Synthesis, SAR elucidations and molecular docking study of newly designed isatin based oxadiazole analogs as potent inhibitors of thymidine phosphorylase

Article abstract of DOI:10.1016/j.bioorg.2018.05.011

Thymidine phosphorylase is an enzyme involved in pyrimidine salvage pathway that is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and gliostatin. It is enormously up regulated in a variety of solid tumors. Furthermore, surpassing of TP level protects tumor cells from apoptosis and helps cell survival. Thus TP is identified as a prime target for developing novel anticancer therapies. A new class of exceptionally potent isatin based oxadiazole (1–30) has been synthesized and evaluated for thymidine phosphorylase inhibitory potential. All analogs showed potent thymidine phosphorylase inhibition when compared with standard 7-Deazaxanthine, 7DX (IC₅₀ = 38.68 ± 1.12 μM). Molecular docking study was performed in order to determine the binding interaction of these newly synthesized compounds, which revealed that these synthesized compounds established stronger hydrogen bonding network with active site of residues as compare to the standard compound 7DX.

Full text of DOI:10.1016/j.bioorg.2018.05.011

Accepted Manuscript
Synthesis, SAR elucidations and molecular docking study of newly designed
Isatin based oxadiazole analogs as potent inhibitors of thymidine phosphorylase
Muhammad Tariq Javid, Fazal Rahim, Muhammad Taha, Mohsan Nawaz,
Abdul Wadood, Muhammad Ali, Ashik Mosaddik, Syed Adnan Ali Shah, Rai
Khalid Farooq
PII:
S0045-2068(18)30156-1
DOI:
https://doi.org/10.1016/j.bioorg.2018.05.011
YBIOO 2364
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 February 2018
6 May 2018
14 May 2018
Please cite this article as: M. Tariq Javid, F. Rahim, M. Taha, M. Nawaz, A. Wadood, M. Ali, A. Mosaddik, S.
Adnan Ali Shah, R. Khalid Farooq, Synthesis, SAR elucidations and molecular docking study of newly designed
Isatin based oxadiazole analogs as potent inhibitors of thymidine phosphorylase, Bioorganic Chemistry (2018), doi:
https://doi.org/10.1016/j.bioorg.2018.05.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, SAR elucidations and molecular docking study of newly designed
Isatin based oxadiazole analogs as potent inhibitors of thymidine phosphorylase
Muhammad Tariq Javida, Fazal Rahima, Muhammad Tahab, Mohsan Nawaza, Abdul
Wadoodc, Muhammad Alid, Ashik Mosaddikb, Syed Adnan Ali Shahe,f, Rai Khalid Farooqg
aDepatment of Chemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan.
bDepartment of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University,
P.O. Box1982, Dammam 3144, Saudi Arabia.
cDepartment of Biohemistry, Abdul Wali Khan University Mardan, Mardan-23200, Pakistan.
dUoN Chair of Oman's Medicinal Plants and Marine Natural Products, University of Nizwa, P.O. Box 33, Birkat Al Mauz, Nizwa 616,
Oman
eAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar
Puncak Alam, Selangor D. E. Malaysia.
fFaculty of Pharmacy, Universiti Tecknologi MARA Puncak Alam, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia.
gDepartment of Neuroscience Research, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal
University, P.O. Box 1982, Dammam 3144, Saudi Arabia.
________________________________________________________________________
Abstract
Thymidine phosphorylase is an enzyme involved in pyrimidine salvage pathway that is
identical to platelet-derived endothelial cell growth factor (PD-ECGF) and gliostatin. It is
enormously up regulated in a variety of solid tumors. Furthermore, surpassing of TP level
protects tumor cells from apoptosis and helps cell survival. Thus TP is identified as a prime
target for developing novel anticancer therapies. A new class of exceptionally potent isatin
based oxadiazole (1-30) has been synthesized and evaluated for thymidine phosphorylase
inhibitory potential. All analogs showed potent thymidine phosphorylase inhibition when
compared with standard 7-Deazaxanthine, 7DX (IC50 = 38.68 ± 1.12 µM). Molecular docking
study was performed in order to determine the binding interaction of these newly synthesized
compounds, which revealed that these synthesized compounds established stronger hydrogen
bonding network with active site of residues as compare to the standard compound 7DX.
Keywords: Isatin, Oxadiazole, Synthesis, TP inhibition, SAR, Molecular docking.
*Corresponding Author: fazalstar@gmail.com, Tel.: 0092-335-9528343
taha_hej@yahoo.com and mtaha@iau.edu.sa (M.T)
1

1.0. Introduction
Cancer is the leading cause of premature mortality worldwide [1], which ultimately led vast
interest in exploring several potential angiogenic modulators in order to develop new
therapies for cancer treatment [2-4]. Among various angiogenic activators, thymidine
phosphorylase (TP, EC 2.4.2.4) has been identified as a crucial angiogenic protein [5]. TP is
found in many normal tissues and cells present in both cytoplasm and nucleus [6]. TP causes
a variety of pathological complications such as psoriasis, rheumatoid arthritis, inflammatory
bowel disease and atherosclerosis etc [7]. TP is a major contributor in tumor angiogenesis as
it facilitates proliferation of endothelial cells during cancer metastasis [7, 8]. TP has been
shown to be identical to platelet-derived endothelial cell growth factor (PDECGF) [9-12],
which has been implicated in angiogenesis and chemotaxis in human tumors [13, 14].
Increased hypoxia correlates with elevated TP activity, and increased levels of this enzyme
have been observed in colorectal, ovarian, pancreatic, and breast tumors [15, 16]. In the last
few years, hybrid drug design has emerged as a prime tool for the discovery of innovative
anticancer therapies that can potentially overcome most of the pharmacokinetic drawbacks
encountered when using conventional anticancer drugs.
Isatin as an indole derivative widely distributed endogenously in human and other
mammalian tissues and fluids probably due to tryptophan metabolic pathway. The versatility
of isatin molecular architecture makes it as an ideal platform for structural modification and
derivatization and many isatin derivatives exhibit a broad range of biological activities such
as anticancer[17], antidepressant [18], anticonvulsant [19], antifungal [20], anti-HIV [21] and
anti-inflammatory activity [22]. In the last several decades, many researchers have embarked
on the development of new isatin-based anticancer agents [23-25].
1,3,4-Oxadiazole have attracted attention due to their remarkable biological and
pharmacological properties. Recently, differently substituted 1,3,4-oxadiazoles have been
reported for their biological activities, such as antibacterial [26], antifungal [27], insecticidal
[28] antiviral [29] inflammatory [30] antitubercular [31] antitumor [32] and analgesic activity
[33].
Our research group has been working on synthesis of different heterocycles such as thiazole,
oxazole, oxadiazole, thiadiazole, thiazolidenone etc and reports various activities of these
compounds [34-36]. Currently we are involved in synthesis of heterocyclic hybrid molecules
2

[37-45]. We have observed that the hybrid compounds are more potent than their individual
counterpart. Keeping in view the great biological potential of isatin and oxadiazole, here in
this study we are reporting synthesis and thymidine phosphorylase inhibitory potential of
isatin based oxadiazole analogs.
2.0. Result and discussions
2.1. Chemistry
Isatin based oxadiazole were synthesized in two steps, first 2-aminooxadiazole was refluxed
by reacting semicarbazide with different substituted benzaldehyde in methanol. After
completion of reaction, the intermediate was further cyclized by treating it with iodine and
potassium carbonate in 1,4-dioxane to form 2-aminooxadiazole. In the second step,
synthesized 2-aminooxadiazole was further heated under reflux with different substituted
isatin in methanol for 3hrs to obtained desired different isatin based oxadiazole.
Scheme: Synthesis of isatin based oxadiazole derivatives (1-30)
3

Figure-1: General structure for compounds (1-30)
Table-1: Synthesis of various analogs of isatin based oxadiazole derivatives (1-30) and its
thymidine phosphorylase inhibition
Comp. No.
R
R1
R2
IC50 (µM ± SEMa)
7.80 ± 0.20
Category-A
N-Isopropyl
H
H
1
2
3
4
5
N-Butyl
9.40 ± 0.20
H
N-Pentyl
H
5-isopropyl
5.30 ± 0.10
H
H
16.50 ± 0.30
19.40 ± 0.40
Category-B
H
5-isopropyl
6.20 ± 0.10
4.70 ± 0.10
5.30 ± 0.10
6
7
8
N-Isopropyl
N-Butyl
H
H
Category-C
4

H
5-isopropyl
14.60 ± 0.3
11.30 ± 0.30
17.60 ± 0.40
9
N-Isopropyl
N-Butyl
H
H
10
11
Category-D
H
5-isopropyl
48.50 ± 1.20
29.10 ± 0.90
38.60 ± .90
46.20 ± 1.20
49.40 ± 1.30
12
13
14
15
16
N-Isopropyl
N-Butyl
N-Pentyl
H
H
H
H
H
Category-E
N-Isopropyl
H
25.10 ± 0.50
19.70 ± 0.40
34.60 ± 0.60
26.80 ± 0.60
18.40 ± 0.40
37.30 ± 0.80
17
18
19
20
21
22
N-Butyl
N-Pentyl
H
H
H
5-isopropyl
N-Isopropyl
N-Butyl
H
H
5

N-Pentyl
H
26.40 ± 0.60
22.10 ± 0.60
34.30 ± 0.60
34.60 ± 0.70
30.10 ± 0.60
18.70 ± 0.40
17.90 ± 0.50
16.40 ± 0.40
23
24
25
26
27
28
29
30
H
5-isopropyl
H
5-isopropyl
N-Pentyl
H
H
H
H
H
N-Butyl
H
H
H
Standard
38.68 ± 1.12
7-Deazaxanthined
2.2. Biological activity
Isatin based oxadiazole derivatives (1-30) were substituted at position-5 as R2 and N-
alkylated as R1 and aryl substituted as R with a diverse range of groups having electron
withdrawing and electron donating effect. On the basis of these groups, compounds are
classified into five categories A-E in order to simplify the SAR. The SAR was elucidated on
the basis of different substitutions. It was assumed that a variation in the inhibitory activities
of compounds in a particular category is attributed by different substitutions on isatin part and
phenyl part of oxadiazole (Figure-2).
6

The isatin based oxadiazole a class of heterocycles should be acknowledged as new lead
inhibitors with much more pronounced inhibitory potentials against thymidine phosphorylase
and angiogenesis as compared to 7DX. While these analogs possess the strong and
convincing potential in the future design of new TP inhibitors. All synthesized compounds
(1-30) were evaluated against thymidine phosphorylase inhibition. All compounds showed
potent thymidine phosphorylase inhibition when compared with standard 7-Deazaxanthine,
7dx (IC50 = 38.68 ± 1.12 µM). Structure activity relationship has been established for all
compounds mainly based on substitution pattern on phenyl ring. In order to simplify the SAR
all compounds were categorized in five categories on the basis of substitution on phenyl ring
of oxadiazole. Five analogs such as 1-5are kept in category A. The compounds 6-8 were kept
in category B. Three compounds such as 9-11 were kept in category C. Compounds such as
12-16 were kept in category D. The remaining compounds 17-30 were kept in category E.
Analog1 (IC50 = 7.80 ± 0.20 µM) having 3-hydroxyl group on phenyl part and N-alkylated
isopropyl, analog 2 (IC50 = 9.40 ± 0.20 µM) having 3-hydroxyl group on phenyl part and N-
butyl, analog 3 (IC50 = 5.30 ± 0.10 µM) having 3-hydroxyl on phenyl part and 5-isopropyl on
Isatin phenyl part and analog 4 (IC50 = 16.50 ± 0.30 µM) having 3-hydroxyl on phenyl part
and N-alkylated pentane showed potent inhibition. Other analog 5 (IC50 = 19.40 ± 0.40 µM)
having 4-hydroxy on phenyl part showed excellent inhibitory potential due to substitution on
phenyl ring and N-alkylation. The greater potential is due to the methoxy group on phenyl
ring. Similar pattern was observed like hydroxy analogs.
Figure-2: Structure of compounds (1-5)
7

Analog 6 (IC50 = 6.20 ± 0.10 µM) a hydroxy, methoxy and isopropyl on isatin phenyl part
and analog 7 (IC50 = 4.70 ± 0.10 µM) a hydroxy, methoxy and N-isopropyl and analog 8(IC50
= 5.30 ± 0.10 µM) a hydroxy, methoxy and N-beutane showed potent inhibitory potential.
Figure-3: Structure of compounds (6-8)
A category-C possessed different substitution on isatin and same substitution on phenyl part.
Analog 9 (IC50 = 14.60 ± 0.3 µM) a chloro, benzyloxy and 5-isopropyl on isatin phenyl part
and analog 10 (IC50 = 11.30 ± 0.30 µM) a chloro, benzyloxy and N-isopropyl showed
excellent potential. Analog 11(IC50 = 17.60 ± 0.40 µM) having 3-benzyloxy-4-chloro on
phenyl part and N-alkylated butane. The slight activity difference among these analogs
showed that the position of isopropyl also affect the inhibition. By comparing analog 11aN-
alkylated butane and analog 9 a 5-isopropylwith analog 10a N-alkylated isopropane, the
analog 10 was found superior than these two analogs 9and 11.
Figure-4: SAR of compounds (9-11)
In category-D, analog 16 possess no substitution on isatin part and other analogs in category-
C possess different substitutions’ on isatin part and same substitution on phenyl part. Analog
13 having 5-bromo-2-methoxy on phenyl part and N-isopropyl and Analog 12, 14and15
having 5-bromo2-methoxy phenyl, 5-isopropyl, n-alkylated butane, and pentane showed good
to moderate thymidine phosphorylase inhibition. Among these analog 13 showed good
inhibition than compared with standard 7DX.
8

Figure-5: SAR of compounds (12-16)
The category-E possessed different substitution on isatin and different substitution on phenyl
part. Analog (17-30) having nitrile group at ortho, meta and para position on phenyl part and
substituted, N-alkylated isatin showed remarkable thymidine phosphorylase inhibition. By
comparing analogs 17-30 was found well than the other analogs. The reason for remarkable
potential of these fourteen (14) analogs is due to the position difference of nitrile group on
phenyl ring and substituted, N-alkylated isatin.
It was concluded from this study that either EWG or EDG on phenyl part exhibited potential
but the slight variance in potential was mainly affected by the position of the substituent as
well as in some cases the number of substituent also play a role.
9

Figure-6: SAR of compounds (20-30)
However, the in vitro thymidine phosphorylase inhibition studies and to understand the
different structural motifs of the compounds in binding with the active site of thymidine
phosphorylase, in silico molecular modeling was also performed.
2.3. Molecular modeling and docking studies
To explore the binding mode of these oxadiazole derivatives, molecular docking was
performed. The docking results showed that all the compounds well accommodated in the
active site of thymidine phosphorylase. From the docking conformation of the most active
compound, compound 7 (IC50 = 4.70 ± 0.10), it was observed that this compound established
six polar interactions and four pi-H interactions with active site residues and good docking
score (-13.1172) as compare to the reference compound having docking score of -8.1034 and
10

biological activity with IC50 of 38.68 ± 1.12. The oxygen and two nitrogen atoms present at
the 1,3,4-oxadiazole moiety of the compound formed one H-acceptor with His 85 and two H-
acceptor bonds with Lys 190 respectively. Furthermore, carbonyl oxygen atom of the 1-
isopropylindolin-2-one moiety formed H-acceptor bonds with active site residue Gln 156
whereas the -OH moiety of the compound formed two H-acceptor interactions with the -NH
group of the Arg 171 residue of the enzyme. Arg 115, Tyr 168 and Ile 183 made pi-H
linkages with the 5-ring and 6-ring moieties of the compound as shown in Figure-7a. This
strong bonding network might be one of the reasons for this compound to show good
biological activity. The docking conformation of the second most active compound,
compound 3 (IC50 = 5.30 ± 0.10), showed that this compound formed five bonds with the
active site residues His 85, Tyr 168, Ser 186 and Lys 190 respectively (Figure-7b) and good
docking score (-12.7026). The nitrogen atom of the compound formed one H-acceptor bond
with His 85 while Tyr 168 residue was observed making two hydrogen bonds with nitrogen
atoms of the 1,3,4-oxadiazole moiety of the compound. Ser 186 and Lys 190 residues formed
polar interactions with the -NH and carbonyl oxygen atom of the 5-isopropylindolin-2-one
moiety of the compound respectively. Arg 115, Ile 183 and Phe 210 formed three pi-H
linkages with the compound. The potency of this compound might be due to the presence of
the electron donating group (-OH). The third most active compound 6 (IC50 = 6.20 ± 0.10)
was observed with docking score of -12.0767 and good interactions with the binding pocket
residues (Figure-7c). His 85 and Lys 190 formed pi-H linkages with the compound. His 85
and Tyr 168 established polar interactions with the -OH and oxygen atom of the MeO
moieties respectively while Arg 115 formed polar bonds with the –NH of the 1,3,4-
oxadiazol-2-amine moiety of the ligand respectively. The electronic cloud system of this
compound might be the reason of its high potency. The forth most one active compound,
compound 1 (IC50 = 7.80 ± 0.20 and docking score = -12.0502) formed two polar
interactions with the His 85 and Lys 190 while Tyr 168 formed two pi-H contacts with the
compound as shown in Figure-7d.
Overall a good correlation was observed between the docking scores and biological activities
of these compounds Figure-8 (see S1). The docking score and report of predicted interaction
of dock confirmation was mention in table (See S2).
11

Figure-7: Docking conformations of compounds on thymidine phosphorylase enzyme. (a)
3D binding mode of compound 7as inhibitor of thymidine phosphorylase enzyme. (b) 3D
binding mode of compound 3. (c) 3Dbinding mode of compound 6. (d) 3D binding mode of
compound 1 in binding cavity of thymidine phosphorylase enzyme. Ligands are shown green
colour.
Conclusion
In conclusion we have synthesized isatin based oxadiazole analogs 1–30, characterized by EI-
1
MS and H-NMR and checked for thymidine phosphorylase inhibitory potentials. Eight
compounds were identified as potent thymidine phosphorylase inhibitor.
3.0. Experimental
3.1. General methods
NMR experiments were performed on Advance Bruker AM 500 MHz machine (France).
DMSO was used for accurate NMR analysis. Light of wavelength 254 and 265 nm were used
to visualize the chromatogram. Electron impact mass spectra (EI MS) were recorded on a
12

Finnigan MAT-311A (Germany) mass spectrometer. Thin layer chromatography (TLC) was
performed on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany).
3.2. General Procedure for the synthesis of isatin based oxadiazole derivatives (1-30)
Isatin based oxadiazole were synthesized in two steps, first semicarbazide (1mmol, 0.71g)
and different substituted benzaldehydes (1mmol) were mixed and reflux in methanol for 2-3
hrs. The completion of reaction was monitored by TLC. After completion of reaction, the
intermidiate was further cyclized by treating it with iodine and potassium carbonate in 1, 4-
dioxane to give a 2-aminooxadiazole moiety. After drying, it was reacted with 5% Na2S2O3
(20 mL) and then it was extracted the desired organic compound from the reaction mixture
using CH2Cl2/MeOH (10:1, 10 mL × 4). The desired organic compound was dried over
anhydrous sodium sulphate and was purified using petroleum ether and ethylacetate mixture
as eluent through silica gel column chromatography afford the synthesis of corresponding
analogs. In the second step, 1mmol of synthesized 2-aminooxadiazole were refluxed with
different substituted isatin in methanol in the presence of few drops of glacial acetic acid for
3hrs. Reaction completion was monitored through TLC. After completion of reaction,
reaction mixture was washed with chloroform/hexane to obtained desired different isatin
based oxadiazole.
3.2.1. (E)-3-(5-(3-hydroxyphenyl)-1,3,4-oxadiazol-2-ylimino)-1-isopropylindolin-2-one
(1)
1
Dark black solid, Yield: 73%; H-NMR: (500 MHz, DMSO-d6): δ 7.81 (d, J = 5.6 Hz, 1H,
Ar), 7.78 (d, J = 5.7 Hz, 1H, Ar), 7.48 (t, J = 5.9 Hz, 1H, Ar), 7.30 (s, 1H, Ar), 7.58 (d, J =
6.2 Hz, 1H, Ar), 7.31 (t, J = 5.6 Hz, 1H, Ar), 7.20 (t, J = 6.0 Hz, 1H, Ar), 6.86 (d, J = 6.2 Hz,
13
1H, Ar), 5.32 (s, 1H, -OH), 1.20 (s, 6H, -CH3), 3.91 (s, 1H); C-NMR (125 MHz, DMSO-
d6): δ 163.8, 163.1, 162.5, 158.3, 157.1, 147.1, 131.0, 130.2, 129.1, 124.0, 127.4, 120.8,
+
118.5, 116.4, 113.5, 110.5, 58.1,23.3, 23.2; HREI-MS: m/z Calcd for C19H16N4O3, [M]
348.1222; found 348.1208.
3.2.2. (E)-1-butyl-3-(5-(3-hydroxyphenyl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one (2)
1
Light black solid, Yield: 71%; H-NMR: (500 MHz, DMSO-d6): δ 7.57 (d, J = 6.4 Hz,
1H,Ar),7.50 (d, J = 5.7 Hz, 1H, Ar), 7.48 (t, J = 7.2 Hz, 1H, Ar), 7.41 (s, 1H, Ar), 7.24 (d, J =
6.3 Hz, 1H, Ar), 7.15 (d, J = 6.9, 1H, Ar), 7.11 (t, J = 6.5 Hz, 1H, Ar), 6.90 (d, J = 6.8, 1H,
Ar), 6.70 (s, 1H, -OH), 3.28 (m, 6H, -CH2), 1.20 (s, 3H, -CH3); 13C-NMR (125 MHz, DMSO-
13

d6): δ 163.8, 163.4, 162.8, 162.3, 157.8, 147.1, 131.5, 130.3, 129.2, 127.1, 124.0, 120.6,
117.9, 116.4, 116.0, 113.1, 43.4, 30.4, 21.4, 16.5; HREI-MS: m/z Calcd for C20H18N4O3, [M]
+ 362.1378; found 362.1365.
3.2.3. (E)-3-(5-(3-hydroxyphenyl)-1,3,4-oxadiazol-2-ylimino)-5-isopropylindolin-2-one
(3)
1
Light brown solid , Yield: 74%; H-NMR: (500 MHz, DMSO-d6): δ 9.58 (s, 1H, -NH), 7.53
(s, 1H, Ar), 7.47(d, J = 6.5 Hz, 1H, Ar), 7.34 (d, J = 6.4 Hz, 1H, Ar), 7.27 (s, 1H, Ar), 7.08 (t,
J = 6.8 Hz, 1H, Ar), 6.89 (d, J = 6.5 Hz, 1H, Ar), 6.77 (d, J = 6.6 Hz, 1H, Ar), 5.54 (s, 1H, -
OH), 2.88 (s, 1H, -CH), 1.82 (s, 6H, -CH3); 13C-NMR (125 MHz, DMSO-d6): δ 165.1, 163.3,
163.1, 157.8, 151.2, 146.5, 139.1, 131.2, 129.4, 128.5, 127.3, 122.4, 120.8, 118.2, 116.4,
+
113.5, 34.5, 24.6, 24.6; HR-EI-MS: m/z Calcd for C19H16N4O3, [M] 348.1221; found
348.1211.
3.2.4. (E)-3-(5-(3-hydroxyphenyl)-1,3,4-oxadiazol-2-ylimino)-1-pentylindolin-2-one (4)
1
Light black solid, Yield: 76%; H-NMR: (500 MHz, DMSO-d6): δ 7.76 (d, J = 7.0 Hz, 1H,
Ar), 7.68 (d, J= 6.7 Hz, 1H, Ar), 7.53 (d, J = 6.4 Hz, 1H, Ar),7.47 (t, J = 5.7 Hz, 1H, Ar),
7.27 (t, J = 6.6 Hz, 1H, Ar), 7.19 (s, 1H, Ar), 7.08 (t, J = 6.5 Hz, 1H, Ar), 6.94 (d, J = 6.7 Hz,
13
1H, Ar), 5.64 (s, 1H, -OH), 3.84 (m, 8H, -CH2), 0.92 (s, 3H, -CH3); C-NMR (125 MHz,
DMSO-d6): δ 165.2, 164.5,163.4, 163.1, 157.9, 148.1, 132.1, 131.2, 130.3, 128.1, 125.2,
121.4, 118.6, 116.7, 116.4, 113.5, 43.8, 30.5, 28.4, 23.7, 16.6; HR-EI-MS: m/z Calcd for
C21H20N4O3, [M] + 376.1534; found 376.1520.
3.2.5. (E)-3-(5-(3-hydroxyphenyl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one (5)
Light red solid, Yield: 77%; 1H-NMR: (500 MHz, DMSO-d6): δ 8.10 (s, 1H, -NH), 7.60 (d, J
= 6.4 Hz, 1H, Ar), 7.49 (d, J = 6.6 Hz, 1H, Ar), 7.59 (t, J = 6.7 Hz, 1H, Ar), 7.32 (d, J = 6.1
Hz, 1H, Ar), 7.28 (s, 1H, Ar), 7.12 (t, J = 6.5 Hz, 1H, Ar), 7.08 (t, J = 6.1 Hz, 1H, Ar), 6.95
13
(d, J = 6.7 Hz, 1H, Ar), 5.55 (s, 1H, Ar); C-NMR (125 MHz, DMSO-d6): δ 165.0, 163.2,
161.2, 157.8, 151.4, 142.5, 131.4, 129.7, 128.5, 127.8, 125.2, 120.9, 120.2, 117.4, 115.5,
113.5; HR-EI-MS: m/z Calcd for C16H10N4O3, [M] + 306.0752; found 306.0739.
3.2.6.
(E)-3-(5-(3-hydroxy-4-methoxyphenyl)-1,3,4-oxadiazol-2-ylimino)-5-
isopropylindolin-2-one (6)
14

Red solid, Yield: 76%; 1H-NMR: (500 MHz, DMSO-d6): δ 8.0 (s, 1H, -NH), 7.75 (s, 1H, Ar),
7.53 (d, 6.4 Hz, 1H, Ar), 7.48 (d, J= 6.2 Hz, 1H, Ar), 7.27 (s, 1H, Ar), 7.02 (d, J = 6.9 Hz,1H,
Ar), 6.87 (d, J = 6.9 Hz, 1H, Ar), 6.14 (s, 1H, -OH), 3.91 (s, 3H, -CH3), 2.87 (s, 1H, -CH),
1.85 (s, 6H, -CH3); 13C-NMR (125 MHz, DMSO-d6): δ 159.3, 156.7, 156.1, 155.9,
154.5,150.6, 149.4, 138.3, 131.0, 127.5, 124.9, 122.7, 117.8, 117.6, 113.8, 111.6, 56.1, 30.6,
25.3, 25.3; HR-EI-MS: m/z Calcd for C20H18N4O4, [M] + 378.1327; found 378.1315.
3.2.7.
(E)-3-(5-(3-hydroxy-4-methoxyphenyl)-1,3,4-oxadiazol-2-ylimino)-1-
isopropylindolin-2-one (7)
Dark brown solid, Yield: 81%; 1H-NMR: (500 MHz, DMSO-d6): δ 7.81 (d, , J = 6.7 Hz, 1H,
Ar), 7.52 (d, , J = 6.6 Hz, 1H, Ar), 7.45 (d, , J = 7.2 Hz, 1H, Ar), 7.31 (d, 6.5 Hz, 1H, Ar),
7.29 (d, J = 6.8 Hz, 1H, Ar), 7.17 (s, 1H, Ar), 6.96 (d, J = 6.1 Hz, 1H, Ar), 5.54 (s, 1H, -OH),
2.52 (s, 1H, -CH), 2.26 (s, 3H, -CH3), 1.67 (s, 6H, -CH3): 13C-NMR (125 MHz, DMSO-d6): δ
174.8, 156.9, 151.5, 149.0, 147.3, 140.1, 133.9, 133.3, 129.8, 127.6, 118.3, 116.1, 113.9,
+
112.6, 111.7, 108.9, 56.6, 55.5, 24.6, 24.6; HR-EI-MS: m/z Calcd for C20H18N4O4, [M]
378.1328; found 378.1320.
3.2.8. (E)-1-butyl-3-(5-(3-hydroxy-4-methoxyphenyl)-1,3,4-oxadiazol-2-ylimino)indolin-
2-one (8)
1
Yellow solid, Yield: 82%; H-NMR: (500 MHz, DMSO-d6): δ 8.22 (d, J = 6.4 Hz, 1H, Ar),
7.87 (d, J = 6.6 Hz, 1H, Ar), 7.77 (d, J = 6.5 Hz, 1H, Ar), 7.65 (d, J = 6.3 Hz, 1H, Ar), 7.47
(s, 1H, Ar), 7.20 (d, J = 6.1 Hz, 1H, Ar), 7.03 (d, J = 6.7 Hz, 1H, Ar), 5.64 (s, 1H, -OH), 3.91
13
(m, 2H, -CH2), 3.82 (s, 3H, -CH3), 1.74 (m, 4H, -CH2), 1.36 (m, 3H, -CH3); C-NMR (125
MHz, DMSO-d6): δ 165.2, 159.9, 156.7, 156.2, 155.7, 150.8, 149.7, 138.4, 131.2, 127.9,
124.8, 122.4, 117.6, 117.3, 113.9, 111.7, 58.2, 54.1, 30.5, 24.5, 18.6; HREI-MS: m/z Calcd
for C21H20N4O4, [M] + 392.1484; found 392.1472.
3.2.9.
(E)-3-(5-(3-(benzyloxy)-4-chlorophenyl)-1,3,4-oxadiazol-2-ylimino)-5-
isopropylindolin-2-one (9)
1
Red solid, Yield: 78%; H-NMR: (500 MHz, DMSO-d6): δ 9.8 (s, 1H, -NH), 7.76 (s, 1H,
Ar),7.55 (d, J = 6.5 Hz, 2H, Ar), 7.47 (m, 3H, Ar), 7.32 (d, J = 6.2 Hz, 1H, Ar), 7.36 (s, 1H,
Ar), 7.27 (d, J = 6.3 Hz, 1H, Ar), 7.13 (d, J = 6.0 Hz, 1H, Ar), 6.86 (d, J = 6.6 Hz, 1H, Ar),
5.13 (s, 2H, -CH2), 3.84 (s, 1H, -CH), 1.92 (s, 6H, -CH3); 13C-NMR (125 MHz, DMSO-d6): δ
164.9, 164.2, 163.7, 151.3, 156.2,146.6, 139.2, 137.4, 131.1, 129.5, 129.5, 127.9, 127.6,
15

127.6, 127.2, 127.0, 125.7, 123.1, 121.7, 121.4, 118.1, 113.3, 71.4, 34.2, 24.5, 24.5; HR-EI-
MS: m/z Calcd for C26H21ClN4O3, [M] + 472.1302; found 472.1297.
3.2.10.
(E)-3-(5-(3-(benzyloxy)-4-chlorophenyl)-1,3,4-oxadiazol-2-ylimino)-1-
isopropylindolin-2-one (10)
1
Silver solid, Yield: 75%; H-NMR: (500 MHz, DMSO-d6): δ 7.82 (d, J = 6.6 Hz, 1H, Ar),
7.52 (d, J = 6.4 Hz, 1H, Ar),7.42 (d, J = 6.5 Hz, 1H, Ar), 7.38 (d, J = 6.6 Hz, 2H, Ar), 7.35 (t,
J = 5.9. Hz, 3H, Ar), 7.23 (s, 1H, Ar), 7.20 (d, J = 6.2 Hz, 1H, Ar), 7.18 (d, J = 6.4 Hz, 1H,
Ar), 7.07 (d, J = 6.8 Hz, 1H, Ar), 5.21 (s, 2H, -CH2), 3.83 (s, 1H, -CH), 2.08 (s, 6H, -CH3);
13C-NMR (125 MHz, DMSO-d6): δ163.5, 157.2, 156.8, 149.5, 148.7, 139.4, 136.8, 128.9,
128.4, 128.3, 128.0, 127.9, 127.7, 123.6, 121.1, 120.6, 118.0, 117.2, 113.5, 113.0, 109.4,
+
108.5, 72.6, 55.6, 24.7, 24.7; HR-EI-MS: m/z Calcd for C26H21ClN4O3, [M] 472.1302;
found 472.1295.
3.2.11.
(E)-3-(5-(3-(benzyloxy)-4-chlorophenyl)-1,3,4-oxadiazol-2-ylimino)-1-
butylindolin-2-one (11)
Light Yellow solid, Yield: 81%; 1H-NMR: (500 MHz, DMSO-d6): δ 7.85 (d, J = 6.6 Hz, 1H,
Ar), 7.64 (d, J = 6.2 Hz, 1H, Ar), 7.50 (t, J = 5.6 Hz, 1H, Ar), 7.45 (t, J = 5.2 Hz, 1H, Ar),
7.44 (m, 3H, Ar), 7.38 (d, J = 6.6 Hz, 1H, Ar), 7.28 (d, J = 6.4 Hz, 1H, Ar), 7.19 (d, J = 6.2
Hz, 2H, Ar), 7.08 (s, 1H, Ar), 5.25 (s, 2H, -CH2), 3.82 (m, 6H, -CH2), 1.69 (m, 3H, -CH3);
13C-NMR (125 MHz, DMSO-d6): δ 162.5, 159.2, 156.9, 151.7, 149.6, 138.4, 137.8, 129.9,
129.4, 128.5, 128.2, 128.1, 127.7, 127.6, 125.2, 124.7, 122.7, 122.1, 118.5, 116.4, 116.4,
113.5, 73.6, 44.7, 31.5, 22.7, 17.6; HR-EI-MS: m/z Calcd for C27H23ClN4O3, [M] + 486.1458;
found 486.1443.
3.2.12.
(E)-3-(5-(5-bromo-2-methoxyphenyl)-1,3,4-oxadiazol-2-ylimino)-5-
isopropylindolin-2-one (12)
1
Dark Yellow solid, Yield: 65%; H-NMR: (500 MHz, DMSO-d6): δ 8.10 (s, 1H, -NH), 7.77
(s, 1H, Ar), 7.47 (d, J = 6.6 Hz, 1H, Ar), 7.38 (s, 1H, Ar), 7.36 (d, J = 6.5 Hz, 1H, Ar), 7.01
(d, J = 7.3 Hz,1H, Ar), 6.85 (d, J = 6.5 Hz, 1H, Ar), 3.86 (s, 3H, -CH3), 2.89 (s, 1H, -CH),
1.26 (s, 6H, -CH3); 13C-NMR (125 MHz, DMSO-d6): δ 159.4, 156.6, 156.0, 155.9, 148.8,
143.1, 136.5, 134.2, 132.4, 131.0, 127.5, 122.2, 117.7, 117.4, 114.8, 112.7, 56.2, 32.6, 23.6,
23.6; HR-EI-MS: m/z Calcd for C20H17N4O3, [M] + 440.0484; found 440.0472.
16

3.2.13.
(E)-3-(5-(5-bromo-2-methoxyphenyl)-1,3,4-oxadiazol-2-ylimino)-1-
isopropylindolin-2-one (13)
1
Crystal solid, Yield: 78%; H-NMR: (500 MHz, DMSO-d6): δ 8.09 (d, J = 6.8 Hz, 1H, Ar),
7.84 (d, J = 6.6 Hz, 1H, Ar), 7.57 (d, J = 6.3Hz, 1H, Ar), 7.45 (d, J= 6.1 Hz, 1H, Ar), 7.12 (s,
1H, Ar), 7.02 (d, J = 6.8 Hz, 1H, Ar), 6.74 (d, J = 6.6 Hz, 1H, Ar), 3.34 (s, 1H, -CH), 2.12 (s,
3H, -CH3), 1.84 (s, 6H, -CH3); 13C-NMR (125 MHz, DMSO-d6): δ 164.9, 160.9, 156.6,
156.1, 147.5, 133.9, 133.4, 133.0, 131.1, 127.5, 125.0, 118.2, 116.4, 115.6, 113.8, 112.7,
+
79.1, 55.9, 30.6, 30.6; HR-EI-MS: m/z Calcd for C20H17BrN4O3, [M] 440.0484; found
440.0471.
3.2.14. (E)-3-(5-(5-bromo-2-methoxyphenyl)-1,3,4-oxadiazol-2-ylimino)-1-butylindolin-
2-one (14)
Light Brown solid, Yield: 80%; 1H-NMR: (500 MHz, DMSO-d6): δ 7.80 (d, J = 6.4 Hz, 1H,
Ar), 7.53 (d, J = 6.5 Hz, 1H, Ar), 7.48 (d, J= 6.6 Hz, 1H, Ar), 7.36 (s, 1H, Ar), 7.29 (d, J =
6.4 Hz, 1H, Ar), 6.95 (d, J = 6.2 Hz, 1H, Ar), 6.87 (d, J = 6.4 Hz, 1H, Ar), 3.83 (s, 3H, -CH3),
13
3.77 (m, 6H, -CH2), 1.65 (m, 3H, -CH3); C-NMR (125 MHz, DMSO-d6): δ164.4, 158.6,
157.0, 156.9, 150.8, 143.1, 136.5, 136.2, 133.4, 131.0, 126.5, 123.2, 117.8, 117.5, 114.9,
112.8, 56.2, 45.3, 31.6, 22.6, 17.8; HR-EI-MS: m/zCalcd for C21H19BrN4O3, [M] + 454.0640;
found 454.0628.
3.2.15. (E)-3-(5-(5-bromo-2-methoxyphenyl)-1,3,4-oxadiazol-2-ylimino)-1-pentylindolin-
2-one (15)
1
Transparent solid, Yield: 79%; H-NMR: (500 MHz, DMSO-d6): δ 7.86 (d, J = 6.3 Hz, 1H,
Ar), 7.67 (d, J = 6.1 Hz, 1H, Ar), 7.51 (d, J= 6.0 Hz, 1H, Ar), 7.47 (d, J = 6.6 Hz,1H, Ar),
7.32 (s, 1H, Ar), 7.29 (d, J = 6.5 Hz, 1H, Ar), 7.02 (d, J = 6.4 Hz, 1H, Ar), 3.92 (s, 3H, -
13
CH3), 2.10 (m, 8H, -CH2), 1.67 (m, 3H, -CH3); C-NMR (125 MHz, DMSO-d6): δ 164.8,
161.7, 156.8, 156.4, 149.5, 148.9, 133.5, 133.1, 131.3, 129.5, 125.0, 118.4, 116.6, 115.4,
115.8, 113.7, 58.2, 44.9, 30.5, 28.9, 25.4, 18.5; HREI-MS: m/z Calcd for C22H21BrN4O3, [M]
+ 468.0796; found 468.0783.
3.3.16.
(16)
(E)-3-(5-(5-bromo-2-methoxyphenyl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one
17

1
Light orange solid, Yield: 79%; H-NMR: (500 MHz, DMSO-d6): δ 8.12 (s, 1H, -NH), 7.87
(d, J = 6.0 Hz, 1H, Ar), 7.77 (d, J = 6.1 Hz, 1H, Ar), 7.67 (t, J = 6.1 Hz, 1H, Ar), 7.59 (d, J =
6.7 Hz, 1H, Ar), 7.35 (s, 1H, Ar), 7.19 (s, 1H, Ar), 7.08 (d, J = 6.6 Hz, 1H, Ar), 3.87 (s, 3H, -
13
CH3); C-NMR (125 MHz, DMSO-d6): δ 163.4, 161.6, 156.2, 151.4, 142.5, 135.6, 134.8,
131.4, 130.4, 125.2, 120.6, 118.6, 118.2, 115.4, 113.8, 56.4, 32.6; HR-EI-MS: m/z Calcd for
C17H11BrN4O3, [M] + 398.0016; found 398.0004.
3.3.17.
(E)-2-(5-(1-isopropyl-2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-
yl)benzonitrile (17)
1
Dark Brown solid, Yield: 71%; H-NMR:(500 MHz, DMSO-d6): δ 8.09 (d, J = 6.4 Hz, 1H,
Ar), 7.94 (d, J = 6.1 Hz, 1H, Ar), 7.84 (d, J = 6.6 Hz, 1H, Ar), 7.73 (t, J = 6.5 Hz, 1H, Ar),
7.64 (t, J = 6.2 Hz, 1H, Ar), 7.53 (t, J = 6.0 Hz, 1H, Ar), 7.46 (d, J = 6.7 Hz, 1H, Ar), 7.35 (t,
13
J = 5.6 Hz, 1H, Ar), 2.12 (s, 1H, -CH), 1.67S (s, 6H, -CH3); C-NMR (125 MHz, DMSO-
d6): δ 165.4, 164.3, 162.8, 161.7, 148.2, 141.1, 134.3, 133.5, 132.2, 130.5, 130.1, 129.2,
125.4, 118.2, 117.8, 116.1, 105.0, 60.1, 21.6, 21.6; HR-EI-MS: m/z Calcd for C20H15N5O2,
[M] + 357.1225; found 357.1212.
3.3.18. (E)-4-(5-(1-butyl-2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-yl)benzonitrile
(18)
1
Brown solid, Yield: 81%; H-NMR:(500 MHz, DMSO-d6): δ 7.93 (d, J = 5.9 Hz, 1H, Ar),
7.83 (d, J = 6.5 Hz, 1H, Ar), 7.76 (d, J= 6.6 Hz, 2H, Ar), 7.56 (d, J = 6.2 Hz, 2H, Ar), 7.49 (t,
J = 6.2 Hz, 1H, Ar), 7.11 (t, J = 5.3 Hz, 1H, Ar), 2.09 (m, 6H, -CH2), 1.68 (m, 3H, -CH3);
13C-NMR (125 MHz, DMSO-d6): δ 165.2, 164.1, 162.5, 161.3, 148.3, 141.1, 134.2, 134.2,
132.1, 130.7, 130.2, 129.1, 129.1, 125.3, 119.5, 118.3, 113.0, 46.1, 30.7, 23.4, 17.5; HR-EI-
MS: m/z Calcd for C21H17N5O2, [M] + 371.1381; found 371.1369.
3.3.19. (E)-4-(5-(2-oxo-1-pentylindolin-3-ylideneamino)-1,3,4-oxadiazol-2-yl)benzonitrile
(19)
1
Dark brown solid, Yield: 81%; H-NMR: (500 MHz, DMSO-d6): δ 7.94 (d, J = 6.4 Hz, 1H,
1H, Ar), 7.87 (d, J= 6.2 Hz, 1H, Ar), 7.65 (d, J= 6.2 Hz, 2H, Ar), 7.55 (d, J = 6.6 Hz, 2H,
Ar), 7.46 (t, J = 5.9 Hz, 1H, Ar), 7.11 (t, J = 5.2 Hz, 1H, Ar), 2.18 (m, 8H, -CH2), 1.76 (m,
3H, -CH3); 13C-NMR (125 MHz, DMSO-d6): δ 165.1, 164.3, 162.2, 161.4, 148.4, 141.2,
134.2, 134.5, 132.4, 130.5, 130.6, 129.3, 129.3, 125.5, 119.4, 118.6, 113.2, 46.0, 30.7, 29.6,
24.4, 18.5; HR-EI-MS: m/z Calcd for C22H19N5O2, [M] + 385.1538; found 385.1530.
18

3.3.20.
(E)-4-(5-(5-isopropyl-2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-
yl)benzonitrile (20)
1
Dark orange solid, Yield: 71%; H-NMR: (500 MHz, DMSO-d6): δ 8.01 (s, 1H, -NH), 7.93
(d, J = 6.2 Hz, 2H, Ar), 7.83 (d, J= 6.5 Hz, 2H, Ar), 7.49 (s, 1H, Ar), 7.36 (d, J = 6.8 Hz,1H,
13
Ar), 6.88 (d, 1H, J = 6.6 Hz, Ar), 2.88 (s, 1H, -CH), 1.74 (m, 6H, -CH3); C-NMR (125
MHz, DMSO-d6): δ 165.5, 164.4, 162.9, 161.6, 148.3, 141.2, 134.4, 133.2, 132.5, 130.4,
129.2,127.2, 123.4, 119.2, 117.9, 117.1, 113.0, 37.1, 25.4, 25.4; HR-EI-MS: m/z Calcd for
C20H15N5O2, [M] + 357.1225; found 357.1217.
3.3.21.
(E)-4-(5-(1-isopropyl-2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-
yl)benzonitrile (21)
1
Light orange solid, Yield: 72%; H-NMR:(500 MHz, DMSO-d6): δ 7.94 (d, J = 6.2 Hz, 2H,
Ar), 7.83 (d, J = 6.6 Hz, 1H, Ar), 7.76 (d, J = 6.5 Hz, 2H, Ar), 7.66 (d, J = 6.2 Hz, 1H, Ar),
7.51 (t, J = 6.1 Hz, 1H, Ar), 7.34 (t, J = 5.8 Hz, 1H, Ar), 3.85 (s, 1H, -CH), 1.68 (m, 6H, -
13
CH3); C-NMR (125 MHz, DMSO-d6): δ 165.4, 164.3, 162.8, 161.7, 148.2, 133.5, 133.5,
132.2, 131.4, 129.2, 129.2, 129.1, 125.3, 119.2, 117.8, 113.1, 105.0, 60.2, 21.8, 21.8; HR-EI-
MS: m/z Calcd for C20H15N5O2, [M] + 357.1225; found 357.1216.
3.3.22. (E)-2-(5-(1-butyl-2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-yl)benzonitrile
(22)
1
Dark Orange solid, Yield: 81%; H-NMR:(500 MHz, DMSO-d6): δ 8.14 (d, J = 6.6 Hz, 1H,
Ar), 8.09 (d, J = 6.5 Hz, 1H, Ar), 7.86 (d, J = 6.5 Hz, 1H, Ar), 7.73 (t, J = 6.6 Hz, 1H, Ar),
7.65 (d, J = 6.4 Hz, 1H, Ar), 7.53 (t, J = 5.6 Hz, 1H, Ar), 7.46 (t, J = 5.2 Hz, 1H, Ar), 7.38 (t,
J = 4.8 Hz, 1H, Ar), 3.64 (m, 6H, -CH2), 2.09 (m, 3H, -CH3);13C-NMR (125 MHz, DMSO-
d6): δ 165.1, 164.0, 162.4, 161.2, 148.1, 141.0, 134.3, 134.4, 132.3, 130.8, 130.4, 129.6,
129.6, 125.2, 119.6, 118.5, 113.1, 46.2, 30.5, 23.2, 17.4; HR-EI-MS: m/z Calcd for
C21H17N5O2, [M] + 371.1381; found 371.1368.
3.3.23. (E)-2-(5-(2-oxo-1-pentylindolin-3-ylideneamino)-1,3,4-oxadiazol-2-yl)benzonitrile
(23)
1
Dark Brown solid, Yield: 81%; H-NMR:(500 MHz, DMSO-d6): δ 8.11 (d, J = 6.6 Hz, 1H,
Ar), 7.86 (d, J = 6.4 Hz, 1H, Ar), 7.73 (t, J = 6.6 Hz, 1H, Ar), 7.70 (d, J= 6.4 Hz, 1H, Ar),
7.53 (t, J = 6.1 Hz, 1H, Ar), 7.49 (t, J = 5.6 Hz, 1H, Ar), 7.16 (t, J = 5.4 Hz, 1H, Ar), 6.78 (d,
19

J = 6.8 Hz, 1H, Ar), 3.44 (m, 8H, -CH2), 1.64 (m, 3H, -CH3); 13C-NMR (125 MHz, DMSO-
d6): δ 165.2, 164.1, 162.5, 161.4, 148.6, 140.5, 134.3, 133.5, 132.2, 130.4, 130.4, 129.1,
125.2, 118.4, 117.8, 116.5, 104.2, 45.0, 30.4, 28.6, 24.1, 17.6; HR-EI-MS: m/z Calcd for
C22H19N5O2, [M] + 385.1538; found 385.1524.
3.3.24.
(E)-2-(5-(5-isopropyl-2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-
yl)benzonitrile (24)
Light Red solid, Yield: 76%; 1H-NMR:(500 MHz, DMSO-d6): δ 8.12 (s, 1H, -NH), 7.92 (d, J
= 6.4 Hz, 2H, Ar), 7.86 (d, J = 6.1 Hz, 2H, Ar), 7.49 (s, 1H, Ar), 7.37 (d, J = 6.8 Hz, 1H, Ar),
13
6.88 (d, 1H, J = 6.6 Hz, Ar), 2.88 (s, 1H, -CH), 1.77 (m, 6H, -CH3); C-NMR (125 MHz,
DMSO-d6): δ 165.4, 164.3, 162.8, 161.7, 148.2, 141.1, 134.3, 133.5, 132.2, 130.5, 130.1,
129.2, 125.4, 118.2, 117.8, 116.1, 105.0, 60.1, 21.6, 21.6; HR-EI-MS: m/z Calcd for
C20H15N5O2, [M] + 357.1225; found 357.1213.
3.3.25.
(E)-3-(5-(5-isopropyl-2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-
yl)benzonitrile (25)
1
Light yellow solid, Yield: 71%; H-NMR:(500 MHz, DMSO-d6): δ 8.10 (s, 1H, -NH), 8.09
(d, J = 6.6 Hz, 1H, Ar), 7.99 (d, J = 5.9 Hz, 1H, Ar), 7.86 (s, 1H, Ar), 7.58 (t, J = 6.5 Hz, 1H,
Ar), 7.48 (d, J = 6.7 Hz, 1H, Ar), 7.38 (s, 1H, Ar), 6.71 (d, J = 6.7 Hz, 1H, Ar), 2.89 (s, 1H, -
CH), 1.22 (m, 6H, -CH3); 13C-NMR (125 MHz, DMSO-d6): δ 165.2, 164.1, 162.9, 161.8,
149.2, 146.1, 139.3, 133.5, 132.3, 130.4, 130.2, 129.2, 127.4, 127.1, 122.6, 119.2, 117.8,
35.6, 25.4, 25.4; HR-EI-MS: m/z Calcd for C20H15N5O2, [M] + 357.1225; found 357.1213.
3.3.26. (E)-3-(5-(2-oxo-1-pentylindolin-3-ylideneamino)-1,3,4-oxadiazol-2-yl)benzonitrile
(26)
1
Light brown solid, Yield: 81%; H-NMR:(500 MHz, DMSO-d6): δ 8.10 (d, J = 6.5 Hz, 1H,
Ar), 7.98 (s, 1H, Ar), 7.84 (d, J = 6.3 Hz, 1H, Ar), 7.77 (d, J = 6.3 Hz, 1H, Ar), 7.58 (t, J =
6.5 Hz, 1H, Ar), 7.49 (t, J = 6.4 Hz, 1H, Ar), 7.16 (t, J = 6.0 Hz, 1H, Ar), 6.70 (d, J = 6.9 Hz,
13
1H, Ar), 3.90 (m, 8H, -CH2), 1.35 (m, 3H, -CH3); C-NMR (125 MHz, DMSO-d6): δ165.6,
164.1, 162.5, 161.0, 148.1, 133.2, 132.8, 132.2, 131.5, 130.4, 129.5, 127.6, 125.3, 119.3,
118.5, 116.4, 113.6, 46.2, 30.6, 29.5, 24.2, 18.1; HR-EI-MS: m/z Calcd for C22H19N5O2, [M]
+ 385.1538; found 385.1529.
20

3.3.27. (E)-3-(5-(1-butyl-2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-yl)benzonitrile
(27)
1
Light brown solid, Yield: 81%; H-NMR:(500 MHz, DMSO-d6): δ 8.09 (d, J = 6.6 Hz, 1H,
Ar), 7.87 (d, J = 6.4 Hz, 1H, Ar), 7.76 (t, J = 6.6 Hz, 1H, Ar), 7.60 (d, J = 6.4 Hz, 1H, Ar),
7.58 (t, J = 6.3 Hz, 1H, Ar), 7.44 (t, J = 5.9 Hz, 1H, Ar), 7.12 (t, J = 5.3 Hz, 1H, Ar), 6.74 (d,
J = 6.8 Hz, 1H, Ar), 3.88 (m, 8H, -CH2), 1.66 (m, 3H, -CH3); 13C-NMR (125 MHz, DMSO-
d6): δ 165.0, 163.4, 162.1, 161.0, 148.2, 133.0, 132.4, 131.8, 130.9, 130.2, 130.1, 127.6,
125.6, 119.2, 118.6, 116.5, 114.1, 45.2, 30.4, 22.2, 16.4; HR-EI-MS: m/z Calcd for
C21H17N5O2, [M] + 371.1381; found 371.1372.
3.3.28. (E)-3-(5-(2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-yl)benzonitrile (28)
Orange solid, Yield: 71%; 1H-NMR:(500 MHz, DMSO-d6): δ 8.14 (s, 1H, -NH), 8.10 (d, J =
6.7 Hz, 1H, Ar), 7.99 (d, J = 6.6 Hz, 1H, Ar), 7.85 (s, 1H, Ar), 7.78(d, J = 6.4 Hz, 1H, Ar),
7.60 (t, J = 6.6 Hz, 1H, Ar), 7.51 (t, J = 5.6 Hz, 1H, Ar), 7.15 (t, J = 5.1 Hz, 1H, Ar), 6.92 (d,
13
J = 6.5 Hz, 1H, Ar); C-NMR (125 MHz, DMSO-d6): δ 165.1, 164.0, 162.4, 150.2, 143.1,
133.0, 133.0, 131.4, 130.3, 130.1, 128.4, 126.6, 125.6, 120.2, 119.4, 118.4, 113.2; HR-EI-
MS: m/z Calcd for C17H9N5O2, [M] + 315.0755; found 315.0741.
3.3.29. (E)-4-(5-(2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-yl)benzonitrile (29)
Orange solid, Yield: 71%; 1H-NMR: (500 MHz, DMSO-d6): δ 8.10 (s, 1H, -NH), 7.97 (d, J =
9.3 Hz, 2H, Ar), 7.84 (d, J = 9.2 Hz, 2H, Ar), 7.60 (d, J = 9.0 Hz, 1H, Ar), 7.52 (d, 1H, J =
13
8.8 Hz, Ar), 7.50 (t, J = 6.5 Hz, 1H, Ar), 7.08 (t, J = 5.6 Hz, 1H, Ar); C-NMR (125 MHz,
DMSO-d6): δ 165.2, 164.1, 162.6, 151.0, 142.2, 133.2, 133.2, 131.7, 130.8, 130.2, 128.6,
+
126.6, 125.4, 120.3, 119.3, 118.2,113.1; HR-EI-MS: m/z Calcd for C17H9N5O2, [M]
315.0755; found 315.0844.
3.3.30. (E)-2-(5-(2-oxoindolin-3-ylideneamino)-1,3,4-oxadiazol-2-yl)benzonitrile (30)
Orange solid, Yield: 71%; 1H-NMR: (500 MHz, DMSO-d6): δ 8.11 (s, 1H, -NH), 7.95 (d, J =
6.4 Hz, 1H, Ar), 7.86 (d, J = 6.4 Hz, 1H, Ar), 7.72 (t, J = 6.5 Hz, 1H, Ar), 7.60 (t, J = 6.0 Hz,
1H, Ar), 7.51 (d, 1H, J = 6.2 Hz, Ar), 7.49 (t, J = 5.7 Hz, 1H, Ar), 7.08 (t, J = 5.2 Hz, 1H,
13
Ar), 6.93 (d, J = 6.5 Hz, 1H, Ar); C-NMR (125 MHz, DMSO-d6): δ 164.1, 163.0, 161.2,
151.3, 142.6, 141.4, 134.6, 133.5, 132.4, 130.6, 130.6, 129.4, 125.6, 120.3, 118.6, 117.8,
105.4; HR-EI-MS: m/z Calcd for C17H9N5O2, [M] + 315.0755; found 315.0840.
21

3.4. Thymidine phosphorylase assay
Since human TP is not easily to get, we used commercially available recombinant E. coli TP.
Main sequence of TP is frequently preserved throughout evolution as mammalian TP that is
reported to share 39% sequence resemblance with the TP of E. coli. The mammalian enzyme
is also shared up to 70% resemblance with the active site residues, and three-dimensional
structure of E. coli TP enzyme. The Thymidine phosphorylase/PD-ECGF (E. coli) activity
was determined by measuring the absorbance at 290 nm spectrophotometrically [46-48]. In
brief, total reaction mixture of 200 µL contained 145 µL of potassium phosphate buffer (pH
7.4), 30 µL of enzyme (E. coli) at concentration 0.05 and 0.002 U, respectively, were
º
incubated with 5 lL of test materials for 10 min at 25 C in microplate reader. After
incubation, pre-read at 290 nm was taken to deduce the absorbance of substrate particles.
Substrate (thymidine, λmax; 265 nm) 20 µL, 1.5 mM, was dissolved in potassium phosphate
buffer, was immediately added to plate and degradation of thymidine continuously read after
10, 20, and 30 min in 96-well ELISA plate reader (spectra max, molecular devices, CA,
USA). The 7-Deazaxanthine was used as positive control. All assays were performed in
triplicate. The extinction coefficient "e" of product was required to know enzyme activity
related to absorbance. Its Beer Lambert Abs = e c l. The l was pathlength of cuvette and c
was concentration of product that appeared or substrate that disappeared.
3.5 Statistical analysis/Calculations
Results were processed using SoftMax Pro 4.8 software (Molecular Devices, CA, USA) and
then by Microsoft Excel. Percent inhibition for above mentioned biological activities was
calculated by following formula:
Percent Inhibition =100 - (OD test compound / OD control) ×100
3.6. Molecular docking
The docking is a significant tool to explore the interactions between an inhibitor and the
target [49]. To find the binding interactions of these compounds in the active sites of the
thymidine phosphorylase, the MOE-Dock program (www.chemcomp.com) was used to
perform molecular docking. The 3D crystal structure of the thymidine phosphorylase (4EAD)
was retrieved from the Protein Databank (PDB). The synthesized compounds were docked
into the active site of the target enzyme in MOE (www.chemcomp.com) by the default
22

parameters i-e Placement: Triangle Matcher, rescoring 1: London dG, Refinement:
Forcefield, Rescoring 2: London dG. For each ligand ten conformations were generated, and
the top ranked conformation based on docking score was selected for further studies in
molecular docking. After the molecular docking, we analyzed the best poses having polar, H-
pi and pi-H interactions by Pymol software.
Acknowledgements
The authors are thankful to Higher Education commission of Pakistan for financial support
under the National Research Program for Universities, project number 5721& 5092.
References
1. J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin,
D. Forman, F. Bray, Int. J. Cancer. 136 (2015) 359.
2. F. Haviv, M. F. Bradley, D. M. Kalvin, A. J. Schneider, D. J. Davidson, S. M. Majest,
L. M. McKay, C. J. Haskell, R. L. Bell, B. Nguyen, K. C. Marsh, B. W. Surber, J. T.
Uchic, J. Ferrero, Y. C. Wang, J. Leal, R. D. Record, J. Hodde, S. F. Badylak, R. R.
Lesniewski, J. Henkin, J. Med. Chem. 48 (2005) 2838.
3. M. T. Conconi, G. Marzaro, L. Urbani, I. Zanusso, R. Di Liddo, I. Castagliuolo, P.
Brun, F. Tonus, A. Ferrarese, A. Guiotto, A. Chilin, Eur. J. Med. Chem. 67 (2013)
373.
4. L. Jia, S. Ma, Eur. J. Med. Chem. 121 (2016) 209.
5. Y. Y. Elamin, S. Rafee, N. Osman, K. J. O′Byrne, K. Gately, Cancer microenviron. 9
(2016) 33.
6. S. B. Fox, A. Moghaddam, M. Westwood, H. Turley, R. Bicknell, K. C. Gatter, A. L.
Harris, J. Pathol. 176 (1995) 183.
7. A. Bronckaers, F. Gago, J. Balzarini and S. Liekens, Med. Res. Rev. 29 (2009) 903.
8. S. I. Akiyama, T. Furukawa, T. Sumizawa, Y. Takebayashi, Y. Nakajima, S.
Shimaoka, M. Haraguchi, Cancer Sci. 95 (2004) 851-857.
9. A. Moghaddam, R. Bicknell, Biochem. 31 (1992) 12141.
10. T. Furukawa, A. Yoshimura, T. Sumizawa, M. Haraguchi, S. Akiyama, K. Fukui, M.
Ishizawa, Y. Yamada, Nature. 356 (1992) 668-668.
11. N. S. Brown, Jones, A. Fujiyama, C. Harris, A. L. Bicknell, Cancer Res. 60 (2000)
6298.
23

12. K. Usuki, J. Saras, J. Waltenberger, K. Miyazono, G. Pierce, A. Thomason, C. H.
Heldin, Biochem. Biophys. Res. Commun. 184 (1992) 1311.
13. F. Ishikawa, K. Miyazono, U. Hellman, H. Drexler, C. Wernstedt, K. Hagiwara, K.
Usuki, F. Takaku, W. Risau, C. H. Heldin, Nature 338 (1989) 557-562.
14. M. Haraguchi, K. Miyadera, K. Uemura, T. Sumizawa, T. Furukawa, K. Yamada, S.
Akiyama, Y. Yamada, Nature. 368 (1994) 198.
15. M. J. Perez-Perez, E. M.Priego, A. I. Hernandez, M. J. Camarasa, J. Balzarini, S.
Liekens, Mini-ReV. Med. Chem. 12 (2005) 1113.
16. A. Moghaddam, H. T. Zhang, T. P. D. Fan, D. E.Hu, V. Lees, H. Turley, S. B. Fox,
K. C. Gatter, A. L. Harris, R. Bicknell, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 998.
17. H. S. Ibrahim, S. M. Abou-Seri, M. Tanc, M. M. Elaasser, H. A. Abdel-Aziz, C. T.
Supuran, Eur. J. med. Chem. 103 (2015) 583.
18. R. Rohini, P. M. Reddy, K. Shanker, K. Kanthaiah, V. Ravinder, A. Hu, Archi.
Pharma. Res. 34 (2011) 1077-1084.
19. M. Verma, S. N. Pandeya, K. N. Singh, J. P. Stables, Actapharma. (Zagreb, Croatia),
54 (2004) 49.
20. S. N. Pandeya, P. Yogeeshwari, D. Sriram, G. Nath, Ind. J. Pharm. Sci. 64 (2002)
209-212.
21. P. Selvam, N. Murugesh, M. Chandramohan, Z. Debyser, M. Witvrouw, Ind. J.
Pharm. Sci. 70 (2008) 779-782.
22. S. K. Sridhar, A. Ramesh, Biolo. Pharm. Bulletin. 24 (2001) 1149.
23. W. M. Eldehna, A. Altoukhy, H. Mahrous, H. A. Abdel-Aziz, Eur. J. med. Chem. 90
(2015) 684-694.
24. P. Sharma, K. R. Senwar, M. K. Jeengar, T. S. Reddy, V. G. Naidu, A. Kamal, N.
Shankaraiah, Eur. J. med. Chem. 104 (2015) 11.
25. H. Pervez, M. Ramzan, M. Yaqub, K. M. Khan, Letter in Drug Design & Discovery.
8 (2011) 452-458.
26. N. N. Farshori, M. R. Banday, A. Ahmad, A. U. Khan, A. Rauf, Bioorg. Med. Chem.
Lett. 20 (2010) 1933.
27. Z. N. Cui, Y. X. Shi, L. Zhang, Y. Ling, B. J. Li, Y. Nishida, X. L. Yang, J. Agric.
Food Chem. 60 (2012) 11649.
28. Y. H. Li, H. J. Zhu, K. Chen, R. Liu, A. Khallaf, X. N. Zhang, J. P. Ni, Org. Biomol.
Chem. 11 (2013) 3979.
24

29. A. A. El-Emam, O. A. Al-Deeb, M. Al-Omar, Lehmann, J. Bioorg. Med. Chem. 12
(2004) 5107.
30. S. Bansal, M. Bala, S. S. Suthar, S. Choudhary, S. Bhattacharya, V. Bhardwaj, S.
Singla, A. Joseph, Eur. J. Med. Chem. 80 (2014) 167.
31. S. G. Kucukguzel, E. E. Oruc, S. Rollas, F. Sahin, A. Ozbek, Eur. J. Med. Chem. 37
(2002) 197.
32. S. Bondock, S. Adel, H. A. Etman, F. A. Badria, Eur. J. Med. Chem. 48 (2012) 192.
33. A. Husain, M. Ajmal, Acta Pharm. 59 (2009) 223.
34. M. Taha, M. T. Javid, S. Imran, M. Selvaraj, S. Chigurupati, H. Ullah, F. Rahim, F.
Khan, J. I. Mohammad, K. M. Khan, Bioorg. Chem. 74 (2017) 179.
35. F. Rahim, H. Ullah, M. T. Javid, A. Wadood, M. Taha, M. Ashraf, A. Shaukat, M.
Junaid, S. Hussain, W. Rehman, R. Mehmood, M. Sajid, M. N. Khan, K. M. Khan.
Bioorg. Chem. 62 (2015) 15.
36. F. Rahim, K. Zaman, H. Ullah, M. Taha, A. Wadood, M. T. Javed, W. Rehman, M.
Ashraf, R. Uddin, I. uddin, H. Asghar, A. A. Khan, K. M. Khan. Bioorg. Chem. 63
(2015) 123.
37. M. Taha, N.H. Ismail, S. Imran, M. Selvaraj, F. Rahim, RSC Adv. 6 (2016) 3003
38. M. Taha, N.H. Ismail, S. Imran, M. Selvaraj, A. Rahim, M. Ali, S. Siddiqui, F.
Rahim, K.M. Khan, Bioorg. Med. Chem., 23, (2015) 7394.
39. M. Taha, H. Ullah, H.; L. M. R. Muqarrabun, M.N. Khan, F. Rahim, N. Ahmat, M.
Ali, S. Perveen, Eur. J. Med. Chem., 143 (2018) 1757.
40. M. Taha, S. Sultan, H.A. Nuzar, F. Rahim, S. Imran, H. Naz, N.H. Ismail, H. Ullah,
Bioorg. Med. Chem., 24 (2016) 3696.
41. M. Taha, N.H. Ismail, S. Imran, H. Khan, A. Wadood F. Rahim, H. Ullah, Bioorg.
Chem., 68 (2016) 56.
42. M. Taha N.H. Ismail, W. Jamil, K.M. Khan, U. Salar, S.M. Kashif, F. Rahim, Y.
Latif, Med. Chem. Res., 24 (2015) 3166.
43. K.A.N.A. Zawawi, M.Taha, N. Ahmat, A. Wadood, N.H. Ismail, F. Rahim, M. Ali
Bioorg. Med. Chem. 23 (2015) 3119.
44. K.M Khan, F. Rahim, A. Wadood, M. Taha, M. Khan, N. Ambreen, S. Perveen, M. I.
Choudhary, Bioorg. Med. Chem. Lett. 24(2014) 1825.
45. K. M. Khan, F. Rahim, S. A. Halim, M. Taha, M. Khan, Shagufta, Z. Qasmi, S.
Perveen, M. I. Choudhary, Bioorg. Med. Chem. 19 (2011) 4286.
25

46. H. Ullah, F. Rahim, M. Taha, I. Uddin, A. Wadood, S. A. A. Shah, R. K. Farooq, M.
Nawaz, Z. Wahab, K. M. Khan, Bioorg. Chem., 78 (2018) 58.
47. M.Taha, S. A. A.Shah, M. Afifi, S. Imran, S. Sultan, F. Rahim, N. H. Ismail, K.
M.Khan, Bioorg. Chem., 78 (2018) 17.
48. I. Uddin, M.Taha, F. Rahim, A. Wadood, 78 (2018) 324.
49. A. R. Leach, B. K. Shoichet, C. Peishoff, J. Med. Chem. 49 (2006) 5851.
26

Synthesis, SAR elucidations and molecular docking study of newly designed
Isatin based oxadiazole analogs as potent inhibitors of thymidine phosphorylase
Muhammad Tariq Javida, Fazal Rahima, Muhammad Tahab, Mohsan Nawaza, Abdul
Wadoodc, Muhammad Alid, Ashik Mosaddikb, Syed Adnan Ali Shahe,f, Rai Khalid Farooqg
aDepatment of Chemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan.
bDepartment of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University,
P.O. Box1982, Dammam 3144, Saudi Arabia.
cDepartment of Biohemistry, Abdul Wali Khan University Mardan, Mardan-23200, Pakistan.
dUoN Chair of Oman's Medicinal Plants and Marine Natural Products, University of Nizwa, P.O. Box 33, Birkat Al Mauz, Nizwa 616,
Oman
eAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar
Puncak Alam, Selangor D. E. Malaysia.
fFaculty of Pharmacy, Universiti Tecknologi MARA Puncak Alam, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia.
gDepartment of Neuroscience Research, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal
University, P.O. Box 1982, Dammam 3144, Saudi Arabia.
*Corresponding Author: fazalstar@gmail.com, Tel.: 0092-335-9528343
taha_hej@yahoo.com and mtaha@iau.edu.sa (M.T)
27

Highlights:
 Synthesis of isatin based oxadiazole analogs
 In vitro thymidine phosphorylase
 Identification of a new of thymidine phosphorylase inhibitor
 Structure Activity Relationship established
 Molecular docking
28