Fragment-based approach to the design of 5-chlorouracil-linked-pyrazolo[1, 5-a][1,3,5]triazines as thymidine phosphorylase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.10.022

5-Chlorouracil-linked-pyrazolo[1,5-a][1,3,5]triazines were designed as new thymidine phosphorylase inhibitors based on the fragment based drug design approach. Multiple-step convergent synthetic schemes were devised to generate the target compounds. The intermediate 5-chloro-6-chloromethyluracil was synthesized by a 4-step reaction. A series of the second bicyclic intermediates, namely pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-one, was obtained from various substituted 3-aminopyrazoles. These two intermediates were coupled finally in the presence of sodium ethoxide and methanol to yield the desirable target compounds. The methylthio coupling spacer was found to be suitable in enabling the interaction of the two fragments at the active site and allosteric site of the enzyme. The best coupled compound (9q) inhibited the thymidine phosphorylase with an IC₅₀ value as low as 0.36 ± 0.1 μM. In addition, 9q demonstrated a mixed-type of enzyme inhibition kinetics, thus suggesting that it might indeed potentially bind at two different sites on the enzyme. Georg Thieme Verlag KG Stuttgart New York ISSN 0935-8943.

Full text of DOI:10.1016/j.ejmech.2013.10.022

European Journal of Medicinal Chemistry 70 (2013) 400e410
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Fragment-based approach to the design of 5-chlorouracil-linked-
pyrazolo[1,5-a][1,3,5]triazines as thymidine phosphorylase inhibitors
Lingyi Sun a, Jiarong Li a, Hriday Bera a, Anton V. Dolzhenko b, Gigi N.C. Chiu a,
,
Wai Keung Chui a
*
a Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore
b School of Pharmacy, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor WA 46150, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Chlorouracil-linked-pyrazolo[1,5-a][1,3,5]triazines were designed as new thymidine phosphorylase
inhibitors based on the fragment based drug design approach. Multiple-step convergent synthetic
schemes were devised to generate the target compounds. The intermediate 5-chloro-6-
chloromethyluracil was synthesized by a 4-step reaction. A series of the second bicyclic intermediates,
namely pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-one, was obtained from various substituted 3-
aminopyrazoles. These two intermediates were coupled finally in the presence of sodium ethoxide
and methanol to yield the desirable target compounds. The methylthio coupling spacer was found to be
suitable in enabling the interaction of the two fragments at the active site and allosteric site of the
enzyme. The best coupled compound (9q) inhibited the thymidine phosphorylase with an IC50 value as
Received 10 May 2013
Received in revised form
1 October 2013
Accepted 7 October 2013
Available online 15 October 2013
Keywords:
5-Chlorouracil-linked-pyrazolo[1,5-a][1,3,5]
triazines
Thymidine phosphorylase inhibitor
Fragment-based drug design
Mixed-type enzyme inhibition kinetics
low as 0.36 ꢀ 0.1
suggesting that it might indeed potentially bind at two different sites on the enzyme.
Ó 2013 Elsevier Masson SAS. All rights reserved.
mM. In addition, 9q demonstrated a mixed-type of enzyme inhibition kinetics, thus
1. Introduction
effective. Hence, a combination use of these therapeutic agents
with a TP inhibitor may be an approach that can improve the
pharmacological efficacy of these agents [4].
It has been reported in literature that TP also possesses other
functions related to cancer biology; it can stimulate tumour
angiogenesis [5], induce tumour metastasis [6,7] and promote
tumour growth by preventing apoptosis [8]. Therefore, based on
these pieces of evidence, it appears to suggest that TP is a druggable
target for developing inhibitors that may be used as therapeutic
agents for chemotherapy.
Thymidine phosphorylase (TP) is an enzyme that catalyses the
reversible phosphorolysis of pyrimidine nucleosides (Fig. 1) [1]. In
the normal intracellular environment, TP plays a key role in
maintaining a balance of the nucleotide pool by controlling nucleic
acid homeostasis so as to ensure an ample supply of deoxy-
ribonucleoside triphosphates (dNTPs) for DNA replication and
repair [2].
Besides the regulation of pyrimidine nucleosides, TP can also
induce the phosphorolysis of several pyrimidine nucleoside de-
rivatives. Some nucleoside derivatives such as capecitabine and 50-
deoxy-5-fluorouridine are designed as prodrugs and they are being
converted to the corresponding parent drugs by TP [3] On the other
hand, some nucleoside derivatives such as 5-fluoro-20-deoxyur-
idine and trifluorothymidine have been used as therapeutic agents
directly and TP may inactivate them thus making them less
Pioneering work on the development of TP inhibitors originated
in the late 1960s [9]. Pyrimidine derivatives such as 6-
aminothymine (6AT), 6-amino-5-chlorouracil (6A5CU) and 6-
amino-5-bromouracil (6A5BU) were the first few TP inhibitors to
be generated. They exhibited IC50 values of around 30 mM and they
have served as reference bioactive compounds for more than 30
years in this field of research [10]. However, their relatively less
favourable IC50 values did not allow them to be developed into drug
candidates. In the year 2000, a very potent inhibitor (IC50
value ¼ 35 nM against human TP) namely 5-chloro-6-[1-(2-
iminopyrrolidinyl)methyl] uracil hydrochloride (TPI) (Fig. 2) was
discovered by Fukushima et al. [11]. TPI was also found to inhibit
the enzyme competitively with the substrate thymidine. In the
development of TP inhibitors, another interesting discovery was
* Corresponding author. Department of Pharmacy, Faculty of Science, Block S4, 18
Science Drive 4, Singapore 117543, Singapore. Tel.: þ65 6516 2933; fax: þ65 6779
1554.
E-mail addresses: lingyisun@gmail.com (L. Sun), g0701228@nus.edu.sg
(H. Bera), dolzhenkoav@gmail.com (A.V. Dolzhenko), phacncg@nus.edu.sg
(G.N.C. Chiu), phacwk@nus.edu.sg (W.K. Chui).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.022

L. Sun et al. / European Journal of Medicinal Chemistry 70 (2013) 400e410
401
Since TP inhibitors containing only the pyrimidine moiety are
competitive inhibitors while TP inhibitors containing a purine-like
moiety without the homophthalimide structure are non-
competitive inhibitors, it can be inferred that the pyrimidine moi-
ety is a competitive fragment while the purine-like moiety without
the homophthalimide structure is a non-competitive fragment.
These two fragments should bind to TP at different sites. Hence-
forth, based on the fragment based drug design approach; this
study has been set up with the hypothesis that a new compound
that possesses both the pyrimidine moiety (competitive fragment)
and the purine-like moiety without the homophthalimide struc-
ture (non-competitive fragment) would still inhibit TP. This frag-
ment combination strategy has not been reported in works related
to the development of TP inhibitors and if it works, it will generate
new chemical entities that may contribute to the existing pool of TP
inhibitors. In order to test this hypothesis, the authors set up a
study plan that contained two objectives. The first objective
involved devising a new synthetic scheme that could chemically
link the pyrimidine scaffold, namely 5-chlorouracil, with the pyr-
azolo[1,5-a][1,3,5]triazine scaffold. The second objective was to
evaluate the TP inhibitory activity of these dual scaffold linked
compounds using a spectrophotometric bioassay. Should active TP
inhibitors were to be found; the inhibitory enzyme kinetics would
also be elucidated.
O
N
O
P
OH
OH
O
HN
O
HN
+
+ Pi
O
O
O
N
H
O
HO
OH
HO
OH
Fig. 1. Enzymatic reaction catalysed by thymidine phosphorylase.
the TP inhibitory activity of purine derivatives such as 7-
deazaxanthine (7DX, IC50 value ¼ 40
mM against Escherichia coli
TP) [12] and 50-O-tritylinosine (KIN59, IC50 value ¼ 67
mM, 44 mM
against human, E. coli TP respectively) [13] (Fig. 2). This finding
further expanded the molecular space for TP inhibitors. It was
found that 7DX is a competitive TP inhibitor with respect to
thymidine (same as most pyrimidine derived inhibitors) [14], while
KIN59 was found to be a non-competitive inhibitor of TP with
respect to thymidine [13]. The authors of this manuscript have
recently reported the synthesis of a series of purine analogues
namely 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones
(Fig. 2). This series demonstrated TP inhibitory activity and the
most active compound inhibited E. coli TP at an IC50 value of
0.04 mM. The most active compound in the 1,3-dihydro-pyrazolo
In the design of these new compounds, choosing the right
spacer to link both the 5-chlorouracil moiety and the pyrazolo[1,5-
a][1,3,5]triazine scaffold is critical to ensure that inhibitory activity
against the enzyme is retained. Since Yano et al. has reported that S-
(5-chlorouracil-6-ylmethyl)thiourea hydrochloride (Fig. 2) inhibi-
[1,5-a][1,3,5]triazin-2-thioxo-4-ones series was found to be a non-
competitive inhibitor as well. Therefore, this observation suggested
that without the homophthalimide moiety, which has been
considered as an important structure for competitive inhibitors by
some workers [1], the purine-like compounds may bind to a site of
TP other than the thymidine binding site.
ted human TP at an IC50 value of 0.35
mM [15], this study hypoth-
esized that a methylthio moiety at position 6 of the uracil ring
Fig. 2. Structures of TPI, 7DX, KIN59, 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones, S-(5-chlorouracil-6-ylmethyl)thiourea hydrochloride and 3H-2-(5-chlorouracil-6-
methylthio)-pyrazolo[1,5-a][1,3,5]triazin-4-ones.

402
L. Sun et al. / European Journal of Medicinal Chemistry 70 (2013) 400e410
might allow the uracil ring in these new compounds to interact
appropriately with the enzyme. This prompted the authors of this
manuscript to use the methylthio moiety as a flexible linker to
connect the pyrazolo[1,5-a][1,3,5]traizine and the 5-chlorouracil
moiety together. Therefore, the target compounds were finally
decided to be 3H-2-(5-chlorouracil-6-methylthio)pyrazolo[1,5-a]
[1,3,5]triazin-4-ones (Fig. 2).
spectroscopic techniques (1H and 13C NMR). In addition, the purity
of the compounds was determined by reverse-phase HPLC and was
found to be in the range of 95% and 100% pure. These purified
compounds were used in the bioassay that determined the inhib-
itory activity against TP.
2.2. Structure activity relationship (SAR) based on results of in vitro
TP enzyme assay
2. Results and discussion
TP inhibitory activity of the synthesized target compounds (7ae
m, 9aeq) was evaluated by an established continuous UV spec-
trophotometric enzyme assay [21] using a commercially available
(Sigma, T-2807) recombinant E. coli. TP (Enzyme Commission (EC)
Number 2.4.2.4) and thymidine as the substrate. In the bioassay, the
rate of decrease in absorbance, monitored at 290 nm, was taken to
be the rate of the enzymatic activity. Triplicate experiments were
conducted and the mean IC50 values of the target compounds were
calculated and presented with the standard deviations. Based on
the results of the in vitro TP bioassay, all the compounds (7aem,
9aeq) exhibited TP inhibitory activity with mean IC50 values
2.1. Chemistry
In planning the synthetic approach of the target compounds, it
was found that the final chemical structure could be dissected into
the 5-chlorouracil moiety and the pyrazolo[1,5-a][1,3,5]triazine
scaffold in three ways as shown in Fig. 3.
Since a few 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-
4-ones were already synthesized in our previous work, it would be
convenient to adopt approach B as the method for preparing the
target compounds. Therefore, the corresponding retrosynthesis
plan was devised in which the designed target compounds would
be generated through coupling reactions using 5-chloro-6-
chloromethyluracil and 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-
2-thioxo-4-ones as starting materials (Fig. 4).
The key intermediate 5-chloro-6-chloromethyluracil was syn-
thesized via a four-step reaction (Scheme 1) beginning with the
oxidation of 6-methyluracil 1 with selenium oxide to generate
uracil-6-carbaldehyde 2 (Yield 35%) [16]. This step was followed by
reduction with sodium borohydride to give 6-hydroxymethyluracil
3 (Yield 79%) [17]. Then the hydroxyl group was replaced with
chloro by refluxing in excess neat thionyl chloride to yield 95% of 6-
chloromethyluracil 4 [18]. Finally, chlorination at C-5 of the uracil
ring was achieved by using sulfuryl chloride to generate 5-chloro-
6-chloromethyluracil 5 (Yield 68%) [19].
ranging from 67.8 mM to 0.36 mM (Table 1). TPI was used as the
reference compound for comparison, its IC50 value under the same
evaluating conditions was determined to be 2.7 nM, which was
consistent with the value reported by Grierson et al. [22] (2.5 nM).
From the data, the idea of combining both the 5-chlorouracil
moiety and the pyrazolo[1,5-a][1,3,5]triazine scaffold with the
methylthio spacer has produced new compounds that retained TP
inhibitory activity. On closer examination of the biological data
with the structures of the test compounds, interesting structure-
activity related observations have emerged.
Compound 7a was the first compound in the library where 5-
chlororacil was linked chemically to pyrazolo[1,5-a][1,3,5]triazine
via a methylthio spacer and it was found to show weak inhibition
against TP as the IC50 value was determined to be 67.8 ꢀ 4.4
mM.
Further modification on 7a was carried out at positions 7 and 8.
When methyl, tert-butyl and trifluoromethyl groups were intro-
duced separately at position 7 of 7a, there were small improve-
ments seen in the IC50 values. Similar trend was observed when
methyl, halogens, nitrile and ethoxycarbonyl groups were inserted
at position 8 of 7a. However, the IC50 values improved significantly
when 7a was substituted with a phenyl group at position 7 (7m,
The other key intermediate 1,3-dihydropyrazolo[1,5-a][1,3,5]
triazin-2-thioxo-4-ones (6 and 8) were prepared by a two-step
synthetic approach as illustrated in our previous reported work
[20]. Finally, target compounds 7 and 9 were generated via coupling
reactions, in refluxing methanol, between 5-chloro-6-
chloromethyluracil 5 and 1,3-dihydropyrazolo[1,5-a][1,3,5]triazin-
2-thioxo-4-ones 6 or 8 (Scheme 2). This coupling reaction has not
been reported hitherto. The presence of sodium ethoxide was
necessary for this type of reaction since it was needed to generate
highly nucleophilic anions through the abstraction of the acidic
proton on the pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-one. The
anionic forms of pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones
would then react with 5-chloro-6-chloromethyluracil via a nucle-
ophilic attack to generate the target compounds in good yields
(Yields 60%e92% for 7aem, yields 40%e96% for 9aeq).
IC50 value ¼ 13.0 ꢀ 4.4
mM) and 8 (9g, IC50 value ¼ 9.7 ꢀ 2.42
mM).
The phenyl effect was seen throughout in series 12 and 13. For
instance, based on a comparison between compounds 7a & 7m, 7b
& 7i, 7c & 7j and 7d & 7h, the results showed that the inclusion of a
phenyl ring had improved the TP inhibition by 5.2, 5.7, 34.1 and 14.8
times respectively. A similar phenyl effect was also observed for
compounds 7a & 9g, 9a & 9l, 9b & 9i and 9c & 9j. The changes in TP
inhibition in these cases were 7.0, 4.8, 17.8 and 33.3 times respec-
tively. Therefore, it is very clear from the results that the presence of
the phenyl ring is important for the inhibitory activity of this type of
compounds, and it seems to suggest that there is a potential hy-
drophobic interaction site close to positions 7 and 8.
A total of 30 compounds with different substituents on the
pyrazolo[1,5-a][1,3,5]triazine scaffold were synthesized. None of
these target compounds has been reported previously and all of
them were systematically characterized by melting points and
In addition to the requirement of a phenyl ring, the substituent
at position 4 of this phenyl ring also appeared to be an important
contributory factor to the inhibitory activity. For compounds with a
phenyl ring at position 7, the fact that inhibitory activity of 7i < 7j
suggested that increasing the hydrophobicity of substituents on the
phenyl ring would improve the potency. This observation is
consistent with the Craig plot where the methyl group and the tert-
butyl group are known to possess similar electron donating prop-
erties while the hydrophobicity of methyl group <tert-butyl group.
The same trend was also observed for compounds with sub-
stituents located in the top right hand quadrant of the Craig plot
O
N
O
N
N
N
Cl
O
HN
N
Cl
O
HN
N
O
O
R
R
S
S
HN
NH
HN
NH
A
B
Fig. 3. Dissection analysis of 3H-2-(5-chlorouracil-6-methylthio)-pyrazolo[1,5-a]
[1,3,5]triazin-4-ones.
(þs
, þp): Potency of 7e < 7f < 7g < 7h while their relative

L. Sun et al. / European Journal of Medicinal Chemistry 70 (2013) 400e410
403
O
N
O
O
N
Cl
N
Cl
O
HN
N
HN
HN
N
+
O
Cl
R
S
R
O
N
H
S
N
H
HN
NH
3H-2-(5-chlorouracil-6-methylthio)
pyrazolo[1,5-a][1,3,5]triazin-4-ones
5-chloro-6-chloro
methyluracil
1,3-dihydro-pyrazolo[1,5-a]
[1,3,5]triazin-2-thioxo-4-ones
Fig. 4. Retrosynthesis of 5-chlorouracil linked pyrazolo[1,5-a][1,3,5]triazines.
substituents, which were fluoro, chloro, bromo, trifluoromethyl
respectively, showed an increase in hydrophobicity. However, the
fact that 7e was less potent than 7m indicated that the electron
withdrawing property of the substituent might reduce the inhibi-
tory activity.
same point. This observation pointed to the fact that 9q exhibited
mixed inhibition kinetics on TP with respect to thymidine being the
substrate (Fig. 5). This was further supported by the fact that the
Vmax values decreased from 8.9
m
M/min to 5.9
M to 2,829
concentration increased from 0 to 2 mM (Km and Vmax values are
m
M/min, while the
Km values increased from 770
m
mM as the inhibitor
For compounds with a phenyl ring at position 8, it was also
found that the inhibitory activity would be improved while
increasing the hydrophobicity of substituents on the phenyl ring
based on the result that potency of methoxy substituted compound
9n <ethoxy substituted compound 9o <methyl substituted com-
pound 9l <tert-butyl substituted compound 9m as well as potency
of fluoro substituted compound 9h <chloro substituted compound
9i <bromo substituted compound 9j and trifluoromethyl
substituted compound 9k. However, different from compounds
with a phenyl ring at position 7, the fact that compound 9m with
the more hydrophobic tert-butyl group was less potent than bromo
substituted compound 9j and trifluoromethyl substituted com-
pound 9k suggested that the electro donating property of the
substituent might reduce the inhibitory activity. This conclusion
was also supported by the fact that methoxy substituted compound
9n was less potent than 9g. Therefore, for compounds with a phenyl
ring at position 8, electron withdrawing and hydrophobic sub-
stituents on position 4 of this phenyl ring were preferred. Further
synthesized compound 9p and 9q with more electron withdrawing
and hydrophobic iodo group and pentafluorosulfur group showed
calculated from the EadieeHofstee plots) This behaviour of the
change in Vmax and Km values of the enzyme is typical of an in-
hibitor that exhibits mixed inhibition kinetics, and it was possible
that 9q might interact with TP at two different sites–the substrate
binding site and an allosteric site. The Ki value of 9q was calculated
as 0.44
mM by a reported method [24], and the Ki/Km value was
0.00057.
2.4. A comparison between the TP inhibitory activities of pyrazolo
[1,5-a][1,3,5]triazines versus their corresponding 5-chlorouracil
linked derivatives
Since
3H-2-(5-chlorouracil-6-methylthio)-pyrazolo[1,5-a]
[1,3,5]triazin-4-ones were generated from the corresponding 1,3-
dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones, it was of
interest to compare these two series of TP inhibitors to investigate
the effect of introducing the 5-chlorouracil ring on TP inhibition.
TP inhibitory activity of 1,3-dihydro-pyrazolo[1,5-a][1,3,5]tri-
azin-2-thioxo-4-ones has been evaluated in our previous studies
[20], and it was found that the SAR of 5-chlorouracil-linked-pyr-
azolo[1,5-a][1,3,5]triazines was similar to the SAR of fused bicyclic
pyrazolo[1,5-a][1,3,5]triazines (Fig. 6). In our previous studies,
fused bicyclic pyrazolo[1,5-a][1,3,5]triazines were found to be non-
competitive inhibitors, and they might interact with TP at an allo-
steric site. Therefore, TP inhibitory activity of 5-chlorouracil-linked-
pyrazolo[1,5-a][1,3,5]triazines should be predominated by the
interaction between the pyrazolo[1,5-a][1,3,5]triazine scaffold and
the allosteric site.
better activity with IC50 values as low as 0.61
repectively.
mM and 0.36 mM
In summary, our results of the in vitro enzyme assay has pro-
vided evidence to support the hypothesis that by using TPI and
KIN59 as leads, our designed target compounds consisting of both
pyrimidine and purine-type moieties linked via a methylthio
spacer would exhibit TP inhibitory activity.
2.3. Enzyme inhibition kinetic studies
When 5-chlorouracil was chemically coupled to the pyrazolo
[1,5-a][1,3,5]triazine scaffold, the 5-chlorouracil moiety would be
expected to affect the binding affinity of the pyrazolo[1,5-a][1,3,5]
triazine scaffold at the allosteric site. On the other hand, the pres-
ence of the 5-chlorouracil moiety would provide binding possibility
at the substrate binding site. Therefore, the resultant biological
effect of introducing the 5-chlorouracil moiety would depend on
Further investigation was carried out to determine the enzyme
inhibition kinetics of 9q using various concentrations of the sub-
strate thymidine. [23] Four concentrations of 9q (0, 0.1
mM, 0.5 mM,
and 2 M) were chosen for performing the enzyme inhibition ki-
m
netic study. It was demonstrated on the LineweavereBurk plot that
the inhibition lines did not intersect the x axis nor the y axis at the
O
O
O
O
O
Cl
a
b
d
c
HN
HN
HN
HN
HN
O
N
H
O
N
H
O
N
H
CHO
O
N
H
O
N
H
Cl
OH
Cl
1
2
3
5
4
Scheme 1. Synthesis of 5-chloro-6-chloromethyluracil. Reagents and conditions: (a) SeO2, AcOH, reflux, yield 35%; (b) NaBH4, MeOH/H2O, 0 ꢁC, yield 79%; (c) SOCl2, reflux, yield
95%; (d) SO2Cl2, AcOH, yield 68%.

404
L. Sun et al. / European Journal of Medicinal Chemistry 70 (2013) 400e410
O
O
O
Cl
Cl
N
N
HN
HN
N
a
Cl
O
HN
N
R
R
+
O
O
N
H
S
N
H
S
N
HN
NH
5
6a-m
7a-m
7a R=H
7b R=CH3
6a R=H
6b R=CH3
7c R=C(CH3)3
7d R=CF3
6c R=C(CH3)3
6d R=CF3
7e R=4-fluorophenyl
7f R=4-chlorophenyl
7g R=4-bromophenyl
7h R=4-trifluoromethylphenyl
7i R=4-methylphenyl
7j R=4-tert-butylphenyl
7k R=4-methoxyphenyl
7l R=4-ethoxyphenyl
7mR=phenyl
6e R=4-fluorophenyl
6f R=4-chlorophenyl
6g R=4-bromophenyl
6h R=4-trifluoromethylphenyl
6i R=4-methylphenyl
6j R=4-tert-butylphenyl
6k R=4-methoxyphenyl
6l R=4-ethoxyphenyl
6mR=phenyl
O
O
O
Cl
Cl
N
N
HN
HN
N
Cl
O
HN
N
a
+
O
O
N
H
S
N
H
S
N
R
R
HN
NH
5
9a-q
8a-q
9a R=CH3
9b R=Cl
9c R=Br
9d R=I
9e R=CN
9f R=COOEt
9g R=phenyl
9h R=4-fluoro-phenyl
9i R=4-chloro-phenyl
9j R=4-bromo-phenyl
9k R=4-trifluoromethyl-phenyl
9l R=4-methyl-phenyl
9mR=4-tert-butyl-phenyl
9n R=4-methoxy-phenyl
9o R=4-ethoxy-phenyl
9p R=4-iodo-phenyl
8a R=CH3
8b R=Cl
8c R=Br
8d R=I
8e R=CN
8f R=COOEt
8g R=phenyl
8h R=4-fluoro-phenyl
8i R=4-chloro-phenyl
8j R=4-bromo-phenyl
8k R=4-trifluoromethyl-phenyl
8l R=4-methyl-phenyl
8mR=4-tert-butyl-phenyl
8n R=4-methoxy-phenyl
8o R=4-ethoxy-phenyl
8p R=4-iodo-phenyl
8q R=4-pentafluorosulfur-phenyl
9q R=4-pentafluorosulfur-phenyl
Scheme 2. Synthesis of 3H-2-(5-chlorouracil-6-methylthio)-pyrazolo[1,5-a]-1,3,5-triazin-4-ones. Reagents and conditions: (a) EtONa, MeOH, reflux, yields 60%e92% (7aem) and
40%e96% (9aeq).
the cooperativity between this moiety and the pyrazolo[1,5-a]
[1,3,5]triazine scaffold. If the compromise in binding affinity of the
pyrazolo[1,5-a][1,3,5]triazine scaffold at the allosteric site could be
compensated by the interaction of 5-chlorouracil moiety at the
substrate binding site, the inhibitory activity would increase upon
linking with the 5-chlorouracil moiety. If such a compromise could
not be compensated by the increase of one more intermolecular
interaction at the substrate binding site, the inhibitory activity
would decrease upon linking with the 5-chlorouracil moiety. Based
on the relative activities calculated (Table 2), it was found that for
almost all of the 7-substituted pyrazolo[1,5-a][1,3,5]triazines (6ae
m), coupling with the 5-chlorouracil moiety gave rise to improved
inhibitory action against TP. Therefore, for these 7-substituted
pyrazolo[1,5-a][1,3,5]triazines, the effect of one more substrate
binding site overcame the effect of less binding affinity at the
allosteric site. However, for pyrazolo[1,5-a][1,3,5]triazines with
substituents located at position 8 (8aeq), the relative inhibitory
activities were not consistent and did not follow a trend. In this
case, it was found that 8 of the 17 linked compounds became less
active upon coupling with 5-chlorouracil. Closer examination of the
data revealed that five of the most potent compounds in this library
that exhibited IC50 values less than 1 mM (viz 8i, 8j, 8k, 8p, and 8q)
had their activity decreased to 0.1e0.5 times after coupling.
Therefore, for these more potent pyrazolo[1,5-a][1,3,5]triazines, the
compromise in the interaction between the pyrazolo[1,5-a][1,3,5]
triazine scaffold and the allosteric site could not be compensated by
one more substrate binding site.
3. Conclusion
In conclusion, the chosen synthetic scheme was successful in
coupling the 5-chlorouracil moiety to 6 and 8 thus generating a
total of 30 compounds (7aem, 9aeq) that inhibited TP to various
extent. The flexible methylthio linker was found to be a suitable
spacer for bridging the 5-chlorouracil moiety with the pyrazolo
[1,5-a][1,3,5]triazine scaffold thus enabling the retention of the

L. Sun et al. / European Journal of Medicinal Chemistry 70 (2013) 400e410
Table 1 (continued )
405
Table 1
TP inhibitory activity of 3H-2-(5-chlorouracil-6-methylthio)-pyrazolo[1,5-a]-1,3,5-
triazin-4-ones..
a
Cpd
R1
R2
IC50 (mM)
O
9i
H
H
H
2.8 ꢀ 0.6
1.2 ꢀ 0.3
1.3 ꢀ 0.3
Cl
Br
N
Cl
O
HN
N
R1
O
S
N
9j
R2
HN
NH
F
F
9k
F
a
Cpd
R1
R2
IC50 (mM)
7a
7b
H
Me
H
H
67.8 ꢀ 4.4
55.5 ꢀ 6.8
9l
H
H
H
H
H
7.3 ꢀ 0.6
1.8 ꢀ 0.1
7c
H
51.2 ꢀ 6.1
9m
9n
9o
9p
9q
F
F
21.3 ꢀ 2.1
12.0 ꢀ 1.0
0.61 ꢀ 0.19
0.36 ꢀ 0.10
OMe
OEt
I
7d
H
31.0 ꢀ 0.5
F
7e
7f
H
H
H
22.6 ꢀ 1.9
10.3 ꢀ 2.2
6.2 ꢀ 1.6
F
Cl
Br
H
SF5
7g
TPI
e
e
0.0027 ꢀ 0.0001
a
Values are mean, n ¼ 3.
F
7h
H
2.1 ꢀ 0.4
F
inhibitory action on TP. The most active compound 9q demon-
strated an IC50 value as low as 0.36 M, and it was found to exhibit
F
m
mixed-type of enzyme inhibition kinetics thus suggesting the
possibility of it binding at two sites in the enzyme. A comparison
between 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones
and 3H-2-(5-chlorouracil-6-methylthio)-pyrazolo[1,5-a][1,3,5]tri-
azin-4-ones revealed non-consistent inhibitory activity changes
which might be attributed to additive effects of increase in number
of binding sites together with weakening of the interaction at the
allosteric site upon linked with 5-chlorouracil. The most significant
outcome of this work has been the successful development of a
mixed type TP inhibitor 9q via the fragment combination approach.
In addition, preliminary cytotoxicity studies on 9q were carried out.
The results showed that cell viability of the breast cancer cell line
MDA-MB-231 was about 67% of the control in the MTT assay where
7i
7j
H
H
9.8 ꢀ 2.0
1.5 ꢀ 0.4
7k
7l
H
H
H
11.8 ꢀ 1.9
10.0 ꢀ 1.3
13.0 ꢀ 4.4
OMe
OEt
9q was used in a concentration of 100 mM. This indicated that 9q
may not exhibit severe cytotoxic effect and that it has the potential
for being developed into a safe anti-angiogenesis agent.
7m
9a
9b
9c
9d
9e
9f
H
H
H
H
H
H
Me
Cl
Br
I
CN
COOEt
35.3 ꢀ 5.4
49.8 ꢀ 1.5
40.0 ꢀ 7.2
30.8 ꢀ 2.2
38.8 ꢀ 5.7
47.2 ꢀ 9.5
4. Experimental section
4.1. Chemistry
All starting materials were obtained from commercial suppliers
and used without further purification. Melting points were deter-
mined on a Gallenkamp melting point apparatus without correc-
9g
9h
H
H
9.7 ꢀ 2.4
tion. NMR spectra were recorded, on
spectrometer, in DMSO-d6 and using TMS as the internal standard.
Chemical shifts ( ) were reported in parts per million downfield
from the internal standard. The signals were quoted as s (singlet),
a Bruker DPX-300
8 ꢀ 1.0
(continued on next page)
F
d

406
L. Sun et al. / European Journal of Medicinal Chemistry 70 (2013) 400e410
the samples were prepared by dissolving them in methanol. The
analysis was performed at 30 ꢁC with a suitable mobile phase at a
flow rate of 1 ml/min, and the ultraviolet detection was made at
wavelength 254 nm. The separations were carried out using
gradient elution. The initial mobile phase consisting of water with
0.1% acetic acid and acetonitrile with 0.1% acetic acid (95:5) was
eluted for 1 min. This was gradually changed to 0:100 within 6 min
and remained at 0:100 for another 1 min. The mobile phase was
then gradually changed back to the starting composition within
6 min and maintained for one more minute. The injection volume
was 5 mL. The purity of all the compounds was found to be more
than 95% which was satisfactory for bioassay.
4.2. General method for preparing 3H-2-(5-chlorouracil-6-
methylthio)-pyrazolo[1,5-a][1,3,5]triazin-4-ones (7, 9)
0.5 mmol pyrazolo[1,5-a]-1,3,5-triazine-2-thioxo-4(1H,3H)-
ones and 0.5 mmol 5-chloro-6-chloromethyluracil were refluxed in
a mixture of 4 ml methanol/ethanol and 0.19 ml 21%wt EtONa
ethanol solution for 0.5 h. Precipitate was filtered, washed with
cold water, recrystallized from dmso/water and dried in oven to
give products, yield 40%e96%.
Fig. 5. LineweavereBurk plots of thymidine phosphorylase inhibition by compound 9q
prepared at different concentrations. All data points are means of three experiments.
4.2.1. 3H-2-(5-chlorouracil-6-methylthio)-pyrazolo[1,5-a][1,3,5]
d (doublet), t (triplet), m (multiplet). Column chromatography was
conducted on silica gel (230e400 mesh). ESI-HRMS was recorded
on a Micromass Q-Tof Premier. HPLC analysis was carried out on all
the compounds used in biological assays, using a HewlettePackard
series 1050 HPLC system equipped with a HP-1050 quaternary
pump, a degasser, diode array detector, a HP-1100 autosampler and
triazin-4-one (7a)
Yield 60%. Mp: 268e270 ꢁC. 1HNMR (DMSO-d6):
d 4.34 (s, 2H,
CH2), 6.37 (d, J ¼ 1.2 Hz, 1H, CH), 8.01 (d, J ¼ 1.2 Hz, 1H, CH), 11.33 (s,
1H, NH), 11.64 (s, 1H, NH), 13.00 (s, 1H, NH). 13CNMR (DMSO-d6):
d
30.3, 98.6, 107.6, 144.5, 146.8, 148.0, 149.0, 150.8, 155.5, 160.7.
HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C10H6ClN6O3S 324.9911, found
a LiChrosorb reversed phase C18 (5
mm) column (4.6 ꢂ 250 mm). All
324.9953.
Fig. 6. Summary of SAR: (a) SAR of fused bicyclic pyrazolo[1,5-a][1,3,5]triazines; (b) SAR of 5-chlorouracil-linked-pyrazolo[1,5-a][1,3,5]triazines.

L. Sun et al. / European Journal of Medicinal Chemistry 70 (2013) 400e410
407
H]e calcd for C16H9ClFN6O3S
Table 2
163.7. HRMSeESI (m/z): [M
419.0129, found 419.0026.
ꢃ
Comparison between the two series of TP inhibitors.
Cpd
IC50
(m
M)
Cpd
IC50
(
mM)
Relative TP inhibitory activitya
4.2.6. 3H-7-(4-chlorophenyl)-2-(5-chlorouracil-6-methylthio)-
6a
6b
6c
6d
6e
6f
6g
6h
6i
68.2 ꢀ 2.7
79.4 ꢀ 8.2
29.1 ꢀ 4.6
32.6 ꢀ 5.7
41.7 ꢀ 0.5
33.5 ꢀ 3.8
34.9 ꢀ 6.3
9.9 ꢀ 1.9
7a
7b
7c
7d
7e
7f
7g
7h
7i
67.8 ꢀ 4.4
55.5 ꢀ 6.8
51.2 ꢀ 6.1
31.0 ꢀ 0.5
22.6 ꢀ 1.9
10.3 ꢀ 2.2
6.2 ꢀ 1.6
2.1 ꢀ 0.4
9.8 ꢀ 2.0
1.5 ꢀ 0.4
11.8 ꢀ 1.9
10.0 ꢀ 1.3
13.0 ꢀ 4.4
35.3 ꢀ 5.4
49.8 ꢀ 1.5
40.0 ꢀ 7.2
30.8 ꢀ 2.2
38.8 ꢀ 5.7
47.2 ꢀ 9.5
9.7 ꢀ 2.4
8.0 ꢀ 1.0
2.8 ꢀ 0.6
1.2 ꢀ 0.3
1.3 ꢀ 0.3
7.3 ꢀ 0.6
1.8 ꢀ 0.1
21.3 ꢀ 2.1
12.0 ꢀ 1.0
0.61 ꢀ 0.19
0.36 ꢀ 0.10
1.0
1.4
0.6
1.1
1.8
3.3
5.6
4.7
2.0
4.4
2.4
0.6
3.6
2.5
0.6
1.1
0.7
w2.6
w2.1
2.7
1.3
0.2
0.5
0.2
0.2
3.1
1.9
1.3
0.3
0.1
pyrazolo[1,5-a][1,3,5]triazin-4-one (7f)
Yield 85%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d 4.36 (s, 2H, CH2),
6.91 (s, 1H, CH), 7.57 (d, 2H, J ¼ 8.4 Hz, CH), 8.00 (d, J ¼ 8.4 Hz, 2H,
CH), 11. 35 (s, 1H, NH), 11.64 (s, 1H, NH), 13.11 (s, 1H, NH). 13CNMR
(DMSO-d6):
d 30.4, 95.7, 107.6, 128.9, 130.0, 131.8, 135.0, 144.5,
148.0, 150.3, 150.8, 155.4, 156.4, 160.7. HRMSeESI (m/z): [M ꢃ H]e
19.3 ꢀ 2.3
6.6 ꢀ 1.6
calcd for C16H9Cl2N6O3S 434.9834, found 434.9770.
6j
6k
6l
7j
7k
7l
28.4 ꢀ 2.0
6.1 ꢀ 0.7
4.2.7. 3H-7-(4-bromophenyl)-2-(5-chlorouracil-6-methylthio)-
pyrazolo[1,5-a][1,3,5]triazin-4-one(7g)
6m
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
46.2 ꢀ 1.8
87.3 ꢀ 6.0
27.6 ꢀ 1.3
44.4 ꢀ 8.6
20.8 ꢀ 2.7
>100
7m
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
Yield 92%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
4.25 (s, 2H, CH2),
6.61 (s,1H, CH), 7.62 (d, J ¼ 8.4 Hz, 2H, CH), 7.92 (d, J ¼ 8.4 Hz, 2H, CH),
11.59 (s, 2H, NH). 13CNMR (DMSO-d6):
29.1, 91.4, 105.4,121.8,127.9,
d
131.5, 132.0, 147.1, 148.5, 149.5, 150.8, 152.5, 159.6, 161.4. HRMSeESI
(m/z): [M ꢃ H]ꢃ calcd for C16H9BrClN6O3S 478.9329, found 478.9447.
>100
26.3 ꢀ 2.0
10.5 ꢀ 3.3
0.54 ꢀ 0.04
0.63 ꢀ 0.12
0.24 ꢀ 0.04
1.7 ꢀ 0.1
4.2.8. 3H-2-(5-chlorouracil-6-methylthio)-7-(4-
trifluoromethylphenyl)-pyrazolo[1,5-a][1,3,5]triazin-4-one(7h)
Yield 72%. Mp: 258e260 ꢁC. 1HNMR (DMSO-d6):
d 4.37 (s, 2H,
CH2), 6.99 (s, 1H, CH), 7.86 (d, J ¼ 8.0 Hz, 2H, CH), 8.22 (d, J ¼ 8.0 Hz,
2H, CH), 11.37 (s, 1H, NH), 11.65 (s, 1H, NH). 13CNMR (DMSO-d6):
5.6 ꢀ 1.3
39.7 ꢀ 9.9
15.5 ꢀ 0.3
0.21 ꢀ 0.03
0.04 ꢀ 0.005
d
29.3, 95.1, 106.5, 122.8, 125.5, 125.8, 125.8, 126.8, 129.2, 129.5,
135.8, 143.6, 147.0, 149.4, 149.7, 153.9, 155.6, 159.7. HRMSeESI (m/z):
[M ꢃ H]e calcd for C17H9ClF3N6O3S 469.0097, found 468.9975.
a
The relative TP inhibitory activity was calculated by taking the ratio of the IC50
value of the pyrazolo[1,5-a][1,3,5]triazine to the IC50 value of the corresponding 5-
chlorouracil coupled compound (eg. IC50 value ratio of 6a to 7a and 8a to 9a etc).
4.2.9. 3H-2-(5-chlorouracil-6-methylthio)-7-(4-methylphenyl)-
pyrazolo[1,5-a][1,3,5]triazin-4-one(7i)
Yield 84%. Mp: 268e269 ꢁC. 1HNMR (DMSO-d6):
d 2.34 (s, 3H,
CH3), 4.29 (s, 2H, CH2), 6.65 (s, 1H, CH), 7.27 (d, J ¼ 8 Hz, 2H, CH),
4.2.2. 3H-2-(5-chlorouracil-6-methylthio)-7-methyl-pyrazolo[1,5-
7.84 (d, J ¼ 8 Hz, 2H, CH), 11.48 (s, 1H, NH), 11.62 (s, broad, 1H, NH).
a][1,3,5]triazin-4-one (7b)
13CNMR (DMSO-d6):
d 20.9, 29.2, 92.7, 105.9, 126.0, 126.1, 129.3,
Yield 79%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
2.29 (s, 3H, CH3),
129.4, 129.6,138.5,147.9, 149.7, 149.9,154.6,159.7. HRMSeESI (m/z):
4.32 (s, 2H, CH2), 6.19 (s, 1H, CH), 11.30 (s, 1H, NH), 11.62 (s, 1H, NH),
[M ꢃ H]ꢃ calcd for C17H12ClN6O3S 415.038, found 415.0350.
12.88 (s,1H, NH). 13CNMR (DMSO-d6):
d 15.2, 30.3, 98.6,107.6,144.1,
148.1, 149.4, 150.8, 155.4, 156.0, 160.7. HRMSeESI (m/z): [M ꢃ H]ꢃ
4.2.10. 3H-2-(5-chlorouracil-6-methylthio)-7-(4-tert-
calcd for C11H8ClN6O3S 339.0067, found 339.0071.
butyllphenyl)-pyrazolo[1,5-a][1,3,5]triazin-4-one(7j)
Yield 74%. Mp: 252e254 ꢁC. 1HNMR (DMSO-d6):
d 1.32 (s, 9H,
4.2.3. 3H-2-(5-chlorouracil-6-methylthio)-7-tert-butyl-pyrazolo
CH3), 4.37 (s, 2H, CH2), 6.85 (s, 1H, CH), 7.51 (d, J ¼ 8.4 Hz, 2H, CH),
[1,5-a][1,3,5]triazin-4-one (7c)
7.91 (d, J ¼ 8.4 Hz, 2H, CH), 11.37 (s, 1H, NH), 11.66 (s, 1H, NH).
Yield 63%. Mp: 208e209 ꢁC. 1HNMR (DMSO-d6):
d
1.29 (s, 9H,
13CNMR (DMSO-d6):
d 29.2, 30.9, 34.4, 94.5, 106.4, 125.5, 125.9,
CH3), 4.29 (s, 2H, CH2), 6.23 (s,1H, CH),11.43 (s,1H, NH),11.62 (s,1H,
129.0, 143.4, 146.9, 149.0, 149.7, 151.9, 154.9, 155.5, 159.6. HRMSeESI
NH). 13CNMR (DMSO-d6):
d 29.1, 29.7, 32.4, 93.6, 106.1, 144.2, 147.4,
(m/z): [M ꢃ H]ꢃ calcd for C20H18ClN6O3S 457.085, found 457.0832.
148.5,149.6,155.7,159.6,166.6. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for
C
14H14ClN6O3S 381.0537, found 381.0407.
4.2.11. 3H-2-(5-chlorouracil-6-methylthio)-7-(4-methoxyphenyl)-
pyrazolo[1,5-a][1,3,5]triazin-4-one (7k)
4.2.4. 3H-2-(5-chlorouracil-6-methylthio)-7-trifluoromethyl-
pyrazolo[1,5-a][1,3,5]triazin-4-one (7d)
Yield 88%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d 3.81 (s, 3H, CH3),
4.24 (s, 2H, CH2), 6.49 (s, 1H, CH), 7.026 (d, J ¼ 8.7 Hz, 2H, CH), 7.86
Yield 67%. Mp: 288e289 ꢁC. 1HNMR (DMSO-d6):
d
4.34 (s, 2H,
CH2), 6.78 (s, 1H, CH), 11.31 (s, 1H, NH), 11.62 (s, 1H, NH). 13CNMR
(DMSO-d6): 13CNMR (DMSO-d6):
29.3, 94.9, 106.6, 116.8, 119.5,
(d, J ¼ 8.7 Hz, 2H, CH), 11. 59 (s, 1H, NH). 13CNMR (DMSO-d6):
d 29.1,
55.1, 90.6, 105.3, 114.0, 125.3, 127.3, 147.6, 148.7, 149.5, 150.8, 153.5,
d
159.6, 159.7, 161.6. HRMSeESI (m/z): [M
C17H12ClN6O4S 431.0329, found 431.0340.
ꢃ
H]ꢃ calcd for
122.2, 124.8, 143.1, 145.1, 145.4, 145.8, 146.2, 146.7, 149.3, 149.6,
157.0, 159.6. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C11H5ClF3N6O3S
392.9784, found 392.9573.
4.2.12. 3H-2-(5-chlorouracil-6-methylthio)-7-(4-ethoxyphenyl)-
pyrazolo[1,5-a][1,3,5]triazin-4-one (7l)
4.2.5. 3H-2-(5-chlorouracil-6-methylthio)-7-(4-fluorophenyl)-
pyrazolo[1,5-a][1,3,5]triazin-4-one (7e)
Yield 76%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
1.35 (t, J ¼ 6.8 Hz,
3H, CH3), 4.07 (q, J ¼ 6.8 Hz, 2H, CH2), 4.20 (s, 2H, CH2), 6.35 (s, 1H,
Yield 90%. Mp: 212e214 ꢁC. 1HNMR (DMSO-d6):
d 4.35 (s, 2H,
CH), 6.98 (d, J ¼ 8.8 Hz, 2H, CH), 7.86 (d, J ¼ 8.8 Hz, 2H, CH), 11.58 (s,
CH2), 6.86 (s, 1H, CH), 7.32 (t, J ¼ 8.7 Hz, 2H, CH), 8.03 (dd,
1H, NH), 11.74 (s, broad, 1H, NH). 13CNMR (DMSO-d6):
d 14.6, 29.0,
J ¼ 8.7 Hz, JHeF ¼ 5.7 Hz, 2H, CH), 11.36 (s, 1H, NH), 11.63 (s, 1H, NH).
48.5, 63.0, 88.9, 104.8, 114.3, 125.7, 127.2, 149.4, 149.6, 151.6, 152.6,
158.7, 159.8, 165.1. HRMSeESI (m/z): [M
H]ꢃ calcd for
18H14ClN6O4S 445.0486, found 445.0728.
13CNMR (DMSO-d6):
d
29.2, 92.1, 105.8, 115.5, 115.7, 128.1, 128.2,
ꢃ
129.1, 129.1, 146.4, 148.3, 149.6, 150.5, 153.3, 159.8, 160.0, 161.3,
C

408
L. Sun et al. / European Journal of Medicinal Chemistry 70 (2013) 400e410
4.2.13. 3H-2-(5-chlorouracil-6-methylthio)-7-phenyl-pyrazolo[1,5-
159.7, 161.9. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C16H10ClN6O3S
a][1,3,5]triazin-4-one (7m)
401.0224, found 401.0307.
Yield 89%. Mp: 156e158 ꢁC. 1HNMR (DMSO-d6):
d
4.36 (s, 2H,
CH2), 6.89 (s, 1H, CH), 7.47 (m, 3H, CH), 7.98 (d, J ¼ 7.2 Hz, 2H, CH),
11.35 (s, 1H, NH), 11.64 (s, 1H, NH). 13CNMR (DMSO-d6):
29.2, 94.5,
4.2.21. 3H-2-(5-chlorouracil-6-methylthio)-8-(4-fluorophenyl)-
pyrazolo[1,5-a][1,3,5]triazin-4-one (9h)
d
106.4, 126.1, 128.8, 129.3, 131.8, 143.4, 146.9, 149.1, 149.7, 155.4,
159.6, 161.1. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C16H10ClN6O3S
401.0224, found 401.0011.
Yield 90%. Mp: 288e289 ꢁC. 1HNMR (DMSO-d6):
d 4.43 (s, 2H,
CH2), 7.23 (t, J ¼ 8.7 Hz, 2H, CH), 8.02 (dd, J ¼ 8.7 Hz, JHeF ¼ 5.7 Hz,
2H, CH), 8.47 (s, 1H, CH), 11.39 (s, 1H, NH), 11.66 (s, 1H, NH), 13.17 (s,
broad, 1H, NH). 13CNMR (DMSO-d6):
d
29.4, 105.8, 108.0, 115.2,
115.5, 127.3, 127.9, 142.8, 144.4, 144.9, 145.1, 147.5, 149.6, 158.0,
158.9, 159.6, 162.1. HRMSeESI (m/z): [M
H]ꢃ calcd for
C16H9ClFN6O3S 419.0129, found 419.0178.
4.2.14. 3H-2-(5-chlorouracil-6-methylthio)-8-methyl-pyrazolo[1,5-
a][1,3,5]triazin-4-one (9a)
ꢃ
Yield 59%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
2.12 (s, 3H, CH3),
4.37 (s, 2H, CH2), 7.86 (s, 1H, CH), 11.32 (s, 1H, NH), 11.63 (s, 1H, NH),
12.78 (s, broad, 1H, NH). 13CNMR (DMSO-d6):
7.1, 29.1105.5, 106.0,
d
4.2.22. 3H-2-(5-chlorouracil-6-methylthio)-8-(4-chlorophenyl)-
144.0, 145.0, 146.2, 147.6, 149.6, 153.9, 159.6. HRMSeESI (m/z):
[M ꢃ H]ꢃ calcd for C11H8ClN6O3S 339.0067, found 339.0207.
pyrazolo[1,5-a][1,3,5]triazin-4-one (9i)
Yield 83%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
4.39 (s, 2H, CH2),
7.41 (d, J ¼ 8.4 Hz, 2H, CH), 7.99 (d, J ¼ 8.4 Hz, 2H, CH), 8.40 (s, 1H,
CH), 11.40 (s, 1H, NH), 11.64 (s, 1H, NH). 13CNMR (DMSO-d6):
29.5,
105.7, 107.0, 127.0, 128.6, 130.0, 130.8, 142.4, 145.4, 146.1, 148.0,
149.7, 159.7, 160.2. HRMSeESI (m/z): [M
H]ꢃ calcd for
16H9Cl2N6O3S 434.9834, found 434.9847.
4.2.15. 3H-8-chloro-2-(5-chlorouracil-6-methylthio)-pyrazolo[1,5-
d
a][1,3,5]triazin-4-one (9b)
Yield 59%. Mp: 162e164 ꢁC 1HNMR (DMSO-d6):
d
4.40 (s, 2H,
ꢃ
CH2), 8.15 (s, 1H, CH), 11.28 (s, 1H, NH),11.62 (s, 1H, NH), 13.04 (s, 1H,
C
NH). 13CNMR (DMSO-d6):
d 30.3, 100.0, 107.5, 144.1, 144.8, 144.9,
147.9, 150.8, 157.3, 160.7. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for
4.2.23. 3H-8-(4-bromophenyl)-2-(5-chlorouracil-6-methylthio)-
C
10H5Cl2N6O3S 358.9521, found 358.9397.
pyrazolo[1,5-a][1,3,5]triazin-4-one (9j)
Yield 93%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d 4.39 (s, 2H, CH2),
4.2.16. 3H-8-bromo-2-(5-chlorouracil-6-methylthio)-pyrazolo[1,5-
7.56 (d, J ¼ 8.4 Hz, 2H, CH), 7.95 (d, J ¼ 8.7 Hz, 2H, CH), 8.42 (s, 1H,
a][1,3,5]triazin-4-one (9c)
CH), 11.43 (s, 1H, NH), 11.64 (s, 1H, NH), 13.18 (s, 1H, NH). 13CNMR
Yield 79%. Mp: 238e240 ꢁC. 1HNMR (DMSO-d6):
d
4.41 (s, 2H,
(DMSO-d6): d 29.4,105.6,107.1, 118.4, 127.3, 131.1, 131.4, 142.3,145.3,
CH2), 8.13 (s, 1H, CH), 11.28 (s, 1H, NH), 11.62 (s, 1H, NH), 13.08 (s, 1H,
145.8,147.8,149.6,159.6,160.0. HRMSeESI (m/z): [M ꢃ H]e calcd for
NH). 13CNMR (DMSO-d6):
d
30.2, 85.2, 107.4, 144.0, 146.4, 146.6,
C16H9BrClN6O3S 478.9329, found 478.9447.
148.0, 150.8, 157.4, 160.8. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for
C
10H5BrClN6O3S 402.9016, found 402.9043.
4.2.24. 3H-2-(5-chlorouracil-6-methylthio)-8-(4-
trifluoromethylphenyl)-pyrazolo[1,5-a][1,3,5]triazin-4-one (9k)
4.2.17. 3H-2-(5-chlorouracil-6-methylthio)-8-iodo-pyrazolo[1,5-a]
Yield 96%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
4.48 (s, 2H, CH2),
7.76 (d, J ¼ 8.4 Hz, 2H, CH), 8.22 (d, J ¼ 8.4 Hz, 2H, CH), 8.63 (s, 1H,
CH), 11.37 (s, 1H, NH), 11.68 (s, 1H, NH). 13CNMR (DMSO-d6):
29.5,
[1,3,5]triazin-4-one (9d)
Yield 63%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
4.44 (s, 2H, CH2),
d
8.08 (s, 1H, CH), 11.29 (s, 1H, NH), 11.63 (s, 1H, NH), 13.19 (s, 1H, NH).
106.1, 108.2, 123.0, 125.4, 125.5, 125.7, 126.0, 126.3, 126.7, 128.4,
135.4, 143.6, 143.8, 144.9, 146.9, 149.6, 159.5. HRMSeESI (m/z):
[M ꢃ H]ꢃ calcd for C17H9ClF3N6O3S 469.0097, found 469.0134.
13CNMR (DMSO-d6):
d 30.1, 53.4, 107.4, 144.1, 148.0, 149.4, 150.5,
150.8, 157.2, 160.7. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C10H5ClI-
N6O3S 450.8877, found 450.8815.
4.2.25. 3H-2-(5-chlorouracil-6-methylthio)-8-(4-methylphenyl)-
4.2.18. 3H-8-carbonitrile-2-(5-chlorouracil-6-methylthio)-pyrazolo
pyrazolo[1,5-a][1,3,5]triazin-4-one (9l)
[1,5-a][1,3,5]triazin-4-one (9e)
Yield 72%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d 2.31 (s, 3H, CH3),
Yield 56%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
4.41 (s, 2H, CH2),
4.47 (s, 2H, CH2), 7.22 (d, J ¼ 4.4 Hz, 2H, CH), 7.88 (d, J ¼ 4.4 Hz, 2H, CH),
8.38 (s, 1H, CH), 11.33 (s, 1H, NH), 11.60 (s, 1H, NH). 13CNMR (DMSO-
8.47 (s,1H, CH),11.37 (s,1H, NH),11.66 (s,1H, NH), 13.03 (s, broad,1H,
d6):
d
30.3, 81.8, 107.4, 114.0, 145.2, 146.7, 148.1, 150.8, 153.7, 160.8,
NH). 13CNMR (DMSO-d6):
d 20.7, 29.3, 106.0, 109.8, 125.6, 128.1, 129.2,
163.8. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C11H5ClN7O3S 349.9863,
135.4, 143.4, 143.7, 144.1, 147.0, 149.7, 155.8, 159.5. HRMSeESI (m/z):
found 349.9759.
[M ꢃ H]ꢃ calcd for C17H12ClN6O3S 415.038, found 415.0576.
4.2.19. 3H-8- carboxylic acid ethyl ester e2-(5-chlorouracil-6-
methylthio)-pyrazolo[1,5-a][1,3,5]triazin-4-one (9f)
4.2.26. 3H-2-(5-chlorouracil-6-methylthio)-8-(4-tert-
butylphenyl)-pyrazolo[1,5-a][1,3,5]triazin-4-one (9m)
Yield 40%. Mp: 266 ꢁC. 1HNMR (DMSO-d6):
d
1.30 (t, J ¼ 6.9 Hz,
3H, CH3), 4.26 (q, J ¼ 7.0 Hz, 2H, CH2), 4.37 (s, 1H, CH2), 8.23, (s, 1H,
CH), 10.99 (s, 1H, NH), 11.65 (s, 1H, NH). 13CNMR (DMSO-d6):
15.3,
Yield 93%. Mp: 264e266 ꢁC. 1HNMR (DMSO-d6):
d 1.32 (s, 9H,
CH3), 4.29 (s, 2H, CH2), 6.67 (s, 1H, CH), 7.49 (d, J ¼ 8.5 Hz, 2H, CH),
d
7.88 (d, J ¼ 8.0 Hz, 2H, CH), 11.50 (s, 1H, NH), 11.61 (s, 1H, NH), 13.04
30.5, 61.1, 103.8, 107.3, 144.2, 146.9, 148.1, 149.5, 150.4, 160.7, 160.9,
162.4. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C13H9ClN6O5S 397.0122,
found 397.0021.
(s, broad, 1H, NH). 13CNMR (DMSO-d6):
d 29.3, 31.0, 34.4, 93.6,
106.3, 125.5, 125.9, 129.3, 144.6, 147.5, 149.5, 149.7, 151.8, 155.1,
156.7, 159.7. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C20H18ClN6O3S
457.085, found 457.0990.
4.2.20. 3H-2-(5-chlorouracil-6-methylthio)-8-phenyl-pyrazolo[1,5-
a][1,3,5]triazin-4-one (9g)
4.2.27. 3H-2-(5-chlorouracil-6-methylthio)-8-(4-methoxyphenyl)-
Yield 84%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
4.49 (s, 2H, CH2),
pyrazolo[1,5-a][1,3,5]triazin-4-one (9n)
7.26 (t, J ¼ 7.5 Hz, 1H, CH), 7.42 (t, J ¼ 7.5 Hz, 2H, CH), 8.01 (d,
Yield 80%. Mp: 292e294 ꢁC. 1HNMR (DMSO-d6):
d 3.77 (s, 3H,
J ¼ 7.5 Hz, 2H, CH), 8.54 (s, 1H, CH), 11.36 (s, 1H, NH), 11.68 (s, 1H,
OCH3), 4.39 (s, 2H, CH2), 6.96 (d, J ¼ 8.5 Hz, 2H, CH), 7.90 (d,
NH), 11.68 (s, broad, 1H, NH). 13CNMR (DMSO-d6):
d 29.4, 105.5,
J ¼ 8.5 Hz, 2H, CH), 8.33 (s, 1H, CH), 11.45 (s, 1H, NH), 11.63 (s, 1H,
107.3, 125.3, 125.5, 128.5, 132.2, 141.8, 145.8, 147.5, 148.6, 149.7,
NH), 13.11 (s, broad, 1H, NH). 13CNMR (DMSO-d6):
d 29.4, 55.0,

L. Sun et al. / European Journal of Medicinal Chemistry 70 (2013) 400e410
409
105.6, 108.1, 114.1, 124.3, 126.7, 142.1, 144.5, 146.4, 148.1, 149.7, 157.5,
4.4. TP enzyme kinetic studies
159.4, 159.7. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C17H12ClN6O4S
431.0329, found 431.0493.
To investigate the inhibitory effect of the most potent compound
9q against TP at varying thymidine concentrations, velocities of the
enzyme catalysed reactions at different concentrations of thymi-
4.2.28. 3H-2-(5-chlorouracil-6-methylthio)-8-(4-ethoxyphenyl)-
pyrazolo[1,5-a][1,3,5]triazin-4-one (9o)
dine (100
using the same procedure as described previously. 9q was used at
concentrations of 0 M, 0.1 M, 0.5 M, and 2 M. Lineweavere
Burk plots of TP inhibition by 9q were then generated using
Microsoft Office Excel 2007 based on data obtained from triplicate
experiments to determine the inhibition type of 9q.
mM, 250 mM, 500 mM and 1000 mM) were determined
Yield 94%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
1.35 (t, J ¼ 7.2 Hz,
3H, CH3), 4.07 (q, J ¼ 7.2 Hz, 2H, CH2), 4.19 (s, 2H, CH2), 6.33 (s, 1H,
m
m
m
m
CH), 6.98 (d, J ¼ 8.8 Hz, 2H, CH), 7.86 (d, J ¼ 8.8 Hz, 2H, CH), 11.58 (s,
1H, NH), 11.76 (s, 1H, NH). 13CNMR (DMSO-d6):
d 14.6, 29.1, 63.1,
88.7, 104.8, 114.4, 125.8, 127.3, 149.5, 149.9, 150.1, 151.8, 152.5, 158.7,
159.9, 165.1. HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C18H14ClN6O4S
445.0486, found 445.0650.
Acknowledgements
4.2.29. 3H-2-(5-chlorouracil-6-methylthio)-8-(4-iodophenyl)-
This work was supported by the National Medical Research
Council Grant (R-148-000-102-275), Singapore. We appreciate Ms.
Sogand Zareisedehizadeh for her assistance in our HRMS work.
pyrazolo[1,5-a][1,3,5]triazin-4-one (9p)
Yield 67%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d
4.27 (s, 2H, CH2),
7.68 (d, J ¼ 8.4 Hz, 2H, CH), 7.81 (d, J ¼ 8.4 Hz, 2H, CH), 8.23 (s, 1H,
CH), 11.57 (s, broad, 2H, NH). 13CNMR (DMSO-d6):
29.4, 48.5, 91.0,
d
References
105.7, 107.4, 127.5, 131.2, 137.2, 142.5, 145.1, 145.4, 147.6, 149.6, 159.6.
HRMSeESI (m/z): [M ꢃ H]ꢃ calcd for C16H9ClIN6O3S 526.919, found
526.9349.
[1] M.-J. Perez-Perez, E.-M. Priego, A.-I. Hernandez, M.-J. Camarasa, J. Balzarini,
S. Liekens, Thymidine phosphorylase inhibitors: recent developments
and potential therapeutic applications, Mini Rev. Med. Chem. 5 (2005) 1113e
1123.
[2] F. Focher, S. Spadari, Thymidine phosphorylase: a two-face janus in anticancer
chemotherapy, Curr. Cancer Drug Tar. 1 (2001) 141e153.
4.2.30. 3H-2-(5-chlorouracil-6-methylthio)-8-(4-
pentafluorosulfurphenyl)-pyrazolo[1,5-a][1,3,5]triazin-4-one (9q)
[3] A.V. Patterson, H. Zhang, A. Moghaddam, R. Bicknell, D.C. Talbot, I.J. Stratford,
A.L. Harris, Increased sensitivity to the prodrug 50-deoxy-5-fluorouridine and
modulation of 5-fluoro-20-deoxyuridine sensitivity in MCF-7 cells transfected
with thymidine phosphorylase, Br. J. Cancer 72 (1995) 669e675.
[4] T. Emura, F. Nakagawa, A. Fujioka, H. Ohshimo, T. Yokogawa, H. Okabe,
K. Kitazato, An optimal dosing schedule for a novel combination antimetab-
olite, TAS-102, based on its intracellular metabolism and its incorporation into
DNA, Int. J. Mol. Med. 13 (2004) 249e255.
[5] D. Cox, R. O’Kennedy, R.D. Thornes, The rarity of liver toxicity in
patients treated with coumarin (1, 2-benzopyrone), Hum. Exp. Toxicol. 8
(1989) 501e506.
[6] S. Takao, S.-i. Akiyama, A. Nakajo, H. Yoh, M. Kitazono, S. Natsugoe,
K. Miyadera, M. Fukushima, Y. Yamada, T. Aikou, Suppression of metastasis by
thymidine phosphorylase inhibitor, Cancer Res. 60 (2000) 5345.
[7] Y. Nakajima, T. Gotanda, H. Uchimiya, T. Furukawa, M. Haraguchi, R. Ikeda,
T. Sumizawa, H. Yoshida, S.-i. Akiyama, Inhibition of metastasis of tumor cells
overexpressing thymidine phosphorylase by 2-deoxy-l-ribose, Cancer Res. 64
(2004) 1794e1801.
[8] H. Uchimiya, T. Furukawa, M. Okamoto, Y. Nakajima, S. Matsushita, R. Ikeda,
T. Gotanda, M. Haraguchi, T. Sumizawa, M. Ono, M. Kuwano, T. Kanzaki, S.-
i. Akiyama, Suppression of thymidine phosphorylase-mediated angiogenesis
and tumor growth by 2-deoxy-l-ribose, Cancer Res. 62 (2002) 2834e2839.
[9] B.R. Baker, J.L. Kelley, Irreversible enzyme inhibitors. CLXXI. Inhibition of FUDR
[5-fluoro-20-deoxyuridine] phosphorylase from Walker 256 rat tumor by 5-
substituted uracils, J. Med. Chem. 13 (1970) 461.
[10] P. Langen, G. Etzold, D. Bärwolff, B. Preussel, Inhibition of thymidine phos-
phorylase by 6-aminothymine and derivatives of 6-aminouracil, Biochem.
Pharmacol. 16 (1967) 1833.
Yield 89%. Mp: >300 ꢁC. 1HNMR (DMSO-d6):
d 4.30 (s, 2H, CH2),
7.84 (d, J ¼ 8.8 Hz, 2H, CH), 8.19 (d, J ¼ 8.8 Hz, 2H, CH), 8.33 (s, 1H,
CH), 11.50 (s, broad, 1H, NH), 11.61 (s, 1H, NH). 13CNMR (DMSO-d6):
d
29.5, 103.3, 105.1, 124.6, 125.86, 125.91, 125.94, 137.4, 140.8, 148.3,
148.8, 148.9, 149.1, 149.2, 149.5, 159.7, 167.3. HRMSeESI (m/z):
[M ꢃ H]ꢃ calcd for C16H9ClF5N6O3S2 526.9786, found 527.0028.
4.3. In vitro TP enzyme assay
Enzyme and substrate were obtained from SigmaeAldrich, and
the activity assay was performed at room temperature in a Hitachi
U-1900 spectrophotometer at a detection wavelength of 290 nm.
This method is based on the manufacturer instruction, which was a
classical method described by Krenitsky [21].
For all compounds, inhibition rate at a certain concentration was
obtained by the following procedure:
To 780
sorption cuvette was subsequently added 10
in pH 7.0 phosphate buffer), 10 l compound solution in DMSO (for
blank, add in 10 l DMSO), and 200 l Thymidine solution (5 mM in
ml 200 mM phosphate buffer (pH 7.4) in the 1.5 ml ab-
ml TP solution (1.5U/ml
m
m
m
pH 7.4 phosphate buffer). After gentle shaking by inversion of the
cuvette, the absorption values at 4 min, 8 min, 12 min, 16 min,
20 min were recorded successively. These absorption values were
plotted against time, and linear regression was performed to obtain
the slope of the line plotted, which was equalled to the velocity of
enzyme catalysed reaction. Then the inhibition rate at this con-
centration could be calculated using the following formula:
[11] M. Fukushima, N. Suzuki, T. Emura, S. Yano, H. Kazuno, Y. Tada, Y. Yamada,
T. Asao, Structure and activity of specific inhibitors of thymidine phosphor-
ylase to potentiate the function of antitumor 20-deoxyribonucleosides, Bio-
chem. Pharmacol. 59 (2000) 1227.
[12] J. Balzarini, A.E. Gamboa, R. Esnouf, S. Liekens, J. Neyts, E. De Clercq, M.-
J. Camarasa, M.-J. Pérez-Pérez, 7-Deazaxanthine, a novel prototype inhibitor of
thymidine phosphorylase, FEBS Lett. 438 (1998) 91.
[13] S. Liekens, A.-I. Hernandez, D. Ribatti, E. De Clercq, M.-J. Camarasa, M.-J. Perez-
Perez, J. Balzarini, The nucleoside derivative 50-O-Trityl-inosine (KIN59) sup-
presses thymidine phosphorylase-triggered angiogenesis via a noncompeti-
tive mechanism of action, J. Biol. Chem. 279 (2004) 29598.
[14] E. Casanova, A.-I. Hernandez, E.-M. Priego, S. Liekens, M.-J. Camarasa,
J. Balzarini, M.-J. Perez-Perez, 50-O-Tritylinosine and analogues as allosteric
inhibitors of human thymidine phosphorylase, J. Med. Chem. 49 (2006) 5562.
[15] S. Yano, H. Kazuno, T. Sato, N. Suzuki, T. Emura, K. Wierzba, J.-i. Yamashita,
Y. Tada, Y. Yamada, M. Fukushima, T. Asao, Synthesis and evaluation of 6-
methylene-bridged uracil derivatives. Part 2: optimization of inhibitors of
human thymidine phosphorylase and their selectivity with uridine phos-
phorylase, Bioorg. Med. Chem. 12 (2004) 3443.
Inhibition rate ¼
velocity without the inhibitorevelocity with the inhibitor
velocity without the inhibitor
ꢂ 100%
For IC50 value determination, inhibition rates of at least 7
different concentrations that span over the estimated IC50 value
were determined in the same way and plotted against logarithmic
concentrations using OriginPro 8 SR0 v8.0724 (B724). IC50 value
was determined as the concentration of the inhibitor that caused
50% inhibition. Results were means of three experiments.
[16] K.-Y. Zee-Chen, C.C. Cheng, Pyrimidines. XX.
A convenient preparation
of orotaldehyde and thymine-6-carboxaldehyde, J. Heterocycl. Chem. 4 (1967)
163.
[17] M. Claesen, H. Vanderhaeghe, Pyrimidines III-Dérivés de l’acide pyrimidone-
6-carboxylique-4 et de l’aldéhyde correspondant, Bull. Soc. Chim. Belg. 66
(1957) 292e302.

410
L. Sun et al. / European Journal of Medicinal Chemistry 70 (2013) 400e410
[18] R.S. Klein, J.J. Fox, Nucleosides. LXXVIII. Synthesis of some 6-substituted ura-
cils and uridines by the Wittig reaction, J. Org. Chem. 37 (1972) 4381.
[19] S. Yano, Y. Tada, H. Kazuno, T. Sato, J. Yamashita, N. Suzuki, T. Emura,
M. Fukushima, T. Asao, Preparation and Formulation of Uracil Derivatives as
Antitumor Activity Potentiators and Antitumor Agents, Taiho Pharmaceutical
Co., Ltd, Japan, 1996, p. 108.
[20] L. Sun, H. Bera, W.K. Chui, Synthesis of pyrazolo[1,5-a][1,3,5]triazine de-
rivatives as inhibitors of thymidine phosphorylase, Eur. J. Med. Chem. 65
(2013) 1e11.
[22] J.R. Grierson, J.S. Brockenbrough, J.S. Rasey, L. Wiens, H. Vesselle, Synthesis
and in vitro evaluation of 5-fluoro-6-[(2-iminopyrrolidin-1-YL)methyl]uracil,
TPI(F): an inhibitor of human thymidine phosphorylase (TP), Nucleosides,
Nucleotides Nucleic Acids 29 (2009) 49.
[23] R.S. Klein, M. Lenzi, T.H. Lim, K.A. Hotchkiss, P. Wilson, E.L. Schwartz, Novel 6-
substituted uracil analogs as inhibitors of the angiogenic actions of thymidine
phosphorylase, Biochem. Pharmacol. 62 (2001) 1257.
[24] X.-Q. Wu, J. Wang, Z.-R. Lü, H.-M. Tang, D. Park, S.-H. Oh, J. Bhak, L. Shi, Y.-
D. Park, F. Zou, Alpha-glucosidase folding during urea denaturation: enzyme
kinetics and computational prediction, Appl. Biochem. Biotech. 160 (2010)
1341.
[21] T.A. Krenitsky, S.R.M. Bushby, Stabilized Thymidine Phosphorylase Formula-
tion, Burroughs Wellcome Co, United States, 1979.