Inhibition of uridine phosphorylase. Synthesis and structure-Activity relationships of aryl-substituted 1-((2-hydroxyethoxy)methyl)-5-(3- phenoxybenzyl)uracil

Article abstract of DOI:10.1021/jm960688j

Structure-activity relationship studies on a series of 1-((2- hydroxyethoxy)methyl)-5-(3-(substituted-phenoxy)benzyl)uracils as inhibitors of murine liver uridine phosphorylase have led to compounds with IC₅₀s as low as 1.4 nM. The two most potent compounds, 10j (3-cyanophenoxy) and 11f (3-chlorophenoxy) were tested in vive for effects on steady-state concentrations of circulating uridine in mice and rats. Both compounds were substantially more efficacious than BAU (5-benzylacyclouridine) both in vitro and in vivo.

Full text of DOI:10.1021/jm960688j

© Copyright 1997 by the American Chemical Society
Volume 40, Number 8
April 11, 1997
Articles
In h ibition of Ur id in e P h osp h or yla se. Syn th esis a n d Str u ctu r e-Activity
Rela tion sh ip s of Ar yl-Su bstitu ted
1-((2-Hyd r oxyeth oxy)m eth yl)-5-(3-p h en oxyben zyl)u r a cil
G. Faye Orr,*,† David L. Musso,† J ames L. Kelley,‡ Suzanne S. J oyner,§ Stephen T. Davis,§ and
David P. Baccanari§
Division of Organic Chemistry and Division of Pharmacology and Molecular Therapeutics, Glaxo Wellcome Inc.,
Research Triangle Park, North Carolina 27709
Received October 3, 1996X
Structure-activity relationship studies on a series of 1-((2-hydroxyethoxy)methyl)-5-(3-
(substituted-phenoxy)benzyl)uracils as inhibitors of murine liver uridine phosphorylase have
led to compounds with IC50s as low as 1.4 nM. The two most potent compounds, 10j
(3-cyanophenoxy) and 11f (3-chlorophenoxy) were tested in vivo for effects on steady-state
concentrations of circulating uridine in mice and rats. Both compounds were substantially
more efficacious than BAU (5-benzylacyclouridine) both in vitro and in vivo.
Uridine phosphorylase (EC 2.4.2.3) (UrdPase) and
thymidine phosphorylase (EC 2.4.2.4)1 are mammalian
enzymes that catalyze the reversible phosphorolysis of
pyrimidine nucleosides. These phosphorylases, which
function as salvage enzymes in pyrimidine metabolism,
are responsible for the degradation of chemotherapeutic
agents such as 5-fluoro-2′-deoxyuridine (FUDR).2 Sev-
eral researchers have studied both the antineoplastic
applications3-5 of UrdPase inhibitors such as BAU (5-
benzylacyclouridine) and their use in combination with
zidovudine as a means of reducing the severity of bone
marrow suppression.6-10
mouse liver.15,16 More recent studies have led to 1′-
hydroxymethyl, 1′-aminomethyl, and barbituric acid
derivatives with potent inhibitory properties.16-19
We recently reported SAR studies of the effect of aryl
substituents on the inhibitory potency of BAU.20 The
most potent inhibitors had IC50s ranging from 27 to 70
nM, and several compounds were found to elevate
plasma uridine concentrations by 3-9-fold in rats. We
now report research on 5-(3-phenoxybenzyl)uracils,
which has led to a series of 1-((2-hydroxyethoxy)methyl)-
5-(3-(substituted-phenoxy)benzyl)uracils with IC50s as
low as 1.4 nM.
Early research on the inhibition of UrdPase led to the
development of 5-benzyluracils (BUs) with good inhibi-
tory potency.11-14 Later work by Niedzwichi, et al.
provided (2-hydroxyethoxy)methyl derivatives of BUs
such as BAU and BBAU (5-(m-(benzyloxy)benzyl)-
acyclouridine) with enhanced potency against the en-
zyme from sarcoma S-180 cytosol and the cytosol of
Ch em istr y Discu ssion
The target inhibitors in Table 1 were prepared in
several steps as illustrated in Schemes 1 and 2. The
intermediate substituted 3-phenoxyphenyl propionates
5 were prepared by alkylation of the appropriate phenol
precursors 1 with 1-bromo-3-fluorobenzene to give the
diphenyl ethers 2 (Scheme 1). Reaction of 2 with ethyl
acrylate under Heck conditions gave the phenoxyphenyl
acrylates 3, which were reduced to the propionates 5
via catalytic hydrogenation. The parent unsubstituted
3-phenoxyphenyl propionate 5 (R ) H) was prepared
* Author to whom correspondence should be addressed.
† Division of Organic Chemistry.
‡ Current address: Krenitsky Pharmaceuticals Inc., Four University
Place, 4611 University Dr Durham, NC 27707.
§ Division of Pharmacology and Molecular Therapeutics.
X Abstract published in Advance ACS Abstracts, March 15, 1997.
S0022-2623(96)00688-7 CCC: $14.00 © 1997 American Chemical Society

1180 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8
Orr et al.
Ta ble 1. Physical Properties of 1-Substituted 5-(3-Phenoxybenzyl)uracils
compd no.
R
R3
method
yield, %
mp, °C
formulaa
IC50, nM
10a
11a
10b
11b
10c
10d
10e
11e
10f
11f
10g
10h
10i
10j
12
H
H
4-F
4-F
4-Cl
4-OCH3
3-F
3-F
3-Cl
3-Cl
3-CF3
3-CH3
3-OCH3
3-CN
BAU
BBAU
H
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
38
23
42
15
55
40
28
15
65
15
36
46
23
40
59
39
93-95
C20H20N2O5
C21H22N2O6
14
10
98
22
84
210
14
8.2
15
1.4
32
13
CH2OH
H
CH2OH
H
H
H
CH2OH
H
CH2OH
H
H
H
H
H
H
139-141
106-109
147-148
109-110
110-112
98-100
C
20H19FN2O5
C21H21FN2O6
20H19ClN2O5
C21H22N2O6
20H19FN2O5
C21H21FN2O6
20H19ClN2O5
C
C
122-123
118-120
118-119
103-105
110-111
98-102
115-117
144-146
111-112
C
C21H21ClN2O6
C21H19F3N2O5
C21H22N2O5
C21H22N2O6
31
C
C
C
21H19N3O5
14H16N2O4
21H22N2O5
5.9
460
84
13
a
All compounds were analyzed for C, H, and N.
Sch em e 1a
5-(3-phenoxybenzyl)uracils 10 and 11 in satisfactory
yields (Table 1).
In h ibition of Ur idin e P h osph or ylase: Str u ctu r e-
Activity Rela tion sh ip s. The 1-substituted 5-(3-phe-
noxybenzyl)uracils in Table 1 were tested for inhibition
of UrdPase from murine liver. The IC50 is the concen-
tration of inhibitor that results in a 50% decrease in
reaction velocity. Our previous work20 focused on the
effects of aryl substituents on the inhibitory potency of
the known UrdPase inhibitor BAU. The most active
analogs were those with a 3-alkoxy substituent on the
aryl moiety with IC50s as low as 27 nm. We subse-
quently found that the 3-phenoxy analogs 10a and 11a
are somewhat more potent than the alkoxy compounds
with IC50s )14 and 10 nm, respectively. The focus of
the work reported herein was to evaluate the effects of
substituents on the 3-phenoxy ring on the UrdPase
inhibitory potency and on the pharmacokinetics of
plasma uridine levels.
a
Reagents: (i) NaOMe, MeOH then m-bromofluorobenzene,
N-methylpyrrolidinone, 180 °C; (ii) ethyl acrylate, Pd(OAc)2, P(o-
tolyl)3, Et3N, MeCN, 125 °C in bomb; (iii) H2, PtO2, EtOH, (iv) H2,
10% Pd-C, EtOH; (v) malonic acid, pyridine, piperidine, 90 °C;
(vi) 1.0 N ethereal HCl, EtOH, reflux.
Substitution in the para position of the phenoxy ring
with a F (10b), Cl (10c), or OCH3 (10d ) substituent, and
with the (2-hydroxyethoxy)methyl side chain at N-1,
resulted in a loss of potency with IC50s of 98, 84, and
210 nm, respectively. The 4-fluoro analog (11b), which
contains the branched side chain at N-1, was more
potent (IC50 ) 22 nm) than 10b.
via the acrylic acid 4 from 3-phenoxybenzaldehyde as
illustrated in Scheme 1.
The intermediate 2-thioxo-4-pyrimidinones 7 (Scheme
2) were prepared by treating the 3-phenoxyphenyl
propionates 5 with potassium tert-butoxide, followed by
reaction of the potassium salt 6 with ethyl formate and
condensation with thiourea21 to give the 2-thioxo-4-
pyrimidinones 7a -7j (Table 3) in 53-84% yields. Hy-
drolysis of the 2-thioxo-4-pyrimidinones 7 in refluxing
aqueous chloroacetic acid22 provided the 5-(3-phenoxy-
benzyl)uracils 8a -8j (Table 3) in 61-94% yield.
The 5-benzyluracils 8a -8j were alkylated via the
O-silylated intermediates, which were formed using bis-
(trimethylsilyl)acetamide, with either (2-acetoxyethoxy)-
methyl bromide23 or 2-(bromomethoxy)-1,3-propanediyl
dibenzoate.24 The intermediate esters 9 were hydro-
lyzed with methanolic ammonia to give the 1-substituted-
Derivatives containing a substituent in the meta
position [F (10e), Cl (10f), CF3 (10g), CH3 (10h ), and
OCH3 (10i)], and the (2-hydroxyethoxy)methyl side
chain, gave compounds with IC50s comparable to that
of the parent 10a (IC50s ranging from 32 to 14 nm). The
3-cyano analog 10j was more potent with an IC50 of 5.9
nm. The 3-F (11e) and 3-Cl (11f) analogs, which contain
the branched side chain, were also more potent than
the parent 10a with IC50s of 8.2 and 1.4 nm, respec-
tively.
To summarize, strongly electron withdrawing groups
in the meta position of the phenoxy moeity result in
more potent analogs, while strongly electron donating
groups result in a loss of potency. Furthermore, branch-

Inhibition of Uridine Phosphorylase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8 1181
Ta ble 2. Physical Properties of Diphenyl Ethers
compd no.
R
X
method
yield %
mp, °C
oil
formulaa
C12H8BrFO
12H8BrClO
C13H11BrO2
2a
2b
2c
2d
2e
2f
2g
2h
2i
4-F
4-Cl
4-OCH3
3-F
3-Cl
3-CF3
3-CH3
3-OCH3
3-CN
2-F
4-F
4-Cl
4-OCH3
3-F
3-Cl
3-CF3
3-CH3
3-OCH3
3-CN
2-F
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
B
C
D, E
E
47
53
30
43
63
31
17
29
30
27
84
67
91
79
79
(b)
89
71
90
58
80
94b
90
88
96
93
87
93
99
91
70
36
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
111-113c
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
C
C
C
12H8BrFO
12H8BrClO
C13H8BrF3O
C13H11BrO
C13H11BrO2
C13H8BrNO
2j
C12H8BrFO
3a
3b
3c
3d
3e
3f
3g
3h
3i
CHdCHCO2Et
CHdCHCO2Et
CHdCHCO2Et
CHdCHCO2Et
CHdCHCO2Et
CHdCHCO2Et
CHdCHCO2Et
CHdCHCO2Et
CHdCHCO2Et
CHdCHCO2Et
CHdCHCO2H
CH2CH2CO2Et
CH2CH2CO2Et
CH2CH2CO2Et
CH2CH2CO2Et
CH2CH2CO2Et
CH2CH2CO2Et
CH2CH2CO2Et
CH2CH2CO2Et
CH2CH2CO2Et
CH2CH2CO2Et
CH2CH2CO2Et
C17H15FO3
C17H15ClO3
C18H18O4
C17H15FO3
C17H15ClO3
C18H18O3
C18H18O4
C18H15NO3
C17H15FO3
C15H12O3
3j
4
H
H
5a
5b
5c
5d
5e
5f
5g
5h
5i
4-F
4-Cl
4-OCH3
3-F
3-Cl
3-CF3
3-CH3
3-OCH3
3-CN
2-F
C17H17FO3‚0.15H2O
C17H17ClO3
C18H20O4
C17H17FO3
C17H17ClO3
C18H17F3O3
C18H20O3‚0.33H2O
C18H20O4
E
E
E
E
E
E
E
5j
5k
Ed
E
C18H17NO3‚0.10H2O
C17H17FO3
a
b
d
All compounds were analyzed for C and H. This compound was used without purification. c Recrystallized from MeCN-water. The
3-cyano analog, 3i, was reduced using 10% palladium-carbon instead of platinum oxide.
Sch em e 2a
a
Reagents: (vii) potassium tert-butoxide in THF, HCO2Et, Et2O; (viii) thiourea, 2-PrOH, reflux; (ix) glacial HOAc, 20% aqueous
ClCH2CO2H, reflux; (x) BSA, ClCH2CH2Cl, reflux then BrCH2OCH(R3)CH2OR2; (xi) MeOH, NH3, room temperature.
ing of the acyclic side chain (R3 ) CH2OCH(CH2OH)-
CH2OH in Table 1) also increases potency. The IC50s
of 10j and 11f represent 4.5-fold and 19-fold increases
in potency, respectively, as compared to our best inhibi-
tors reported previously.20
Eleva tion of P la sm a Ur id in e Levels. Plasma
uridine concentration is maintained at a steady-state
level via a balance between synthesis and catabo-
lism.25,26 When catabolism is inhibited, continued bio-
synthesis results in a time-dependent increase in plasma
uridine levels. The pharmacodynamic effects of 10j
(IC50 ) 5.9 nM) and 11f (IC50 ) 1.4 nM) were compared
to those of BAU (IC50 ) 460 nM) in BDF1 mice (Figure
1A). Following treatment with 30 mg/kg of 10j, plasma
uridine levels increased over 6 h and achieved a
maximum concentration of 28 µM, a 10-fold elevation

1182 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8
Orr et al.
Ta ble 3. Physical Properties of 5-(3-Phenoxybenzyl)-2-thiouracils and 5-(3-Phenoxybenzyl)uracils
compd no.
R
X
method
yield, %
mp, °C
formulaa
C17H14N2O2S
17H14N2O3
C17H13FN2O2S
17H13FN2O3
C17H13ClN2O2S
17H13ClN2O3
7a
8a
7b
8b
7c
8c
7d
8d
7e
8e
7f
8f
7g
8g
7h
8h
7i
H
H
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
F
G
F
G
F
G
F
G
F
G
F
G
F
G
F
72
72
76
92
80
88
63
93
53
94
54
88
84
61
62
85
79
78
58
90
195-197
281-283
199-201
260-263
187-189
255-257
179-181
238-240
169-171
243-245
163-165
209-211
149-151
203-205
161-163
229-230
165-168
188-190
210-213
266-268
C
4-F
4-F
4-Cl
C
4-Cl
C
4-OCH3
4-OCH3
3-F
3-F
3-Cl
C18H16N2O3S
C18H16N2O4
C17H13FN2O2S
C
17H13FN2O3
C17H13ClN2O2S
17H13ClN2O3
3-Cl
C
3-CF3
3-CF3
3-CH3
3-CH3
3-OCH3
3-OCH3
2-F
C18H13F3N2O2S
C18H13F3N2O3
C18H16N2O2S
C18H16N2O3
C18H16N2O3S
C18H16N2O4
O
S
O
S
G
F
G
F
8i
7j
8j
C17H13FN2O2S
C17H13FN2O3
2-F
O
G
a
All compounds were analyzed for C, H, and N.
over the predose value. Compound 11f produced 20 µM
uridine (peak at 2 h postdose), whereas BAU elevated
plasma uridine to 11 µM (peak at 1 h postdose). The
enhanced effects of 10j and 11f compared to BAU were
evident within the first 2 h after dosing, at a time when
the plasma drug concentrations were similar (Figure
1B). Thus both compounds were more potent than BAU
in vivo. In addition, 10j and 11f exhibited biphasic
elimination kinetics, and the somewhat more pro-
nounced pharmacodynamic effect of 10j may be due to
its higher plasma levels at the latter time points.
with chemotherapeutic agents such as 5-fluorouracil
and 5-fluoro-2′-deoxyuridine.
Exp er im en ta l Section
Melting points were taken in capillary tubes using a
Thomas-Hoover Unimelt and are uncorrected. The NMR
spectra were recorded on a Varian XL-100-15-FT or a Varian
FT-80A spectrometer. The UV absorption spectra were mea-
sured on a Cary 118 UV-vis spectrophotometer. Each ana-
lytical sample had spectral data compatible with its assigned
structure and moved as a single spot on thin-layer chroma-
tography (TLC). The TLC’s were developed on Whatman 200
The perturbation of plasma uridine by BAU is species-
dependent, and the rat is a stringent test for the efficacy
of UrdPase inhibitors in vivo .27 When 10j or 11f (90
mg/kg, po) was administered to rats, plasma uridine
levels were increased 4-8-fold for 4 h (Figure 2A). The
elevation in plasma uridine levels produced by 10j and
11f in rats persisted for at least 1.3 times longer than
the elevation produced by one of our best inhibitors re-
ported previously.20 In contrast, the same dose of BAU
elevated plasma uridine 2-4-fold (peak at 30 min post-
dose), and plasma uridine was back to control level with-
in 5 h. The enhanced pharmacodynamic effects of 10j
and 11f were produced by plasma drug concentrations
that were 2-10-fold less than BAU for the first 3 h.
m
MK6F plates of silica gel with fluorescent indicator.
Preparative flash chromotography as performed on silica gel
60 (40-63 mm, E. Merck No. 9385) using the method of Still
et al.28 Elemental analyses were performed by Atlantic
Microlab, Inc.
Meth od A. 3-Br om o-3'-flu or od ip h en yl Eth er (2d ). A
solution of 3-fluorophenol (8.25 g, 74 mmol) and 25% sodium
methoxide in methanol (18.5 mL, 81 mmol) was stirred for 3
h at ambient temperature. The solution was concentrated in
vacuo to a tan solid. The phenoxide was dissolved in N-meth-
yl-2-pyrrolidinone (50 mL) and heated in a 180 °C oil bath.
1-Bromo-3-fluorobenzene (12.95 g, 74 mmol) was added, and
the mixture was stirred for 24 h at 180 °C. After being cooled
to room temperature, the reaction mixture was diluted with
water (100 mL) and extracted with dichloromethane (2 × 100
mL). The organic layer was concentrated to dryness in vacuo,
redissolved in diethyl ether (100 mL), washed with water (100
mL) and a saturated aqueous sodium bicarbonate solution (100
mL), dried over sodium sulfate, filtered, and concentrated in
vacuo to a brown oil (12.5 g). The oil was chromatographed
on silica gel 60, eluting with hexane. The fractions containing
only 3-bromo-3′-fluorodiphenyl ether were combined and con-
centrated in vacuo to give 8.42 g (43%) of 2d as a clear,
colorless oil; NMR (DMSO-d6): δ 7.14-6.87 (m, 4H, Ar), 7.53-
7.29 (m, 4H, Ar).
Con clu sion s
A systematic study of the effect of aryl substituents
on potency has led to 5-(3-phenoxybenzyl)uracils 10j and
11f, which have IC50s of 5.9 and 1.4 nM, respectively,
for inhibition of UrdPase. Compound 10j exhibited the
greater effect on circulating uridine levels in BDFI mice.
This compound was substantially more active than BAU
at equivalent doses and elevated circulating uridine
levels for a longer period of time. Compound 10j is a
good candidate for antineoplastic studies in combination
Meth od B. Eth yl 3-(3-(3-F lu or op h en oxy)p h en yl)a cr yl-
a te (3d ). A mixture of 2d (5.30 g, 19.8 mmol), ethyl acrylate
(2.48 g, 24.8 mmol), triethylamine (2.51 g, 24.8 mmol), pal-
ladium acetate (44.5 mg, 0.2 mmol), tri-o-tolylphosphine (244
mg, 0.8 mmol), and acetonitrile (20 mL) were added to a heavy

Inhibition of Uridine Phosphorylase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8 1183
F igu r e 2. Plasma profiles of uridine and UrdPase inhibitors
in rats following oral administration of 90 mg/kg BAU (squares),
10i (circles), or 11f (triangles). Blood samples were collected
at the times indicated and assayed for (A) plasma uridine and
(B) plasma drug concentration. Points are the average value
from two to three rats. Control plasma uridine concentration
ranged from 0.6 to 1.1 mM in the various experiments.
F igu r e 1. Plasma profiles of uridine and UrdPase inhibitors
in mice following intraperitoneal administration of 30 mg/kg
BAU (squares), 10i (circles), or 11f (triangles). Blood samples
were collected at the times indicated and assayed for (A)
plasma uridine and (B) plasma drug concentration. Each point
is the average value from three mice.
glass-walled bottle, flushed with nitrogen, sealed, and heated
in a 100 °C oil bath for 5 h. Cold 0.1 N hydrochloric acid (40
mL) was added to the cooled reaction mixture. The lower layer
was collected and concentrated in vacuo to a dark-green oil
and chromatographed on silica gel 60 with 0-3% ethyl acetate/
hexanes as eluent. The fractions containing only ethyl 3-(3-
(3-fluorophenoxy)phenyl)acrylate were combined and concen-
trated in vacuo to give 4.48 g (79%) of 3d as a clear, colorless
oil: NMR (DMSO-d6): δ 7.66 (d, 1H, J ) 16 Hz, dCH), 7.60-
7.31 (m, 4H, Ar), 7.15-6.83 (m, 4H, Ar), 6.68 (d, 1H, J ) 16
Hz, dCH), 4.20 (q, 2H, J ) 7 Hz, CH2), 1.26 (t, 3H, J ) 7 Hz,
CH3).
Meth od C. (E)-3-(3-P h en oxyp h en yl)-2-p r op en oic Acid
(4). A solution of 3-phenoxybenzaldehyde (25.0 g, 126 mmol),
malonic acid (26.2 g, 252 mmol), and piperidine (2.0 mL, 20
mmol) in pyridine (50 mL) was stirred in an oil bath heated
at 90 °C for 18 h. After being cooled to ambient temperature,
the solution was poured into cold water (1 L). The pH of the
aqueous mixture was adjusted to pH 2 with concentrated
hydrochloric acid. The solids which formed were collected by
suction filtration, washed with water, and recrystallized from
acetonitrile/water to give 24.18 g (80%) of 4 as a white solid:
mp 111-113 °C; TLC, methanol: dichloromethane (1:19), one
spot with Rf ) 0.25; NMR (CDCl3) δ 7.71 (d, 1H, J ) 16 Hz,
ArCH), 7.40-7.00 (m, 9H, ArH), 6.38 (d, 1H, J ) 16 Hz,
CHCO2).
Meth od D. (E)-Eth yl 3-(3-P h en oxyp h en yl)-2-p r op e-
n oa te (5a ). A solution of 4 (14.51 g, 60.4 mmol) and 1.0 M
ethereal hydrochloric acid (40 mL) in absolute ethanol (150
mL) was refluxed with stirring under nitrogen for 24 h. The
ethanol was removed in vacuo and the residue taken up in
ethyl acetate (150 mL) and washed with a saturated aqueous
sodium bicarbonate solution (2 × 75 mL). The washes were
back-extracted with ethyl acetate (50 mL) and the combined
extracts washed with brine, dried over anhydrous sodium
sulfate, and filtered. The filtrate was evaporated in vacuo to
give 15.31 g (94%) of 5a as a yellow oil, which was used without
further purification: NMR (DMSO-d6) δ 7.62 (d, 1H, ArCH),
7.12 (m, 9H, Ar), 6.36 (d, 1H, CHCO2), 4.24 (q, 2H, CH2),1.32
(t, 3H, CH3).
Meth od E. Eth yl 3-(3-(3-F lu or op h en oxy)p h en yl)p r o-
p ion a te (5e). To a solution of 3d (6.33 g, 22 mmol) and
absolute ethanol (100 mL) was added platinum oxide hydrate
(100 mg, 0.44 mmol). The mixture was shaken on a Parr
apparatus under hydrogen atmosphere (30 psi) for 6 h. The
mixture was filtered to remove the catalyst and concentrated
in vacuo to give 5.91 g (93%) of 5e as a clear, colorless oil:
NMR (DMSO-d6): δ 7.48-7.30 (m, 2H, Ar), 7.10-6.78 (m, 6H,
Ar), 4.03 (q, 2H, J ) 7.0 Hz, CH3CH2O), 2.87 (t, 2H, J ) 7.6
Hz, CH2), 2.63 (t, 2H, J ) 7.4 Hz, CH2Ar), 1.14 (t, 3H, J ) 7.0
Hz, CH3CH2O).

1184 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8
Orr et al.
Meth od F . 1,2-Dih yd r o-5-(3-(3-flu or op h en oxy)ben zyl)-
2-th ioxo-4(3H)-p yr im id in on e (7e). A solution of 5e (5.51
g, 19.0 mmol) and ethyl formate (3.07 g, 41.4 mmol) in diethyl
ether (35 mL) was added dropwise with stirring to a solution
of potassium tert-butoxide (1 M in THF, 47.0 mL, 47.0 mmol)
in diethyl ether (125 mL) cooled in an ice bath under nitrogen.
The solution was stirred at ambient temperature for 18 h, and
the solvent was removed in vacuo. The residue was dissolved
in 2-propanol (50 mL), thiourea (2.89 g, 38.0 mmol) was added,
and the mixture was refluxed under nitrogen for 5 h. The
solvent was removed in vacuo, and the solid residue was
washed with cold diethyl ether and dissolved in cold water,
and the pH was adjusted to 4 with glacial acetic acid. The
beige precipitate was collected on a filter, washed several times
with water and diethyl ether, and dried under a vacuum at
ambient temperature for 18 h to give 3.29 g (53%) of 7e, mp
170-172 °C dec. Recrystallization of 0.40 g from methanol
gave 0.20 g of an analytically pure sample: NMR (DMSO-d6)
δ 12.45 (s, 1H, NH), 12.25 (s, 1H, NH), 7.11 (m, 9H, Ar and
H-6), 3.55 (s, 2H, CH2Ar); MS: m/ e 329 (M+).
Meth od G. P r ep a r a tion of 5-(3-(3-F lu or op h en oxy)-
ben zyl)u r a cil (8e). A suspension of 7e (2.70 g, 8.2 mmol) in
glacial acetic acid (40 mL) and 20% aqueous chloroacetic acid
(40 mL) was refluxed with stirring for 6 h. After being cooled
to ambient temperature and then in an ice bath, the mixture
was filtered and the solids were washed with water and ether
and dried in a vacuum oven at 80 °C for 18 h to give 2.34 g
(91%) of 8e as an off-white solid, mp 243-245 °C. Recrystal-
lization of 0.400 g from acetic acid-water gave 0.161 g of an
analytically pure sample: NMR (DMSO-d6): δ 11.10 (s, 1H,
NH), 10.75 (s, 1H, NH), 7.10 (m, 9H, Ar and H-6), 3.50 (s, 2H,
CH2Ar); MS m/ e 313 (M+).
Meth od H. P r ep a r a tion of 1-((2-Acetoxyeth oxy)m eth -
yl)-5-(3-(3-flu or op h en oxy)ben zyl)u r a cil (9e). Bis(trimeth-
ylsilyl)acetamide (1.38 mL, 5.6 mmol) was added to a stirred
suspension of 8e (1.00 g, 3.2 mmol) in dichloroethane (35 mL)
under nitrogen. The mixture was refluxed with stirring for 1
h, the heat was removed, and the solution which formed was
cooled in an ice bath. A solution of (2-acetoxyethoxy)methyl
bromide (0.55 g, 2.8 mmol) in acetonitrile (4 mL) was added
to the cooled solution, and the resulting solution was allowed
to warm to ambient temperature and stirred under nitrogen
for 18 h. The solvents were removed in vacuo, and the residue
was dissolved in dichloromethane (75 mL) and washed with
water (3 × 25 mL) and brine. The solvents were removed in
vacuo, and the residual oil was introduced onto a column of
silica gel 60 wetted with dichloromethane. The column was
eluted with dichloromethane:2-propanol (100:2), and the frac-
tions containing product were combined. The solvents were
removed in vacuo to give 0.58 g (48%) of 9e as a clear oil: NMR
(DMSO-d6) δ 11.42 (s, 1H, NH), 7.69 (s, 1H, H-6), 7.11 (m, 8H,
Ar), 5.08 (s, 2H, NCH2O), 4.07 (t, 2H, OCH2), 3.68 (t, 2H,
CH2CH2O), 3.54 (s, 2H, CH2Ar), 1.96 (s, 3H, CH3); MS m/ e
429.
Meth od I. P r ep a r a tion of 1-((2-Hyd r oxyeth oxy)m eth -
yl)-5-(3-(3-flu or op h en oxy)ben zyl)u r a cil (10e). A solution
of 0.53 g (1.2 mmol) of 9e in methanol (75 mL) saturated with
ammonia gas was stirred in a stoppered flask for 24 h at
ambient temperature. The methanol was removed in vacuo,
and the residue was recrystallized from 2-propanol:hexane and
dried in a vacuum oven at 80 °C to give 0.282 g (74%) of 10e
as a white solid: mp 98-100 °C; NMR (DMSO-d6) δ 11.40 (s,
1H, NH), 7.69 (s, 1H, H-6), 7.11 (m, 8H, ArH), 5.08 (s, 2H,
NCH2O), 4.68 (s, 1H, OH), 3.54 (s, 2H, CH2Ar), 3.49 (s, 4H,
(CH2)2) MS m/ e 387.
An im a l Dosin g a n d Blood Collection . Pharmacokinetic
studies were conducted in mice and rats. UrdPase inhibitors
were solubilized in saline by adjusting to pH 9 with 0.1 N
NaOH and administered in a volume of 10 mL/kg. Mice
(groups of 18-21 BDF1 females) were dosed ip, and blood was
collected from three mice at each time point. Whole blood was
obtained by cardiac puncture of CO2-anesthetized mice with
a syringe containing 50 µL of 5% EDTA, and plasma was
isolated by centrifugation (3000g for 10 min). Pharmacokinetic
studies were performed on male CD rats implanted with a
jugular vein cannula. The animals were placed in individual
metabolic cages and fed chow and water overnight. Food was
removed 6 h before the start of the experiment. Rats (three/
group) were dosed po, and whole blood samples (0.35 mL) were
removed from the cannula using a 1 cm3 syringe containing
50 µL of 5% EDTA. This volume of blood was replaced by the
injection of 0.35 mL of saline at each time point. The ability
to take sequential blood samples from the same rat resulted
in a standard error of approximately 10% between replicates
compared to 35% in the mouse studies where the pharmaco-
kinetic curves were constructed from single blood samples
taken from individual mice. Plasma was frozen at -20 °C and
stored for HPLC analyses.
HP LC An a lyses. Plasma concentrations of uridine and the
UrdPase inhibitors were determined using reverse-phase
HPLC.27 Briefly, protein was removed from the samples by
ultrafiltration using the Centrifree micropartition system
(Amicon Division, W. R. Grace and Co., Beverly, MA) or by
acetonitrile extraction. HPLC was performed on a reverse-
phase Microsorb C18 column (250 mm × 4.6 mm i.d.; Rainin
Instrument Co., Woburn, MA) with a Dynamax Axial Com-
pression guard column. An isocratic elution (1 mL/min) in 50
mM ammonium acetate buffer, pH 4.8, 0.5% acetonitrile was
followed by a linear gradient to 36% acetonitrile in the same
mobile phase. The exact time of each segment was dependent
upon the elution properties of the UrdPase inhibitor being
analyzed. The effluent was monitored by UV absorption at
265 nm. Uridine eluted as a distinct peak under the HPLC
conditions used for the analysis of each inhibitor. Control (t
) 0) plasma uridine concentrations in BDF1 mice and CD rats
were 3.3 ( 1.4 µM (n ) 9) and 0.8 ( 0.3 µM (n ) 29),
respectively.
Ack n ow led gm en t. We thank Robert Tansik and
Hattie Roberts for assistance with the UrdPase inhibi-
tion assays and Donna Staton and Sherron Paris for
assistance with the manuscript preparation.
Refer en ces
(1) (a) Krenitsky, T. A.; Barclay, M.; J acquez, J . A. Specificity of
Mouse Uridine Phosphorylase. Chromatography, Purification,
and Properties. J . Biol. Chem. 1964, 239, 805-812. (b) Kren-
itsky, T. A.; Mellors, J . W.; Barclay, R. K. Pyrimidine Nucleosi-
dases. Their Classification ond Relationship to Uric Acid Ribo-
nucleoside Phosphorylase. J . Biol. Chem. 1965, 240, 1281-1286.
(2) el Kouni, M. H.; el Kouni, M. M.; Naguib, F. N. M. Differences
in Activities and Substrate Specificity of Human and Murine
Pyrimidine Nucleoside Phosphorylases: Implications for Che-
motherapy with 5-Fluoropyrimidines. Cancer Res. 1993, 53,
3687-3693.
(3) Chu, M. Y. W.; Naguib, F. N. M.; Iltzsch, M. H.; el Kouni, M.
H.; Chu, S. H.; Cha, S.; Calabresi, P. Potentiation of 5-Fluoro-
2′-epoxyuridine Antineoplastic Activity by the Uridine Phospho-
rylase Inhibitors Benzylacyclouridine and Benzyloxybenzylacy-
clouridine. Cancer Res. 1984, 44, 1852-1856.
(4) Darnowski, J . W.; Handschumacher, R. E. Tissue-specific En-
hancement of Uridine Utilization and 5-Fluorouracil Therapy
in Mice by Benzylacyclouridine. Cancer Res. 1985, 45, 5364-
5368.
En zym e Assa ys. Inhibition of UrdPase from mouse liver
was quantitated using a radiochemical assay previously
described.27 Briefly, the conversion of [2-14C]uridine to [2-14C]-
uracil was determined in 20 mM potassium phosphate buffer,
pH 8.0, 1 mM EDTA, 170 µM [2-14C]uridine (specific activity
7.1 µCi/µmol), 1 mM dithiothreitol, and enzyme ( inhibitor.
After 30 min at 37 °C, the assay was terminated by heating
in a 100 °C water bath for 1 min, and uridine and uracil were
separated via TLC. The amount of enzyme used in the assay
was chosen to catalyze 10% conversion of uridine to uracil in
the uninhibited reaction.
(5) Martin, D. S.; Stolfi, R. L.; Sawyer, R. C. Use of Oral Uridine as
a
Substitute for Parental Uridine Rescue of 5-Fluorouracil
Therapy, with and without the Uridine Phosphorylase Inhibitor
5-Benzylacyclouridine. Cancer Chemother. Pharmacol. 1989, 24,
9-14.
(6) Richman, D. D.; Fischl, M. A.; Grieco, M. H.; Gottlieb, M. S.;
Volberding, P. A.; Laskin, O. L.; Leedom, J . M.; Groopman, J .
E.; Mildvan, D.; Hirsch, M. S.; J ackson, G. G.; Durack, D. T.;
Nusinoff-Lehrman, S.; and the AZT Collaborative Working
Group, The Toxicity of Azidothymidine (AZT) in the Treatment

Inhibition of Uridine Phosphorylase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8 1185
of Patients with AIDS and AIDS-Related Complex. N. Engl. J .
Med. 1987, 317, 192-197.
(17) Naguib, F. N.; Levesque, D. L.; Wang, E.-C.; Panzica, R. P.; el
Kouni, M. H. 5-Benzylbarbituric Acid Derivatives, Potent and
Specific Inhibitors of Uridine Phosphorylase. Biochem. Phar-
macol. 1993, 46, 1273-1283.
(18) Pan, B.-C.; Chu, S.-H. Synthesis of Enantiomers of 2′-Amino-
methyl-5-benzylacyclouridine (AM-BAU) and 2′-Aminomethyl-
5-benzyloxybenzylacyclouridine (AM-BBAU), Potent Inhibitors
of Uridine Phosphorylase. J . Heterocycl. Chem. 1988, 25, 1587-
1592.
(7) Yarchoan, R.; Weinhold, K. J .; Lyerly, H. K.; Gelmann, E.; Blum,
R. M.; Shearer, G. M.; Mitsuya, H.; Collins, J . M.; Myers, C. E.;
Klecker, R. W.; Markham, P. D.; Durack, D. T.; Lehrman, S. N.;
Barry, D. W.; Fischl, M. A.; Gallo, R. C.; Bolognesi, D. P.; Broder,
S. Administration of 3′-Azido-3′-deoxythymidine, an Inhibitor of
HTLV-III/LAV Replication, to Patients with AIDS or AIDS-
Related Complex. Lancet 1986, 575-580.
(8) Sommadossi, J .-P.; Carlisle, R.; Schinazi, R. F.; Zhou, Z. Uridine
Reverses the Toxicity of 3′-Azido-3′-deoxythymidine in Normal
Human Granulocyte-Macrophage Progenitor Cells In Vitro
without Impairment of Antiretroviral Activity. Antimicrob.
Agents Chemother. 1988, 32, 997-1001.
(9) Calabresi, P.; Falcone, A.; St. Clair, M. H.; Wiemann, M. C.; Chu,
S. H.; Darnowski, J . W. Benzylacyclouridine Reverses Azidot-
hymidine-Induced Marrow-Suppression without Impairment of
Anti-HIV Activity. Blood 1990, 76, 2210-2215.
(10) Falcone, A.; Darnowski, J . W.; Ruprecht, R. M.; Chu, S. H.;
Brunetti, I.; Calabresi, P. Differential Effect of Benzylacyclou-
ridine on the Toxic and Therapeutic Effects of Azidothymidine
in Mice. Blood 1990, 76, 2216-2221.
(11) Baker, B. R. Irreversible Enzyme Inhibitors. LXXV. Inhibitors
of Thymidine Phosphorylase. I. Mode of Ribofuranose Binding.
J . Med. Chem. 1967, 10, 297-301.
(12) Baker, B. R.; Kelley, J . L. Irreversible Enzyme Inhibitors.
CLXIX. Inhibition of FUDR Phosphorylase from Walker 256 Rat
Tumor by 6-Substituted Uracils. J . Med. Chem. 1970, 13, 456-
458.
(13) Baker, B. R.; Kelley, J . L. Irreversible Enzyme Inhibitors.
CLXXI. Inhibition of FUDR Phosphorylase from Walker 256 Rat
Tumor by 5-Substituted Uracils. J . Med. Chem. 1970, 13, 461-
467.
(14) Kelley, J . L.; Baker, B. R. Irreversible Enzyme Inhibitors. 202.
Candidate Active-Site-Directed Irreversible Inhibitors of FUDR
Phosphorylase from Walker 256 Rat Tumor Derived from
1-Benzyl-5-(3-ethoxybenzyl)uracil. J . Med. Chem. 1982, 25, 600-
603.
(15) Niedzwicki, J . G.; Chu, S. H.; El Kouni, M. H.; Rowe, E. C.; Cha,
S. 5-Benzylacyclouridine and 5-Benzyloxybenzylacyclouridine,
Potent Inhibitors of Uridine Phosphorylase. Biochem. Pharma-
col. 1982, 31, 1857-1861.
(19) For a comprehensive review of phosphorylase ligands, see:
Niedzwicki, J . G.; El Kouni, M. H.; Chu, S. H.; Cha, S. Structure-
Activity Relationship of Ligands of the Pyrimidine Nucleoside
Phosphorylases. Biochem. Pharmacol.1983, 32, 399-415.
(20) Orr, G. F.; Musso, D. L.; Boswell, G. E.; Kelley, J . L.; J oyner, S.
S.; Davis, S. T.; Baccanari, D. P. Inhibition of Uridine Phos-
phorylase: Synthesis and Structure-Activity Relationships of
Aryl-Substituted 5-Benzyluracils and 1-[(2-Hydroxyethoxy)meth-
yl]-5-benzyluracils. J . Med. Chem. 1995, 38, 3850-3856.
(21) Orr, G. F.; Musso, D. L. Improved synthesis of 5-Benzyl-2-
thiouracils. Synth. Commun. 1996, 26 (1), 179-189.
(22) J ohnson, T. B.; Hemingway, E. H. Researches on Pyrimidines.
CLIX. Synthesis of 4-Phenylcytosine. J . Am. Chem. Soc., 1915,
37, 378-383.
(23) Elion, G. B.; Furman, P. A.; Fyfe, J . A.; de Miranda, P.;
Beauchamp, L.; Schaeffer, H. J . Selectivity of Action of an
Antiherpectic Agent, 9-(2-Hydroxyethoxymethyl)guanine. Proc.
Natl. Acad. Sci. U.S.A. 1977, 74, 5716-5720.
(24) Schaeffer, H. J .; Beauchamp, L.; de Miranda, P.; Elion, G. B.;
Bauer, D. J .; Collins, P. 9-(2-Hydroxyethoxymethyl)guanine
Activity Against Viruses of the Herpes Group. Nature 1978, 272,
583.
(25) Gasser, T. J .; Moyer, D.; Handschumacher, R. E. Novel Single
Pass Exchange of Circulating Uridine in Rat Liver. Science 1981,
213, 777-779.
(26) Monks, A.; Cysyk, R. L. Uridine Regulation by the Isolated Rat
Liver: Perfusion with an Artificial Oxygen Carrier. Am. J .
Physiol. 1982, 242, 465-470.
(27) Davis, S. T.; J oyner, S. S.; Chandrasurin, P.; Baccanari, D. P.
Species-Dependent Differences in the Biochemical Effects and
Metabolism of 5-Benzylacyclouridine. Biochem. Pharmacol. 1993,
45, 173-181.
(28) Still, W. C.; Kahn, M.; Mutra, A. Rapid Chromatographic Tech-
nique for Preparative Separations with Moderate Resolution. J .
Org. Chem. 1978, 43, 2923-2925.
(16) Naguib, F. N.; El Kouni, M. H.; Chu, S. H.; Cha, S. New
Analogues of Benzylacyclouridines, Specific and Potent Inhibi-
tors of Uridine Phosphorylase from Human and Mouse Livers.
Biochem. Pharmacol. 1987, 36, 2195-2201.
J M960688J