Synthesis and enzymatic evaluation of pyridinium-substituted uracil derivatives as novel inhibitors of thymidine phosphorylase.

Article abstract of DOI:10.1016/S0968-0896(01)00309-1

A series of water soluble N(1)- and C(6)-substituted uracil pyridinium compounds were prepared as potential inhibitors of thymidine phosphorylase (TP). The C(6)-uracil substituted derivatives were the most active. 1-[(5-Chloro-2,4-dihydroxypyrimidin-6-yl)methyl]pyridinium chloride, was identified as the best inhibitor being 5-fold more potent than the known inhibitor, 6-amino-5-bromouracil.

Full text of DOI:10.1016/S0968-0896(01)00309-1

Bioorganic & Medicinal Chemistry 10 (2002) 525–530
Synthesis and Enzymatic Evaluation of Pyridinium-Substituted
Uracil Derivatives as Novel Inhibitors of
Thymidine Phosphorylase
Paul E. Murray, Virginia A. McNally, Stacey D. Lockyer, Kaye J. Williams,
Ian J. Stratford, Mohammed Jaffar and Sally Freeman*
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK
Received21 March 2001; revised19 July 2001; accepted15 August 2001
Abstract—A series of water soluble N(1)- and C(6)-substituted uracil pyridinium compounds were prepared as potential inhibitors
of thymidine phosphorylase (TP). The C(6)-uracil substituted derivatives were the most active. 1-[(5-Chloro-2,4-dihydroxy-
pyrimidin-6-yl)methyl]pyridinium chloride, was identified as the best inhibitor being 5-fold more potent than the known inhi-
bitor, 6-amino-5-bromouracil. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
chemotactic stimulation of endothelial cell migration
from tissue surrounding the tumour.9 The precise
mechanisms are not fully understood but are thought to
involve either the down-regulation of extracellular thy-
midine levels, which are inhibitory to angiogenesis,10 or
via the production of angiogenic 2-deoxyribose (the
dephosphorylated form of 2-deoxyribose-1-phos-
phate).11 The role of TP in tumour development has led
to its identification as a potentially important chemo-
therapeutic target in the treatment of cancer.2,8 It has
been proposedthat specific inhibition of TP couldsig-
nificantly reduce tumour growth by disruption of
angiogenesis or metastasis andpromotion of apoptosis.
The growth andmetastasis of solidtumours is depen-
dent on the formation of new blood vessels (angiogen-
esis).1 Many solidtumours are able to promote
angiogenic signals via gene-expression pathways.2 This
leads to the production of angiogenic growth factors
such as platelet-derived endothelial cell growth factor
(PD-ECGF).3 Substantially increasedlevels of PD-
ECGF have been reportedwithin several tumour types
relative to surrounding non-neoplastic regions.4 In
addition, recent studies have shown that PD-ECGF
may also play a significant role in tumour metastasis5
andthe inhibition of hypoxia-inducedapoptosis within
certain tumours.6 Furukawa andco-workers have
demonstrated that PD-ECGF is identical to thymidine
phosphorylase (TP, dThdPase, EC 2.4.2.4).7
Early examples of TP inhibitors consist of simple 5/6-
12
substituteduracil derivatives,
the most potent being
6-amino-5-bromouracil (6A5BU).13 Recently, imino-
pyrrolidinyl-substituted uracils and multi-substrate
alkylphosphonate analogues have also been identified as
TP inhibitors.8,14,15 In this paper we wish to report the
syntheses of a novel series of bicyclic pyridinium-sub-
stituteduracil andthymine analogues andtheir in vitro
TP Escherichia coli inhibitory activity.16
TP is an important enzyme involvedin the biochemical
catabolic/salvage pathway of pyrimidine-based 2-deoxy-
ribonucleosides. Its principal function is the reversible
phosphorolysis of thymidine to thymine and 2-deoxy-
ribose-1-phosphate (Scheme 1). The enzyme is also
known to activate 5-fluorouracil (5-FU) andits many
prodrugs, the first step in their metabolism to 5-fluoro-
20-deoxyribonucleotides which act as thymidylate syn-
thetase inhibitors.8 TP derives its angiogenic activity by
Results and Discussion
Chemistry
The compounds prepared were categorised into two
groups according to the position at which the pyri-
dinium group was substituted on the uracil/thymine
*Corresponding author. Tel.: +44-161-275-2366; fax: +44-161-275-
2396; e-mail: sally.freeman@man.ac.uk
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00309-1

526
P.E.Murray et al./ Bioorg.Med.Chem.10 (2002) 525–530
Scheme 1. The reversible phosphorolysis of thymidine catalysed by thymidine phosphorylase (TP).
heterocyclic ring. They were either C(6)-substituted( 5–
7, 11–15 and 21) or N(1)-substitutedderivatives ( 25 and
27).
N(1)-Alkylpyridinium thymine analogues 25 and 27
were preparedby two different synthetic routes from
thymine 22 (Scheme 5). Here, various synthetic approa-
ches were undertaken in the preparation of N(1)-
methylpyridinium compound 25, but resultedin low
yields (>10%).25ꢀ28 An alternative synthetic route was
thus usedin which the N(1)-chloromethyl adduct 24 was
preparedin situ from 23,29 which was subsequently
quenched with pyridine to give the novel pyridinium
compound 25. The synthesis of N(1)-ethylpyridinium
derivative 2730 (Scheme 5) andwas achievedin two
steps via 1-(2-bromoethyl)thymine 26.31
Compounds 5–7, where the pyridinium group was
directly fused to the uracil ring, were prepared from
2,4,6-trichloropyrimidine 1 (Scheme 2). 6-Chlorouracil
2 was preparedby base-catalysedhyrdolysis of
117.
Initial attempts to synthesise pyridinium 5 devised by
Schmidt and co-workers18 using pyridine in chlorobenzene
resultedin low yields. However, the omission of chloro-
benzene and the use of pyridine as solvent afforded 5 in
moderate yield (52%). Ring halogenation of 2 using
either NCS/AcOH19 or bromine water20 gave 5-halo
substitutedderivatives 3 and 4, respectively. By heating
at reflux in pyridine, these compounds were converted
to the novel pyridinium analogues 6 and 7.
Evaluation as inhibitors of TP
All pyridinium compounds prepared were tested for
their TP inhibitory activity against the known inhibitor
6-amino-5-bromouracil (6A5BU)13 using a modification
of a standard TP assay described by Evrard and co-
workers (Table 1).32 The IC50 values were obtained
using Escherichia coli TP (which shows 34% homology
in their amino acidsequence, and69% homology of the
active site of human TP).33 The data show that the
N(1)-alkyl-substitutedthymine analogues ( 25 and 27)
Compound 11, the first in a series of methylene bridged
bicyclic compounds, was prepared from 6-(chloro-
methyl)uracil 8 andneat pyridine at room temperature
(Scheme 3).21 Novel C(6)-methylenepyridinium deriva-
tives (12–15) were preparedby ring halogenation of
compound 8 using either NCS/AcOH18 to give chloro
compound 9,22 or NBS/AcOH to give the novel bromo
adduct 10. Compounds 12 and 13 were synthesisedby
reacting 9 or 10 with neat pyridine at room temperature,
whereas the aminopyridinium compounds 14 and 15
were preparedby reacting 9 with 2- and3-aminopyr-
idine, respectively, in refluxing chlorobenzene.
The thymine derivative 21 was preparedfrom 5,6-dime-
thylpyrimidine-2,4-dione 16 (Scheme 4). The first step
involvedselective oxidation of the 6-methyl group to the
corresponding aldehyde 17, followedby reduction with
Scheme 3. Synthesis of methyleneperidinium derivatives 11–15.
Reagents andconditions: (a) NCS, AcOH, 60 ꢁC (82%); (b) NBS,
AcOH, 60 ꢁC (71%).
NaBH4 to give the hydroxymethyl compound 18 in
23,24
excellent yield(85%).
Ring andside-chain chlor-
ination in refluxing POCl3 gave 19, which upon acid-
catalysed hydrolysis afforded the chloromethyl com-
pound 2024 in goodyield. Nucleophilic substitution of
the chloro group of 20 with neat pyridine at room tem-
perature gave the pyridinium compound 21.
Scheme 4. Synthesis of methylenepyridinium derivative 21. Reagents
andconditions: (a) SeO 2, AcOH, Á (70%); (b) NaBH4, rt (85%); (c)
POCl3, Á (91%); (d) concd HCl, MeOH, Á (74%); (e) pyridine, rt
(43%).
Scheme 2. Synthesis of pyridinium derivatives 5–7. Reagents and
conditions: (a) NaOH (aq), Á; (b) concdHCl, rt (74%); (c) pyridine,
Á; (d) NCS, AcOH, Á (40%), (e) Br2, H2O, 60 ꢁC (71%).

P.E.Murray et al./ Bioorg.Med.Chem.10 (2002) 525–530
527
Scheme 5. Synthesis of thymine analogues 25 and 27. Reagents andconditions: (a) (Me 3Si)2NH, Me3SiCl, Á; (b) CH3SCH2Cl, CH3CN, Á (67%);
(c) SO2Cl2, DCM, ꢀ78 ꢁC; (d) pyridine, rt; (e) BrCH2CH2Br, DMF, 80 ꢁC (28%).
andthe 6-pyridinium substitutedderivatives ( 5–7) were
not as active as the reference inhibitor 6A5BU. How-
ever, with the exception of the 5-unsubstituteduracil
analogue 11, the 6-methylenepyridinium compounds
were more active than 6A5BU. The most active com-
pound 12 displays a 5-fold increase in potency over
6A5BU. Derivatives 12–15 and 21 containing the addi-
tional monomethylene bridging unit between the hetero-
cyclic rings were the most potent in the series. This unit
will increase molecular flexibility allowing the inhibitor
to bindto residues within the active site of TP (in con-
trast, 5–7 exist as planar compounds). In addition, the
above observations seem to support the fact that the
uracil N(1) group may be important for binding within
Halogen substitution at the 5-position of the uracil ring
also confers significant inhibitory activity, with 12–15
being more active than 11 (an observation which agrees
with other literature reports).8,12ꢀ14 It has been sug-
gested that this may be due to the increased acidity of
the uracil N(1)H group within these compounds as a
result of the halogen’s electron-withdrawing cap-
ability—increasing the polarity of this group may
strengthen hydrogen bonding of uracil with residues in
the active site.12a This observation, coupledwith the fact
that thymine derivative 21 is at least twice as potent as
uracil derivative 11, may also support the proposed
existence of lipophilic interactions of the inhibitor with
the hydrophobic region of the pyrimidine-binding
pocket.33,35
the enzyme’s active site33 andthat the best TP inhibitors
8,12ꢀ14,34
are the 6-substitituteduracil analogues.
More-
over, amino substitution of the pyridinium ring in com-
pounds 14 and 15 resultedin a 4-foldincrease in
potency.
Conclusion
A series of water soluble N(1)- andC(6)-substituted
uracil pyridinium compounds have been prepared, the
most potent of which are the novel 6-methylenepyri-
dinium derivatives 12–15 and 21. These compounds
represent the only heteroaromatic ring substitutedura-
cil-basedinhibitors of thymidine phosphorylase (TP). In
agreement with literature reports, structure–activity
relationships show that C(5)-halogeno substitution and
additional ‘side-group’ uracil substitution at the C(6)
position [vs N(1)-substitution] are favorable. The most
active pyridinium compound in the series was 1-[(5-
chloro-2,4-dihydroxypyrimidin-6-yl)methyl]pyridinium
chloride 12.
Table 1. Inhibition of TP by pyridinium-substituted uracil analogues
and 6A5BU
1
CompoundType
R
R2
n
IC50 (mM)a
5
6
7
A
A
A
A
A
A
A
A
A
B
H
Cl
Br
H
Cl
Br
Cl
H
H
H
H
H
0
0
0
1
1
1
1
1
1
>1
>1
>1
>1
0.23
0.50
0.50
0.30
0.50
>1
Experimental
11
12
13
14
15
21
25
27
6A5BU
General methods
H
The 1H and 13C NMR spectra were recorded on a JEOL
JNM-EX270 spectrometer at 270 and67.5 MHz,
respectively. Chemical shifts are expressedin ppm ( d).
The NMR spectra were obtained in deuterium oxide
(D2O) unless otherwise stated. Melting points were
determined using a Gallenkamp capillary tube appara-
tus andare uncorrected. Mass spectra andelemental
analyses were performedat the Department of Chem-
istry (University of Manchester). All solvents usedwere
2-NH2
3-NH2
H
—
—
Cl
CH3
CH3
CH3
—
1
2
—
B
—
>1
1.10b
—
aIC50 values represent the result of the means of three assay experi-
ments andare accurate to ꢂ10%.
8,13,15
bIC50 value obtainedwas approximately 30-foldgreater than that
reportedin the literature using different assay techniques.

528
P.E.Murray et al./ Bioorg.Med.Chem.10 (2002) 525–530
dried via standard procedures. The reactions were per-
formed under a dry nitrogen atmosphere.
1-[(2,4-Dihydroxypyrimidin-6-yl)methyl]pyridinium
chloride (11). 6-(Chloromethyl)uracil (0.10 g,
8
0.62 mmol) andpyridine (5 mL) was stirredfor 12 h at
room temperature. The cloudy suspension obtained was
concentratedin vacuo to give a gray–green solidwhich
was recrystallised(MeOH) to afford0.06 g (40%) of the
title compound 11. Mp 280–282 ꢁC (lit.21 280–282 ꢁC)
1H NMR d 5.62 (1H, s, uracil–CH), 5.73 (2H, s,
NCH2N+), 8.20 (2H, t, J=7.0 Hz, 2ꢃpyr–CH), 8.72
(1H, t, J=9.0 Hz, pyr–CH), 8.96 (2H, d, J=7.0 Hz,
2ꢃpyr–CHN+). MS (ES+) 204 (cation M+).
1-(2,4-Dihydroxypyrimidin-6-yl)pyridinium chloride (5).
6-Chlorouracil17 2 (0.50 g, 3.41 mmol) was dissolved in
pyridine (10 mL) and heated at reflux for 6 h. On cool-
ing, the orange solidformedwas filtered, washedwith
dichloromethane (30 mL) and dried. The product was
recrystallisedfrom methanol to ꢁgive 0.51 g (52%) of the
title compound 5. Mp >300 C (lit.18 >300 ꢁC). 1H
NMR d 6.27 (1H, s, uracil–CH), 8.33 (2H, t, J=7.1 Hz,
2ꢃpyr–CH), 8.89 (1H, t, J=8.4 Hz, pyr–CH), 9.26 (2H,
d, J=8.3 Hz, 2ꢃpyr–CHN+). MS (ES+) 190 (cation
M+).
1-[(5-Chloro-2,4-dihydroxypyrimidin-6-yl)methyl]pyridi-
nium chloride (12). 5-Chloro-6-(chloromethyl)uracil 9
(0.10 g, 0.51 mmol) andpyridine (5 mL) was stirredfor
24 h at room temperature. The cloudy suspension
obtainedwas concentratedin vacuo to give a white solid
which was recrystallised(MeOH) to afford0.12 g (86%)
of the title compound 12. Mp >300 ꢁC. 1H NMR d 5.89
(2H, s, NCH2N+), 8.18 (2H, t, J=7.1 Hz, 2ꢃpyr–CH),
8.69 (1H, t, J=9.1 Hz, pyr–CH), 8.97 (2H, d, J=6.9 Hz,
2ꢃpyr–CHN+); 13C NMR d 57.5, 110.9, 127.5, 142.0,
144.3, 147.0, 150.7, 161.3. MS (ES+) 238, 240 (cation
M+). Anal. calcdfor C 10H9Cl2N3O2: C, 43.82; H, 3.31;
N, 15.33; Cl, 25.87. Found: C, 43.80; H, 3.60; N, 15.05;
Cl, 25.90%.
1 - (5 - Chloro - 2,4 - dihydroxypyrimidin - 6- yl)pyridinium
chloride (6). 5,6-Dichlorouracil 3 was preparedin a
40% yieldfrom 6-chlorouracil 2 andNCS as described
by West andBarrett. 19 Compound 3 (0.10 g, 0.55 mmol)
was dissolved in pyridine (2 mL) and heated at reflux for
6 h. On cooling, the yellow solidformedwas filtered,
washed with dichloromethane (30 mL) and dried to give
0.07 g (41%) of the pyridinium compound 6. Mp
>300 ꢁC. H NMR d 8.35 (2H, t, J=7.1 Hz, 2ꢃpyr–
1
CH), 8.90 (1H, t, J=8.2 Hz, pyr–CH), 9.26 (2H, d,
J=8.4 Hz, 2ꢃpyr–CHN+); 13C NMR d 98.0, 127.6,
143.1, 148.4, 156.6, 157.5, 164.2. MS (ES+) 224, 226
(cation M+).
1-[(5-Bromo-2,4-dihydroxypyrimidin-6-yl)methyl]pyridi-
nium chloride (13). 5-Bromo-6-(chloromethyl)uracil 10
(0.10 g, 0.42 mmol) andpyridine (3 mL) was stirredfor
72 h at room temperature. The cloudy suspension
obtainedwas filter,ed washedwith chloroform
1 - (5 - Bromo - 2,4 - dihydroxypyrimidin - 6- yl)pyridinium
chloride (7). 5-Bromo-6-chlorouracil20
4
(0.20 g,
0.89 mmol) was dissolved in pyridine (5 mL) and heated
at reflux for 5.5 h. On cooling, the yellow solidformed
was filtered, washed with dichloromethane (30 mL) and
dried to afford 0.20 g (82%) of the title compound 7.
Mp >300 ꢁC. 1H NMR d 8.33 (2H, t, J=7.1 Hz,
2ꢃpyr–CH), 8.89 (1H, t, J=8.4 Hz, pyr–CH), 9.26 (2H,
d, J=8.3 Hz, 2ꢃpyr–CHN+); 13C NMR d 97.7, 127.7,
143.0, 148.2, 156.4, 157.3, 164.2. MS (ES+) 268, 270
(cation M+).
(25 mL) andrdiedto afford0.11 g (83%) of pyri-
dinium compound 13. Mp >300 ꢁC. 1H NMR d
5.80 (2H, s, NCH2N+), 8.17 (2H, t, J=6.8 Hz,
2ꢃpyr–CH), 8.65 (1H, t, J=8.7 Hz, pyr–CH), 8.94
(2H, d, J=5.9 Hz, 2ꢃpyr–CHN+); 13C NMR
d
57.7, 109.3, 17.5, 141.7, 144.7, 147.0, 151.3, 161.4.
MS (ES+) 282, 284 (cation M+). Anal. calcdfor
C10H9BrClN3O2: C, 37.70; H, 2.85; N, 13.19; Br, 25.08;
Cl, 11.13. Found: C, 38.07; H, 2.90; N, 13.03; Br, 24.99;
Cl, 10.96%.
5-Chloro-6-(chloromethyl)-2,4-dihydroxypyrimidine (9).
Compound 9 was preparedfrom 6-(chloromethyl)uracil
8 andNCS in 82% yield. Mp 272–273 ꢁC (lit.22 270–
2-Amino-1-[(5-chloro-2,4-dihydroxypyrimidin-6-yl)me-
thyl]pyridinium chloride (14). 5-Chloro-6-(chloro-
275 ꢁC). H NMR (DMSO-d6) d 4.49 (2H, s, CH2Cl),
1
11.60 (1H, s, NH), 11.75 (1H, s, NH).
methyl)uracil
9
(0.10 g,
0.51 mmol)
and2-
aminopyridine (0.25 g, 2.66 mmol) were suspended in
chlorobenzene (10 mL) andheatedat reflux for 48 h.
The solid obtained was filtered, washed with dichloro-
methane (30 mL) and dried. The crude solid was dis-
solvedin ethanol/water/concdHCl (8:2:1, 50 mL),
concentratedin vacuo andwashedwith hot methanol to
yield0.04 g (29%) of the title compound 14 as a black
5-Bromo-6-(chloromethyl)-2,4-dihydroxypyrimidine (10).
A solution of 6-(chloromethyl)uracil 8 (1.0 g, 6.2 mmol)
in glacial acetic acid (15 mL) and acetic anhydride
(1 mL) was heatedto 80 ꢁC for 30 min. The temperature
of the solution was loweredto 60 ꢁC and N-bromo-
succinimide (NBS, 1.4 g, 7.8 mmol) was added. The
reaction mixture was stirredat this temperature for 3 h.
On cooling, the suspension was pouredinto icedwater
(50 mL), andthe white solidwas filteredanddriedto
give 1.1 g (71%) of 10. Mp 260–261 ꢁC. 1H NMR
(DMSO-d6) d 4.47 (2H, s, CH2Br), 11.62 (1H, s,
NH), 11.68 (1H, s, NH); 13C NMR (DMSO-d6) d
41.4, 97.4, 149.0, 150.3, 160.3. MS (CI) 127, 161,
206, 238 (M+). Anal. calcdfor C 5H4BrClN2O2: C,
25.08; H, 1.68; N, 11.70. Found: C, 25.53; H, 1.52; N,
11.62%.
crystalline solid. Mp >300 ꢁC. H NMR (DMSO-d6) d
1
5.30 (2H, s, NCH2N+), 7.17 (1H, t, J=6.6 Hz, pyr–
CH), 7.44 (1H, d, J=8.9 Hz, pyr–CH), 8.00 (1H, t,
J=7.4 Hz, pyr–CH), 8.12 (1H, d, J=6.9 Hz, pyr–
CHN+), 11.40 (2H, s, NH2), 11.53 (1H, s, NH), 11.78
(1H, s, NH); 13C NMR (DMSO-d6) d 49.4, 107.3, 115.5,
116.2, 127.3, 139.7, 142.9, 146.6, 149.7, 157.5. MS
(ES+) 95, 217, 239, 253 (cation M+). Anal. calcdfor
C10H10Cl2N4O2: C, 41.54; H, 3.49; N, 19.38. Found: C,
41.38; H, 3.95; N, 20.37%.

P.E.Murray et al./ Bioorg.Med.Chem.10 (2002) 525–530
529
3-Amino-1-[(5-chloro-2,4-dihydroxypyrimidin-6-yl)me-
thyl]pyridinium chloride (15). 5-Chloro-6-(chloro-
methyl)uracil 9 (0.10 g, 0.51 mmol) and3-aminopyridine
(0.25 g, 2.66 mmol) were suspended in chlorobenzene
(10 mL) andheatedat reflux for 16 h. A brown solidwas
obtained which was filtered, washed with dichloro-
methane (30 mL) and dried. The solid was dissolved in
ethanol/water/concdHCl (8:2:1, 50 mL), concentrated
in vacuo andwashedwith hot methanol to yield0.07 g
(46%) of the title compoundas tan crystalline flakes.
Mp >300 ꢁC. 1H NMR (DMSO-d6) d 5.55 (2H, s,
NCH2N+), 7.70 (2H, m, 2ꢃpyr–CH), 8.06 (1H, s, pyr–
CHN+), 8.12 (1H, d, J=6.1 Hz, pyr–CHN+); 13C
NMR (DMSO-d6) d 57.7, 109.6, 127.4, 128.6, 129.0,
132.1, 142.6, 149.0, 150.8, 160.2. MS (ES+) 219, 253
(cation M+). Anal. calcdfor C 10H10Cl2N4O2: C, 41.54;
H, 3.49; N, 19.38. Found: C, 41.18; H, 3.75; N, 18.92%.
151.9, 166.4. MS (ES+) 218 (cation M+). Anal. calcd
for C11H12ClN3O2: C, 52.08; H, 4.77; N, 16.56; Cl,
13.98. Found: C, 51.75; H, 4.63; N, 16.22; Cl, 13.84%.
1-[(2,4-Dihydroxy-5-methylpyrimidin-1-yl)methylene]pyr-
idinium chloride (25). 1-(Methylthiomethyl)thymine27 23
(0.25 g, 1.34 mmol) was dissolved in dry dichloro-
methane (5 mL) at ꢀ78 ꢁC. Sulfuryl chloride (0.2 mL,
2.50 mmol) was added with stirring under a dry nitrogen
atmosphere. The white suspension was slowly warmed
to room temperature and dry pyridine (2 mL) was
added. The yellow solution obtained was stirred for a
further 24 h. The redsolution affordedwas concentrated
in vacuo to give a dark red gum. Addition of chloro-
form resultedin the precipitation of a white solidwhich
was filtered, washed with chloroform (25 mL) and dried
to yield0.14 g (42%) of the title compound 25. Mp 225–
227 ꢁC. 1H NMR d 1.92 (3H, s, CH3), 6.46 (2H, s,
NCH2N+), 7.81 (1H, s, thymine–CH), 8.17 (2H, t,
J=7.1 Hz, 2ꢃpyr–CH), 8.69 (1H, t, J=7.9 Hz, pyr–
CH), 9.07 (2H, d, J=5.9 Hz, 2ꢃpyr–CHN+); 13C NMR
d 10.9, 69.5, 112.4, 128.1, 139.9, 143.8, 147.6, 151.5,
166.0. MS (ES+) 139, 218 (cation M+). Anal. calcdfor
C11H12ClN3O2: C, 52.08; H, 4.77. Found: C, 47.40; H,
4.96%.
6-(Hydroxymethyl)-2,4-dihydroxy-5-methylpyrimidine
(18). Compound 18 was preparedby the reduction of
thymine-6-carboxaldehyde23 17 with NaBH4 in 85%
yield. Mp 225–226 ꢁC (lit.24 223–224 ꢁC). 1H NMR
(DMSO-d6) d 1.74 (3H, s, CH3), 4.24 (2H, d, J=5.3 Hz,
CH2O), 5.44 (1H, bs, OH), 10.20 (1H, bs, NH), 11.00
(1H, bs, NH).
6-(Chloromethyl)-2,4-dichloro-5-methylpyrimidine (19).
6-(Hydroxymethyl)-2,4-dihydroxy-5-methylpyrimidine 18
(0.25 g, 1.62 mmol) was suspended in phosphorus oxy-
chloride (5 mL) and heated at reflux under nitrogen for
12 h. The solution was cooledandconcentratedin
vacuo to yielda dark redoil. Icedwater (25 mL) was
slowly added and the product was extracted with
chloroform (3ꢃ25 mL). The organic phase was washed
with brine, dried (MgSO4), andevaporatedto give
1-[2-(2,4-Dihydroxy-5-methylpyrimidin-1-yl)ethyl]pyridi-
nium bromide (27). 1-(2-Bromoethyl)thymine31 26
(0.65 g, 0.28 mmol) was dissolved in pyridine (2 mL) and
stirredat room temperature for 48 h. The suspension
was filteredandoven-driedto afford0.43 g (49%) of the
title compound 27 as a white solid. Mp 298–300 ꢁC. H
1
NMR d 1.83 (3H, s, CH3), 4.39 (2H, t, J=5.6 Hz,
CH2N), 4.97 (2H, t, J=5.6 Hz, CH2N+), 7.37 (1H, s,
thymine–CH), 8.10 (2H, t, J=7.3 Hz, 2ꢃpyr–CH), 8.62
(1H, t, J=7.9 Hz, pyr–CH), 8.87 (2H, d, J=5.3 Hz,
2ꢃpyr–CHN+). MS (ES+) 153, 232 (cation M+). Anal.
calcdfor C 12H14BrN3O2: C, 46.17; H, 4.52; N, 13.46;
Br, 25.60. Found: C, 46.02; H, 4.71; N, 13.17; Br,
24.82%.
1
0.31 g (91%) of 19 as a yellow oil. H NMR (CDCl3) d
2.47 (3H, s, CH3), 4.59 (2H, s, CH2Cl); 13C NMR
(CDCl3) d 14.0, 43.1, 127.6, 157.0, 163.5, 166.0. MS (EI)
84, 139, 176, 210 (M+); AMM: 209.9517, C6H5Cl3N2
requires 209.9518.
6-(Chloromethyl)-2,4-dihydroxy-5-methylpyrimidine (20).
6-(Chloromethyl)-2,4-dichloro-5-methylpyrimidine 19
(1.1 g, 4.70 mmol) was dissolved in methanol (5 mL) and
concdhyrdochloric acid(1 mL). The mixture was
heatedat reflux for 2 h. The white solidformedwas fil-
tered, washed with dichloromethane (20 mL) and dried
to afford0.61 g (74%) of the title compound 20. Mp
TP Assays
Materials
E.coli TP (600 units/mL) was obtainedfrom the Sigma
Chemical Company. Spectrophotometric measurements
were obtainedusing a Beckman 650 spectrophometer.
ꢁ
ꢁ
1
244–245 C (lit.24 241–244 C). H NMR (DMSO-d6) d
1.82 (3H, s, CH3), 4.43 (2H, s, CH2Cl), 10.90 (1H, bs,
NH), 11.19 (1H, bs, NH).
Methods
1-[(2,4-Dihydroxy-5-methylpyrimidin-6-yl)methyl]pyridi-
nium chloride (21). 6-(Chloromethyl)-2,4-dihydroxy-5-
methylpyrimidine 20 (0.08 g, 0.46 mmol) andpyridine
(3 mL) was stirredfor 48 h at room temperature. The
creamy suspension obtainedwas concentratedin vacuo
to yielda tan solid. Recrystallisation (MeOH) afforded
0.06 g (40%) of the title compound 21. Mp >300 ꢁC. 1H
NMR d 2.03 (3H, s, CH3), 5.77 (2H, s, NCH2N+), 8.16
(2H, t, J=7.1 Hz, 2ꢃpyr–CH), 8.67 (1H, t, J=7.8 Hz,
pyr–CH), 8.88 (2H, d, J=5.9 Hz, 2ꢃpyr–CHN+); 13C
NMR d 9.1, 57.5, 113.4, 128.5, 139.9, 144.0, 146.9,
The compounds were prepared to the required final
concentrations in a 1:1 substrate buffer mixture of thy-
midine (10 mM) and potassium dihydrogen phosphate
(KH2PO4, 10 mM, pH=8.4). To each test sample 1.5
units of TP was added. The samples (final volu-
me=300 mL) were incubatedfor 1 h at 37 ꢁC, then
quenchedwith ice-cold0.5 M NaOH solution (700 mL).
Inhibitory activity was determined by measuring the
rate of thymidine to thymine conversion spectro-
photometrically at an absorbance level of 300 nm. Thy-
mine levels within the test samples (in nmoles/min) were

530
P.E.Murray et al./ Bioorg.Med.Chem.10 (2002) 525–530
obtainedby interpolation of the above readings via a
linear standard curve generated using 0.2–1.0 mM solu-
tions of thymine (Abs300 readings versus log[thymine]
values were plottedto the best fit polynomial). The IC 50
value was defined as the compound concentration
requiredto decrease the Abs 300 reading obtained using a
control sample of TP in substrate buffer alone by 50%.
Enzyme Inhibitors; J. Wiley: New York, 1967; p 79. (b) Baker,
B. R.; Kelley, J. L. J.Med.Chem. 1970, 13, 458, andrefer-
ences therein. (c) Niedzwicki, J. G.; El Kouni, M. H.; Chu,
S. H.; Cha, S. Biochem.Pharmacol. 1983, 32, 399.
13. Langen, P.; Etzold, G.; Barwolff, D.; Preussel, B. Bio-
¨
chem.Pharmacol. 1967, 16, 1833.
14. (a) Shigeto, M.; Nitanda, T.; Furukawa, T.; Sumizawa,
T.; Tani, A.; Nishimoto, K.; Akiba, S.; Miyadera, K.; Hara-
guchi, M.; Miyadera, K.; Yamada, Y.; Yoshida, H.; Kanzaki,
T.; Akiyama, S.-I. Cancer Res. 1999, 59, 1911. (b) Fukushima,
M.; Suzuki, N.; Emura, T.; Yano, S.; Kazuno, H.; Tada, Y.;
Yamada, Y.; Asao, T. Biochem.Pharmacol. 2000, 59, 1227. (c)
Takao, S.; Akiyama, S.; Nakajo, A.; Yoh, H.; Kitazono, M.;
Natsugoe, S.; Miyadera, K.; Fukushima, M.; Yamada, Y.;
Aikou, T. Cancer Res. 2000, 60, 5345.
Acknowledgements
We are grateful to the Association of International
Cancer Research (PEM) andthe Meidcal Research
Council (SDL) for financial support of this project.
PEM thanks the American College of Radiology for
Junior Investigator Award(11th International Chemical
Modifiers Conference—Tumor Physiology and Cancer
Treatment).
15. (a) Gamboa, A. E.; Balzarini, J.; Esnouf, R.; De Clercq,
E.; Camarasa, M.-J.; Pe
´
43, 971. (b) Balzarini, J.; Degre
rez-Pe
´
`
R.; De Clercq, E.; Engelborghs, Y.; Camarasa, M.-J.; Pe
rez, M.-J. J.Med.Chem. 2000,
ve, B.; Gamboa, A. E.; Esnouf,
´
rez-
Perez, M.-J. FEBS Lett. 2000, 483, 181.
´
16. Ishikawa, F.; Miyazono, K.; Hellman, U.; Drexler, H.;
Wernstedt, C.; Hagiwara, K.; Usuki, K.; Takaku, F.; Risau,
W.; Heldin, C.-H. Nature 1989, 338, 557.
References and Notes
17. Cresswell, R. M.; Wood, H. C. S. J.Chem.Soc. 1960, 4768.
18. Schmidt, A.; Kindermann, M. K.; Vainiotalo, P.; Nieger,
M. J.Org.Chem. 1999, 64, 9499.
19. West, R. A.; Barrett, H. W. J.Amer.Chem.Soc. 1954, 76,
3146.
20. Pfleiderer, W.; Deiss, H. Israel J.Chem. 1968, 6, 603.
21. Nagpal, K. L.; Jain, P. C.; Srivastava, P. C.; Dhar, M. M.;
Anand, N. Indian J.Chem. 1968, 6, 762.
1. (a) Baillie, C. T.; Winslet, M. C.; Bradley, N. J. Br.J.
Cancer 1995, 72, 257. (b) Battegay, E. J. J.Mol.Med. 1995,
73, 333.
2. Griffiths, L.; Stratford, I. J. Br.J.Cancer 1997, 76, 689.
3. (a) Miyazono, K.; Okabe, T.; Urabe, A.; Takaku, F.; Hei-
din, C. H. J.Biol.Chem. 1987, 262, 4098. (b) Brown, S. B.;
Bicknell, R. Biochem.J. 1998, 334, 1.
4. (a) Bicknell, R.; Moghaddam, A.; Zhang, H. T.; Harris,
A. L.; Hu, D. E.; Fan, T. P. Proc.Am.Assoc.Cancer.Res.
1994, 35, 567. (b) Griffiths, L.; Stratford, I. J. Int.J.Radiation
Oncology, Biol., Phys. 1998, 42, 877.
5. Takao, S.; Akiyama, S.; Nakajo, A.; Yoh, H.; Kitazano,
M.; Natsugoe, S.; Miyadera, K.; Fukushima, M.; Yamada, Y.;
Aikou, T. Cancer Res. 2000, 60, 5345.
6. Kitazano, M.; Takebayashi, Y.; Ishitsuka, K.; Takao, S.;
Tani, A.; Furukawa, T.; Miyadera, K.; Yamada, Y.; Aikou, T.;
Akiyama, S.-I. Biochem.Biophys.Res.Commun. 1998, 253, 797.
7. Furukawa, T.; Yoshimura, A.; Sumizawa, T.; Haraguchi,
M.; Akiyama, S.-I.; Fukui, K.; Ishizawa, M.; Yamada, Y.
Nature 1992, 356, 668.
22. Cornforth, J. W.; Huang, H. T. J.Chem.Soc 1948, 1988.
23. Zee-Cheng, K.-Y.; Cheng, C. C. J.Heterocycl.Chem.
1967, 4, 163.
24. Johnson, T. B.; Chernoff, L. H. J.Amer.Chem.Soc. 1913,
35, 585.
25. Kundu, N. G.; Khatri, S. G. Synthesis 1985, 323.
26. Ahmad, S.; Ozaki, S.; Nagase, T.; Iigo, M.; Tokuzen, R.;
Hoshi, A. Chem.Pharm.Bull. 1987, 35, 4137.
27. Zagorodnii, S. G.; Malyshev, A. A.; Konstantinova, I. D.;
Kuznetsov, S. A.; Miroshnikov, A. I. Russ.J.Bioorg.Chem.
(Engl.Transl). 1997, 23, 550.
28. Ozaki, S.; Nagase, T.; Tamai, H.; Mori, H.; Hoshi, A.;
Iigo, M. Chem.Pharm.Bull. 1987, 35, 3894.
8. Cole, C.; Foster, A. J.; Freeman, S.; Jaffar, M.; Murray,
P. E.; Stratford, I. J. Anti-Cancer Drug Des. 1999, 14, 383.
9. Moghaddam, A.; Zhang, H. T.; Fan, T. P.; Hu, D. E.;
Lees, V. C.; Turley, H.; Fox, S.; Gatter, K. C.; Harris, A. L.;
Bicknell, R. Proc. Natl. Acad. Sci. U.S.A. 1995, 998.
10. Finnis, C.; Dodsworth, N.; Pollitt, C. E.; Carr, G.; Sleep,
D. Eur.J.Biochem. 1993, 212, 201.
29. Edstrom, E. D.; Feng, X.; Tumkevicius, S. Tetrahedron
Lett. 1996, 37, 759.
30. Shimidzu, T.; Murakami, A.; Konishi, Y.; Minami, M.
Bull.Chem.Soc.Jpn. 1978, 5, 821.
31. Ciapetti, P.; Taddei, M. Tetrahedron 1998, 54, 11305.
32. Evrard, A.; Cuq, P.; Robert, B.; Vian, L.; Pe
Cano, J.-P. Int.J.Cancer 1999, 80, 465.
`
legrin, A.;
11. (a) Haraguchi, M.; Miyadera, K.; Uemera, K.; Sumizawa,
T.; Furukawa, T.; Yamada, K.; Akiyama, S.-I.; Yamada, Y.
Nature 1994, 368, 198. (b) Dada, M. A.; Boshoff, C. H.;
Comley, M. A.; Turley, H.; Schneider, J. W.; Chetty, R.;
Gatter, K. C. J.Clin.Pathol. 1996, 49, 400.
33. Cole, C.; Marks, D. S.; Jaffar, M.; Stratford, I. J.; Dou-
glas, K. T.; Freeman, S. Anti-Cancer Drug Des. 1999, 14, 411.
34. Focher, F.; Ubiali, D.; Pregnolato, M.; Zhi, C.; Gambino,
J.; Wright, G. E.; Spadari, S. J.Med.Chem. 2000, 43, 2601.
35. Erion, M. D.; Stoeckler, J. D.; Guida, W. C.; Walter,
R. L.; Ealick, S. E. Biochemistry 1997, 36, 11735.
12. (a) Baker, B. R. Design of Active-site-directed Irreversible