5'-[2-(2-Nitrophenyl)-2-methylpropionyl]-2'-deoxy-5-fluorouridine as a potential bioreductively activated prodrug of FUDR: Synthesis, stability and reductive activation

Article abstract of DOI:10.1016/S0960-894X(00)00108-6

5'-[2-(2-Nitrophenyl)-2-methylpropionyl]-2'-deoxy-5-fluorouridine was synthesized as a potential bioreductively activated prodrug of 5-fluoro-2'-deoxyuridine (FUDR). The target compound was stable in both phosphate buffer and human serum and was found to

Full text of DOI:10.1016/S0960-894X(00)00108-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 797±800
50-[2-(2-Nitrophenyl)-2-methylpropionyl]-20-deoxy-5-¯uorouridine
as a Potential Bioreductively Activated Prodrug of FUDR:
Synthesis, Stability and Reductive Activation
Longqin Hu,* Bin Liu and Douglas R. Hackingy
Department of Pharmaceutical Chemistry, College of Pharmacy, Rutgers, The State University of New Jersey,
160 Frelinghuysen Road, Piscataway, NJ 08854, USA
Received 14 January 2000; accepted 9 February 2000
AbstractÐ50-[2-(2-Nitrophenyl)-2-methylpropionyl]-20-deoxy-5-¯uorouridine was synthesized as a potential bioreductively activated
prodrug of 5-¯uoro-20-deoxyuridine (FUDR). The target compound was stable in both phosphate bu€er and human serum and was
found to release quickly the parent drug FUDR in quantitative yield upon mild chemical reduction. # 2000 Elsevier Science Ltd.
All rights reserved.
5-Fluoro-20-deoxyuridine (FUDR, 1) is a ¯uoropyr-
imidine nucleoside used in the treatment of a variety of
tumors.1 It exerts its anticancer e€ects mainly by sup-
pression of DNA synthesis. It is converted by a single
thymidine kinase to the active metabolite 5-¯uoro-20-
deoxyuridine-50-monophosphate (2), which disrupts
DNA synthesis via its inhibition of thymidylate syn-
thase and its incorporation into DNA.2 The simplicity
of this pathway provides a strong rationale for the use
of FUDR in preference to 5-¯uorouracil (5-FU, 3),
which undergoes extensive metabolism to both active
¯uoropyrimidine nucleotides and other inactive degra-
dation products.2 Unfortunately, FUDR has not shown
consistently superior therapeutic results.3 It su€ers from
a number of drawbacks including high toxicity, rapid
blood clearance, rapid conversion to 5-FU, poor oral
activity and lack of selectivity.
Because of the primitive state of tumor vasculature,
solid tumors often develop regions of chronic or acute
hypoxia as a result of chronic or transient de®ciencies of
blood ¯ow. Such oxygen de®ciency often leads to resis-
tance to ionizing radiation and to many chemothera-
peutic drugs.4 6 Hypoxia also appears to accelerate
malignant tumor progression and increase metastasis.6
Hypoxic tumor cells are known to have a greater capa-
city for reductive reactions as compared to well-oxyge-
nated normal cells.6 This unique feature of solid tumors
provides an attractive target for selective anticancer
chemotherapy. Several bioreductively activated nitro
compounds, quinones and aromatic N-oxides are cur-
rently in clinical trials as hypoxia-selective cytotoxins
and could potentially be developed into selective anti-
cancer prodrugs.7
Prodrug design is an important strategy that has been
proven to work for many drugs in improving their
undesirable physico-chemical and biological proper-
ties.8 10 Several strategies based on intramolecular
cyclization reactions have been reviewed.11 Recently,
prodrug strategies have also been used in targeted drug
delivery including antibody-directed enzyme prodrug
therapy (ADEPT) and gene-directed enzyme prodrug
therapy (GDEPT). In these approaches, an enzyme is
delivered site-speci®cally by chemical conjugation or
genetic fusion to a tumor speci®c antibody or by
enzyme gene delivery systems into tumor cells. This is
then followed by the administration of a prodrug, which
is selectively activated by the delivered enzyme at the
tumor cells. A number of these systems are in develop-
ment and have been reviewed.12 16 Among the enzymes
*Corresponding author. Tel.: +1-732-445-5291; fax: +1-732-445-
6312; e-mail: longhu@rci.rutgers.edu
yDouglas R. Hacking was an undergraduate pharmacy student (P-3) in
L.H.'s laboratory at the University of Oklahoma College of Phar-
macy. Present address: University of Oklahoma College of Pharmacy,
Oklahoma City, OK 73117, USA.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00108-6

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L. Hu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 797±800
under evaluation is a bacterial nitroreductase from
Escherichia coli B. This FMN-containing ¯avoprotein is
capable of reducing certain aromatic nitro groups to the
corresponding amines or hydroxylamines in the pre-
sence of a cofactor NADH or NADPH.17 19
(SOCl2) in methanol, the corresponding methyl ester
was dialkylated using methyl iodide and sodium hydride
in the presence of catalytic amount of 18-crown-6 to
give methyl 2-nitrophenyl-2-methylpropionate (8).
Sodium hydroxide-mediated hydrolysis converted the
methyl ester 8 to the corresponding acid 9. Coupling of
the acid 9 to the 50-primary hydroxyl group of FUDR
was accomplished by a Mitsunobu reaction (treatment
with diethyl azodicarboxylate (DEAD) and triphenyl
phosphine (PPh3)).21,22 A major advantage of this
approach is that the condensation reaction can be
e€ected under mild and neutral conditions. Further-
more, the reaction is selective for the 50-primary
hydroxyl over the 30-secondary hydroxyl of the 20-deoxy-
ribonucleosides, thus avoiding the requirement for pro-
tection and subsequent deprotection of the 30-hydroxyl
group.21 All new compounds were fully characterized by
1H NMR and high-resolution MS.23
To improve the oral bioavailability and tumor-selectivity
of FUDR, we designed and synthesized 50-[2-(2-nitro-
phenyl)-2-methylpropionyl]-20-deoxy-5-¯uorouridine (4)
as a bioreductively activated FUDR prodrug that could
be used to target hypoxic cells or used in the enzyme
prodrug therapies mentioned above. Scheme 1 shows
the potential mechanism of activation of the target
compound 4. After reduction in the hypoxic tumor cells
or by an enzyme such as the bacterial nitroreductase,
the resulting hydroxylamine 5a (RˆOH) or amine 5b
(RˆH) could undergo facile cyclization reaction form-
ing the lactam 6a or 6b and, at the same time, releasing
the active drug FUDR (1). The two methyl groups
attached to the a-position of the carbonyl are designed
to restrict the rotational freedom of the conformation of
the molecule and place the carbonyl group in a more
favorable position with respect to the nucleophilic
amine or hydroxylamine.20 In addition, the two methyl
groups might inhibit the esterase-catalyzed hydrolysis
reaction of the ester bond and provide the necessary
stability in human serum, which is required for the
compound to be useful. In this communication, we
report the synthesis, stability in phosphate bu€er and in
human serum, and the reductive activation of target
compound 4.
Stability Test
One concern in using compound 4 as a prodrug is the
presence of the ester linkage that might make it vulner-
able to hydrolysis by esterases present in the blood. We,
therefore, tested the stability of compound 4 by incu-
bating it in sodium phosphate bu€er (100 mM, pH 7.4)
and in human serum at 37 ꢀC. Acetonitrile was used to
help dissolve compound 4 and the concentration of
acetonitrile in the incubation mixture was kept below
5%. At di€erent time intervals, aliquots (25 mL) were
withdrawn and analyzed by HPLC on a C18 reversed
phase column using acetonitrile/water/TFA as the
mobile phase. In the case of human serum, the esterase
activity was quenched with 7% HClO4 (100 mL) imme-
diately after the aliquots were withdrawn. Our results
indicate that compound 4 is stable in both phosphate
bu€er and human serum and no signi®cant hydrolysis
was observed after three days of incubation. Derivatives
Chemical Synthesis
The synthesis of 50-[2-(2-nitrophenyl)-2-methylpropio-
nyl]-20-deoxy-5-¯uorouridine (4) is shown in Scheme 2
starting from the commercially available 2-nitrophenyl-
acetic acid (7). After treatment with thionyl chloride
Scheme 1. Proposed mechanism of activation of the FUDR prodrug 4.
Scheme 2. Synthesis of FUDR prodrug 4. (a) SOCl2, MeOH (97%); (b) Mel, NaH, 18-Crown-6, 5 ꢀC (89%); (c) NaOH, MeOH (90%); (d) DEAD,
PPh3, FUDR, dioxane, rt (57%).

L. Hu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 797±800
799
Scheme 3. Chemical reduction of FUDR prodrug 4. (a) H2 balloon, 10% Pd/C, MeOH, h to overnight; (b) NaBH4, 10% Pd/C, MeOH-H2O, 20 min.
without the two methyl groups at the a-position of the
ester carbonyl or without the FUDR moiety (such as in
a methyl ester) showed the same stability in phosphate
bu€er, but were signi®cantly less stable than compound
4 in human serum (data not shown). We attribute this
unusual stability of the ester bond in 4 to the steric
hindrance provided by the two methyl groups a to the
ester carbonyl as well as the deoxy-ribose sugar ring in
FUDR.
calculation of a kinetic constant impossible. On the
other hand, in a derivative without the two methyl
groups, the cyclization process can be accurately mon-
itored by observing the change in absorbance at 249 nm
and has a calculated half life of 14 min at 37 ꢀC (data
not shown). These results suggest that the two methyl
groups at the a-position of the ester carbonyl serve to
restrict the rotational freedom of the conformation of
the molecule and facilitate the intramolecular cycliza-
tion process as proposed in Scheme 1.
In summary, 50-[2-(2-nitrophenyl)-2-methylpropionyl]-
20-deoxy-5-¯uorouridine (4) was synthesized as a poten-
tial prodrug of FUDR to target hypoxic tumor cells
or to be activated by a nitroreductase. Compound 4
is stable and resists the hydrolysis by human serum
esterases present in the blood. The release of FUDR
from 4 is very fast after conversion of the nitro group to
the amino group. Compound 4 could potentially be
used to treat hypoxic solid tumors or used in combina-
tion with a nitroreductase in antibody-directed or gene-
directed enzyme prodrug therapy to improve the phy-
sico-chemical properties and the therapeutic e€ective-
ness of FUDR in the treatment of cancer. Further work
is underway to study its cytotoxicity towards hypoxic
cancer cells or cancer cells in the presence of a nitror-
eductase and NAD(P)H.
Chemical Reduction
To test the feasibility of reductive release of the anti-
cancer drug FUDR from compound 4, we selected two
mild chemical reduction conditions that mimick the
bioreduction in hypoxic tumor cells and the enzymatic
action of a nitroreductase. One was hydrogenation
under normal atmospheric pressure in the presence of
10% Pd/C and the other was sodium borohydride
(NaBH4) reduction in the presence of 10% Pd/C.24 Both
reduction conditions were mild and neutral; and both
would allow selective reduction of the nitro group
without a€ecting other functional groups in the mole-
cule. Both of these chemical reduction methods would
only convert the nitro group to the ®nal amino group.
Bioreduction in hypoxic tumor cells or reduction by a
nitroreductase would more likely stop at the inter-
mediate hydroxylamine before reaching the ®nal amino
product. Kinetic studies have shown that cyclization of
2-nitroarylamides via the hydroxylamine intermediate is
actually faster than that via the amino product.25,26
Thus, the hydrogenation and NaBH4 reduction reactions
are simple, practical chemical tests of the cyclization-
activation system.
Acknowledgements
Part of this work was done at the University of Okla-
homa College of Pharmacy. This research was sup-
ported in part by an OCAST health research grant
(H97-013) and a grant from the US Army Department
of Defense Prostate Cancer Research Program (DAMD
17-98-1-8544). We thank Dr. Larry M. Russon (OU
Mass Spectrometry Laboratory) for providing mass
spectrometry data.
Hydrogenation was found to be slower than sodium
borohydride reduction. The former required hours of
incubation to complete the reaction while the latter took
only 20 min to ®nish. The only isolated products under
both reduction conditions were the lactam 6b and the
parent drug 1 (Scheme 3). Both products were identi®ed
References and Notes
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directly after partial hydrogenation (4 min) to test the
cyclization process in pH 7.4 phosphate bu€er at 37 ꢀC,
the cyclization was so fast that within seconds the
absorbance at 249 nm became constant, making the
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