Synthesis, anticancer, and computational studies of 1, 3, 4-oxadiazole-purine derivatives

Article abstract of DOI:10.1002/jhet.3987

Theophylline-7-acetic acid (acefylline) (3) and its derivatives are pharmacologically active compounds and generally recognized as bronchodilators for the treatment of respiratory diseases like acute asthma for over 70 years. In this article, synthesis of

Full text of DOI:10.1002/jhet.3987

Received: 30 September 2019
DOI: 10.1002/jhet.3987
Revised: 6 February 2020
Accepted: 18 March 2020
A R T I C L E
Synthesis, anticancer, and computational studies of 1, 3,
4-oxadiazole-purine derivatives
Irum Shahzadi1 | Ameer F. Zahoor1
| Azhar Rasul2 | Nasir Rasool1
|
Zohaib Raza3 | Shahla Faisal4 | Bushra Parveen1 | Shagufta Kamal5
|
Muhammad Zia-ur-Rehman6 | Faisal M. Zahid4
1Department of Chemistry, Government
Abstract
College University Faisalabad, Faisalabad,
Pakistan
Theophylline-7-acetic acid (acefylline) (3) and its derivatives are pharmacologi-
cally active compounds and generally recognized as bronchodilators for the
treatment of respiratory diseases like acute asthma for over 70 years. In this
article, synthesis of 2-((5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-
2Department of Zoology, Government
College University Faisalabad, Faisalabad,
Pakistan
3Department of Pharmacology,
Government College University
Faisalabad, Faisalabad, Pakistan
4Department of Statistics, Government
College University Faisalabad, Faisalabad,
Pakistan
5Department of Biochemistry,
Government College University
Faisalabad, Faisalabad, Pakistan
yl)methyl)-1,3,4-oxadiazol-2-yl)thio)-N-arylacetamides
(10a-j)
has
been
reported. All the synthesized derivatives (10a-j) were structurally verified by
1
FT-IR, H NMR, 13C NMR and evaluated for their anti-cancer (using MTT
assay), hemolytic and thrombolytic potential. N-(4-Chlorophenyl)-2-(5-((1,-
3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-1,3,4-oxadiazol-2-
ylthio)acetamide (10g) was found to be the most active against human liver
cancer cell lines (Huh7) having cell viability 53.58 1.28 using 100 μg/mL
concentration of compound which was further in-silico modelled to describe
the possible mechanistic insights for its anti-proliferative activity. The results
of hemolytic and thrombolytic activities indicated that these derivatives were
less toxic and hold considerable potential as a drug candidate. 2-(5-((1,3-
Dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-1,3,4-oxadiazol-2-
ylthio)-N-(2-fluorophenyl)acetamide (10c) of the series was found to be least
toxic with 0.1% hemolysis relative to ABTS (95.5%) as positive control. 2-(5-
((1,3-Dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-1,3,4-oxadiazol-
2-ylthio)-N-(tetrahydro-2H-pyran-4-yl)acetamide (10j) exhibited potent clot lysis
activity (90%) as compared to negative control DMSO (0.57%).
6Applied Chemistry Research Centre,
PCSIR Laboratories Complex, Lahore,
Pakistan
Correspondence
Ameer F. Zahoor, Department of
Chemistry, Government College
University Faisalabad, 38000 Faisalabad,
Pakistan.
Email: fawad.zahoor@gcuf.edu.pk; fawad.
zahoor@gmail.com
1 | INTRODUCTION
hypotensive agents on cardiovascular system,[4,5] whereas
its alkyl substituted derivatives in 7- and 8-positions
Purine based natural products produce a variety of biologi-
cally and pharmacologically active derivatives via structural
modifications.[1] Various purine-based drugs are currently
used for the treatment of different diseases including
respiratory and neurodegenerative diseases, cancer, and
diabetes.[2] Theophylline is a purine-based xanthene which
exhibits multidimensional biological and pharmacologi-
cal properties.[3] 7, 8-disubstituted theophylline act as
increase its activity at adenosine receptors and act as anti-
cancer, anti-microbial, anti-inflammatory, and vascular
relaxing agents[6,7] (Figure 1). Some of its derivatives are
found active against hyperglycemia which causes diabetic
complications in NIDDM (noninsulin dependent diabetes
mellitus) patients.[8] Its amide and Schiff base derivatives
have been reported as bronchodilators for therapy of
respiratory diseases like chronic pulmonary disease or
J Heterocyclic Chem. 2020;1–13.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals LLC
1

2
SHAHZADI ET AL.
asthma,[9–12] whereas few others such as caffeine and pen-
toxyfylline are well known micromolar inhibitors of
18 chitinases family of bacteria with greater DNA binding
ability through hydrogen bonding.[13]
relevant molecules,[28,29] herein, we aimed to synthesize
some novel acefylline-oxadiazole hybrids and investigate
their anti-cancer, hemolytic, and thrombolytic activities.
Theophylline and nitric oxide play an important role
in inflammatory pathways. Interaction of theophylline
with nitric oxide modulators makes it more active against
inflammatory autoimmune disorder called rheumatoid
arthritis.[14] Cancer is among the major global health
problems leading to mortality following cardiovascular
diseases. Nucleoside and amide analogues of theophyl-
line containing 1,2,3-triazoles are among the most prom-
ising and complex anti-cancer drugs.[15,16]
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
The synthetic route for the novel series of acefylline
derivatives is shown in Scheme 1. Theophylline-7-
acetic acid (3) was subjected to esterification with
methanol using sulfuric acid as catalyst (with 68%
yield) followed by treatment with hydrazine hydrate to
get theophylline-7-acetohydrazide (5) in 98% yield.[8,30]
Hydrazide (5) was further reacted with carbon disulfide
in ethanol in basic media to prepare 1,3,4 oxadiazole-
2-thiol (6) in 62% yield.[31] Various 2-bromo-N-phenyl
acetamides (9a-j) (obtained by reacting various aro-
matic amines (7a-j) with bromo acetyl bromide (8)[32]
and were coupled with 1,3,4 oxadiazole-2-thiol (6) in
the presence of pyridine and dichloromethane to attain
the target compounds (10a-j) in 60%-80% yields (Scheme 1,
Table 1).
Acefylline derivatives are known to possess many
other utilities in literature such as cardiac stimulant,[17]
anti-asthmatic,[18] anti-bacterial[19] adenosine antagonist
receptor,[20] and as anti-cancer agents.[21] Literature reveals
that introduction of bulky alkyl group in acefylline may
lead to compounds with potential anti-cancer activity.[22]
Similarly, oxadiazole-2-thiols have attained much
interest in pharmaceutical industry due to their remark-
able biological activities including anti-inflammatory,
anti-edema, anti-microbial, anti-convulsant, analgesic,
anti-hepatitis B, anti-tumor, anti-HIV, anti-malarial, anti-
tuberculosis, as well as anti-parkinson activities.[23–25]
A
number of oxadiazole based compounds (Figure 2) have
been discovered and are being used as drugs including
Ataluren 2A for cystic fibrosis treatment, an anti-cancer
drug Zibotentan 2B and an anti-retroviral drug Raltegravir
2C used for HIV infection treatment.[26]
2.2 | Spectral interpretation of
representative molecule (10 g)
N-(4-Chlorophenyl)-2-(5-((1,3-dimethyl-2,6-dioxo-2,3-
dihydro-1H-purin-7(6H)-yl)methyl)-1,3,4-oxadiazol-2-ylthio)
acetamide (10g) was synthesized as a yellow amorphous
In continuation of our research towards the develop-
ment of novel synthetic routes/synthesis[27] of biologically
O
O
O
H
N
O
N
N
N
N
N
Cl
N
N
N
N
N
S
N
N
O
N
O
N
O
Anti-cancer 1B
Anti-asthmatic 1A
1C
Phosphodiasterase Inhibitor
FIGURE 1 Bioactive theophylline compounds
N
O
O
N
F
OH
O
OH
H
O
O
N
N
N
H
F
O
O
N S
N
N
N
N
N
N
N
O
O
Raltegravir 2C
Ataluren 2A
Zebotentan 2B
FIGURE 2 Structures of oxadiazole containing compounds in late stage clinical development A and B, or launched drug (2C)

SHAHZADI ET AL.
3
NHNH2
O
O
OH
O
O
N
O
N
O
N
O
N
N
N
N
N2H4 . H2O
N
N
N
N
H2SO4, MeOH
reflux, 6 h
N
r.t, overnight
O
O
O
5, 98%
3
4, 68%
10a, R = C6H5
10b, R = 3-Cl-C6H4
10c
reflux
CS2
KOH
, R = 2-F-C6H4
10d, R = 2,4-Me2-C6H3
10e
6 h
N
N
N
, R = 3,4-Cl2-C6H3
10f, R = 2-Cl-C6H4
10g, R = 4-Cl-C6H4
N
H
N
O
O
N
DCM/Py
R
SH
O
S
O
r.t, 24-48 h
N
N
N
N
O
N
10h
, R = 4-F-C6H4
10i, R = 2-OMe-C6H4
H
N
R
N
Br
O
N
O
10j
, RNH =
O
9a-j
10a-j, 60-80%
6, 62%
N
SCHEME 1 Reaction strategy for the synthesis of compounds (10a-j)
solid. Structure of the molecule was verified by molecular
signals two at δ 119 and two at δ 131. The substituted phe-
nyl carbons showed their signals at δ 133 for C Cl and δ
140 for C N (Figure. 3B) Spectroscopic studies confirmed
the structure of 10g and named as N-(3-chlorophenyl)-
2-(5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-
yl)methyl)-1,3,4-oxadiazol-2-ylthio)acetamide. Other syn-
thetic derivatives were structurally characterized by
similar approach.
ion peak in EI-MS spectrum at m/z 461, further it was
1
confirmed by H-, 13C NMR and IR spectroscopy. In IR
spectrum, absorption bands were observed at v 3351 (str.,
N H); 1600-1650 (str., CO-xanthene); 1700 (str., CO-
amide); 1453 (str., C C); 1545 (str., C N); 1331 (str.,
C N); 1473 (str., CH2); 1218 (C O C); 801 (C H aro-
matic out of plane); 600-700 (C S) cm−1. In 1H-NMR
spectrum at δ 8.22, the most downfield signal for N H of
the purine heterocyclic ring was observed. 2H-1 of methy-
lene group resonated at δ 5.79 and 3H-3 whereas 3H-4 of
purine ring resonated at 3.30 as singlet. Two signals of the
C and N of acetamide group appeared at δ 4.28 and δ
10.54. Aromatic protons H-20 and H-60 reverberated as a
doublet at δ 7.33 (J = 8 Hz) while H-30 and H-50resonated
as doublet at δ 7.13 exhibited coupling (J = 6 Hz) with
H-20 and H-60, respectively (Figure. 3A).
The carbon scaffold of this compound was also con-
firmed by 13C NMR. All the 18 carbons depicted their sig-
nals in the spectrum and the downfield signals of
quaternary carbon at δ 163.5 and 164 confirmed the for-
mation of 1,3,4 oxadiazole ring. The other two signals at δ
152 and 155 belonged to 2C O of xanthene core. One
methine signal of theophylline ring appeared at δ 144 and
the two signals at δ 107 and 148 confirmed the C C in the-
ophylline ring. Two resonance signals confirmed the C and
N-substituted acetamide groups, one at δ 166 for carbonyl
group and the other at δ 38.5 for methylene group. One sig-
nal in the spectrum at δ 43 was characteristic for methy-
lene linker in the molecule. The 4-chlorophenyl ring joined
with acetamide group was confirmed having four methine
2.3 | Anti-cancer activity
All the compounds 10a-j were assessed for their anti-
cancer activities for human liver cells (Huh7) (Table 1).
The better activity was displayed by compound 10g hav-
ing cell viability 53.58 1.28 which was lower than the
other derivatives as well as acefylline itself (cell viabil-
ity = 86.32 11.75). The molecules 10j, 10e, 10i, and 10a
also showed significant results but were less as compared
to 10g against cancer cell line (cell viability = 65.24
2.72, 79.93 3.80, 83.27 5.45, and 87.34 2.03
respectively). All other compounds of this series were
found to be inactive against cancer cell lines with rela-
tively high values of cell viability. Compound 10g was
further screened for its anti-cancer activity by applying
different concentrations (0.9-500 μg). Best results were
observed with 500 μg concentration (cell viabil-
ity = 28.65 2.46) as compared to 0.9 μg concentration
(cell viability = 63.67 1.75) (Table 2). A structure-
activity relationship is explained on the basis of distinc-
tive cytotoxic behavior of all the synthesized compounds.

4
SHAHZADI ET AL.
TABLE 1
Anti-cancer activity (cell viability), cytotoxic potential through thrombolytic and hemolytic activity of N-phenylacetamide
derivatives of acefylline (10a-j)
Compounds
10a
R/Amine
aCell viability Huh7 (liver)
% Hemolysis
% Thrombolysis
81.91 4.05
0.49
57.5
10b
125.95 2.78
0.35
60
10c
10d
130.86 2.53
0.10
11.3
60
80
149.50
4
10e
79.93 3.8
28.5
64
10f
114.81 2.93
3.59
0.71
50
57
10g
53.58 1.28
10h
N.D
5.78
55

SHAHZADI ET AL.
5
TABLE 1 (Continued)
Compounds
10i
R/Amine
aCell viability Huh7 (liver)
% Hemolysis
% Thrombolysis
83.27 5.45
8.98
58
10j
Morpholine
87.61 2.02
86.32 11.75
20.88
44.08
90
Acefylline
6.85
Standards
DMSO
100
0
0.01
95.9
0.57
86
(−ve control)
ABTS
(+ve control)
Abbreviation: N.D, Not Determined.
aCell viability: (Mean SD, in triplicate with 100 μg/mL concentration).
(d, J = 8 Hz)
10.54
H
7.33
4.28
H
N
N
H
H
(d, J = 6 Hz)
H
7.13
119
5.79
H
163.5
43
140
N
N
164
S
166
38.5
2''1'
N
131
133
Cl
H
O
2'
H
9
3'
1''
10
N
O
H
O
27.9
155
107
H
N
1
N
3.30 4
H
S
119
O
O
11
152
N
4' Cl
7
N
144
131
5
O
2
6'
5'
H
H
8.22
N
6
O
N
148
7.33
H
N
8
O
N
(d, J = 8 Hz)
7.13
(d, J = 6 Hz)
H
29.6
3
H
H
3.30
(A)
(B)
FIGURE 3 1H NMR (A), 13C NMR (B) Chemical shifts of compound 10g
TABLE 2
different concentrations
Anti-cancer activity of compound 10g by using
2.4 | Structure-activity relationship
Structure-activity relationship (SAR) regarding anti-
cancer activity can be explained on the basis of substitu-
ents present on phenyl ring. An insight into the struc-
tures and activities of the compounds under investigation
reveals that the incorporation of substituents with elec-
tron donating effect generally enhanced the anti-cancer
activity, for example, activity of compound 10a having
unsubstituted phenyl ring (cell viability = 87.61 2.03)
was comparable to acefylline (cell viability = 86.32
11.75) while compounds 10j (cell viability 65.2 2.72)
and 10i (cell viability = 83.27 5.45) exhibited greater
anticancer activities than 10a due to the presence of mor-
pholine ring (instead of phenyl ring) and methoxy group
at ortho position of phenyl ring, respectively. The pres-
ence of chloro substituent at different positions of phenyl
group showed interesting results such as compound 10g
(cell viability 53.58 1.28) bearing chloro group at para
position of phenyl ring showed less cell viability and
found to be the most active anti-cancer agent among
all the synthesized compounds while the activity of
S.No
Concentrations (μg)
aCell viability SD
28.65 2.01
32.62 1.17
41.14 1.41
42.63 2.89
47.45 4.55
50.48 3.84
52.75 4.03
57.83 6.33
60.09 3.35
63.67 1.75
1
500
2
250
3
125
4
62.5
31.25
15.6
7.8
5
6
7
8
3.9
9
1.9
10
11
0.9
Control
100
0
aCell viability: (Mean SD, n = 3 in triplicate).
compound 10e (79.93 3.8) decreased when phenyl ring
was substituted with chloro group at meta as well as
para position. Similarly, compounds 10f and 10b bearing

6
SHAHZADI ET AL.
chloro group at ortho position and at meta position of
phenyl ring even showed no toxicity towards cancer cell
lines with high cell viability values (114.81 2.93) and
(125.95 2.78), respectively. It shows that the presence
of chloro substituent at para positon of phenyl ring as
compared to ortho and meta may have greater interaction
with the cancer cell lines and is responsible for its greater
activity. Compounds 10c with flouro substituent at ortho
position and 10d having methyl substituent at ortho
and para position of phenyl ring showed least activities
with very high cell viability values (130.86 4) and
(149.5 4) respectively.
negative control DMSO (0.57%) and acefylline (6.85%).
This compound dissolved almost 90% clots by releasing
plasmin enzyme which dissolved cell membrane cross
links leading to lysis of thrombs. All the other synthe-
sized derivatives showed more than 50% thrombolysis,
10a (60%), 10b (60%), 10c (60%), 10d (80%) and 10e
(64%), 10f (50%), 10g (57%), 10h (55%), and 10i (58%).
The compounds (10j and 10d) with high clot dissolving
property could be employed for cardio related drug
designing.
Same trend was observed for hemolytic and thrombo-
lytic activities; compound 10j (hemolysis 20.88% and
thrombolysis 90%) with morpholine ring having tertiary
nitrogen is found as the most active compound while
compound 10e with chloro substituent at meta and para
positions (hemolysis 28.5% and thrombolysis 64%) and
10d having methyl substituent at ortho and para posi-
tions of phenyl ring (hemolysis 11.3% and thrombolysis
80%) showed greater activities. All other synthesized
derivatives having unsubstituted or mono substituted
phenyl ring as a substituent exhibited lesser activities as
in case of 10c (bearing flouro group at ortho position)
showed 0.1% hemolysis.
2.7 | In-silico modelling of
representative compound (10g)
The in-silico modelling was performed to investigate
the potential mechanism of action of compounds under
study that may be responsible for their anti-cancer activi-
ties. The PASS prediction indicated the STAT3 (Pa
approximately 0.75) as potential anti-cancer therapeutic
target for compound 10g. STAT3 (Signal transducer and
activator of transcription 3) is a crucial transcription fac-
tor that relay the upstream signals from various cytokines
and growth factors to regulate the cell proliferation, dif-
ferentiation and survival. The inhibition of STAT3 dimer-
ization is an attractive anti-cancer strategy to limit its
aberrant activation in development, progression and
maintenance of variety of malignancies.[33] There has
been evidence that suggests the theophylline in modula-
tion of STAT3 signaling.[34–36] In this study, compound
10g was found to dock at STAT3 hotspot with −8.72
Kcal/mol conformational energy as a function of its
inhibitory potential. The compound 10g interacted with
STAT3 hotspot with superior binding affinity as it signifi-
cantly exceeded the −5.5 Kcal/mol conformational
energy of acefylline (Table 3). The docking studies simu-
late the spatial configuration of ligand—protein complex-
ation and their interactions to explain the ligand's
inhibitory potential.
Although, acefylline oriented itself into the configura-
tion that it interacted with conserved residues for com-
pound 10g interaction at the hotspot of STAT3 (Figure 4).
However, its conformation was unable to attain the bind-
ing affinity comparable to compounds 10g. In addition to
share the contact with conserved residues for acefylline
activity, Compound 10g was found to interact with
additional residues of STAT3 hotspot that, owing to its
structural diversity, may justify its superior inhibitory
potential.
2.5 | Hemolytic activity
All the compounds (10a-j) were also tested for hemolytic
activity. Percentage hemolysis was calculated and the
results are shown in Table 1. All the synthesized com-
pounds exhibited moderate to low toxicity towards RBCs
(red blood cells). Highest toxicity was observed for the
derivative 10e (28.5%) which showed maximum binding
with hemoglobin by rupturing the RBC's cell membrane
but it was even low as compared to acefylline itself having
44.08% hemolytic activity. Compound 10j (20.8%) was
found to be the second most toxic compound among the
list of compounds. Lower toxicity was exhibited by com-
pounds 10a (4.1%), 10d (11.3%), 10f (3.5%), 10h (5.9%) and
10i (8.9%) respectively. Least toxicity was observed for mol-
ecules, 10c (0.1%), 10b (0.3%), and 10g (0.7%) as compared
to standard 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid (ABTS) having 95.9% of hemolysis.
2.6 | Thrombolytic activity
All the synthesized compounds were analyzed for their
thrombolytic activity. The analysis of structure-activity
relationship revealed that the highest activity was
observed for the compound 10j (90%) as compared to
Acefylline formed the H-bond with LYS351 and ionic
interaction with LYS352 of STAT3 hotspot and its com-
plexation was further stabilized by the additional interac-
tions with TYR306, ARG126, ASP311, and GLU72 at the

SHAHZADI ET AL.
7
interface of STAT3 complex (Figure 5). It is noteworthy,
that compound 10g inhibited the STAT3 hotspot by inter-
acting with its conserved residues (ie, LYS351 and
LYS352) for acefylline. It significantly inhibited the
STAT3 hotspot by H-bond, π-alkyl with LYS351 and ionic
interaction with LYS351 and LYS352. Consistently, the
TABLE 3
Analysis of parameters in induced fit docking simulation
Binding score
Target Ligands
(ΔG) Kcal/mol Interacting residues
−5.5 LYS351, LYS352, TYR306,
ASP311, ARG126, GLU72
LYS351, LYS352, TYR353, TYR306, ARG126, H-bonding, π—Cation, π—Anion, π—π
ASP311, GLU72, TYR119, TYR122, Stacked, π—Alkyl
Type of interaction
STAT3 Acefylline
H-bonding, π—Cation, π—Anion, Attractive
Charge, Salt bridge, π—Alkyl
Compound 10g −8.72
FIGURE 4 Conformational analysis of acefylline and compound 10g at the hotspot of STAT3. Simulated best binding mode of
acefylline A, and compound 10g B, interaction in three dimensional (3D) space of binding pocket
FIGURE 5 Acefylline and compound 10g interactions with residues at the hotspot of STAT-3. Acefylline A, and compound 10g B, in
two-dimensional (2D) space to visualize their interactions with residues shown as balls colored by type of interactions

8
SHAHZADI ET AL.
compound 10g disrupted the complexation of STAT3
with key residues at the interface. Moreover, the struc-
tural diversity allowed the compound 10g to further
inhibit the complexation of STAT-3 by additional π-π
stacked interaction with TYR353 at its hotspot that may
further justify its superior inhibitory potential and anti-
cancer property as compared to acefylline.
4.2 | Synthesis of methyl
2-(1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-
purin-7(6H)-yl) acetate (4)
Theophylline 7-acetate 4 was synthesized by Fischer
esterification method. Theophylline 7-acetic acid
3 (0.021 mol) was dissolved in methanol (50 mL) and
temperature was dropped to 0ꢀC, catalytic amount of
H2SO4 (1.5 mL) was added and heated under reflux for
6 hours. The reaction mixture was cooled and product
was extracted with ethyl acetate (50 mL × 3). The col-
lected organic layer was dried over by using anhydrous
Na2SO4. The solvent was removed by rotary evaporator
and crude product was obtained which was purified by
column chromatography. White powder; Yield: 72%; mp
275ꢀC; IR: v 3291 (Ar-H); 1746 (CO-ester); 1600-1650
(CO-xanthene); 1551 (C C); 1545 (C N); 1473 (CH2);
3 | CONCLUSION
In this paper, synthesis of series of novel N-
phenylacetamide derivatives of oxadiazole of theophylline
7-acetic acid (10a-j) and their evaluation for anti-cancer,
hemolytic and thrombolytic activities are reported. Com-
pound 10g showed significant anticancer activity even at
very low concentration (0.9 μg) cell viability value was
63.67 1.75, it was then in-silico studied. The in-silico
study of molecule 10g was in good agreement with the
anticancer activity results. The cytotoxic profile of these
compounds (10a-j) was also tested via hemolytic and
thrombolytic activities and the resulting data indicates
low toxicity and high clot lysis activities of all molecules.
Compounds 10c, 10b, and 10g exhibited least hemolytic
activities (0.1%, 0.3%, and 0.7%). The clot lysis evalua-
tions were also highly remarkable presenting 10d (80%
clot lysis) and 10j (90% clot lysis) as most potent throm-
bolytic agents. Based on our structure based activity find-
ings, further modifications on oxadiazole derivatives of
acefylline can be done in order to develop potent and
highly selective thrombolytic, anti-cancer, and hemolytic
agents.
1
1218 (C O C). H-NMR (400 MHz, DMSO-d6, δ/ppm):
3.18, 3.30 (s, 6H, 2NCH3 CO), 3.68 (s, OCH3), 5.79 (s, 2H,
NCH2), 8.22 (s, 1H, N CH). 13C NMR (100 MHz, DMSO-
d6, δ/ppm): 27.9 (CH3), 29.6 (CH3), 43, (NCH2), 51.6
(OCH3), 107, 123, 131, 144, 148, (Ar-C), 152, 155 (2C O),
166.2 (COO).
4.3 | Synthesis of 2-(1,3-dimethyl-
2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)
acetohydrazide (5)
Theophylline 7-acetate 4 (0.0079 mol) was dissolved in
methanol (50 mL). Dropped the temperature to 0ꢀC,
hydrazine monohydrate (2 mL) was added and stirred
overnight. Solvent was evaporated and solid pure acet-
ohydrazide of acefylline 5 was obtained. Light grey pow-
der; Yield: 90%; mp 320ꢀC; IR: v 3300 (N H); 1696 (CO-
amides); 1600-1650 (CO-xanthene); 1551 (C C); 1545
(C N); 1473 (CH2). 1H-NMR (400 MHz, DMSO-d6,
δ/ppm): 3.17, 3.38 (s, 6H, 2NCH3 CO), 4.22 (d, NH2), 5.79
(s, 2H, NCH2), 8.22 (s, 1H, N CH), 9.08 (d, NH). 13C
NMR (100 MHz, DMSO-d6, δ/ppm): 27.9 (CH3), 29.6
(CH3), 43, (NCH2), 107, 123, 131, 144, 148, (Ar-C),
151, 154 (2C O).
4 | EXPERIMENTAL
4.1 | Materials and methods
The reagents used for the research work were purchased
from E. Merk Germany. For drying of organic phases,
anhydrous Na2SO4 was used. IR spectra (ν, cm−1) were
recorded in KBr pellets on Bruker FTIR spectrometer.
NMR spectra were obtained on a Bruker Spectrometer
model AV-400 at 400 Mega Hertz The solvent peak was
calibrated at 2.50 ppm. Chemical shifts (δ) were mea-
sured in ppm with most commonly used solvent DMSO.
Abbreviations used are s for singlet, d for doublet, t for
triplet, dd for doublet of doublet, and m for multiplet.
The J values (coupling constant) are given in Hertz. Melt-
ing points were found in glass capillaries by using Gal-
lenkamp equipment. TLC performed on pre-coated silica
gel 60 F254 plates using a mixture of DCM and methanol.
For detection of spots on TLC plates, UV light was used.
4.4 | Synthesis of 7-((5-mercapto-
1,3,4-oxadiazol-2-yl)methyl)-1,3-dimethyl-
1H-purine-2,6(3H,7H)-dione (6)
A mixture of acetohydrazide 5 (0.00317 mol) and potas-
sium hydroxide (0.00317 mol) were dissolved in absolute
ethanol (50 mL), carbon disulfide (0.64 mL) was added
and reaction mixture was allowed to heat under reflux
for 6 hours. Through TLC completion of reaction was

SHAHZADI ET AL.
9
monitored and after completion reaction mixture was
cooled and excess chilled water was added, acidified with
dilute hydrochloric acid (10%) and precipitates of
1,3,4-oxadiazole derivative of acefylline 6 were formed
which were filtered off and purified by recrystallization
method. Light brown powder; Yield: 71%; mp 254ꢀC; IR:
v 3291 (Ar-H); 1600-1650 (CO-xanthene); 1551 (C C);
1545 (C N); 1473 (CH2); 1218 (C O C), 600-700 (C S).
1H-NMR (400 MHz, DMSO-d6, δ/ppm): 3.0 (s, SH), 3.18,
3.30 (s, 6H, 2NCH3 CO), 5.79 (s, 2H, NCH2), 8.22 (s, 1H,
N CH). 13C NMR (100 MHz, DMSO-d6, δ/ppm): 27.9
(CH3), 29.6 (CH3), 43, (NCH2), 107, 123, 131, 144,
148, (Ar-C), 152, 155 (2C O), 163.5164 (C O C).
δ/ppm): 27.9 (CH3), 29.6 (CH3), 38.5 (SCH2), 43, (NCH2),
106.45, 121.6, 121.6, 128, 128.9, 128.9, 140, 144, 148, (Ar-
C), 151.18, 154.27 (2C O), 163.5164 (C O C), 165.2
(CONH). MS m/z (ES+) 427.1066 (M+) (100%). Anal.
Calcd. For C18H17N7O4S: C, 50.58; H, 4.01; N, 22.94;
Found; C, 50.60; H, 4.31; N, 22.98.
4.6.2 | N-(3-Chlorophenyl)-
2-(5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-
1H-purin-7(6H)-yl)methyl)-1,3,4-oxadiazol-
2-ylthio)acetamide (10b)
Light grey amorphous solid; Yield: 66%; mp 195ꢀC; IR
(KBr):
υ
3300 (N H); 1600-1650 (CO-xanthene);
4.5 | Preparation of 2-bromo-N-
(un/substituted-phenyl) acetamides (10a-j)
1660-1700 (CO-amide) 1549 (C C); 1545 (C N); 1309
(C N); 1473 (CH2); 1218 (C O C); 750 (C H aromatic
out of plane); 600-700 (C S). 1H-NMR (400 MHz,
DMSO-d6, δ/ppm): 3.17, 3.42 (s, 6H, NCH3-3,4), 4.28 (s,
Reactions of equimolar (0.012 mol) quantities of various
aryl amines 7a-j with 2-bromo acetyl bromides 8 were
carried out in the presence of DCM and pyridine with
2 hours stirring to synthesize various 2-bromo-N-
substituted-phenyl acetamides 9a-j.
0
0
2H, SCH2-100), 5.83 (s, 2H, NCH2-1), 7.12 (d, J4 ,5 = 6 Hz,
0
0
0
0
1H, H-40), 7.39 (dd, J5 ,4 = J5 ,6 = 6.4 Hz, 1H, H-50), 7.39
(d, J5 ,6 = 6.4 Hz, 1H, H-50), 7.75 (s,1H, H-20), 8.72 (s, 1H,
0
’
N CH), 10.57 (s, 1H, CONH). 13C NMR (100 MHz,
DMSO-d6, δ/ppm): 27.96 (CH3), 29.94 (CH3), 37.27
(SCH2), 41.14, (NCH2), 106.46, 117.99, 119.07, 123.85,
131, 133.62, 140.46, 143.67, 148.73, (Ar-C), 151.40, 154.74
(2C O), 163.67, 164.43 (C O C), 165.48 (CONH). MS
m/z (ES+) 461.0682 (M+) (100%). Anal. Calcd. For
C18H16ClN7O4S: C, 46.81; H, 3.49; N, 21.23; Found; C,
46.87; H, 3.82; N, 21.28.
4.6 | Synthesis of N-phenylacetamide
derivatives of oxadiazole of theophylline
7-acetic acid (10a-j)
Oxadiazole analogue 6 (68.0 mmol) along with pyridine
(2.3 mmol) were dissolved in dichloromethane and
stirred the reaction mixture for 15 minutes. After that 2-
bromo-N-phenylacetamide 9a-j (3.0 mmol) were added
and allowed to stir the mixture at room temperature for
24-48 hours. After completion of reaction as monitored
by TLC, n-hexane was added to it. The resulting precipi-
tates of thio N-phenylacetamide derivatives of acefylline
10a-j were collected and recrystallized from ethanol.
4.6.3 | 2-(5-((1,3-Dimethyl-2,6-dioxo-
2,3-dihydro-1H-purin-7(6H)-yl)methyl)-
1,3,4-oxadiazol-2-ylthio)-N-(2-fluorophenyl)
acetamide (10c)
Off white amorphous solid; Yield: 69%; mp 189ꢀC; IR
(KBr):
υ
3364 (N H); 1600-1650 (CO-xanthene);
1660-1724 (CO-amide) 1551 (C C); 1553 (C N); 1473
(CH2); 1328 (C N); 1218 (C O C); 750 (C H aromatic
out of plane); 600-700 (C S). 1H-NMR (400 MHz,
DMSO-d6, δ/ppm): 3.18, 3.42 (s, 6H, NCH3-3, 4), 4.34 (s,
2H, SCH2-100), 5.84 (s, 2H, NCH2-1), 7.15-7.87 (m, 4H, Ar-
H), 8.23 (s, 1H, N CH), 10.18 (s, 1H, CONH). 13C NMR
(100 MHz, DMSO-d6, δ/ppm): 27.96 (CH3), 29.94 (CH3),
36.81 (SCH2), 41.14, (NCH2), 106.47, 115.90, 116.06,
124.17, 124.85, 126.04, 143.67, 148.73, 151.42, (Ar-C),
152.86, 154.75 (2C O), 163.67, 164.44 (C O C), 165.71
(CON). MS m/z (ES+) 445.0989 (M+) (100%). Anal. Calcd.
For C18H16FN7O4S: C, 48.54; H, 3.12; N, 22.01; Found; C,
48.9; H, 3.05; N, 22.04.
4.6.1 | 2-(5-((1,3-Dimethyl-2,6-dioxo-
2,3-dihydro-s-2-ylthio)-N-
phenylacetamide (10a)
Light yellow powder; Yield: 73%; mp 215ꢀC; IR (KBr): υ
3290 (N H); 1600-1650 (CO-xanthene); 1662 (CO-amide)
1597 (C C); 1545 (C N); 1309 (C N); 1473 (CH2); 1218
(C O C); 750 (C H aromatic out of plane); 600-700
1
(C S). H-NMR (400 MHz, DMSO-d6, δ/ppm): 3.14, 3.42
(s, 6H, NCH3-3,4), 4.28 (s, 2H, SCH2-100), 5.79 (s, 2H,
NCH2-1), 7.19-7.61 (m, 5H, Ar-H), 8.22 (s, 1H, N CH),
10.54 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6,

10
SHAHZADI ET AL.
4.6.4 | 2-(5-((1,3-Dimethyl-2,6-dioxo-
2,3-dihydro-1H-purin-7(6H)-yl)methyl)-
1,3,4-oxadiazol-2-ylthio)-N-
1660-1706 (CO-amide) 1551 (C C); 1531 (C N); 1473
(CH2); 1347 (C N); 1218 (C O C); 750 (C H aromatic
out of plane); 600-700 (C S). 1H-NMR (400 MHz,
DMSO-d6, δ/ppm): 3.18, 3.42 (s, 6H, NCH3-3,4), 4.35 (s,
2H, SCH2-100), 5.85 (s, 2H, NCH2-1), 7.20 (s, 1H, H-40),7.32
(2,4-dimethylphenyl)acetamide (10d)
0
0
Off white amorphous solid; Yield: 72%; mp 191ꢀC; IR
(s, 1H, H-50), 7.48 (d, J3 ,4 = 8 Hz, 1H, H-30), 7.70 (d,
0
0
(KBr):
υ
3284 (N H); 1600-1650 (CO-xanthene);
J6 ,5 = 4 Hz, 1H, H-60), 8.23 (s, 1H, N CH), 9.93 (s, 1H,
CONH). 13C NMR (100 MHz, DMSO-d6, δ/ppm): 27.96
(CH3), 29.94 (CH3), 36.72 (SCH2), 41.15, (NCH2), 106.46,
126.10, 126.58, 127.01, 127.93, 129.99, 134.85, 143.67,
148.72, (Ar-C), 151.41, 154.73 (2C O), 163.71, 164.40
(C O C), 165.80 (CONH). MS m/z (ES+) 461.068 (M+)
(100%). Anal. Calcd. For C18H16ClN7O4S: C, 46.81; H,
3.49; N, 21.23; Found; C, 46.81; H, 3.65; N, 21.38.
1660-1700 (CO-amide) 1551 (C C); 1545 s(C N); 1473
(CH2); 1331 (C N); 1218 (C O C); 750 (C H aromatic
out of plane); 600-700 (C S). 1H-NMR (400 MHz,
DMSO-d6, δ/ppm): 2.13, 2.24 (6H, Ar-CH3), 3.19, 3.43 (s,
6H, NCH3-3,4), 4.28 (s, 2H, SCH2-100), 5.85 (s, 2H, NCH2-
0
0
1), 6.96 (d, J5 ,6 = 6 Hz, 1H, H-50), 7.01 (s, 1H, H-30), 7.21
0
0
(d, J6 ,5 = 6.4 Hz, 1H, H-60), 8.24 (s, 1H, N CH), 9.63 (s,
1H, CONH). 13C NMR (100 MHz, DMSO-d6, δ/ppm):
18.10 (CH3), 20.92 (CH3), 27.96 (CH3), 29.95 (CH3), 36.75
(SCH2), 41.16, (NCH2), 106.47, 125.22, 126.92, 131.30,
132.02, 133.63, 135.06, 143.69, 148.74, (Ar-C), 151.43,
154.74 (2C O), 163.66, 164.55 (C O C), 165.21
(CONH). MS m/z (ES+) 455.1385 (M+) (100%). Anal.
Calcd. For C20H21N7O4S: C, 52.74; H, 4.55; N, 21.58;
Found; C, 52.79; H, 4.85; N, 21.62.
4.6.7 | N-(4-Chlorophenyl)-
2-(5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-
1H-purin-7(6H)-yl)methyl)-1,3,4-oxadiazol-
2-ylthio)acetamide (10g)
Light yellow amorphous solid; Yield: 61%; mp 220ꢀC;
IR (KBr): υ 3351 (N H); 1600-1650 (CO-xanthene);
1700 (CO-amide) 1453 (C C); 1545 (C N); 1331
(C N); 1473 (CH2); 1218 (C O C); 801 (C H aro-
4.6.5 | N-(3,4-Dichlorophenyl)-
2-(5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-
1H-purin-7(6H)-yl)methyl)-1,3,4-oxadiazol-
2-ylthio)acetamide (10e)
1
matic out of plane); 600-700 (C S). H-NMR (400 MHz,
DMSO-d6, δ/ppm): 3.19, 3.35 (s, 6H, 2NCH3-3,4), 4.28
(s, 2H, SCH2-100), 5.79 (s, 2H, NCH2-1), 7.13 (d,
0
0
0
0
0
0
0
0
J3 ,2 = J5 ,6 6 Hz, 2H, H-30and H-50), 7.39 (d, J2 ,3 = J6 ,5
Cream solid; Yield: 79%; mp 210ꢀC; IR (KBr): υ 3322
(N H); 1600-1650 (CO-xanthene); 1660-1720 (CO-amide)
1551 (C C); 1532 (C N); 1473 (CH2); 1314 (C N); 1218
(C O C); 750 (C H aromatic out of plane); 600-700
(C S). 1H-NMR (400 MHz, DMSO-d6, δ/ppm): 3.17, 3.41 (s,
6H, NCH3-3,4), 4.28 (s, 2H, SCH2-100), 5.83 (s, 2H, NCH2-1),
8 Hz, 2H, H-20and H-60), 8.22 (s, 1H, N CH), 10.54 (s,
1H, CONH). 13C NMR (100 MHz, DMSO-d6, δ/ppm):
27.9 (CH3), 29.6 (CH3), 38.5 (SCH2), 43, (NCH2), 106.43,
118.6, 119.7, 123, 131, 133, 140, 144, 148, (Ar-C), 151.2,
154.5 (2C O), 163.5164 (C O C), 165.27 (CONH). MS
m/z (ES+) 461.068 (M+) (100%). Anal. Calcd. For
C18H16ClN7O4S: C, 46.81; H, 3.49; N, 21.23; Found; C,
46.81; H, 3.65; N, 21.38.
7.43 (d, J5 ,6 = 8 Hz, 1H, H-50), 7.54 (d, J6 ,5 = 8 Hz, 1H,
H-60), 7.91 (s, 1H, H-20), 8.21 (s, 1H, N CH), 10.66 (s, 1H,
CONH). 13C NMR (100 MHz, DMSO-d6, δ/ppm): 27.94
(CH3), 29.91 (CH3), 37.15 (SCH2), 41.11, (NCH2), 106.42,
119.58, 120.75, 125.62, 131.20, 131.56, 139.07, 143.64,
148.69, (Ar-C), 151.36, 154.70 (2C O), 163.66, 164.39
(C O C), 165.63 (CONH). MS m/z (ES+) 495.0323 (M+)
(100%). Anal. Calcd. For C18H15Cl2N7O4S: C, 43.56; H,
3.05; N, 19.75; Found; C, 43.69; H, 3.35; N, 19.85.
0
0
0
0
4.6.8 | 2-(5-((1,3-Dimethyl-2,6-dioxo-
2,3-dihydro-1H-purin-7(6H)-yl)methyl)-
1,3,4-oxadiazol-2-ylthio)-N-(4-fluorophenyl)
acetamide. (10h)
Grey amorphous; Yield: 64%; mp 205ꢀC; IR (KBr): υ 3364
(N H); 1600-1650 (CO-xanthene); 1724 (CO-amide) 1553
(C C); 1545 (C N); 1328 (C N); 1473 (CH2); 1218
(C O C); 772 (C H aromatic out of plane); 600-700
4.6.6 | N-(2-Chlorophenyl)-
2-(5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-
1H-purin-7(6H)-yl)methyl)-1,3,4-oxadiazol-
2-ylthio)acetamide (10f)
1
(C S). H-NMR (400 MHz, DMSO-d6, δ/ppm): 3.18, 3.34
(s, 6H, NCH3-3,4), 4.28 (s, 2H, SCH2-100), 5.79 (s, 2H,
0
0
0
0
NCH2-1), 7.22 (d, J3 ,2 = J5 ,6 6 Hz, 2H, H-30 and H-50),
0
0
0
0
Light grey amorphous solid; Yield: 67%; mp 190ꢀC;
IR (KBr): υ 3275 (N H); 1600-1650 (CO-xanthene);
7.60 (d, J2 ,3 = J6 ,5 8 Hz, 2H, H-20 and H-60), 8.22 (s, 1H,
N CH), 10.54 (s, 1H, CONH). 13C NMR (100 MHz,

SHAHZADI ET AL.
11
DMSO-d6, δ/ppm): 27.9 (CH3), 29.6 (CH3), 38.5 (SCH2),
43, (NCH2), 106.47, 118.6, 119.7, 123, 131, 133, 140,
144, 148, (Ar-C), 151.17, 154.20 (2C O), 163.5164
(C O C), 165.42 (CONH). MS m/z (ES+) 445.0989 (M+)
(100%). Anal. Calcd. For C18H16FN7O4S: C, 48.54; H,
3.01; N, 22.01; Found; C, 48.9; H, 3.02; N, 22.04.
C17H21N7O5S: C, 44.03; H, 4.62; N, 25.67; Found; C,
44.33; H, 4.77; N, 25.54.
4.7 | Cell viability assay
4.7.1 | Cell culture and treatment
4.6.9 | 2-(5-((1,3-Dimethyl-2,6-dioxo-
2,3-dihydro-1H-purin-7(6H)-yl)methyl)-
1,3,4-oxadiazol-2-ylthio)-N-
The prepared analogues were analyzed against Huh7 can-
cer cell lines grown in DMEM (Dulbecco's Modified
Eagle's Medium) supplemented with penicillin (100 units/
mL) and streptomycin (100 μg/mL along with10% FBS
(fatal bovine serum) with 5% carbon dioxide (CO2) in
humidified air at 37ꢀC. Compounds were dissolved in
DMSO and cells were treated with a final DMSO concen-
tration of 0.05%. Dimethyl sulfoxide treated cells were
used as control in all the experiments.
(2-methoxyphenyl)acetamide (10i)
Light grey amorphous solid; Yield: 77%; mp 175ꢀC;
IR (KBr): υ 3275 (N H); 1600-1650 (CO-xanthene);
1660-1706 (CO-amide) 1551 (C C); 1531 (C N); 1473
(CH2); 1347 (C N); 1218 (C O C); 750 (C H aro-
1
matic out of plane); 600-700 (C S). H-NMR (400 MHz,
DMSO-d6, δ/ppm): 3.18, 3.43 (s, 6H, NCH3-3,4), 3.83
(Ar-OCH3) 4.34 (s, 2H, SCH2-100), 5.84 (s, 2H, NCH2-1),
4.7.2 | Determination of cell viability
0
0
0
0
6.89 (t, J5 ,4 = 8 Hz, J5 ,6 = 4 Hz, 1H, H-50), 7.05 (d,
0
0
0
0
J4 ,3 = 4 Hz, 2H, H-3,40), 7.91 (d, J6 ,5 = 8 Hz, 1H, H-
60), 8.23 (s, 1H, N=CH), 9.61 (s, 1H, CONH). 13C NMR
(100 MHz, DMSO-d6, δ/ppm): 27.96 (CH3), 29.94 (CH3),
37.07 (SCH2), 41.15, (NCH2), 56.18 (Ar-OCH3), 106.47,
111.72, 120.72, 121.88, 125.16, 127.33, 143.67, 148.73,
149.86, (Ar-C), 151.42, 154.75 (2C O), 163.67, 164.53
(C O C), 165.37 (CONH). MS m/z (ES+) 457.1218
(M+) (100%). Anal. Calcd. For C19H19N7O5S: C,
49.88; H, 4.19; N, 21.43; Found; C, 50.18; H, 4.24;
N, 21.61.
Cellular viability was evaluated by employing MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay as standard.[37] Cells were plated for over-
night at 96-well plates. With different concentrations of
compounds Huh7 cells were treated for 48 hours and cells
were further cultivated for a period of 4 hours at 37ꢀC
followed by addition of MTT reagent (500 μg/mL).
Finally, 150 μL DMSO was added to each plate shaked
well and absorbance was recorded in a Thermo Scientific
microplate reader at 490 nm. The % age of cell viability
was calculated.
4.6.10 | 2-(5-((1,3-Dimethyl-2,6-dioxo-
2,3-dihydro-1H-purin-7(6H)-yl)methyl)-
1,3,4-oxadiazol-2-ylthio)-N-(tetrahydro-2H-
pyran-4-yl)acetamide (10j)
4.8 | Hemolysis assay
Hemolytic potential of synthesized compounds was eval-
uated by following a literature method.[38] Blood sample
(3 mL) from albino rat of either sex was taken and was
centrifuged at 1000×g for 5 minutes. Red blood cells were
separated, washed thrice with chilled sterile phosphate
buffer saline (PBS) solution of pH 7.4 (5 mL). Sample
(10 mg/mL in DMSO) solution (20 μL) was mixed with
blood suspension (180 μL) and incubated at 37ꢀC for
35 minutes. The samples were again kept in ice for
5 minutes and recentrifuged for 5 minutes at 1000×g.
From each tube supernatant (100 μL) was collected and
diluted 10 times with chilled PBS (4ꢀC). DMSO was
used as negative control and ABTS (2,20-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid) as positive control.
Absorbance was observed at 576 nm and % hemolysis was
calculated by applying the formula:
Brown amorphous solid; Yield: 70%; mp 150ꢀC; Mol.
Formula: C17H21N7O5S; IR (KBr): υ 3290 (N H);
1600-1650 (CO-xanthene); 1662 (CO-amide) 1597
(C C); 1545 (C N); 1309 (C N); 1473 (CH2); 1218
(C O C); 750 (C H aromatic out of plane); 600-700
1
(C S). H-NMR (400 MHz, DMSO-d6, δ/ppm): 1.70 (t,
2H), 3.17, 3.34 (s, 6H, NCH3-3, 4), 2.9-3.65 (m, 8H,
morpholine), 4.28 (s, 2H, SCH2-100), 5.79 (s, 2H, NCH2-
1), 8.22 (s, 1H, N CH), 10.54 (s, 1H, CONH). 13C
NMR (100 MHz, DMSO-d6, δ/ppm): 27.94 (CH3), 29.65
(CH3), 38.5 (SCH2), 43, (NCH2), 122, 125 156, (Ar-C),
36.7, 36.7, 51.6, 65.2, (aliphatic cyclic), 151.45,
154.28 (2C O), 163.5164 (C O C), 165.2 (CONH). MS
m/z (ES+) 436.1279 (M+) (100%). Anal. Calcd. For

12
SHAHZADI ET AL.
Absorbance of sample−Absorbance of negative control ðDMSOÞ
Absorbance of positive control ðABTSÞ
%ageof hemolysis =
4.9 | Thrombolysis assay
The X-ray crystallized 3D structures of STAT3 (PDB ID:
5AX3, 2.984 Å resolution) was recovered from RCSB
(Research Collaboratory for Structural bioinformatics)
Protein Data Bank (PDB) (https://www.rcsb.org/). The
macromolecule was prepared and optimized to fix the
structural problems in Structure Preparation application
of MOE. Protonate 3D application was used to correct the
tautomeric and ionization states of residues. The struc-
ture was further energy minimized by Charmm27
forcefield to relieve the geometrical constraints of resi-
dues and partial charge was calculated. The binding site
was defined by Site Finder application and molecular sur-
face was created around the binding pocket. The Dock
application of MOE was used to dock the ligand into
binding site by Triangle Matcher placement with subse-
quent induced fit refinement using GBVI/WSA dG and
London dG and scoring function. Total 30 poses were
refined and the pose with lowest conformational energy
The synthesized derivatives were analyzed for thrombo-
lytic activity by prescribed method.[39] Blood sample
(1 mL) taken from rat of either sex was transferred in dif-
ferent pre-weighed eppendorf tubes and incubated for
45 minutes at 37ꢀC. Serum was eliminated completely
after clot formation and each eppendorf was again
weighed to find out the weight of clot. (Clot weight =
weight of eppendorf with clot − weight of eppendorf
alone). 100 μL Sample was added and incubated for
3 hours at 37ꢀC and observed for clot lysis. DMSO was also
added to one of the clot containing tubes which serves as
negative thrombolytic control. Fluid was separated after
incubation. Eppendorfs were again weighed and weight
difference was observed after clot lysis which was indicated
as percentage of thrombolysis. Positive control was ABTS
and negative control was DMSO.
Initial clot weight−Final clot weight
Percentage of clot lysis ð%Þ =
× 100
Initial weight of clot
4.10 | In-silico modelling
(ie, ΔG) was used to investigate the ligand conformation
and interactions within the binding site of receptor using
Accelrys discovery studio visualizer v17.2.
The test compound, with superior anti-cancer activity,
was further in-silico modelled to investigate its potential
mechanism of action. The spectrum of biological poten-
tial of this derivative was predicted by the PASS predic-
tion tool that utilizes Bayesian approach-based algorithm
to estimate therapeutic targets with 95% accuracy of pre-
diction.[40] The anti-cancer therapeutic targets with
Pa > 50% were selected as potential target and further
evaluated in molecular docking against. The inhibitory
potential of experimental compound was investigated for
these targets by the function of induced fit docking in
Molecular Operating Environment (MOE) 2015.10. The
compound was sketched in ChemDraw ultra and geomet-
rical constraints were relieved by energy minimization
using CHARMm forcefield with MMFF94x partial charge
in Accelrys discovery studio visualizer 17.2. The three-
dimensional (3D) conformers of acefylline (PubChem
CID: 69550) was fetched from PubChem database and
optimized in Accelrys discovery studio visualizer 17.2.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the facilities pro-
vided by Government College University Faisalabad,
Pakistan.
ORCID
Ameer F. Zahoor https://orcid.org/0000-0003-2127-
8848
Zohaib Raza https://orcid.org/0000-0001-7789-4530
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How to cite this article: Shahzadi I, Zahoor AF,
Rasul A, et al. Synthesis, anticancer, and
computational studies of 1, 3, 4-oxadiazole-purine
derivatives. J Heterocyclic Chem. 2020;1–13. https://
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