5-Phenylthioacyclouridine: A potent and specific inhibitor of uridine phosphorylase

Article abstract of DOI:10.1016/S0006-2952(00)00410-X

5-Phenylthioacyclouridine (PTAU or 1-[(2-hydroxyethoxy)methyl]-5-phenylthiouracil) was synthesized as a highly specific and potent inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3). PTAU has inhibition constant (K(is)) values of 248 and 353 nM towa

Full text of DOI:10.1016/S0006-2952(00)00410-X

Biochemical Pharmacology, Vol. 60, pp. 851–856, 2000.
© 2000 Elsevier Science Inc. All rights reserved.
ISSN 0006-2952/00/$–see front matter
PII S0006-2952(00)00410-X
5-Phenylthioacyclouridine: A Potent and Specific
Inhibitor of Uridine Phosphorylase
Mahmoud H. el Kouni,*† Naganna M. Goudgaon,‡§ʈ Mohammad Rafeeq,‡
Omar N. Al Safarjalani,* Raymond F. Schinazi§ʈ and Fardos N. M. Naguib*
*DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY AND COMPREHENSIVE CANCER CENTER, UNIVERSITY OF
ALABAMA AT BIRMINGHAM, BIRMINGHAM, AL 35294; ‡DEPARTMENT OF CHEMISTRY, GULBARGA UNIVERSITY,
GULBARGA, INDIA; §VETERANS AFFAIRS MEDICAL CENTER (ATLANTA), DECATUR, GA 30033; AND
ʈ LABORATORY OF BIOCHEMICAL PHARMACOLOGY, EMORY UNIVERSITY SCHOOL OF MEDICINE,
ATLANTA, GA 30322, U.S.A.
ABSTRACT. 5-Phenylthioacyclouridine (PTAU or 1-[(2-hydroxyethoxy)methyl]-5-phenylthiouracil) was
synthesized as a highly specific and potent inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3). PTAU has
inhibition constant (Kis) values of 248 and 353 nM towards UrdPase from mouse and human livers, respectively.
PTAU was neither an inhibitor nor a substrate for thymidine phosphorylase (EC 2.4.2.4), uridine–cytidine
kinase (EC 2.7.1.48), thymidine kinase (EC 2.7.1.21), dihydrouracil dehydrogenase (EC 1.3.1.2), orotate
phosphoribosyltransferase (EC 2.4.2.10), or orotidine 5Ј-monophosphate decarboxylase (EC 4.1.2.23), the
enzymes that could utilize the substrate (uridine or thymidine) or products (uracil or thymine) of UrdPase.
Different isomers of 5-tolylthiouracil also were synthesized and tested as inhibitors of UrdPase. The meta-
substituted isomer was 3- to 4-fold more potent as an inhibitor of UrdPase than the para- or ortho-substituted
isomers. These data indicate that the hydrophobic pocket in the active site of UrdPase adjacent to the 5-position
of the pyrimidine ring can accommodate the meta-substituted 5-phenyluracils better than the other isomers,
leading to improved inhibition. Therefore, it is anticipated that the potency of PTAU can be increased further
by the addition of certain hydrophobic groups at the meta position of the phenyl ring. PTAU has potential
usefulness in the therapy of cancer and AIDS as well as other pathological and physiological disorders that can
be remedied by the administration of uridine. BIOCHEM PHARMACOL 60;6:851–856, 2000. © 2000 Elsevier
Science Inc.
KEY WORDS. 5-phenylthioacyclouridine; inhibitor; uridine phosphorylase; chemotherapy
The enzyme UrdPase¶ (EC 2.4.2.3) plays a crucial role in
the chemotherapy of cancer and AIDS. The importance of
UrdPase in cancer chemotherapy stems from the fact that
UrdPase is the enzyme responsible for the activation and
deactivation of several chemotherapeutic analogues, most
notably the 5-fluoropyrimidines [1–7], as several human
tumors are devoid of dThdPase (EC 2.4.2.4) activity [1, 5,
7]. Moreover, UrdPase exhibits a circadian rhythm in mice
[8, 9], which is opposite to that observed for the anticancer
efficacy of FdUrd [10]. In addition, host toxicities of some
of these anticancer (e.g. FUra) [11–14] as well as anti-HIV
(e.g. AZT) [15, 16] drugs are antagonized by uridine, the
availability and concentration of which are controlled by
UrdPase [17–25]. The important role played by UrdPase in
the chemotherapy of cancer and AIDS has generated a
strong interest in developing inhibitors for this enzyme.
Such inhibitors would enhance the chemotherapeutic effi-
cacy of these drugs by preventing their degradation and/or
host toxicity.
Acyclouridines were shown to be potent inhibitors of
UrdPase from various sources [1, 5–7, 26–32]. They were
shown to potentiate the efficacy of FdUrd in vitro and in
vivo [5, 7], to increase the level and duration of uridine in
plasma [17–25] and heart perfusate [33], to enhance the
salvage of uridine by various tissues [18–20], and to reduce
host toxicity of FUra [18, 20, 34], FdUrd [35], and AZT [15,
36]. In this study we report the synthesis of PTAU as an
inhibitor of UrdPase. PTAU is lipophilic, and therefore it is
anticipated that its uptake by the liver and intestine, the
principal organs involved in pyrimidine catabolism, would
be facilitated. A preliminary report has been presented [37].
† Corresponding author. Tel. (205) 934-1132; FAX (205) 934-8240;
E-mail: m.elkouni@ccc.uab.edu
¶ Abbreviations: AZT, 3Ј-azido-3Ј-deoxythymidine; BAU, 5-benzylacy-
clouridine; BBAU, 5-(benzyloxybenzyl)acyclouridine; dThdPase, thymi-
dine phosphorylase; DTT, dithiothreitol; FdUrd, 5-fluoro-2Ј-deoxyuri-
dine; FUra, 5-fluorouracil; PTAU, 5-phenylthioacyclouridine or 1-[(2-
hydroxyethoxy)methyl]-5-phenylthiouracil; and UrdPase, uridine
phosphorylase.
MATERIALS AND METHODS
Chemicals
[2-14C]Uridine (56 Ci/mol) and [2-14C]thymidine (56 Ci/
mol) were obtained from Moravek Biochemicals, Inc., and
silica gel G/UV254 polygram TLC plates from Brinkmann.
Received 6 December 1999; accepted 3 March 2000.

852
M. H. el Kouni et al.
5-PHENYLTHIOURACIL. 5-Phenylthiouracil was synthe-
1
sized as described previously [31, 38]. H NMR (CDCl3) ␦
11.35 (brs, 2H, NH), 7.88 (s, 1H, 6-H), 7.12–7.30 (m, 5H,
SPh).
2-ACETOXYETHYL ACETOXYMETHYL ETHER. 2-Acetoxy-
ethyl acetoxymethyl ether was synthesized as previously
1
described [39]. H NMR (CDCl3) ␦ 5.3 (s, 2H, OCH2O),
4.08 (A2B2 pattern, 4H, OCH2CH2O), 3.08 (s, 6-H,
COCH3).
SCHEME 1.
1-[(2-ACETOXYETHOXY)METHYL]-5-PHENYLTHIOURACIL.
N,O-Bis(trimethylsilyl)-acetamide (6.40 mL, 25 mmol)
was added to a mixture of 5-phenylthiouracil (2.20 g, 10
mmol) and 2-acetoxyethyl acetoxymethyl ether (2.15 g, 12
mmol) in dry CH2Cl2 (40 mL) with stirring under a
nitrogen atmosphere. After 2 hr of stirring at room tem-
perature, the clear solution was cooled to 0°, and SnCl4 (10
mL, 1.0 M CH2Cl2 solution, 10 mmol) was added. Then
the reaction mixture was warmed to room temperature and
stirred overnight. The solution was poured slowly into a
mixture of CHCl3 (100 mL) and saturated aqueous
NaHCO3 (100 mL) solution. The resulting emulsion was
separated by filtration, the aqueous fraction was extracted
further with EtOAc (3 ϫ 50 mL), and the combined
organic fractions were dried over Na2SO4 and concentrated
under reduced pressure. Trituration of the oily residue with
diethyl ether (40 mL) afforded the product as a white solid
The protein assay kit was purchased from Bio-Rad Labora-
tories. All other chemicals were obtained from the Sigma
Chemical Co. or the Aldrich Chemical Co.
Chemical Synthesis
5-Phenylthiouracil and the other 5-substituted uracils were
prepared from 5-chlorouracil by the method outlined in
Scheme 1. PTAU or 1-[(2-hydroxyethoxy)methyl]-5-phe-
nylthiouracil was prepared by the method outlined in
Scheme 2. Melting points were determined on an Electro-
1
thermal melting point apparatus and are uncorrected. H
NMR spectra were recorded on a General Electric QE-300
(300 MHz) spectrometer. Experiments were monitored
using TLC analysis performed on Kodak Chromatogram
sheets precoated with silica gel and a fluorescent indicator;
column chromatography employing silica gel (60–200
mesh; Fisher Scientific) was used for the purification of
products. Microanalyses were performed at Galbraith Lab-
oratories, Inc.
1
(2.05 g, 61%), m.p. 96–99°. H NMR (CDCl3) ␦ 1.87 (s,
3H, COCH3), 3.61–4.05 (A2B2 pattern, 4H,
OCH2CH2O), 5.02 (s, 2H, OCH2O), 7.05–7.19 (m, 5H,
SPh), 7.50 (s, 1H, 6-H), 8.88 (s, 1H, NH, D2O exchange-
SCHEME 2.

5-Phenylthioacyclouridine, an Inhibitor of UrdPase
853
able). Anal. Calc. for C15H16N2O5S: C, 53.94; H, 4.79; N,
8.33. Found: C, 53.36; H, 4.73; N, 8.50.
Enzyme Studies
PREPARATION OF ENZYME EXTRACTS. Human liver samples
were obtained from the Tissue Procurement Facility of the
Comprehensive Cancer Center of the University of Ala-
bama at Birmingham. Mouse livers were obtained from
female CD-1 mice (18–20 g) (Charles River Laboratories).
The samples were minced and homogenized in ice-cold
(3:1, v/w) buffer [20 mM potassium phosphate (pH 8.0), 1
mM DTT, 1 mM EDTA] using a Polytron homogenizer
(Brinkmann Instruments). The homogenates were centri-
fuged at 105,000 g for 1 hr at 4°. The supernatant fluids
(cytosol) were collected. UrdPase and dThdPase were
isolated from the cytosol by column chromatography as
previously described [6].
1-[(2-HYDROXYETHOXY)METHYL]-5-PHENYLTHIOURACIL
(5-PHENYLTHIOACYCLOURIDINE OR PTAU). To a solution of
1-[(2-acetoxyethoxy)methyl]-5-phenylthiouracil (1.68 g, 5
mmol) in MeOH (20 mL) was added 10 mL of 1 N NaOMe
in MeOH. After 4 hr at room temperature, the pH was
adjusted to 4.0 with 1 N HCl, and the solvent was removed
under reduced pressure to obtain a solid. The residue was
loaded onto a silica gel column, and the product was
obtained by elution with CH2Cl2/MeOH (9:1). The TLC
pure fractions were pooled and concentrated, and the
residue was recrystallized from absolute ethanol to yield the
desired title product as a white crystalline solid (1.18 g,
1
80%), m.p. 131–133°. H NMR (DMSO-d6) ␦ 3.31–3.57
URDPASE AND DTHDPASE ASSAYS. Assays were conducted
at 37° under conditions where enzyme activity was linear
with respect to time and enzyme concentration. Nucleoside
(uridine or thymidine) cleavage was measured isotopically
by following the formation of nucleobases from their
respective nucleosides as previously described [6]. Five
concentrations of the inhibitor were used, ranging from 8 to
900 ␮M. The reaction mixture contained 20 mM potassium
phosphate (pH 8), 1 mM EDTA, 1 mM DTT, 1 mM
[2-14C]uridine or [2-14C]thymidine (6 Ci/mol), and 25 ␮L
cytosol in a final volume of 50 ␮L. Reactions were started
by the addition of extract and stopped by boiling in a water
bath for 2 min followed by freezing. Precipitated proteins
were removed by centrifugation. Uridine and thymidine
were separated from their respective nucleobases on silica
gel TLC plates developed with chloroform:methanol:acetic
acid (90:5:5, by vol.), and the radioactivity in the spots was
determined on a percentage basis using a Berthold LB-2821
Automatic TLC-Linear Analyzer. The Rf values were:
uridine, 0.07; uracil, 0.43; thymidine, 0.14; and thymine,
0.62.
(m, 4H, OCH2CH2O), 4.66 (t, 1H, OH, D2O exchange-
able), 5.16 (s, 2H, NCH2O), 7.13–7.31 (m, 5H, SPh), 8.29
(s, 1H, 6-H), 11.67 (s, 1H, NH, D2O exchangeable). Anal.
Calc. for C13H14N2O4S: C, 53.03; H, 4.79; N, 9.52. Found:
C, 52.64; H, 4.80; N, 9.52.
5-O-TOLYLTHIOURACIL (5-ORTHO-TOLYLTHIOURACIL OR
2-METHYL-5-PHENYLTHIOURACIL). Reaction of 5-chloroura-
cil (730 mg, 5 mmol) with o-thiocresol (620 mg, 5 mmol)
in 25 mL of ethylene glycol in the presence of potassium
carbonate (700 mg, 5 mmol) as described for the prepara-
tion of 5-phenylthiouracil yielded the title compound (680
mg, 67%), m.p. 263–265°. 1H NMR (CDCl3) ␦ 11.32 (brs,
2H, NH), 7.82 (s, 1H, 6-H), 6.90–7.20 (m, 4H, SPh), 2.32
(s, 3H, PhCH3). Anal. Calc. for C11H10N2O2: C, 56.31; H,
4.30; N, 11.96. Found C, 56.11; H, 4.30; N, 11.96.
5-M-TOLYLTHIOURACIL (5-META-TOLYLTHIOURACIL OR
3-METHYL-5-PHENYLTHIOURACIL). Reaction of 5-chloroura-
cil (730 mg, 5 mmol) with m-thiocresol (620 mg, 5 mmol)
in 25 mL of ethylene glycol in the presence of potassium
carbonate (700 mg, 5 mmol) as described for the prepara-
tion of 5-phenylthiouracil yielded the title compound (570
mg, 56%), m.p. 265–268°. 1H NMR (CDCl3) ␦ 11.30 (brs,
2H, NH), 7.85 (s, 1H, 6-H), 6.95–7.18 (m, 4H, SPh), 2.25
(s, 3H, PhCH3). Anal. Calc. for C11H10N2O2: C, 56.31; H,
4.30; N, 11.96. Found C, 55.76; H, 4.26; N, 12.03.
Kinetic Studies
Inhibition constants (Kis) and apparent Ki values were
estimated from the data using computer programs with least
squares fitting. The program was written by Dr. Sungman
Cha and modified to fit IBM BASIC by Dr. F. N. M.
Naguib.
5-P-TOLYLTHIOURACIL
(5-PARA-TOLYLTHIOURACIL
OR
DETERMINATION OF KIS VALUES. Kis values were estimated
from the slopes and intercepts of the double-reciprocal plots
by the methods of Wilkinson [40] and Cleland [41], using
uridine concentrations ranging from 30 to 700 ␮M.
4-METHYL-5-PHENYLTHIOURACIL). Reaction of 5-chloroura-
cil (730 mg, 5 mmol) with p-thiocresol (620 mg, 5 mmol)
in 25 mL of ethylene glycol in the presence of potassium
carbonate (700 mg, 5 mmol) as described for the prepara-
tion of 5-phenylthiouracil yielded the title compound (610
mg, 60%), m.p. 270–273°. 1H NMR (CDCl3) ␦ 11.30 (brs,
2H, NH), 7.85 (s, 1H, 6-H), 6.90–7.22 (m, 4H, SPh), 2.25
(s, 3H, PhCH3). Anal. Calc. for C11H10N2O2: C, 56.31; H,
4.30; N, 11.96. Found C, 56.11; H, 4.40; N, 11.92.
DETERMINATION OF APPARENT KI VALUES. Using uridine
(125 ␮M) and five different concentrations of the inhibitor
ranging from 50 to 900 ␮M, apparent Ki values were
estimated from Dixon plots (1/v vs [I]) [42] of the data.

854
M. H. el Kouni et al.
TABLE 2. Apparent Ki values of 5-thio-substituted uracil
analogues for UrdPase from human liver
Compound
Apparent Ki* (␮M)
5-(Phenylthio)uracil
meta-Tolylthiouracil
para-Tolylthiouracil
ortho-Tolylthiouracil
175.2 Ϯ 66.3
28.6 Ϯ 4.3
100.1 Ϯ 48.5
119.9 Ϯ 53.2
*Values are means Ϯ standard error of estimation from at least three determinations
measured at 125 ␮M uridine and 20 mM inorganic phosphate.
from mouse and human livers. The results in Table 1
demonstrate that PTAU was more potent than its benzyl-
acyclouridine counterpart, BAU, and comparable in po-
tency to BBAU, a more potent inhibitor of UrdPase than
BAU [26, 27].
To determine the specificity of PTAU, the compound
was tested against various enzymes that could utilize the
substrate (uridine or thymidine) or products (uracil or
thymine) of UrdPase as previously described [28]. PTAU
was neither an inhibitor nor a substrate for dThdPase,
uridine–cytidine kinase (EC 2.7.1.48), thymidine kinase
(EC 2.7.1.21), dihydrouracil dehydrogenase (EC 1.3.1.2),
orotate phosphoribosyltransferase (EC 2.4.2.10), or oroti-
dine 5Ј-monophosphate decarboxylase (EC 4.1.2.23), as
there was no significant effect on their activity in the
presence of these compounds. These experiments indicate
that PTAU is a specific inhibitor of UrdPase.
FIG. 1. Inhibition of human liver UrdPase by PTAU. Plots of
1/velocity versus 1/[uridine] at various fixed concentrations of
PTAU. Each point represents at least three determinations.
Protein Determination
Protein concentrations were determined spectrophoto-
metrically by the method of Bradford [43], using bovine
␥-globulin as a standard.
Consistent with the notion that increased hydrophobic-
ity at the 5-position of the pyrimidine ring enhances
binding of pyrimidines to UrdPase by interacting with a
hydrophobic pocket on the enzyme adjacent to the 5-posi-
tion of the pyrimidine ring [1, 26–30, 32, 49, 50], we
synthesized and tested different isomers of 5-tolylthiouracil
as inhibitors of UrdPase. Table 2 compares the apparent Ki
values of these different isomers of tolylthiouracil. The
meta-substituted isomer was 3- to 4-fold more potent as an
inhibitor of UrdPase than the para- or ortho-substituted
isomers. Similar results were observed with meta isomers of
5-benzyluracil [51] and 5-(benzyloxy)benzylbarbituric acid
[52]. These data clearly indicate that the meta isomer of
5-phenyl-substituted uracils provides a significant inhibi-
tory effect on UrdPase over the ortho or para isomers. It also
provides evidence that the hydrophobic pocket in the
active site of UrdPase [1, 26–30, 32, 49, 50] can accom-
modate the meta-substituted 5-phenyluracils better than
the other isomers, leading to better inhibition. Therefore, it
is expected that further hydrophobic substitutions (e.g.
alkyl, aryl groups) at the meta position of the phenyl ring of
PTAU would yield even more potent inhibitors of UrdPase
than PTAU itself.
RESULTS AND DISCUSSION
In the present study, we synthesized PTAU as a new, highly
specific, and potent inhibitor of UrdPase. PTAU was
subjected to kinetic studies to determine the mechanism of
inhibition and the Kis value, and also was compared with
BAU and BBAU. All three inhibitors (PTAU, BAU, and
BBAU) showed competitive inhibition with UrdPase from
mouse and human livers. The liver was chosen as the source
of UrdPase because it is generally believed that it is the
organ that regulates pyrimidine metabolism in the body [17,
44–48]. Figure 1 shows the Lineweaver–Burk plot of
PTAU with human liver UrdPase. PTAU had Kis values of
248 and 353 nM towards UrdPase from mouse and human
livers, respectively (Table 1). Table 1 also compares the Kis
values of PTAU with those of its 5-benzyl-substituted
counterparts (BAU and BBAU) as inhibitors of UrdPase
TABLE 1. Inhibition constants (Kis) of PTAU compared with
those of BAU and BBAU for UrdPase from mouse and
human livers
Kis* (nM)
Inhibitor
Mouse liver
Human liver
PTAU has an excellent potential in various aspects of
chemotherapy. The usefulness of UrdPase inhibitors has
already been established in the field of experimental che-
motherapy of cancer and AIDS. We previously developed
various 5-substituted derivatives of acyclouridine and acy-
clobarbituric acid as specific inhibitors of UrdPase [1,
PTAU
BAU
BBAU
248 Ϯ 46
420 Ϯ 40
170 Ϯ 00
353 Ϯ 76
1190 Ϯ 200
220 Ϯ 29
*Values are means Ϯ standard error of estimation from at least three determinations
measured at 20 mM inorganic phosphate.

5-Phenylthioacyclouridine, an Inhibitor of UrdPase
855
phorylase activity and plasma concentration of uridine in
mice. Biochem Pharmacol 40: 2479–2485, 1990.
26–28, 31]. These inhibitors were shown to enhance the
efficacy of FdUrd against human tumors in vitro and in vivo
[5, 7]. Furthermore, the compounds were shown to elevate
the concentration and prolong the half-life of uridine in the
plasma [17–25], as well as increase the salvage of uridine by
various tissues [18, 19]. Therefore, these inhibitors were
used to protect against or rescue from the host toxicity of
anticancer (i.e. FUra) [18, 20, 34) and anti-HIV (i.e. AZT)
[16, 36] drugs, the toxicities of which were shown to be
antagonized by administration of exogenous uridine. The
new acyclonucleoside PTAU is likely to share the various
chemotherapeutic characteristics of the older inhibitors.
Furthermore, it is anticipated that the lipophilicity of
PTAU will enhance its uptake by the liver and intestine,
the main organs responsible for pyrimidine metabolism, and
hopefully its chemotherapeutic efficacy.
9. Naguib FNM, Soong S-j and el Kouni MH, Circadian rhythm
of orotate phosphoribosyltransferase, pyrimidine nucleoside
phosphorylases and dihydrouracil dehydrogenase in mouse
liver. Possible relevance to chemotherapy with 5-fluoropy-
rimidines. Biochem Pharmacol 45: 667–673, 1993.
10. von Roemeling R and Hrushesky W, Determination of the
therapeutic index of floxuridine by its circadian infusion
pattern. J Natl Cancer Inst 82: 386–393, 1990.
11. Martin DS, Stolfi RL, Sawyer RC, Spiegeleman S and Young
CW, High-dose 5-fluorouracil with delayed uridine “rescue”
in mice. Cancer Res 42: 3964–3970, 1982.
12. Klubes P, Cerna I and Meldon MA, Uridine rescue from
lethal toxicity of 5-fluorouracil in mice. Cancer Chemother
Pharmacol 8: 17–21, 1982.
13. Klubes P and Cerna I, Use of uridine rescue to enhance
antitumor selectivity of 5-fluorouracil. Cancer Res 43: 3182–
3186, 1983.
14. van Groeningen CJ, Peters GJ, Leyva A, Laurensse E and
Pinedo HM, Reversal of 5-fluorouracil-induced myelosuppres-
sion by prolonged administration of high-dose uridine. J Natl
Cancer Inst 81: 157–162, 1989.
15. Sommadossi JP, Carlisle R, Schinazi RF and Zhou Z, Uridine
reverses the toxicity of 3Ј-azido-3Ј-deoxythymidine in normal
human granulocyte-macrophage progenitor cells in vitro with-
out impairment of antiretroviral activity. Antimicrob Agents
Chemother 32: 997–1001, 1988.
16. Sommadossi JP, Zhu Z, Carlisle R, Xie MY, Weidner DA and
el Kouni MH, Novel pharmacologic approaches for the
treatment of AIDS and potential use of uridine phosphorylase
inhibitors. In: Advances in Chemotherapy of AIDS (Eds. Diasio
RB and Sommadossi JP), pp. 63–73. Pergamon Press, New
York, 1990.
In conclusion, we have synthesized PTAU as a specific
and potent inhibitor of UrdPase. Its potency may be
increased further by the addition of hydrophobic groups at
the meta position of its phenyl ring. PTAU could have a
wide application in the therapy of cancer, AIDS, as well as
other pathological and physiological disorders where ad-
ministration of uridine has been shown to be useful [cf.
Refs. 23 and 25 and references therein].
We thank Dr. Raymond P. Panzica for his help and critical reading of
the manuscript.
17. Monks A, Ayers O and Cysyk RL, Effect of 5-benzylacyclouri-
dine, a potent inhibitor of uridine phosphorylase, on the
metabolism of circulating uridine by the isolated rat liver.
Biochem Pharmacol 32: 2003–2009, 1983.
18. Darnowski JW and Handschumacher RE, Tissue-specific
enhancement of uridine utilization and 5-fluorouracil therapy
in mice by benzylacyclouridine. Cancer Res 45: 5364–5368,
1985.
19. Peters GJ, van Groeningen CJ, Laurensse EJ, Lankelma J,
Leyva A and Pinedo HM, Uridine-induced hypothermia in
mice and rats in relation to plasma and tissue levels of uridine
and its metabolites. Cancer Chemother Pharmacol 20: 101–
108, 1987.
References
1. Niedzwicki JG, el Kouni MH, Chu SH and Cha S, Pyrimidine
acyclonucleosides, inhibitors of uridine phosphorylase. Bio-
chem Pharmacol 30: 2097–2101, 1981.
2. Woodman PW, Sarrif AM and Heidelberger C, Specificity of
pyrimidine nucleoside phosphorylases and the phosphorolysis
of 5-fluoro-2Ј-deoxyuridine. Cancer Res 40: 507–511, 1980.
3. Ishitsuka H, Miwa M, Takemoto K, Fukuoka K, Itoga A and
Maruyama HB, Role of uridine phosphorylase for antitumor
activity of 5Ј-deoxy-5-fluorouridine. Gann 71: 112–123,
1980.
20. Martin DS, Stolfi RL and Sawyer C, Use of oral uridine as a
substitute for parenteral uridine rescue of 5-fluorouracil ther-
apy, with and without the uridine phosphorylase inhibitor
5-benzylacyclouridine. Cancer Chemother Pharmacol 24:
9–14, 1989.
21. Davis ST, Joyner SS, Chandrasurin P and Baccanari DP,
Species-dependent differences in the biochemical effects and
metabolism of 5-benzylacyclouridine. Biochem Pharmacol 45:
173–181, 1993.
4. Veres Z, Szabolcs A, Szinai I, De´nes G and Jeney A,
Enzymatic cleavage of 5-substituted-2Ј-deoxyuridines by py-
rimidine nucleoside phosphorylases. Biochem Pharmacol 35:
1057–1059, 1986.
5. Chu MYW, Naguib FNM, Iltzsch MH, el Kouni MH, Chu
SH, Cha S and Calabresi P, Potentiation of 5-fluoro-2Ј-
deoxyuridine antineoplastic activity by the uridine phospho-
rylase inhibitors benzylacyclouridine and benzyloxybenzylacy-
clouridine. Cancer Res 44: 1852–1856, 1984.
22. Sommadossi J-P, Cretton EM, Kidd LB, McClure HM,
Anderson DC and el Kouni MH, Effects of 5-benzylacyclouri-
dine, an inhibitor of uridine phosphorylase, on the pharma-
cokinetics of uridine in rhesus monkeys: Implications for
chemotherapy. Cancer Chemother Pharmacol 37: 14–22, 1995.
23. Ashour OM, Naguib FNM and el Kouni MH, 5-Benzyloxy-
benzylbarbituric acid acyclonucleoside, a uridine phospho-
rylase inhibitor, and 2Ј,3Ј,5Ј-triacetyluridine, a prodrug of
uridine, as modulators of plasma uridine concentration. Im-
plications for chemotherapy. Biochem Pharmacol 51: 1601–
1612, 1996.
6. el Kouni MH, el Kouni MM and Naguib FNM, Differences in
activities and substrate specificity of human and murine
pyrimidine nucleoside phosphorylases: Implications for che-
motherapy with 5-fluoropyrimidines. Cancer Res 53: 3687–
3693, 1993.
7. Ashour OM, Naguib FNM, Khalifa MMA, Abdel-Raheem
MH, Panzica RP and el Kouni MH, Enhancement of 5-fluoro-
2Ј-deoxyuridine antitumor efficacy by the uridine phospho-
rylase inhibitor 5-(benzyloxybenzyl)barbituric acid acyclo-
nucleoside. Cancer Res 55: 1092–1098, 1995.
8. el Kouni MH, Naguib FNM, Park KS, Cha S, Darnowski JW
and Soong S-j, Circadian rhythm of hepatic uridine phos-
24. Pizzorno G, Yee L, Burtness BA, Marsh JC, Darnowski JW,

856
M. H. el Kouni et al.
Chu MYW, Chu HS, Chu E, Leffert JJ, Handschumacher RE
and Calabresi P, Phase I clinical and pharmacological studies
of benzylacyclouridine, a uridine phosphorylase inhibitor. Clin
Cancer Res 4: 1165–1175, 1998.
37. Goudgaon NM, Lee NM and el Kouni MH, 5-Arylthioacyc-
louridines: Potent inhibitors of uridine phosphorylase. Proc
Am Assoc Cancer Res 36: 1716, 1995.
38. Roth B and Bunnett JF, 5-Arylthiopyrimidines. V: Kinetics of
the cyclization of 4-oxo derivatives to 10H- and 10-alkyl-
pyrimido[5,4-b][1,4]benzothiazines (1,3-diazaphenothiazines).
J Am Chem Soc 87: 340–349, 1965.
39. Rosowsky A, Kim SH and Wick M, Synthesis and antitumor
activity of an acyclonucleoside derivative of 5-fluorouracil.
J Med Chem 24: 1171–1181, 1981.
40. Wilkinson GN, Statistical estimations in enzyme kinetics.
Biochem J 80: 324–332, 1961.
41. Cleland WW, The statistical analysis of enzyme kinetic data.
Adv Enzymol 29: 1–32, 1967.
42. Dixon M and Webb EC, Enzymes. Academic Press, New
York, 1979.
43. Bradford MM, A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the princi-
ple of protein-dye binding. Anal Biochem 72: 248–254, 1976.
44. Gasser T, Moyer JD and Handschumacher RE, Novel single-
pass exchange of circulating uridine in rat liver. Science 213:
777–778, 1981.
45. Holstege A, Leser H-G, Pausch J and Gerok W, Uridine
catabolism in Kupffer cells, endothelial cells, and hepatocytes.
Eur J Biochem 149: 169–173, 1985.
25. Ashour OM, Al Safarjalani ON, Naguib FNM, Goudgaon
NM, Schinazi RF and el Kouni MH, Modulation of plasma
uridine concentration by 5-(phenylselenenyl)acyclouridine,
an inhibitor of uridine phosphorylase: Relevance to chemo-
therapy. Cancer Chemother Pharmacol 45: 351–361, 2000.
26. Niedzwicki JG, Chu SH, el Kouni MH, Rowe EC and Cha S,
5-Benzylacyclouridine and 5-benzyloxybenzylacyclouridine,
potent inhibitors of uridine phosphorylase. Biochem Pharmacol
31: 1857–1861, 1982.
27. Naguib FNM, el Kouni MH, Chu SH and Cha S, New
analogues of benzylacyclouridines, specific and potent inhib-
itors of uridine phosphorylase from human and mouse livers.
Biochem Pharmacol 36: 2195–2201, 1987.
28. Naguib FNM, Levesque DL, Wang E-C, Panzica RP and el
Kouni MH, 5-Benzylbarbituric acid derivatives, potent and
specific inhibitors of uridine phosphorylase. Biochem Pharma-
col 46: 1273–1283, 1993.
29. Park KS, el Kouni MH, Krenitsky TA, Chu SH and Cha S,
Inhibition of uridine phosphorylase from Escherichia coli by
benzylacyclouridines. Biochem Pharmacol 35: 3853–3855,
1986.
30. el Kouni MH, Naguib FNM, Niedzwicki JG, Iltzsch MH and
Cha S, Uridine phosphorylase from Schistosoma mansoni.
J Biol Chem 263: 6081–6086, 1988.
31. Goudgaon NM, Naguib FNM, el Kouni MH and Schinazi RF,
Phenylselenenyl- and phenylthio-substituted pyrimidines as
inhibitors of dihydrouracil dehydrogenase and uridine phos-
phorylase. J Med Chem 36: 4250–4254, 1993.
46. Holstege A, Pausch J and Gerok W, Effect of 5-diazouracil on
the catabolism of circulating pyrimidines in rat liver and
kidney. Cancer Res 46: 5576–5581, 1986.
47. Monks A, and Cysyk RL, Uridine regulation by the isolated
rat liver: Perfusion with an artificial oxygen carrier. Am J
Physiol 242: R465–R470, 1982.
32. el Kouni MH, Naguib FNM, Panzica RP, Otter BA, Chu SH,
Gosselin G, Chu CK, Schinazi RF, Shealy YF, Goudgaon N,
Ozerov AA, Ueda T and Iltzsch MH, Effect of modifications
in the pentose moiety and conformational changes on the
binding of nucleoside ligands to uridine phosphorylase from
Toxoplasma gondii. Biochem Pharmacol 51: 1687–1700, 1996.
33. Chua BHL, Watson P, Kleinhans B and Morgan HE, Effect of
5- benzyloxybenzylacyclouridine on the labeling of UTP and
RNA in perfused rat heart. Fedn Proc 44: 1773, 1985.
34. Ashour OM, Naguib FNM, Panzica RP, Al Safarjalani ON
and el Kouni MH, Modulation of 5-fluorouracil host toxicity
by 5-(benzyloxybenzyl)barbituric acid acyclonucleoside, a uri-
dine phosphorylase inhibitor, and 2Ј,3Ј,5Ј-tri-o-acetyluridine,
a prodrug of uridine. Biochem Pharmacol 60: 427–431, 2000.
35. Chu S-H, Wiemann MC, Chu MY and Calabresi P, Modu-
lation of FUdR toxicity by two benzylacyclouridine analogs.
Proc Am Assoc Cancer Res 28: 410, 1987.
48. Moyer JD, Oliver JT and Handschumacher RE, Salvage of
circulating pyrimidine nucleosides in the rat. Cancer Res 41:
3010–3017, 1981.
49. Niedzwicki JG, el Kouni MH, Chu SH and Cha S, Structure-
activity relationship of ligands of the pyrimidine nucleoside
phosphorylases. Biochem Pharmacol 32: 399–415, 1983.
50. el Kouni MH, Naguib FNM, Chu SH, Cha S, Ueda T,
Gosselin G, Imbach J-L, Shealy F and Otter BA, Effect of the
N-glycosidic bond conformation and modifications in the
pentose moiety on the binding of nucleoside ligands to
uridine phosphorylase. Mol Pharmacol 34: 104–110, 1988.
51. Orr GF, Musso DL, Boswell GE, Keeley JL, Joyner SS, Davis
ST and Baccanari DP, Inhibition of uridine phosphorylase:
Synthesis and structure-activity relationships of aryl-substi-
tuted 5-benzyluracils and 1-[(2-hydroxyethoxy)methyl]-5-
benzyluracils. J Med Chem 38: 3850–3856, 1995.
52. Guerin DJ, Mazeas D, Musale MS, Naguib FNM, Al Safar-
jalani ON, el Kouni MH and Panzica RP, Uridine phospho-
rylase inhibitors. Chemical modification of benzyloxybenzyl-
barbituric acid and its effect on UrdPase inhibition. Bioorg
Med Chem Lett 9: 1477–1480, 1999.
36. Calabresi P, Falcone A, St. Clair MH, Wiemann MC, Chu
SH and Darnowski JW, Benzylacyclouridine reverses azido-
thymidine-induced bone marrow suppression without impair-
ment of anti-human immunodeficiency virus activity. Blood
76: 2210–2215, 1990.