Potential tumor-selective nitroimidazolylmethyluracil prodrug derivatives: Inhibitors of the angiogenic enzyme thymidine phosphorylase

Article abstract of DOI:10.1021/jm020964w

Thymidine phosphorylase (TP) is an angiogenic growth factor and a target for anticancer drug design. Molecular modeling suggested that 2′-aminoimidazolylmethyluracils would be potent inhibitors of TP. The novel 5-halo-2-amino-imidazolylmethyluracils (4b/4c) were very potent inhibitors of E. coli TP (IC₅₀ ～ 20 nM). Contrastingly, the corresponding 2′-nitroimidazolylmethyluracil (as bioreductively activated) prodrugs (3b/3c) were 1000-fold less active (IC₅₀ 22-24 μM. This approach may be used to selectively deliver TP inhibitors into hypoxic regions of solid tumors where TP is overexpressed.

Full text of DOI:10.1021/jm020964w

J . Med. Chem. 2003, 46, 207-209
207
P oten tia l Tu m or -Selective
Nitr oim id a zolylm eth ylu r a cil P r od r u g
Der iva tives: In h ibitor s of th e An giogen ic
En zym e Th ym id in e P h osp h or yla se
Christian Cole, Philip Reigan, Abdul Gbaj,
Philip N. Edwards, Kenneth T. Douglas,
Ian J . Stratford, Sally Freeman, and
Mohammed J affar*
F igu r e 1. Known TP inhibitors.
As TP is expressed at high levels in platelets and
other normal tissues, such as the brain, there would be
substantial advantage in selectively inhibiting TP in the
tumors where it is generating its angiogenic effects by
School of Pharmacy & Pharmaceutical Sciences, University
of Manchester, Oxford Road, Manchester M13 9PL, UK
1
3
promoting tumor growth. Here, we report the design
and synthesis of novel tumor-activated TP prodrugs that
are likely to meet this criterion. From a homology model
of human TP based on the Escherichia coli structure
Received J une 14, 2002
Abstr a ct: Thymidine phosphorylase (TP) is an angiogenic
growth factor and a target for anticancer drug design. Molec-
ular modeling suggested that 2′-aminoimidazolylmethyluracils
would be potent inhibitors of TP. The novel 5-halo-2-amino-
imidazolylmethyluracils (4b/4c) were very potent inhibitors
of E. coli TP (IC50 ∼ 20 nM). Contrastingly, the corresponding
(in the open conformation), we identified important
residues such as Ser 217, Asp 209, and Leu 148 in the
cavity where thymine/thymidine is presumed to bind.
14
Nitroimidazolylmethyluracil analogues (3, as potentially
hypoxia-mediated bioreductively activated TP inhibi-
tors) and their corresponding amino derivatives (4) have
been synthesized. The presence of hypoxia in tumors
will cause bioreductive “activation” of the nitro prodrug
to form the active amino species (Table 1) in areas of
the tumor where TP is most highly expressed. The
activation mechanism in hypoxia is catalyzed by reduc-
tive enzymes such as cytochrome P450 reductase. There
are many examples in the literature of bioreductive
activation of a nitro prodrug to the active amino
derivative for selective delivery of cytotoxic agents,
2
′-nitroimidazolylmethyluracil (as bioreductively activated)
prodrugs (3b/3c) were 1000-fold less active (IC50 22-24 µM).
This approach may be used to selectively deliver TP inhibitors
into hypoxic regions of solid tumors where TP is overexpressed.
In tr od u ction . An essential stage in the growth and
metastasis of solid tumors is the development of new
blood vessels (angiogenesis). Platelet-derived endothelial
cell growth factor (PD-ECGF) has been implicated in a
variety of angiogenic effects by promoting endothelial
1
cell proliferation in a range of tumor cell types. PD-
1
5-17
ECGF is identical to the enzyme thymidine phospho-
providing confidence in such an approach.
2
rylase (TP, dThdPase, EC 2.4.2.4). TP catalyses the
The compounds were designed using molecular mod-
eling by quantitative docking studies on the proposed
compounds, and their energies were calculated in the
docked states. To prove the principle that TP can
discriminate between the nitro and amino analogues,
the compounds were synthesized and evaluated for
inhibition of recombinant purified E. coli TP (which
shows a 69% sequence similarity to the active site of
human TP) (purchased from Sigma Chemical Company,
reversible phosphorylation of thymidine to thymine and
2
-deoxyribose-1-phosphate (see Supporting Informa-
tion), and it is proposed that the dephosphorylated
-deoxyribose is responsible for the angiogenic stimulus
2
3
of TP.
TP/PD-ECGF has chemotactic activity in vitro, and
angiogenic effects in vivo, and its expression is an
adverse prognostic indicator in breast, bladder, ovarian,
4
and colorectal tumors. Griffiths and colleagues showed
14
UK). The experimental IC50 values were compared
that TP/PD-ECGF is regulated by hypoxia and is focally
expressed in the hypoxic regions of solid tumors. Thus,
the hypoxic up-regulation of TP in many tumors and
its angiogenic activity makes TP an attractive target
for cancer chemotherapy.6 By inhibiting TP, tumor
growth can be inhibited.
with those of the known inhibitors 6A5BU and TPI.
Molecu la r Mod elin g. As previously described, the
known inhibitors 6A5BU (1) and TPI (2) showed good
positioning within the active site of TP, with several
H-bonds formed to active site residues. In particular,
interactions with Ser 217, Leu 148, and Asp 209
appeared important for achieving low binding energy
5
The development of TP inhibitors has primarily
centered on uracil-type analogues with various substit-
14
by 2.
7
-10
uents at the C-5 and/or C-6 positions.
The 6-amino-
The amino compounds 4 (X ) H, Cl, Br) were designed
from docking studies on the E. coli enzyme and a human
5
-bromouracil (1, 6A5BU) is commonly used as the
1
1
benchmark, and the potent inhibitor 5-chloro-6-[(2-
iminopyrrolidin-1-yl)methyl]uracil hydrochloride (2, TPI)
caused a substantial reduction in tumor growth rate
when given continuously to mice carrying experimental
tumors (for structures, see Figure 1).12
14
similarity model. The Autodock energies for the bound
amino derivatives revealed that they were more highly
stabilized than their corresponding nitro prodrug de-
rivatives 3 (X ) H, Cl, Br) within the active site of TP.
The most highly populated binding conformation of 4c
generated by Autodock (see Figure 2) had several
H-bonds with active-site residues including Ser 217 and
Asp 209, which was similar to TP I. The docking
*
To whom correspondence should be addressed. Tel: +44-(0)161-
75-2429. Fax: +44-(0)161-275-2396. E-mail: mohammed.jaffar@
man.ac.uk.
2
1
0.1021/jm020964w CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/12/2002

2
08 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Letters
the 5-substituted-6-[(2-nitroimidazol-1-yl)methyl]uracil
derivatives (3a ; X ) H, 3b; X ) Cl, 3c; X ) Br) in
reasonable yields. Reduction of the nitro derivatives
with NaBH4/Pd gave the desired 6-[(2-aminoimidazol-
1
4
-yl)methyl]uracil conjugates (4a ; X ) H, 4b; X ) Cl,
c; X ) Br) in good yields. Alternatively, the amino
derivatives were synthesized from the appropriate
5
2
-substituted-6-(chloromethyl)uracils by coupling with
-aminoimidazole. TPI and 6A5BU were synthesized
7
,18
using literature methods.
Eva lu a tion a s TP In h ibitor s. The TP inhibition
assay was carried out using a continuous spectropho-
tometric assay adapted for a 96-well plate Molecular
Devices Spectromax instrument at 355 nm with 5-nitro-
2
′-deoxyuridine (0.13 mM) as the substrate in the
presence of the enzyme (45 nM, 25 °C). The IC50 values
Table 1) were calculated as the concentration at which
0% reduction of the initial rate (V0) was attained (see
(
5
1
9
Supporting Information). The inhibition selectivity
ratio (ISR) is obtained by dividing the IC50 of a nitro
compound by those for the corresponding amino com-
pound.
F igu r e 2. Binding mode of 5-bromo-6-[(2-aminoimidazol-1-
yl)methyl]uracil (4c) in the active site of human TP.
Sch em e 1. Synthesis of Nitro- and Aminoimidazolyl-
uracil Conjugatesa
Ta ble 1. TP Inhibition Data
compound
TPI
X
IC50 (µM)
ISRa
-
0.02 ( 0.002
1.60 ( 0.17
1.60 ( 0.08
0.56 ( 0.02
21.7 ( 1.2
0.021 ( 0.014
24.4 ( 1.0
0.019 ( 0.002
-
-
2.8
6
3
4
3
4
3
4
A5BU
-
a
a
b
b
c
c
H
H
Cl
Cl
Br
Br
1033
1284
a
ISR is the inhibition selectivity ratio (IC50 nitro/IC50 amino).
a
Reagents and conditions: (i) NCS or NBS, AcOH, 60 °C; (ii)
The results in Table 1 show that the 5-halo-6-[(2-
aminoimidazol-1-yl)methyl]uracils (4b; X ) Cl 4c; X )
Br) were significantly (100-fold) more potent than
1
-potassio-2-nitroimidazole, DMF, rt, N2, 24 h; (iii) 1-potassio-2-
nitroimidazole, DMF, 75 °C, N2, 2 h; (iv) NaBH4, Pd/C, MeOH/
H2O, N2, 24 h; (v) 2-aminoimidazole sulfate, NaOEt, DMF, rt,
2
6 h.
6
A5BU, and were as potent as TPI. The very strong
inhibition by 4b, 4c, and TP I is noteworthy, and our
preliminary analysis indicates tight-binding inhibition,
so that for these compounds with E. coli TP, the IC50
values are limited by the enzyme concentration. More
importantly, in all cases the 2-aminoimidazolyl com-
pounds 4 were markedly more potent than their nitro
congeners 3. For the 5-halo analogues, there was at least
a 1000-fold increase in potency for the amino derivatives
(4b; X ) Cl and 4c; X ) Br) compared with their nitro
analogues (3b; X ) Cl and 3c; X ) Br). Thus, under
bioreductive conditions, the nitro compounds could act
as prodrugs to their amino counterparts. The 5-unsub-
stituted aminoimidazolyl conjugate (4a) showed a marked
decrease in potency compared to the 5-halo conjugates.
This may be due either to the fact that bulky substit-
uents at the C-5 position results in the imidazolyl ring
adopting a favorable conformation in the active site of
TP, or to an electronic effect. The presence of the amino
group enhances potency further, possibly due to hydro-
gen bonding with Asp 209 (this interaction is absent for
the nitro derivatives) (see Figure 2).
energies for TP I and 4c (X ) Br) for the human enzyme
-
1
were also similar (-47.90 and -45.96 kcal mol
,
respectively). Thus, on the basis of the active site
structures of either the E. coli or human TP, it was
predicted that the enzyme would strongly discriminate
the nitro from the amino forms of the appropriate
imidazolyl-substituted uracil derivatives (3 and 4).
The modeling studies also demonstrated that TP has
an affinity for an amino group four bond-lengths from
the 6-position of uracil, which can mimic an oxycarbe-
nium ion transition-state. The conformational restric-
tion afforded by a ring system aids the binding of the
amino-substituent to active site residues (Figure 2).
Ch em ica l Syn th esis. The nitrouracil prodrugs were
synthesized from 6-(chloromethyl)uracil (5) (Scheme 1).
Reaction of 5 with either N-chlorosuccinimide (NCS) or
N-bromosuccinimide (NBS) gave the desired 5-halo-6-
(
chloromethyl)uracil derivatives (6b and 6c, respec-
1
0
tively). The 6-(chloromethyl)uracils (5, 6b, and 6c)
were coupled with 1-potassio-2-nitroimidazole to give

Letters
Con clu sion . Docking studies of the modeled TP
predicted that the binding of the amino derivatives was
energetically more favored than that of their corre-
sponding nitro counterparts. This has been confirmed
experimentally for the E. coli TP enzyme. For the
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 209
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1
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(
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Ack n ow led gm en t. This work was funded, in part,
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(
Su p p or tin g In for m a tion Ava ila ble: The H NMR, 13C
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