Discovery of 5-substituted-6-chlorouracils as efficient inhibitors of human thymidine phosphorylase

Article abstract of DOI:10.1021/jm070644i

Thymidine phosphorylase plays an important role in angiogenesis, which is an attractive target for therapy of cancer and other diseases. In our continuous effort to develop novel inhibitors of thymidine phosphorylase, we have discovered that 6-halouracils substituted at position C5 by certain hydrophobic groups exhibit significant inhibitory activity against this enzyme. The most potent compounds bear a five- or six-membered cyclic substituent containing a π-electron system at C5 and a chlorine atom attached at C6. 6-Chloro-5-cyclopent-1-en-1-yluracil 7a is the most efficient derivative in this study, with K_i = 0.20 ± 0.03 μM (K_i/ ^dThdK_m = 0.0017) for thymidine phosphorylase expressed in V79 cells and K_i = 0.29 ± 0.04 μM (K_i/ ^dThdK_m = 0.0024) for the enzyme purified from placenta.

Full text of DOI:10.1021/jm070644i

6016
J. Med. Chem. 2007, 50, 6016-6023
Discovery of 5-Substituted-6-chlorouracils as Efficient Inhibitors of Human Thymidine
Phosphorylase
Radim Nencka,* Ivan Votruba, Hubert Hˇrebabecky´, Petr Jansa, Eva Tlousˇt’ova´, Kveˇta Horska´, Milena Masoj´ıdkova´, and
Anton´ın Holy´
Gilead Sciences & IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Centre for New AntiVirals and Antineoplastics (IOCB), FlemingoVo na´m. 2, CZ-166 10, Prague 6, Czech Republic
ReceiVed June 5, 2007
Thymidine phosphorylase plays an important role in angiogenesis, which is an attractive target for therapy
of cancer and other diseases. In our continuous effort to develop novel inhibitors of thymidine phosphorylase,
we have discovered that 6-halouracils substituted at position C5 by certain hydrophobic groups exhibit
significant inhibitory activity against this enzyme. The most potent compounds bear a five- or six-membered
cyclic substituent containing a π-electron system at C5 and a chlorine atom attached at C6. 6-Chloro-5-
cyclopent-1-en-1-yluracil 7a is the most efficient derivative in this study, with Ki ) 0.20 ( 0.03 µM
(Ki/dThdKm ) 0.0017) for thymidine phosphorylase expressed in V79 cells and Ki ) 0.29 ( 0.04 µM
(Ki/dThdKm ) 0.0024) for the enzyme purified from placenta.
Introduction
2-deoxy-D-ribose and, thus, with tumor vascularisation or
progression of angioproliferative disorders.1,4 In addition, TP
seems to inhibit tumor cell apoptosis.5 Also, it is involved in
degradation of pyrimidine nucleoside analogue drugs such as
BVDU [(E)-5-(2-bromovinyl)-2′-deoxyuridine] and 2′-deoxy-
5-(trifluoromethyl)uridine.6 Therefore, the search for inhibitors
of human TP has attracted considerable attention in recent years.
Angiogenesis is the complex process in which new blood
vessels are formed from existing vasculature. The proliferation
of a network of blood vessels occurs physiologically during
embryogenesis, female menstrual cycle, and wound healing.
Nevertheless, angiogenesis is relatively infrequent event in a
healthy adult. Thus, increased angiogenic activity could be a
promising drug target for several pathological processes, includ-
ing solid tumor growth and metastasis, rheumatoid arthritis,
psoriasis, diabetic retinopathy, and hyperproliferation of vasa
vasorum in atherosclerosis. Thymidine phosphorylase (TP)a was
identified as one of the enzymes that play an important role in
this process.1-3
TP (identical to platelet-derived endothelial cell growth factor
PD-ECGF and gliostatin) was discovered 50 years ago as an
enzyme participating in the thymidine salvage pathway. TP
catalyzes the cleavage of thymidine into thymine and 2-deoxy-
D-ribose-1-phosphate, which is subsequently dephosphorylated
to give 2-deoxy-D-ribose. This simple monosaccharide has
recently been proved to possess significant chemotactic activity
for endothelial cells and to be one of the angiogenesis-inducing
factors. The expression of TP is considerably elevated in various
solid tumors, as well as in diverse chronic inflammatory
diseases, and corresponds roughly with the level of produced
In the past few years, numerous potential inhibitors of human
TP have been identified. Nevertheless, only few structural types
can be distinguished. The most potent inhibitors are uracil
derivatives bearing a small group at C5 (usually an atom of
chlorine or bromine) and an amino or methylene amino group
at C6.7-9 TPI [5-chloro-6-(2-iminopyrrolidin-1-yl)methyl-2,4-
(1H,3H)-pyrimidine] 1 (Figure 1) developed by Taiho Pharma-
ceutical Co. seems to be the most promising compound, so far.8
Other interesting TP inhibitor types include 7-deazaxanthine10
and 5′-O-tritylinosine11 analogues. Also, specific compounds
containing phosphonate group have shown significant inhibitory
activity against TP from physiological human tissues or rat SD-
lymphoma.12,13 However, none of the above-mentioned com-
pounds has passed clinical trials, yet.
The positive influence of a hydrophobic group on the
inhibitory activity of several uracil derivatives against TP has
been known for almost 40 years.14,15 On the other hand, the
hydrophobic substitution at C5 was underestimated and the main
attention focused on functionalization at C6 is probably due to
the fact that 5-phenyluracil and its homologues showed very
modest activity against mammalian TP in early studies.14
However, practically no attention was paid to the potency of
6-halouracils.
* Corresponding author. Phone: +420 220183323. Fax: +420 220183560.
E-mail: nencka@uochb.cas.cz.
a Abbreviations: %FA, fraction absorption; Ac, acetyl; BisTris, 2-(bis-
(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol; Bn, benzyl;
n-BuLi, n-butyllithium; BVDU, [(E)-5-(2-bromovinyl)-2′-deoxyuridine];
DMF, dimethylformamide; DTT, dithiothreitol; EDTA, ethylenediamine-
tetraacetic acid; Et, ethyl; Ki, dissociation equilibrium constant of the
inhibitor/enzyme complex; log D6.5, decimal logarithm of distribution
coefficient at pH 6.5; log P, decimal logarithm of partition coefficient; log
In our study, we report the synthesis and evaluation of a novel
class of human TP inhibitors based on 6-chlorouracil having a
hydrophobic substituent at C5. An exploration of other func-
tional groups at position C6 was also carried out.
S
cal, calculated decimal logarithm of solubility in mol/L; log Sexp, experi-
mentally determined decimal logarithm of solubility in mol/L; Pd(PPh3)4,
tetrakis(triphenylphosphine)palladium(0); PD-ECGF, platelet-derived en-
dothelial cell growth factor; Ph, phenyl; pKa, minus the decimal logarithm
of acid dissociation constants; QM, quantum mechanics; QSAR, quantitative
structure-activity relationship; SD-lymphoma, Sprague-Dawley rats lym-
phoma; THF, tetrahydrofuran; dTmdKm, dissociation equilibrium constant
of the thymidine/enzyme complex; TMS, trimethylsilane; TMSI, iodotri-
methylsilane; TP, thymidine phosphorylase; TPI, [5-chloro-6-(2-iminopy-
rrolidin-1-yl)methyl-2,4(1H,3H)-pyrimidine]; TPSA, topological polar sur-
face area.
Chemistry
The description of the synthesis of the title compounds was
categorized in accordance with whether the change of substituent
was accomplished at C5 or C6. The first part was dedicated to
10.1021/jm070644i CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/27/2007

Human Thymidine Phosphorylase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6017
chromatography and finally deprotected by concentrated hy-
drochloric acid in acetic acid to obtain 13a-c (Scheme 4).
The second part of our work was devoted to the 5-phenylu-
racil derivatives substituted at C6 by various groups. The uracil
bearing the phenyl group at C5 was chosen because of its high
stability under diverse chemical conditions and high inhibitory
activities against human TPs in the preliminary assay. The
6-methyl analogue was prepared from 5-bromo-2,4-dimethoxy-
6-methylpyrimidine.20 The transformation was started by halo-
gen-lithium exchange by n-butyllithium at -78 °C, followed
by lithium/zinc transmetalation at room temperature. Subsequent
Negishi cross-coupling with iodobenzene was carried out using
Pd(PPh3)4 as catalyst at 60 °C in THF.
The deprotection step accomplished by concentrated hydro-
chloric acid in THF/dioxane at reflux provided the desired
product in a good yield (Scheme 5). Our effort to synthesize
6-fluoro derivative by a route similar that used for preparation
of 6-chloro derivative was unsuccessful. Therefore, this product
was achieved in a rather classical way, as shown in Scheme 6.
In the first step of the sequence, 5-phenylbarbituric acid 17 was
chlorinated with POCl3 to afford 2,4,6-trichloro-5-phenylpyri-
midine 18 by the procedure introduced by Hendry and co-
workers.21 Further fluorination was performed by potassium
fluoride in sulfolane in the presence of 18-crown-6 at 160 °C.
The resulting trifluoro derivative 19 was treated with 2 equiv
of lithium benzylate and subsequently deprotected by BBr3 in
dichloromethane to give the desired 6-fluoro-5-phenyluracil 21.
6-Bromo analogue 23 was obtained by bromination of the
barbituric acid 17 followed by basic hydrolysis.
Figure 1. The structure of TPI.
the 6-chlorouracil derivatives bearing the hydrophobic group
at C5, and the second to 5-phenyluracils with diverse function-
ality at C6.
5-Substituted-6-chlorouracils were prepared by four synthetic
routes. All of them started from commercially available
4-chloro-2,6-dimethoxypyrimidine 2 by direct ortho-lithiation
with butyllithium at -78 °C.
5-Alkyl-4-chloro-2,6-dimethoxypyrimidines 4a-g were ob-
tained by reaction of lithiated intermediate 3 with appropriate
alkyl halide in yields varying from 74 to 92%. The reaction
conditions were similar to those described by Cushman and co-
workers.16
The crucial part of the synthesis of the compounds 5a-g was
the hydrolysis of methoxy groups. After screening a variety
conditions, e.g., TMSI in CH2Cl2, BBr3 in CH2Cl2, and aqueous
HBr in AcOH, the best results were obtained by employing 35%
aqueous hydrochloric acid either in acetic acid or in a mixture
of H2O/THF/dioxane. Under these conditions, the yields were
relatively high and the products were obtained simply by
crystallization (Scheme 1).
Indeed, compounds 5a,17 5b,17 5c,18 and 5g18 have been
already reported by different synthetic methods, but their
inhibitory potency remained undiscovered. These compounds
were included in our study to comprehensively illustrate the
structure-activity relationship.
The intermediate alcohols 6a-d were synthesized simply by
quenching of the lithiated reaction mixture with the appropriate
ketone followed by a slow rise in temperature. By this synthetic
procedure we have obtained satisfactory yields of products from
cyclohexanone (6b) and acetone (6c).
However, the results obtained in reactions with cyclopen-
tanone (preparation of 6a) and 3-pentanone (preparation of 6d)
were rather modest, probably due to increased enolization. With
cyclopentanone, this drawback can be partially overcome by
addition of cerium(III) chloride. The deprotection step was
accomplished again by refluxing derivatives 6a-d with hydro-
chloric acid either in THF/dioxane or in acetic acid (Scheme
2).
The lithiated intermediate 3 was readily transmetalated with
zinc chloride in THF at room temperature to give the corre-
sponding organozinc reagent 8. Subsequent Negishi couplings,
with the use of aryl halides and Pd(PPh3)4 as the catalyst, were
used for syntheses of derivatives bearing aromatic substituents
at C5. Compounds 9a-f were obtained in high yields (62-
97%) and deprotected to give the final products 10a-f (Scheme
3).
The potential of aromatic nucleophilic substitution of the
chlorine atom at C6 was demonstrated by the synthesis of
5-phenyl-6-pyrrolidin-1-yluracil 24 and 6-[(2-aminoethyl)-
amino]-5-phenyluracil 25 (Scheme 7). In these cases, pyrrolidine
and ethane-1,2-diamine were used as the nucleophiles.
Results and Discussion
All the prepared compounds were evaluated for inhibitory
activity against recombinant human TP expressed in V79
Chinese hamster cells and TP purified from human placenta.
As shown in the Table 1, most of the presented derivatives
possess significant activity against both of the enzymes. Also,
the agreement of the results in both enzymatic assays is
noteworthy.
The first stage of exploring the impact of the substitution
consisted of introducing alkyl groups of various lengths to
position C5 of 6-chlorouracil. It is evident that the activity of
the homologues rapidly increases from the ethyl to the butyl
derivative. Interestingly, when pentyl is attached, remarkable
setback is observed. The inhibitory potency is recovered by
further elongation of the side chain to heptyl. We assume that
one of the possible reasons for such fluctuation could be the
limited space in the hydrophobic pocket of the enzyme
compensated by more convenient distortion of the side chain
in the case of heptyl analogue 5f.
The initial step in the synthesis of 5-[(1E)-alk-1-en-1-yl]-4-
chloro-2,6-dimethoxypyrimidines 12a-c was the preparation of
aldehyde 11. The previously reported protocol19 was altered by
addition of DMF instead of ethyl formate in order to simplify
the removal of the side products by column chromatography.
The resulting carbonyl compound 11 reacted smoothly with
phosphorus ylides to give the mixtures of E/Z isomers. The E/Z
ratio was determined by GC (47:53 for 12a:Z-isomer, 47:53
for 12b:Z-isomer, 49:51 for 12c:Z-isomer). The desired E-
isomers 12a-c were purified by preparative reversed-phase
Generally, introduction of the double bond into the side chain
results in significant increase of potency. All compounds bearing
a linear alkenyl group instead of the corresponding aliphatic
chain exhibit dramatically higher inhibitory efficacy. Further-
more, replacement of the linear chain by a cyclic alkenyl ring
leads to even more potent derivatives. 6-Chloro-5-cyclopent-
1-en-1-yluracil (7a) exhibits the highest inhibitory activity
against both TPs in this group as well as in the whole study.
Apparently, the cyclopentenyl group suitably accommodates the
hydrophobic pocket of the enzyme. In contrast, compound 7d,

6018 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Nencka et al.
Scheme 1a
a Conditions: (i) n-BuLi, THF, -78 °C; (ii) R-X, -78 °C; (iii) concd aq HCl, AcOH, reflux.
Scheme 2a
hydrophobic interaction explains the high affinity of 5-substituted-
6-chloruracils for human TP.
This docking study shows a large hydrophobic pocket (which
is theoretically able to accept substituent with 26 carbon atoms),
which offers a design opportunity for further development of
existing human TP inhibitors based on uracil moiety. QSAR
and QM studies are now under way and will be presented soon.
During our study, we have also measured the water solubility
and dissociation properties of the leading compound 7a to
predict its possible parenteral applicability. The experimentally
determined value log Sexp ) -2.46 is in excellent agreement
with the value calculated by ALOGPS 2.128,29 software (log
Scal ) -2.42). The acid dissociation constant, pKa, was
determined by UV spectroscopy to be 5.80 ( 0.10. The data
show that the compound possesses acceptable water solubility,
which can be easily increased by salt formation.30
Moreover, we have calculated several descriptors for estima-
tion of the intestinal absorption, an important component of oral
bioavailability. Topological polar surface area, TPSA, was
calculated by method described by Ertl et al.31 to be 53.72 Å.
The distribution coefficient at pH ) 6.5, log D6.5 ) 0.40 (
0.38, was predicted from log P calculated by ACD/LogP
Freeware 10.0532 (log P ) 1.18 ( 0.30) and the experimental
pKa value by the method of Scherrer and Howard.33 Hydrogen-
bond-donor count is 2, hydrogen-bond-acceptor count is 4, and
the number of rotatable bonds is 1.34 Also, the Lipinski’s rule-
of-five35 is not violated for compound 7a. According to the
recent models of Hou et al.36 and on the basis of our calculations,
we assume that the fraction absorption of our compound 7a,
%FA, (defined as total mass of drug absorbed divided by the
given dose of drug),36 will be with high probability more than
30%.
a Conditions: (i) ketone, -78 °C to rt; (ii) concd HCl, THF, dioxane,
reflux; (iii) concd HCl, AcOH, reflux.
with a branched side chain that closely resembles an “open”
cyclopentene, shows at least 2-fold lower activity.
Also, high affinities toward the enzyme (although lower than
for 7a) were shown for compounds 10a and 10e, bearing phenyl
and thiophenyl substituent at C5, respectively. Any further
substitution of the phenyl ring, replacement by a bulkier
aromatic group, or insertion of the methylene bridge between
uracil and phenyl moiety causes significant loss of activity. The
reduction of the potency may be owing to the unfavorable steric
interaction with the enzyme. As can be expected, introduction
of a more polar, electron-deficient, heteroaromatic group is also
unfavorable.
All modifications performed at position C6 resulted in a
decrease of inhibitory activity in both tested lines. 6-Fluoro and
6-bromo derivatives exhibit activity 2- and 5-fold lower
compared to their 6-chloro counterpart, respectively. The
substitution of the chlorine atom by a methyl group causes
complete loss of affinity. Surprisingly, introduction of a
substituent containing one or two amino functionalities, which
was shown to enhance significantly the inhibitory potency of
5-bromouracil derivatives in our previous study,7 exerts no
activity at all.
We have shown that the potency of the presented compounds
strongly depends on the hydrophobicity of the substituent at
position C5. We have used in silico techniques for better
understanding of this hydrophobic interaction. The structure of
human TP is now well-known.24-26 We have used the X-ray
structure of human TP in complex with TPI24 for our docking
study. For the first approximation, the docking studies were done
using Chem3D Ultra 10.0 software.27 We have used the
implemented dock algorithm with exported distances between
uracil moiety and its binding amino acids (H116, K221, S217,
R202) of human TP from the X-ray structure of human TP in
complex with TPI.24
Conclusion
In conclusion, a series of 5-substituted-6-chlorouracil deriva-
tives has been identified to possess significant activity against
human thymidine phosporylases. The most effective inhibitor
is compound 7a, which inhibits the enzyme expressed in V79
cells competitively with Ki ) 0.20 ( 0.03 µM (Ki/dThdKm
)
0.0017) and the enzyme purified from placenta with Ki ) 0.29
( 0.04 µM (Ki/dThdKm ) 0.0024). Obviously, the potency of
the presented compounds strongly depends on the nature of the
substituent at position C5. A cyclic group with a π-electron
system seems to be essential for ideal inhibitory activity.
Furthermore, our results demonstrate that the efficacy of
inhibition of uracil derivatives does not require an amino or
related group at position C6. In this manner, our study changes
the traditional view on uracil-based TP inhibitors and provides
a novel lead for further research.
A strong contribution to the tight interactions between human
TP and 5-substituted-6-chloruracil comes from the hydrophobic
substituent at position C5, which is held in the huge hydrophobic
pocked formed by V208, L211, L213, I214, I218, and L251 of
the R domain and L75, I78, L148, V232, V234, V241, F242,
and L255 of the R/â domain (Figure 2). This multiple
Experimental Section
Melting points are uncorrected and were determined on a Kofler
block or on a Bu¨chi B-540 melting point apparatus. NMR spectra
were recorded on Bruker Avance 500 (1H at 500 MHz, 13C at 125.8
MHz) and Bruker Avance 400 (1H at 400 MHz, 13C at 100.6 MHz)

Human Thymidine Phosphorylase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6019
Scheme 3a
a Conditions: (i) ZnCl2, THF, rt; (ii) R-X, Pd(PPh3)4, THF, 60 °C; (iii) concd HCl, THF, dioxane, reflux.
Scheme 4a
a Conditions: (i) DMF, -78 °C; (ii) Ph3PdCH-R, THF, 0 °C; (iii) concd HCl, AcOH, reflux.
Scheme 5a
and refilled with argon three times. Anhydrous THF (35 mL) was
added followed, after several minutes, by a ketone (11.5 mmol).
The resulting mixture was stirred for 15 min and cooled to -78
°C. Concurrently, a solution of 4-chloro-2,6-dimethoxypyrimidine
(2 g, 11.5 mmol) in dry THF (27 mL) was treated with n-BuLi
(8.2 mL, 13.1 mmol, 1.6 M solution in hexanes) for 0.5 h at -78
°C under argon atmosphere and this mixture was subsequently
transferred to the flask containing the ketone and CeCl3. This
slurry solution was stirred for 1 h at -78 °C, 2 h at -20 °C, 1.5
h at 0 °C, and 16 h at room temperature. The reaction was
quenched by saturated aqueous solution of NH4Cl (50 mL) and
extracted with Et2O (3 × 150 mL). The combined organic phases
were washed with brine, dried over Na2SO4, and evaporated to
dryness. The residue was purified by column chromatography on
silica gel.
5-Aryl-4-chloro-2,6-dimethoxypyrimidines, 9a-f. A solution
of 4-chloro-2,6-dimethoxypyrimidine (0.7 g, 4 mmol) in anhydrous
THF (20 mL) was cooled to -78 °C and treated with n-BuLi (3
mL, 4.8 mmol, 1.6 M solution in hexanes). After stirring for 30
min at -78 °C, a solution of dry ZnCl2 (1.36 g, 10 mmol) in THF
(10 mL) was added, the dry ice bath was removed, and the mixture
was allowed to warm to room temperature and stirred for further 1
h. Then, a degassed solution of Pd[(C6H5)3P]4 (115 mg, 0.1 mmol)
and aryl iodide (4.5 mmol) in THF (10 mL) was added via syringe
and the resulting mixture was heated to 65 °C for 16 h. The reaction
mixture was partitioned between NH4Cl (50 mL) and Et2O (3 ×
100 mL). The organic layer was washed with a saturated solution
of EDTA (40 mL) and water (50 mL), dried (Na2SO4), and
concentrated in vacuo. Products were purified by silica gel
chromatography.
a Conditions: (i) (a) n-BuLi, THF, -78 °C; (b) ZnCl2, THF, rt; (c)
iodobenzene, Pd(PPh3)4, THF, 60 °C; (ii) concd HCl, THF, dioxane, reflux.
spectrometers with TMS as internal standart or referenced to the
residual solvent signal. Mass spectra were measured on a ZAB-
EQ (VG Analytical) spectrometer. The chemicals were obtained
from commercial sources (Sigma-Aldrich) or prepared according
to the published procedures. 4-Chloro-2,6-dimethoxypyrimidine was
sublimed under reduced pressure before use. THF was distilled from
sodium benzophenone ketyl under argon.
5-Alkyl-4-chloro-2,6-dimethoxypyrimidines, 4a-g. To a solu-
tion of 4-chloro-2,6-dimethoxypyrimidine (2 g, 11.5 mmol) in
anhydrous THF (30 mL) was slowly added n-BuLi (8.5 mL, 13.6
mmol, 1.6 M solution in hexanes) at -78 °C under an argon
atmosphere. Stirring was continued for 0.5 h at the same temper-
ature. Alkyl iodide (14 mmol) was then added neat. Temperature
was allowed to rise spontaneously to room temperature and stirring
was continued for another 16 h. The resulting mixture was
hydrolyzed with saturated aqueous NH4Cl (50 mL) and extracted
with Et2O (3 × 100 mL). The combined organic extract were
washed with water (40 mL), dried (Na2SO4), and evaporated. Crude
products were purified by column chromatography on silica gel or
flash chromatography.
2-(4-Chloro-2,6-dimethoxypyrimidin-5-yl)alcohols, 6a-d.
Method A. To a solution of 4-chloro-2,6-dimethoxypyrimidine
(5.35 mmol) in anhydrous THF (20 mL) was added n-BuLi (4 mL,
6.41 mmol, 1.6 M solution in hexanes) at -78 °C under argon
atmosphere. The mixture was stirred for 0.5 h, ketone was added
via syringe, and stirring was continued for 0.75 h at -78 °C, 2 h
at -20 °C, and 2 h at 0 °C. The mixture was partitioned between
saturated aqueous NH4Cl solution (30 mL) and ether (3 × 60 mL).
The combined organic layers were washed with brine, dried
(Na2SO4), and evaporated to dryness. The residue was purified by
chromatography on a silica gel column.
Method B. This method was similar to method A, except that
the slow increase of temperature after addition of ketone was
followed by additional stirring for 16 h at room temperature.
Method C. CeCl3·7H2O (6.4 g, 17.2 mmol) was dried for 3 h at
120-130 °C under high vacuum. The reaction flask was evacuated
4-Chloro-2,6-dimethoxypyrimidine-5-carbaldehyde, 11. An
oven-dried flask was charged with 4-chloro-2,6-dimethoxypyrimi-
dine (1.9 g, 10.9 mmol) and then evacuated and backfilled with
argon. Anhydrous THF (40 mL) was added through a rubber
septum. The mixture was cooled to -78 °C and a 1.6 M solution
of n-BuLi in hexanes (7.5 mL, 12 mmol) was added dropwise. The
mixture was stirred for an additional 0.5 h. Then, DMF (2 mL, 26
mmol) was added and stirring was continued for 2 h at the same
temperature. The reaction was quenched by addition of aqueous
HCl (1.6 M, 50 mL) and the mixture was extracted with ether (3
× 70 mL). The combined organic layers were washed with aqueous
HCl (1.6 M, 50 mL) and water (40 mL), dried (Na2SO4), and
evaporated to dryness. The residue was purified by column
chromatography on silica gel (ethyl acetate-toluene, 1:6) to afford
the pure product (1.5 g, yield 70%). The characterizations of the
product fully agreed with those previously reported.19

6020 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Nencka et al.
Scheme 6a
a Conditions: (i) POCl3, N,N-dimethylaniline, reflux; (ii) KF, sulfolane, 18-crown-6, 160 °C; (iii) BnOLi, THF, -78 °C to rt; (iv) BBr3, CH2Cl2, -78 °C
to rt; (v) POBr3, N,N-dimethylaniline, toluene, reflux; (vi) NaOH, H2O.
Scheme 7a
General Methods for Deprotection of Final Uracil Derivatives
5a-g, 7a-d, 10a-f, and 13a-c. Method I. A mixture of the
appropriate dimethoxypyrimidine (1 mmol) in concentrated hydro-
chloric acid (2 mL) and of acetic acid (2 mL) was refluxed for 2
h. The cooled slurry was treated with water (2 mL), heated to the
reflux for 1-2 min, and then refrigiated for 2 h. The resulting
precipitate was filtered and washed with water and ether.
Method II. A mixture of the appropriate dimethoxypyrimidine
(1 mmol) in concentrated hydrochloric acid (2 mL), THF (1.5 mL),
and dioxane (1.5 mL) was heated to reflux for 2 h. The resulting
mixture was cooled and half of the volume was removed by
evaporation. Water (2 mL) was then added. The mixture was boiled
for 1-2 min, and after refrigerating for 2 h, the crystals were filtered
and washed with water and ether.
a Conditions: (i) R-H, reflux.
Table 1. Inhibitory Activities of Selected Derivatives against
Human TPs
Ki (µM)a
human TP,
expressed
in V79 cells
human TP,
purified from
placenta
6-Chloro-5-ethylpyrimidine-2,4(1H,3H)-dione, 5a, was pre-
compd
pared by method I: yield 82%, white crystals; mp 216 °C (lit.17
5ab
5bb
5cc
5d
>20
>20
1
mp 227-228 °C); H NMR (DMSO-d6) δ 0.96 (t, 3H, J ) 7.4
5.81 ( 0.82
1.03 ( 0.10
3.67 ( 1.25
1.83 ( 0.39
1.06 ( 0.20
4.65 ( 0.89
0.20 ( 0.03
0.42 ( 0.07
3.34 ( 0.45
0.49 ( 0.06
0.40 ( 0.04
1.74 ( 0.11
4.59 ( 0.99
0.71 ( 0.11
0.28 ( 0.06
3.01 ( 0.59
1.17 ( 0.19
0.63 ( 0.09
0.41 ( 0.06
>20
4.02 ( 0.83
1.65 ( 0.34
3.09 ( 0.42
1.79 ( 0.26
1.60 ( 0.25
4.55 ( 0.56
0.29 ( 0.04
0.75 ( 0.10
5.10 ( 0.88
0.91 ( 0.14
0.43 ( 0.09
1.27 ( 0.22
3.94 ( 0.49
0.97 ( 0.18
0.54 ( 0.10
3.99 ( 0.74
1.21 ( 0.20
0.92 ( 0.10
0.91 ( 0.14
>20
Hz), 2.30 (q, 2H), 11.30 (s, 1H), 11.80 (bs, 1H); 13C NMR (DMSO-
d6) δ 12.75 (C2′), 18.78 (C1′), 111.55 (C5), 140.81(C6), 149.80
(C2), 162.87(C4). Anal. (C6H7ClN2O2) C, H, N.
5e
5f
6-Chloro-5-propylpyrimidine-2,4(1H,3H)-dione, 5b, was pre-
5gc
7a
pared by method I: yield 84%, white crystals; mp 221 °C (lit.17
1
mp 238-240 °C); H NMR (DMSO-d6) δ 0.84 (t, 3H, J ) 7.4
7b
7c
7d
Hz), 1.40 (sext, 2H, J ) 7.5 Hz), 2.26 (bt, 2H, J ) 7.5 Hz), 11.30
(s, 1H), 11.80 (bs, 1H); 13C NMR (DMSO-d6) δ 13.78 (C3′), 21.18
(C2′), 27.19 (C1′), 110.14 (C5), 141.22 (C6), 149.83 (C2), 163.09-
(C4). Anal. (C7H9ClN2O2) C, H, N.
10a
10b
10c
10d
10e
10f
13a
13b
13c
16
5-Butyl-6-chloropyrimidine-2,4(1H,3H)-dione, 5c, was pre-
pared by method I: yield 88%, white crystals; mp 194-196 °C
1
(lit.18 mp 197-198 °C); H NMR (DMSO-d6) δ 0.86 (t, 3H, J )
7.4 Hz), 1.28 (m, 2H), 1.35 (m, 2H), 2.29 (bt, 2H, J ) 7.6 Hz),
11.29 (s, 1H), 11.80 (bs, 1H); 13C NMR (DMSO-d6) δ 13.96 (C4′),
22.04 (C3′), 24.97 (C2′), 30.07 (C1′), 110.32 (C5), 141.15 (C6),
149.85 (C2), 163.07 (C4). Anal. (C8H11ClN2O2) C, H, N.
21
23
24
0.50 ( 0.08
1.21 ( 0.16
>20
0.47 ( 0.10
1.21 ( 0.19
>20
6-Chloro-5-pentylpyrimidine-2,4(1H,3H)-dione, 5d, was pre-
pared by method I: yield 94%, white crystals; mp 191-192 °C
(water); 1H NMR (DMSO-d6) δ 0.87 (t, 3H, J ) 7.5 Hz), 1.25 (m,
4H), 1.37 (m, 2H), 2.28 (bt, 2H, J ) 7.5 Hz), 11.30 (s, 1H), 11.80
(bs, 1H); 13C NMR (DMSO-d6) δ 14.06 (C5′), 22.10 (C4′), 25.18
(C3′), 27.51 (C2′), 31.06 (C1′), 110.39 (C5), 141.05 (C6), 149.82
(C2), 163.06 (C4). Anal. (C9H13ClN2O2) C, H, N.
25
>20
>20
TPId
0.0013 ( 0.00017
0.0021 ( 0.00023
a Km(thymidine) ) 121 ( 19 µmol L-1
.
b Reference 17. c Reference 18.
d Reference 8.
6-Chloro-5-hexylpyrimidine-2,4(1H,3H)-dione. 5e, was pre-
pared by method I: yield 89%, white crystals; mp 186 °C (water);
1H NMR (DMSO-d6) δ 0.85 (t, 3H, J ) 7.0 Hz), 1.25 (m, 6H),
1.36 (m, 2H), 2.28 (t, 2H, J ) 7.6 Hz), 11.29 (s, 1H), 11.80 (bs,
1H); 13C NMR (DMSO-d6) δ 14.11 (C6′), 22.20 (C5′), 25.23 (C4′),
27.80 (C3′), 28.52 (C2′), 31.06 (C1′), 110.34 (C5), 141.11 (C6),
149.825 (C2), 163.05 (C4). Anal. (C10H15ClN2O2) C, H, N.
6-Chloro-5-heptylpyrimidine-2,4(1H,3H)-dione, 5f, was pre-
pared by method I: yield 92%, white crystals; mp 178-179 °C
(water); 1H NMR (DMSO-d6) δ 0.85 (t, 3H, J ) 7.4 Hz), 1.25 (m,
8H), 1.36 (m, 2H), 2.28 (bt, 2H, J ) 7.6 Hz), 11.30 (s, 1H), 11.80
5-(Alk-1-en-1-yl)-4-chloro-2,6-dimethoxypyrimidines, 12a-
c. The mixture of alkyltriphenylphosphonium bromide (0.64 mmol)
and n-BuLi (0.4 mL, 0.64 mmol, 1.6 M in hexanes) in THF (5
mL) was stirred for 30 min at 0 °C. Then, the solution of 11 (100
mg, 0.49 mmol) in THF was added and this mixture was stirred
for an additional 2 h at the same temperature. The reaction was
quenched by addition of aqueous NH4Cl (5 mL). Extraction with
ether (3 × 15 mL) and drying of organic layer over Na2SO4 was
followed by evaporation to dryness. Chromatography on silica gel
(petroleum ether-ethyl acetate, 10:1) afforded mixtures of E and
Z isomers. The E isomers were isolated by reversed-phase HPLC.

Human Thymidine Phosphorylase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6021
Figure 2. Docking model of 7a to the active site of human TP; molecular surface of human TP (H, white; C, gray; N, blue; O, red) and 7a in ball
and stick model (C, gray; N, blue; O, red; Cl, green).
(bs, 1H); 13C NMR (DMSO-d6) δ 14.15 (C7′), 22.26 (C6′), 25.24
(C5′), 27.85 (C4′), 28.67 (C3′), 28.84 (C2′), 31.41 (C1′), 110.34
(C5), 141.16 (C6), 149.86 (C2), 163.07 (C4). Anal. (C11H17ClN2O2)
C, H, N.
6-Chloro-5-phenylpyrimidine-2,4(1H,3H)-dione, 10a, was pre-
pared by method I (yield 77%) or method II (yield 91%), white
crystals; mp 310 °C (water); 1H NMR (DMSO-d6) δ 7.27 (m, 2H),
7.38 (m, 3H), 11.49 (s, 1H), 12.10 (bs, 1H); 13C NMR (DMSO-d6)
δ 112.04 (C5), 127.94, 128.09 (2C), 130.92 (2C), 131.95, 142.32
(C6), 149.82 (C2), 162.49 (C4). Anal. (C10H7ClN2O2) C, H, N.
6-Chloro-5-(3,5-dimethylphenyl)pyrimidine-2,4(1H,3H)-di-
one, 10b, was prepared by method II: yield 93%, white crystals;
5-Benzyl-6-chloropyrimidine-2,4(1H,3H)-dione, 5g, was pre-
pared by method II: yield 97%, white crystals; mp 247 °C (lit.18
1
mp 235-236 °C); H NMR (DMSO-d6) δ 3.65 (s, 2H), 7.20 (m,
3H), 7.25 (m, 2H), 11.42 (s, 1H), 11.95 (bs, 1H); 13C NMR (DMSO-
d6) δ 31.05 (CH2), 109.76 (C5), 126.59 (C4′), 128.42 (2C, C3′),
128.78 (2C, C2′), 139.34 (C1′), 142.81 (C6), 150.08 (C2), 163.45
(C4). Anal. (C11H9ClN2O2) C, H, N.
1
mp 288 °C (water); H NMR (DMSO-d6) δ 2.26 (q, 6H, J ) 0.8
Hz), 6.84 (dt, 2H, J ) 0.8 Hz, J ) 1.7 Hz), 6.96 (sept, 1H, J ) 0.8
Hz), 11.45 (s, 1H), 12.07 (bs, 1H); 13C NMR (DMSO-d6) δ 21.04
(2C) 112.25 (C5), 128.46 (2C), 129.41, 131.80, 137.05 (2C), 142.11
(C6), 149.81 (C2), 162.51 (C4). Anal. (C12H11ClN2O2) C, H, N.
6-Chloro-5-(2-naphthyl)pyrimidine-2,4(1H,3H)-dione, 10c, was
prepared by method II: yield 82%, white crystals; mp >325 °C
6-Chloro-5-cyclopent-1-en-1-ylpyrimidine-2,4(1H,3H)-di-
one, 7a, was prepared by method II: yield 49%, pale orange
1
crystals; mp 276-277 °C (EtOH); H NMR (DMSO-d6) δ 1.86
(pent, 2H, J ) 7.4 Hz), 2.39 (m, 2H), 2.47 (m, 2H), 5.89 (pent,
1H, J ) 2.2 Hz), 11.46 (s, 1H), 12.06 (bs, 1H); 13C NMR (DMSO-
d6) δ 23.09 (C4′), 32.71 (C5′), 34.86 (C3′), 109.08 (C5), 133.00
(C2′), 134.11 (C1′), 140.93 (C6), 149.59 (C2), 161.99 (C4). Anal.
(C9H9ClN2O2) C, H, N.
1
(water); H NMR (DMSO-d6) δ 7.37 (m, 1H), 7.52 (m, 3H), 7.73
(m, 1H), 7.96 (m, 2H), 11.53 (s, 1H), 12.25 (bs, 1H); 13C NMR
(DMSO-d6) δ 110.45 (C5), 125.35, 125.68, 126.13, 126. 52, 128.39,
128.68, 129.15, 130.07, 132.08, 133.40, 143.64 (C6), 150.22 (C2),
162.66 (C4). Anal. (C14H9ClN2O2) C, H, N.
6-Chloro-5-cyclohex-1-en-1-ylpyrimidine-2,4(1H,3H)-dione,
7b, was prepared by method II: yield 76%, white crystals; mp
6-Chloro-5-(4-fluorophenyl)pyrimidine-2,4(1H,3H)-dione, 10d,
was prepared by method II: yield 89%, white crystals; mp 298-
1
279 °C (EtOH); H NMR (DMSO-d6) δ 1.54 (m, 2H), 1.61 (m,
1
2H), 2.00 (m, 2H), 2.06 (m, 2H), 5.57 (sept, 1H, J ) 1.7 Hz),
11.30 (s, 1H), 11.87 (bs, 1H); 13C NMR (DMSO-d6) δ 21.65(C5′),
22.44 (C4′), 25.03 (C3′), 27.62 (C6′), 114.02 (C5), 129.61 (C2′),
129.96 (C1′), 140.98 (C6), 149.79 (C2), 162.23 (C4). Anal. (C10H11-
ClN2O2) C, H, N.
299 °C (water); H NMR (DMSO-d6) δ 7.21 (m, 2H), 7.32 (m,
2H), 11.51 (s, 1H), 12.15 (bs, 1H); 13C NMR (DMSO-d6) δ 111.13
(C5), 115.03 (d, 2C, JCF ) 21.5 Hz, C3′), 128.02 (d, JCF ) 3.2 Hz,
C1′), 133.07 (d, 2C, JCF ) 8.3 Hz, C2′), 142.48 (C6), 149.77 (C2),
161.83 (d, JCF ) 3.2 Hz, C4′), 162.47 (C4). Anal. (C10H6ClFN2O2)
C, H, N.
6-Chloro-5-(1-methylethenyl)pyrimidine-2,4(1H,3H)-dione, 7c,
was prepared by method II: yield 44%, white crystals; mp 256 °C
6-Chloro-5-(2-thienyl)pyrimidine-2,4(1H,3H)-dione, 10e, was
prepared by method II: yield 70%, white crystals; mp 291-292
°C (water, EtOAc/EtOH); 1H NMR (DMSO-d6) δ 7.19 (dd, 1H, J
) 3.6 Hz, J ) 5.2 Hz), 7.34 (dd, 1H, J ) 1.2 Hz, J ) 3.6 Hz),
7.71 (dd, 1H, J ) 1.2 Hz, J ) 5.2 Hz), 11.72 (s, 1H), 12.39 (bs,
1H); 13C NMR (DMSO-d6) δ 105.82 (C5), 126.47 (C5′), 127.43
(C4′), 129.22 (C3′), 131.72 (C2′), 142.88 (C6), 149.29 (C2), 161.97
(C4). Anal. (C8H5ClN2O2S) C, H, N.
1
(water); H NMR (DMSO-d6) δ 1.84 (bt, 3H, J ) 1.3 Hz), 4.90
(dq, 1H, J ) 1.0 Hz, Jgem) 2.0 Hz), 5.26 (bpent, 1H, J ∼ Jgem
)
2.0 Hz), 5.57 (sept, 1H, J ) 1.7 Hz), 11.30 (s, 1H), 11.87 (bs,
1H); 13C NMR (DMSO-d6) δ 22.26 (CH3), 113.44 (C5), 119.12
(dCH2), 136.73 (Cd), 140.85 (C6), 149.77 (C2), 161.88 (C4). Anal.
(C7H7ClN2O2) C, H, N.
6-Chloro-5-[(1E)-1-ethylprop-1-en-1-yl]pyrimidine-2,4(1H,3H)-
dione, 7d, was prepared by method II: yield 40%, white crystals;
mp 226-229 °C (water); 1H NMR (DMSO-d6) δ 0.84 (t, 3H, J )
6-Chloro-5-pyridin-3-ylpyrimidine-2,4(1H,3H)-dione Hydro-
chloride, 10f. The compound was prepared by method I, except
the 2 mL of water was not added after refluxing and the crystalline
product was filtered directly from the cooled reaction mixture
and washed with EtOH and ether: yield 76%, white crystals; mp
7.6 Hz), 1.67 (d, 3H, J ) 6.8 Hz), 2.21 (bq, 2H, J ) 7.6 Hz),
‘
5.33 (qt, 1H, Je 1.0 Hz), 11.29 (s, 1H), 11.87 (bs, 1H); 13C NMR
(DMSO-d6) δ 12.46, 13.39, 22.68, 113.76 (C5), 126.93 (dCH),
133.74 (Cd), 141.71 (C6), 149.86 (C2), 162.47 (C4). Anal. (C9H11-
ClN2O2) C, H, N.
1
>325 °C; H NMR (DMSO-d6) δ 8.02 (dd, 1H, J ) 5.0 Hz, J )
7.8 Hz), 8.44 (d, 1H, J ) 7.8 Hz), 8.85 (m, 2H), 11.80 (bs, 1H),

6022 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Nencka et al.
12.00 (bs, 1H); 13C NMR (DMSO-d6) δ 106.94 (C5), 126.28 (C5′),
130.96 (C3′), 142.53 (C4′), 144.36 (C6), 144.78 (C6′), 146.34 (C2′),
149.60 (C2), 161.99 (C4). Anal. (C9H6ClN3O2. HCl) C, H, N.
6-Chloro-5-[(1E)-prop-1-en-1-yl]pyrimidine-2,4(1H,3H)-di-
one, 13a, was prepared by method I: yield 43%, pale beige crystals;
mp 268 °C (water); 1H NMR (DMSO-d6) δ 1.79 (dd, 3H, J ) 1.7
Hz, J ) 6.8 Hz,), 6.09 (dq, 1H, J ) 1.7 Hz, J ) 15.7 Hz), 6.73
(dq, 1H, J ) 6.8 Hz, J ) 15.7 Hz), 11.38 (s, 1H), 12.04 (bs, 1H);
13C NMR (DMSO-d6) δ 19.55 (C3′), 107.12 (C5), 120.74 (C2′),
130.11 (C1′), 140.93 (C6), 149.05 (C2), 162.09 (C4). Anal. (C7H7-
ClN2O2) C, H, N.
was stirred for 2 h at -78 °C and 16 h at room temperature. The
reaction was quenched with saturated NH4Cl (25 mL) and
extracted with ethyl acetate (3 × 75 mL). The organic phase was
dried (Na2SO4), evaporated in vacuo, and chromatographed on silica
gel (petroleum ether-EtOAc, 20:1). Crystallization from cyclo-
hexane gave 528 mg of 20 as white needles (yield 72%; mp 87-
88 °C).
6-Fluoro-5-phenylpyrimidine-2,4(1H,3H)-dione, 21. A solution
of BBr3 (3 mL, 1 M in CH2Cl2) was added to a solution of 20 (180
mg, 0.47 mmol) in CH2Cl2 (5 mL) at -78 °C. The mixture was
stirred overnight at room temperature and partitioned between
aqueous saturated NaHCO3 (50 mL) and ethyl acetate (75 mL).
Aqueous phase was acidified by diluted aqueous HCl and extracted
with ethyl acetate (3 × 75 mL). The organic layers were combined,
dried (Na2SO4), and evaporated under reduced pressure. Chroma-
tography on silica gel (EtOAc-acetone-EtOH-H2O, 17:3:3:2)
afforded 30 mg of product 21: yield 31%; mp 282 °C (water); 1H
NMR (DMSO-d6) δ 7.35 (m, 5H) 11.40 (bs, 2H); 13C NMR
(DMSO-d6) δ 94.55 (d, JCF ) 10.9 Hz, C5), 127.58, 128.48 (d, JCF
) 26.0 Hz), 130.33 (2C), 148.94 (d, JCF ) 11.4 Hz, C2), 159.30
(d, JCF ) 266.4 Hz, C6), 164.00 (d, JCF ) 15.1 Hz, C4). Anal.
(C10H7FN2O2) C, H, N.
5-[(1E)-But-1-en-1-yl]-6-chloropyrimidine-2,4(1H,3H)-di-
one, 13b, was prepared by method I: yield 59%, beige crystals;
1
mp 248 °C (water); H NMR (DMSO-d6) δ 0.99 (t, 3H, J ) 1.7
Hz), 1.43 (m, 2H), 6.07 (dt, 1H, J ) 1.2 Hz, J ) 15.8 Hz), 6.79
(dt, 1H, J ) 6.7 Hz, J ) 15.8 Hz), 11.38 (bs, 1H), 12.05 (bs, 1H);
13C NMR (DMSO-d6) δ 13.72 (C4′), 26.77 (C3′), 107.06 (C5),
118.68 (C2′), 136.667 (C1′), 141.14 (C6), 149.04 (C2), 162.08 (C4).
Anal. (C8H9ClN2O2) C, H, N.
6-Chloro-5-[(1E)-pent-1-en-1-yl]pyrimidine-2,4(1H,3H)-di-
one, 13c, was prepared by method I: yield 55%, white crystals;
1
mp 231 °C (water); H NMR (DMSO-d6) δ 0.88 (t, 3H, J ) 7.3
2,4,6-Tribromo-5-phenylpyrimidine, 22. In a flame-dried flask
flushed with argon, the 5-phenylbarbituric acid 1721 (2.4 g, 11.75
mmol) and POBr3 (13.45 g, 47 mmol) were mixed with toluene
(23 mL, freshly distilled from sodium) and N,N-dimethylaniline
(2.7 mL, 21.3 mmol), and the mixture was refluxed for 6 h. The
mixture was poured onto crushed ice and extracted with ether (3
× 100 mL). The organic phase was dried (Na2SO4) and evaporated
to dryness. Crystallization from ethyl acetate afforded 2.21 g of 22
as pale yellow crystals (yield 48%; mp 208 °C). Further product
was recovered from mother liquors by chromatography on silica
gel (petroleum ether-ethyl acetate, 20:1) (0.51 g, 11%).
6-Bromo-5-phenylpyrimidine-2,4(1H,3H)-dione, 23. The finely
powdered compound 22 (500 mg, 1.27 mmol) was refluxed with
NaOH (0.21 g, 5.25 mmol) in H2O (3.5 mL) for 1.5 h. The resulting
solution was cooled, neutralized with diluted hydrochloric acid, and
adsorbed onto silica gel. Chromatography on silica gel (ethyl
acetate-toluene, 20:3) and subsequent crystallization from aqueous
ethanol afforded 73 mg of 23 as white crystals: yield 22%; mp
308-309 °C; 1H NMR (DMSO-d6) δ 7.23 (m, 2H), 7.33 (m, 1H),
7.38 (m, 2H), 11.46 (s, 1H), 12.02 (s, 1H); 13C NMR (DMSO-d6)
δ 115.17 (C5), 127.88 (C4′), 128.05 (2C, C3′), 130.93 (2C, C2′),
133.89 (C1′), 133.98 (C6), 150.13 (C2), 161.86 (C4). Anal. (C10H7-
BrN2O2) C, H, N.
5-Phenyl-6-pyrrolidin-1-ylpyrimidine-2,4(1H,3H)-dione, 24.
The mixture of 10a (676 mg, 3.03 mmol) and pyrrolidine (10 mL)
was refluxed for 6 h. After evaporation to dryness, ethyl acetate
(15 mL) was added and the suspension was heated to reflux for 1
min. Then, the mixture was diluted by EtOH (5 mL) and the heating
was repeated. The precipitate was filtrated, suspended in water (5
mL), and boiled for 1 min. The crystalline product was collected
and recrystallized from methanol to give 647 mg of 24 as white
crystals: yield 83%; mp 311 °C; 1H NMR (DMSO-d6) δ 1.62 (m,
8H), 2.96 (m, 4H), 7.15 (m, 3H), 7.24 (m, 2H); 13C NMR (DMSO-
d6) δ 89.71 (C5), 125.54 (C1′), 127.08 (2C, C3′), 132.50 (2C, C2′),
136.52 (C1′), 151.86 (C2), 152.74 (C6), 164.17 (C4). Anal.
(C14H15N3O2) C, H, N.
Hz), 1.40 (sext, 2H, J ) 7.3 Hz), 2.10 (qd, 2H, J ) 1.5 Hz, J )
7.2 Hz), 6.08 (dt, 1H, J ) 1.5 Hz, J ) 15.7 Hz), 6.74 (dt, 1H, J )
7.2 Hz, J ) 15.7 Hz), 11.38 (s, 1H), 12.05 (bs, 1H); 13C NMR
(DMSO-d6) δ 13.75 (C5′), 22.21 (C4′), 35.84 (C3′), 107.015 (C5),
119.71 (C2′), 135.02 (C1′), 141.11 (C6), 149.03 (C2), 162.07 (C4).
Anal. (C9H11ClN2O2) C, H, N.
2,4-Dimethoxy-6-methyl-5-phenylpyrimidine, 15. In a flame-
dried flask, a 1.6 M solution of n-BuLi in hexanes (4.3 mL, 6.9
mmol) was added dropwise to a solution of 1420 (1.34 g, 5.57 mmol)
in THF (30 mL) at -78 °C under argon atmosphere. The mixture
was stirred for 30 min. Then a solution of ZnCl2 (1.8 g, 13.2 mmol)
in THF (10 mL) was added, and the mixture was allowed to warm
to room temperature and stirred for an additional 1 h. The resulting
mixture was treated with a solution of phenyl iodide (1.4 g, 0.78
mL, 6.9 mmol) and Pd(PPh3)4 (133 mg, 0.11 mmol) in THF (10
mL) at 60 °C for 16 h. The mixture was diluted saturated aqueous
NH4Cl (25 mL) and extracted with ether (3 × 75 mL). The extract
was washed with brine (45 mL) and concentrated under reduced
pressure. Chromatography on silica gel (petroleum ether-ethyl
acetate, 10:1) afforded 15 (356 mg, 28%) as white crystals; mp 92
°C (cyclohexane). The analytical sample was sublimed under
reduced pressure.
6-Methyl-5-phenylpyrimidine-2,4(1H,3H)-dione, 16. The mix-
ture of 15 (184 mg, 0.8 mmol), THF (1 mL), dioxane (1 mL), and
concentrated aqueous HCl (1.3 mL) was heated to reflux for 3.5 h,
cooled down, and refrigerated for 30 min. The precipitate was
collected by filtration, washed with water, and ether/petroleum ether
mixture and dried to give 131 mg of analytically pure white
crystals: yield 81%; mp 323-325 °C; 1H NMR (DMSO-d6) δ 1.93
(s, 3H), 7.20 (d, 2H), 7.30 (t, 1H), 7.36 (t, 2H) 10.93 (bs, 1H),
11.10 (bs, 1H); 13C NMR (DMSO-d6) δ 22.40 (CH3), 111.77 (C5),
127.50, 128.31 (2C), 131.33 (2C), 133.83, 149.53 (C6), 151.19 (C2),
163.98 (C4). Anal. (C11H10N2O2) C, H, N.
2,4,6-Trifluoro-5-phenylpyrimidine, 19. Under reduced pres-
sure (5 mBar) at 140 °C, the solvent (2 mL) was removed from a
mixture of KF (1.47 g, 25.30 mmol) and sulfolane (22 mL).
Subsequently, compound 1821 (2 g, 7.7 mmol) and 18-crown-6 (150
mg) were added. The resulting mixture was heated to 160 °C for
5 h, cooled down, and partitioned between water (75 mL) and
petroleum ether (100 mL). Aqueous phase was extracted with ether
(2 × 200 mL). The combined organic phases were washed with
water (3 × 400 mL), dried over Na2SO4, and evaporated. Chro-
matography on silica gel (hexanes-toluene, 20:3) afforded 19 as
colorless oil (720 mg, 44%).
2,4-Bis(benzyloxy)-6-fluoro-5-phenylpyrimidine, 20. The mix-
ture of benzyl alcohol (432 mg, 3.99 mmol), THF (5 mL), and 1.6
M n-BuLi in hexanes (2.5 mL, 3.99 mmol) prepared at -78 °C
was added to a solution of 19 (400 mg, 1.9 mmol) in THF (10
mL) over 40 min at the same temperature. The resulting mixture
6-[(2-Aminoethyl)amino]-5-phenylpyrimidine-2,4(1H,3H)-di-
one, 25. A mixture of 10a (500 mg, 2.25 mmol) and ethylenedi-
amine (6 mL) was heated to reflux for 5 h. The mixture was left to
stand overnight at room temperature. The precipitate was collected
by filtration, washed with EtOH, and recrystallized from water to
1
give 467 mg of 25 as white crystals: yield 84%; mp 251 °C; H
NMR (DMSO-d6) δ 2.26 (m, 2H), 3.15 (m, 2H), 5.97 (bs, 2H),
7.20 (m, 1H), 7.22 (m, 2H), 7.32 (m, 2H), 9.60-10.20 (bs, 3H);
13C NMR (DMSO-d6) δ 41.69, 44.68, 88.03 (C5), 125.97, 128.31
(2C), 131.74 (2C), 134.37 (C1), 152.61 (C2), 154.92 (C6), 163.21
(C4). Anal. (C12H14N4O2·H2O) C, H, N.
Thymidine Phosphorylases. The recombinant human thymidine
phosphorylase (V79TP) expressed in V79 Chinese hamster cells

Human Thymidine Phosphorylase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6023
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was a commercial product (Sigma, T-9319) and the enzyme from
human placenta (hpTP) was purified using a combination of
described procedures.22,23
Enzyme Assay. The standard reaction mixture (50 µL) contained
20 mM BisTris-HCl (pH 6.4), 1 mM EDTA, 2 mM DTT, 200 µM
potassium phosphate (pH 6.7), various concentration of [3H-methyl]-
thymidine, and tested compounds. The reaction was started by the
addition of 44 nU of enzyme, incubated at 37 °C for 8 min, and
stopped by spotting a 2 µL aliquot onto a silica gel 60 F254 plate
that had been prespotted with 0.01 µmol of each thymine and
thymidine. The plate was developed in the nonaqueous phase of
the solvent system ethyl acetate-water-formic acid (60:35:5). The
spots were visualized under UV light (254 nm) and cut out for
radioactivity determination in toluene-based scintillation cocktail.
Kinetic constants Km and Ki were determined from the Lin-
eweaver-Burk and Dixon plots. Data based on results from at least
four independent experiments were evaluated by the nonlinear
regression method (GOSA, Bio-Log).
Acknowledgment. We thank Jaroslav Gu¨nter for separation
of E/Z isomers by preparative HPLC and Dr. Josef Cvacˇka for
GC measurements. We would like to thank to Dr. Jiˇr´ı Hanusek
for measurement of pKa. This study, a part of the research project
#Z4 055 0506, was supported by the Ministry of Education of
the Czech Republic (Centre for New Antivirals and Antine-
oplastics #1M0508), by the Program of Targeted Projects of
Academy of Sciences of the Czech Republic (1QS400550501)
and by Gilead Sciences, Inc. (Foster City, CA).
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Supporting Information Available: Spectroscopic data for new
compounds and elemental analysis results. This material is available
free of charge via the Internet at http://pubs.acs.org.
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