Synthesis and enzymatic evaluation of xanthine oxidase-activated prodrugs based on inhibitors of thymidine phosphorylase

Article abstract of DOI:10.1016/j.bmcl.2004.08.036

A series of xanthine oxidase-activated prodrugs of known inhibitors of thymidine phosphorylase are described. These prodrugs were oxidised by xanthine oxidase at C-2 and/or C-4 of the uracil ring to generate the desired TP inhibitor. The scheme shows the prodrug of TPI. A series of xanthine oxidase-activated prodrugs of known inhibitors of thymidine phosphorylase has been designed and synthesised to introduce tumour selectivity. These prodrugs were oxidised by xanthine oxidase at C-2 and/or C-4 of the uracil ring to generate the desired TP inhibitor.

Full text of DOI:10.1016/j.bmcl.2004.08.036

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5247–5250
Synthesis and enzymatic evaluation of xanthine oxidase-activated
prodrugs based on inhibitors of thymidine phosphorylase
Philip Reigan, Abdul Gbaj, Edwin Chinje, Ian J. Stratford,
*
Kenneth T. Douglasand Sally Freeman
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK
Received 25 June 2004; revised 16 August 2004; accepted 17 August 2004
Available online 17 September 2004
Abstract—A series of xanthine oxidase-activated prodrugs of known inhibitors of thymidine phosphorylase has been designed and
synthesised to introduce tumour selectivity. These prodrugs were oxidised by xanthine oxidase at C-2 and/or C-4 of the uracil ring to
generate the desired TP inhibitor.
Ó 2004 Elsevier Ltd. All rights reserved.
Thymidine phosphorylase (TP, EC 2.4.2.4) catalyses the
reversible phosphorolysis of thymidine (1) to thymine
(2) and a-D-2-deoxyribose-1-phosphate (3, Scheme 1).
The dephosphorylated sugar, D-2-deoxyribose, has been
shown to have chemotactic activity in vitro and angio-
genic activity in vivo1,2 TP iselevated in several hypoxic
tumours, promoting both angiogenesis and metastasis,
and suppressing apoptosis.3 TP isalso known asplate-
let-derived endothelial cell growth factor (PD-ECGF).4
TP is distinct from other angiogenic growth factors,
since it exertsitsactionsthrough itsenzymatic activity.
the design of more potent inhibitors.6 7-Deazaxanthine
(5, 7-DX) was the first purine derivative to display inhi-
bition of both E.coli TP and angiogenesis in chorioal-
lantoic membrane.7 One of the most potent inhibitors
is5-chloro-6-[(2-iminopyrrolidin-1-yl)methyl]-uracil hy dro-
chloride (6, TPI), which causes a reduction of tumour
growth, promotes apoptosis and inhibits angiogenesis.8
TPI in combination with the anti-tumour agent 20-
deoxy-(5-trifluoromethyl)uridine iscurrently in phase 1
clinical studies (Fig. 1).9
Since TP is expressed at high levels in human blood
platelets and normal tissues (e.g., ovaries, salivary
glands, brain), there is an advantage in selectively inhib-
iting TP at the site of the tumour.
Inhibitorsof human TP are of interest in cancer chemo-
therapy.5 The ÔclassicalÕ inhibitor 6-amino-5-bromoura-
cil (4, 6A5BU) istypically uesd asthe benchmark for
Scheme 1. The reaction catalysed by TP and proposed transition state.
Keywords: Thymidine phosphorylase; Xanthine oxidase; Prodrug; Inhibitor.
*
Corresponding author. Tel.: +44 (0)161 275 2366; fax: +44 (0)161 275 2396; e-mail: sally.freeman@man.ac.uk
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.036

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P.Reigan et al./ Bioorg.Med.Chem.Lett.14 (2004) 5247–5250
Figure 1. Known TP inhibitors.
Scheme 2. The enzymatic activity of XO.
Scheme 3. Synthesis of XO-activated prodrugs of 6A5BU (10–12).
Xanthine oxidase (XO, EC 1.1.3.22) catalyses the oxida-
tion of hypoxanthine (7) and xanthine (8) to uric acid (9,
Scheme 2). XO-mediated prodrug activation hasbeen
used to increase the bioavailability, solubility and
selectivity of a number of antivirals.10,11 XO exhibits
broad substrate specificity12 the known TP inhibitors
being uracil-based heterocycles are attractive targets for
the XO-activated prodrug strategy. Furthermore, both
TP and XO are expressed at high levels in hypoxic tis-
sue,13,14 breast,15 colorectal14,16 and brain tumours.17,18
Stille coupling21 of 11 with Z-1-ethoxy-2-(tributylstan-
nyl)ethane followed by deprotection of the resulting enol
ether with concomitant cyclisation gave the novel XO
prodrug 14.
The novel XO-activated prodrug of TPI, 5-chloro-6-[(2-
iminopyrrolidin-1-yl)methyl]-3H-pyrimidin-4-one hydro-
chloride (16, Scheme 5) was synthesised by radical
halogenation22 of 5-chloro-6-methyl-3H-pyrimidin-4-
one, using N-chlorosuccinimide/benzoyl peroxide, with
subsequent coupling to 2-iminopyrrolidine hydrochlo-
ride (Scheme 5).
Here, we report the design, synthesis and enzymatic
evaluation of a series of potential tumour-selective
XO-activated prodrugsof the known TP inhibitors
6A5BU (4), 7-DX (5) and TPI (6). These prodrugs lack
the carbonyl substitution at C-2 and/or C-4 of the uracil
ring.
The TP inhibition assay, using purified recombinant E.
coli TP (69% sequence similarity to human TP), was
accomplished using a continuous spectrophotometric
assay,23 adapted for a spectrophotometer with a 96-well
microplate reader, at 355nm and 25°C. The reaction
mixture (0.2mL) contained 0.13mM 5-nitro-20-deoxy-
uridine as the substrate in potassium phosphate buffer
(0.1M, pH7.4). Reaction wasinitiated by addition of
TP (0.00883U/0.2mL). The IC50 valuesfor both the
TP inhibitorsand their XO-activated prodrugsare given
in Table 1. Asanticipated and required, the parent TP
inhibitors( 4–6) were significantly more potent TP
inhibitorsthan their correpsonding prodrug.s Indeed,
prodrugs 10–16 showed no inhibition of E.coli TP even
at high concentrations, suggesting that C-2 and C-4 car-
bonyl substitution of the uracil ring moiety is essential
for TP inhibition.
The 6-amino-5-bromopyrimidine series of XO-activated
prodrugsof 6A5BU ( 10–12) was synthesised by electro-
philic substitution of the appropriate 6-aminopyrim-
idine with molecular bromine (Scheme 3).19
The 7H-pyrrolo[2,3-d]pyrimidine series of XO-activated
prodrugsof 7-DX ( 13–15) was synthesised as outlined in
Scheme 4. Prodrug 13 wasprepared via cyclocondensa-
tion, followed by acid-catalysed hydrolysis of the acetal
and cyclisation and finally desulfurisation with Raney
Nickel.20 The deoxygenation of 13 via the chlorinated
intermediate afforded prodrug 15.20 Palladium-catalysed

P.Reigan et al./ Bioorg.Med.Chem.Lett.14 (2004) 5247–5250
5249
Scheme 4. Synthesis of XO-activated prodrugs of 7-DX (13–15).
Scheme 5. Synthesis of XO-activated prodrug of TPI (16).
The kinetic constants (Km and Vmax) for the XO-activa-
tion of the prodrugswere determined using a continuous
spectrophotometric assay.25 The reaction mixture
(3.0mL) contained potassium phosphate buffer
(0.05M, pH7.5) and the prodrug substrates (25–
150lM) at 25°C; reaction wasinitiated by addition of
XO (0.9U/mL). Prodrug oxidation wasmonitoring at
a wavelength pre-determined for the appropriate TP
inhibitor. The XO substrate data are given in Table 1.
The 6-amino-5-bromopyrimidine prodrugs 10–12 were
good substrates for XO and the formation of 6A5BU
(4) was observed in each case. The 7H-pyrrolo[2,3-d]-
pyrimidines 13–15 were also good substrates for XO
10,25
yielding 7-DX (5) asthe oxidation product.
Simi-
larly prodrug 16 was selectively oxidised at C-2 by XO
to give TPI (6).
In summary, XO-activated prodrugs of three known TP
inhibitors have been investigated. In all cases, the TP
inhibitor wasa more potent inhibitor of E.coli TP, than
the corresponding prodrug. All prodrugs were oxidised
by XO at C-2 and/or C-4 of the uracil ring resulting in
the formation of the appropriate TP. The XO prodrug
approach providesan opportunity for the development
of tumour-selective inhibitors of TP.
Table 1. Inhibition data for E.coli TP for the TP inhibitors( 4–6) and
their prodrugs( 10–16), and bovine XO-mediated activation data for
prodrugs( 10–16)
Compound E.coli TP IC50 (lM)
Bovine XO
Acknowledgements
Km (lM) Vmax (mM/min)
4, 6A5BU
10
1.6
––
––
Thiswork wasfunded in part by the School of Phar-
macy, University of Manchester, the MRC and the Lib-
yan Government.
No inhibition at 148 23.8
No inhibition at 137 32.5
No inhibition at 213 44.4
1.25
1.20
0.98
––
11
12
5, 7-DX
13
14
6.5
––
No inhibition at 207 45.1
No inhibition at 148 29.8
No inhibition at 163 67.2
0.02a
No inhibition at 47.2 64.9
0.51
0.48
0.24
––
References and notes
15
6, TPI
16
––
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a Thisisan upper limit for IC
24
asthe inhibition isstoichiometric.
50

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