Synthesis, anti-thymidine phosphorylase activity and molecular docking of 5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-ones

Article abstract of DOI:10.1016/j.bioorg.2013.07.004

In our lead finding program, a series of 5-thioxo-[1,2,4]triazolo[1,5-a][1, 3,5]triazin-7-ones and their 5-thio-alkyl derivatives were designed and synthesized which contained different substituents at ortho-position of 2-phenyl ring attached to the fused

Full text of DOI:10.1016/j.bioorg.2013.07.004

Bioorganic Chemistry 50 (2013) 34–40
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, anti-thymidine phosphorylase activity and molecular docking
of 5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-ones
b
b
Hriday Bera a,b, , Min Huey Lee , Lingyi Sun , Anton V. Dolzhenko b,c, Wai Keung Chui 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 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:
Received 22 June 2013
Available online 2 August 2013
In our lead finding program, a series of 5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-ones and their 5-
thio-alkyl derivatives were designed and synthesized which contained different substituents at ortho-
position of 2-phenyl ring attached to the fused ring structure. The preliminary pharmacological evalua-
tion demonstrated that the synthesized compounds exhibited a varying degree of inhibitory activity
towards thymidine phosphorylase (TP), comparable to reference compound, 7-Deazaxanthine (7-DX,
Keywords:
Heterobicyclic system
Annulation reaction
In vitro enzyme assay
Thymidine phosphorylase inhibitors
Angiogenesis
2) (IC50 value = 42.63 lM). The study also inferred that the ortho-substituted group at the phenyl ring
and 5-thio-alkyl moiety imparted steric hindrance effects in the binding site of the enzyme, leading to
a reduced inhibitory response. In addition, compound 3a was identified as a mixed-type inhibitor of
TP. Moreover, computational docking study was performed to illustrate the important structural infor-
mation on the plausible ligand-enzyme binding interactions.
Molecular docking
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
inhibition of thymidine phosphorylase may be viewed as a plausi-
ble strategy to overcome its pathological effects. Pioneering re-
During the last few decades, target-based drug design for
anticancer therapy has gained increasing attention [1]. This ap-
proach has produced rationally designed inhibitors of dihydrofo-
late reductase [2], thymidylate synthase [3], purine nucleoside
phosphorylase [4], glycinamide ribonucleotide formyltransferase
[5] and matrix metalloproteases [6] that can either inhibit DNA
replication or block critical biochemical pathways, resulting in pre-
vention of growth of cancer cells. Continuous efforts in this field
identified thymidine phosphorylase (TP, EC 2.4.2.4) as a potential
therapeutic target for cancer therapy [7]. Several studies demon-
strated that thymidine phosphorylase (TP), also known as platelet
derived endothelial cell growth factor (PD-ECGF) and gliostatin, is
overexpressed in many solid tumors and plays a crucial role in can-
cer angiogenesis [8]. TP exerts angiogenesis via the release of its
search in this field has discovered several potent TP inhibitors,
most of these inhibitors are derivatives of pyrimidin-2,4-dione
with only a few that are fused bicyclic heterocycles [11]. In this
context, TPI and 7-DX have emerged as leading TP inhibitor candi-
dates (Fig. 1) [12,13].
Recently, based on structural similarities with the reference
compounds, a series of 1,3,5-triazin-2,4-dione and their fused
analogues was designed, synthesized and their in vitro TP inhibi-
tory potential was evaluated. Among them, 2-phenyl-5-thioxo-
[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-one showed inhibitory activ-
ity against TP [14]. The lead structure was modified by introducing
different substituents at meta- and/or para- positions of 2-phenyl
ring attached to the fused ring scaffold with the aim to improve the
TP inhibition potency. The generated compounds exhibited varying
degrees of inhibitory activity towards TP, comparable or better
than parent compound [15].
The success of these projects has led us to investigate the
hypothesis that the structural modification of 2-phenyl-5-thioxo-
[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-one by inserting various elec-
tron withdrawing substituents on the ortho-position of the phenyl
ring would exhibit TP inhibitory action. Moreover, it was assumed
that the corresponding 5-thio-alkyl derivatives of these com-
pounds would demonstrate inhibitory properties towards TP. To
address these hypotheses, herein, a library of 1,2,4-triazolo[1,5-
a][1,3,5]triazines (Fig. 2) was synthesized via a practical synthetic
metabolic product, 2-deoxy-D-ribose that stimulates the secretion
and/or expression of many angiogenic factors, including MMP-9,
MMP-2, VEGF, IL-8 and others [9]. Subsequently, it stimulates a
cascade of events, including the migration of endothelial cells
and rapid formation of long-lasting functional neo-vessels, lead-
ing to increased metastasis and tumour growth [8,10]. Therefore,
⇑
Corresponding authors. Address: Gokaraju Rangaraju College of Pharmacy,
Bachupally, Hyderabad 500090, India (H. Bera), Department of Pharmacy, National
University of Singapore, Singapore 117543 (W.K. Chui).
E-mail addresses: hriday.bera@gmail.com (H. Bera), phacwk@nus.edu.sg
(W.K. Chui).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.07.004

H. Bera et al. / Bioorganic Chemistry 50 (2013) 34–40
35
O
O
O
N
N
HN
N
A
Cl
HN
HN
S
N
H
N
R
N
H
O
N
H
O
N
H
NH.HCl
TPI, 1
7-DX, 2
O
N
O
N
Fig. 1. Structures of known TP inhibitors.
H
N
N
HN
N
B
HN
N
N
H
N
S
S
R
R
O
c
O
N
N
N
N
HN
N
Scheme 2. The prototropic interconversion between tautomeric forms of com-
pounds 3a–3e.
HN
N
R1
S
N
H
S
N
R
R
3a-3e
cyclocondensation in water (Scheme 1). A substantial improve-
ment in yield of key intermediates (8) was observed for this
approach. Moreover, the overall reaction time was reduced to
8–15 min as compared to several hours by the conventional heat-
ing process.
All the synthesized compounds (3a–3e and 4a–4j) were charac-
terized by melting points and different spectroscopic techniques
(1H NMR, 13C NMR and MS). Interestingly, the 13C NMR spectros-
copy was used as an important tool to confirm and distinguish
the structures of the target compounds. The 13C peak of the thio-
carbonyl (C@S) carbon of 3a was found to appear at around
175.8 ppm. The appearance of the peak at about 13.9 ppm assigned
to SMe in the 13C NMR spectrum confirmed the formation of prod-
uct 4a. The purity of the compounds was examined by reverse
phase HPLC method and elemental analysis. The purity of all com-
pounds was satisfactory (above 95%).
4a-4j
Fig. 2. Structures of target compounds to be synthesized.
approach and the target compounds were evaluated for TP inhibi-
tion using an in vitro enzyme assay to explore potential lead TP
inhibitors. In addition,
performed to illustrate the binding site and the plausible ligand-
enzyme binding interactions of the compounds.
a computational docking study was
2. Results and discussion
2.1. Chemistry
The synthesis of the title compounds (3a–3e and 4a–4j) was
achieved via annulation of 1,3,5-triazine ring onto 3(5)-amino-
1,2,4-triazoles as described in Scheme 1 [14,15]. The adopted
synthetic method was previously disclosed by Bokaldere and
co-workers [16].
The reaction of 5-amino-1,2,4-triazoles (8) with ethoxycarbonyl
isothiocyanate in DMF gave the thiourea (9) derivatives which
underwent base catalyzed intramolecular heterocyclization, lead-
ing to the generation of target compounds (3a–3e). Compounds
4a–4j, corresponding 5-thio-alkyl derivatives of 3a–3e, were
subsequently produced by treating 3a–3e with alkyl halide in
aqueous alkali. This approach afforded products of acceptable
yields (32–86%) and high purity. Moreover, an environmental
benign and ease workup protocol was utilized to produce 5-ami-
no-1,2,4-triazoles (8) [17,18]. The two-step reaction involved the
synthesis of amidoguanidines (7) from commercially available
substituted acid chlorides (5) followed by microwave-assisted
Due to annular tautomerism, target compounds 3a–3e can exist
in dynamic equilibrium of (A) 4-, (B) 3- and (C) 1H-forms
(Scheme 2). The prototropic interconversion between these tauto-
meric forms resulted in appearance of a broad 4-N(H) signal in 1H
NMR spectra of compounds in DMSO. The broad signal was found
to disappear on heating, due to rapid exchange of the NH proton.
2.2. Anti-thymidine phosphorylase activity
Fifteen compounds (3a–3e and 4a–4j) were evaluated for TP
inhibitory activity by a spectrophotometric assay that used recom-
binant human thymidine phosphorylase, expressed in E. coli
(T2807 – Sigma Aldrich) as the enzyme and thymidine as the
substrate. The original method developed by Krenitsky and Bushby
[19] was modified and adopted. The results of the enzyme
O
O
R
H
NH
Heat at 180 o
60-90 min
C
N
NH
N
NH2
NH
Microwave
180 oC for 8-15 min
N
H
Cl
H2N
+
NH2
N
H
NH2
N
R
8
R
5
7
6
1. DMF
2. Ethoxycarbonyl isothiocyanate
3. Stir at rt for 5 h
O
O
O
S
N
NH
N
N
OEt
N
N
HN
N
H
HN
N
N
H
N
R1
N
S
N
1. Alkyl halides
2. Stir at rt for 60-90 min
S
N
H
1. NaOH
R
R
R
2. Ethanol (80 %)
9
4a-4j
3. Heat at 100 oC for 30 min
3a-3e
Scheme 1. Synthesis of target compounds (3a–3e and 4a–4j).

36
H. Bera et al. / Bioorganic Chemistry 50 (2013) 34–40
Table 1
Thymidine phosphorylase inhibitory activity of the synthesized compounds (3–4).
.
Entry
Cpd.
R
R1
TP inhibition activitya IC50
(lM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
7-DX
H
F
Cl
Br
NO2
H
–
–
–
–
39.56 1.76
45.45 2.45
75.30 4.51
80.41 3.12
88.55 2.38
95.05 4.42
107.11 5.23
145.32 4.37
160.08 4.45
102.25 2.49
110.12 3.57
155.82 4.43
164.8 2.98
104.6 3.13
111.2 4.22
42.63 5.23
–
CH3
CH3
CH3
CH3
C2H5
C2H5
C2H5
C2H5
C3H7
CH2Ph
–
F
Br
NO2
H
F
Br
NO2
H
H
–
a
Experiments carried out in triplicate.
inhibitory potency were expressed in terms of IC50 values and were
compared with that of 7-DX (IC50 = 42.63 M) (Table 1).
Compounds 3a–3e and 4a–4j demonstrated varying degrees of
TP inhibition with IC50 values ranging between 40 and 165 M and
compound 3a was observed to have best anti-TP activity
(IC50 = 39.56 M) among the two groups of compounds evaluated
in this study. The structural modification of compound 3a with
the introduction of electron withdrawing substituents at ortho-po-
sition of phenyl ring resulted in a decrease in the binding interac-
tions. The ortho-substituted groups were assumed to impart a
steric hindrance effect in the binding pocket of enzyme, leading
to a decreased inhibitory effect. Moreover, the inhibition activity
of the compounds bearing substituents at ortho-position was in
the descending order of H > F > Cl > Br > NO2. This observation
clearly implied that the activity was dependent on the relative size
of substituents. The inhibitory potency of compounds was found to
decrease gradually with increasing size of the moiety.
Further structural modification of 3a–3e by replacing 5-thioxo
moiety with thiomethyl group, resulting in subsequent elimination
of active hydrogen from N4, led to dramatic decrease in inhibition
l
l
property, as observed in compounds 4a–4d (IC50 = 95–160 lM). In
addition, as the alkyl chain length and bulk increased, a substantial
reduction in TP inhibitory activity was observed. Therefore, it can
be inferred that the 5-thioxo moiety (C@S) was optimal in the struc-
ture of the TP inhibitors. In other word, TP inhibitors having triazol-
o[1,5-a][1,3,5]triazine scaffold, must carry a C(@O)ANHAC(@S)
fragment in the structure.
A brief kinetic study of compound 3a was performed to eluci-
date the mechanism of inhibition according to previously disclosed
method [15]. Lineweaver–Burk plots were constructed using the
data obtained from the study (Fig. 3). The results demonstrated
that compound 3a was a mixed-type inhibitor of TP in the presence
of thymidine as a variable substrate since the straight lines corre-
sponding to different concentrations of the inhibitor (0, 30 and
l
40 lM) intersected in the left panel of the reciprocal plot. A similar
type of inhibition mechanism was observed with a structurally
resemble TP inhibitor [15].
2.3. Molecular docking
To investigate the plausible binding orientations and molecular
interactions of the synthesized compounds, a molecular docking
study was performed using geometry optimized structure of the
compounds. The compounds were allowed to dock at the active
site of TP (PDB code: 2WK6) [20] using SYBYL-X 1.3. To analyze
the docking results, the lowest energy conformation of the com-
pounds was adopted. Initially, the docking protocol was validated
by comparing the binding interactions of TPI in the active site of
TP with the previously reported results. The predicted binding
mode and interaction pattern of TPI generated by our software
were found to be in accordance with the reported crystallographic
study [21] with a RMS deviation of 0.5 Å (Fig. 5a and 5b in supple-
mentary material).
Fig. 3. Lineweaver–Burk plots of TP inhibition by 3a with respect to thymidine
(dThd) as a variable substrate. Results are presented as means SD; SD denoted by
error bars (Experiments carried out in triplicate).

H. Bera et al. / Bioorganic Chemistry 50 (2013) 34–40
37
Fig. 4. Graphical representation of interactions between TP and its inhibitors analyzed by computational docking: (a) hydrogen bonding of 7-DX (colored ball and stick) with
Lys221, Thr118 (green colored ball and stick); (b) hydrophobic interaction of 7-DX (colored ball and stick) with amino acid (orange colored sphere); (c) hydrogen bonding of
3a (colored ball and stick) with Thr118 (green colored ball and stick); (d) hydrophobic interaction of IVb (colored ball and stick) with amino acid (orange colored sphere); (e)
hydrogen bonding of 4a (colored ball and stick) with Lys221 and Thr118 (green colored ball and stick); and (f) hydrophobic interaction of 4a (colored ball and stick) with
amino acid (orange colored sphere). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
The analysis of docking interactions between TP and reference
compound, 7-DX revealed that the carbonyl group at C4 and NH-
7 of 7-DX created strong hydrogen bonding contacts with Lys221
and Tyr118 respectively, located in the active site of enzyme
(Fig. 4a) [21]. The NH group neighboring to the thiocarbonyl
moiety in compound 3a displayed hydrogen bonding interaction
with Tyr118, while the thiocarbonyl moiety positioned itself into
a hydrophobic pocket formed by some amino acid residues like

38
H. Bera et al. / Bioorganic Chemistry 50 (2013) 34–40
Ile218, Ile214, Val 208, Val241, Val208 and Leu148 (Fig. 4c and 4d).
As a result, compound 3a showed inhibitory activity with an IC50
value in micromolar range being comparable to7-DX (Table 1).
Moreover, compound 3a showed a different binding orientation
as compared to 7DX and consequently it demonstrated a mixed
mode of inhibition with respect to thymidine. It is noteworthy that
reference inhibitor, 7-DX, behaved as a competitive or mixed-type
inhibitor in the presence of variable concentrations of thymine
[22].
It is also evident from the interaction pattern that the
compounds 4a and other 5-thio-alkyl derivatives exhibited poor
penetration to the active site probably due to the increased steric
bulkiness of their thiomethyl group resulted in diminished
inhibitory effect (Fig. 4e and 4f).
precipitated products (3a–3e) was filtered off, recrystallized with
suitable solvents and dried under vacuum [14,15].
4.1.3. 2-Phenyl-5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7(4H)-
one (3a)
Yield: 78.2%; mp: 258–259 °C (Ethanol); ESI-MS m/z 244.1
(Mꢀ1)+; purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 7.53–7.55
(m, 3H, H-30, H-40 and H-50), 8.04–8.07 (m, 2H, H-20 and H-60),
13.12 (s, 1H, NH), 14.31 (br. s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6): d 127.1 (C-20 and C-60), 129.5 (C-30 and C-50), 129.7
(C-40), 131.3 (C-10), 141.7 (C-2), 151.9 (C-9), 162.4 (C-7), 175.8
(C-5); Anal. calcd. for C10H7N5OS: C, 48.97; H, 2.88; N, 28.55.
Found: C, 47.99; H, 2.71; N, 28.21.
4.1.4. 2-(2-Fluorophenyl)-5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-
7-one (3b)
3. Conclusion
Yield: 50.8%; mp: 235 °C (Acetic acid); ESI-MS m/z 262.3
(Mꢀ1)+; purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 7.20–7.25
(m, 2H, ArAH), 7.32–7.42 (m, 1H, ArAH), 7.86–7.91 (m, 1H, Ar-H)
12.15 (s, 1H, NH), 14.12 (br. s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6): d 117.3 (C-30), 125.4 (C-10), 180.4 (C-50), 133.2 (C-60),
141.7 (C-40), 158.7 (C-20), 159.3 (C-2), 162.11 (C-9), 175.9 (C-7),
179.9 (C-5); Anal. calcd. for C10H6FN5OS: C, 45.62; H, 2.30; N,
26.60. Found: C, 45.99; H, 2.21; N, 26.28.
We have synthesized a small library of 1,2,4-triazolo[1,5-
a][1,3,5]triazines via ring annelation reaction and evaluated these
compounds for their thymidine phosphorylase inhibitory activity.
The biological evaluation identified a number of new TP inhibitors
and several compounds in particular those that bear ortho-substi-
tutions on the phenyl ring, viz., compounds 3a-3e. These com-
pounds exhibited inhibitory activity against TP with IC50 values
being well comparable to 7-DX. In addition, it was found that the
C5 thioxo moiety is essential for interaction with the enzyme.
Compound 3a exhibited a mixed mode of inhibition towards TP
with respect to thymidine as a variable substrate. The molecular
docking studies suggested a justified binding mode of the active
compounds in the binding site of TP. Therefore, these compounds
constitute a new direction for design and synthesis of fused-ring
TP inhibitors.
4.1.5. 2-(2-Chlorophenyl)-5-thioxo-[1,2,4]triazolo[1,5-
a][1,3,5]triazin-7-one (3c)
Yield: 60.1%; mp: 166 °C (Acetic acid); ESI-MS m/z 278.0
(Mꢀ1)+; purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 7.46–7.58
(m, 2H, ArAH), 7.63 (d, 1H, ArAH, J = 7.1584), 7.92 (dd, 1H, Ar-H,
J = 1.88 and 7.15 Hz), 13.1 (s, 1H, NH), 14.2 (br. s, 1H, NH); 13C
NMR (75 MHz, DMSO-d6): d 131.8 (C-50), 128.1 (C-60), 130.8 (C-
30), 131.6 (C-40), 131.7 (C-20), 131.8 (C-10), 141.1 (C-2), 150.8 (C-
9), 160.7 (C-7), 175.3 (C-5); Anal. calcd. for C10H6ClN5OS: C,
42.94; H, 2.16; N, 25.04. Found: C, 41.99; H, 2.01; N, 24.72.
4. Experimental
4.1. Chemistry
4.1.6. 2-(2-Bromophenyl)-5-thioxo-[1,2,4]triazolo[1,5-
a][1,3,5]triazin-7-one (3d)
Yield: 59.5%; mp: 144 °C (Acetic acid); ESI-MS m/z 324.0 (M-1)+;
purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 7.38–7.65 (m, 2H,
ArAH), 7.69–7.92 (m, 2H, ArAH) 13.15 (s, 1H, NH), 14.21(br. s,
4.1.1. General procedures
All reagents were purchased from Sigma–Aldrich or Alfa Aesar
and were used without further purification. Melting points were
determined on a Gallenkamp melting point apparatus and were
uncorrected. 1H NMR and 13C NMR spectra were recorded on a Bru-
ker 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 spec-
trometer using the electrospray ionization (ESI) mode. Reactions
were monitored by TLC on silica gel (60 F254) coated aluminum
plate.
13
1H, NH); C NMR (75 MHz, DMSO-d6): d 120.9 (C-20), 127.8 (C-
50), 130.3 (C-60), 131.7 (C-40), 131.8 (C-30), 134.0 (C-10), 141.1 (C-
2), 150.7 (C-9), 161.6 (C-7), 175.2 (C-5); Anal. calcd. for C10H6BrN5-
OS: C, 37.05; H, 1.87; N, 21.60. Found: C, 36.81; H, 1.75; N, 20.94.
4.1.7. 2-(2-Nitrophenyl)-5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-
7-one (3e)
Yield: 40.1%, mp: 211 °C (Acetic acid), ESI-MS m/z 289.1
(Mꢀ1)+; purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 7.64–8.08
(m, 3H, ArAH), 8.81–8.99 (m, 1H, ArAH), 13.21 (s, 1H, NH),
4.1.2. General procedure for the synthesis of 5-thioxo-(1,2,4)-
triazolo[1,5-a](1,3,5)triazin-7-ones and its derivatives (3a–3e)
Initially, amidoguanidines (7) were prepared via the reaction of
substituted benzoyl chloride (5) (10 mmol) and aminoguanidine
hydrochloride (6) (20 mmol). The obtained intermediates (7) were
then treated with microwave irradiation power (100 W) for
8–15 min in water to generate 5-amino-1,2,4-triazoles (8) [18].
Subsequently, 5-amino-1,2,4-triazoles (8) (3 mmol) were reacted
with ethoxycarbonyl isothiocyanate (3.3 mmol) in anhydrous
DMF (4 ml) at room temperature, leading to formation of thiourea
derivatives (9).
In a well stirred solution of sodium hydroxide (9 mmol) in eth-
anol (80%, 20 ml), thiourea derivatives (9) were added and heated
on a water bath for 20 min. After cooling, the solvent was evapo-
rated under vacuum and the residue was re-suspended in water
(25 ml). It was acidified up to pH 1–3 using 2.5 M HCl. The
13
14.21(br. s, 1H, NH); C NMR (75 MHz, DMSO-d6): d 122.8 (C-30),
124.6 (C-10), 131.2 (C-40), 132.4 (C-60), 133.2 (C-50), 141.6 (C-20),
149.5 (C-2), 151.8 (C-9), 159.5 (C-7), 175.9 (C-5); Anal. calcd. for
C10H6N6O3S: C, 41.38; H, 2.08; N, 28.95. Found: C, 41.34; H, 2.04;
N, 28.22.
4.1.8. General procedure for the synthesis of thioalkyl derivatives of
3a-3e (4a–4j)
To a stirred solution of 3a–3e (5 mmol) in 4 ml of 2.5 M NaOH,
iodomethane/ethyl iodide, propyl iodide/benzyl bromide
(7.5 mmol) was added, the stirring was continued for 2 h. A solid
precipitate was observed which upon acidification (pH 1–3) with
2.5 M HCl afforded another solid. The precipitated solid (4a–4j)
was filtered, washed with ice cold water and recrystallized by
water–ethanol system.

H. Bera et al. / Bioorganic Chemistry 50 (2013) 34–40
39
4.1.9. 2-Phenyl-5-methylthioxo- [1,2,4]triazolo[1,5-a][1,3,5]triazin-7-
one (4a)
4.1.15. 2-(2-Bromophenyl)-5-ethylthioxo-[1,2,4]triazolo[1,5-
a][1,3,5]triazin-7-one (4g)
Yield: 58.2%; mp: 292–294 °C (Ethanol–water); ESI-MS m/z
Yield: 76.6%; mp: 239 °C (Ethanol–water); ESI-MS m/z 350.1
(Mꢀ1)+; purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 1.25 (t,
3H, CH3, J = 7.4 Hz), 3.23 (q, 2H, CH2, J = 7.3 Hz), 7.23–7.56 (m,
1H, ArAH), 7.61–7.73 (m, 3H, ArAH), 13.86 (br. s, 1H, NH); 13C
NMR (75 MHz, DMSO-d6): d 14.7 (CH3), 25.5 (CH2), 121.6 (C-20),
128.3 (C-50), 131.6 (C-60), 132.1 (C-40), 132.4 (C-30), 134.5 (C-10),
143.8 (C-2), 157.4 (C-9), 163.1 (C-7), 164.8(C-5); Anal. calcd. for
258.0 (Mꢀ1)+; purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 2.62
(s, 3H, SMe), 7.54–7.55 (m, 3H, H-30, H-40 and H-50), 13.15 (br. s,
13
1H, NH); C NMR (75 MHz, DMSO-d6): d 13.9 (SMe), 127.1 (C-20
and C-60), 129.4 (C-30and C-50), 130.4 (C-40), 131.0 (C-10), 143.8
(C-2), 157.9 (C-9), 163.1 (C-5), 165.3 (C-7); Anal. calcd. for C11H9N5-
OS: C, 50.95; H, 3.50; N, 27.01. Found: C, 50.26; H, 3.41; N, 26.73.
C12H10BrN5OS: C, 40.92; H, 2.86; N, 19.88. Found: C, 40.28; H,
2.81; N, 19.26.
4.1.10. 2-(2-Fluorophenyl)-5-methylthioxo-[1,2,4]triazolo[1,5-
a][1,3,5]triazin-7-one (4b)
4.1.16. 2-(2-Nitrophenyl)-5-ethylthioxo-[1,2,4]triazolo[1,5-
a][1,3,5]triazin-7-one (4h)
Yield: 86.3%; mp: 231 °C (Ethanol–water); ESI-MS m/z 277.0
(Mꢀ1)+; purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 2.52 (s,
3H, CH3), 7.30–7.46 (m, 4H, ArAH), 13.94 (br. s, 1H, NH); 13C
NMR (75 MHz, DMSO-d6): d 117.3 (C-30), 118.3 (C-10), 125.3 (C-
50), 130.9 (C-60), 132.9 (C-40), 143.8 (C-20), 157.5 (C-2), 158.7 (C-
9), 159.9 (C-7), 162.1 (C-5); Anal. calcd. for C11H8FN5OS: C, 47.65;
H, 2.91; N, 25.26. Found: C, 46.77; H, 2.82; N, 26.11.
Yield: 40.2%; mp: 188 °C; ESI-MS m/z 318.0 (Mꢀ1)+; pur-
ity > 95%; 1H NMR (300 MHz, DMSO-d6): d 1.37 (t, 3H, CH3), 3.24
(q, 2H, CH2), 7.69–7.91 (m, 2H, ArAH), 7.99–8.13 (m, 2H, ArAH),
13.87 (br. s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d 14.7 (CH3),
25.5 (CH2), 123.4 (C-30), 124.4 (C-10), 130.9 (C-40), 132.1 (C-60),
133.0 (C-50), 143.7 (C-20), 149.7 (C-2), 157.8 (C-9), 160.1 (C-7),
165.3 (C-5); Anal. calcd. for C12H10N6O3S: C, 45.28; H, 3.17; N,
26.40. Found: C, 44.34; H, 3.05; N, 26.88.
4.1.11. 2-(2-Bromophenyl)-5-methylthioxo-[1,2,4]triazolo[1,5-
a][1,3,5]triazin-7-one (4c)
4.1.17. 2-Phenyl-5-propylthioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-
one (4i)
Yield: 59.2%; mp: 138 °C (Ethanol–water), ESI-MS m/z 337.2
(Mꢀ1)+; purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 2.63 (s,
1H, CH3), 7.36–7.61 (m, 2H, ArAH), 7.71–7.93 (m, 2H, ArAH),
13.71 (br. s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d 13.9 (CH3),
121.6 (C-20), 128.3 (C-50), 131.6 (C-60), 132.1 (C-40), 132.4 (C-30),
134.5 (C-10), 143.7 (C-2), 157.3 (C-9), 163.1 (C-7), 165.5 (C-5); Anal.
calcd. for C11H8BrN5OS: C, 39.07; H, 2.38; N, 20.71. Found: C, 38.42;
H, 2.29; N, 20.13.
Yield: 73.1%; mp: 214 °C (Ethanol–water); ESI-MS m/z 287.0
(Mꢀ1)+; purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 1.02 (t,
3H, CH3), 1.76 (m, 2H, CH2), 3.21 (t, 2H, SCH2), 7.43–7.62 (m, 3H,
ArAH), 8.02–8.22 (m, 1H, ArAH), 13.23 (br. s, 1H, NH); 13C NMR
(75 MHz, DMSO-d6): d 13.6 (CH3), 22.4 (CH2), 32.7 (SCH2), 127.1
(C-20 and C-60), 129.4 (C-30 and C-50), 130.4(C-40), 131.0(C-10),
143.9 (C-2), 158.0 (C-9), 163.0 (C-7), 164.7 (C-5); Anal. calcd. for
C
13H13N5OS: C, 54.34; H, 4.56; N, 24.37. Found: C, 53.98; H, 4.45;
4.1.12. 2-(2-Nitrophenyl)-5-methylthioxo-[1,2,4]triazolo[1,5-
a][1,3,5]triazin-7-one (4d)
N, 24.11.
Yield: 67.8%, mp: 253 °C, ESI-MS m/z 303.8 (Mꢀ1)+; pur-
ity > 95%; 1H NMR (300 MHz, DMSO-d6): d 2.50 (s, 3H, CH3),
7.70–7.89 (m, 2H, Ar-H), 7.99 (d, 1H, ArAH, J = 7.5 Hz), 8.08 (d,
1H, ArAH, J = 7.53 Hz), 13.50 (br. s, 1H, NH); 13C NMR (75 MHz,
4.1.18. 2-Phenyl-5-benzylthioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-
one (4j)
Yield: 38.9%; mp: 231 °C (Ethanol); ESI-MS m/z 334.5 (Mꢀ1)+;
purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 3.20 (s, 2H, CH2),
7.21–7.30 (m,1H, ArAH), 7.36–7.84 (m, 3H, ArAH), 7.91–8.01 (m,
2H, ArAH), 8.12–8.23 (m, 2H, ArAH), 13.78 (br. s, 1H, NH); 13C
NMR (75 MHz, DMSO-d6): d 34.7 (CH2), 127.0 (C-20 and C-60),
127.8 (C-400), 129.0 (C-300 and C-500), 129.3 (C-30 and C-50),
129.5(C-200 and C-600), 130.6 (C-40), 131.0 (C-10), 137.6 (C-100),
146.3 (C-2), 158.8 (C-9), 162.2 (C-7), 167.3 (C-5); Anal. calcd. for
DMSO-d6):
d
13.9 (CH3), 123.4(C-30), 124.4(C-10), 130.9(C-40),
132.1(C-60), 133.0(C-50), 143.7(C-20), 149.7 (C-2), 157.8 (C-9),
160.1 (C-7), 166.0 (C-5); Anal. calcd. for C11H8N6O3S: C, 43.42; H,
2.65; N, 27.62. Found: C, 43.12; H, 2.56; N, 27.02.
4.1.13. 2-Phenyl-5-ethylthioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-
one (4e)
C17H13N5OS: C, 60.88; H, 3.91; N, 20.88. Found: C, 59.15; H, 3.78;
N, 20.23.
Yield: 31.8%; mp: 249 °C (Ethanol); ESI-MS m/z 272.2 (Mꢀ1)+;
purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 1.37 (t, 3H, CH3,
J = 7.34 Hz), 3.22 (q, 2H, CH2, J = 7.5 Hz), 7.49–7.57 (m, 3H, ArAH),
8.07–8.17 (m, 1H, ArAH), 13.65 (br. s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6): d 14.8 (CH3), 25.5(CH2), 127.1 (C-20 and C-60), 129.4 (C-
30 and C-50), 130.4 (C-40), 130.0 (C-10), 143.9 (C-2), 157.9 (C-9),
163.1 (C-7), 164.6 (C-5); Anal. calcd. for C12H11N5OS: C, 52.73; H,
4.06; N, 25.62. Found: C, 51.37; H, 4.01; N, 25.11.
4.2. In vitro thymidine phosphorylase enzyme assay
A spectrophotometric assay method, originally developed by
Krenitsky and Bushby [19], was adopted to evaluate in vitro TP
inhibitory activity of the synthesized compounds. Briefly, thymi-
dine (substrate) and recombinant thymidine phosphorylase, ex-
pressed in E. coli (T2807-Sigma Aldrich) were used for this assay.
Absorbance at 290 nm was recorded on a Shimadzu UV Mini
1240 UV–Vis Spectrophotometer. The enzymatic reaction was ini-
tiated by addition of substrate (200
taining 780 l of potassium phosphate buffer (pH 7.4), 10
enzyme at concentration of 1.5 U and 10 l of test compounds dis-
4.1.14. 2-(2-Fluorophenyl)-5-ethylthioxo-[1,2,4]triazolo[1,5-
a][1,3,5]triazin-7-one (4f)
ll, 5 mM) into a cuvette con-
Yield: 53.4%; mp: 189 °C (Ethanol); ESI-MS m/z 291.0 (Mꢀ1)+;
purity > 95%; 1H NMR (300 MHz, DMSO-d6): d 1.40 (t, 3H, CH3,
J = 7.2 Hz), 3.25 (q, 2H, CH2, J = 7.0 Hz), 7.27–7.47 (m, 2H, ArAH),
7.96–8.23 (m, 2H, ArAH), 7.97–8.23 (m, 1H, ArAH), 13.73 (br. s,
l
l
l of
l
solved in DMSO. The decrease in absorbance due to conversion of
thymidine to thymine was followed after 4, 8, 12, 16 and 20 min
and from the slope of the change in absorbance, the initial reaction
rate was determined. The same experiments were performed using
13
1H, NH), C NMR (75 MHz, DMSO-d6): d 117.4 (C-30), 118.4 (C-
10), 125.3 (C-50), 131.1 (C-60), 132.9 (C-40), 143.8 (C-20), 162.1 (C-
2), 164.8 (C-9), 175.8 (C-7), 179.9 (C-5); Anal. calcd. for C12H10FN5-
OS: C, 49.48; H, 3.46; N, 24.04. Found: C, 48.32; H, 3.35; N, 23.29.
10
ll of DMSO to calculate slope of uninhibited enzyme. The initial
rates of the change in absorbance at different concentrations of

40
H. Bera et al. / Bioorganic Chemistry 50 (2013) 34–40
inhibitors were converted to % inhibition of enzyme and plotted
against inhibitor concentrations using Graphpad Prism vs 4.0 to
give the IC50 at 50% inhibition.
The inhibitory activity of each compound was calculated by the
following formulae:
Funds of National university of Singapore for their financial assis-
tance. We also thank Gokaraju Rangaraju Educational Society for
providing essential laboratory facilities and support for this
project.
Appendix A. Supplementary material
Slope of inhibited enzyme
Slope of uninhibited enzyme
Activ
ity ¼
ꢁ 100%
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2013.
07.004.
Inhibition ¼ 100% ꢀ Activity
All the experiments were carried out in triplicate.
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Acknowledgments
The authors are grateful to the National Medical Research Coun-
cil grant, Singapore (R-148-000-102-275) and Academic Research