Discovery of mixed type thymidine phosphorylase inhibitors endowed with antiangiogenic properties: Synthesis, pharmacological evaluation and molecular docking study of 2-thioxo-pyrazolo[1,5-a][1,3,5]triazin-4-ones. Part II

Article abstract of DOI:10.1016/j.ejmech.2014.03.063

In our drug discovery program, a series of 2-thioxo-pyrazolo[1,5-a][1,3,5] triazin-4-ones were designed, synthesized and evaluated for their TP inhibitory potential. All the synthesized analogues conferred a varying degree of TP inhibitory activity, comparable or better than positive control, 7-deazaxanthine (7-DX, 2) (IC₅₀ value = 42.63 μM). A systematic approach to the lead optimization identified compounds 3c and 4a as the most promising TP inhibitors, exhibiting mixed mode of enzyme inhibition. Moreover, selected compounds demonstrated the ability to attenuate the expression of the angiogenic markers (viz. MMP-9 and VEGF) in MDA-MB-231 cells at sublethal concentrations. In addition, molecular docking studies revealed the plausible binding orientation of these inhibitors towards TP, which was in accordance with the experimental results. Taken as a whole, these compounds would constitute a new direction for the design of novel TP inhibitors with promising antiangiogenic properties.

Full text of DOI:10.1016/j.ejmech.2014.03.063

European Journal of Medicinal Chemistry 78 (2014) 294e303
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of mixed type thymidine phosphorylase inhibitors endowed
with antiangiogenic properties: Synthesis, pharmacological evaluation
and molecular docking study of 2-thioxo-pyrazolo[1,5-a][1,3,5]triazin-
4-ones. Part II
,
b
,
c
b
b
b,d
Hriday Bera a , Probir kumar Ojha , Bee Jen Tan , Lingyi Sun , Anton V. Dolzhenko
**
,
*
Wai-Keung Chui b , Gigi Ngar Chee Chiu
,
b
a Gokaraju Rangaraju College of Pharmacy, Bachupally, Hyderabad 500090, India
b Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore
c Jadavpur University, Kolkata 700032, India
d 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:
In our drug discovery program, a series of 2-thioxo-pyrazolo[1,5-a][1,3,5]triazin-4-ones were designed,
synthesized and evaluated for their TP inhibitory potential. All the synthesized analogues conferred a
varying degree of TP inhibitory activity, comparable or better than positive control, 7-deazaxanthine (7-
Received 4 February 2014
Received in revised form
18 March 2014
Accepted 20 March 2014
Available online 21 March 2014
DX, 2) (IC50 value ¼ 42.63
mM). A systematic approach to the lead optimization identified compounds 3c
and 4a as the most promising TP inhibitors, exhibiting mixed mode of enzyme inhibition. Moreover,
selected compounds demonstrated the ability to attenuate the expression of the angiogenic markers (viz.
MMP-9 and VEGF) in MDA-MB-231 cells at sublethal concentrations. In addition, molecular docking
studies revealed the plausible binding orientation of these inhibitors towards TP, which was in accor-
dance with the experimental results. Taken as a whole, these compounds would constitute a new di-
rection for the design of novel TP inhibitors with promising antiangiogenic properties.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Pyrazolo[1,5-a][1,3,5]triazine
Thymidine phosphorylase inhibitors
Mixed-type inhibition
Breast cancer
Antiangiogenesis
1. Introduction
Folkman postulated that antiangiogenesis might be an effective
cancer treatment strategy [5]. Since then, there was an increasing
Carcinoma is the leading cause of premature death in the world
[1]. Due to the rapid advancement in cancer diagnosis and therapy,
the life expectancy and survival rate of cancer patients have shown
improvement, however, the overall clinical outcome of cancer is
still unsatisfactory due to its high invasive and metastatic proper-
ties. In order to achieve therapeutic success in cancer patients,
there is still room to develop new treatment strategies that target at
various stages of tumour progression. A flurry of scientific in-
vestigations has identified angiogenesis as one of the important
processes in cancer development. Angiogenesis was found to pro-
mote tumour growth and metastasis [2e4]. Moreover, in 1971,
interest in exploring novel antiangiogenic agents for cancer therapy
[6e9].
Among several angiogenic activators, thymidine phosphorylase
(TP, EC 2.4.2.4) has been recognized as an important angiogenic
protein that is frequently overexpressed within solid tumours [10].
In recent years, emerging data provided convincing evidence that
TP and its metabolic product, 2-deoxy-D-ribose (2DDR) stimulate
the secretion and/or expression of many angiogenic factors, such as
MMPs and VEGF, which trigger a signalling cascade to induce
endothelial cell migration, proliferation, elongation and sprouting.
These events eventually lead to angiogenesis and cancer metastasis
[11]. Therefore, TP has been implicated as a potential target for the
development of chemotherapeutic agents [12,13]. An increasing
number of studies in this field have discovered several potent TP
inhibitors. Structurally, most of them are pyrimidin-2,4-dione de-
rivatives. To date, only a few TP inhibitors incorporating hetero-
bicyclic templates have been designed and reported [14e17].
Among them, TPI and 7-DX (Fig. 1) have emerged as leading TP
* Corresponding author. Gokaraju Rangaraju College of Pharmacy, Bachupally,
Hyderabad 500090, India.
** Corresponding author.
E-mail addresses: hriday.bera1@gmail.com (H. Bera), phacwk@nus.edu.sg
(W.-K. Chui).
http://dx.doi.org/10.1016/j.ejmech.2014.03.063
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

H. Bera et al. / European Journal of Medicinal Chemistry 78 (2014) 294e303
295
Fig. 1. Chemical structures of known TP inhibitors.
inhibitor candidates [18,19]. Although several potent inhibitors
were generated and tested, very few TP inhibitors have progressed
beyond the preclinical stage [20]. Therefore, the development of TP
inhibitors possessing antiangiogenic activity is assumed to improve
TP-targeted therapy.
Scheme 1. Synthesis of target compounds (3e3c). Reagents and conditions: (a) NaCN,
HCl, aqueous-ethanol, r.t. (b) NH2NH2.H2O, methanol, MW, 140 ꢀC, 1 h (c) EtO2CN]O,
DMF, r.t., 5 h (d) NaOH, 80% ethanol (aq.), 100 ꢀC, 20 min.
The close structural similarity between pyrazolo[1,5-a][1,3,5]
triazine and purine nuclei vindicated the development of pyrazolo
[1,5-a][1,3,5]triazines as medicinally active compounds that would
target several enzymes involved in the metabolism of biogenic
purines and G-protein coupled purinergic signalling receptors [21].
A wide variety of attractive pharmacological effects of this scaffold
has led us to investigate the anti-TP effects of pyrazolo[1,5-a][1,3,5]
triazin-2,4-diones and their thioxo analogues which are bio-
isosterically modified derivatives of 7-DX. In this context, a small
library of compounds bearing substituents at position C7 or C8 of
the core ring structure was synthesized. Among various com-
pounds synthesized, 2-thioxo analogues of pyrazolo[1,5-a][1,3,5]
triazin-2,4-diones displayed various extent of TP inhibitory activity
[16].
The success of the project inspired us to design molecules
modified at both C7 and C8 positions of the fused ring structure for
further biological evaluation. To study the effects of various sub-
stituents on the TP inhibitory activity, Craig plot directed approach
[22] was employed. In the present study, a series of pyrazolo[1,5-a]
[1,3,5]triazin-2-thioxo-4-one derivatives (Fig. 2) with differently
substituted aromatic rings were synthesized. The synthesized
compounds were subsequently evaluated for their in vitro TP
inhibitory potential. To elucidate the mechanism of enzyme inhi-
bition, a brief kinetic study was attempted. Moreover, selected
compounds were exploited to investigate their antiangiogenic
properties by performing experiments that demonstrate an inhi-
bition of MMP-9 and VEGF expression in MDA-MB-231 cells. We
also performed in silico molecular docking studies to explore the
ligand binding site and possible interactions mode of the synthe-
sized compounds with TP.
afforded the thiourea derivatives (8 and 12) which underwent
intramolecular heterocyclization in the presence of base, resulting
in the formation of target compounds (3e3c and 4e4n
respectively).
Our synthetic strategy also employed two efficient and expe-
ditious protocols to produce two key intermediates viz., 5-
aminopyrazoles (7) and 3,5-diaminopyrazoles (11). The reactions
of commercially available substituted bromoacetophenones (5)
with sodium cyanide gave cyanoacetophenones (6), which under-
went a cyclocondensation reaction upon treatment with hydrazine
hydrate under microwave irradiation to furnish 7 (Scheme 1)
[26,27]. On the other hand, the preparation of 3,5-
diaminopyrazoles (11) comprised the synthesis of mono-
substituted malononitriles (10) via reductive alkylation of malo-
nonitrile with benzaldehydes (9) [28]. Subsequently, the reactions
of intermediates (10) with hydrazine hydrate in refluxing ethanol
yielded corresponding 3,5-diaminopyrazoles (11) (Scheme 2).
All the synthesized compounds (3e3c and 4e4n) were char-
acterized by melting points and various spectroscopic techniques
(1H NMR, 13C NMR and MS). The purity of the compounds was
assessed using reverse phase HPLC method and elemental analysis.
Interestingly, the structures of compounds 3, 4 were readily
confirmed by 13C NMR spectroscopy. The analysis of the 13C NMR
spectral data of compound 3 revealed that two sharp signals of
carbonyl (C]O) and thiocarbonyl (C]S) carbon appeared at
around 155.9 and 173.6 ppm respectively. Similarly, the character-
istic signals of one carbonyl (C]O) and two thiocarbonyl (C]S)
2. Results and discussion
2.1. Chemistry
The synthesis of the title compounds (3, 4) was achieved via
annulation of 1,3,5-triazine ring onto aminopyrazoles, as outlined
in Schemes 1 and 2. The adopted synthetic procedures were based
on previously disclosed methods [16,23e25]. The reaction of ami-
nopyrazoles (7 and 11) with ethoxycarbonyl isothiocyanate in DMF
Scheme 2. Synthesis of target compounds (4e4n). Reagents and conditions: (a)
mandalonitrile, aqueous-ethanol, NaHB4, 1.0 M HCl, r.t. (b) NH2NH2.H2O, Ethanol,
reflux, 5e8 h (c) Ethoxycarbonyl isothiocyanate, DMF, r.t., 5 h (d) NaOH, 80% ethanol
(aq.), 100 ꢀC, 20 min.
Fig. 2. Chemical structures of target compounds.

296
H. Bera et al. / European Journal of Medicinal Chemistry 78 (2014) 294e303
carbon of compound 4 appeared around 151.9, 173.5 and 179.8 ppm
respectively in 13C NMR spectra. The purity of all compounds was
satisfactory (above 95%).
compound 4, a number of substituents scattered around all the four
quadrants of Criag plot were selected in this study and they were
introduced at the para position of the phenyl ring of compound 4.
This approach generated a variety of analogues of 4 which exhibi-
ted significantly different levels of TP inhibitory activity, with IC50
2.2. Evaluation of biological activities
values ranging from 4.0 to 52.0 mM (Table 1).
In series 4, with few exceptions (4g, 4h and 4i), substituted
compounds, no matter having electron-withdrawing or electron-
donating groups, conferred a significantly higher activity than the
2.2.1. Anti-thymidine phosphorylase (Anti-TP) activity
Nineteen compounds (3, 4) were tested for their TP inhibitory
activity by a spectrophotometric coupled enzyme assay that used
recombinant human thymidine phosphorylase, expressed in E. coli
(T2807 e Sigma Aldrich) as the enzyme and thymidine as the
substrate. The adopted protocol is a modification to the original
method developed by Krenitsky (Krenitsky et al., 1979) [29]. The
inhibitory potencies of compounds were expressed in terms of IC50
values and were compared with the reference inhibitor, 7-DX
parent compound 4 (Table 1). Compound 4e (IC50 ¼ 19.98
carrying a methyl moiety (þ , ꢁ ), showed almost a 2.6-fold in-
crease in inhibitory activity as compared to 4g (IC50 ¼ 51.44 M)
that had a methoxy substituent (ꢁ , ꢁ ). In addition, compound 4f
(IC50 value ¼ 12.56 M), bearing an ethyl group, displayed more
mM),
p
s
m
p
s
m
effective inhibition than compound 4e having a methyl substituent.
Moreover, among all the compounds evaluated, compound 4a
bearing a CF3 moiety was observed to have the best anti-TP activity
and it exhibited 12.8, 7.8 and 11.2-fold enhancement in inhibitory
activity as compared to compounds 3, 4 and 7-DX respectively. The
highest TP inhibitory activity of compound 4a was possibly due to
its high lipophilicity, imparted by introduction of a CF3 group.
Overall, the lipophilicity of compounds was considered to be an
important prerequisite to elicit a prominent TP inhibitory effect.
These findings inspired us to modify compound 4 by inserting
various hydrophobic motifs viz., biphenyl, phenoxyphenyl and
naphthalene groups at the C8 position. Interestingly, compounds
4j, 4k and 4l demonstrated an improved inhibition profile towards
TP relative to compound 4. Our investigation was further extended
by introducing 2-furyl and 2-thienyl moieties at the C8 position of
the core scaffold in order to explore the influence of different ar-
omatic heterocycles in activity. However, such effort did not reveal
any significant improvement in potency, as evident in compounds
4m and 4n.
(IC50 ¼ 42.63
mM) (Table 1). The preliminary enzyme inhibition
study of compounds (3e3c) suggested following structureeactivity
relationships: (1) Replacement of the phenyl ring of compound 3
(IC50 ¼ 46.21
nificant reduction in affinity for TP, as evident in compound 3a
M). This result indicated that the t-butyl group might
mM) by a bulky t-butyl group at C7 resulted in sig-
(IC50 ¼ 55.70
m
present a negative steric hindrance effect to the binding site of the
enzyme. (2) Introduction of a substituent (CN) into the structure of
compound 3 demonstrated substantial improvement in binding
interactions, as observed in compound 3b (IC50 ¼ 30.32
mM). (3)
M) exhibited
Moreover, disubstituted analogue 3c (IC50 ¼ 9.07
m
significantly enhanced inhibition profile relative to mono
substituted compound 3b.
Based on the preliminary enzyme inhibition results, we were
prompted to modify both C7 and C8 positions of the fused ring
structure using similar synthetic protocol. The resulting disubsti-
tuted compound 4 (IC50 ¼ 29.92
mM) was found to be more
favourable for TP inhibitory activity than the mono substituted
analogue 3. With the aim of enhancing the inhibition properties of
2.2.2. Mechanism of enzyme inhibition
In an attempt to explore the mode of enzyme inhibition, a brief
enzyme inhibition kinetic study was performed. Compounds 3c
and 4a, endowed with promising TP inhibitory activity were
selected for kinetic analysis. In this study, compounds 3c and 4a
were found to exhibit mixed- type inhibition with respect to
thymidine as the variable substrate, since the reciprocal plots for
compounds showed straight lines that intersected each other in the
left panel (Fig. 3). This was further confirmed by the fact that
increased inhibitor concentration was accompanied by an increase
in the Km values and a decrease in the Vmax values.
Table 1
Thymidine phosphorylase inhibitory activity of the synthesized compounds (3, 4).
Entry
Cpd
R
TP inhibition activitya,b
IC50 M)
The model of Scheme 3 was adopted to explain the mixed-type
inhibition. Compounds 3c and 4a were assumed to interact with
the enzyme (E) as well as enzyme-substrate (ES) complex without
affecting substrate binding [30]. We also calculated the values of
(
m
1
2
3
4
5
6
7
8
3
Ph
C(Me)3
4-CNC6H4e
3,4-Cl2C6H3e
Ph
46.21 ꢂ 1.82
55.70 ꢂ 5.13
30.32 ꢂ 2.74
9.07 ꢂ 1.23
29.92 ꢂ 4.39
3.82 ꢂ 0.78
10.54 ꢂ 2.50
14.78 ꢂ 1.86
22.65 ꢂ 4.65
19.98 ꢂ 4.26
12.56 ꢂ 1.53
51.44 ꢂ 3.95
37.38 ꢂ 4.93
51.53 ꢂ 2.69
6.11 ꢂ 0.83
4.46 ꢂ 0.42
4.29 ꢂ 0.88
36.72 ꢂ 5.65
27.69 ꢂ 4.02
42.63 ꢂ 5.25
3a
3b
3c
4
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
inhibition constants (Ki and aKi) by replotting the slopes and in-
tercepts of the double reciprocal plots against the inhibitor con-
centrations (Table 2). The data clearly indicated that the inhibitors
had stronger affinity for the free enzyme than enzyme-substrate
4-CF3C6H4-
4-ClC6H4-
4-BrC6H4-
4-FC6H4-
4-MeC6H4-
4-EtC6H4-
4-MeOC6H4-
4-OHC6H4-
4-CNC6H4-
4-PhC6H4-
4-OPhC6H4-
1-naphthyl
2-furyl
9
complex
exhibited reduced affinity for substrate in comparison to free
enzyme ( > 1) (Scheme 3). Due to structural variability in the core
(a > 1). Moreover, the enzymeeinhibitor complex
10
11
12
13
14
15
16
17
18
19
20
a
scaffold, compounds 3c and 4a showed different modes of inter-
action as compared to 7-DX, which behaved as a competitive or
mixed-type inhibitor of the enzyme in the presence of variable
thymine concentrations [31].
4m
4n
7-Dx
2.2.3. Antiangiogenic activity: inhibition of MMP-9 and VEGF
expression
Matrix metallopeptidase 9 (MMP-9) belongs to a family of zinc-
dependent endopeptidases. It plays a pivotal role in the accelerated
2-thienyl
a
Values are means of three experiments.
Values are presented as means ꢂ SD.
b

H. Bera et al. / European Journal of Medicinal Chemistry 78 (2014) 294e303
297
Table 2
A summary of the kinetic parameters of 3c and 4a.
Cpd
Substrate
Ki value (
m
M)a,b
a
Type of inhibition
3c
4a
dThd
dThd
1.77 ꢂ 0.35
1.65 ꢂ 0.10
4.10
5.20
Mixed type
Mixed type
a
Values are means of three experiments.
b
Values are presented as means ꢂ SD.
To explore the inhibitory effects of the selected compounds on
PMA-induced MMP-9 expression, gelatine zymography [35] was
conducted. In this study, MDA-MB-231 cells were treated with the
indicated concentrations of compounds (25e100
mM) and the
proteolytic activity of MMP-9 (band corresponded to the MW of
92 kDa) was investigated. The results conferred that all compounds
dose dependently attenuated MMP-9 expression (Fig. 4a). Quanti-
tative densitometric analysis of the zymograms showed that all
compounds (except 4a) significantly inhibited MMP-9 expression
at concentrations of 50 and 100 mM (p < 0.05) (Fig. 4b). Moreover,
the inhibitory effects of these compounds on VEGF expression were
investigated by enzyme-linked immunosorbent assay (ELISA) [15].
As depicted in Fig. 4c, the expression of VEGF was markedly
reduced by all the tested compounds (3c, 4a, 4j, 4k and 4l) at doses
ranging from 25 to 100
mM, as compared to vehicle control
(p < 0.05). In contrast, the reference TP inhibitor (7-DX) did not
exert any significant inhibitory effect on MMP-9 and VEGF
expression even at very high concentration (100 mM) (Fig. 4ec). In
order to ascertain the inhibitory effects of the compounds on MMP-
9 and VEGF expression were not due to their cytotoxic effects, a cell
viability assay [36] was conducted on MDA-MB-231 cells. An
insignificant growth-inhibitory effect (cell viability 80e100%) was
observed with all doses of 7-DX and each compound tested
(Fig. 4d).
It is noteworthy that the compounds (4a, 4j, 4k and 4l) bearing
substitutions at positions C7 and C8, displayed a higher inhibitory
activity against MMP-9 and VEGF as compared to mono substituted
analogue (3c). Most TP inhibitors discovered so far are substituted
uracil analogues that compete with the substrate for access to the
catalytic cleft of TP [11]. In contrast, compounds (3, 4) represented a
unique structural class of TP inhibitors and were assumed to
interact to the enzyme at distinct sites, leading to attenuation of TP-
induced upregulation of MMP-9 and VEGF. The kinetic behaviour of
these compounds strongly supported our assumption.
Fig. 3. LineweavereBurk plots of TP inhibition by 3c (A) and 4a (B), in the presence of
variable concentrations of dThd, demonstrating mixed type enzyme inhibition. Results
are presented as means ꢂ SD; SD denoted by error bars (Experiments carried out in
triplicate).
breakdown of extracellular matrix surrounding the tumour cells
that eventually results in increased tumour cell migration, invasion
and metastasis [32]. On the other hand, vascular endothelial
growth factor (VEGF) plays a paradoxical role in tumour angio-
genesis by inducing the proliferation, migration and survival of
endothelial cells [33]. A large number of studies reported that TP is
associated with the induction of MMP-9 and VEGF expression in
tumour cells [10]. Moreover, a significant correlation was observed
between VEGF and TP expression in solid tumours, as both
proangiogenic factors share the same transcription site [34].
Compounds (3c, 4a, 4j, 4k and 4l) that showed attractive in vitro TP
2.3. Molecular docking study
To gain a deeper insight into the binding mode and to explore
the detailed molecular interactions of the synthesized compounds,
the geometry optimized structures of the inhibitors were docked
into the binding sites of TP (PDB code: 2WK6) [37] using Discovery
Studio 2.1 (Accelrys Inc, San Diego, CA, USA) [38]. Before docking,
the docking protocol was validated by comparing the predicted
binding orientation of bound ligand, 5-iodouracil (5-IUR) with the
reported crystallographic interactions [37]. The observed binding
modes of 5-IUR in the crystal structure were strikingly well-aligned
inhibition profile (IC50 less than 10 mM), were examined for their
ability to suppress MMP-9 and VEGF production in MDA-MB-231
cells.
ꢀ
with the predicted docking poses (rmsd ¼ 0.497A) (Fig. S1, in
Supplementary Information).
To illustrate the kinetic behaviour of the synthesized inhibitors,
two binding pockets viz. site A (5-IUR binding site) and site B
(allosteric site proximal to the 5-IUR binding site) were identified
and the docked conformations at two different sites were
compared with respect to their docking scores. The site B is
composed of several amino acid residues (Leu423, Val425, Asp426,
Val427, Ala469, Arg279, Gly278, Pro472, Asp304, Ser471, Asp301,
Pro300, Pro299, Ala468, Lys275 and Pro276) located in the vicinity
Scheme 3. Enzyme kinetic model explaining mixed type inhibition.

298
H. Bera et al. / European Journal of Medicinal Chemistry 78 (2014) 294e303
Fig. 4. Effect of selected compounds (3c, 4a, 4j, 4k and 4l) and 7-DX on MMP-9 and VEGF expression and cell growth in MDA-MB-231 cells: (A) suppression of PMA induced MMP-9
expression and their dose response; (B) densitometry analysis of MMP expression; (C) attenuation of VEGF expression in MDA-MB-231 cells after 24 h treatment; (D) cell viability
(%) of cells after 72 h treatment. Results are presented as means ꢂ SD; SD denoted by error bars (Experiments carried out in triplicate). *p < 0.05.
of the active site (site A). A direct comparison of the dock scores at
two different sites (i.e., site A and site B) implied that all the in-
hibitors exhibited a much higher dock score towards site B as
compared to site A, indicating a reduced affinity of compounds for
site A (Table S1, in Supplementary Information). Overlay of docked
pose of all the inhibitors at site B revealed nearly identical binding
orientations. Multiple weak interactions including H-bonding,
studied, compounds 4j and 4k with lipophilic groups (e.g. biphenyl
and phenoxyphenyl) displayed the highest docking scores at site B
and established themselves as potent TP inhibitors. Overall, the
docking scores at site B were corroborating well with the experi-
mental binding affinities of the compounds tested. Furthermore,
compounds 3c and 4a conferred a better interaction than that of 7-
DX (dock score ¼ 62.56 and 65.16 in site A and site B respectively).
The reference inhibitor, 7-DX was found to construct a hydrogen
bonding network with Gly 271, Lys275 and Val 227, located at site B
(Fig. S2, in Supplementary Information). By considering the results
of the molecular modelling study, we can speculate that the com-
pounds 3c and 4a may interact with the amino acid residues
located at (site A) or adjacent (site B) to the active cleft [39] with a
strong bonding force towards site B. Consequently, they showed a
mixed mode of inhibition with respect to thymidine.
cation-p, p-s, van der Waals and hydrophobic interactions stabi-
lized the protein-ligand association. In particular, the core scaffold
of the compounds created a hydrogen-bonding network, whereas
the phenyl rings were favoured to form hydrophobic interactions
with the amino acid residues.
The docking orientations of the two most active compounds (3c
and 4a) were analyzed and compared with that of reference in-
hibitor, 7-DX. The identified binding modes of 3c and 4a are
depicted in Fig. 5a and b respectively. Compounds 3c and 4a
exhibited a remarkably low binding affinity for site A (dock score
2.144 and 17.997 respectively) relative to site B (dock score 85.477
and 105.824 respectively). Compound 3c appeared to form three
hydrogen bonding interactions with Lys275, Ser471 and Ala469. On
the other hand, compound 4a constructed six hydrogen bonding
contacts with different amino acid residues, including Ser471,
3. Conclusion
In conclusion, a small library of pyrazolo[1,5-a][1,3,5]triazine
derivatives were synthesized via ring annelation reaction and
assessed for their anti-TP activity. The biological evaluation iden-
tified a number of structurally distinct TP inhibitors. The most
promising candidates (3c and 4a) exhibited a mixed mode of in-
hibition towards TP. Moreover, selected TP inhibitors attenuated
MMP-9 and VEGF expression in MDA-MB-231 cells, illustrating the
Asp301, Asp304, Ala469 and Val427. It also created two cation-
p
interactions with Arg279 and Lys275, explaining its highest po-
tency against enzyme. In addition, among all the compounds

H. Bera et al. / European Journal of Medicinal Chemistry 78 (2014) 294e303
299
Fig. 5. Docked conformation of compounds 3c (A) and 4a (B) along with important amino acid residues in site B of TP (green dashed line indicates hydrogen bonding contacts, while
brick red line implies
of this article.)
p-cationic interactions with amino acid residues). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
anti-angiogenic potential of these compounds. In silico docking
study elucidated the distinct binding poses and interactions of
these compounds to enzyme. Therefore, this study presents a
starting point for the discovery of clinically useful novel TP
inhibitors.
aldehydes (9) (10 mmol) with malononitrile (10 mmol), as
described previously [28]. The resulting mono-substituted malo-
nonitriles (10) (10 mmol) were subsequently treated with hydra-
zine hydrate (20 mmol) in refluxing ethanol to obtain 3,5-
diaminopyrazoles (11). In the next step, the reactions between 5-
aminopyrazoles (7)/3,5-diaminopyrazoles (11) (3 mmol) and
ethoxycarbonyl isothiocyanate (3.3 mmol) in anhydrous DMF
(4 mL) at room temperature yielded corresponding thiourea (8 and
12) derivatives.
4. Experimental
4.1. Chemistry
To a stirred solution of sodium hydroxide (9 mmol) in ethanol
(80%, 20 mL), carbethoxythiourea derivatives (8 and 12) (3 mmol)
were added and heated on a water bath for 20 min. After cooling,
the solvent was slowly evaporated under vacuum, and the residue
was suspended in water (25 mL). The resulting suspension was then
acidified up to pH 1e3 using 2.5 M HCl. The precipitated products
(3-3c and 4-4n) was filtered off, purified with an appropriate sol-
vent, and dried under vacuum (Schemes 1 and 2).
4.1.1. General procedures
Commercially available reagents and solvents were used
without further purification. Melting points were determined on a
Gallenkamp melting point apparatus and were presented uncor-
rected. 1H NMR and 13C NMR spectra were recorded on a Bruker
DPX-300 spectrometer at 300 MHz and 75 MHz respectively using
DMSO-d6 as solvent and TMS as internal standard. Mass spectra
were obtained on a Finnigan MAT LCQ LC-MS mass spectrometer
using electrospray ionization (ESI) mode. Reactions were moni-
tored by TLC on silica gel (60 F254) coated aluminium plate. The
HPLC method was performed using HewlettePackard series 1050
HPLC system equipped with an HP-1050 quaternary pump, a
degasser, diode array detector, an HP-1100 autosampler and a
4.1.3. 7-Phenyl-2-thioxo-2,3-dihydro-1H-pyrazolo[1,5-a][1,3,5]
triazin-4-one (3)
White powder (71% yield); Mp 285e286 ꢀC (Ethanol-water);
ESI-MS m/z 243.0 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 6.39 (s,
1H, CH), 7.38e7.54 (m, 3H, H-30, H-40 and H-50), 7.88e8.00 (m, 2H,
H-20 and H-60), 12.75 (s, 1H, NH), 13.56 (s, 1H, NH); 13C NMR
LiChrosorb reversed phase C18 (5
m
m) column (4.6 ꢃ 250 mm). The
(75 MHz, DMSO-d6):
d
87.3 (C-8), 126.8 (C-30 and C-50), 129.4 (C-20
separations were carried out using gradient elution. The experi-
ment started using mobile phase composition of 95:5 (aqueous
phase:acetonitrile) which gradually changed to 0:100 within 7 min.
The flow rate was set at 1 mL/min and the ultraviolet detection was
made at wavelength 254 nm. Elemental analyses were carried out
on a Vario EL-III C, H, N, S analyzer (Germany) and values were
within ꢂ0.5% of the calculated values. The purity of all synthesized
compounds was found to be more than 95%.
and C-60), 130.1 (C-40), 131.7 (C-10), 142.0 (C-7), 142.1 (C-9), 155.9 (C-
4), 173.6 (C-2); Anal. calcd. for C11H8N4OS: C, 54.09; H, 3.30; N,
22.94. Found: C, 53.78; H, 3.24; N, 22.74.
4.1.4. 7-t-Butyl-2-thioxo-2,3-dihydro-1H-pyrazolo[1,5-a][1,3,5]
triazin-4-one (3a)
White powder (55% yield); Mp 268e270 ꢀC (Ethanol-water);
ESI-MS m/z 223.1 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 1.23 (s,
9H, t-but), 5.83 (s, 1H, CH), 12.59 (s, 1H, NH), 13.37 (s, 1H, NH). 13
C
4.1.2. General procedure for the synthesis of 2-thioxo-pyrazolo[1,5-
a][1,3,5]triazin-4-ones (3-3c and 4-4n)
NMR (75 MHz, DMSO-d6):
d 30.1 (3CH3), 33.0 (t-but), 87.1 (C-8),
141.1 (C-7), 142.0 (C-9), 167.5 (C-4), 173.4 (C-2); Anal. calcd. for
C9H12N4OS: C, 48.20; H, 5.39; N, 24.98. Found: C, 47.42; H, 5.32; N,
24.52.
Initially, cyanoacetophenones (6) were accomplished through
the reaction of substituted bromoacetophenones (5) (10 mmol)
with sodium cyanide (20 mmol). 5-aminopyrazoles (7) were then
prepared from the cyclocondensation of cyanoacetophenones (6)
(10 mmol) with hydrazine (20 mmol) under microwave irradiation
[26,27]. Moreover, 3,5-diaminopyrazoles (11) were synthesized via
an initial formation of mono-substituted malononitriles (10) from
the condensation of substituted aromatic or heteroaromatic
4.1.5. 7-(4-Cyano-phenyl)-2-thioxo-2,3-dihydro-1H-pyrazolo[1,5-
a][1,3,5]triazin-4-one (3b)
White powder (61% yield); Mp > 300 ꢀC (Ethanol-water); ESI-
MS m/z 287.2 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 6.50 (s,

300
H. Bera et al. / European Journal of Medicinal Chemistry 78 (2014) 294e303
1H, CH), 7.97e8.16 (m, 4H, AreH), 12.82 (s, 1H, NH), 13.64 (s, 1H,
NH). 13C NMR (75 MHz, DMSO-d6):
87.6e87.7 (C-8), 126.6e126.8
DMSO-d6): d
23.8 (CH2), 91.6 (C-8), 118.9 (C-40), 130.2 (C-30 and C-
d
50), 131.0 (C-2’and C-60), 138.8 (C-10), 139.2 (C-7), 140.8 (C-9), 151.9
(C-4), 173.6 (C-2), 179.9 (NH(C]S)NH2); Anal. calcd. for
(C-20 and C-60), 128.6e130.4 (C-30 and C-50), 131.9 (C-40), 134.3 (CN),
135.5e135.8 (C-10), 142.0e142.3 (C-7), 154.9e155.1 (C-9), 167.4e
167.9 (C-4), 173.6 (C-2); Anal. calcd. for C12H7N5OS: C, 53.52; H,
2.62; N, 26.01. Found: C, 52.88; H, 2.57; N, 26.42.
C13H11BrN6OS2: C, 37.96; H, 2.70; N, 20.43. Found: C, 37.32; H, 2.65;
N, 20.52.
4.1.11. [8-(4-Fluoro-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4d)
4.1.6. 7-(3,4-Dichloro-phenyl)-2-thioxo-2,3-dihydro-1H-pyrazolo
[1,5-a][1,3,5]triazin-4-one (3c)
White powder (65% yield); Mp 221e223 ꢀC (Ethanol-water);
White powder (63% yield); Mp 287e289 ꢀC (Ethanol-water);
ESI-MS m/z 349.0 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 4.09 (s,
ESI-MS m/z 312.9 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d
6.52 (s,
2H, CH2), 7.10 (dd, 2H, H-30 and H-50, J ¼ 8.9, 8.9 Hz), 7.20 (dd, 2H, H-
20 and H-60, J ¼ 8.7, 5.6 Hz), 8.94 and 9.05 (2 s, 2H, (C]S) NH2),10.09
(s, 1H, (C]S) NH), 12.69 (s, 1H, NH), 13.55 (br. s, 1H, NH); 13C NMR
1H, CH), 7.71 (d, 1H, H-60, J ¼ 8.3 Hz), 7.94 (dd, 1H, H-50, J ¼ 8.5 Hz,
J ¼ 2.1 Hz), 8.18 (d, 1H, H-20, J ¼ 1.9 Hz), 12.80 (s, 1H, NH), 13.63 (s,
1H, NH). 13C NMR (75 MHz, DMSO-d6):
d
87.7 (C-8), 126.7 (C-60),
(75 MHz, DMSO-d6): d
23.4 (CH2), 92.7 (C-8), 114.8 (d, C-30 and C-50,
128.4 (C-20), 131.6 (C-50), 132.3 (C-40), 132.4 (C-30), 132.5 (C-10), 141.9
(C-7), 142.3 (C-9), 153.6 (C-4), 173.6 (C-2); Anal. calcd. for
J ¼ 21.3 Hz), 129.6 (d, C-20 and C-60, J ¼ 7.3 Hz), 135.7 (d, C-10,
J ¼ 2.9 Hz), 138.6 (C-7), 140.8(C-9), 151.8(C-4), 160.6 (d, C-40,
J ¼ 241.4 Hz), 173.5(C-2), 179.8 (NH(C]S)NH2); Anal. calcd. for
C
11H6Cl2N4OS: C, 42.19; H, 1.93; N, 17.89. Found: C, 42.65; H, 1.88; N,
17.42.
C13H11FN6OS2: C, 44.56; H, 3.16; N, 23.98. Found: C, 43.26; H, 3.22;
N, 22.76.
4.1.7. (8-Benzyl-4-oxo-2-thioxo-1,2,3,4-tetrahedro-pyrazolo[1,5-a]
[1,3,5]triazin-7-yl)-thiourea (4)
4.1.12. [8-(4-Methyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4e)
White powder (73% yield); Mp 239e241 ꢀC (Ethanol-water);
ESI-MS m/z 331.1 (Mꢁ1)þ; 1H NMR (30 MHz, DMSO-d6):
d
4.11(s,
White powder (76% yield); Mp 254e256 ꢀC (Ethanol-water);
2H, CH2), 712e7.33 (m, 5H, Ph), 8.94 and 9.07 (2 s, 2H, (C]S) NH2),
ESI-MS m/z 345.1 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 2.25 (s,
.
10.06 (s, 1H, (C]S) NH), 12.70 (s, 1H, NH), 13.57 (br. s, 1H, NH); 13C
3H, Me), 4.05 (s, 2H, CH2), 7.04e7.09 (m, 5H, AreH), 8.93 and 9.03
(2 s, 2H, (C]S) NH2), 9.98 (s, 1H, (C]S) NH), 12.69 (s, 1H, NH), 13.55
NMR (75 MHz, DMSO-d6):
d
24.2 (CH2), 92.0 (C-8), 125.8 (C-40),
127.8 (C-30 and C-50), 128.2 (C-2’and C-60), 138.5 (C-10), 139.6 (C-7),
140.7 (C-9), 151.9 (C-4), 173.5 (C-2), 179.8 (NH(C]S)NH2); Anal.
calcd. for C13H12N6OS2: C, 46.97; H, 3.64; N, 25.28. Found: C, 45.85;
H, 3.52; N, 24.89.
(br. s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d 20.5 (Me), 23.7
(CH2), 92.4 (C-8), 112.7 (C-30 and C-50), 128.7 (C-2’and C-60), 134.8
(C-40), 136.4 (C-10), 138.3 (C-7), 140.7 (C-9), 151.9 (C-4), 173.5 (C-2),
179.7 (NH(C]S)NH2); Anal. calcd. for C14H14N6OS2: C, 48.54; H,
4.07; N, 24.26. Found: C, 47.26; H, 3.98; N, 24.54.
4.1.8. [8-(4-Trifluoromethyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-
tetrahedro-pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4a)
White powder (71% yield); Mp 189e191 ꢀC (Ethanol-water);
4.1.13. [8-(4-Ethyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4f)
ESI-MS m/z 399. (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d
4.21 (s,
White powder (73% yield); Mp 240 ꢀC (Ethanol-water); ESI-MS
2H, CH2), 7.39 (d, 2H, H-30 and H-50, J ¼ 7.9 Hz), 7.65 (d, 2H, H-20 and
H-60, J ¼ 8.1 Hz), 8.95 and 9.10 (2 s, 2H, (C]S) NH2), 10.21 (s, 1H,
(C]S) NH), 12.65 (s, 1H, NH), 13.53 (br. s, 1H, NH); 13C NMR
m/z 359.1 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 1.14 (t, 3H, CH3,
J ¼ 7.6 Hz), 2.55 (q, 2H, CH2, J ¼ 7.6 Hz), 4.05 (s, 2H, CH2), 7.07 (d, 2H,
H-30 and H-50, J ¼ 8.3 Hz), 7.11 (d, 2H, H-20 and H-60, J ¼ 8.5 Hz), 8.94
and 9.07 (2 s, 2H, (C]S) NH2), 10.03 (s, 1H, (C]S) NH), 12.63 (s, 1H,
(75 MHz, DMSO-d6):
d 24.4 (CH2), 91.0 (C-8), 124.4 (q, CF3,
J ¼ 271.9 Hz), 124.9 (q, C-30 and C-50, J ¼ 3.7 Hz), 126.7 (q, C-40,
J ¼ 31.5 Hz), 128.6 (C-2’and C-60), 139.3 (C-10), 140.9 (C-7), 144.8 (C-
9), 151.9 (C-4), 173.6 (C-2), 179.9 (NH(C]S)NH2); Anal. calcd. for
NH), 13.54 (br. s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d 15.7
(Me), 23.8 (CH2), 27.7 (CH2), 92.4 (C-8), 127.5 (C-30 and C-50), 127.8
(C-2’and C-60), 136.8 (C-40), 138.6 (C-10), 140.8 (C-7), 141.2 (C-9),
151.9 (C-4), 173.5 (C-2), 179.8 (NH(C]S)NH2); Anal. calcd. for
C
14H11F3N6OS2: C, 42.00; H, 2.77; N, 20.99. Found: C, 43.24; H, 2.68;
N, 20.79.
C15H16N6OS2: C, 49.98; H, 4.47; N, 23.31. Found: C, 48.84; H, 4.42; N,
23.02.
4.1.9. [8-(4-Chloro-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4b)
4.1.14. [8-(4-Methoxy-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4g)
White powder (78% yield); Mp 243e245 ꢀC (Ethanol-water);
ESI-MS m/z 365.1 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d
4.09 (s,
White powder (69%); Mp 252e254 ꢀC (Ethanol-water); ESI-MS
2H, CH2), 7.18 (d, 2H, H-30 and H-50, J ¼ 8.4 Hz), 7.33 (d, 2H, H-20 and
H-60, J ¼ 8.5 Hz), 8.94 and 9.05 (2 s, 2H, (C]S) NH2), 10.11 (s, 1H,
(C]S) NH), 12.69 (s, 1H, NH), 13.53 (br. s, 1H, NH); 13C NMR
m/z 361.1 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 3.71 (s, 3H,
OMe), 4.03 (s, 2H, CH2), 6.84 (d, 2H, H-30 and H-50, J ¼ 8.8 Hz), 7.10
(d, 2H, H-20 and H-60, J ¼ 8.7 Hz), 8.95 and 9.04 (2 s, 2H, (C]S) NH2),
9.99 (s, 1H, (C]S) NH), 12.70 (s, 1H, NH), 13.56 (br. s, 1H, NH); 13C
(75 MHz, DMSO-d6):
d
23.7 (CH2), 91.6 (C-8), 128.0 (C-30 and C-50),
129.7 (C-2’and C-60), 130.4 (C-40), 138.7 (C-10), 138.7 (C-7), 140.7 (C-
9), 151.8 (C-4), 173.5 (C-2), 179.8 (NH(C]S)NH2); Anal. calcd. for
NMR (75 MHz, DMSO-d6): d 23.3 (CH2), 54.9 (MeO), 92.7 (C-8),
113.6 (C-30 and C-50), 128.8 (C-20 and C-60), 131.4 (C-40), 138.2 (C-10),
140.7 (C-7), 151.9 (C-9), 157.5 (C-4), 173.5 (C-2), 179.7 (NH(C]S)
NH2); Anal. calcd. for C14H14N6O2S2: C, 46.40; H, 3.89; N, 23.19.
Found: C, 46.12; H, 3.82; N, 23.11.
C
13H11ClN6OS2: C, 42.56; H, 3.02; N, 22.91. Found: C, 42.12; H, 3.11;
N, 22.08.
4.1.10. [8-(4-Bromo-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4c)
4.1.15. [8-(4-Hydroxy-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4h)
White powder (82% yield); Mp 220 ꢀC (Ethanol-water); ESI-MS
m/z 411.1 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d
4.07 (s, 2H,
White powder (80% yield); Mp 219e221 ꢀC (Ethanol-water);
CH2), 7.13 (d, 2H, H-30 and H-50, J ¼ 8.4 Hz), 7.46 (d, 2H, H-20 and H-
60, J ¼ 8.4 Hz), 8.93 and 9.05 (2 s, 2H, (C]S) NH2), 10.09 (s, 1H, (C]
S) NH), 12.67 (s, 1H, NH), 13.52 (br. s, 1H, NH); 13C NMR (75 MHz,
ESI-MS m/z 347.1 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 3.97 (s,
2H, CH2), 6.66 (d, 2H, H-30 and H-50, J ¼ 8.5 Hz), 6.96 (d, 2H, H-20 and
H-60, J ¼ 8.5 Hz), 8.94 and 9.03 (2 s, 2H, (C]S) NH2), 9.17 (s, 1H, OH),

H. Bera et al. / European Journal of Medicinal Chemistry 78 (2014) 294e303
301
9.90 (s, 1H, (C]S) NH), 12.68 (s, 1H, NH), 13.54 (br. s, 1H, NH); 13C
NMR (75 MHz, DMSO-d6):
23.3 (CH2), 92.7 (C-8), 114.9 (C-30 and
2H, CH2), 6.00 (d, 1H, H-30, J ¼ 3.1 Hz), 6.34 (dd, 1H, H-40, J ¼ 3.1,
1.9 Hz), 7.55 (d, 1H, H-50, J ¼ 1.0 Hz), 8.98 and 9.09 (2 s, 2H, (C]S)
NH2), 10.12 (s, 1H, (C]S) NH),12.72 (s, 1H, NH), 13.54 (br. s, 1H, NH);
d
C-50), 128.7 (C-2’and C-60), 129.5 (C-40), 138.2 (C-10), 140.7 (C-7),
151.9 (C-9), 155.4 (C-4), 173.5 (C-2), 179.7 (NH(C]S)NH2); Anal.
calcd. for C13H12N6O2S2: C, 44.82; H, 3.47; N, 24.12. Found: C, 44.26;
H, 3.39; N, 23.76.
13C NMR (75 MHz, DMSO-d6):
d
18.5 (CH2), 89.2 (C-8), 105.5 (C-40),
110.4 (C-30), 138.5 (C-50), 140.7 (C-20), 141.6 (C-7), 151.8 (C-9), 152.9
(C-4), 173.5 (C-2), 179.8 (NH(C]S) NH2); Anal. calcd. for
C11H10N6O2S2: C, 40.98; H, 3.13; N, 26.07. Found: C, 40.18; H, 3.21; N,
4.1.16. [8-(4-Cyano-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4i)
27.24.s
White powder (62% yield); Mp 252e254 ꢀC (Ethanol-water);
4.1.21. (4-Oxo-8-thiophen-2-ylmethyl-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4n)
ESI-MS m/z 356.1 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 4.20 (s,
2H, CH2), 7.34 (d, 2H, H-30 and H-50, J ¼ 8.3 Hz), 7.46 (d, 2H, H-20 and
H-60, J ¼ 8.3 Hz), 8.96 and 9.06 (2 s, 2H, (C]S) NH2), 10.22 (s, 1H,
(C]S) NH), 12.74 (s, 1H, NH), 13.55 (br. s, 1H, NH); 13C NMR
White powder (72% yield); Mp 230e232 ꢀC (Ethanol-water);
ESI-MS m/z 337.0 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 4.30 (s,
2H, CH2), 6.81 (d, 1H, H-30, J ¼ 2.8 Hz), 6.92 (dd, 1H, H-40, J ¼ 5.0,
3.5 Hz), 7.32 (d, 1H, H-50, J ¼ 5.0 Hz), 8.97 and 9.06 (2 s, 2H, (C]S)
NH2), 10.19 (s, 1H, (C]S) NH), 12.72 (s, 1H, NH), 13.61 (br. s, 1H, NH);
(75 MHz, DMSO-d6):
d 24.7 (CH2), 90.6 (C-8), 108.7 (CN), 118.9 (C-
40), 128.9 (C-30 and C-50), 132.0 (C-2’and C-60), 138.9 (C-10), 140.8 (C-
7), 145.8 (C-9), 151.8 (C-4), 173.6 (C-2), 179.7 (NH(C]S)NH2); Anal.
calcd. for C14H11N7OS2: C, 47.05; H, 3.10; N, 27.43. Found: C, 46.76;
H, 3.14; N, 27.98.
13C NMR (75 MHz, DMSO-d6):
d
19.4 (CH2), 92.1 (C-8), 124.0 (C-40),
124.5 (C-50), 126.7 (C-30), 138.2 (C-20), 140.7 (C-7), 142.6 (C-9), 151.6
(C-4), 173.5 (C-2), 179.8 (NH(C]S) NH2); Anal. calcd. for
C11H10N6OS3: C, 39.04; H, 2.98; N, 24.83. Found: C, 40.22; H, 3.92; N,
4.1.17. (8-Biphenyl-4-ylmethyl-4-oxo-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl)-thiourea (4j)
24.06.
White powder (70%); Mp 246e248 ꢀC (Ethanol-water); ESI-MS
4.2. Biological characterisation
m/z 407.1 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 4.16 (s, 2H,
CH2), 7.27 (d, 2H, H-300 and H-500, J ¼ 8.2 Hz), 7.33 (t, 1H, H-400,
J ¼ 7.3 Hz), 7.44 (t, 2H, H-200 and H-600, J ¼ 7.7 Hz), 7.58 (d, 2H, H-30
and H-50, J ¼ 8.2 Hz), 7.63 (d, 2H, H-20 and H-60, J ¼ 7.5 Hz), 8.98 and
9.12 (2 s, 2H, (C]S) NH2), 10.15 (s, 1H, (C]S) NH), 12.67 (s, 1H, NH),
4.2.1. In vitro thymidine phosphorylase enzyme assay
A spectrophotometric assay method [29] was adopted to eval-
uate in vitro TP inhibitory activity of the synthesized compounds.
Briefly, thymidine (substrate) and recombinant thymidine phos-
phorylase, expressed in E. coli (T2807-Sigma) were utilized for this
assay. The enzymatic reaction was initiated by the addition of
substrate (200
tassium phosphate buffer (pH 7.4), 10
tion of 1.5 U, and 10 L of test compounds dissolved in DMSO. The
conversion of thymidine to thymine was determined at 290 nm
after 4, 8, 12, 16 and 20 min on a Shimadzu UV Mini 1240 UVeVis
Spectrophotometer. The initial rates of change in absorbance at
different concentrations of inhibitor were converted to % inhibition
of enzyme. Plotting the data as % inhibition as a function of inhibitor
concentration using Graphpad Prism vs 4.0 yielded the IC50 values.
The slope of uninhibited enzyme was estimated by conducting
13.52 (br. s, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
d 23.9 (CH2),
92.0 (C-8), 126.4 (C-30 and C-50), 126.5 (C-20 and C-60), 127.1 (C-40),
128.4 (C-300 and C-500), 128.8 (C-200and C-600), 137.8 (C-10), 138.8 (C-
400), 139.0 (C-100), 139.9 (C-7), 140.8 (C-9), 151.9 (C-4), 173.6 (C-2),
179.8 (NH(C]S)NH2); Anal. calcd. for C19H16N6OS2: C, 55.86; H,
3.95; N, 20.57. Found: C, 54.78; H, 3.87; N, 21.28.
m
L, 5 mM) into a cuvette containing 780
ml of po-
mL of enzyme at concentra-
m
4.1.18. [4-Oxo-8-(4-phenoxy-benzyl)-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4k)
White powder (81%); Mp 210e212 ꢀC (Ethanol-water); ESI-MS
m/z 423.2 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 4.09 (s, 2H,
CH2), 6.81e7.45 (m, 9H, AreH), 8.96 and 9.07 (2 s, 2H, (C]S) NH2),
10.07 (s, 1H, (C]S) NH), 12.68 (s, 1H, NH), 13.53 (br. s, 1H, NH); 13
NMR (75 MHz, DMSO-d6):
23.6 (CH2), 92.1 (C-8), 118.1 (C-30 and
C
same experiments using 10 mL of DMSO. The percentage activity of
each inhibitor was calculated by the following equations-
d
C-50), 118.6 (C-20 and C-60), 123.0 (C-40), 129.4 (C-300 and C-500), 129.9
(C-200and C-600), 134.8 (C-10), 138.6 (C-400), 140.8 (C-100), 151.9 (C-7),
154.6 (C-9), 156.9 (C-4), 173.5 (C-2), 179.8 (NH(C]S)NH2); Anal.
calcd. for C19H16N6O2S2: C, 53.76; H, 3.80; N, 19.80. Found: C, 54.22;
H, 3.72; N, 20.21.
(i) Activity ¼ (Slope of inhibited enzyme/Slope of uninhibited
enzyme) ꢃ 100%
(ii) Inhibition ¼ 100% ꢁ Activity
All the experiments were carried out in triplicate.
4.1.19. (8-Naphthalen-2-ylmethyl-4-oxo-2-thioxo-1,2,3,4-
tetrahedropyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4l)
White powder (63% yield); Mp 230e233 ꢀC (Ethanol-water);
4.2.2. Enzyme inhibition kinetics study
The enzyme inhibition was evaluated in the presence of
different concentrations of inhibitor at varying concentrations of
ESI-MS m/z 381.2 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 4.56 (s,
2H, CH2), 6.85e6.26 (m, 11H, AreH), 9.00 and 9.26 (2 s, 2H, (C]S)
NH2),10.22 (s,1H, (C]S) NH),12.72 (s,1H, NH),13.48 (br. s,1H, NH);
thymidine (1000, 500, 300, 200, 100 mM). The inorganic phosphate
was fixed at the saturating concentration (25 mM). The conversion of
thymidide (dThd) to thymine was recorded at 290 nm. All the ex-
periments were conducted in triplicate.
13C NMR (75 MHz, DMSO-d6):
d 22.2 (CH2), 90.0 (C-8), 123.1 (AreC),
123.8 (AreC), 125.5 (AreC), 125.6 (AreC), 125.7 (AreC), 126.2 (Are
C), 128.2 (AreC), 131.5 (AreC), 133.1 (AreC), 135.4 (AreC), 139.1 (C-
7), 140.8 (C-9), 152.6 (C-4), 173.5 (C-2), 179.9 (NH(C]S) NH2); Anal.
calcd. for C17H14N6OS2: C, 53.39; H, 3.69; N, 21.97. Found: C, 52.75;
H, 3.62; N, 21.45.
4.2.3. Gelatine zymography
Gelatine zymography was preformed following reported pro-
cedures with some modifications (Liotta and Stetler-Stevenson
1990) [35]. Briefly, the MDA-MB-231 cells were seeded onto six-
well plates in RPMI with 10% FBS and allowed to grow to 80%
confluence. The cells were passaged into serum-free medium for at
least 24 h before being treated with compounds and PMA (80 nM).
After incubation, the conditioned medium was harvested and then
4.1.20. (8-Furan-2-ylmethyl-4-oxo-2-thioxo-1,2,3,4-tetrahedro-
pyrazolo[1,5-a][1,3,5]triazin-7-yl]-thiourea (4m)
White powder (74% yield); Mp 227e229 ꢀC (Ethanol-water);
ESI-MS m/z 321.0 (Mꢁ1)þ; 1H NMR (300 MHz, DMSO-d6):
d 4.13 (s,

302
H. Bera et al. / European Journal of Medicinal Chemistry 78 (2014) 294e303
assayed for protein content. The volume of each samples having
equal amount of protein was adjusted by adding PBS to samples.
The resulting samples were mixed with 3ꢃ loading buffer and
subjected to electrophoresis on a 7.5% SDS-PAGE gel containing 0.1%
(w/v) gelatine using 1ꢃ Tris-Glycin SDS running buffer at 100 V for
90e120 min. Subsequently, the gels were placed in renaturing
buffer (2.5% Triton X-100), washed thoroughly to remove traces of
SDS and equilibrated in 1ꢃ zymogram developing buffer (50 mM
tris, 10 mM CaCl2, 0.15 M NaCl, pH-7.5). The gels were then incu-
bated overnight at 37 ꢀC in fresh developing buffer to allow gelatine
digestion. The gelatinolytic activity of MMPs was visualized by
staining the gels with 1% coomassie blue R-250 and destained
repeatedly in 50% methanol, 10% acetic acid, 40% water (v/v) until
the bands were clearly visible.
structures were removed and options for tautomer generation,
ionization change, isomer generation and 3D-generator were set to
the default values. Subsequently, molecular docking of the pre-
processed ligands was performed and various binding conforma-
tions for ligands were generated by Monte Carlo trial method. The
final energy refinement of the ligand pose or pose optimization in
ligandfit was estimated with Broyden-Flecher Gold Farbshanno
(BFGS) method. The best-scoring docking pose of each ligand was
identified and the dock scores were compared with the experi-
mental binding affinities.
Acknowledgements
This work was financially supported by National Medical
Research Council Grant, Singapore (R-148-000-102-275) and Aca-
demic Research Funds of National University of Singapore. The
authors are also thankful to Gokaraju Rangaraju Educational Soci-
ety and Jadavpur University for constant encouragement and for
providing essential laboratory facilities for this project.
4.2.4. VEGF assay
The VEGF expression in MDA-MB-231 cells was assessed with a
colorimetric enzyme-linked immunosorbent assay (ELISA) kit
following the manufacturer’s instructions. (Abfrontier) [15]. After
treatment with the indicated doses of compounds or DMSO, the cell
supernatant was collected and subjected to centrifugation (5000 g
for 5 min) to remove cells and debris. The diluted samples and VEGF
standard solutions were placed in the precoated 96-well plate and
incubated at 37 ꢀC for 90 min. Subsequently, the plate contents
were discarded and 0.1 mL of biotinylated anti-human VEGF anti-
body working solution was introduced and incubated for another
60 min. The plates were then thoroughly washed with 0.01 M TBS
and 0.1 mL of avidin-biotin-peroxidase complex working solution
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.063. These data include MOL
files and InChiKeys of the most important compounds described in
this article.
was added to each well. After 30 min of incubation, 90
mL of pre-
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