Design, synthesis, biological evaluation and molecular docking of new uracil analogs-1,2,4-oxadiazole hybrids as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2020.127438

A new series of uracil analogues-1,2,4-oxadiazole hybrid derivatives were synthesized by a new, simple, and efficient method using for the first time HAP-SO₃H as an heterogenous acid catalyst for the condensation and cyclization between amidoxime and aldehyde. The new derivatives were characterized by HRMS, FT-IR, ¹H NMR, and ¹³C NMR spectroscopy techniques. The synthesized 1,2,4-oxadiazole hybrids were evaluated for their cytotoxic activity in five human cancer cell lines: melanoma (A-375), fibrosarcoma (HT-1080), breast (MCF-7 and MDA-MB-231), and lung carcinoma (A-549). Data showed that compounds 22 and 23 were potent cytotoxic agents against HT-1080 and MFC-7 cells with IC₅₀ inferior to 1 μM. The possible mechanism of apoptosis induction by the derivatives was investigated using Annexin V staining, caspase-3/7 activity, mitochondrial membrane potential measurement, and analysis cell cycle progression. The compound 22 induced apoptosis through caspase-3/7 activation and S-phase arrest in HT-1080 and A549 cells. The molecular docking showed that compound 22 activated the caspase-3 by forming a stable protein-ligand complex.

Full text of DOI:10.1016/j.bmcl.2020.127438

Journal Pre-proofs
Design, synthesis, biological evaluation and molecular docking of new uracil
analogs-1,2,4-oxadiazole hybrids as potential anticancer agents
Az-Eddine El Mansouri, Ali Oubella, Mohamed Maatallah, Moulay Youssef
AitItto, MohamedZahouily, Hamid Morjani, Hassan B. Lazrek
PII:
S0960-894X(20)30549-7
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127438
BMCL 127438
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
14 June 2020
3 July 2020
21 July 2020
Please cite this article as: El Mansouri, A-E., Oubella, A., Maatallah, M., Youssef AitItto, M.,
MohamedZahouily, Morjani, H., Lazrek, H.B., Design, synthesis, biological evaluation and molecular docking of
new uracil analogs-1,2,4-oxadiazole hybrids as potential anticancer agents, Bioorganic & Medicinal Chemistry
Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127438
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Design, synthesis, biological evaluation and molecular docking of new uracil analogs-
1,2,4-oxadiazole hybrids as potential anticancer agents
Az-Eddine El Mansouria,b,Ali Oubellac,Mohamed Maatallahd,Moulay Youssef AitIttod,
MohamedZahouilya,e*, Hamid Morjanif*, Hassan B. Lazrekb*
aLaboratoire de Materiaux, Catalyse & Valorisation des Ressources Naturelles, URAC 24, Faculte des
Sciences et Techniques, Universite Hassan II, Casablanca, Morocco.
bLaboratory of Biomolecular and Medicinal Chemistry, Department of Chemistry, Faculty of Science
Semlalia, BP 2390, Marrakech 40001, Morocco.
cLaboratoire de Synthese Organique et de Physico-Chimie Moleculaire, Departement de Chimie, Faculte´
des Sciences, Semlalia BP 2390, Marrakech 40001, Morocco,
dLaboratoire de Chimie théorique Faculty of Science SemlaliaBP 2390, Marrakech 40001, Morocco.
eMoroccan Foundation for Advanced Science, Innovation and Research (MAScIR), VARENA Center,
Rue Mohamed El Jazouli, Madinat Al Irfane, 10100 Rabat, Morocco
fBioSpecT - EA7506 UFR de Pharmacie, Univ-Reims 51, rue Cognacq Jay 51096 Reims cedex.
Hassan B. Lazrek: Email address- hblazrek50@gmail.com, Hamid Morjani: Email address-
hamid.morjani@univ-reims.fr, MohamedZahouily: Email address-mzahouily@gmail.com
Highlights
- Novel synthetic method for preparation of 1, 2, 4-oxadiazole derivatives was developed
- A novel series of uracil analogues-1,2,4-oxadiazole hybrids were synthesized and evaluated for their
anticancer activity against 5 cancer cells.
- The compounds 22 and 23 exhibited better antiproliferative activities than reference drug DOX
against MCF-7.
- Compound 22 induced apoptosis through activation of caspase 3/7 and arrested S-phase in cell cycle
- Molecular docking suggested that compound 22 activate caspase-3
Abstract
A new series of uracil analogues-1,2,4-oxadiazole hybrid derivatives were synthesized by a new,
simple, and efficient method using for the first time HAP-SO3H as an heterogenous acid catalyst for the
condensation and cyclization between amidoxime and aldehyde. The new derivatives were characterized
1
13
by HRMS, FT-IR, H NMR, and C NMR spectroscopy techniques. The synthesized 1,2,4-oxadiazole
hybrids were evaluated for their cytotoxic activity in five human cancer cell lines: melanoma (A-375),
fibrosarcoma (HT-1080), breast (MCF-7 and MDA-MB-231), and lung carcinoma (A-549). Data showed
that compounds 22 and 23were potent cytotoxic agents against HT-1080 and MFC-7 cells with IC50
inferior to 1 µM. The possible mechanism of apoptosis induction by the derivatives was investigated
using Annexin V staining, caspase-3/7 activity, mitochondrial membrane potential measurement, and
analysis cell cycle progression. The compound 22 induced apoptosis through caspase-3/7 activation and
S-phase arrest in HT-1080 and A549 cells. The molecular docking showed that compound 22 activated
the caspase-3 by forming a stable protein-ligand complex.

Graphical abstract
5-Fluorouracil
O
F
HN
O
N
Biphenyl
1,2,4-oxadiazole
N
R
N
O
Linker
In-vitro
Cytotoxic Activity
MCF-7: IC50=0.89 µM (R=OCH3) < IC50=1.93 µM (DOX)
HT-1080: IC50= 0.88 µM (R=H), 0.89 µM (R=OCH3)
Key words: hybrid molecules, uracil analogues,1,2,4-oxadiazole, heterogeneous catalyst, anticancer
activity, apoptosis, molecular docking.
Due to the high number of death, cancer treatment has attracted the attention of medicinal chemists to
prepare novel efficient anticancer drugs, with minimal side effects and active at a small
dose[1].Moreover, the discovery of new anticancer drugs has become a challenge against the failure of
several treatment strategies due to the development of drug resistance, toxicity, and reduced
bioavailability[2]. Therefore, apoptosis has become one of the attractive targets for the design and
development of new anticancer agents [3]. Apoptosis is a natural form of a programmed cell death
process that is essential to maintain tissue homeostasis [4]. This mode of action is recognized by
characteristic changes in the cells, such as caspase cascade activation [5]. Indeed, caspases (cysteinyl-
aspartate-specific proteases) cleave an aspartic acid from their respective peptide substrates in the
presence of a histidine residue, which leads to cell death [6]. Among these, caspases-3 and -7 are the key
effector enzymes of the apoptosis execution [7].
On the other hand, the caspase activation in apoptosis is considered as an attractive target for the
development of new selective anticancer drugs [8].Indeed, several uracil analogs, such as compound 1
(Figure 1)and 5-Fluorouracil derivatives have been described as a promising class of anticancer agents
mediated by the mechanism of apoptosis induction, by activation of the caspase-3 in renal cell carcinoma,
and cell cycle arrest[9-11]. In addition, the U.S. Food and Drug Administration (FDA) has approved 5-
fluoro-2'-deoxyuridine (Floxuridine 2, Figure 1) since 1970 as an anticancer drug[12]. Also, it was
discovered that 6-azauracil and 6-azauridine3 are potent inhibitors of tumor growth [13-15].

Over the past years, heterocyclic oxadiazole derivatives have been developed and widely studied for
their anticancer[16], antiviral[17], antioxidant[18], anti-inflammatory [19], antifungal activity [20], and
antibacterial activities [21]. In particular, 1,2,4-oxadiazole analogues react as a hydrolysis-resistant ester
or amide bioisostere, which can be used in the treatment of a wide range of diseases [22-25].The high
biological potential of 1,2,4-oxadiazole derivatives against cancer cells has been explained by different
modes of action. For instance, Khan et al. have synthesized some promising candidates (product 4as an
example: Fig. 1), which induces apoptosis with activities against several breast and colorectal cancer cell
lines in vitro and in vivo [26]. Moreover, new 1, 2, 4-oxadiazole derivatives (5 and 6as examples) have
been prepared for the treatment of caspase-mediated cancer diseases [27-31].
These findings motivated us to synthesize new uracil analogues-1,2,4-oxadiazole hybrids as
potentialanticancer agents. The compounds were designed by the incorporation of the nucleobases
(Uracil, 5-Fluorouracil, and 6-Azauracil) and 1,2,4-oxadiazole via benzyl linker to combine the benefits
of their effects to give a compact structure with promising anticancer activity. The benzyl group allows
greater flexibility around a CH2–nucleobase bond and could make π-interactions [32, 33]. Also, to gain
more information about the structure-activity relationship, various phenyl derivatives were introduced to
the oxadiazole ring (Figure 1).The desired hybrids were prepared using for the first time HAP-SO3H as a
heterogeneous acid catalyst. The cytotoxic effect of these hybrid molecules was studied first inthe A375
cell line. Therefore, the most two active compounds (22, 23) were investigated in HT-1080, A-549, MCF-
7, and MDA-MB-231 cells. Then, the possible mechanism of apoptosis induction through the activation
of caspase was run down. Finally, molecular docking was carried out to characterize the ability of
compound 22toactivate thecaspase-3 protein.

O
6-Azauracil
5-Fluorouracil
O
Uracil
O
NH
F
NH
N
NH
N
O
(CH2)6
N
O
O
N
O
HO
HO
H
O
O
O
H
H
OH OH
OH
HO
2
1
3
CF3
Cl
Cl
Br
1,2,4-oxadiazole
N
N
S
N
O
S
N
O
N
O
O
N
N
O
1,2,4-oxadiazole
1,2,4-oxadiazole
4
6
5
Molecular
Hybridization
Uracil: R1= H, Y = CH
nucleobase moity
5-Fluorouracil: R1= F, Y = CH
6-Azauracil: R1= F, Y = N
O
R1
HN
Y
O
N
Benzyl
N
R2
linke
N
O
r
1,2,4-oxadiazole
Figure 1: Design strategy of new uracil analogs-1, 2, 4-oxadiazole hybrids as potential anticancer
agents
The most used methods for the synthesis of 1,2,4-oxadiazole are 1,3-dipolar cycloaddition (2 + 3
strategy) of nitrile/nitrile oxide and hetero-cyclization of amidoxime with carboxylic or aldehyde
derivatives. In the latter method, the condensation of amidoxime with carboxylic or aldehyde derivatives
give respectively O-acylamidoxime or N–hydroxyl imidamide, followed by intramolecular cyclization to
afford 1,2,4-oxadiazole. However, carboxylic derivatives have several drawbacks: (i) acyl chlorides are
very toxic, few derivatives are available and hard to handle. (ii) while carboxylic acids need a coupling
reagent such as DCC, EDC, CDI, TBTU, HOBt, or HBTU [34-41]to react with amidoximes. On the other
hand, many catalysts and reagents were developed in the literature to prepare 1,2,4-oxadiazole from

aldehyde as EtOH/H2O/MnO2 or NaOCl [42], Cs2CO3 [43], AcOH [44]. Herein, we report a new, simple,
and efficient acid-mediated Hydroxyapatite-sulfonic acid (HAP-SO3H) one-pot synthesis of new 1,2,4-
oxadiazole hybrid analogues from amidoximes and aldehydes.
The synthetic route of the target new1,2,4-oxadiazole hybrid derivatives 17-26 is shown in scheme
1and table 2. First, the intermediates 4-6were prepared through the N1-alkylation of pyrimidin-2,4-diones
with 4-(bromomethyl) benzonitrile using modified Hilbert-Jonhson reaction [45, 46]. The treatment of 4-
6with hydroxylamine hydrochloride and trimethylamine in ethanol at reflux gave amidoximes 7-9 [47].
On the other hand, biphenyl aldehydes derivatives 13-14 were prepared by the C-C coupling reaction of
boronic acids 10-11 with 4-bromobenzaldehyde12 using Na2PdP2O7as a heterogeneous catalyst in water
[48].
As illustrated in Table1, the target compound 17was prepared from amidoxime 7 and benzaldehyde 15
at 80° for 24 hours. The optimal conditions were examined by changingthe catalyst, oxidant,and solvent.
Firstly, we applied literature conditions for the preparation of 1,2,4-oxadiazole in the basic medium
Cs2CO3[43] or Cs2CO3 in the presence of I2 as oxidant [49], but no reaction has occurred (entry1 and 2,
table1). Meanwhile, Teimouri et al. have developed a strategy for the synthesis of 1,3,4-oxadiazoles via
alumina and nano-γ-alumina Sulfuric Acid [50]. Thus, to obtain desired products, the reaction conditions
were changed to acidic medium. To do this, we used acetic acid in ethanol to afforded product 17 in 31%
yields. Moreover, we replaced acetic acid by a heterogeneous acid catalyst K10/CF3SO3H [51] to give
compound 17 with 36%. Next, the use of the new heterogeneous catalyst Hydroxyapatite-sulfonic acid
(HAP-SO3H) increased the yield to 49% (table1, entry7 vs 8). Therefore, we studied the influence of
iodine as anoxidant on the course of the reaction [37]. As a result, the addition of 1 equivalent of iodine
enhanced the yield of desired product 17 to 71% (table1, entry 10). It is interesting to notice when the
reaction was carried out using 0.5 equivalents of iodine, or without HAP-SO3H, the yield of compound 17
decreased to 54% (table1, entry9 vs 12). Screening of solvents was also carried out and it was found that
the reaction required polar and protic solvent. The best results were found using the MeOH/DMSO
couple (entry7, table1).
The optimal conditions (table1, entry10) were generalized to prepare desired hybrid derivatives17-26
in 49-72% yields, as showed in table 2. All products were characterized by High-Resolution Mass
Spectrometry (HRMS), Nuclear Magnetic Resonance (1H-NMR; 13C-NMR) and Fourier Transform
Infrared (FTIR) spectroscopy methods (Experimental data).

O
N
O
N
R1
R1
O
NH
NH
R1
1) HMDS, (NH4)2SO4, 120°C, 3h
2) R-Br, KI, CH3CN, 80°C, 12h
Y
(NH2OH, HCl), Et3N
EtOH, Reflux, 3h
NH
Y
O
O
Y
N
H
1-3
O
H2N
NC
4-6
N
7-9
HO
1, 4, 7: R1= H, Y= CH
2, 5, 8: R1= F, Y= CH
3, 6, 9: R1= H, Y= N
R2
Br
O
H
Na2PdP2O7, K2CO3
Water, MW,100°C, 15min
R2
H
B(OH)2
O
10, 11
12
10, 13: R2= H
13, 14
11, 14: R2= OCH3
Scheme 1: synthetic route for starting compounds amidoxime 7-9 and biphenyl aldehydes 13-14
Table 1: Optimization of reaction conditions
O
N
O
N
HN
HN
O
H
O
O
Solvent, Catalyst
+
Oxidant, 80°C, 24h
NH2
OH
N
15
N
N
O
7
17
Entry
Solvent
Catalyst
Oxidant
Yield (%)
1
2
3
MeOH/DMSO
MeOH/DMSO
Ethanol
Cs2CO3
Cs2CO3
AcOH
---
I2 (1eq)
---
No reaction
No reaction
31
4
5
Ethanol
MeOH/Dioxane
K10/CF3SO3H
K10/CF3SO3H
---
---
36
No reaction
6
7
8
9
10
11
12
MeOH/Dioxane/DMSO K10/CF3SO3H
---
---
---
41
41
49
54
71
73
50
MeOH/DMSO
MeOH/DMSO
MeOH/DMSO
MeOH/DMSO
MeOH/DMSO
MeOH/DMSO
K10/CF3SO3H
HAP-SO3H
HAP-SO3H
HAP-SO3H
HAP-SO3H
------
I2 (0.5eq)
I2 (1eq)
I2 (1.5eq)
I2 (1eq)
*Optimal conditions: 17 (0.13mmole, 35mg), benzaldehyde 15 (0.26mmole, 0.03ml), HAP-SO3H (8mg),
I2 (0.13mmole, 34mg), MeOH/DMSO (1.5ml/0.5ml), 80°C, 24h.

Table 2: Generalization of the method on different amidoximes and aldehydes
O
O
R1
R1
NH
O
H
NH
Y
Y
N
O
N
O
R2
N
NH2
OH
R2
N
N O
17-26
13-16
7-9
O
O
O
F
HN
O
HN
HN
O
N
N
N
N
O
R2
R2
R2
N
N
N
N O
N O
N O
2
2
2
24
R =H: , 71%
17
R =H: , 71%
20
R =H: , 72%
2
2
2
25
R =4-OCH3: , 62%
18
21
R =OCH3: , 65%
R =4-OCH3: , 59%
2
2
2
26
R =4-OCH3Ph: , 47%
19
22
R =Ph: , 60%
R =Ph: , 60%
2
23
R =4-OCH3Ph: , 64%
*Condition: Amidoxime 7-9 (0.13mmole), aldehyde 13-16 (0.26mmole), HAP-SO3H (8mg), I2 (0.13mmole, 34mg),
MeOH/DMSO (1.5ml/0.5ml), 80°C, 24h.
First, a preliminary study was conducted to determine the most potent compounds based on the cell
viability evaluation of the synthesized compounds at 5 and 10 µM on melanoma human cell line A375.
Then, the selected two compounds were deeply studied for their cytotoxic effect onHT-1080, A-549,
MCF-7, and MDA-MB-231 at different concentrations, which gave the IC50 values.
As shown in table 4, an inspection of the results according to structural variations between the
molecules showed the following remarks: structural modification of the amidoxime moiety (compound9)
into heterocyclic 1,2,4-oxadiazole (compound24) increased the anti-proliferative activity (Entry 2 vs
Entry 10, Table 4). Hence, the 1,2,4-oxadiazole ring was kept, and we examined the effect of pyrimidine
nucleobase, three bases were studied uracil, 5-fluorouracil, and 6-azauracil. As indicated in table 4 (Entry
3 vs. Entry 6 vs. Entry 10), the best activity was obtained when 5-fluorouracil was used as a nucleobase.
Finally, by keeping the 5-fluorouracil ring unchanged in position 3, we explored the modifications on
position 5 of the oxadiazole ring. Four groups were selected phenyl, 4-methoxyphenyl, biphenyl, and 4’-
methoxybiphenyl. It was noticed that the substitution of the hydrogen atom at position 4 of phenyl by
methoxy decreased the anti-proliferative activity (table 4, Entry 6 and 7). While the introduction of the
pharmacophore biphenyl instead of phenyl increased the cytotoxic activity. For instance, the cell viability
decreased from 56% to 29% at 5µM when we compared the compound 20 (phenyl) with 22 (biphenyl).
Similarly, compound 23 bearing 4’-methoxybiphenyl showed better cytotoxicity (Cell viability 29%) than
molecule 21 having the 4-methoxyphenyl group (50%). This result is in accordance with the literature
which stated that the incorporation of this pharmacophore enhanced cell growth inhibition[52, 53].As a
result, the coupling of 5-fluorouracil, 1,2,4-oxadiazole, and substituted biphenyl produced the most

promising activities. In particular, the compounds 22 and 23showed the highest activity with cell viability
inferior to 29 % at 10 and 5µM.
In the next step, the anticancer activities of compounds 22 and 23were extended to the human cell lines
HT-1080, A-549, MCF-7, and MDA-MB-231. Data reported on table 5 shows thatcompound 22exhibited
significant cytotoxicitywith an IC50 value of 0.88µM against HT-1080 cells.The introduction of the
methoxy group in position para of the biphenyl decreased the activity of compound 23to 0.98µM.
Interestingly, whentheMCF-7 cell line was used, the oxadiazole 23 showed higher cytotoxicity than22
with IC50 values of 0.89 and 1.19µM, respectively. Finally, the cancer cell lines HT-1080 and MDA-
MB-231 were less sensitive to these molecules with IC50 values between 3.12 and 8.37µM.
Table 3: in vitro cytotoxic activity (Cell viability) of synthetic compounds 17-26 and 9 against A375
Entry
1
Compound Cell viability (%) at 5µm Cell viability (%) at 10µm
DMSO
9
100
100
76
100
94
72
55
82
30
82
17
23
62
55
44
2
3
4
5
6
7
8
9
17
18
59
19
85
20
56
21
50
22
29
23
29
10
11
12
24
25
100
53
26
76
Table 4: in vitro cytotoxic activity (IC50) of 1, 2, 4-oxadiazoles 22 and 23 against HT1080, A549,
MCF7 and MDA-MB231
IC50 (µM)
Compound
HT-1080
0.88
A-549
3.12
4.02
MCF-7
1.19
0.89
MDA-MB-231
22
23
8.37
6.52
3.41
0.98
DOX
0.93
2.67
1.83
IC 50 values are the concentrations that cause 50 % inhibition of cancer cell growth (µM) after 48h.
To quantify the percentage of apoptotic cells after treating HT-1080 and A-549 cells with 5 µM for
24h of compound 22 annexin-V/7-AAD dual staining analysis was carried out [54]. As shown in Figure
5, the rate of apoptotic cells (early and late apoptosis) increased from 1.34 to 13.81% (10-fold) for HT-
1080 cells and from 9.08 to 29.01 (3-fold) for A-549 cells. In particular, the rate of early apoptotic cells
increased by 3.5-fold for HT-1080 and 1.5-fold for A-549. Also, the percentage of late apoptotic cells
increased from 0.12 to 9.56% in HT-1080 cells and from 3.01 to 14.72% in A-549. These data suggest
that compound22could induce the early event of apoptosis, which is characterized by significant positive
staining of annexin V.

Late Apoptosis
Early Apoptosis
30
20
10
0
Control (HT1080)
22 (HT1080)
)
)
)
)
0
0
l
9
9
8
8
4
4
0
5
0
5
-
1
1
A
-
(
A
T
2
(
T
H
(
H
o
2
(
r
l
2
t
o
2
n
r
t
o
n
C
o
C
Control (A549)
22 (A549)
Figure 5: Annexin V-FITC/7-AAD double staining for detection of apoptosis in HT-1080 and A549
cells after treatment with 5 µM of compound 22 as well as control (DMSO) for 24 h. Compound 22
induces apoptosis.
Dysfunction of mitochondria membrane potential (MMP) has also been reported to be involved in
drug-induced cell death and particularly to contribute to triggering apoptosis. In this study, mitochondrial
dysfunction was determined based on the MMP index that was measured by flow cytometry using the
Muse MitoPotential kit (Millipore-Merck). HT-1080 and A-549 cells were treated with 5 µM of
compound 22 at concentration for 24 h. As shown in Figure 6, compound 22 did not induce a marked
decrease in the MMP index. In fact, in the case of HT-1080 cells, the rate of live cells with high MMP
decreased from 90.10 to 78.67. Similarly, in A-549 cells, this rate decreased from 86.64 to 78.05%. This
suggests that compound 22 is able to induce cell death independently of the mitochondrial pathway.

HT-1080
A-549
100
80
60
40
20
0
Control (HT-1080)
22 (HT-1080)
Control 22
Control 22
Control (A-549)
22 (A-549)
Figure 6: Flow cytometric analysis (FACS) of the mitochondrial membrane potential in HT-1080 and
A-549 cells treated with compound 22 at 5 µM concentration for 24 h.
Caspase-3 and -7 belong to eleven caspases characterized in humans, they are the main downstream
effector caspases that play fundamental roles in drug-induced apoptosis [55].In order to confirm that
compound 22 exerts its cytotoxic effect in HT-1080 and A-549 cells by inducing apoptosis through a
caspase pathway. We investigated the proteolytic activity of caspase-3/7 in HT-1080 and A-549 cell lines
when treated with 5 µM of the compound 22 for 24 hours. Figure 7 indicates that compound 22 increased
3- to 3.5-fold the rate of cells presenting positive activity of caspase 3/7 when compared to the control
cells. These data are in agreement with those obtained on annexin V staining and suggest that compound
22 induced apoptosis through caspase-3/7 activation in fibrosarcoma HT-1080 and adenocarcinoma A-
549 cancer cells.

****
30
20
10
0
***
Control (HT-1080)
22 (HT-1080)
Control 22
Control 22
HT-1080
A-549
Control (A-549)
22 (A-549)
Figure 7: Induction of caspase 3/7 activity in response to compound 22. HT-1080 and A-549 Cells
were treated with 5 µM of desired compound as well as DMSO (control) for 24h.
To determine whether the compounds were able to induce alterations in the cell cycle progression, we
next evaluated the effect of the compound 22 on the cell cycle progression by using flow cytometric
analysis in HT-1080 and A-549 cells after treatment with 5 µM of the compound for 24h (Fig. 8). As
shown in Figure 8, there is an increase in S phase against HT-1080 and A-549 cell lines, while a slight
decrease in G2/M when compared to control cells. These experimental data suggest that the product 22
tested on HT-1080 and A-549 cells induced S phase cell cycle arrest. We observed that the proportion of
cells in the G0/G1 phase was slightly decreased in the presence of compound 22 when compared to
control cells. On the other hand, an increase in the rate of cells in the S phase was observed. G2/M rate
decreased slightly in HT-1080 cells, it remained identical between control and treated cells in the case of
A-549 cells.

G2/M
S
G0/G1
100
50
0
Control (HT-1080)
22 (HT-1080)
)
)
)
)
0
0
l
9
9
8
8
4
4
0
5
0
5
-
1
1
A
-
(
A
T
2
(
T
H
(
H
o
2
(
r
l
2
t
o
2
n
r
t
o
Control (A-549)
22 (A-549)
n
C
o
C
Figure 8: Flow cytometry analysis of cell cycle phase distribution inHT-1080 and A-549 cells after
treatment of 5 µM of compound 22in comparison with control for 24. Compound 22 induced S phase
arrest.
As can be identified from the caspase activation assay, compound 22 induced apoptosis through
caspase-3/7 in fibrosarcoma HT-1080 and adenocarcinoma A-549 cancer cells. Thus, we decided to
continue investigating the caspase activation mechanism of this compound using molecular docking
simulation. Recently, the docking study of 1,2,4-oxadiazole derivatives as potentcaspase-3 activators was
carried out [28]. As shown in Figure 9, the compound 22 was positioned in the binding pocket of caspase-
3 (PDB ID: 1RE1) based on previously published docking studies of this protein [28, 56]. Before that, the
self-docking experiment was carried out. The root-mean-square deviation (RMSD) between the predicted
and the native poses were found to be 0.95Å (1RE1; NA3). This result indicated that our docking
protocol is good for the reproduction of the native poses (<2Å).
The protein-ligand complex analysis shows that the ligand is bound deep inside the active site with the
estimated free energy of binding-8.42kcal/mol. The docked pose of 22 was surrounded by a partially
hydrophobic cavity especially, the biphenyl moiety (Figure 11). The complex is stabilized by the
formation offive hydrogen bondsASN342---O=C4 (2.113Å), TRP348---O=C4 (2.265Å), TRP348---FC5
(2.765Å), ARG341---Noxadiazole(2.585Å), and SER343—O=C2 (2.141Å). The compound 22 also showed
additional Pi-Cation, Pi-Anion,Pi-Donor Hydrogen Bond, Pi-Pi T-shaped, and Pi-Alkyl interactions
(figure9, 10,and Table 1 in supplementary data).These resultsare in accordance with the literature which
stated that several compounds incorporated 1,2,4-oxadiazole, or 5-fluorouracil activated caspase-3[28,

57]. Overall, these results provide a worthy explanation for the anticancer activity of the synthesized
compound 22 as an activator of caspase-3.
Figure 9: The position and the 3D interactions of compound 22 into the binding pocket of caspase-3
Figure 10: 2D interactions of compound 22 with the amino acid residues of caspase-3

Figure 11: The position of ligand 22 in the hydrophobic cavity of caspase-3
In summary, a new, simple, and efficient synthesis strategy using HAP-SO3H as a heterogeneous acid
catalyst was developed in the preparation of novel uracil analogues-1, 2, 4-oxadiazole hybrid molecules.
The procedure offers several advantages, including simple reaction conditions as well as simple
experimental and product isolation procedures. Teen compounds were synthesized and evaluated for their
anticancer activity against A-375, HT-1080, A-549, MCF-7, and MDA-MB-231 cell lines. The
compounds 22 and 23 were the most active in terms of cytotoxic effect. Moreover, these compounds were
more actives than positive control drug DOX, particularly against HT-1080 and MCF-7 cells.The
mechanism of the anti-proliferative effect was studied on HT-1080 and A-549. Data suggest that the
compound 22functionally induced at least two apoptosis markers (annexin V staining and caspase 3/7
activity), whereas, slight changes were observed in mitochondrial membrane potential (MMP) and cell
cycle progression. The molecular docking study also demonstrated the potent anticancer activity of 22 by
providing the strong interaction of this compound with caspase-3 protein. Thus, from the present study, it
can be concluded that the compounds 22 and 23 could be considered as a new molecular template and as
a promising lead candidate for further study.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
Acknowledgements
This paper is dedicated to the memory of Professor W. Pfleiderer (University of Konstance, Germany)
deceased on January 2018. The authors would like to thank Professor R. Benhida, Professeur S. Rocchi
and Dr. H. NedraTekaya (Nice University, France) and Dr. Y. Sanghvi (President of Rasaym Inc.

Encinitas, CA, USA) for their help on the realization of this work. Also, we would like to acknowledge
the technical staff of the CAC (Centre of Analysis and Characterization) University Cadi Ayyad
Marrakech and Faculty of Sciences and Technologies Mohammedia, University Hassan II Casablanca for
running the spectroscopic analysis.
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Highlights Al Mansouri et al.
Highlights
- Novel synthetic method for preparation of 1, 2, 4-oxadiazole derivatives was developed
- A novel series of uracil analogues-1, 2, 4-oxadiazole hybrids were synthesized and evaluated for their
anticancer activity against 5 cancer cells.
- Compound 22 induced apoptosis through activation of caspase 3/7 and arrested S-phase in cell cycle
- Molecular docking suggested that compound 22 activate caspase-3
El Mansouri et al 13.06.20
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.
Graphical abstract

5-Fluorouracil
O
F
HN
O
N
Biphenyl
1,2,4-oxadiazole
N
R
N
O
Linker
In-vitro
Cytotoxic Activity
MCF-7: IC50=0.89 µM (R=OCH3) < IC50=1.93 µM (DOX)
HT-1080: IC50= 0.88 µM (R=H), 0.89 µM (R=OCH3)
List of schemes
O
N
O
R1
R1
O
NH
NH
R1
1) HMDS, (NH4)2SO4, 120°C, 3h
2) R-Br, KI, CH3CN, 80°C, 12h
Y
(NH2OH, HCl), Et3N
EtOH, Reflux, 3h
NH
Y
O
N
O
Y
N
H
1-3
O
H2N
NC
4-6
N
7-9
HO
1, 4, 7: R1= H, Y= CH
2, 5, 8: R1= F, Y= CH
3, 6, 9: R1= H, Y= N
R2
Br
O
H
Na2PdP2O7, K2CO3
Water, MW,100°C, 15min
R2
H
B(OH)2
O
10, 11
12
10, 13: R2= H
13, 14
11, 14: R2= OCH3
Scheme 1: synthetic route for starting compounds amidoxime 7-9 and bis-phenyl aldehydes 13-14
List of Tables
Table 5: Optimization of reaction conditions

O
N
O
N
HN
HN
O
H
O
O
Solvent, Catalyst
Oxidant,80°C, 24h
+
NH2
OH
N
15
N
N
O
7
17
Entry
Solvent
Catalyst
Oxidant
Yield (%)
1
2
3
MeOH/DMSO
MeOH/DMSO
Ethanol
Cs2CO3
Cs2CO3
AcOH
---
I2 (1eq)
---
No reaction
No reaction
31
4
5
6
7
8
9
10
11
12
Ethanol
MeOH/Dioxane
K10/CF3SO3H
K10/CF3SO3H
K10/CF3SO3H
K10/CF3SO3H
HAP-SO3H
HAP-SO3H
HAP-SO3H
HAP-SO3H
------
---
---
---
---
36
No reaction
MeOH/Dioxane/DMSO
MeOH/DMSO
MeOH/DMSO
MeOH/DMSO
MeOH/DMSO
MeOH/DMSO
MeOH/DMSO
41
41
49
54
71
73
50
---
I2 (0.5eq)
I2 (1eq)
I2 (1.5eq)
I2 (1eq)
*Optimal conditions: 17 (0.13mmole, 35mg), benzaldehyde 15 (0.26mmole, 0.03ml), HAP-SO3H (8mg), I2 (0.13mmole, 34mg), MeOH/DMSO (1.5ml/0.5ml),
80°C, 24h.
Table 6: Generalization of the method on different amidoximes and aldehydes
O
O
R1
R1
NH
O
H
NH
Y
Y
N
O
N
O
R2
N
NH2
OH
R2
N
N O
17-26
13-16
7-9
O
O
O
F
HN
O
HN
HN
O
N
N
N
N
O
R2
R2
R2
N
N
N
N O
N O
N O
2
2
2
24
R =H: , 71%
17
R =H: , 71%
20
R =H: , 72%
2
2
2
25
R =4-OCH3: , 62%
18
21
R =OCH3: , 65%
R =4-OCH3: , 59%
2
2
2
26
R =4-OCH3Ph: , 47%
19
22
R =Ph: , 60%
R =Ph: , 60%
2
23
R =4-OCH3Ph: , 64%
*Condition: Amidoxime 7-9 (0.13mmole), aldehyde 13-16 (0.26mmole), HAP-SO3H (8mg), I2 (0.13mmole, 34mg), MeOH/DMSO (1.5ml/0.5ml), 80°C, 24h.
Table 3: in vitro anticancer activity (Cell viability) of synthetic compounds 17-26 and 9 against A375
Entry
Compound
Cell viability (%) at 5 µm
Cell viability (%) at 10 µm
1
2
3
4
5
6
DMSO
9
17
18
19
100
100
76
59
85
100
94
72
55
82
30
20
56

7
8
9
10
11
12
21
22
23
24
25
26
50
29
29
100
53
76
82
17
23
62
55
44
Table 4: in vitro anticancer activity (IC50) of 1, 2, 4-oxadiazoles 22 and 23 against HT1080, A549,
MCF7 and MDA-MB231
IC50 (µM)
Compound
HT-1080
0.88
0.98
A-549
3.12
4.02
MCF-7
1.19
0.89
MDA-MB-231
22
23
8.37
6.52
3.41
DOX
0.93
2.67
1.83
IC 50 values are the concentrations that cause 50 % inhibition of cancer cell growth (µM) after 48h.