Synthesis of pyrazolo[1,5-a][1,3,5]triazine derivatives as inhibitors of thymidine phosphorylase

Article abstract of DOI:10.1016/j.ejmech.2013.03.063

Thymidine phosphorylase (TP) is an enzyme that promotes tumor growth and metastasis and therefore is an attractive druggable target. Using a reported TP inhibitor, 7-deazaxanthine (7DX), as the lead compound; this study was set up to evaluate whether pyrazolo[1,5-a][1,3,5]triazin-2,4-diones and pyrazolo[ 1,5- a][1,3,5]triazin-2-thioxo-4-ones would exhibit TP inhibitory activity. The pyrazolo[1,5-a][1,3,5] triazine nucleus was constructed using a reaction that annulated the 1,3,5-triazine ring onto a pyrazole scaffold. Among the 52 compounds synthesized and tested, it was found that 1,3-dihydro-pyrazolo[1,5-a] [1,3,5]triazin-2-thioxo-4-ones exhibited various extent of inhibitory activity against TP. The best compound 17p, which bears a para-substituted pentafluorosulfur group, showed an IC₅₀ value of 0.04 μM, which was around 800 times more potent than the 7DX (IC₅₀ = 32 μM) under the same bioassay conditions. The results of the study suggested that a substituent with +σ and +π properties inserted at position 4 of a phenyl ring that is attached to position 8 of the pyrazolo[1,5-a][1,3,5]triazin- 2-thioxo-4-one scaffold would give excellent TP inhibitory action. In addition, 17p was found to be a non-competitive inhibitor thus suggested that it might interact with TP at a position different from the substrate binding site.

Full text of DOI:10.1016/j.ejmech.2013.03.063

European Journal of Medicinal Chemistry 65 (2013) 1e11
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of pyrazolo[1,5-a][1,3,5]triazine derivatives as inhibitors of
thymidine phosphorylase
*
Lingyi Sun, Hriday Bera, Wai Keung Chui
Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
Thymidine phosphorylase (TP) is an enzyme that promotes tumor growth and metastasis and therefore
is an attractive druggable target. Using a reported TP inhibitor, 7-deazaxanthine (7DX), as the lead
compound; this study was set up to evaluate whether pyrazolo[1,5-a][1,3,5]triazin-2,4-diones and pyr-
azolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones would exhibit TP inhibitory activity. The pyrazolo[1,5-a][1,3,5]
triazine nucleus was constructed using a reaction that annulated the 1,3,5-triazine ring onto a pyrazole
scaffold. Among the 52 compounds synthesized and tested, it was found that 1,3-dihydro-pyrazolo[1,5-a]
[1,3,5]triazin-2-thioxo-4-ones exhibited various extent of inhibitory activity against TP. The best com-
Received 28 November 2012
Received in revised form
21 March 2013
Accepted 28 March 2013
Available online 10 April 2013
Keywords:
pound 17p, which bears a para-substituted pentafluorosulfur group, showed an IC50 value of 0.04
which was around 800 times more potent than the 7DX (IC50 ¼ 32 M) under the same bioassay con-
and þ properties inserted at
mM,
Pyrazolo[1,5-a][1,3,5]triazines
Thymidine phosphorylase
Enzyme inhibitor
m
ditions. The results of the study suggested that a substituent with þ
s
p
position 4 of a phenyl ring that is attached to position 8 of the pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-
one scaffold would give excellent TP inhibitory action. In addition, 17p was found to be a non-competitive
inhibitor thus suggested that it might interact with TP at a position different from the substrate binding
site.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
nucleoside analog therapeutic agents, the clinical implication of
inhibiting TP has broadened to include controlling the develop-
Thymidine phosphorylase (TP) is an enzyme that catalyzes the
reverse phosphorolysis of pyrimidine nucleosides. It plays a key
role in maintaining the balance of the nucleotide pool and con-
trolling nucleic acid homeostasis by ensuring an ample supply of
deoxyribonucleoside triphosphates (dNTPs) for DNA replication
and repair [1].
ment of tumor.
Structurally, most of the active TP inhibitors are derivatives of
pyrimidine and its analogs which were designed to interact with
the thymidine binding site. The best inhibitor found to date is 5-
chloro-6-[1-(2-iminopyrrolidinyl)methyl] uracil hydrochloride
(TPI) [7] which exhibits strong inhibitory activity as denoted by a
However, besides pyrimidine nucleosides, TP can also induce
the phosphorolysis of several nucleoside analog therapeutic
agents thus reduce their bioavailability [2]. Therefore, it has been
proposed that a co-administration of a nucleoside analog thera-
peutic agent together with a TP inhibitor might improve the bio-
logical efficacy of the therapeutic agent. TP has been also found to
be associated with tumor angiogenesis [3] and metastasis [4,5],
and it can promote tumor growth by preventing apoptosis as well
[6]. Therefore, in addition to improving the biological efficacy of
reported IC50 value of 0.035 mM [8]. Besides pyrimidines, some
purine analogs such as 7-deazaxanthine (7DX) [9] (Fig. 1) has been
found to demonstrate inhibitory activity against TP too. However,
7DX (IC50 value ¼ 40
mM [9]) is not as potent as TPI, and further
structural modifications may lead to more potent purine-like TP
inhibitors. In addition, some multisubstrate inhibitors designed for
interacting with both the thymidine and phosphate binding sites
have been reported in the literature [10e12]. However, these
multisubstrate inhibitors showed weaker inhibition against TP, and
they inhibited the enzyme in its opened conformation.
The pyrazolo[1,5-a][1,3,5]triazine heterocyclic system is recog-
nized as an analog of purine in which the 9-N atom is translocated
to position 5 of the bicyclic ring system. This heterocycle has been
substituted for purine in the area of nucleoside chemistry and many
biologically active agents have been developed [13].
* Corresponding author. Postal Address: 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: g0801101@nus.edu.sg (L. Sun), g0701228@nus.edu.sg
(H. Bera), 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.2013.03.063

2
L. Sun et al. / European Journal of Medicinal Chemistry 65 (2013) 1e11
Fig. 1. 7DX and proposed substituted pyrazolo[1,5-a][1,3,5]triazine derivatives.
Previous investigations on this scaffold have discovered
triazines can be categorized according to how the bicyclic fused
ring system is formed. The following approaches have been re-
ported in the literature, namely, (A) annulation of the 1,3,5-triazine
ring onto a pyrazole scaffold; (B) annulation of the pyrazole ring
onto a 1,3,5-triazine scaffold; (C) concurrent formation of both the
1,3,5-triazine and pyrazole ring; and (D) ring transformation re-
actions [13]. In the selection of the synthetic approach to be
adopted for this study, commercial availability of the starting ma-
terials and synthetic practicality were important consideration
factors. Since many 4- and 5-substituted 3-amino pyrazoles are
either commercially available or can be easily synthesized; there-
fore it is believed that 7 or 8 substituted pyrazolo[1,5-a][1,3,5]tri-
azines can be conveniently generated through the use of approach
A. Hence this approach was adopted for synthesizing the target
compounds in this study. By reacting respectively the substituted
3-amino pyrazoles with either ethoxycarbonyl isocyanate or
ethoxycarbonyl isothiocyanate; both the 2,4-dione series and the 2-
thioxo-4-one series can be readily synthesized in two steps. For the
required aryl substituted 3-amino pyrazoles which were unavai-
lable commercially, reported methods from the literature were
used to synthesize them. The method of synthesis depended on
positions where the final aryl substituents were located.
3-amino pyrazoles with aryl substituents located at position 4
was prepared via a three step synthetic scheme [23e25] (Scheme
1). 4-Substituted-aryl acetonitriles (2) were synthesized by treat-
ing correspondingly substituted bromomethylbenzenes 1 with
potassium cyanide (Yield 56% for 2a; 69% for 2b) and then subse-
quently reacted with dimethoxymethyldimethylamine to give 3-
dimethylamino-2-aryl-acrylonitriles 3 (Yield 80% for 3a, 85% for
3b). The target 4-aryl-3-amino pyrazoles 4 were obtained via a
reaction between hydrazine and the corresponding acrylonitriles
(Yield 29% for 4a, 19% for 4b).
numerous compounds that exhibit various therapeutic properties.
In particular, many enzyme inhibitors were found to carry the
pyrazolo[1,5-a][1,3,5]triazine scaffold, and a few of them have been
approved as drugs. Some of the examples are included herein to
illustrate the range of targeted enzymes. In 1985, Robins et al. [14]
developed 8-phenyl-5-aza-9-deazahypoxanthine as a potent in-
hibitor of xanthine oxidase for the treatment of gout. Cyclin-
dependent kinase, which is involved in the regulation of the cell
cycle, was found to be inhibited by suitably substituted 2,4-
diaminopyrazolo[1,5-a][1,3,5]triazines [15e17]. The class of cyclic
nucleotide phosphodiesterases, which hydrolyze second messen-
gers such as cAMP and cGMP, is another group of enzymes that can
be inhibited by pyrazolo[1,5-a][1,3,5]triazines [18e21]. Finally, DNA
gyrase, which is present in bacteria but not in human, and therefore
a good target for antibacterial therapy, can also be selectively
inhibited by some pyrazolo[1,5-a][1,3,5]triazines [22].
In summary, the pyrazolo[1,5-a][1,3,5]triazine scaffold has
served as a good template for developing enzyme inhibitors as
therapeutic agents. With appropriate structural modifications, this
scaffold can be readily used to fulfill pharmacophoric requirements
in the design stage of drug discovery. Together with the established
approaches to chemical synthesis, libraries of compounds can be
readily generated and evaluated for different biological activities.
In this study, using 7DX as the lead compound, it is hypothesized
that 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2,4-dione as well as
1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-one (Fig. 1)
may possess TP inhibitory activity comparable to 7DX. In addition,
the secondary aim of the study is to investigate whether separate
substitution on positions 7 and 8 may lead to the enhancement of
TP inhibitory activity of the above mentioned pyrazolo[1,5-a][1,3,5]
triazines.
5-Phenyl-1H-pyrazol-3-ylamine 7 was prepared by a two-step
synthetic reaction [26] (Scheme 2). 3-Oxo-3-phenylpropionitrile 6
was first synthesized by treating 2-bromo-1-phenylethanone 5
with potassium cyanide (Yield 76%) and followed by a reaction with
hydrazine to generate the target amine 7 (Yield 16%).
2. Results and discussion
2.1. Chemistry
Target compounds 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-
2,4-diones 10 and 13 were synthesized via a two-step synthetic
scheme [27] as presented in Scheme 3. The corresponding amines 7,
For the past 50 years, several methods have been reported for
the construction of the pyrazolo[1,5-a][1,3,5]triazine ring system.
The common approaches to the synthesis of pyrazolo[1,5-a][1,3,5]
Scheme 1. Synthesis of 4-aryl-1H-pyrazol-3-ylamines. Reagents and conditions: (a) KCN, ethanol, reflux, yields 56% (2a) and 69% (2b); (b) dimethoxymethyldimethylamine, DMF,
reflux, yields 80% (3a) and 85% (3b); (c) NH2NH2, ethanol, reflux, yields 29% (4a) and 19% (4b).

L. Sun et al. / European Journal of Medicinal Chemistry 65 (2013) 1e11
3
reactions between amines and ethoxycarbonyl isothiocyanate took
shorter time to complete and gave higher yields. This could be due
to the presence of the higher electrophilic center in the reactant
ethoxycarbonyl isothiocyanate. An intramolecular ring annulation
reaction of compounds 14 and 16 was carried out under the
catalysis of sodium ethoxide to produce the eventual target com-
pounds of 15 and 17 (Yields 54%e89% for 15ael, Yields 43%e94% for
17aep).
Scheme 2. Synthesis of 5-phenyl-1H-pyrazol-3-ylamine. Reagents and conditions: (a)
KCN, ethanol, 50 ꢀC, yield 76%; (b) NH2NH2, ethanol, reflux, yield 16%.
Among the 52 target compounds synthesized; compounds 10a
[27], 10f [28], 10i [28], 10k [28], 10l [29], 13e [30], 15a [27], 15l [29],
17a [15] and 17e [14] have been previously reported; however, full
characterization of these compounds were not disclosed and their
TP inhibitory activity was never investigated. The rest of the 42
target compounds, which have not hitherto been reported in
literature, were synthesized and fully characterized by 1H, 13C NMR
and mass spectrometry. Their purity was also determined by
reverse phase HPLC (to be >95% pure) and found to be suitable for
biological evaluation.
8, and 11 were reacted separately with ethoxycarbonyl isocyanate
at room temperature in anhydrous DMF to give N-ethoxycarbonyl-
N0-(pyrazol-3-yl) ureas 9 and 12 in good yields (Yields 41%e91% for
9ael, Yields 40%e76% for 12ael). This was followed subsequently
by an intramolecular ring annulation reaction under the catalysis of
sodium ethoxide to generate the target compounds (Yields 41%e
91% for 10ael, Yields 51%e87% for 13ael).
On the other hand, target compounds1,3-dihydropyrazolo[1,5-
a][1,3,5]triazin-2-thioxo-4-ones 15 and 17 were synthesized simi-
larly by a two-step synthesis as illustrated in Scheme 4 [27]. The
corresponding amines 4, 7, 8, and 11 were reacted separately with
ethoxycarbonyl isothiocyanate instead at room temperature in
anhydrous DMF to give N-ethoxycarbonyl-N0-(pyrazol-3-yl)thio-
ureas 14 and 16 in good yields (Yields 82%e98% for 14ael, Yields
64%e95% for 16aep). Compared to the same reaction between
amines and ethoxycarbonyl isocyanate, it was found that the
2.2. Structure activity relationship (SAR) based on results of in vitro
TP enzyme assay
All the 1,3-dihydropyrazolo[1,5-a][1,3,5]triazin-2,4-diones (10
and 13) and 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-
Scheme 3. Synthesis of 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2,4-diones. Reagents and conditions: (a) ethoxycarbonyl isocyanate, DMF, rt, yields 41%e91% (9ael) and 40%e76%
(12ael); (b) EtONa, ethanol, reflux, yields 41%e91% (10ael) and 49%e87% (13ael).

4
L. Sun et al. / European Journal of Medicinal Chemistry 65 (2013) 1e11
Scheme 4. Synthesis of 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones. Reagents and conditions: (a) ethoxycarbonyl isothiocyanate, DMF, rt, yields 82%e98% (14ael) and
64%e95% (16aep); (b) EtONa, ethanol, reflux, yields 32%e89% (15ael) and 43%e94% (17aep).
ones (15 and 17) were evaluated for inhibitory activity using a
modified TP bioassay. A continuous UV spectrophotometric enzyme
assay [14] was employed in the evaluation. The bioassay used re-
combinant Escherichia coli TP (EC Number 2.4.2.4), expressed in E.
coli as the enzyme 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. A comparison of the rate of
enzymatic reaction in the absence and presence of an inhibitor or
test compound would give the relative extent of enzyme inhibition.
First, compounds 10ael, 13ael, 15ael and 17ael were screened
the pyrazolo[1,5-a][1,3,5]triazine scaffold was important for the TP
inhibitory activity.
To further investigate the structure and TP inhibitory activity
relationship of the 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-
thioxo-4-ones (15 and 17), the IC50 values for TP inhibition of all
the compounds in this series were subsequently determined. The
two libraries of 26 compounds (15ael, 17aen) exhibited IC50 values
ranging from 87.3
mM to 0.24
mM, with 8 out of the 26 compounds
having IC50 values below 10
m
M (Table 1).
Depending on the substituents at position 7 and position 8, the
inhibitory activity of the pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-
2ones can vary significantly. In comparison, compound 15a, which
did not possess any substituent at either position 7 or position 8,
was found to be about half as active as the reference compound
7DX. However, with the introduction of certain substituents on 15a
at either 7 or 8 position, the inhibitory activity was found to be
better than 7DX with respect to IC50 values.
at concentration of 50 mM for their inhibitory effects against the
enzymatic activity. The screening results, expressed as percent in-
hibition, showed that none of the 1,3-dihydropyrazolo[1,5-a][1,3,5]
triazin-2,4-diones (10 and 13) exhibited more than 50% inhibition
at the concentration of 50 mM, thus these two series were consid-
ered not active against TP. However, 21 out of the 24 1,3-dihydro-
pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones (15 and 17) were
found to exhibit a range of 50%e100% inhibition at the concentra-
In particular, placing a phenyl ring at either position 7 or 8
appeared to enhance the TP inhibition. For instance, based on a
comparison between compounds 15a & 15l,15b & 15i, 15c & 15j and
tion of 50
mM. Therefore, this screening experiment suggested that
the bioisosteric substitution of oxygen with sulfur at position 2 of

L. Sun et al. / European Journal of Medicinal Chemistry 65 (2013) 1e11
Table 1 (continued )
5
Table 1
TP inhibitory activity of 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones.
a
Compound
R1
R2
IC50 (mM)
17m
17n
17o
17p
H
H
H
H
15.5 ꢁ 0.3
0.58 ꢁ 0.02
0.21 ꢁ 0.03
OEt
NO2
I
a
Compound
R1
R2
IC50
(
m
M)
15a
15b
H
Me
H
H
68.2 ꢁ 2.7
79.4 ꢁ 8.2
0.040 ꢁ 0.005
32.2 ꢁ 5.1
SF5
15c
15d
H
H
29.1 ꢁ 4.6
32.6 ꢁ 5.7
F
F
7DX
F
a
Values are mean, n ¼ 3.
15d & 15h, the results showed that the inclusion of a phenyl ring
had improved the TP inhibition by 1.5, 4.1, 4.4 and 3.3 times
respectively. A similar phenyl effect was also observed for com-
pounds 17a & 17j, 17b & 17g and 17c & 17h. The changes in TP in-
hibition in these cases were even greater; registering an
enhancement in TP inhibition of 51.3, 51.1 and 70.5 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. Based on the
comparison above, it showed that position 8 was possibly closer to
the hydrophobic site.
In addition to the requirement of a phenyl ring at position 8, the
substituents on position 4 of this phenyl ring also appeared to be an
important contributory factor to the inhibitory activity. On the one
hand, the fact that inhibitory activity of 17l < 17m < 17j suggested
that increasing the hydrophobicity of substituents on the phenyl
ring would improve the potency. This is consistent with the Craig
plot where the methoxy group (OCH3), the ethoxy group
(OCH2CH3), and the methyl group (CH3) are known to possess
similar electron donating properties while the hydrophobicity of
methoxy group < ethoxy group < methyl group. However, such
requirement for increase in hydrophobicity appeared to have a
limit. The fact that 17k was about 3.3 times less potent than 17j
demonstrated that when the bulkiness got too large, the inhibitory
activity would not increase accordingly. On the other hand, the fact
that 17n was 18.1 times more potent than 17f suggested that
greater electron withdrawing effect would lead to more active
compounds, since the fluoro group (F) and the nitro group (NO2)
possess similar hydrophobicity constant while the electron with-
drawing property of fluoro group was weaker than that of nitro
group. Therefore, it could be deduced that the more hydrophobic
and electron withdrawing substituents inserted on para position of
the phenyl ring, the compounds would become more potent. This
conclusion was consistent with the observation that potency of
17i > 17g, 17h > 17f > 17e.
15e
15f
H
H
H
H
41.7 ꢁ 0.5
33.5 ꢁ 3.8
34.9 ꢁ 6.3
9.9 ꢁ 1.9
F
Cl
Br
15g
15h
F
F
F
15i
15j
15k
15l
H
H
H
H
19.3 ꢁ 2.3
6.6 ꢁ 1.6
28.4 ꢁ 2.0
46.2 ꢁ 1.8
OMe
17a
17b
17c
17d
H
H
H
H
Me
Cl
Br
I
87.3 ꢁ 6.0
27.6 ꢁ 1.3
44.4 ꢁ 8.6
20.8 ꢁ 2.7
17e
17f
17g
17h
17i
H
H
H
H
H
26.3 ꢁ 2.0
10.5 ꢁ 3.3
0.54 ꢁ 0.04
0.63 ꢁ 0.12
0.24 ꢁ 0.04
F
Cl
Br
F
Based on the above SAR derived from the results of the 50
compounds tested, two more compounds, namely, 17o and 17p
were synthesized to validate the structural requirements for TP
inhibition. The SAR results appeared to suggest that substituents
F
F
17j
17k
17l
H
H
H
1.7 ꢁ 0.1
5.6 ꢁ 1.3
39.7 ꢁ 9.9
located in the top right hand quadrant of the Craig plot (þ
s
, þ
p)
would give good TP inhibitory activity. Therefore, two more com-
pounds bearing the iodo group (I) and the pentafluorosulfur group
(SF5) at position 4 of the phenyl ring (17o and 17p respectively)
were synthesized as per the method described above and their TP
inhibitory action was evaluated. 17o inhibited TP at an IC50 value of
OMe
0.21 mM while 17p exhibited 50% inhibition at a concentration of

6
L. Sun et al. / European Journal of Medicinal Chemistry 65 (2013) 1e11
a larger extent. Lastly, it was also found that when substituents
6
5
0
which had a larger hydrophobic constant and more positive Ham-
mett constant (ie. more electron withdrawing) were introduced at
position 4 of the attached phenyl ring, the IC50 values would
decrease further. This was validated by compounds 17o and 17p
where their inhibitory activities were around 150 times and 800
times more potent than 7DX respectively. Further kinetic studies
revealed that 17p was a non-competitive inhibitor thus it might
interact with the enzyme in a way different from 7DX, which was
reported as a competitive inhibitor [33].
0.01µM
0.1µM
0.2µM
4
3
2
1
0
-0.008
-0.003
0.002
0.007
0.012
-1
-2
-3
-4
1/[S] (µM-1)
4. Experimental section
Fig. 2. LineweavereBurk plots of thymidine phosphorylase inhibition by compound
17p prepared at different concentrations. All data points are means of three
experiments.
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-
0.04 mM. Therefore, 17p was found to be around 800 times more
potent than 7DX under the evaluating conditions used for the
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
a Bruker DPX-300
bioassay.
d
2.3. Enzyme inhibition kinetic studies
from the internal standard. The signals were quoted as s (singlet),
d (doublet), t (triplet), m (multiplet). Thin-layer chromatography
(TLC) was developed on aluminum-supported pre-coated silica gel
plates (Merck, 60 F254). Column chromatography was conducted on
silica gel (230e400 mesh). ESI-MS was recorded on a Finnigan MAT
LC-MS. HPLC analysis was carried out on all target compounds used
in biological assays, using a HewlettePackard series 1050 HPLC
system equipped with an HP-1050 quaternary pump, a degasser,
diode array detector, an HP-1100 autosampler and a LiChrosorb
Some reported TP inhibitors have been investigated for their
action modes [31,32]. With the identification of 17p as the most
active compound, it was of interest to determine whether 17p would
interact with TP in any way that would be different from 7DX.
Therefore, based on a reported method [32], further investigation
was carried out to determine the enzyme inhibition kinetics of 17p
using four different concentrations of the substrate thymidine and
four different concentrations of 17p (0, 0.01 mM, 0.1 mM, and 0.2 mM).
reversed phase C18 (5
mm) column (4.6 ꢂ 250 mm). All the samples
It was demonstrated on the LineweavereBurk plot that the lines
generated by 17p at different concentrations had different gradients
and they converged at a point on the negative side of the x axis. This
observation pointed to the fact that 17p exhibited non-competitive
inhibition kinetics on TP with respect to thymidine being the sub-
strate (Fig. 2). This was further supported by the fact that the Vmax
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 main-
values decreased from 9.9
17p, while the Km values did not change significantly as the inhibitor
concentration increased from 0 to 0.2 M. (Km and Vmax values were
mM/min to 2.6 mM/min in the presence of
m
calculated from the EadieeHofstee plots) This characteristic Vmax
and Km values of the enzyme is typical in the presence of an inhibitor
that exhibits non-competitive inhibition kinetics.
tained for one more minute. The injection volume was 5 ml. The
purity of all target compounds was found to be more than 95%
which was satisfactory for bioassay.
3. Conclusion
4.2. General procedure for the preparation of aryl substituted
The selected synthetic strategy of annulating the 1,3,5-triazine
ring onto a pyrazole scaffold was proven to be quite an efficient
way to synthesize the pyrazolo[1,5-a][1,3,5]triazines as designed.
The two series of 2,4-diones and 2-thioxo-4-ones were synthesized
with ease and all the final target compounds were prepared in
generally good yields. The hypothesis that 10a and 15a might
possess TP inhibitory activity comparable to 7DX was not supported
by the experimental results since both of them were obviously less
active than 7DX. However, it was found that many substituted 1,3-
dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones were more
active than 7DX. Therefore, the bioisosteric replacement of oxygen
with sulfur at position 2 of the pyrazolo[1,5-a][1,3,5]triazine scaffold
was found to be essential for the TP inhibitory activity of this type of
compounds; and proper substituents on position 7 or 8 of this
scaffold would enhance the inhibitory activity. In particular, the
insertion of a phenyl ring at either position 7 or 8 was enough to
enhance the TP inhibition, especially when the aryl group was
located at position 8; the inhibitory activity was found to increase by
acetonitriles (2)
To 8 mmol of the correspondingly substituted bromome-
thylbenzene in 40 ml of ethanol was added 2 ml of 6 M potassium
cyanide solution. The reaction mixture was refluxed for 1 h. On
cooling, the mixture was concentrated by rotary evaporation under
vacuum to remove the ethanol. The residue was washed with
deionized water and recrystallized from ethanol/water to give the
product.
4.2.1. (4-Iodophenyl)acetonitrile (2a)
Yield 56%. Mp: 50e52 ꢀC (lit [23]: 56e58 ꢀC). 1H NMR (DMSO-
d6):
2H, CH).
d
4.02 (s, 2H, CH2), 7.17 (d, J ¼ 8.4 Hz, 2H, CH), 7.76 (d, J ¼ 8.4 Hz,
4.2.2. (4-Pentafluorosulfurphenyl)acetonitrile (2b)
Yield 69%. Mp: 53e54 ꢀC. 1H NMR (DMSO-d6):
d
4.21 (s, 2H, CH2),
7.60 (d, J ¼ 8.4 Hz, 2H, CH), 7.95 (d, J ¼ 8.4 Hz, 2H, CH).

L. Sun et al. / European Journal of Medicinal Chemistry 65 (2013) 1e11
7
4.3. General procedure for the preparation of 3-dimethylamino-2-
(lit [34]: 126e127 ꢀC). 1H NMR (DMSO-d6):
d
4.77 (s, 2H, NH2), 5.76
aryl substituted-acrylonitriles (3)
(s,1H, CH), 7.25 (t, J ¼ 7.2 Hz,1H, CH), 7.37 (t, J ¼ 7.2 Hz, 2H, CH), 7.64
(d, J ¼ 7.2 Hz, 2H, CH), 11.75 (s, 1H, NH).
To 3 mmol of the corresponding aryl substituted acetonitrile
dissolved in 5 ml of anhydrous DMF was added 4.5 mmol of
dimethoxymethyldimethylamine. The reaction mixture was
refluxed for 5 h. On cooling, the mixture was poured into 50 ml of
cold water. The precipitate that formed was filtered to give the
product.
4.5. General procedure for the preparation of N-ethoxycarbonyl-N0-
(pyrazol-3-yl) ureas (9, 12)
To a fine suspension of 1 mmol of the corresponding amine in
3 ml of anhydrous DMF was added 1 mmol of ethoxycarbonyl
isocyanate. After stirring the mixture for 12 h at room temperature,
30 ml of cold water was added. The precipitated product was
filtered, washed with cold water and recrystallized from
acetonitrile.
4.3.1. 3-Dimethylamino-2-(4-iodophenyl)acrylonitrile (3a)
Yield 80%. Mp: 91e92 ꢀC. 1H NMR (DMSO-d6):
d 3.20 (s, 6H, CH3),
7.13 (d, J ¼ 8.8 Hz, 2H, CH), 7.50 (s, 1H, CH), 7.60 (d, J ¼ 8.8 Hz, 2H,
CH).
4.6. General procedure for the preparation of 1,3-dihydro-pyrazolo
4.3.2. 3-Dimethylamino-2-(4-pentafluorosulfurphenyl)acrylonitrile
(3b)
[1,5-a][1,3,5]triazin-2,4-diones (10, 13)
Yield 85%. Mp: 144e145 ꢀC. 1H NMR (DMSO-d6):
d
3.25 (s, 6H,
1 mmol of the corresponding N-ethoxycarbonyl-N0-(pyrazol-3-
yl) urea was refluxed in a mixture of 4 ml of ethanol and 0.76 ml
of 21%wt EtONa (2 mmol) ethanol solution for 0.5 h. The precipitate
that formed was collected by filtration, then dissolved in water and
acidified with HCI until pH 4. The precipitate formed was filtered
and recrystallized from methanol/water.
CH3), 7.48 (d, J ¼ 8.8 Hz, 2H, CH), 7.71 (s, 1H, CH), 7.76 (d, J ¼ 8.8 Hz,
2H, CH).
4.4. General procedure for the preparation of 4-aryl substituted-
1H-pyrazol-3-ylamines (4)
To
5
mmol of the corresponding 3-dimethylamino-2-aryl
4.6.1. 1,3-Dihydro-pyrazolo[1,5-a][1,3,5]triazin-2,4-dione (10a)
Yield 64%. Mp: 310e311 ꢀC (lit [27]: 330 ꢀC). ESI-MS 150.5 m/z
substituted acrylonitriles in 80 ml of ethanol was added 13 ml of
80%wt hydrazine water solution (200 mmol). The reaction mixture
was refluxed for 8 h. On cooling, the reaction mixture was
concentrated by rotary evaporation under vacuum. The residue was
extracted with ethyl acetate (20ml ꢂ 2) and purified by column
chromatography (HEX/EA (1:1) was first used to remove impurities
followed by flashing out the product with CHCl3/MeOH (5:1)) to
give the product.
(M ꢃ 1). 1H NMR (DMSO-d6):
d
5.79 (d, J ¼ 1.8 Hz, 1H, CH), 7.88 (d,
J ¼ 1.5 Hz, 1H, CH), 11.58 (s, 1H, NH), 11.88 (s, 1H, NH). 13C NMR
(DMSO-d6):
d 89.6, 141.8, 145.4, 146.3, 149.4.
4.6.2. 1,3-Dihydro-7-methylpyrazolo[1,5-a][1,3,5]triazin-2,4-dione
(10b)
Yield 55%. Mp: 317e318 ꢀC. ESI-MS 165.0 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d 2.19 (s, 3H, CH3), 5.65 (s, 1H, CH), 11.49 (s, 1H, NH),
4.4.1. 4-(4-Iodophenyl)-1H-pyrazol-3-ylamine (4a)
11.78 (s, 1H, NH). 13C NMR (DMSO-d6):
d 14.9, 90.0, 141.9, 145.1,
Yield 29%. Mp: 168e170 ꢀC. 1H NMR (DMSO-d6):
d
4.63 (s, 1H,
149.5, 155.8.
CH), 5.19 (s, broad, 1H, NH), 7.33 (d, J ¼ 6.4 Hz, 2H, CH), 7.64 (d,
J ¼ 8.4 Hz, 2H, CH), 7.77 (s, 1H, NH), 11.79 (s, 1H, NH).
4.6.3. 1,3-Dihydro-7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-2,4-
dione (10c)
4.4.2. 4-(4-Pentafluorosulfurphenyl)-1H-pyrazol-3-ylamine (4b)
Yield 41%. Mp: 258e259 ꢀC. ESI-MS 207.1 m/z (M ꢃ 1). 1H NMR
Yield 19% as black oil. 1H NMR (DMSO-d6):
d
5.05 (s, 2H, NH2),
(DMSO-d6): d 1.25 (s, 9H, CH3), 5.74 (s, 1H, CH), 11.45 (s, 1H, NH),
7.70 (d, J ¼ 8.4 Hz, 2H, CH), 7.79 (d, J ¼ 8.4 Hz, 2H, CH), 7.81 (s, 1H,
11.81 (s, 1H, NH). 13C NMR (DMSO-d6):
d 29. 5, 32.4, 85.7, 140.7,
CH), 11.83 (s, broad, 1H, NH).
144.2, 148.3, 166.6.
4.4.3. 3-Oxo-3-phenylpropionitrile (6)
4.6.4. 1,3-Dihydro-7-trifluoromethylpyrazolo[1,5-a][1,3,5]triazin-
2,4-dione (10d)
To 1.99 g of 2-bromo-1-phenyl-ethanone (10 mmol) dissolved in
6 ml of ethanol was added 6 ml of 5 M potassium cyanide solution.
The reaction mixture was stirred at 50 ꢀC for 2 h and then poured
into 50 ml of water. The pH was adjusted to 3 with 2 M HCl, the
precipitate that formed was filtered and recrystallized from
ethanol/water to give 1.1 g product. Yield 76%. Mp: 77e78 ꢀC (lit
1 mmol of N-ethoxycarbonyl-N0-(5-trifluoromethyl-pyrazol-3-
yl) urea was refluxed in a mixture of 4 ml of ethanol and 0.76 ml
of 21%wt EtONa (2 mmol) ethanol solution for 0.5 h. The precipitate
that formed was collected by filtration, then dissolved in 20 ml of
water and acidified with HCI until pH 4. The resulting solution was
extracted by ethyl acetate (20ml ꢂ 3) and the organic phase was
rotary evaporated under vacuum to dryness to yield 0.16 g product.
Yield 73%. Mp: 228e230 ꢀC. ESI-MS 218.9 m/z (M ꢃ 1). 1H NMR
[26]: 80 ꢀC). 1H NMR (DMSO-d6):
d 4.76 (s, 2H, CH2), 7.57 (t,
J ¼ 7.2 Hz, 2H, CH), 7.71 (t, J ¼ 7.2 Hz, 1H, CH), 7.95 (d, J ¼ 7.2 Hz, 2H,
CH).
(DMSO-d6):
(DMSO-d6):
d
d
6.21 (s, 1H, CH), 12.02 (s, broad, 2H, NH). 13C NMR
86.3, 116.5, 119.2, 121.9, 124.6, 142.5, 143.8, 144.8,
4.4.4. 5-Phenyl-1H-pyrazol-3-ylamine (7)
To 1.45 g of 3-oxo-3-phenyl-propionitrile (10 mmol) in 25 ml of
ethanol was added 6.4 ml of 80%wt hydrazine water solution
(100 mmol). The reaction mixture was refluxed for 3 h. On cooling,
the precipitate that came out was filtered. The filtrate was then
concentrated by rotary evaporation under vacuum to remove the
ethanol. The residue was extracted with ethyl acetate (20ml ꢂ 2)
and purified by column chromatography (HEX/EA (1:1) was first
used to remove impurities followed by CHCl3/MeOH (3:1) to flash
out the product) to give 0.25 g product. Yield 16%. Mp: 122e123 ꢀC
145.2, 145.6, 146.0, 148.1.
4.6.5. 1,3-Dihydro-7-(4-fluorophenyl)pyrazolo[1,5-a][1,3,5]triazin-
2,4-dione (10e)
Yield 80%. Mp: 316e318 ꢀC. ESI-MS 245.0 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
6.31 (s, 1H, CH), 7.30 (t, J ¼ 6.6 Hz, 2H, CH), 7.98 (dd,
J ¼ 6.6 Hz, JHeF ¼ 4.2 Hz, 2H, CH), 11.63 (s, 1H, NH), 12.03 (s, 1H, NH).
13C NMR (DMSO-d6):
d 86.02, 115.9, 128.3, 141.9, 144.3, 148.4, 154.3,
161.6, 164.0.

8
L. Sun et al. / European Journal of Medicinal Chemistry 65 (2013) 1e11
4.6.6. 1,3-Dihydro-7-(4-chlorophenyl)pyrazolo[1,5-a][1,3,5]triazin-
2,4-dione (10f)
4.6.15. 1,3-Dihydro-8-bromopyrazolo[1,5-a][1,3,5]triazin-2,4-dione
(13c)
Yield 64%. Mp: 296e298 ꢀC. ESI-MS 261.2, 263.2 m/z (M ꢃ 1). 1H
Yield 66%. Mp: 306e308 ꢀC. ESI-MS 228.9, 230.9 m/z (M ꢃ 1). 1H
NMR (DMSO-d6):
d
6.33 (s, 1H, CH), 7.53 (d, J ¼ 8.8 Hz, 2H, CH), 7.95
NMR (DMSO-d6): d 7.91 (s, 1H, CH), 11.72 (s, 1H, NH), 12.25 (s, 1H,
(d, J ¼ 8.8 Hz, 2H, CH), 11.64 (s, 1H, NH), 12.04 (s, 1H, NH). 13C NMR
NH). 13C NMR (DMSO-d6):
d 75.1, 140.0, 144.8, 146.2, 149.5.
(DMSO-d6): d 86.0,127.8,128.8,130.4,133.9,141.9,144.2,148.3,154.0.
4.6.16. 1,3-Dihydro-8-iodopyrazolo[1,5-a][1,3,5]triazin-2,4-dione
(13d)
4.6.7. 1,3-Dihydro-7-(4-bromophenyl)pyrazolo[1,5-a][1,3,5]triazin-
2,4-dione (10g)
Yield 61%. Mp: 292e294 ꢀC. ESI-MS 276.9 m/z (M ꢃ 1). 1H NMR
Yield 58%. Mp: 310e312 ꢀC. ESI-MS 305.2, 307.2 m/z (M ꢃ 1). 1H
(DMSO-d6):
NMR (DMSO-d6): d 143.2, 144.6, 149.6, 150.2.
d C
7.82 (s, 1H, CH), 11.67 (s, 1H, NH), 12.01 (s, 1H, NH). 13
NMR (DMSO-d6):
d
6.33 (s, 1H, CH), 7.53 (d, J ¼ 8.8 Hz, 2H, CH), 7.95
(d, J ¼ 8.8 Hz, 2H, CH), 11.64 (s, 1H, NH), 12.04 (s, 1H, NH). 13C NMR
(DMSO-d6):
d
86.0,122.6,128.1,130.8,131.7,141.9,144.1,148.3,154.0.
4.6.17. 1,3-Dihydro-8-phenylpyrazolo[1,5-a][1,3,5]triazin-2,4-dione
(13e)
4.6.8. 1,3-Dihydro-7-(4-trifluoromethylphenyl)pyrazolo[1,5-a]
[1,3,5]triazin-2,4-dione (10h)
Yield 54%. Mp: 268e269 ꢀC. ESI-MS 227.0 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
7.3 (t, J ¼ 7.2 Hz, 1H, CH), 7.42(t, J ¼ 7.2 Hz, 2H, CH),
Yield 91%. Mp: 302e304 ꢀC. ESI-MS 295.2 m/z (M ꢃ 1). 1H NMR
7.53 (d, J ¼ 7.2 Hz, 2H, CH), 8.13 (s, 1H, CH), 11.7 (s, 1H, NH), 11.88 (s,
(DMSO-d6):
d
6.43 (s, 1H, CH), 7.83 (d, J ¼ 8.4 Hz, 2H, CH), 8.16 (d,
broad, 1H, NH). 13C NMR (DMSO-d6):
d 104.3, 126.6, 127.1, 128.6,
J ¼ 8.4 Hz, 2H, CH), 11.70 (s, 1H, NH), 12.09 (s, 1H, NH). 13C NMR
129.6, 136.2, 144.1, 144.5, 148.8.
(DMSO-d6):
d 86.4, 122.7, 125.4, 125.6, 125.7, 126.8, 128.1, 128.8,
129.1, 129.4, 129.7, 135.5, 142.1, 144.1, 148.3, 153.6.
4.6.18. 1,3-Dihydro-8-(4-fluorophenyl)pyrazolo[1,5-a][1,3,5]triazin-
2,4-dione (13f)
4.6.9. 1,3-Dihydro-7-(4-methylphenyl)pyrazolo[1,5-a][1,3,5]triazin-
2,4-dione (10i)
Yield 79%. Mp: 280e282 ꢀC. ESI-MS 245.0 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
7.24 (t, J ¼ 6.6 Hz, 2H, CH), 7.55 (dd, J ¼ 6.6 Hz, JHe
Yield 83%. Mp: 308e309 ꢀC. ESI-MS 241.3 m/z (M ꢃ 1). 1H NMR
¼ 4.2 Hz, 2H, CH), 8.10 (s, 1H, CH),11.7 (s,1H, NH),11.92 (s,1H, NH).
F
(DMSO-d6):
d
2.35 (s, 3H, CH3), 6.26 (s, 1H, CH), 7.28 (d, J ¼ 8.0 Hz,
13C NMR (DMSO-d6):
d 103.5, 115.3, 115.5, 126.0, 129.2, 129.3, 136.2,
2H, CH), 7.80 (d, J ¼ 8.0 Hz, 2H, CH), 11.59 (s, 1H, NH), 11.98 (s, 1H,
144.2, 144.5, 148.8, 159.8, 162.3.
NH). 13C NMR (DMSO-d6):
d 20.9, 85.8, 126.0, 128.8, 129.4, 138.9,
141.7, 144.2, 148.4, 155.2.
4.6.19. 1,3-Dihydro-8-(4-chlorophenyl)pyrazolo[1,5-a][1,3,5]
triazin-2,4-dione (13g)
4.6.10. 1,3-Dihydro-7-(4-tert-butyl-phenyl)-pyrazolo[1,5-a][1,3,5]
triazin-2,4-dione (10j)
Yield 57%. Mp: 296e297 ꢀC. ESI-MS 261.3, 263.1 m/z (M ꢃ 1). 1H
NMR (DMSO-d6):
d
7.45 (d, J ¼ 8.4 Hz, 2H, CH), 7.55 (d, J ¼ 8.4 Hz, 2H,
Yield 85%. Mp: 307e309 ꢀC. ESI-MS 283.3 m/z (M ꢃ 1). 1H NMR
CH), 8.14 (s, 1H, CH), 11.75 (s, 1H, NH), 11.95 (s, 1H, NH). 13C NMR
(DMSO-d6): 103.2,128.5,128.6,128.9,131.1,136.5,144.1,144.4,148.8.
(DMSO-d6):
d
1.31 (s, 9H, CH3), 6.26 (s, 1H, CH), 7.49 (d, J ¼ 8.8 Hz,
d
2H, CH), 7.84 (d, J ¼ 8.8 Hz, 2H, CH), 11.60 (s, 1H, NH), 11.99 (s, 1H,
NH). 13C NMR (DMSO-d6):
30.9, 34.4, 85.8, 125.5, 125.8, 128.7,
141.6, 144.2, 148.3, 151.9, 155.0.
d
4.6.20. 1,3-Dihydro-8-(4-bromophenyl)pyrazolo[1,5-a][1,3,5]
triazin-2,4-dione (13h)
Yield 49%. Mp: 278e280 ꢀC. ESI-MS 306.9 m/z (M ꢃ 1). 1H NMR
4.6.11. 1,3-Dihydro-7-(4-methoxyphenyl)pyrazolo[1,5-a][1,3,5]
triazin-2,4-dione (10k)
(DMSO-d6):
d
7.49 (d, J ¼ 8.8 Hz, 2H, CH), 7.59 (d, J ¼ 8.8 Hz, 2H, CH),
8.14 (s, 1H, CH), 11.71 (s, 1H, NH), 11.93 (s, 1H, NH). 13C NMR (DMSO-
d6): 103.2, 119.6, 128.9, 129.1, 131.4, 136.5, 144.1, 144.3, 148.8.
Yield 78%. Mp: 260e262 ꢀC. ESI-MS 257.2 m/z (M ꢃ 1). 1H NMR
d
(DMSO-d6):
d
3.81 (s, 3H, CH3), 6.22 (s, 1H, CH), 7.02 (d, J ¼ 8.8 Hz,
2H, CH), 7.85 (d, J ¼ 8.8 Hz, 2H, CH), 11.56 (s, 1H, NH), 11.96 (s, 1H,
4.6.21. 1,3-Dihydro-8-(4-trifluoromethylphenyl)pyrazolo[1,5-a]
[1,3,5]triazin-2,4-dione (13i)
NH). 13C NMR (DMSO-d6):
d 55.1, 85.5, 114.1, 124.0, 127.5, 141.6,
144.1, 148.3, 154.9, 160.1.
Yield 77%. Mp: 288e289 ꢀC. ESI-MS 295.3 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d 7.61e8.13 (m, 4H, CH), 8.23 (s, 1H, CH), 11.74 (s, 1H,
4.6.12. 1,3-Dihydro-7-phenylpyrazolo[1,5-a][1,3,5]triazin-2,4-dione
(10l)
NH), 12.05 (s, broad, 1H, NH). 13C NMR (DMSO-d6):
d 103.0, 122.9,
125.3, 125.3, 125.3, 125.4, 125.6, 126.5, 126.8, 127.5, 134.0, 137.4,
144.1, 144.3, 148.9.
Yield 89%. Mp: 308e310 ꢀC (lit [29]: 327e329 ꢀC). ESI-MS 227.0
m/z (M ꢃ 1). 1H NMR (DMSO-d6):
d 6.31 (s, 1H, CH), 7.39e7.53 (m,
3H, CH), 7.93 (d, J ¼ 6.8 Hz, CH, 2H), 11.63 (s, 1H, NH), 12.02 (s, 1H,
4.6.22. 1,3-Dihydro-8-(4-methylphenyl)pyrazolo[1,5-a][1,3,5]
triazin-2,4-dione (13j)
NH). 13C NMR (DMSO-d6):
d 86.0, 126.1, 128.8, 129.3, 131.5, 141.8,
144.2, 148.3, 155.1.
Yield 87%. Mp: 289e290 ꢀC. ESI-MS 241.3 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
2.32 (s, 3H, CH3), 7.21 (d, J ¼ 8.0 Hz, 2H, CH), 7.41 (d,
4.6.13. 1,3-Dihydro-8-methylpyrazolo[1,5-a][1,3,5]triazin-2,4-dione
(13a)
J ¼ 8.0 Hz, 2H, CH), 8.09 (s, 1H, CH), 11.69 (s, 1H, NH), 11.81 (s, 1H,
NH). 13C NMR (DMSO-d6):
d 20.7, 104.3, 126.6, 126.9, 129.1, 135.8,
Yield 51%. Mp: 318e319 ꢀC. ESI-MS 164.5 m/z (M ꢃ 1). 1H NMR
135.9, 144.1, 144.5, 148.8.
(DMSO-d6): d 1.96 (s, 3H, CH3), 7.65 (s,1H, CH),11.47 (s,1H, NH),11.81
(s,1H, NH).13C NMR (DMSO-d6):
d
6.52, 97.1,137.2,144.2,146.5,148.6.
4.6.23. 1,3-Dihydro-8-(4-tert-butylphenyl)pyrazolo[1,5-a][1,3,5]
triazin-2,4-dione (13k)
4.6.14. 1,3-Dihydro-8-chloropyrazolo[1,5-a][1,3,5]triazin-2,4-dione
(13b)
Yield 74%. Mp: 301e303 ꢀC. ESI-MS 283.0 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
1.31 (s, 9H, CH3), 6.25 (s, 1H, CH), 7.48 (d, J ¼ 8.4 Hz,
Yield 53%. Mp: 299e300 ꢀC. ESI-MS 185.0, 186.9 m/z (M ꢃ 1). 1H
2H, CH), 7.84 (d, J ¼ 8.4 Hz, 2H, CH), 11.59 (s, 1H, NH), 11.98 (s, 1H,
NH). 13C NMR (DMSO-d6):
30.9, 34.4, 85.8, 125.5, 125.8, 128.7,
141.6, 144.2, 148.3, 151.9, 155.0.
NMR (DMSO-d6):
d
7.94 (s, 1H, CH), 11.72 (s, 1H, NH), 12.33 (s, 1H,
d
NH). 13C NMR (DMSO-d6):
d 91.2, 138.4, 144.4, 144.8, 149.4.

L. Sun et al. / European Journal of Medicinal Chemistry 65 (2013) 1e11
9
4.6.24. 1,3-Dihydro-8-(4-methoxyphenyl)pyrazolo[1,5-a][1,3,5]
triazin-2,4-dione (13l)
J ¼ 8.7 Hz, JHeF ¼ 5.7 Hz, 2H, CH), 12.76 (s, 1H, NH), 13.59 (s, broad,
1H, NH). 13C NMR (DMSO-d6):
d 86.7, 115.9, 128.5, 141.4, 141.6, 154.4,
Yield 75%. Mp: 264e266 ꢀC. ESI-MS 257.3 m/z (M ꢃ 1). 1H NMR
161.6, 164.1, 173.0.
(DMSO-d6):
d
3.78 (s, 3H, CH3), 6.97 (d, J ¼ 8.4 Hz, 2H, CH), 7.44 (d,
J ¼ 8.4 Hz, 2H, CH), 8.06 (s, 1H, CH), 11.66 (s, 1H, NH), 11.81 (s, 1H,
4.8.6. 1,3-Dihydro-7-(4-chlorophenyl)pyrazolo[1,5-a][1,3,5]triazin-
2-thioxo-4-one (15f)
NH). 13C NMR (DMSO-d6):
d 55.1, 104.2, 114.1, 121.9, 128.5, 135.5,
144.2, 144.6, 148.8, 158.1.
Yield 80%. Mp: 288e290 ꢀC. ESI-MS 277.0, 279.3 m/z (M ꢃ 1). 1H
NMR (DMSO-d6):
d
6.40 (s, 1H, CH), 7.53 (d, J ¼ 8.4 Hz, 2H, CH), 7.97
4.7. General method for preparation of N-ethoxycarbonyl-N0-
(pyrazol-3-yl)thioureas (14, 16)
(d, J ¼ 8.4 Hz, 2H, CH), 12.71 (s, broad, 1H, NH). 13C NMR (DMSO-d6):
d 87.9, 129.0, 130.0, 131.2, 135.2, 142.5, 142.8, 155.3, 174.1.
To a fine suspension of 1 mmol of the corresponding amine in
3 ml of anhydrous DMF was added 1 mmol of ethoxycarbonyl
isothiocyanate. After stirring the mixture for 1 h at room temper-
ature, 30 ml of cold water was added. The precipitated product was
filtered, washed with cold water and recrystallized from ethanol.
4.8.7. 1,3-Dihydro-7-(4-bromophenyl)pyrazolo[1,5-a][1,3,5]triazin-
2-thioxo-4-one (15g)
Yield 64%. Mp: 293e294 ꢀC. ESI-MS 320.9, 322.9 m/z (M ꢃ 1). 1H
NMR (DMSO-d6):
d
6.43 (s, 1H, CH), 7.68 (d, J ¼ 8.4 Hz, 2H, CH), 7.91
(d, J ¼ 8.4 Hz, 2H, CH), 12.78 (s, 1H, NH), 13.59 (s, broad, 1H, NH). 13
C
NMR (DMSO-d6): d 86.7, 122.8, 128.2, 130.4, 131.8, 141.3, 141.7, 154.2,
4.8. General method for preparation of 1,3-dihydro-pyrazolo[1,5-a]
173.0.
[1,3,5]triazin-2-thioxo-4-ones (15, 17)
4.8.8. 1,3-Dihydro-7-(4-trifluoromethylphenyl)pyrazolo[1,5-a]
[1,3,5]triazin-2-thioxo-4-one (15h)
1 mmol of the corresponding N-ethoxycarbonyl-N0-(pyrazol-3-
yl)thiourea was refluxed in a mixture of 4 ml of ethanol and
0.76 ml of 21%wt EtONa (2 mmol) ethanol solution for 0.5 h. Pre-
cipitate that came out was collected by filtration, then dissolved in
water and acidified with HCI until pH 4. Precipitate that formed was
filtered and recrystallized from methanol/water.
Yield 85%. Mp: 277e278 ꢀC. ESI-MS 311.2 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
6.53 (s, 1H, CH), 7.84 (d, J ¼ 8.4 Hz, 2H, CH), 8.19 (d,
J ¼ 8.4 Hz, 2H, CH), 12.84 (s, 1H, NH), 13.65 (s, 1H, NH). 13C NMR
(DMSO-d6):
d 87.1, 120.0, 122.7, 125.4, 125.7, 126.9, 128.1, 129.0,
129.3, 129.6, 129.9, 135.1, 141.4, 141.8, 153.8, 173.1.
4.8.1. 1,3-Dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-one
(15a)
4.8.9. 1,3-Dihydro-7-(4-methylphenyl)pyrazolo[1,5-a][1,3,5]triazin-
2-thioxo-4-one (15i)
Yield 54%. Mp: 284e286 ꢀC. (lit [27]: 298e300 ꢀC) ESI-MS 167.0
Yield 81%. Mp: 295 ꢀC. ESI-MS 257.2 m/z (M ꢃ 1). 1H NMR
m/z (M ꢃ 1). 1H NMR (DMSO-d6):
d
5.90 (d, J ¼ 1.5 Hz, 1H, CH), 7.88
(DMSO-d6):
d
2.35 (s, 3H, CH3), 6.34 (s, 1H, CH), 7.27 (d, J ¼ 8.0 Hz,
(d, J ¼ 1.5 Hz, 1H, CH), 12.72 (s, 1H, NH), 13.46 (s, 1H, NH). 13C NMR
2H, CH), 7.81 (d, J ¼ 8.0 Hz, 2H, CH),12.73 (s,1H, NH),13.53 (s, broad,
(DMSO-d6):
d
90.5, 141.5, 142.6, 146.6, 174.0.
1H, NH). 13C NMR (DMSO-d6):
d 20.9, 86.5, 126.1, 128.4, 129.4, 139.2,
141.4, 141.5, 155.4, 173.0.
4.8.2. 1,3-Dihydro-7-methylpyrazolo[1,5-a][1,3,5]triazin-2-thioxo-
4-one (15b)
4.8.10. 1,3-Dihydro-7-(4-tert-butylphenyl)pyrazolo[1,5-a][1,3,5]
triazin-2-thioxo-4-one (15j)
Yield 79%. Mp: 281e282 ꢀC. ESI-MS 181.0 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
2.23 (s, 3H, CH3), 5.77 (s,1H, CH),12.61 (s,1H, NH),13.33
Yield 67%. Mp: 289 ꢀC. ESI-MS 299.1 m/z (M ꢃ 1). 1H NMR
(s,1H, NH).13C NMR (DMSO-d6):
d
15.0, 90.7,141.8,142.3,155.8,173.9.
(DMSO-d6):
d
1.31 (s, 9H, CH3), 6.37 (s, 1H, CH), 7.50 (d, J ¼ 8.8 Hz,
2H, CH), 7.87 (d, J ¼ 8.8 Hz, 2H, CH),12.76 (s,1H, NH),13.57 (s, broad,
4.8.3. 1,3-Dihydro-7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-2-
thioxo-4-one (15c)
1H, NH). 13C NMR (DMSO-d6):
d 30.9, 34.4, 86.5, 125.5, 125.9, 128.4,
141.4, 141.4, 152.1, 155.3, 172.9.
Yield 56%. Mp: 258e260 ꢀC. ESI-MS 223.3 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
1.26 (s, 9H, CH3), 5.85 (s, 1H, CH), 12.62 (s, 1H, NH),
4.8.11. 1,3-Dihydro-7-(4-methoxyphenyl)pyrazolo[1,5-a][1,3,5]
triazin-2-thioxo-4-one (15k)
13.41 (s, 1H, NH). 13C NMR (DMSO-d6):
d 29.5, 32.5, 86.5, 140.5,
141.4, 166.9, 172.8.
Yield 58%. Mp: 288 ꢀC. ESI-MS 273.2 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
3.81 (s, 3H, CH3), 6.30 (s, 1H, CH), 7.02 (d, J ¼ 8.4 Hz,
4.8.4. 1,3-Dihydro-7-trifluoromethylpyrazolo[1,5-a][1,3,5]triazin-2-
thioxo-4-one (15d)
2H, CH), 7.88 (d, J ¼ 8.4 Hz, 2H, CH),12.57 (s, broad,1H, NH),13.53 (s,
broad, 1H, NH). 13C NMR (DMSO-d6):
d 56.3, 87.6, 115.2, 124.9, 128.7,
1 mmol of N-ethoxycarbonyl-N0-(5-trifluoromethyl-pyrazol-3-
yl) thiourea was refluxed in a mixture of 4 ml of ethanol and
0.76 ml of 21%wt EtONa (2 mmol) ethanol solution for 0.5 h. The
precipitate that formed was collected by filtration, then dissolved in
20 ml of water and acidified with HCI until pH 4. The resulting
solution was extracted by ethyl acetate (20ml ꢂ 3) and concen-
trated by rotary evaporation under vacuum. The residue was
recrystallized from EA/HEX to give 75 mg product. Yield 32%. Mp:
142.7, 143.2, 156.3, 161.3, 174.0.
4.8.12. 1,3-Dihydro-7-phenylpyrazolo[1,5-a][1,3,5]triazin-2-thioxo-
4-one (15l)
Yield 69%. Mp: 285e286 ꢀC (lit [29]: 304e305 ꢀC). ESI-MS 243.3
m/z (M ꢃ 1). 1H NMR (DMSO-d6):
d 6.41 (s, 1H, CH), 7.37e7.58 (m,
3H, CH), 7.95 (d, J ¼ 6.6 Hz, 2H, CH), 12.78 (s, 1H), 13.58 (s, 1H). 13
C
NMR (DMSO-d6): d 87.8,127.3,129.9,130.6,132.3,142.5,142.7,156.4,
236e237 ꢀC. ESI-MS 234.9 m/z (M ꢃ 1). 1H NMR (DMSO-d6):
d
6.27
87.0,
174.1.
(s, 1H, CH), 13.02 (s, broad, 1H, NH). 13C NMR (DMSO-d6):
d
116.4, 119.1, 121.8, 124.5, 141.0, 142.2, 144.9, 145.3, 145.7, 146.1, 173.6.
4.8.13. 1,3-Dihydro-8-methylpyrazolo[1,5-a][1,3,5]triazin-2-thioxo-
4-one (17a)
4.8.5. 1,3-Dihydro-7-(4-fluorophenyl)pyrazolo[1,5-a][1,3,5]triazin-
2-thioxo-4-one (15e)
Yield 47%. Mp: 276 ꢀC. ESI-MS 180.6 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d 2.04 (s, 3H, CH3), 7.76 (s, 1H, CH), 12.61 (s, 1H, NH),
Yield 89%. Mp: 286e287 ꢀC. ESI-MS 261.0 m/z (M ꢃ 1). 1H NMR
13.41 (s, 1H, NH). 13C NMR (DMSO-d6):
d 6.7, 98.4, 137.1, 141.4, 146.9,
172.9.
(DMSO-d6):
d
6.41 (s, 1H, CH), 7.31 (t, J ¼ 8.7 Hz, 2H, CH), 8.01 (dd,

10
L. Sun et al. / European Journal of Medicinal Chemistry 65 (2013) 1e11
4.8.14. 1,3-Dihydro-8-chloropyrazolo[1,5-a][1,3,5]triazin-2-thioxo-
4-one (17b)
4.8.23. 1,3-Dihydro-8-(4-tert-butylphenyl)pyrazolo[1,5-a][1,3,5]
triazin-2-thioxo-4-one (17k)
Yield 45%. Mp: 258e260 ꢀC. ESI-MS m/z 200.9, 203.0 (M ꢃ 1).
Yield 94%. Mp: 288e289 ꢀC. ESI-MS 299.1 m/z (M ꢃ 1). 1H NMR
1H NMR (DMSO-d6):
d
8.02 (s, 1H, CH), 12.80 (s, 1H, NH), 13.79 (s,
(DMSO-d6):
d
1.31 (s, 9H, CH3), 6.34 (s, 1H, CH), 7.49 (d, J ¼ 8.5 Hz,
1H, NH). 13C NMR (DMSO-d6):
d
91.9, 138.2, 142.0, 144.8, 174.7.
2H, CH), 7.86 (d, J ¼ 8.5 Hz, 2H, CH), 12.72 (s, 1H, NH), 13.54 (s, 1H,
NH). 13C NMR (DMSO-d6):
30.9, 34.4, 86.5, 125.5, 125.0, 128.4,
141.4, 141.5, 152.1, 155.3, 172.9.
d
4.8.15. 1,3-Dihydro-8-bromopyrazolo[1,5-a][1,3,5]triazin-2-thioxo-
4-one (17c)
Yield 56%. Mp: >360 ꢀC. ESI-MS 245.0, 246.9 m/z (M ꢃ 1). 1H
4.8.24. 1,3-Dihydro-8-(4-methoxyphenyl)pyrazolo[1,5-a][1,3,5]
triazin-2-thioxo-4-one (17l)
NMR (DMSO-d6):
d 8.00 (s, 1H, CH), 12.78 (s, 1H, NH), 13.70 (s, 1H,
NH). 13C NMR (DMSO-d6):
d
75.9, 139.9, 142.0, 146.7, 174.9.
Yield 85%. Mp: 227e228 ꢀC. ESI-MS 273.2 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
3.79 (s, 3H, OCH3), 6.98 (d, J ¼ 8.5 Hz, 2H, CH), 7.45 (d,
4.8.16. 1,3-Dihydro-8-iodopyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-
one (17d)
J ¼ 8.5 Hz, 2H, CH), 8.10 (s, 1H, CH), 12.62 (s, 1H, NH). 13C NMR
(DMSO-d6): d 55.1,105.1,113.8,121.3,129.4,135.8, 141.4,145.0,158.4,
Yield 60%. Mp: 199e201 ꢀC. ESI-MS 292.7 m/z (M ꢃ 1). 1H NMR
173.6.
(DMSO-d6):
NMR (DMSO-d6):
d C
7.92 (s, 1H, CH), 12.77 (s, 1H, NH),13.50 (s, 1H, NH). 13
d
42.5, 141.9, 143.1, 151.0, 174.9.
4.8.25. 1,3-Dihydro-8-(4-ethoxyphenyl)pyrazolo[1,5-a][1,3,5]
triazin-2-thioxo-4-one (17m)
4.8.17. 1,3-Dihydro-8-phenylpyrazolo[1,5-a][1,3,5]triazin-2-thioxo-
Yield 91%. Mp: 286e287 ꢀC. ESI-MS 287.2 m/z (M ꢃ 1). 1H NMR
4-one (17e)
(DMSO-d6):
d
1.35 (t, J ¼ 7.2 Hz, 3H, CH3), 4.08 (q, J ¼ 7.2 Hz, 2H,
Yield 60%. Mp: 222e224 ꢀC (lit [14]: 251e253 ꢀC). ESI-MS 243.3
CH2), 6.33 (s, 1H, CH), 7.01 (d, J ¼ 8.8 Hz, 2H, CH), 7.86 (d, J ¼ 8.8 Hz,
m/z (M ꢃ 1). 1H NMR (DMSO-d6):
d
7.32 (t, J ¼ 7.2 Hz,1H, CH), 7.41 (t,
2H, CH), 12.72 (s, 1H, NH), 13.52 (s, broad, 1H, NH). 13C NMR (DMSO-
J ¼ 7.2 Hz, 2H, CH), 7.51 (d, J ¼ 7.2 Hz, 1H, CH), 8.17 (s, 1H, CH), 12.80
d6):
d 14.6, 63.1, 86.3, 114.6, 123.5, 127.7, 141.4, 141.4, 155.3, 159.6,
(s, NH, 1H). 13C NMR (DMSO-d6):
d 106.5, 128.1, 129.2, 129.5, 130.2,
172.9.
137.2, 142.6, 146.2, 174.9.
4.8.26. 1,3-Dihydro-8-(4-nitrophenyl)pyrazolo[1,5-a][1,3,5]triazin-
2-thioxo-4-one (17n)
4.8.18. 1,3-Dihydro-8-(4-fluorophenyl)pyrazolo[1,5-a][1,3,5]triazin-
2-thioxo-4-one (17f)
Yield 81%. Mp: 261e263 ꢀC. ESI-MS 288.1 m/z (M ꢃ 1). 1H NMR
Yield 68%. Mp: 310 ꢀC. ESI-MS 261.3 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
7.83 (d, J ¼ 8.4 Hz, 2H, CH), 8.25 (d, J ¼ 8.4 Hz, 2H, CH),
(DMSO-d6):
d
7.25 (t, J ¼ 8.7 Hz, 2H, CH), 7.55 (dd, J ¼ 8.7 Hz, JHe
8.32 (s, 1H, CH), 12.89 (s, 1H, NH). 13C NMR (DMSO-d6):
d 103.5,
¼ 5.7 Hz, 2H, CH), 8.14 (s, 1H, CH), 12.75 (s, NH, 1H). 13C NMR
123.5, 128.6, 136.4, 137.8, 141.4, 144.7, 145.7, 174.2.
F
(DMSO-d6):
d 105.4, 116.1, 116.4, 126.66, 126.7, 131.3, 131.4, 137.6,
142.6, 146.1, 160.8, 164.1, 174.9.
4.8.27. 1,3-Dihydro-8-(4-iodophenyl)pyrazolo[1,5-a][1,3,5]triazin-
2-thioxo-4-one (17o)
4.8.19. 1,3-Dihydro-8-(4-chlorophenyl)pyrazolo[1,5-a][1,3,5]
triazin-2-thioxo-4-one (17g)
Yield 86%. Mp: 369 ꢀC. ESI-MS 321.1, 322.9 m/z (M ꢃ 1). 1H NMR
(DMSO-d6):
d
7.33 (d, J ¼ 7.2 Hz, 2H, CH), 7.76 (d, J ¼ 7.2 Hz, 2H, CH),
Yield 90%. Mp: 262e263 ꢀC. ESI-MS 276.7, 279.3 m/z (M ꢃ 1).
8.16 (s, 1H, CH), 12.79 (s, 1H, NH). 13C NMR (DMSO-d6):
d 92.8, 104.3,
1H NMR (DMSO-d6):
d
7.47 (d, J ¼ 8.4 Hz, 2H, CH), 7.53 (d,
128.7, 130.2, 136.4, 137.1, 141.3, 144.6, 173.8.
J ¼ 8.4 Hz, 2H, CH), 8.16 (s, 1H, CH), 12.81 (s, 1H, NH). 13C NMR
(DMSO-d6):
d
104.2, 128.0, 128.3, 129.9, 131.7, 136.5, 141.4, 144.9,
4.8.28. 1,3-Dihydro-8-(4-pentafluorosulfurphenyl)-pyrazolo[1,5-a]
[1,3,5]triazin-2-thioxo-4-one (17p)
173.9.
Yield 43%. Mp: 268e269 ꢀC. ESI-MS 369.0 m/z (M ꢃ 1). 1H NMR
4.8.20. 1,3-Dihydro-8-(4-bromophenyl)pyrazolo[1,5-a][1,3,5]
triazin-2-thioxo-4-one (17h)
(DMSO-d6):
d
7.72 (d, J ¼ 8.4 Hz, 2H, CH), 7.93 (d, J ¼ 8.0 Hz, 2H, CH),
8.24 (s, 1H, CH), 12.86 (s, 1H, NH). 13C NMR (DMSO-d6):
d 103.4,
Yield 81%. Mp: 239 ꢀC. ESI-MS 321.1, 322.9 m/z (M ꢃ 1). 1H
125.7, 128.8, 133.4, 137.5, 141.4, 144.7, 151.0, 174.0.
NMR (DMSO-d6):
d
7.48 (d, J ¼ 8.4 Hz, 2H, CH), 7.60 (d,
J ¼ 8.4 Hz, 2H, CH), 8.17 (s, 1H, CH), 12.79 (s, 1H, NH). 13C NMR
4.9. In vitro TP enzyme assay
(DMSO-d6):
d 105.3, 121.2, 129.6, 131.3, 132.3, 137.8, 142.6, 145.9,
175.0.
The recombinant E. coli thymidine phosphorylase (EC Number
2.4.2.4) expressed in E. coli was purchased from a commercial
supplier (Sigma, T-2807). The enzymatic activity assay was per-
formed at room temperature in a Hitachi U-1900 spectrophotom-
eter using a detection wavelength of 290 nm. This method is based
on the manufacturer’s instruction which is a classical method
described by Krenitsky [35]. For all the compounds, the percentage
inhibition at a certain concentration was obtained by the following
procedure:
4.8.21. 1,3-Dihydro-8-(4-trifluoromethylphenyl)pyrazolo[1,5-a]
[1,3,5]triazin-2-thioxo-4-one (17i)
Yield 85%. Mp: 256e257 ꢀC. ESI-MS 311.0 m/z (M ꢃ 1). 1H
NMR (DMSO-d6):
d 7.72e7.79 (m, 4H, CH), 8.24 (s, 1H, CH), 12.78
(s, 2H, NH). 13C NMR (DMSO-d6):
d 104.0, 120.3, 123.0, 125.2,
125.7, 126.7, 127.0, 127.3, 127.6, 128.4, 128.7, 133.6, 137.4, 141.5,
144.8, 174.0.
10
the test compound solution in DMSO (for blank, 10
added) and 200 l of thymidine solution (5 mM in pH 7.4 phosphate
buffer) were added to 780 l of phosphate buffer (pH 7.4) in a 1.5 ml
cuvette. After gentle shaking by inversion of the cuvette, the
absorbance values at 4 min, 8 min, 12 min, 16 min and 20 min were
recorded successively. These absorbance values were plotted
against time and linear regression was performed to obtain the
m
l of TP solution (1.5 U/ml in pH 7.0 phosphate buffer), 10
ml of
4.8.22. 1,3-Dihydro-8-(4-methylphenyl)pyrazolo[1,5-a][1,3,5]
triazin-2-thioxo-4-one (17j)
m
l DMSO was
m
Yield 94%. Mp: 241e242 ꢀC. ESI-MS 257.0 m/z (M ꢃ 1). 1H NMR
m
(DMSO-d6):
d
2.34 (s, 3H, CH3), 7.22 (d, J ¼ 8.4 Hz, 2H, CH), 7.40 (d,
J ¼ 8.4 Hz, 2H, CH), 8.12 (s, 1H, CH), 12.77 (s, 1H, NH), 13.37 (s, broad,
1H, NH). 13C NMR (DMSO-d6):
d 20.7, 105.3, 126.1, 127.9, 129.0,135.9,
136.3, 141.5, 145.0, 173.4.

L. Sun et al. / European Journal of Medicinal Chemistry 65 (2013) 1e11
11
evaluation of multisubstrate bicyclic pyrimidine nucleoside inhibitors of hu-
man thymidine phosphorylase, J. Med. Chem. 49 (2006) 7807e7815.
[12] S.A. Diab, S.C. De, M. Muzard, R. Plantier-Royon, E. Pfund, T. Lequeux, Fluo-
rophosphonylated nucleoside derivatives as new series of thymidine phos-
phorylase multi-substrate inhibitors, J. Med. Chem. 55 (2012) 2758e2768.
[13] A.V. Dolzhenko, A.V. Dolzhenko, W.-K. Chui, Pyrazolo[1,5-a][1,3,5]triazines(5-
aza-9-deazapurines): synthesis and biological activity, Heterocycles 75 (2008)
1575e1622.
slope of the line plotted which was taken to be the velocity of
enzyme catalyzed reaction. The percentage inhibition at this con-
centration was calculated by dividing the difference between the
enzymatic velocity of the blank and the enzymatic velocity in the
presence of the inhibitor (or test compound) by the enzymatic
velocity of the blank.
[14] R.K. Robins, G.R. Revankar, D.E. O’Brien, R.H. Springer, T. Novinson, A. Albert,
K. Senga, J.P. Miller, D.G. Streeter, Purine analog inhibitors of xanthine oxidase
e structure activity relationships and proposed binding of the molybdenum
cofactor, J. Heterocycl. 22 (1985) 601e634.
[15] Z. Nie, C. Perretta, P. Erickson, S. Margosiak, R. Almassy, J. Lu, A. Averill,
K.M. Yager, S. Chu, Structure-based design, synthesis, and study of pyrazolo
[1,5-a][1,3,5]triazine derivatives as potent inhibitors of protein kinase CK2,
Bioorg. Med. Chem. Lett. 17 (2007) 4191e4195.
[16] T.J. Guzi, K. Paruch, Preparation of Pyrazolotriazines as Kinase Inhibitors for
Treating Cancer and Other Diseases Associated with a Kinase, Schering Cor-
poration, USA, 2005, p. 45.
[17] G. Prevost, M.-O. Lonchampt, S. Kim, B. Morgan, G. Ulibarri, C. Thurieau,
Pyrazolo[1,5-a]-1,3,5-triazine Derivatives with Activity as Cyclin-dependent
Kinase (CDK) and Glycogen Synthase Kinase-3 (GSK-3) Inhibitors, and their
Preparation, Pharmaceutical Compositions, and Use as, e.g., Antiproliferative
Agents, Societe De Conseils De Recherches Et D’applications Scientifiques
S.C.R.A.S., Fr, 2002, p. 49.
[18] K. Senga, D.E. O’Brien, M.B. Scholten, T. Novinson, J.P. Miller, R.K. Robins,
Synthesis and enzymic activity of various substituted pyrazolo[1,5-a]-1,3,5-
triazines as adenosine cyclic 30,50-phosphate phosphodiesterase inhibitors,
J. Med. Chem. 25 (1982) 243e249.
[19] T. Sullivan, B. Duemler, N. Kuttesch, T. Keravis, J. Wells, Irreversible inhibition
of calmodulin-sensitive cyclic nucleotide phosphodiesterase, J. Cyclic Nucle-
otide Protein Phosphor. Res. 11 (1986) 355e364.
To determine the IC50 value of a certain compound, percentage
inhibitions of at least 7 different concentrations that span over the
estimated IC50 values were determined by the method mentioned
above and plotted against logarithmic concentration using Origin-
Pro 8 SR0 v8.0724 (B724). The IC50 value was determined as the
concentration of the inhibitor that caused 50% inhibition. Results
were means of three experiments.
4.10. TP enzyme kinetic studies
To investigate the inhibitory effect of the most potent compound
17p against TP at varying thymidine concentrations, velocities of
the enzyme catalyzed reactions at different concentrations of
thymidine (100
mined using the same procedure as described previously. 17p was
used at concentrations of 0 M, 0.01 M, 0.1 M, and 0.2 M.
mM, 250 mM, 500 mM and 1000 mM) were deter-
m
m
m
m
LineweavereBurk plots of TP inhibition by 17p were then generated
using Microsoft Office Excel 2007 based on data obtained from
triplicate experiments to determine the inhibition type of 17p.
[20] J. Kobe, R.H. Springer, D.E. O’Brien, Pyrazolo(1,5a)1,3,5-triazines, ICN Phar-
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Acknowledgments
This work was supported by the National Medical Research
Council Grant (R-148-000-102-275), Singapore.
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