Design, synthesis, and cytotoxicity screening of 5-aryl-3-(2-(pyrrolyl) thiophenyl)-1, 2, 4-oxadiazoles as potential antitumor molecules on breast cancer MCF-7 cells

Article abstract of DOI:10.1016/j.bioorg.2019.01.067

The work representing the design and the cytotoxic screening of synthetic small molecules (SSMs) such as carbonitriles 3a-c, carboximidamides 4a-c, and oxadiazoles 5–19 as antitumor molecules. Molecules 4c, 9, 12, and 14 show promising cytotoxicity profiles against two cell lines higher than prodigiosin (PG). The results of topoisomerase enzyme inhibition assay show that compounds 4c and 14 display potent inhibitory activity in nano-molar concentration. In addition, DNA-flow cytometry and annexin V analysis also display that compounds 4c, 9, 12, and 14 exhibit antiproliferative activities over MCF-7 cells by cell cycle arrest at G₁ phase and apoptosis-inducing activity by increasing cell percentages at pre G₁ phase. Moreover, Elisa measurement of p53 and apoptosis mediators, show that carboximidamide 4c and oxadiazoles 9, 12, and 14 significantly up-regulate p53 and cell death mediators as puma and Bax/Bcl-2 ratio levels. Subsequently, pro-apoptotic activities are confirmed by active caspase 3/7 percentages green fluorescence assay.

Full text of DOI:10.1016/j.bioorg.2019.01.067

Accepted Manuscript
Design, synthesis, and cytotoxicity screening of 5-aryl-3-(2-(pyrrolyl) thio-
phenyl)-1, 2, 4-oxadiazoles as potential antitumor molecules on breast cancer
MCF-7 cells
Mohammed K. Abd el hameid, Manal R. Mohammed
PII:
S0045-2068(18)30895-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.067
YBIOO 2771
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
18 August 2018
9 January 2019
30 January 2019
Please cite this article as: M.K. Abd el hameid, M.R. Mohammed, Design, synthesis, and cytotoxicity screening of
5
-aryl-3-(2-(pyrrolyl) thiophenyl)-1, 2, 4-oxadiazoles as potential antitumor molecules on breast cancer MCF-7
cells, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.01.067
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Design, synthesis, and cytotoxicity screening of 5-aryl-3-(2-(pyrrolyl) thiophenyl)-1,
, 4-oxadiazoles as potential antitumor molecules on breast cancer MCF-7 cells.
2
a
b
Mohammed K. Abd el hameid , Manal R. Mohammed
a
Organic Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo
b
University, Egypt . Department of Radiation Biology, National Center for Radiation
Research and Technology, Cairo, Egypt.
Dedicated to the memory of Dr. Ibrahim Abouleish
Abstract:
Keywords:
The work representing the design and the cytotoxic screening of synthetic small
molecules (SSMs) such as carbonitriles 3a-c, carboximidamides 4a-c, and oxadiazoles 5-
1
9 as antitumor molecules. Molecules 4c, 9, 12, and 14 show promising cytotoxicity
profiles against two cell lines higher than prodigiosin (PG). The results of topoisomerase
enzyme inhibition assay show that compounds 4c and 14 display potent inhibitory
activity in nano-molar concentration. In addition, DNA-flow cytometry and annexin V
analysis also display that compounds 4c , 9, 12, and 14 exhibit antiproliferative activities
over MCF-7 cells by cell cycle arrest at G
increasing cell percentages at pre G phase . Moreover, Elisa measurement of p53 and
apoptosis mediators, show that carboximidamide 4c and oxadiazoles 9, 12,
1
phase and apoptosis-inducing activity by
1
and 14 significantly up-regulate p53 and cell death mediators as puma and Bax/Bcl-2
ratio levels. Subsequently, pro-apoptotic activities are confirmed by active caspase 3/7
percentages green fluorescence assay.
DNA intercalators, topoisomerase, thiophene, oxadiazole, breast cancer
1
-Introduction:
DNA synthesis has been regarded as one of the most effective targets in cancer
cell growth inhibition and apoptosis induction [1]. Generally, all cancer cells are
characterized by increasing DNA synthesis that has been mainly referred to up-regulation
of DNA topoisomerase (topo) enzymes. Mechanistically, DNA interactive molecules
exert their mode of action as DNA intercalators and topo inhibitors or by dual mode of
action actions. Structurally, the common structural basis for DNA synthesis inhibitors is

the polycyclic molecular skeleton either orthogonal or planar that required for DNA base
pair interaction. Besides that, the molecular skeletons also have to carry side chains that
anchor DNA base pairs or enzyme binding sites by non-covalent interaction [2]. Natural
products are important utility in drug discovery as the diversity of their structures inspires
the discovery of many drugs [3]. Terthiophene (TER), terpyridine and prodigiosin (PG)
(
Fig. 1) are natural compounds with an orthogonal tri-arylated molecular skeleton and
exhibit potent cytotoxic and pro-apoptotic properties toward a variety of cancer cell lines.
The mode of action of these compounds is mainly DNA synthesis inhibition [4-8].
Obatoclax (Teva Pharmaceuticals) (Fig. 1) is an apoptotic inducer agent in clinical trials
over different cancer cell lines and is synthetically derived from prodigiosin [8].
The oxazole, isoxazole and oxadiazole rings are a privileged core in numerous
antitumor molecules like MX 74420(Maxim Pharmaceuticals), SEW 2871(EPI
Corporation), VA-62784 and molecule I (Fig. 1) [9-13]. Interestingly, oxadiazole
derivatives exhibited promising antitumor activities with different modes of actions [14-
1
8]. The mentioned heterocyclic rings represent promising scaffolds in the design of
DNA interacting agents due to the following reasons. They have a similar configurational
ability to act as a rigid spacer between two pharmacophoric arms. In addition, these rings
are hydrogen bond (H-bond) acceptor (A) forming moieties that facilitate binding with
DNA and enzyme binding sites [19]. The mentioned cytotoxic molecules show a
common configurational resemblance in their molecular structures as curved or
orthogonal poly-aryl ring system composed of a core part equipped with two symmetrical
or unsymmetrical aryl groups as side chains. The resistance of cancer cells to apoptosis
induction by antitumor drugs is appeared mainly due to dysfunction in tumor suppressor
gene (p53) and up-regulated expression of cell death modulators as the Bcl-2 protein
family [20]. P53 upregulated modulator of apoptosis (puma) is a downstream protein of
p53 and is expressed as a secondary effect of DNA damage to induce apoptosis in cancer
cells. Puma plays a crucial role in the determination of the Bax/Bcl-2 ratio level in the
cancer cells [21]. The resistance of cancer cells to apoptosis induction for the
chemotherapy is appeared mainly to be due to dysfunction in tumor suppressor gene
(
p53) and up-regulated expression of cell death modulators such as the Bcl-2 protein

family. Therefore, the discovery of novel antitumor molecules enhancing apoptosis-
inducing activities represents a significant challenge in the oncology research.
[
Please insert Fig. 1 about here]
Based on these aforementioned structural similarities and a bio-isosteric relation
between benzene, thiophene and azole rings, synthetic small molecules
(
SSMs) are designed as antitumor agents using the utility of fragment-based drug design
strategy (Fig. 2). The basic structural skeleton of the lead template is formed from
orthogonal 2-(pyrrol-1-yl) thiophene-3-carbonitrile 3a-c. In addition, thiophene
fragments are decorated at C-4 and C-5 positions with three different lipophilic and
conformational moieties as dimethyl and tetra-methylene and 4-methoxyphenyl groups to
give variable skeletal extensions. Furthermore, the thiophene-3-carbonitriles 3a-c are
grafted with a carboximidamide group as (H-bond) acceptor-donner (A-D) pair forming
group via chemical modification of carbonitrile group to reinforce the interaction with the
DNA and/or the enzyme. Besides that, a carboximidamide group also is capable of
forming a pseudo intramolecular H-bond ring that extended the structural skeleton
(
model A, the carboximidamides 4a-c). Finally, the amidoxime group in the
conformers 4a-c is rigidified into oxadiazole with the addition of aryl side chain at the C-
5
5
of the core (model B, oxadiazoles 5-19). The final molecules 5-19 are formed from 3,
-diaryl oxadiazole skeleton with oxadiazole as core ring and unsymmetrical C-3 and C-5
diaryl groups, C-3 moieties are equipped with 2-(pyrrol-1-yl) thiophen-3yl fragments as
bulky side chains and C-5 moieties are substituted phenyl groups) (Fig. 2).
[
Please insert Fig. 2 about here]
2
2
- Discussion:
.1. Chemistry:
The steps for the synthesis of starting materials, intermediates and the target
compounds 3a-c, 4a-c and 5-19 are illustrated in Schemes 1-3. The rationale of using
microwave-assisted organic synthesis (MAOS) in the synthesis of organic molecules is to
create a green road towards sustainable development in the chemical industry [22]. Open
vessel microwave-assisted synthesis (OVMAS) could be used in the large-scale synthesis
and, therefore is suitable for industry [22]. Since Gewald reported his synthetic protocol

for the preparation of 2-aminothiophenes from ketones, active methylene derivatives
and sulfur, several reports were published to improve the reaction conditions [23-26]. In
the current work, 2-aminothiophenes 2a-c were prepared in three component reaction via
reacting ketones 1a-c with propane dinitrile and sulfur with using morpholine as a
catalyst in n-butanol, (high boiling solvent), instead of ethanol under OVMAS conditions
(
Scheme1). The structure of thiophene derivatives 2a-c was confirmed by their reported
physical and spectral data [23-25]. Synthesis of pyrrole ring from primary amines
using dimethoxytetrahydrofuran (DMTHF) under acid catalyst was known as Clauson-
Kaas reaction [27]. In the current work, 2-(pyrrol-1-yl) thiophenes 3a-c were prepared
by reacting 2-aminothiophenes 2a-c with DMTHF in acetic acid as a dual solvent and
catalyst under OVMAS conditions (Scheme 1). Thiophene 3a was reported as a patent
molecule [28], while 2-(pyrrolyl) thiophene 3b is known in the literature by synthesis
under conventional heating with incomplete structural characterization [29]. The
synthesis of 1, 2, 4-oxadiazole ring was reported from the reaction of
carbonitrile, hydroxylamine and acylating agents as carboxylic acids and acid derivatives.
The reaction sequence involves the following mechanistic steps, the hydroxylamine was
added to aryl nitriles to form amidoximes under base catalyzed conditions. After that, the
amidoximes were coupled with the acylating agents to form oxadiazole ring [30-32]. In
this work, oxadiazoles 5-19 (Fig. 3) were prepared by reacting thiophene-3-
carbonitriles 3a-c with hydroxylamine hydrochloride using cesium carbonate as a
catalyst instead of potassium carbonate, (unstable in M.W irradiation), in n-butanol under
OVMAS conditions to produce thiophene-3-carboximidamides 4a-c (Scheme 2).
[
Please insert Fig. 3 about here]
The structure of novel amidoximes 4a-c was established using IR, NMR
spectroscopy, in IR spectra of carboximidamides 4a-c, they show absence of absorption
-1
-1
band at 2218 cm characteristic to nitrile group cm and appearance of absorption bands
-1
1
at the range 3413-3041 cm corresponding to NH
2
and OH groups, while H-NMR
confirm the presence of signals of NH and OH at 4.90-4.95 and 10-63-10.77 ppm. In the
2
second step, amidoximes 4a-c were acylated with appropriate acid chlorides in ethylene
glycol (high boiling solvent) under OVMAS conditions to give 3,5-diaryl 1, 2, 4-
oxadiazoles 5-19 (Scheme 3). Several synthetic trails were performed to prepare

oxadiazoles 10-14 from the nitrile 3b, hydroxylamine, cesium carbonate and acid
chlorides in three-component reaction conditions but the results of all synthetic trails
produced the amide of corresponding nitrile 3b.
2
2
.2-In vitro anti-proliferative studies:
.2.1-In vitro cell growth inhibition:
The molecules 3a-c, 4a-c, and 5-19 are evaluated for anti-proliferative activity
against breast cancer MCF-7 cell line using MTT assay [33] and prodigiosin (PG) is used
as positive control [7,8]. The obtained results are shown in (Table 1and Fig. 4).
[
Please insert Table. 1 about here]
Carboximidamides 4b, 4c and oxadiazoles 9, 11, 12, and 14 with IC50 less than (5
uM) against MCF-7 cells were selected for further cytotoxic evaluation on human colon
cancer HCT-116 cell lines. The results over MCF-7 cells show that SSMs 4c, 9,
1
2, and 14 exhibited cytotoxic activity in sub-micromolar concentration (IC50: 0.19-0.83
μ M). Inspection of the results according to structural variations between the molecules
showed the following remarks. Structural modification of thiophene -3-carbo nitrile 3a-
c into thiophene-3-carboximidamides 4a-c led to improvement in cytotoxicity results,
that may be explained by increasing hydrogen bond formation. Regarding groups present
at C-4 and C-5 of thiophene ring, carbonitrile 3c and carboximidamide 4c exhibited
higher activity within SSMs 3a-c and SSMs 4a-c, indicating that common C-4 (4-
methoxyphenyl) has more contribution in the cytotoxicity than the other two groups.
Meanwhile, in model B, 3-( tetrahydrobenzothiophenyl) oxadiazoles 11, 12, and 14
displayed higher cytotoxicity among oxadiazoles 5-19, suggesting that tetra-methylene
group at thiophene ring has higher cytotoxicity compared with other C-4 and C-5
substituents at thiophene ring. Also, restriction of rotation in amidoxime moiety in
thiophene-3-carboximidamides 4a-c with the addition of different aryl extension may
lead to an increase in cytotoxicity as oxadiazoles 10-14 or decrease in cytotoxic activity
as in SSMs 15-19. In respect to the C-5 aryl groups of oxadiazoles 5-19, SSMs 9, 11,
1
2 and 14 are the most cytotoxic compounds, indicating that 4-methoxyphenyl, 4-
chlorophenyl, and 4-bromophenyl have a positive effect on cytotoxicity. Interestingly, the
molecular skeleton of 3,5-diaryl oxadiazoles 5-19 tolerates only one 4-methoxyphenyl
group for good cytotoxic activity. This observation was confirmed by the low

cytotoxicity results exhibited by SSMs 15-19, where their precursor 4c is a potent
cytotoxic agent. In respect to sensitivity of MCF-7 and HCT-116 cells toward the tested
molecules, MCF-7 cells were more sensitive than HCT-116 cells to the tested
compounds, except 4c (as indicated by the IC50 values). Moreover, thiophene-3-
carboximidamide 4c is more potent than oxadiazole 14 and PG by 2.1- and 4.96- fold
over HCT-116 cells. Besides that, oxadiazoles 12 and 14 with C-5 4-bromophenyl and 4-
methoxyphenyl groups exhibited good cytotoxic effect over HCT-116 cells. According to
these observations, it is concluded that an orthogonal tri- and tetra-orthogonal aryl
molecules 4c and 14 have antitumor activity over both cell lines.
[
Please insert Fig. 4 about here]
2
.2.2. In vitro topoisomerase inhibition assay:
Further topo 2β enzymatic inhibition at 5 different doses was performed for the
selected molecules carboximidamide 4b, 4c and oxadiazoles 9, 11, 12, and 14 to evaluate
their topo 2β IC50 values using PG as a reference. The results are presented in (Table
1
and Fig. 5). The results show that SSMs 4c, 9, 12, and 14 were more potent topo
inhibitors than control that correlate the cytotoxic activity of the compounds to topo
enzyme inhibition. In respect to structural activity correlation, 4-(4-methoxyphenyl)
thiophene 4c displayed
higher
topo
inhibition than tetrahydrobenzothiophene 4b
indicating that, 4-methoxyphenyl is more active than tetramethylene group at thiophene
ring in enzyme inhibition in respect to model A. In addition, oxadiazole 14 is more
enzyme inhibitor than carboximidamide 4b by 4.6- fold, suggesting that, the rigid model
B
displayed higher activity than rotatable model A. Furthermore, 3-
(
(
tetrahydrobenzothiophenyl) oxadiazole 14 showed higher topo enzyme inhibition than 3-
4,5-dimethylthiophenyl) oxadiazole 9, indicating that tetra-methylene group shows
higher activity than dimethyl moieties at thiophene ring in model B. Although 4-(4-
methoxphenyl) thiophene -3-carboximidamide 4c and 5-(4-methoxyphenyl)
oxadiazole 9 are from different models they were nearly equipotent in topo
inhibition, that confirm the role of the 4-methoxyphenyl group in the enzyme inhibition
activity. Oxadiazole 14 with C-5(4-methoxyphenyl) group was more potent than other
oxadiazoles 11 and 12 with C-5 (chlorophenyl and bromophenyl), confirming the
previous finding. Finally, 5-(4-bromophenyl) oxadiazole 12 exhibited higher enzyme

inhibition than 5-(4-chlorophenyl) oxadiazole 11, indicating that the halide nature
contributes to enzyme inhibition activity.
[
Please insert Fig. 5 about here
2
.2.3. In vitro DNA immunofluorescence assay:
On structural basis for SAR studies, carboximidamide 4b, 4c and oxadiazoles 9,
and 14 were selected for DNA binding affinity compared with PG, indirect binding pico
green immunofluorescence assay was performed (Fig. 6). In this assay, the fluorescent
-
pico-green dye reversibly binds DNA to form a persistent fluorescent colored complex.
When the DNA intercalators are added, the dye is displaced from DNA leading to a
decrease in fluorescence [34]. The results showed that model A (SSMs 4b and 4c) had
good binding affinity than model B (9 and 14). The obtained results exhibited a
difference from the results of structural correlation between cytotoxicity, topo enzyme
inhibition assay that may be referred to good abilities of 4b and 4c to form a higher
hydrogen bond with DNA base pair and hence have bitter DNA binding activity
than 9 and 14.
[
Please insert Fig. 6 about here]
2
.2.4. In vitro DNA- flow cytometry and Annexin V/PI staining analysis:
To explore the effect of molecules 4b, 4c, 9, and 14 on cell growth inhibition and
pro-apoptotic characters, the molecules were subjected to DNA flow cytometry cell cycle
analysis. The results are shown and summarized in (Fig. 7). The distribution pattern of
cell cycle phases for MCF-7 cells is a significantly changed after incubation with tested
molecules at their IC50 compared with PG and untreated cells [35]. The molecules
induced a significant accumulation percentage of the cells in G
5 - fold and G /M phases by 1.4-, 1.8-, 1.5- and 1.8- fold respectively, compared with
untreated cells, indicating that the tested molecules have cell cycle arrest at G phase and
0
phase by 8-, 13-, 9.5- and
1
2
1
pro-apoptotic activities. In addition, only, SSMs 4c, 14 and PG induced a significant
decrease in the cell population at S phase by 2 -, 1.6- and 1.3- fold respectively,
compared with untreated cells. It could be concluding that, tested molecules 4c and 14
exhibited potent anti-proliferative activity than compounds 4b and 9.
[
Please insert Fig. 7 about here]

The annexin-V/PI double staining assay further confirm that apoptosis events
occurred in MCF-7 cells. The results were shown and summarized in (Fig. 8). Results of
the assay showed both stages of apoptotic MCF-7 cells increase by 4b, 4c, 9, and 14 by 8
-
, 11-, 9 -, and 15 - fold compared with untreated cells. Moreover, SSM 14 exhibited a
profile of strong apoptotic inducer over MCF-7 cells by 1.2- fold than PG. In conclusion,
data indicate that SSM 4b, 4c, 9, and 14 were a potent inducer of apoptosis over MCF-7
cells and compound 14 could induce apoptosis percentage higher than PG, SSM 4c was
slightly less potent than PG, meanwhile SSMs 4b and 9 showed equal apoptotic inducing
activity to each other and lower than PG.
[
Please insert Fig. 8 about here]
2
.2.5. In vitro ElISA immunoassay for p53 and cell death modulators:
P53 is the main regulator of the intrinsic apoptotic pathway induced by different
stimulations via expression of Puma. Therefore, ELISA-immunoassay for p53, puma, Bax
and Bcl-2 measurement in MCF-7 cells for the effect of molecules 4b, 4c, 9, and 14 at
their IC50 compared with untreated cells and PG is performed. The results were presented
in (Fig. 9 A-E). The data indicated that SSMs 4b, 4c, 9, and 14 up-regulated p53 levels
by 12-, 17-, 19.5-, and 25.7- fold, respectively, higher than untreated cells (Fig. 9A).
Molecules 4c and 9 were nearly equivalent in their up-regulation and both are less than
1
4 and PG. Molecule 14 increased p53 level by 1.1- fold higher than PG. Similarly,
SSMs 4b, 4c, 9, and 14 increased puma level by 4.5-, 6.5-, 5-, and 7.0-, fold, respectively
more than untreated cells (Fig. 9B). Meanwhile, SSM 14 increased level of puma higher
than PG by 1.2-fold and 4c is equipotent compound to PG. This increasing in p53 and
puma levels most probably caused by DNA damage effect of the tested compounds.
Subsequently, to increase in p53 and puma levels, the data showed that, an
increase in Bax and a decrease in Bcl-2 levels (Fig. 9C, D and E). Compounds 4b, 4c, 9,
and 14 increase Bax/Bcl-2 ratio by 4.3-, 8.5-, 3.5, and 9.5- fold to untreated cells. SSM
1
4 has higher Bax/Bcl-2 ratio by 1.3-fold than PG (Fig. 9E). The data obtained from p53,
puma and Bax/Bcl-2 ratio support the previous elucidated structure activity correlation
between the tested compounds.
[
Please insert Fig. 9 about here]

2
.2.6. In vitro green flow cytometry assay for active caspase 3/7:
Caspase-3/7 enzymes are the terminal downstream of caspases activation that
catalyzed the apoptosis process either intrinsic or extrinsic pathways. Treated MCF-7
cells with compounds 4b, 4c, 9, 14, and PG at their IC50 are subjected to green
immunofluorescence to measure active caspase-3/7 percentages. The data show that
carboximidamides 4b,4c and oxadiazoles9, 14 increased active caspase-3/7 percentages
by 3-, 8.6-, 6.4-, 10-fold, respectively, more than untreated MCF-7 cells as seen in (Figs.
1
0). Additionally, carboximidamide 4c increased caspase 3/7 higher than oxadiazole 9.
Moreover, carboximidamide 4c and oxadiazole14 increased caspase-3/7 percentages by
.1- and 1.3- fold than PG. As a final conclusion from biological screening results,
molecules 4b, 4c, 9, and 14 are cytotoxic agents and trigger apoptosis induction in MCF-
cells as a secondary effect to DNA damage with subsequent secondary increasing in
1
7
p53 and Puma levels that overcome the resistance conferred by Bcl-2 proteins via an
increase in Bax/Bcl-2 ratio and activation of terminal caspase 3/7 proteins.
[
Please insert Fig. 10 about here]
3
-Conclusion:
Briefly, a small library of carbonitriles 3a-c, carboximidamides 4a-c and
oxadiazoles 5-19 is designed as potential anticancer molecules. The following steps
demonstrate the synthetic protocol, where thiophene derivatives 2a-c are synthesized in
three component Gewald reaction. In addition, the amino group of thiophene-3-
carbonitrile 2a-c is cyclized into pyrrole ring (thiophenes 3a-c) using DMTHF.
Furthermore, thiophene-3-nitriles 3a-c were converted to thiophene-3-carboxmidoximes
4
a-c via reaction with hydroxyl amine. Finally, carboximidamides 4a-c were cyclized
into oxadiazoles 5-19 by reaction with different aryl acid chlorides. The cytotoxicity
results regarding MCF-7 cells show that, carboximidamide 4c and oxadiazoles 9, 12 and
1
4 show IC50 in sub-micmolar concentration and more potent by 2.3-, 4-, 2.5-, and 10 -
fold than PG. Meanwhile, molecules 4c, 12, and 14 exhibited higher cytotoxic activity by
-, 1.8-, and 2.4- fold, respectively than control on HCT-116 cells. The anti-proliferative
5
activity of tested molecules is correlated to topoisomerase enzyme inhibition, where
carboximidamide 4c and oxadiazoles 9 and 14 were potent enzyme inhibitors by 2-, 2 -,

and 2.7 -fold, respectively than PG. Moreover, the cell cycle flow cytometry analysis
indicated that compounds 4c, 9, 11, and 14 induce cell cycle arrest at G phase of MCF-7
1
cells. In addition, molecule 14 is stronger apoptotic inducer than PG, as demonstrated by
the results of Annexin assay. ELISA measurements for p53 and cell death modulators
(
Puma, Bax and Bcl-2) show that carboximidamide 4c and oxadiazoles 9, 12, and 14
induce upregulation of p53 and increased Puma, and Bax/Bcl-2 ratio levels. The results
of the green fluorescence assay exhibited that carboximidamide 4c and oxadiazoles 9, 11,
and 14 trigger apoptosis induction via increase active caspase 3/7 percentage. Finally,
carboximidamide 4c and oxadiazole 14 represent promising antitumor and apoptotic
agents.
4
4
4
- Experimental:
.1. Chemistry:
. 1. 1. General:
Melting points of the synthesized molecules are measured with a Stuart melting
point apparatus and are uncorrected. The NMR spectra of molecules are measured by
Varian Gemini-300BB 400 MHz FT-NMR spectrometers (Varian Inc., Palo Alto, CA).
1
13
H
and
C
spectra are run at 400 and 100MHz, respectively, in
). IR spectra of molecules are recorded with a
deuterateddimethylsulphoxide (DMSO-d
6
Bruker FT-IR spectrophotometer. Electron impact (EI) mass spectra of molecules are
measured on Hewlett Packard 5988 spectrometer. The analysis of elements is carried out
at The Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt.
Reactions progresses monitored by thin layer chromatography (TLC) on silica gel
precoated plates. The solvent system used for TLC is hexane–ethyl acetate (8.5: 1.5 ml)
mixture and the appeared spots are visualized by UV lamp. Silica gel (200–300) mesh is
used for Column chromatography. Unless otherwise noted, all solvents and reagents are
commercially available and used without further purification. The M.W. work synthesis
workstation is Sieno-Mass-II microwave (M.W.) with the specification (2.45GMHz,
1
000 W).
4
.1.2. Synthesis of 2-amino-thiophene-3-carbonitriles 2a-c: starting materials 2a-c were
prepared via Gewald three component reaction using n-butanol as high boiling solvent

instead of ethanol, in brief, a mixture of appropriate ketones 1a-c (20.0 mmol), propane
dinitrile (3.39 g, 30.0 mmol), and sulfur (3.2 g 10.0mmol) in n-butanol (15 ml) was
stirred and morpholine (2.61 g, 30.0 mmol) was added dropwise at room temperature
0
with cooling in ice bath. After that, the mixture was irradiated at 150 C for 10 min in a
M.W. reactor. The mixture was cool to room temperature and diluted with petroleum
ether (20 ml). The formed brown precipitate is crystallized from ethanol and afford
thiophenes 2a-c. The structure of 2-aminothiophenes 2a-c was confirmed by their
reported physical and spectral data 2a [24], 2b [23] and 2c [25].
4
.1.3. Synthesis of 2-(1H-pyrrol-1-yl) thiophene-3-carbonitriles 3a-c: A mixture of
dimethoxytetrahydrofuran (DMTHF) (0.82 g, 6.2 mmol) and glacial acetic (3 ml) was
stirred for 5 min at room temperature, followed by the addition of thiophene-3-
0
carbonitriles 2a-c (0.62 mmol). The reaction mixture was irradiated at 100 C for 15 min
in the M.W. reactor. After cooling, the mixture is diluted with H
2
O (20 ml) and the
precipitate formed is crystallized from ethanol to furnish thiophenes 3a-c.
4
.1.3.1. 4, 5-Dimethyl-2-(1H-pyrrol-1-yl) thiophene-3-carbonitrile (3a): Yield: 0.9 g (74
o
1
%
) White crystal; m.p. 50-53 C. HNMR (DMSO-d
6
), δ:2.24 (s, 3H, CH ), 2.27 (s, 3H,
3
1
3
CH
3
), 6.27-6.49 (t, 2H, ArH), 6.52-6.59 (d, 2H, 8.7 Hz, ArH) ppm. C-NMR (DMSO-
-
1
d
2
1
4
6
), δ: 9.9, 12.1, 84.6, 108.4, 115.4, 116.0, 123.2, 135.0, 158.7. IR (KBr, ν cm ): 2984-
896 (aliph.CH) , 2215 (CN). Anal.Calcd.for C11
H
10
N S (202.28): C, 65.32; H, 4.98; N,
2
3.85; Found: C, 65.29; H, 4.84 ; N, 13.72.
.1.3.2. 2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrbenzo[b]thiopthene-3-carbonitrile (3b):
o
o
1
Yield: 1.1g (81 %) White crystal ; m.p. 73-76 C (lit ,63-65 C)[24]. HNMR (300 MHz,
DMSO-d ), δ: 1.73-1.79 (m, 4H, 2CH ), 2.65-2.69 (m, 4H, 2CH ), 6.18-6.19 (m, 2H,
ArH), 6.91-6.92 (m, 2H, ArH) ppm. C-NMR (DMSO-d ), δ: 20.4, 22.9, 23.4, 24.5,
6
2
2
1
3
6
8
4
1
6
4.0, 108.5, 115.3, 123.2, 134.5, 141.0, 158.1.
.1.3.3. 3-(4-Methoxyphenyl)-2-(1H-pyrrol-1-yl) thiophene-3-carbonitrile (3c) : Yield:
o
1
.2 g( 76 %), White crystal; m.p. 122-123 C. H-NMR (DMSO-d
.40-6.50(m, 3H, ArH), 6.91-7.05 (m, 4H, ArH), 7.32-7.45 (m, 2H, ArH) ppm. C-NMR
), δ: 56.40, 82.3, 108.5, 115.9, 117.9, 123.7, 124.3, 127.8, 128.0, 129.4, 141.2,
6
), δ: 3.71(s, 3H, CH ),
3
1
3
(
DMSO-d
6
-1
1
60.2. IR (KBr, ν cm ): 3023 (arom.CH), 2983-2892 (aliph.CH), 2218(CN);

Anal.Calcd.for C16
H
12
2
N OS (280.34): C, 68.55; H, 4.31; N, 9.99; Found: C, 68.29; H,
4
4
3
.64 ; N, 10.12.
.1.4. N-Hydroxy-2-(1H-pyrrol-1-yl) thiopthene-3-carboximidamides 4a-c. Thiophene
-carbonitrile derivatives 3a-c (8 mmol) were dissolved in n-butanol (15 ml) and treated
with Cs CO
3
(5.2 g, 16 mmol) and hydroxyl amine hydrochloride (1.0 g, 14.8 mmol) with
0
stirring for 5 min at room temperature. The reaction mixture was irradiated at 140 C for
2
0 min in M.W. reactor. After cooling to room temperature, the mixture was diluted with
diethyl ether (20 ml) and the precipitate formed is collected by filtration, washed with
ethanol, and dried under vacuum to give carboximidamides 4a-c.
4
.1.4.1. N-Hydroxy-4, 5-dimethyl-2-(1H-pyrrol-1-yl) thiopthene-3-carboximidamide
o
1
(
4a): Yield: 1.1 g (59 %) brown solid; m.p. 250-253 C. H-NMR (DMSO-d
6
), δ: 2.25 (s,
O exchangeable), 6.23-6.38 (m, 1H, ArH),
.44-6.49 (d, 1H, 8.7 Hz, ArH), 6.95-6.97 (d, 2H,ArH), 10.63 (s,1H, D O exchangeable),
), δ: 10.4, 10.9, 108.3, 120.7,
23.0, 131.5, 134.0, 136.0, 163.2. IR (KBr, ν cm ): 3412-3090 (NH & OH), 3054
OS (235.31): C, 56.15; H,
3
6
1
1
H, CH
3
), 2.31 (s, 3H, CH
3
), 4.95 (s, 1H, D
2
2
1
3
0.73 (s,1H, D
2
O exchangeable) ppm. CNMR (DMSO-d
6
-
1
(
arom.CH), 2983-2891 (aliph.CH). Anal.Calcd.for C11
H
13
N
3
5
4
.57; N, 17.86; Found: C, 56.28; H, 5.71 ; N, 17.61.
.1.4.2. N-Hydroxy-2-(1H-pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiopthene-3-
o
1
carboximidamide (4b): Yield: 1.40g (67 %), yellow solid; m.p. 262-265 C. H-NMR
(
DMSO-d
6
), δ: 1.60-1.78 (m, 4H, 2CH
2
), 2.72-2.79, (m, 4H, 2CH
2
), 4.90 (s, 2H, D
2
O
O
exchangeable), 6.14-6.27 (m, 2H, ArH), 6.91-6.97(m, 2H, ArH), 10.77 (s, 1H, D
2
1
3
exchangeable) ppm. ; C-NMR (DMSO-d ), δ: 20.4, 23.4, 24.5, 24.6, 108.3, 119.6, 123.1,
6
-
1
1
2
1
4
27.7, 135.6, 137.4, 163.2. IR (KBr, ν cm ): 3413-3110 (-NH &OH), 3054 (arom.CH),
984- 2890 (aliph.CH). Anal.Calcd.for C13
6.08; Found: C, 59.82.; H, .93 ; N, 16.11.
H
15
3
N OS (261.09): C, 59.74; H, 5.79; N,
.1.4.3.N-Hydroxy-4-(4-methoxyphenyl)-2-(1H-pyrrol-1-yl)
thiopthene-3-
o
1
carboximidamide (4c): Yield: 1.45g (58 %), yellow solid; m.p. 286-289 C. H-NMR
300 MHz, DMSO), δ : 3.86 (s, 3H, CH ), 4.92 (s, 2H, D O exchangeable), 6.19-6.24 (m,
H, ArH), 6.34-6.46 (m, 1H, ArH), 6.80-6.85 (s, 2H, ArH), 6.91-7.07 (s, 2H, ArH), 7.39-
(
3
2
2
7
5
1
3
.47 (m, 2H, ArH), 10.76 (s, 1H, D
2
O exchangeable) ppm. CNMR (DMSO-d ), δ :
6
6.4, 108.2, 114.8, 118.3, 123.1, 123.3, 127.0, 128.5, 137.4, 138.2, 160.6, 163.4. IR

-1
(
KBr, ν cm ): 3373-3041 (-NH &OH), 3054 (arom.CH), 2997-2983 (aliph.CH).
Anal.Calcd.for C16 S (313.37): C, 61.32; H, 4.82; N, 13.41; Found: C, 61.86; H,
H
15
N
3
O
2
4
.94 ; N, 13.33.
.1.5. Synthesis of 5-aryl-3-(2-(1H-pyrrol-1-yl)-thiophen-3-yl)-1, 2, 4-oxadiazoles 5-19.
4
Thiophene-3-carboximidoximes 4a-c (0.8 mmol) were dissolved in ethylene glycol (10
ml), then the appropriate acid chloride (0.8 mmol) was added dropwise with cooling and
the reaction mixture was stirred at room temperature for 2 min. The reaction mixture was
0
irradiated at 200 C for 5 min. After cooling, the mixture was diluted with a saturated
aqueous NaHCO
3
solution (10 ml) and extracted with ethyl acetate (3 × 15 ml). The
SO and evaporated under reduced pressure.
combined organic layers were dried over Na
2
4
The obtained residue was purified by flash chromatography using a mixture of ethyl
acetate: pet. ether (1:9) to give oxadiazoles 5-19.
4
.1.5.1. 3-[4, 5-Dimethyl-2-(1H-pyrrol-1-yl) thiophen-3-yl]-5-phenyl-1, 2, 4-oxadiazole
o
1
(
5): Yield: 0.19 g (75 %), yellow powder; m.p. 98-100 C. HNMR(DMSO-d
H, CH ), 2.33 (s, 3H, CH ), 6.31-6.46 (m, 2H, ArH), 6.50-6.61 (m, 3H, ArH), 6.65-7.28
m, 2H, ArH), 7.29-7.39 (m, 2H, Ar H) ppm. C-NMR (DMSO-d
17.3, 124.0, 124.2, 127.5, 128.7, 129.2, 131.7, 134.7, 139.4, 143.5, 166.5, 176.9 ppm.
6
), δ: 2.23 (s,
3
3
3
1
3
(
6
), δ: 11.1, 13.2, 110.4,
1
-1
+
IR (KBr, ν cm ): 3021 (arom.CH) , 2993-2981 (aliph.CH). MS (m/z, %): 321.06 (M ,
5.21), 315.19 (100). Anal.Calcd.for C18 OS (321.4): C, 67.27; H, 4.70; N, 13.07;
Found: C, 67.28; H, 4.74 ; N, 13.31.
.1.5.2. 5-(4-Chlorophenyl)-3-[4, 5-dimethyl-2-(1H-pyrrol-1-yl)thiophen-3-yl]-1, 2, 4-
1
H
15
N
3
4
o
1
oxadiazole (6):Yield: 0.16 g (55%), brown powder. m.p. 153-157 C ; H-NMR (DMSO-
d
7
1
6
), δ: 2.28 (s, 3H, CH
.38-7.40 (d, 2H, ArH), 7.73 (s, 2H, ArH) ppm. C-NMR (DMSO-d
10.4, 124.0, 124.2, 127.5, 128.7, 129.2, 131.7, 134.7, 139.5, 143.5, 166.4, 176.9 ppm.
3
), 2.31 (s, 3H, CH ), 6.54 (s, 2H, ArH), 7.05-7.08 (d, 2H, ArH),
3
1
3
6
) δ: 11.1, 13.2,
-1
+
IR (KBr,νcm ) : 3021 (arom.CH), 2997-2979 (aliph.CH). MS (m/z, %): 355.22 (M ,
1
1
4
0.25), 331.32 (100). Anal.Calcd.for C18
H
14ClN OS (355.84): C, 60.76; H, 3.97; N,
3
1.81; Found: C, 60.70; H, 3.84 ; N, 11.71.
.1.5.3. 5-(4-Bromophenyl)-3-[4, 5-dimethyl-2-(1H-pyrrol-1-yl) thiophen-3-yl]1, 2, 4-
o
1
oxadiazole (7): Yield: 0.19 g (59 %), yellowish white powder. m.p. 132-135 C. H-NMR
DMSO-d ) , δ : 2.26 (s, 3H, CH ), 2.30 (s, 3H, CH ), 6.55-6.96 (m,4H, ArH), 7.25-7.66
(
6
3
3

1
3
(
m, 4H, ArH) ppm. C NMR (DMSO-d
6
), δ: 11.1, 13.2, 110.4, 124.0, 125.4, 126.4,
-1
1
27.5, 129.3, 131.7, 134.8, 139.5, 143.5, 166.5, 176.9 ppm. IR (KBr, νcm ): 3019
+
(
arom.CH), 2995-2980 (aliph.CH) . MS (m/z, %): 399.12 (M , 15.28), 345.51 (100)
.
Anal.Calcd.for C18 OS (400.29) : C, 54.01; H, 3.53; N, 10.50, found : C, 54.22;
H
14BrN
3
H, 3.61; N, 10,38.
4
.1.5.4. 3-[4, 5-Dimethyl-2-(1H-pyrrol-1-yl)thiophen-3-yl]-5-(4-tolyl)1, 2, 4-oxadiazole
o
1
(
8): Yield: 0.13 g (49 %), yellow powder ; m.p. 88-90 C . H-NMR (DMSO-d
s, 3H, CH ), 2.34 (s, 3H, CH ), 2.40 (s, 3H, CH ), 6.55-6.89 (m, 4H, ArH), 7.21-6.26 (m,
H, ArH), 7.54-7.63 (m, 2H, ArH) ppm. C-NMR (DMSO-d ) δ: 11.1, 13.2, 22.1,
10.4, 124.0, 124.1, 127.5, 128.7, 129.2, 131.7, 134.7, 139.4, 143.5, 166.5, 175.8 ppm.
6
), δ : 2.30
(
3
3
3
1
3
2
1
6
-1
+
IR (KBr, νcm ): 3027 (arom.CH), 2997-2984 (aliph.CH) . MS (m/z, %): 334.11 (M ,
5.51), 301.54 (100). Anal.Calcd.for C19 OS (335.42): C, 68.03; H, 5.11;N, 12.53;
Found: C, 68.14; H, 5.23 ; N, 12.63.
.1.5.5.3-[4,5-Dimethyl-2-(1H-pyrrol-1-yl)thiophen-3-yl]-5-(4-methoxyphenyl)-1, 2, 4-
3
H
17
N
3
4
o
1
oxadiazole (9): Yield: 0.16 g (56 %) ,yellow powder; m.p. 167-170 C ; H-NMR (DMSO-
), δ: 2.24 (s, 3H, CH ), 2.38 (s, 3H, CH ), 3.80 (s, 3H, -OCH ), 6.33-6.42 (m, 2H,
ArH), 6.44-6.60 (m, 1H, ArH), 6.61-6.68 (m, 3H, ArH), 6.82-6.93 (m, 2H, Ar H) ppm,
d
6
3
3
3
1
3
C-NMR (DMSO-d ), δ: 11.1, 13.2, 57.4, 110.4, 124.1, 125.2, 126.4, 127.5, 129.2,
6
-
1
1
2
31.7, 134.7, 139.4, 143.4, 166.4, 176.8 ppm. IR (KBr,νcm ): 3020 (arom.CH), 2996-
+
982 (aliph.CH). MS (m/z, %): 351.17 (M , 60.22), 315.41 (100). Anal.Calcd.for
C
19
H
17
N
3
2
O S (351.42): C, 64.94; H, 4.88;N, 11.96; Found: C, 64.74; H, 4.68 ; N, 11.76.
4
4
.1.5.6.3-[2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiophen-3-yl]-5-phenyl-1, 2,
o
1
-oxadiazole (10): Yield: 0.22 g (79 %), orange powder; m.p. 141-146 C. H-NMR
), δ: 1.73-1.82 (m, 4H, 2CH ), 2.71-2.91 (m, 4H, 2CH ), 6.86-7.01 (m, 2H,
ArH), 7.13-7.29 (m, 3H, ArH), 7.37-7.53 (m, 4H, ArH) ppm; C-NMR (DMSO-d ), δ :
2.3, 22.6, 25.7, 25.9, 115.0, 117.6, 127.0, 128.3, 131.7, 132.3, 133.4, 134.0, 135.7,
(
DMSO-d
6
2
2
1
3
6
2
1
-1
45.6, 164.0, 176.6 ppm. IR (KBr, νcm ) : 3015 (arom.CH), 2993-2980 (aliph.CH). MS
+
(
m/z, %): 347.13 (M , 10.58), 151.22 (100). Anal.Calcd.for C20
H
17
3
N OS (347.43): C,
6
4
9.14; H, 4.93;N, 12.09; Found: C, 69.34; H, 4.90 ; N, 12.11.
.1.5.7.3-[2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiophen-3-yl]-5-(4-chloro
o
phenyl)-1, 2, 4-oxadiazole (11): Yield: 0.19 g (63 %), yellow powder; m.p. 152-156 C.

1
H-NMR (DMSO-d
m, 1H, ArH), 6.67-6.79 (m, 4H, ArH), 7.30-7.43 (m, 3H, ArH) ppm. C-NMR
DMSO-d ):δ: 22.5, 22.7, 25.8, 26.0, 111.2, 122.3, 123.1, 124.2, 125.1, 127.3, 128.1,
29.3, 140.3, 142.4, 166.7, 176.8 ppm. IR (KBr, νcm ): 3034 (arom.CH), 2997-2981
6
), δ: 1.75-1.80 (m, 4H, 2CH
2
), 2.61-2.91 (m, 4H, 2CH
2
), 6.47-6.52
1
3
(
(
6
-1
1
+
(
aliph.CH). MS (m/z, %): 381.16 (M , 10.83), 187.32 (100). Anal.Calcd.for
OS (381.88): C, 62.90; H, 4.22; N, 11.00; Found: C, 62.80; H, 4.04 ; N,
C
20
H16ClN
3
1
0.81.
4
.1.5.8. 3-[2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiophen-3-yl]-5-(4-bromo
o
1
phenyl)-1, 2,4-oxadiazole (12): Yield: 0.23 g (66 %), red powder; m.p.121-125 C. H-
NMR (DMSO-d ), δ: 1.74-1.80 (m, 4H, 2CH ), 2.67-2.73 (m, 4H, 2CH ), 6.16-6.20 (m,
H, ArH), 6.38 (s, 3H, ArH), 6.95-6.98 (m, 2H, ArH), 7.24 (s, 1H, ArH), 7.45 (s, 1H,
6
2
2
1
1
3
ArH) ppm; C-NMR (DMSO-d
6
):δ: 22.6, 22.8, 25.2, 26.0, 106.0, 121.2, 121.8, 129.5,
-1
1
30.7, 132.0, 133.7, 134.8, 136.1, 140.0 , 166.2, 175.4 ppm. IR (KBr, νcm ): 3034
+
(
arom.CH), 2994-2983 (aliph.CH). MS (m/z, %): 425.44 (M , 5.98), 331.61 (100).
Anal.Calcd.for C20 OS (426.33): C, 56.34; H, 3.78;N, 9.86; Found: C,56.44; H,
H16BrN
3
3
4
2
.84 ; N, 9.71.
.1.5.9. 3-[2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiophen-3-yl]-5-(4-tolyl)-1,
o
1
, 4-oxadiazole (13): Yield: 0.17 g (60 %), red powder; m.p. 142-146 C. H-NMR
), δ: 1.76-1.95 (m, 4H, 2CH ), 2.15 (s, 3H, CH ), 2.65-2.91 (m, 4H, 2CH ),
.44-6.60 (m, 2H, ArH), 6.62-6.84 (m, 3H, ArH), 6.99-7.05 (m, 2H, ArH), 7.16-7.46(m,
(
DMSO-d
6
2
3
2
6
1
1
3
H, ArH) ppm. C-NMR (DMSO-d
6
) , δ: 21.4, 22.6 , 22.8, 25.2, 26.0, 112.2, 121.0,
-
1
24.6, 125.8, 128.8, 131.4, 133.5, 138.1, 140.9, 141.0, 167.7, 173.1 ppm. IR (KBr, νcm
1
+
)
: 3031 (arom.CH), 2995-2980 (aliph.CH). MS (m/z, %): 361.21 (M , 35.65), 257.51
(
100). Anal.Calcd.for C21
H
19
3
N OS (361.46): C, 69.78; H, 5.39; N, 11.63; Found: C,
6
4
9.88; H, 5.44 ; N, 11.71.
.1.5.10. 3-[2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiophen-3-yl]-5-(4-
methoxyphenyl)-1, 2, 4-oxadiazole (14): Yield: 0.18g (58 %) , buff solid; m.p. 133-
o
1
1
37 C; H-NMR (DMSO-d
s, 3H, -OCH ), 6.16-6.20 (m, 2H, ArH), 6.21-6.38 (m, 2H, ArH), 6.95-6.98 (m, 2H,
ArH), 7.23-7.56 (m, 2H, ArH) ppm. C-NMR (DMSO-d
05.9, 112.2, 121.4, 126.0, 128.7, 132.6, 133.6, 135.6, 145.7, 153.1, 166.0, 176.7 ppm.
6
), δ: 1.74-1.80 (m, 4H, 2CH
2
), 2.60-2.69 (m, 4H, 2CH ), 3.84
2
(
3
1
3
6
), δ: 22.2, 22.9, 25.4, 25.6, 56.5,
1

-1
+
IR (KBr, νcm ): 3024 (arom.CH) , 2990-2982 (aliph.CH) . MS (m/z, %): 377.12 (M ,
0.05), 343.56 (100). Anal.Calcd.for C21 S (377.46): C, 66.82; H, 5.07;N,11.13;
Found: C, 66.77; H, 5.14 ; N, 11.10.
.1.5.11. 5-Phenyl-3-(4-methoxyphenyl-2-(1H-pyrrol-1-yl)thiophen-3-yl)-1, 2, 4-
1
H
19
N
3
O
2
4
o
1
oxadiazole (15): Yield: 0.23 g (71%), orange powder; m.p. 122-126 C. H-NMR
DMSO-d ), δ: 3.84 (s, 3H, -OCH ), 6.20-6.34 (m, 2H, ArH), 6.36-6.66 (m, 2H, ArH),
.70-6.73 (m, 3H, ArH), 6.98-7.08 (m, 3H, ArH), 7.14-7.18 (m, 2H, ArH), 7.53-7.67 (m,
(
6
3
6
2
1
1
3
H, ArH) ppm. C-NMR (DMSO-d
6
):δ: 55.8, 111.7, 112.0, 117.1, 118.7, 119.5, 121.5,
21.6, 122.7, 129.5, 130.7, 132.5, 134.8, 135.9, 136.8, 149.0, 165.9, 176.6 ppm. IR
-1
+
(
KBr, νcm ): 3028 (arom.CH), 2998-2980 (aliph.CH) .MS (m/z, %): 399.10 (M , 39.23),
77.16 (100). Anal.Calcd.for C23 S (399.46): C, 69.15; H, 4.29; N, 10.52; Found:
C, 69.23; H, 4.50 ; N,10.71.
3
H
17
N
3
O
2
4
1
.1.5.12. 5-(4-Chlorophenyl)-3-[4-(4-methoxyphenyl)-2-(1H-pyrrol-1-yl)thiophen-3-yl)-
o
1
, 2, 4-oxadiazole (16): Yield: 0.20 g (59%), red powder; m.p. 166-170 C. H-NMR
), δ: 3.82 (s, 3H, OCH ), 6.26-6.36 (m, 3H, ArH), 6.76-6.79 (m, 4H, ArH),
.99-7.29 (m, 4H, ArH), 7.32-7.39(m, 2H, ArH) ppm. C-NMR (DMSO-d
), δ: 56.5, 106.0, 112.3, 113.2, 121.2, 129.5, 130.6, 132.0, 132.1, 134.9,
(
DMSO-d
6
3
1
3
13
6
6
): C NMR
(
DMSO-d
6
-
1
1
2
36.8, 140.0, 146.0, 149.0, 153.3, 167.0, 175.4 ppm. IR (KBr, νcm ): 3031 (arom.CH) ,
+
993-2982 (aliph.CH) . MS (m/z, %): 433.10 (M , 5.25), 367.33 (100). Anal.Calcd.for
C
23
H16ClN
3
O
2
S (433.91): C, 63.66; H, 3.72; N, 9.68; Found: C, 63.88; H, 3.84 ; N, 9.71.
4
1
.1.5.13. 5-(4-Bromophenyl)-3-[4-(4-methoxyphenyl)-2-(1H-pyrrol-1-yl)thiophen-3-yl)-
o
1
, 2, 4-oxadiazole (17): Yield: 0.25 g (65%) ,brown solid; m.p. 172-173 C. H-NMR
), δ: 3.81 (s, 3H, -OCH ), 6.20-6.21 (m, 2H, ArH), 6.53-6.56 (m, 2H, ArH),
.88-7.09 (m, 5H, ArH), 7.21-7.23 (m, 2H, ArH), 7.40-7.42 (m, 2H, ArH)ppm. C-NMR
), δ: 56.2, 105.4, 106.2, 107.2, 112.3, 113.0, 118.3, 122.0, 127.9, 128.0,
(
DMSO-d
6
3
1
3
6
(
DMSO-d
6
-
1
1
2
29.4, 132.2, 135.0, 139.8, 146.0, 165.4, 176.8 ppm. IR (KBr, νcm ): 3028 (arom.CH) ,
+
992-2980 (aliph.CH). MS (m/z, %): 477.12 (M , 35.21), 413.23 (100). Anal.Calcd.for
C
23
H16BrN
3
2
O S (478.36): C, 57.75; H, 3.37; N, 8.78; Found: C, 57.65; H, 3.30 ; N, 8.70.
4
.1.5.14. 5-(4-Methylphenyl)-3-[4-(4-methoxyphenyl)-2-(1H-pyrrol-1-yl)thiophen-3-yl]-
o
1
, 2, 4-oxadiazole (18): Yield: 0.19 g (58 %), yellowish white powder; m.p. 152-156 C.
1
H-NMR (DMSO-d
6
):δ: 2.38 (s, 3H, CH
3
), 3.78 (s, 3H, -OCH ), 6.33-6.36 (m, 3H, ArH),
3

6
.68-6.94 (m, 2H, ArH), 7.05-7.18(m, 3H, ArH), 7.20-7.44 (m, 3H, ArH), 7.55(s, 1H,
1
3
ArH, 7.58 (s, 1H, ArH). C-NMR (DMSO-d
6
), δ: 20.6, 56.6, 106.0, 117.7, 120.0, 124.6,
1
27.0, 128.6, 133.6, 135.7, 139.1, 141.1, 142.5, 145.6, 153.2, 153.7, 167.7, 174.7 ppm.IR
-1
+
(
KBr, νcm ) : 3012 (arom.CH), 2996-2987 (aliph.CH). MS (m/z, %): 413.11 (M ,
5.22), 347.16 (100). Anal.Calcd.for C24 S (413.49): C, 69.71; H, 4.63; N, 10.16;
Found: C, 69.88; H, 4.44 ; N, 10.17.
2
H
19
N
3
O
2
4
2
.1.5.15. 5-(4-Methoxyphenyl)-3-(4-methoxyphenyl-2-(1H-pyrrol-1-yl)thiophen-3-yl)-1,
o
1
, 4-oxadiazole (19): Yield: 0.16 g (46%), red powder; m.p. 190-193 C. H-NMR
(
DMSO-d
6
), δ : 3.67 (s, 3H, -OCH ), 3.78 (s, 3H, -OCH ), 6.26-6.38 (m, 4H, ArH), 6.41-
3
3
-
1
6
3
.53 (m, 3H, ArH), 6.56-6.95 (m, 3H, ArH), 7.07-7.55 (m, 3H, ArH). IR (KBr, νcm ) :
+
022 (arom.CH) , 2991-2976 (aliph.CH). MS (m/z, %): 429.46 (M , 10.21), 347(100).
Anal.Calcd.for C24
H
19
N
3
3
O S (429.49): C, 67.12; H, 4.46; N, 9.78; Found: C, 67.34; H,
4
4
4
.74 ; N, 9.71.
.2- In vitro Antiproliferative studies
.2.1. In vitro cell growth inhibition
4
.2.1.1. Materials: The human breast adenocarcinoma (MCF-7) and colorectal cancer
(
HCT-116) cell lines were obtained from american type culture collection. Prodigiosin as
positive control and all other chemicals and reagents were purchased from Sigma or
Invitrogen.
4
.2.1.2. Methodology of MTT assay: The cells are grown on RPMI-1640 medium
supplemented with 10% inactivated fetal calf serum and 50µg/ml gentamycin. The cells
were maintained at 37ºC in a humidified atmosphere with 5% CO and are subculture two
2
to three times a week. For antitumor assays, the MCF-7 cells were suspended in medium
4
at concentration 5x10 cell/well in Corning® 96-well tissue culture plates and then
incubated for 24 hr. The tested molecules 3a-c, 4a-c, 5-19 and PG were then added into
9
6-well plates to achieve six concentrations for each molecule (50, 20, 10, 1.0, 0.1, 0.001
µM), respectively. Six vehicle controls with media or 0.5 % DMSO are run for each 96
well plate as a control. After incubating for 24 h, the numbers of viable cells were
determined by the tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-
diphenyltetrazolium bromide) assay [33]. Briefly, the media was removed from the 96
well plate and replaced with 100 µl of fresh culture RPMI 1640 medium without phenol

red then 10 µl of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of PBS) to each
well including the untreated controls. The 96 well plates were then incubated at 37°C and
5
% CO
DMSO is added to each well and mixed thoroughly with the pipette and incubated at
7°C for 10 min. Then, the optical density is measured at 590 nm with the microplate
2
for 4 h. An 85 µl aliquot of the media is removed from the wells, and 50 µl of
3
reader (Sun Rise, TECAN, Inc, USA) to determine the number of viable cells. The
relation between surviving cells and drug concentration was plotted to get the survival
curve of each tumor cell line after treatment with the specified compound. The 50%
inhibitory concentration (IC50), the concentration required to cause toxic effects in 50%
of intact cells, was estimated from graphic plots of the dose response curve for each
concentration using Graph Pad Prism software. The experiment is repeated three times.
4
4
.2.2. In vitro topoisomerase 2 enzymes assay:
.2.2.1. Materials :The in vitro enzyme inhibition determination for the synthesized
molecule tested is carried out in BPS Bioscience Corporation, San Diego, CA, USA
www.bpsbioscience.com). The following served as the enzyme sources, Poly (Glu, Tyr)
(
sodium salt (4:1, Glu: Tyr) (Sigma#P7244) served as the standardized substrate &
Kinase-Glo Plus Luminescence kinase assay kit (Promega # V3772) is used. The assay is
performed using Kinase-Glo Plus luminescence kinase assay kit (Promega).
4.2.2.2. Methodology :The tested molecules 4b, 4c, 9, 11, 12, 14 and PG were diluted to
1
00 µl in 10% DMSO and 5 µl of the dilution is added to a 50 µl reaction so that the final
concentration of DMSO is 1% in all of reactions. All of the enzymatic reactions were
o
conducted at 30 C for 40 minutes. The 50 µl reaction mixture contains 40 mM Tris, pH
7
.4, 10 mM MgCl , 0.1 mg/ml BSA, 1 mM DTT (Dithiothreitol), 10 µM ATP, enzyme
2
substrate and respective kinases. After the enzymatic reaction, 50 µl of enzyme-Glo Plus
luminescence topoisomerase assay solution (Promega) was added to each reaction and
the plate is incubated for 5 minutes at room temperature. Luminescence signal was
measured using a BioTek Synergy 2 microplate reader.The luminescence data were
analyzed using the computer software, Graphpad Prism. The difference between
luminescence intensities in the abscence of topoIIβ (Lu
Lu ) is defined as 100% activity (Lu -Lu ).The values of topo IIβ activity percentages
versus a series of tested molecules concentrations (1 nM , 10 nM, 100 nM, 1 µM, 10 µM)
t
) and in the prescence of topo IIβ
(
c
t
c

are then plotted using non-linear regression analysis of sigmoidal dose-response curve
((logEC50-X)xHill Slope)
generated with the equation: Y: B+(T-B)/1+10
, where Y: percent
activity,B: minimum percent activity, T: maximum percent activity, X: logarithm of
compound and Hill Slope : slope factor or Hill coefficient. The IC50 value was
determined by the concentration causing a half-maximal percent activity using Graph pad
Prism software. The experiment is repeated three times.
4
.2.3. In vitro DNA immunofluorescence binding assay:
Histochemical immunofluorescence assay for establishing indirect DNA binding affinity
is performed [34]. Briefly, slides of fixed MCF-7 cells were rinsed in three changes of
PBS. Non-specific binding is prevented by incubation in blocking solution (10% fetal
bovine serum in PBS) at 37◦C for 30 min. Slides were incubated for 30 min at 37◦C with
pico-green dye solution (Abcam Inc., USA). Slides were rinsed again with PBS to
remove excess dye, then treated with ethanolic solution of the tested compounds 4b, 9,
1
1, 14 and PG at concentration of their IC50 at 37 ◦C for 24 h. After washing, images
were visualized at 642-645 nm using a fluorescence microscope (Axiostar Plus, Zeiss,
Goettingen, Germany) equipped with image analyzer and digital camera (Power Shot
A20, Canon, USA). The experiment is repeated three times.
4
.2.4. In vitro Cell cycle DNA flow cytometry analysis:
6
The MCF-7 cells ( 3 x10 cells/mL ) are seeded in RPMI-1640 medium in T-75 flasks for
2
4 h and then treated with tested molecules 4b, 9, 11, 14 and PG at their IC50 (µM) for 24
h. The MCF-7 cells were then collected by trypsinization, wash with PBS and fix in ice-
cold absolute alcohol. After that, cells were stained using the cycle test™ Plus DNA
Reagent Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s
instructions [35]. Cell cycle distribution was calculated using CELLQUEST software
(
Becton Dickinson Immuno-cytometry Systems, San Jose, CA, USA). The experiment is
repeated three times.
4
.2.5. In vitro Annexin-V/PI staining analysis.
6
The MCF-7 (1 × 10 ) cells are seeded to a plate and cultured for 24 h, after which
indicated dose of tested molecules 4b, 9, 11, 14 and PG were added and incubated for
up 24 h. Cells were then washed with PBS and detached by trypsin followed by

neutralization with culture medium. The detached cells were collected into a 15-mL
centrifuged tube, wash with ice-cold PBS twice, and centrifuge at 1200 rpm for 5 min to
remove supernatant. A volume of 0.1 mL binding buffer, was added to the MCF-7 cells,
followed by adding 5 μL of annexin V-FITC and 5 μL of 50 μg/mL PI staining reagents.
After mixing homogeneously and reacting at 25 °C for 15 min in the dark, the apoptotic
cell population was analyzed by a flow cytometer [FACS Calibur flow cytometer (BD
Biosciences)]. The experiment is repeated three times.
4
4
.2.6. In vitro ElISA immunoassay for p53 and cell death modulators:
.2.6.1. Materials: The levels of p53, Puma, Bax, Bcl-2 were measured in MCF-7 cells
treated with the molecules 4b, 4c, 9, 11, 12, 14 and PG for 24h using ELISA colorimetric
kits. All the procedures were performed according to the manufacturer’s instructions,
.
DRG
Sigma Puma ELISA Human (RAB0500)
Germany. Bcl-2 ELISA Kit Invitrogen Corporation 1600 Faraday Avenue Carlsbad.
.2.6.2. Methodology: The levels of the anti-apoptotic marker Bcl-2 as well as the p53
® Human Bax ELISA (EIA-4487) Marburg,
4
and apoptotic markers Puma, Bax, Bcl-2 was measured using ELISA colorimetric kits
per the manufacturer’s instructions. The optical densities were measured at 450 nm using
ROBONIK P2000 SPECTROPHOTOMETER wl 450 nm, reference 630 nm. The
intensity of the color is proportional to the amount of the corresponding parameter bound
in the initial step. The experiment is repeated three times.
4
.2.7. In vitro green flow cytometry assay for active caspase 3/7:
The enzymatic activities of caspase 3/7 are measured in cell lysates (100 ug protein in 50
™
uL lysis buffer) using Cell Event Caspase-3/7 Green Flow Cytometry Assay Kit,
Catalog Number (C10427) per the manufacturer's instructions. Briefly, control and
5
treated MCF-7 cell(2.5x10 /mL) with molecules 4b, 4c, 9, 11, 12, 14 and PG for 24 h are
washed with ice cold PBS, MCF-7 cell lysates are combined with reaction buffer and
o
incubated with specific colorimetric substrates (Caspase 3/7 Detection Reagent) at 37 C
for 6 h. The samples are measured at 488 nm in BD FACS Calibur flow cytometer. The
experiment is repeated three times.
5
-Acknowledgments:

The author is filled with respect to the memory of Dr. Ibrahim Abouelish, founder of
Sekem Corporation for supporting this work. The author also grateful Dr. Esam
Rashwan, Head of the confirmatory diagnostic unit VACSERA-EGYPT, for carrying out
the cytotoxicity screening and other cell death modulators
6
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Table 1: IC50 of carbonitriles 3a-c, carboximidamide 4a-c and oxadiazoles 5-19 on
MCF-7 cells (uM) and topo enzyme (nM).
1
2
3
a
b
c
Code
R
R
R
MCF-7
IC50(uM)
19.45±1.61
HCT-116
Topo
d
e
IC50 (nM)
3
3
3
4
4
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
a
b
c
a
b
c
-CH
3
-CH
3
-
NT
NT
NT
NT
NT
NT
NT
NT
-(CH
2
)
)
4
4
-
-
--
16.10±1.10
13.40±1.18
5.98±0.41
4.52±0.43
0.83±0.06
13.43±0.91
9.82±0.47
8.64±0.81
12.43±1.46
0.48±0.07
12.94±0.92
1.98±0.09
0.78±0.06
10.51±0.82
0.19±0.05
13.76±1.10
12.11±1.11
15.22±1.32
15.62±1.31
21.80±1.71
1.93±0.18
4-CH
3
OC
-CH
6
H
4
H
-CH
-
-
-
3
3
-(CH
2
10.52±1.03 123.49±9.41
0.57±0.06
NT
4-CH
-CH
-CH
-CH
-CH
-CH
3
OC
6
H
4
H
-
35.32±4.08
NT
3
-CH
-CH
-CH
-CH
3
H
4-Cl
4-Br
4-CH
4-OCH
H
4-Cl
4-Br
4-CH
4-OCH
H
4-Cl
3
3
NT
NT
NT
NT
NT
NT
3
3
3
3
3
3
-CH
3
3
5.13±0.26
NT
2.51±0.53
1.54±0.08
NT
36.31±1.16
NT
87.46±9.21
69.56±1.14
NT
0
1
2
3
4
5
6
7
8
9
-(CH
-(CH
-(CH
CH
-(CH
2
)
)
)
4
-
-
-
2
4
4
-
2
2
)
4
3
2
)
4
-
3
1.17±0.09
NT
27.28±1.11
NT
4-CH
4-CH
4-CH
4-CH
4-CH
3
OC
OC
OC
OC
OC
6
6
6
6
6
H
H
4
H
4
H
4
4
H
H
H
H
H
--
3
NT
NT
NT
NT
NT
NT
NT
NT
3
4-CH
4-Br
4-OCH
3
3
3
H
4
3
PG
-
-
2.84±0.26
73.14±6.23
a
b
c
MCF7, Human breast, HCT116, Human colon cancer cell lines, Topo , topoisomerase
2
β enzyme, d, values are expressed as+ SEM (n: 3) in micromolar concentration and e ,
values mean + SEM (n=3) in nanomolar concentration , NT - Not tested.

Fig. 1: Chemical structure of antitumor agents as orthogonal tricyclic molecules. Core
rings is are shown in black color, bulky aryl groups are in red color and other small aryl
groups are in blue color.

Fig. 2: Diagrammatic sketch illustrate the design strategy. Orthogonal substituted
polyaryl lead moieties are shown in red color, H-bond forming group and rigid
oxadiazole core ring are in black color and small aryl fragments at C-5 of the core are in
blue color.

Fig. 3: Sketch diagram represent the mechanism of formation of 3,5-diaryl oxadiazoles
-19 from amidoximes 4a-c.
5