Design, Synthesis, and Screening of 5-Aryl-3-(2-(pyrrolyl)thiophenyl)-1,2,4-oxadiazoles as Potential Antitumor Molecules on Breast Cancer MCF-7 Cell Line

Article abstract of DOI:10.1248/cpb.c18-00636

The work reported the design and cytotoxic screening of synthetic small molecules: 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 carboximidamide 4c and oxadiazole 14 display potent inhibitory activity in nano-molar concentration higher than PG. In addition, carboximidamide 4c and oxadiazoles 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 population percentages at pre G₁ and G₂/M phases as shown by DNA-flow cytometry assay and annexin V analysis. Moreover, measurement of p53 and cell death mediators, show that carboximidamide 4c and oxadiazoles 9, 12, and 14 significantly up-regulate p53, 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:10.1248/cpb.c18-00636

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c18-00636
Advance Publication
October 5, 2018
Chem. Pharm. Bull.
Regular Article
Design, Synthesis, and Screening of 5-Aryl-3-(2-(pyrrolyl) thiophenyl)-1, 2,
4-oxadiazoles As Potential Antitumor Molecules on Breast Cancer MCF-7
Cell Line.
Mohammed K. Abd el hameid
Organic Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University,
Egypt,
Dedicated to the memory of Dr. Ibrahim Abouleish
Ⓒ 2018 The Pharmaceutical Society of Japan

Abstract:
The work reported the design and cytotoxic screening of synthetic small molecules:
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 carboximidamide 4c and oxadiazole 14 display potent inhibitory activity in nano-molar
concentration higher than PG. In addition, carboximidamide 4c and oxadiazoles 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 population percentages at pre G1 and G2/M
phases as shown by DNA-flow cytometry assay and annexin
V
analysis.
Moreover, measurement of p53 and cell death mediators, show that carboximidamide 4c and
oxadiazoles 9, 12, and 14 significantly up-regulate p53, Puma and Bax/Bcl-2 ratio levels.
Subsequently, pro-apoptotic activities are confirmed by active caspase 3/7 percentages
green fluorescence assay.
Keywords:
DNA intercalators, topoisomerase, thiophene, oxadiazole, breast cancer
Chemical and Pharmaceutical Bulletin Advance Publication

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 synthesis inhibitors either as intercalators or topo inhibitors. Structurally,
the common structural basis of these molecules are the polycyclic molecular skeleton either
orthogonal or planar required for DNA base pair interaction and enzyme binding. Besides
that, the molecular skeletons also have to carry side chains that anchor DNA base pairs and
enzyme binding sites by non-covalent interactions [2]. Natural products are important utility
in drug discovery as the diversity of their structures inspire the design and 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. Their mode of cytotoxicity
is mainly DNA synthesis inhibition [4-8]. Obatoclax (Teva Pharmaceuticals) (Fig. 1) is an
apoptotic inducer agent over different cancer cell lines and is synthetically derived from PG
[8].
Interestingly, the oxazole, isoxazole, and oxadiazole are a privileged core rings in
numerous antitumor molecules like MX 74420(Maxim Pharmaceuticals), SEW 2871(EPI
Corporation), VA-62784, and molecule I (Fig. 1) [9-13]. These rings represent promising
scaffolds in the design of orthogonal DNA interacting agents due to the following reasons.
They have a similar configurational ability to act as a rigid spacer or core between two
pharmacophoric arms. In addition, these rings are hydrogen bond acceptor (H-bond -A)
forming moieties that facilitate binding of molecules with DNA and enzyme binding sites
[14]. The mentioned cytotoxic molecules show a common configurational resemblance in
their molecular structures as curved or orthogonal poly-aryl ring system composed of core
part equipped with two symmetrical or unsymmetrical aryl groups as side chains. P53
upregulated modulator of apoptosis (Puma) is a downstream protein of p53 and is expressed
as a secondary effect to DNA damage to induce apoptosis in cancer cells. Puma plays a vital
role in the determination of the levels of Bax and Bcl-2 proteins in the cancer cells [15,
16]. Cancer cells resistance to apoptosis induction for the first-line chemotherapy 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. Therefore, the discovery of
Chemical and Pharmaceutical Bulletin Advance Publication

novel antitumor molecules enhanced 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 (Fig. 2). The basic structural skeleton of the lead template is formed from
orthogonal 2-(pyrrol-1-yl) thiophene-3-carbonitriles 3a-c as lead compounds. 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 carboximidamide group as H-bond acceptor-donor (A-D) pair forming group via
chemical modification of carbonitrile group to reinforce the interaction with the DNA and/or
the enzyme binding sites. Besides that, carboximidamide group also is capable of forming a
pseudo intramolecular H-bond ring that extended the structural skeleton (model
A, carboximidamides 4a-c). As a final structural modification, the amidoxime group in the
conformers 4a-c is rigidified into oxadiazole ring with the addition of aryl side chains at the
C-5 of the core (model B, oxadiazoles 5-19). The final molecules 5-19 are formed from diaryl
oxadiazole skeleton with oxadiazole as core ring with 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- Discussion:
2.1. Synthesis:
The steps for synthesis of key starting molecules, intermediates, and the final
compounds 3a-c, 4a-c and 5-19 are illustrated in Charts 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 of the chemical industry [17]. Since
Gewald reported his synthetic protocol for the preparation of 2-aminothiophenes from
ketones, active methylene derivatives and sulphur, several reports are published to improve
the reaction conditions [18-21]. In the current work, 2-aminothiophenes 2a-c are 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
MAOS conditions (Chart 1).
Chemical and Pharmaceutical Bulletin Advance Publication

{Please insert Chart 1 about here]
The structure of thiophene derivatives 2a-c is confirmed by their reported physical
and spectral data [18-20]. Synthesis of pyrrole ring from primary amines
using dimethoxytetrahydrofuran (DMTHF) under acid catalyst is known as Clauson-Kaas
reaction [22]. In the current work, 2-(pyrrol-1-yl) thiophenes 3a-c are prepared by reacting 2-
aminothiophenes 2a-c with DMTHF in acetic acid as a dual solvent and catalyst under
MAOS conditions (Chart 1). Thiophene derivative 3a is reported as a patent molecule [23],
while 2-(pyrrolyl) thiophene 3b is known in the literature by synthesis under conventional
heating with incomplete structural characterization [24]. The preparation of 1, 2, 4-oxadiazole
ring is 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 is added to aryl nitriles to form amidoximes under base
catalyzed conditions. Secondary to that, the amidoximes are coupled with the acylating
agents to form oxadiazole ring [25-27].
[Please insert Fig.3 about here]
In this work, oxadiazoles 5-19 (Fig. 3) are prepared as the following, thiophene-3-
carbonitriles 3a-c are reacted with hydroxylamine hydrochloride using cesium carbonate as a
catalyst instead of potassium carbonate, (unstable in M.W irradiation), in n-butanol under
MAOS conditions to produce thiophene-3-carboximidamides 4a-c (Chart 2).
[Please insert Chart 2 about here]
The IR spectra of carboximidamides 4a-c, they show absence of absorption band at
2218 cm-1 characteristic to nitrile group cm-1 and appearance of absorption bands at the range
3413-3041 cm-1 corresponding to NH2 and OH groups, while 1H-NMR confirm the presence
of signals of NH2 and OH at 4.90-4.95 and 10-63-10.77 ppm. In the second step,
amidoximes 4a-c are acylated with appropriate acid chlorides in ethylene glycol (high boiling
solvent) under MAOS conditions to give 3,5-diaryl 1, 2, 4-oxadiazoles 5-19 (Chart 3).
Several synthetic trails are performed to conduct the preparation of 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 produce the amide of corresponding
nitrile 3b.
[Please insert Chart 3 about here]
2.2-In vitro Antiproliferative assay
2.2.1-In vitro cell growth inhibition:
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The molecules 3a-c, 4a-c, and 5-19 are evaluated for antiproliferative activity against
breast cancer MCF-7 cells by MTT assay [28] and PG is used as a control compound [7,8].
The obtained results are shown in (Table 1 and Fig. 4).
[Please insert Table. 1 about here]
Carboximidamides 4b, 4c and oxadiazoles 9, 11, 12, and 14 with IC50 less than (5
μM) against MCF-7 cells are selected for further cytotoxic evaluation on human colon cancer
HCT-116 cell lines. The obtained data concerning MCF-7 cells show that molecules 4c, 9,
12, and 14 exhibit cytotoxic activity in sub-micromolar concentration (IC50: 0.19-0.83 μ
M). Inspection of the results according to structural variations between the molecules show
the following remarks. Structural modification of thiophene -3-carbo nitrile 3a-c into
thiophene-3-carboximidamides 4a-c lead to improvement in cytotoxicity results, that may be
explained by increasing probability of hydrogen bond formation. Regarding groups present at
C-4 and C-5 of thiophene ring, carbonitrile 3c and carboximidamide 4c exhibit higher activity
within molecules 3a-c and 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 display higher cytotoxicity among
oxadiazoles 5-19, suggesting that tetra-methylene group at thiophene ring has higher
participation in cytotoxicity compared with other C-4 and C-5 substituents at thiophene ring.
At the same time, 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 molecules 15-19.
This result may be structurally rationalized as the following, the molecular skeleton of the 2-
(pyrrolyl) thiophenes 4a-c and 3,5-diaryl oxadiazoles 5-19 tolerates only one 4-
methoxyphenyl group for good cytotoxic activity. This observation is confirmed by the low
cytotoxicity results exhibited by molecules 15-19, where their precursor 4c is a potent
cytotoxic agent. In respect to the C-5 aryl groups of oxadiazoles 5-19, molecules 9, 11,
12 and 14 are the most cytotoxic compounds, indicating that 4-methoxyphenyl, 4-
chlorophenyl, and 4-bromophenyl have a positive effect on cytotoxicity. In respect to
sensitivity of MCF-7 and HCT-116 cells toward the tested molecules, MCF-7 cells are 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 exhibit good
Chemical and Pharmaceutical Bulletin Advance Publication

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 topisomerase assay:
Further topo 2β enzymatic inhibition at 5 different doses is performed for the selected
molecules: carboximidamide 4b, 4c, oxadiazoles 9, 11, 12, and 14 to evaluate their topo 2β
IC50 values using PG as a reference. The obtained data show that molecules 4c, 9, 12,
and 14 are higher enzyme 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 displays higher topo inhibition than tetrahydrobenzothiophene
derivative 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
display higher activity than rotatable model A. Furthermore, 3-(tetrahydrobenzothiophenyl)
oxadiazole 14 shows 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-methoxy phenyl) oxadiazole 9 are from different models
they are nearly equipotent in topo inhibition, that confirm the role of 4-methoxyphenyl group
in the enzyme inhibition activity. Oxadiazole 14 with C-5(4-methoxyphenyl) group is more
potent than other oxadiazoles 11 and 12 with C-5 (chlorophenyl and bromophenyl),
confirming the previous finding. Finally, 5-(4-bromophenyl) oxadiazole 12 exhibits 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 are selected for DNA binding affinity compared with PG, indirect binding pico -green
immunofluorescence assay is 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
[29]. The results show that model A (molecules 4b and 4c) have good binding affinity than
Chemical and Pharmaceutical Bulletin Advance Publication

model B (9 and 14). Unfortunately, the obtained results of histochemical immunoassay are
not in agreement with the data of MCF-7 cells IC50 and topo enzyme inhibition, so it will be
excluded from the predication of final structural activity relationship.
[Please insert Fig. 6 about here]
2.2.4. In vitro DNA- flow cytometry and Annexin staining analysis:
To explore the effect of molecules 4b, 4c, 9, and 14 on cell growth inhibition and pro-
apoptotic characters, the molecules are subjected to DNA flow cytometry cell cycle analysis.
The results are shown 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 [30]. The molecules induce accumulation percentage of the cells
in G0 phase by 8-, 13-, 9.5- and 15 - fold and G2/M phases by 1.4-, 1.8-, 1.5-, and 1.8- fold
respectively, in comparison with untreated MCF-7 cells, indicating that the tested molecules
have cell cycle arrest at G1 phase and pro-apoptotic activities. In addition, only, molecules 4c,
14, and PG induce 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 exhibits potent antiproliferative activity than compounds 4b and 9.
[Please insert Fig. 7 about here]
The annexin assay further confirms the occurrence of apoptosis induction in MCF-7
cells by the tested molecules. The results are shown in (Fig. 8). Results of the assay exhibit
that both stages of apoptotic MCF-7 cells (early and late) increase by molecules 4b, 4c, 9,
and 14 by 8 -, 11-, 9 -, and 15 - fold compared with untreated cells. Moreover, compound 14
exhibits a profile of strong apoptotic inducer over MCF-7 cells by 1.2- fold than PG. In
conclusion, data indicate that molecules 4b, 4c, 9, and 14 are the potent inducer of apoptosis
over MCF-7 cells and 14 can induce apoptosis percentage higher than PG, molecule 4c is
slightly less potent than PG, meanwhile compounds 4b and 9 show 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 are shown in (Fig. 9 A-
E). The data indicate that compounds 4b, 4c, 9, and 14 up-regulated p53 levels by 12-, 17-,
Chemical and Pharmaceutical Bulletin Advance Publication

19.5-, and 25.7- fold, respectively, higher than untreated cells (Fig. 9A). Molecules 4c and 9
are nearly equivalent in their up-regulation and both are less than 14 and PG. Molecule 14
increase p53 level by 1.1- fold higher than PG. Similarly, compounds 4b, 4c, 9, and 14
increase Puma level by 4.5-, 6.5-, 5-, and 7.0-, fold, respectively more than untreated cells
(Fig. 9B). Meanwhile, compound 14 increase 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, 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. Molecule 14 has higher Bax/Bcl-2 ratio by 1.3-fold than PG
(Fig. 9C, D, and E). 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 percentage:
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 oxadiazoles 9, 14 increase active caspase-3/7 percentages by 3-,
8.6-, 6.4-, 10-fold, respectively, more than untreated MCF-7 cells as seen in (Figs. 10).
Additionally, carboximidamide 4c increase caspase 3/7 higher than oxadiazole 9. Moreover,
carboximidamide 4c and oxadiazole 14 increase caspase-3/7 percentages by 1.1- 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-7 cells as a secondary effect to
DNA damage with subsequent secondary increasing in 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
Chemical and Pharmaceutical Bulletin Advance Publication

into pyrrole ring (thiophenes 3a-c) using DMTHF. Furthermore, thiophene-3-nitriles 3a-c are
converted to thiophene-3-carboxmidoximes 4a-c via reaction with hydroxyl amine. Finally,
carboximidamides 4a-c are 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 14 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 exhibit higher cytotoxic
activity by 5 -, 1.8-, and 2.4- fold, respectively than control on HCT-116 cells. The
antiproliferative activity of tested molecules is correlated to topoisomerase enzyme inhibition,
where carboximidamide 4c and oxadiazoles 9 and 14 are potent enzyme inhibitors by 2-, 2 -,
and 2.7 -fold, respectively than PG. Moreover, the cell cycle flow cytometry analysis displays
that compounds 4c, 9, 11, and 14 induce cell cycle arrest at G1 phase of MCF-7 cells. In
addition, molecule 14 is stronger apoptotic inducers than PG, as demonstrated by the results
of Annexin assay. ELISA measurement 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 increase Puma, and Bax/Bcl-2 ratio levels. The results of the green fluorescence
assay exhibit that carboximidamide 4c and oxadiazoles 9, 11, and 14 trigger apoptosis
induction via increase active caspase 3/7 percentage. Finally, compounds carboximidamide
4c and oxadiazole 14 represent promising antitumor and apoptosis-inducing agents.
4- Experimental:
4.1. Synthesis:
4. 1. 1. Apparatus:
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
13
Gemini-300BB 300 MHz FT-NMR spectrometers (Varian Inc., Palo Alto, CA). 1H and C
spectra are run at 400 and 100 MHz, respectively, in deuterated dimethyl sulphoxide
(DMSO-d6). IR spectra of molecules are recorded with a 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 progress is 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,
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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, 1000 W).
4.1.2. Synthesis of 2-amino-thiophene-3-carbonitriles 2a-c:
Starting materials 2a-c are 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) is stirred and morpholine (2.61 g, 30.0 mmol) is added dropwise at room
temperature with cooling in ice bath. After that, the mixture is heated at 150 0C for 10 min in
a M.W. reactor. The reaction mixture is subjected to cooling to room temperature. After
cooling, petroleum ether (20 ml) is added to the mixture and the formed brown precipitate is
crystallized from ethanol and afford thiophenes 2a-c. The structure of 2-aminothiophenes 2a-
c is confirmed by their reported physical and spectral data 2a [19], 2b [18] and 2c [20].
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) is stirred for 5 min at room temperature, followed by the addition of thiophene-
0
3-carbonitriles 2a-c (0.62 mmol). The reaction mixture is heated at 100 C for 15 min in the
M.W. reactor. After cooling, the reaction mixture is diluted with H2O (20 ml) and the solid
formed is crystallized from ethanol to give thiophenes 3a-c.
4.1.3.1. 4, 5-Dimethyl-2-(1H-pyrrol-1-yl) thiophene-3-carbonitrile (3a): Yield: 0.9 g
(74 %) white crystal; m.p. 50-53oC. 1H-NMR (DMSO-d6), δ:2.24 (s, 3H, CH3), 2.27 (s, 3H,
CH3), 6.27-6.49 (t, 2H, ArH), 6.52-6.59 (d, 2H, 8.7 Hz, ArH) ppm. 13C-NMR (DMSO-d6), δ:
9.9, 12.1, 84.6, 108.4, 115.4, 116.0, 123.2, 135.0, 158.7. IR (KBr, ν cm-1): 2984-2896
(aliph.CH) , 2215 (CN). Anal.Calcd.for C11H10N2S (202.28): C, 65.32; H, 4.98; N, 13.85;
Found: C, 65.29; H, 4.84 ; N, 13.72.
4.1.3.2. 2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrbenzo[b]thiopthene-3-carbonitrile (3b):
1
Yield: 1.1g (81 %) white crystal ; m.p. 73-76oC (lit ,63-65oC)[24]. H-NMR (300 MHz,
DMSO-d6), δ: 1.73-1.79 (m, 4H, 2CH2), 2.65-2.69 (m, 4H, 2CH2), 6.18-6.19 (m, 2H, ArH),
6.91-6.92 (m, 2H, ArH) ppm. 13C-NMR (DMSO-d6), δ: 20.4, 22.9, 23.4, 24.5, 84.0, 108.5,
115.3, 123.2, 134.5, 141.0, 158.1.
4.1.3.3. 3-(4-Methoxyphenyl)-2-(1H-pyrrol-1-yl) thiophene-3-carbonitrile (3c) : Yield:
1.2 g( 76 %), white crystal; m.p. 122-123oC. 1H-NMR (DMSO-d6), δ: 3.71(s, 3H, CH3), 6.40-
6.50(m, 3H, ArH), 6.91-7.05 (m, 4H, ArH), 7.32-7.45 (m, 2H, ArH) ppm. 13C-NMR (DMSO-
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d6), δ: 56.40, 82.3, 108.5, 115.9, 117.9, 123.7, 124.3, 127.8, 128.0, 129.4, 141.2, 160.2. IR
(KBr, ν cm-1): 3023 (arom.CH), 2983-2892 (aliph.CH), 2218(CN); Anal.Calcd.for
C16H12N2OS (280.34): C, 68.55; H, 4.31; N, 9.99; Found: C, 68.29; H, 4.64 ; N, 10.12.
4.1.4. N-Hydroxy-2-(1H-pyrrol-1-yl) thiopthene-3-carboximidamides 4a-c.
Thiophene 3-carbonitrile derivatives 3a-c (8 mmol) are dissolved in n-butanol (15
ml) and treated with Cs CO3 (5.2 g, 16 mmol) and hydroxyl amine hydrochloride (1.0 g, 14.8
mmol), then the reaction mixture is stirred for 5 min at room temperature. The reaction
mixture is heated at 140 0C for 20 min in M.W. reactor. After cooling, diethyl ether (20 ml) is
added to the reaction mixture and the solid formed is subjected to filtration and dried under
vacuum to produce carboximidamides 4a-c.
4.1.4.1. N-Hydroxy-4, 5-dimethyl-2-(1H-pyrrol-1-yl) thiopthene-3-carboximidamide
(4a): Yield: 1.1 g (59 %) brown solid; m.p. 250-253oC. 1H-NMR (DMSO-d6), δ: 2.25 (s, 3H,
CH3), 2.31 (s, 3H, CH3), 4.95 (s, 1H, D2O exchangeable), 6.23-6.38 (m, 1H, ArH), 6.44-6.49
(d, 1H, 8.7 Hz, ArH), 6.95-6.97 (d, 2H,ArH), 10.63 (s,1H, D2O exchangeable), 10.73 (s,1H,
D2O exchangeable) ppm. 13C-NMR (DMSO-d6), δ: 10.4, 10.9, 108.3, 120.7, 123.0, 131.5,
134.0, 136.0, 163.2. IR (KBr, ν cm-1): 3412-3090 (NH & OH), 3054 (arom.CH), 2983-2891
(aliph.CH). Anal.Calcd.for C11H13N3OS (235.31): C, 56.15; H, 5.57; N, 17.86; Found: C,
56.28; H, 5.71 ; N, 17.61.
4.1.4.2. N-Hydroxy-2-(1H-pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiopthene-3-
1
carboximidamide (4b): Yield: 1.40g (67 %), yellow solid; m.p. 262-265oC. H-NMR
(DMSO-d6), δ: 1.60-1.78 (m, 4H, 2CH2), 2.72-2.79, (m, 4H, 2CH2), 4.90 (s, 2H, D2O
exchangeable), 6.14-6.27 (m, 2H, ArH), 6.91-6.97(m, 2H, ArH), 10.77 (s, 1H, D2O
exchangeable) ppm. ;13C-NMR (DMSO-d6), δ: 20.4, 23.4, 24.5, 24.6, 108.3, 119.6, 123.1,
127.7, 135.6, 137.4, 163.2. IR (KBr, ν cm-1): 3413-3110 (NH &OH), 3054 (arom.CH), 2984-
2890 (aliph.CH). Anal.Calcd.for C13H15N3OS (261.09): C, 59.74; H, 5.79; N, 16.08; Found:
C, 59.82.; H, .93 ; N, 16.11.
4.1.4.3.
N-Hydroxy-4-(4-methoxyphenyl)-2-(1H-pyrrol-1-yl)
thiopthene-3-
1
carboximidamide (4c): Yield: 1.45g (58 %), yellow solid; m.p. 286-289oC. H-NMR (300
MHz, DMSO), δ : 3.86 (s, 3H, CH3), 4.92 (s, 2H, D2O exchangeable), 6.19-6.24 (m, 2H,
ArH), 6.34-6.46 (m, 1H, ArH), 6.80-6.85 (s, 2H, ArH), 6.91-7.07 (s, 2H, ArH), 7.39-7.47 (m,
2H, ArH), 10.76 (s, 1H, D2O exchangeable) ppm. 13C-NMR (DMSO-d6), δ : 56.4, 108.2,
114.8, 118.3, 123.1, 123.3, 127.0, 128.5, 137.4, 138.2, 160.6, 163.4. IR (KBr, ν cm-1): 3373-
Chemical and Pharmaceutical Bulletin Advance Publication

3041 (-NH &OH), 3054 (arom.CH), 2997-2983 (aliph.CH). Anal.Calcd.for C16H15N3O2S
(313.37): C, 61.32; H, 4.82; N, 13.41; Found: C, 61.86; H, 4.94 ; N, 13.33.
4.1.5. Synthesis of 5-aryl-3-(2-(1H-pyrrol-1-yl)-thiophen-3-yl)-1, 2, 4-oxadiazoles 5-19.
Thiophene-3-carboximidoximes 4a-c (0.8 mmol) are dissolved in ethylene glycol (10
ml), then the appropriate acid chloride (0.8 mmol) is added with continuous cooling and
0
stirring of the reaction mixture. The reaction mixture is heated at 200 C for 5 min. After
cooling, saturated aqueous NaHCO3 solution (10 ml) is added to the reaction mixture. Then,
the mixture is extracted with ethyl acetate (3 × 15 ml) and the combined organic layers are
dried and evaporated under reduced pressure. The obtained solid is subjected by column
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-1-yl]-5-phenyl-1, 2, 4-
oxadiazole(5): Yield: 0.19 g (75 %), yellow powder; m.p. 98-100oC.1H-NMR(DMSO-d6), δ:
2.23 (s, 3H, CH3), 2.33 (s, 3H, CH3), 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.13C-NMR (DMSO-d6), δ: 11.1, 13.2, 110.4,
117.3, 124.0, 124.2, 127.5, 128.7, 129.2, 131.7, 134.7, 139.4, 143.5, 166.5, 176.9 ppm. IR
(KBr, ν cm-1): 3021 (arom.CH) , 2993-2981 (aliph.CH). MS (m/z, %): 321.06 (M+, 15.21),
315.19 (100). Anal.Calcd.for C18H15N3OS (321.4): C, 67.27; H, 4.70; N, 13.07; Found: C,
67.28; H, 4.74 ; N, 13.31.
4.1.5.2. 5-(4-Chlorophenyl)-3-[4, 5-dimethyl-2-(1H-pyrrol-1-yl)thiophen-3-yl]-1, 2, 4-
oxadiazole (6):Yield: 0.16 g (55%), brown powder. m.p. 153-157oC ;1H-NMR (DMSO-d6),
δ: 2.28 (s, 3H, CH3), 2.31 (s, 3H, CH3), 6.54 (s, 2H, ArH), 7.05-7.08 (d, 2H, ArH), 7.38-7.40
(d, 2H, ArH), 7.73 (s, 2H, ArH) ppm. 13C-NMR (DMSO-d6) δ: 11.1, 13.2, 110.4, 124.0,
124.2, 127.5, 128.7, 129.2, 131.7, 134.7, 139.5, 143.5, 166.4, 176.9 ppm. IR (KBr,νcm-1) :
3021 (arom.CH), 2997-2979 (aliph.CH). MS (m/z, %): 355.22 (M+, 10.25), 331.32 (100).
Anal.Calcd.for C18H14ClN3OS (355.84): C, 60.76; H, 3.97; N, 11.81; Found: C, 60.70; H,
3.84 ; N, 11.71.
4.1.5.3. 5-(4-Bromophenyl)-3-[4, 5-dimethyl-2-(1H-pyrrol-1-yl) thiophen-3-yl]1, 2, 4-
1
oxadiazole (7): Yield: 0.19 g (59 %), yellowish white powder. m.p. 132-135oC. H-NMR
(DMSO-d6) , δ : 2.26 (s, 3H, CH3), 2.30 (s, 3H, CH3), 6.55-6.96 (m,4H, ArH), 7.25-7.66 (m,
4H, ArH) ppm. 13C-NMR (DMSO-d6), δ: 11.1, 13.2, 110.4, 124.0, 125.4, 126.4, 127.5, 129.3,
131.7, 134.8, 139.5, 143.5, 166.5, 176.9 ppm. IR (KBr, νcm-1): 3019 (arom.CH), 2995-2980
(aliph.CH) . MS (m/z, %): 399.12 (M+, 15.28), 345.51 (100) .Anal.Calcd.for C18H14BrN3OS
(400.29) : C, 54.01; H, 3.53; N, 10.50, found : C, 54.22; H, 3.61; N, 10,38.
Chemical and Pharmaceutical Bulletin Advance Publication

4.1.5.4. 3-[4, 5-Dimethyl-2-(1H-pyrrol-1-yl)thiophen-3-yl]-5-(4-tollyl)1, 2, 4-oxadiazole
(8): Yield: 0.13 g (49 %), yellow powder ; m.p. 88-90oC . 1H-NMR (DMSO-d6), δ : 2.30 (s,
3H, CH3), 2.34 (s, 3H, CH3), 2.40 (s, 3H, CH3), 6.55-6.89 (m, 4H, ArH), 7.21-6.26 (m, 2H,
ArH), 7.54-7.63 (m, 2H, ArH) ppm.13C-NMR (DMSO-d6) δ: 11.1, 13.2, 22.1, 110.4, 124.0,
124.1, 127.5, 128.7, 129.2, 131.7, 134.7, 139.4, 143.5, 166.5, 175.8 ppm. IR (KBr, νcm-1):
3027 (arom.CH), 2997-2984 (aliph.CH) . MS (m/z, %): 334.11 (M+, 35.51), 301.54 (100).
Anal.Calcd.for C19H17N3OS (335.42): C, 68.03; H, 5.11;N, 12.53; Found: C, 68.14; H, 5.23 ;
N, 12.63.
4.1.5.5.3-[4,5-Dimethyl-2-(1H-pyrrol-1-yl)thiophen-3-yl]-5-(4-methoxyphenyl)-1, 2, 4-
oxadiazole (9): Yield: 0.16 g (56 %) ,yellow powder; m.p. 167-170oC ;1H-NMR (DMSO-d6),
δ: 2.24 (s, 3H, CH3), 2.38 (s, 3H, CH3), 3.80 (s, 3H, -OCH3), 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, 13C-NMR
(DMSO-d6), δ: 11.1, 13.2, 57.4, 110.4, 124.1, 125.2, 126.4, 127.5, 129.2, 131.7, 134.7, 139.4,
143.4, 166.4, 176.8 ppm. IR (KBr,νcm-1): 3020 (arom.CH), 2996-2982 (aliph.CH). MS
(m/z, %): 351.17 (M+, 60.22), 315.41 (100). Anal.Calcd.for C19H17N3O2S (351.42): C, 64.94;
H, 4.88;N, 11.96; Found: C, 64.74; H, 4.68 ; N, 11.76.
4.1.5.6.3-[2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiophen-3-yl]-5-phenyl-1, 2,
4-oxadiazole (10): Yield: 0.22 g (79 %), orange powder; m.p. 141-146oC.1H-NMR (DMSO-
d6), δ: 1.73-1.82 (m, 4H, 2CH2), 2.71-2.91 (m, 4H, 2CH2), 6.86-7.01 (m, 2H, ArH), 7.13-7.29
(m, 3H, ArH), 7.37-7.53 (m, 4H, ArH) ppm; 13C-NMR (DMSO-d6), δ : 22.3, 22.6, 25.7, 25.9,
115.0, 117.6, 127.0, 128.3, 131.7, 132.3, 133.4, 134.0, 135.7, 145.6, 164.0, 176.6 ppm. IR
(KBr, νcm-1) : 3015 (arom.CH), 2993-2980 (aliph.CH). MS (m/z, %): 347.13 (M+, 10.58),
151.22 (100). Anal.Calcd.for C20H17N3OS (347.43): C, 69.14; H, 4.93;N, 12.09; Found: C,
69.34; H, 4.90 ; N, 12.11.
4.1.5.7.3-[2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiophen-3-yl]-5-(4-chloro
phenyl)-1, 2, 4-oxadiazole (11): Yield: 0.19 g (63 %), yellow powder; m.p. 152-156oC. 1H-
NMR (DMSO-d6), δ: 1.75-1.80 (m, 4H, 2CH2), 2.61-2.91 (m, 4H, 2CH2), 6.47-6.52 (m, 1H,
ArH), 6.67-6.79 (m, 4H, ArH), 7.30-7.43 (m, 3H, ArH) ppm.13C-NMR (DMSO-d6):δ: 22.5,
22.7, 25.8, 26.0, 111.2, 122.3, 123.1, 124.2, 125.1, 127.3, 128.1, 129.3, 140.3, 142.4, 166.7,
176.8 ppm. IR (KBr, νcm-1): 3034 (arom.CH), 2997-2981 (aliph.CH). MS (m/z, %): 381.16
(M+, 10.83), 187.32 (100). Anal.Calcd.for C20H16ClN3OS (381.88): C, 62.90; H, 4.22; N,
11.00; Found: C, 62.80; H, 4.04 ; N, 10.81.
Chemical and Pharmaceutical Bulletin Advance Publication

4.1.5.8. 3-[2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiophen-3-yl]-5-(4-bromo
phenyl)-1, 2,4-oxadiazole (12): Yield: 0.23 g (66 %), red powder; m.p.121-125oC. 1H-NMR
(DMSO-d6), δ: 1.74-1.80 (m, 4H, 2CH2), 2.67-2.73 (m, 4H, 2CH2), 6.16-6.20 (m, 1H, ArH),
6.38 (s, 3H, ArH), 6.95-6.98 (m, 2H, ArH), 7.24 (s, 1H, ArH), 7.45 (s, 1H, ArH) ppm; 13C-
NMR (DMSO-d6):δ: 22.6, 22.8, 25.2, 26.0, 106.0, 121.2, 121.8, 129.5, 130.7, 132.0, 133.7,
134.8, 136.1, 140.0 , 166.2, 175.4 ppm. IR (KBr, νcm-1): 3034 (arom.CH), 2994-2983
(aliph.CH). MS (m/z, %): 425.44 (M+, 5.98), 331.61 (100). Anal.Calcd.for C20H16BrN3OS
(426.33): C, 56.34; H, 3.78;N, 9.86; Found: C,56.44; H, 3.84 ; N, 9.71.
4.1.5.9. 3-3-[2-(1H-Pyrrol-1-yl)-4, 5, 6, 7-tetrahydrobenzo[b]thiophen-3-yl]-5-(4-tolyl)-1,
2, 4-oxadiazole (13): Yield: 0.17 g (60 %), red powder; m.p. 142-146oC. 1H-NMR (DMSO-
d6), δ: 1.76-1.95 (m, 4H, 2CH2), 2.15 (s, 3H, CH3), 2.65-2.91 (m, 4H, 2CH2), 6.44-6.60 (m,
2H, ArH), 6.62-6.84 (m, 3H, ArH), 6.99-7.05 (m, 2H, ArH), 7.16-7.46(m, 1H, ArH) ppm.13C-
NMR (DMSO-d6) , δ: 21.4, 22.6 , 22.8, 25.2, 26.0, 112.2, 121.0, 124.6, 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 C21H19N3OS
(361.46): C, 69.78; H, 5.39; N, 11.63; Found: C, 69.88; H, 5.44 ; N, 11.71.
4.1.5.10. 3-3-[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. 133-137oC;
1H-NMR (DMSO-d6), δ: 1.74-1.80 (m, 4H, 2CH2), 2.60-2.69 (m, 4H, 2CH2), 3.84 (s, 3H, -
OCH3), 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. 13C-NMR (DMSO-d6), δ: 22.2, 22.9, 25.4, 25.6, 56.5, 105.9, 112.2, 121.4,
126.0, 128.7, 132.6, 133.6, 135.6, 145.7, 153.1, 166.0, 176.7 ppm. IR (KBr, νcm-1): 3024
(arom.CH) , 2990-2982 (aliph.CH) . MS (m/z, %): 377.12 (M+, 10.05), 343.56 (100).
Anal.Calcd.for C21H19N3O2S (377.46): C, 66.82; H, 5.07;N,11.13; Found: C, 66.77; H, 5.14 ;
N, 11.10.
4.1.5.11.
5-Phenyl-3-(4-methoxyphenyl-2-(1H-pyrrol-1-yl)thiophen-3-yl)-1,
2,
4-
oxadiazole (15): Yield: 0.23 g (71%), orange powder; m.p. 122-126oC. 1H-NMR (DMSO-d6),
δ: 3.84 (s, 3H, -OCH3), 6.20-6.34 (m, 2H, ArH), 6.36-6.66 (m, 2H, ArH), 6.70-6.73 (m, 3H,
ArH), 6.98-7.08 (m, 3H, ArH), 7.14-7.18 (m, 2H, ArH), 7.53-7.67 (m, 2H, ArH) ppm.13C-
NMR (DMSO-d6):δ: 55.8, 111.7, 112.0, 117.1, 118.7, 119.5, 121.5, 121.6, 122.7, 129.5,
130.7, 132.5, 134.8, 135.9, 136.8, 149.0, 165.9, 176.6 ppm. IR (KBr, νcm-1): 3028 (arom.CH),
2998-2980 (aliph.CH) .MS (m/z, %): 399.10 (M+, 39.23), 377.16 (100). Anal.Calcd.for
C23H17N3O2S (399.46): C, 69.15; H, 4.29; N, 10.52; Found: C, 69.23; H, 4.50 ; N,10.71.
Chemical and Pharmaceutical Bulletin Advance Publication

4.1.5.12. 5-(4-Chlorophenyl)-3-[4-(4-methoxyphenyl)-2-(1H-pyrrol-1-yl)thiophen-3-yl)-1,
1
2, 4-oxadiazole (16): Yield: 0.20 g (59%), red powder; m.p. 166-170oC. H-NMR (DMSO-
d6), δ: 3.82 (s, 3H, OCH3), 6.26-6.36 (m, 3H, ArH), 6.76-6.79 (m, 4H, ArH), 6.99-7.29 (m,
4H, ArH), 7.32-7.39(m, 2H, ArH) ppm. 13C-NMR (DMSO-d6): 13C NMR (DMSO-d6), δ: 56.5,
106.0, 112.3, 113.2, 121.2, 129.5, 130.6, 132.0, 132.1, 134.9, 136.8, 140.0, 146.0, 149.0,
153.3, 167.0, 175.4 ppm. IR (KBr, νcm-1): 3031 (arom.CH) , 2993-2982 (aliph.CH) . MS
(m/z, %): 433.10 (M+, 5.25), 367.33 (100). Anal.Calcd.for C23H16ClN3O2S (433.91): C,
63.66; H, 3.72; N, 9.68; Found: C, 63.88; H, 3.84 ; N, 9.71.
4.1.5.13. 5-(4-Bromophenyl)-3-[4-(4-methoxyphenyl)-2-(1H-pyrrol-1-yl)thiophen-3-yl)-1,
1
2, 4-oxadiazole (17): Yield: 0.25 g (65%) ,brown solid; m.p. 172-173oC. H-NMR (DMSO-
d6), δ: 3.81 (s, 3H, -OCH3), 6.20-6.21 (m, 2H, ArH), 6.53-6.56 (m, 2H, ArH), 6.88-7.09 (m,
5H, ArH), 7.21-7.23 (m, 2H, ArH), 7.40-7.42 (m, 2H, ArH)ppm. 13C-NMR (DMSO-d6), δ:
56.2, 105.4, 106.2, 107.2, 112.3, 113.0, 118.3, 122.0, 127.9, 128.0, 129.4, 132.2, 135.0, 139.8,
146.0, 165.4, 176.8 ppm. IR (KBr, νcm-1): 3028 (arom.CH) , 2992-2980 (aliph.CH). MS
(m/z, %): 477.12 (M+, 35.21), 413.23 (100). Anal.Calcd.for C23H16BrN3O2S (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]-1,
1
2, 4-oxadiazole (18): Yield: 0.19 g (58 %), yellowish white powder; m.p. 152-156oC. H-
NMR (DMSO-d6):δ: 2.38 (s, 3H, CH3), 3.78 (s, 3H, -OCH3), 6.33-6.36 (m, 3H, ArH), 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, ArH, 7.58 (s,
1H, ArH). 13C-NMR (DMSO-d6), δ: 20.6, 56.6, 106.0, 117.7, 120.0, 124.6, 127.0, 128.6,
133.6, 135.7, 139.1, 141.1, 142.5, 145.6, 153.2, 153.7, 167.7, 174.7 ppm.IR (KBr, νcm-1) :
3012 (arom.CH), 2996-2987 (aliph.CH). MS (m/z, %): 413.11 (M+, 25.22), 347.16 (100).
Anal.Calcd.for C24H19N3O2S (413.49): C, 69.71; H, 4.63; N, 10.16; Found: C, 69.88; H,
4.44 ; N, 10.17.
4.1.5.15. 5-(4-Methoxyphenyl)-3-(4-methoxyphenyl-2-(1H-pyrrol-1-yl)thiophen-3-yl)-1,
1
2, 4-oxadiazole (19): Yield: 0.16 g (46%), red powder; m.p. 190-193oC. H-NMR (DMSO-
d6), δ : 3.67 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 6.26-6.38 (m, 4H, ArH), 6.41-6.53 (m, 3H,
ArH), 6.56-6.95 (m, 3H, ArH), 7.07-7.55 (m, 3H, ArH). IR (KBr, νcm-1) : 3022 (arom.CH) ,
2991-2976 (aliph.CH). MS (m/z, %): 429.46 (M+, 10.21), 347(100). Anal.Calcd.for
C24H19N3O3S (429.49): C, 67.12; H, 4.46; N, 9.78; Found: C, 67.34; H, 4.74 ; N, 9.71.
4.2- In vitro Antiproliferative assay:
4.2.1. In vitro cell growth inhibition:
Chemical and Pharmaceutical Bulletin Advance Publication

4.2.1.1. Materials:
MCF-7 and HCT-116 cell lines are obtained from american type culture collection.
Tumor cells are cultured using DMEM (Invitrogen/Life Technologies) supplemented with10
ug/ml of insulin (Sigma), 10% FBS (Hyclone), and 1% penicillin-streptomycin. Prodigiosin
as positive control and all other chemicals and reagents are purchased from Sigma or
Invitrogen.
4.2.1.2. Methodology of cell growth inhibition assay:
For antitumor activity, the MCF-7 cells are incubated in the medium in Corning® 96-
well tissue culture plates for 24 hr. The tested molecules 3a-c, 4a-c, 5-19 and PG are then
added into 96-well plates at different concentrations for each molecule. Different vehicle
controls with media are run for each 96 well plates as a control. After incubating for 24 h, the
numbers of viable cells are determined by the tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-
2, 5-diphenyltetrazolium bromide) assay [28]. The optical density is recorded at 590 nm with
the microplate reader (Sun Rise, TECAN, Inc, USA) to determine the number of viable cells.
The relation between the percentages of surviving cells and drug concentration is plotted to
get the survival curve of MCF-7 cells after treatment with the specified compound, IC50 is
estimated from graphic plots of the dose -response curve for each concentration using Graph
Pad Prism software. The assay is repeated three times.
4.2.2. In vitro topoisomerase 2β enzymes assay:
4.2.2.1. Materials:
The following materials 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 are diluted to 100 µl in DMSO (10%) and
5 µl of the solution is added to a 50 µl enzyme reaction until e final concentration of DMSO
is 1% in all of the reactions. The enzymatic reactions are conducted at 30oC for 40 minutes.
The reaction mixture contains enzyme substrate and respective kinases. After the enzymatic
reaction, 50 µl of enzyme-Glo Plus luminescence topoisomerase assay solution (Promega) is
added to each reaction and the plate is incubated for 5 minutes at room temperature.
Luminescence signal is measured using a BioTek Synergy 2 microplate reader.The
Chemical and Pharmaceutical Bulletin Advance Publication

luminescence data are analyzed using the computer software, Graphpad Prism. The
difference between luminescence intensities in the absence of topoisomerase (Lut) and in the
presence of an enzyme (Luc) is defined as 100% activity (Lut-Luc).The values of topo IIβ
activity percentages versus a series of molecules concentrations are then plotted using non-
linear regression analysis of sigmoidal dose-response curve. The IC50 value is determined
using Graph pad Prism software. All experiments are repeated three times.
4.2.3. In vitro DNA immunofluorescence binding assay:
Histochemical immunofluorescence assay for establishing indirect DNA binding
affinity is performed [29]. Briefly, slides of fixed MCF-7 cells are 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 are incubated for 30 min at 37◦C with pico-green
dye solution (Abcam Inc., USA). Slides are rinsed again with PBS to remove excess dye,
then treated with an ethanolic solution of the tested compounds 4b, 9, 11, 14 and PG at the
concentration of their IC50 at 37 ◦C for 24 h. After washing, images are visualized at 642-645
nm by a fluorescence microscope (Axiostar Plus, Zeiss, Goettingen, Germany) installed with
a camera (Power Shot A20, Canon, USA).
4.2.4. In vitro DNA- flow cytometry and Annexin staining analysis:
4.2.4a.DNA -flow cytometry analysis:
The MCF-7 cells are incubated at a density of 3 x106 cells/mL RPMI-1640 medium in
T-75 flasks for 24 h and then treated with tested molecules 4b, 9, 11, 14 and PG at their IC50
(µM) for 24 h. The MCF-7 cells are then collected by trypsinization, washed with PBS and
fixed. After that, treated cells are stained using the cycle test plus DNA reagent kit (BD
Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions [30]. The
percentage of cell cycle distribution is calculated using CELLQUEST software (Becton
Dickinson Immuno-cytometry Systems, San Jose, CA, USA).
4.2.4b. Annexin staining apoptosis analysis.
The cells are cultured in a 10-cm plate for 24 h, after which IC50 concentration of
tested molecules 4b, 9, 11, 14 and PG are added and incubated for up 24 h. Then, the cells are
washed with PBS and detached by trypsin. The detached cells are collected into a 15-mL
centrifuged tube, washed with ice-cold PBS twice, and centrifuge at 1200 rpm for 5 min. A
volume of 0.1 mL binding buffer, is added to the treated MCF-7 cells, followed by adding 5
μL of annexin V-FITC and 5 μL of 50 μg/mL PI staining reagents. After mixing and reacting
Chemical and Pharmaceutical Bulletin Advance Publication

at 25 °C for 15 min in the dark, the apoptotic cell percentages are analyzed by a flow
cytometer. All experiments are done in triplicates.
4.2.5. In vitro ElISA immunoassay for p53 and cell death modulators:
The concentration of p53 protein and cell death modulators as Puma, Bax, Bcl-2 are
measured using p53 ELISA kit, Puma ELISA Human (RAB0500), Human, Bcl-2 Elisa kit
and Bax ELISA kit. The procedure is done according to the manufacturer’s instructions.
Briefly, lysates of MCF-7 cells are prepared from control and treated cells treated with IC50
concentration of the tested molecules. Then equal amounts of cell lysates are loaded then
probed with specific antibodies. The optical densities are measured at 450 nm in ROBONEK
P2000 ELISA reader. The experiments are done in triplicates.
4.2.6. In vitro green flow cytometry assay for active caspase 3/7 percentage:
The caspase 3/7 percentages are measured for the effect of tested molecules over
MCF-7 cells in cell lysates using caspase-3/7 green flow cytometry assay kit (Cell Event),
Catalog Number (C10427) per the manufacturer's instructions.
5-Acknowledgments:
The author is grateful to Dr. Al-ShorbagyMY. associate professor Pharmacology and
Toxicology Department, Faculty of Pharmacy, Cairo University for statistical analysis of the
data, Dr. Esam Rashwan, Vacsera, EGYPT, for performing p53 and cell death modulators
assay, Dr. Enas. Ahmed Mohamed, associate professor of anatomy and embryology. Faculty
of Medicine, Cairo University for measuring DNA histochemical immunofluorescence assay
and BPS Bioscience Corporation (USA) for enzyme assay.
6- Conflict of Interest :
The author declares no conflict of interest.
7- Supplementary Materials :
The online version of this article contains supplementary materials.
Chemical and Pharmaceutical Bulletin Advance Publication

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Chemical and Pharmaceutical Bulletin Advance Publication

Table 1: IC50 of carbonitriles 3a-c, carboximidamides 4a-c and oxadiazoles 5-19 on MCF-7
cells (μM) and topo enzyme (nM).
Code
3a
3b
3c
4a
4b
4c
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
PG
R1
R2
R3
MCF-7a
HCT-116b
Topoc
IC50 (nM)e
NT
IC50(μM)d
-CH3
-CH3
-
--
-
-
-
19.45±1.61
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
NT
NT
NT
NT
-(CH2)4-
NT
NT
NT
4-CH3OC6H4
-CH3
H
-CH3
-(CH2)4-
10.52±1.03 123.49±9.41
0.57±0.06
NT
4-CH3OC6H4
-CH3
H
-
H
35.32±4.08
NT
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
4-Cl
4-Br
4-CH3
4-OCH3
H
4-Cl
4-Br
4-CH3
4-OCH3
H
4-Cl
4-CH3
4-Br
4-OCH3
-
NT
NT
NT
NT
NT
NT
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
-(CH2)4-
-(CH2)4-
-(CH2)4-
CH2)4-
-(CH2)4-
1.17±0.09
NT
27.28±1.11
NT
4-CH3OC6H4
H
H
H
H
H
--
4-CH3OC6H4
4-CH3OC6H4
4-CH3OC6H4
4-CH3OC6H4
-
NT
NT
NT
NT
NT
NT
NT
NT
2.84±0.26
73.14±6.23
MCF7, a Human breast, HCT116, b Human colon cancer cell lines, Topo c , topoisomerase 2β
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.
Chemical and Pharmaceutical Bulletin Advance Publication

H3CO
NH
N
N
S
HN
S
C5H11
H3C
S
N
N
Terpyridine
Prodigiosin(PG)
Terthiophene(TER)
H3CO
NH
N
O
N
O
Cl
H3C
N
N
N
HN
Cl
S
S
CF3
H3C
F3C
SEW-2871
MX-74420
Obatoclax
O
N
O
H3CO2C
O
N
O
I
VA-62784
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.
(Color figure can be accessed in the online version.)
Chemical and Pharmaceutical Bulletin Advance Publication

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.
(Color figure can be accessed in the online version.)
Chemical and Pharmaceutical Bulletin Advance Publication

O
Cl
R3
R3
O
O
N
R3
O
N
HON
N
NH2
NH2
N
R1
R2
R1
R2
R1
R2
N
N
S
S
S
5-19
4a-c
4a, R1, R2 = 2 CH3, 4b, R1, R2 = - (CH2)4-, 4c R1, R2 =H, 4-CH3O-C6H4
R3 = H, Cl, Br, CH3, OCH3
Fig. 3: Sketch diagram represent the mechanism of formation of 3,5-diaryl oxadiazoles 5-19
from amidoximes 4a-c.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 4: Dose response curves for cell percentage viability of MCF-7cells, HCT-116 cells after
treatment with tested molecules determined by MTT assay. A -D) Growth Inhibition effect of
molecules on MCF-7 cells, E) Growth Inhibition effect of tested molecules on HCT-116 cells,
F) Graphical presentation of the IC50 of tested molecules. Data is shown as mean + SEM (n:
3).
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 5: A, B) In vitro 5 dose response curves for the determination of topo 2- β enzyme IC50
(nM) for molecules 4b, 4c, 9, 11, 12, 14 compared with PG, C) Graphical presentation for
comparison of IC50 for topo 2- β enzyme (nM) of the tested molecules and PG. The obtained
data and are expressed as mean (n: 3) ± SEM.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 6: Fluorescence intensity (IFU) of DNA indirect binding assay in MCF-7 cells for the
effect of compounds 4b, 4c, 9 and 14 at their IC50 compared with PG as control for 24h. A)
Untreated cells, B) Cells treated with of 4b (4.52 μM), C) Cells treated with molecule 4c
(0.83 μM), D) Cells treated with molecule 9 (0.48 μM), E) Cells treated with molecule 14
(0.19 μM), F) Cells treated with PG (1.93 μM).
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 7: DNA Flow cytometric analysis of the effect of molecule 4b, 4c, 9 and 14 at their IC50
on MCF-7 cells for 24h compared with PG, A) Control cells, B) Cells treated with of
molecule 4b (4.52 μM), C) Cells treated with 4c (0.83 μM), D) cells treated with 9 (0.48 μM),
E) Cells treated with 14 (0.19 μM), F) Cells treated with PG (1.93 μM). G) Statically analysis
of the obtained data and are expressed as mean (n: 3 experiments) ± SEM and statistical
comparisons are carried out using one-way analysis of variance (ANOVA) followed by
Tukey multiple comparisons.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 8: Annexin V/PI analysis by flow cytometry of MCF-7 cells treated with molecules 4b,
4c, 9 and 14 at their IC50 compared with PG as e control for 24h. A) Control cells, B) Cells
treated with of molecule 4b (4.52 μM), C) Cells treated with 4c (0.83 μM), D) cells treated
with 9 (0.48 μM), E) Cells treated with 14 (0.19 μM), F) Cells treated with PG (1.93 μM). G)
Statistical analysis, data are expressed as mean (n: 3) ± SEM and statistical comparisons are
carried out using one-way analysis of variance (ANOVA) followed by Tukey multiple
comparisons. The red color represents the stained Annexin V cells.
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