
H, s), 1.63 (3 H, s), 1.59 (3 H, s); MS (FAB) m/ z 702 (M+ + 1).
Gen er a l Meth od II for P r ep a r a tion of Am id es. All
glassware was flame dried, and the reaction was kept under
argon atmosphere at all times. To anhydrous CH2Cl2 (50 mL)
was added 3-[2-(dibenzylphosphono)oxy-4′,6′-dimethylphenyl)-
3,3-dimethylpropionic acid (6) (200 mg, 0.415 mmol), and the
(36) Khorana, H. G.; Todd, A. R. J . Chem. Soc. 1953, 2257-2260.
(37) Fieser, L. F.; Fieser, M. Reagents for Organic Synthesis; J ohn
Wiley and Sons, Inc: New York, 1967; Vol. 1, p 142.

Phosphate Prodrugs for Amines
J . Org. Chem., Vol. 61, No. 24, 1996 8641
Anal. Calcd for C39H44NO9P: C, 66.76; H, 6.28; N, 1.99.
Found: C, 66.93; H, 6.10; N, 1.90.
in an ice bath. EDC (1.55 g, 8.06 mmol) was added, and the
reaction mixture was stirred for 15 min. CH3OH (396 mg, 12.4
mmol) was then added to the mixture, and stirring was
continued for 12 h. Solvent was removed under reduced
pressure rotary evaporation, and EtOAc (50 mL) and H2O (50
mL) were added to the residue. The organic layer was
separated and further washed with a saturated aqueous NaCl
solution (20 mL) and dried over MgSO4. The desired product
was recrystallized from EtOAc and hexane (1.34 g, 64%): 1H
NMR (CDCl3) δ 7.35 (5 H, m), 5.49 (1 H, d, J ) 8.31 Hz), 5.17-
5.09 (2 H, m), 4.61-4.50 (1 H, m), 3.70 (3 H, s), 3.09-3.01 (1
H, m), 3.08-2.83 (1 H, m), 1.44 (9 H, s); MS (FAB) m/ z 338
(M+ + 1). Anal. Calcd for C17H23NO6: C, 60.53; H, 6.83; N,
4.15. Found: C, 60.50; H, 6.98; N, 4.00.
Gen er a l P r oced u r e for Hyd r ogen a tion . Compounds
7a -e were dissolved in either THF or CH3OH. Ten percent
Pd catalyst on activated carbon was added to the solution, and
the reaction was placed under H2 atmosphere. The reaction
was kept under vigorous stirring for 12 h. The insoluble
catalyst was removed by filtration, followed by solvent removal
under reduced pressure rotary evaporation. The final solid
product was obtained by either trituration in an anhydrous
Et2O and hexane mixture or lyophilization from a frozen
aqueous solution.
p -An isid in e-3-[2′-(d ih yd r oxyp h osp h on o)oxy-4′,6′-d i-
m eth ylp h en yl]-3,3-d im eth ylp r op ion ic Am id e (8a ). Com-
pound 7a (150 mg, 0.255 mmol) was dissolved in THF (20 mL)
and treated according to the general procedure for Hydrogena-
tion described above. The final product was obtained as a
white amorphous powder after vigorous trituration in anhy-
drous Et2O and hexane (80 mg, 77%): 1H NMR (DMSO) δ 9.64
(1 H, s), 7.30-7.27 (2 H, m), 7.05 (1 H, S), 6.78 (2 H, d, J )
9.12 Hz), 6.62 (1 H, s), 3.68(3 H, s), 2.74 (2 H, s), 2.40 (3 H, s),
2.14 (3 H, m), 1.62 (6 H, s); MS (FAB) m/ z 408 (M+ + 1). Anal.
Calcd for C20H26NO6P: C, 58.96; H, 6.39; N, 3.44. Found: C,
58.48; H, 6.10; N, 3.24.
GlyOMe-3-[2′-(d ih yd r oxyp h osp h on o)oxy-4′,6′-d im eth -
ylp h en yl]-3,3-d im eth ylp r op ion ic Am id e (8b). Compound
7b (85 mg, 0.153 mmol) was dissolved in CH3OH (10 mL) and
treated according to the general procedure for hydrogenation
described above. The final product was obtained as a white
amorphous powder (38 mg, 67%): 1H NMR (CD3OD) δ 7.16 (1
H, s), 6.69 (1 H, s), 3.77 (2 H, s), 3.65 (3 H, s), 2.83 (2 H, s),
2.49 (3 H, s), 2.21 (3 H, s), 1.66 (6 H, s); MS (FAB) m/ z 374
(M+ + 1). Anal. Calcd for C16H24NO7P: C, 51.47; H, 6.43; N,
3.75. Found: C, 51.25; H, 6.57; N, 3.88.
Asp -r-OMe-â-Bzl. t-Boc-Asp-â-Bzl-R-OMe (500 mg, 1.48
mmol) was dissolved in 50 mL of CH2Cl2 and 1 mL of TEA.
The reaction mixture was stirred for a period of 2 h. After all
solvents were removed under reduced pressure rotary evapo-
ration, the oily residue was suspended in 50 mL of 1 M HCl
in Et2O solution. After a few minutes, white crystals of the
final product precipitated out. The final product was obtained
as a crystalline white solid (303 mg, 86% yield): 1H NMR
(CDCl3) δ 7.30 (5 H, m), 5.16 (2 H, s), 4.61 (1 H, m), 3.66 (3 H,
s), 3.42-3.23 (2 H, s); MS (FAB) m/ z 238 (M+ + 1); HRMS
(FAB) m/ z 238.1077 (M+ + 1, Calcd 238.1079).
Kin etics of En zym e-Med ia ted Hyd r olysis of P r od r u g
8a . The kinetics of enzymatic hydrolysis of prodrug 8a were
determined by measuring the rate of formation of the lactone
2 after incubating a solution of 8a (45 µmol) with human
placenta AP (32.9 µg) in 100 mM MOPS buffer containing 1
mM MgCl2 and 1 mM ZnCl2, pH 7.4 (2.24 mL) at 37 °C. At
specific time intervals, aliquots of the reaction mixture (200
µL) were removed and quenched by addition of 1 M HClO4
solution in CH3CN (200 µL). The quenched mixture was
analyzed for both the disappearance of 8a and appearance of
the lactone 2 using an ODS hypersil HPLC column (C18, 150
× 24 mm). For 8a , the column was equilibrated and eluted
with 40% CH3CN in 20 mM aqueous phosphate buffer, pH 3.0,
containing 20 mM tetrabutylammonium (TBA) phosphate, the
flow rate was 1 mL/min. For 2, the quenched mixture was
analyzed using the same column and flow rate, but the column
was equilibrated and eluted with 60% CH3CN in H2O. Rep-
resentative chromatograms are shown in Figure 2.
L-P h e OMe -3-[2′-(d ih yd r oxyp h osp h on o)oxy-4′,6′-d i-
m eth ylp h en yl]-3,3-d im eth ylp r op ion ic Am id e (8c). Com-
pound 7c (160 mg, 0.25 mmol) was dissolved in THF (20 mL)
and treated according to the general procedure for hydro-
genenation described above. The final product was obtained
as a white amorphous powder by trituration in Et2O/hexane
(96 mg, 83%): 1H NMR (CD3OD) δ 7.28-7.09 (6 H, m), 6.68
(1 H, s), 4.53-4.48 (1 H, m), 3.59 (3 H, s), 3.02-2.95 (1 H, m),
2.81-2.70 (3 H, m), 2.39 (3 H, s), 2.21 (3 H, s), 1.57 (3 H, s),
1.54 (3 H, s); MS (FAB) m/ z 464 (M+ + 1); HRMS (FAB) m/ z
464.1823 (M+ + 1, Calcd 464.1838). Anal. Calcd for C23H30
-
NO7P × 0.5 H2O: C, 58.48; H, 6.56; N, 2.97. Found: C, 58.50;
H, 6.76; N, 2.85.
Mich aelis-Men ten Kin etics. For determination of Michae-
lis-Menten constants, various concentrations of substrates
8a -d were incubated with AP (1.95 µg for 8a , 8b, and 8d ;
2.51 µg for 8c) in 100 mM MOPS buffer, pH 7.4, in a total
volume of 220 µL for 3 min at 25 °C. Each reaction was
quenched with 1 M HClO4 solution in CH3CN (220 µL). AP
solution was first prepared in H2O containing 1 mM ZnCl2 and
1 mM MgCl2 and then diluted with 100 mM MOPS buffer, pH
7.4, to the desired enzyme concentration. The initial rates of
formation of the lactone 2 were measured by HPLC as
described above, except that the analysis of 2 was monitored
at 225 nm to improve detection limits. It should be noted that
since 8e was not stable in MOPS buffer, pH 7.4, for prolonged
periods of time, solutions of this phosphate prodrug at various
concentrations were prepared in H2O (100 µL) and the reaction
was initiated by mixing the substrate solution with an equal
volume of enzyme solution (1.78 µg in AP) in MOPS buffer
(100 mM, pH 7.4). After incubation for 3 min, the reaction
was quenched by addition of 2 M acetic acid in CH3CN (200
µL) to avoid degradation prior to HPLC sampling. The
Michaelis-Menten parameters, Km and Vmax values, were
obtained by fitting the data to the Michaelis-Menten equation
using a computer program.
Lys-E-CBZ-r-OMe-3-[2′-(d ih yd r oxyp h osp h on o)oxy-4′,6′-
dim eth ylph en yl]-3,3-dim eth ylpr opion ic Am ide (8d). Com-
pound 7d (120 mg, 0.16 mmol) was dissolved in CH3OH (20
mL) and treated according to the general procedure for
hydrogenation described above. The final product was ob-
tained as a white amorphous solid after the obtained residue
was dissolved in H2O and subsequently lyophilized (43 mg,
80%):1H NMR (D2O) δ 7.14 (1 H, s), 6.74 (1 H, s), 4.07-405 (1
H, m), 3.63 (3 H, s), 3.02 (1 H, d, J ) 13.2 Hz), 2.85 (2 H, t, J
) 6.65 Hz), 2.60 (1 H, d, J ) 13.08 Hz), 2.41 (3 H, s), 2.19 (3
H, s), 1.62 (3 H, s), 1.57 (3 H, s), 1.49-1.40 (4 H, m), 1.02-
0.88 (2 H, m); MS (FAB) m/ z 445 (M+ + 1); HRMS (FAB) m/ z
445.2120 (M+ + 1, Calcd 445.2104). Anal. Calcd for C20H33
-
N2O7P × 1 H2O: C, 51.95; H, 7.14; N, 6.06. Found: C, 52.10;
H, 7.40; N, 5.80.
Asp -r-OMe-â-ben zyl-3-[2′-(d ih yd r oxyp h osp h on o)oxy-
4′,6′-d im eth ylp h en yl]-3,3-d im eth ylp r op ion ic Am id e (8e).
Compound 7e (160 mg, 0.23 mmol) was dissolved in THF (20
mL) and treated according to the general procedure for
hydrogenation described above. The final product was ob-
tained as a white amorphous powder after the obtained residue
was dissolved in H2O and subsequently lyophilized (60 mg,
61%):1H NMR (D2O) δ 7.11 (1 H, s), 6.72 (1 H, s), 4.59 (1 H,
m), 3.62 (3 H, s), 2.94 (1 H, d, J ) 14.28 Hz), 2.69 (1 H, d, J )
13.47 Hz), 2.54 (2 H, m), 2.42 (3 H, s), 2.18 (3 H, s), 1.60 (3 H,
s), 1.59 (3 H, s); MS (FAB) m/ z 432 (M+ + 1). Anal. Calcd
for C18H26NO9P: C, 50.12; H, 6.03; N, 3.24. Found: C, 49.98;
H, 6.30; N, 3.00.
Ack n ow led gm en t. The authors thank Drs. Teruna
J. Siahaan and Sanjeev Gangwar for helpful discussions.
t-Boc-Asp -â-Bzl-r-OMe. t-Boc-Asp-â-Bzl (2 g, 6.2 mmol)
was dissolved in CH2Cl2 (50 mL) and allowed to cool to 0 °C
J O961477P