Glioblastoma-specific anticancer activity of newly synthetized 3,5-disubstituted isoxazole and 1,4-disubstituted triazole-linked tyrosol conjugates

Article abstract of DOI:10.1016/j.bioorg.2021.105071

Two series of 3,5-disubstituted isoxazoles (6a–e) and 1,4-disubstituted triazoles (8a–e) derivatives have been synthesized from tyrosol (1), a natural phenolic compound, detected in several natural sources such as olive oil, and well-known by its wide spectrum of biological activities. Copper-catalyzed microwave-assisted 1,3-dipolar cycloaddition reactions between tyrosol-alkyne derivative 2 and two series of aryl nitrile oxides (5a–e) and azides (7a-e) regiospecifically afforded 3,5-disubstituted isoxazoles (6a–e) and 1,4-triazole derivatives (8a–e), respectively in quantitative yields. Synthesized compounds were purified and characterized by spectroscopic means including 1D and 2D NMR techniques and HRMS analysis. The newly prepared hybrid molecules have been evaluated for their anticancer and hemolytic activities. Results showed that most derivatives displayed significant antiproliferative activity against human glioblastoma cancer cells (U87) in a dose-dependent manner. Compounds 6d (IC₅₀ = 15.2 ± 1.0 μg/mL) and 8e (IC₅₀ = 21.0 ± 0.9 μg/mL) exhibited more potent anticancer activity. Moreover, most derivatives displayed low hemolytic activity, even at higher concentrations which suggested that these classes of compounds are suitable candidates for further in vivo investigations. The obtained results allow us to consider the newly synthesized isoxazole- and triazole-linked tyrosol derivatives as promising scaffolds for the development of effective anticancer agents.

Full text of DOI:10.1016/j.bioorg.2021.105071

Bioorganic Chemistry 114 (2021) 105071
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Glioblastoma-specific anticancer activity of newly synthetized 3,5-disub-
stituted isoxazole and 1,4-disubstituted triazole-linked tyrosol conjugates
,
,c,
Imen Aissaa 1, Zaineb Abdelkafi-Koubaab 1, Karim Chouaïba, Maroua Jaloulid, Amine Assela,
Anis Romdhanea, Abdel Halim Harrathd, Naziha Marrakchib e, Hichem Ben Janneta
,
c,
,*
a University of Monastir, Faculty of Science of Monastir, Laboratory of Heterocyclic, Chemistry, Natural Products and Reactivity, TeamMedicinal Chemistry and Natural,
Products (LR11ES39), Department of Chemistry, Avenue of Environment, 5019 Monastir, Tunisia
b Pasteur Institute of Tunis, LR20IPT01, Laboratory of Biomolecules, Venoms and Theranostic Applications, 1002 Tunis, Tunisia
c University of Tunis El Manar, 1068 Tunis, Tunisia
d King Saud University, Department of Zoology, College of Science, Riyadh, Saudi Arabia
e University of Tunis El Manar, Faculty of Medicine of Tunis, 1068 Tunis, Tunisia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two series of 3,5-disubstituted isoxazoles (6a–e) and 1,4-disubstituted triazoles (8a–e) derivatives have been
synthesized from tyrosol (1), a natural phenolic compound, detected in several natural sources such as olive oil,
and well-known by its wide spectrum of biological activities. Copper-catalyzed microwave-assisted 1,3-dipolar
cycloaddition reactions between tyrosol-alkyne derivative 2 and two series of aryl nitrile oxides (5a–e) and
azides (7a-e) regiospecifically afforded 3,5-disubstituted isoxazoles (6a–e) and 1,4-triazole derivatives (8a–e),
respectively in quantitative yields. Synthesized compounds were purified and characterized by spectroscopic
means including 1D and 2D NMR techniques and HRMS analysis. The newly prepared hybrid molecules have
been evaluated for their anticancer and hemolytic activities. Results showed that most derivatives displayed
significant antiproliferative activity against human glioblastoma cancer cells (U87) in a dose-dependent manner.
Tyrosol
Isoxazoles
Triazoles
Microwave-assisted synthesis
Antiproliferative activity
Compounds 6d (IC50 ¼ 15.2 ± 1.0 μg/mL) and 8e (IC50 ¼ 21.0 ± 0.9 μg/mL) exhibited more potent anticancer
activity. Moreover, most derivatives displayed low hemolytic activity, even at higher concentrations which
suggested that these classes of compounds are suitable candidates for further in vivo investigations. The obtained
results allow us to consider the newly synthesized isoxazole- and triazole-linked tyrosol derivatives as promising
scaffolds for the development of effective anticancer agents.
1. Introduction
(Antibacterial), Cycloserine (Antitubercular), Risperidone (Antipsy-
chotic), Leflunomide (Antirheumatic) and Acivicin (Antitumor) [5]. In
Hybrid constructs from entities with known biological activity using
“click” chemistry can be an important source for molecular diversity,
making this a promising approach for the development of leads for
medicinal chemistry applications [1,2]. Among these hybrids five
membered heterocycles, such as 1,2,3-triazole/ isoxazole derivatives,
have attracted special attention over the past decade and found wide
applications in medicinal chemistry [3,4]. Isoxazoles have attracted
considerable attention from organic and medicinal chemists due to their
significant biological activities [4]. Successful applications of devel-
oping isoxazole compounds have resulted in multiple marketed drugs
addition, more than thirty patents have been published describing the
possible use of isoxazole compounds to treat several diseases, in
particular cancer [4]. For example, Luminespib (resorcinylic isoxazole
amide NVP-AUY922), an experimental anticancer drug candidate, is
currently under 28 phase I/II clinical trials which most of them are
completed [6]. It has shown promising activity in preclinical testing
against several different tumor types by inhibiting the heat shock pro-
tein 90 (Hsp90), a chaperone protein that plays a role in the modifica-
tion of a variety of proteins implicated in oncogenesis [7]. Others
isoxazole derivatives showed an anticancer activity through apoptosis
induction and cell cycle arrest [8–11].
with diverse
therapeutic activities such
Sulfamethoxazole
* Corresponding author.
E-mail address: hichem.bjannet@gmail.com (H. Ben Jannet).
1
These authors contribute equally in this work.
https://doi.org/10.1016/j.bioorg.2021.105071
Received 16 November 2020; Received in revised form 10 May 2021; Accepted 5 June 2021
Available online 8 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

I. Aissa et al.
Bioorganic Chemistry 114 (2021) 105071
On the other hand, triazoles are a class of N-heterocyclic compounds
Carboxyamidotriazole, have already been used in clinics or under clin-
ical evaluation for cancer treatment [15]. Mechanistic exploration
revealed that triazole derivatives induced apoptosis and cell cycle arrest
[15,19]. Recently, several studies have reported that new triazole/iso-
xazole derivatives, with different pattern of substitution, exhibited sig-
nificant anticancer activity against various malignant glioma cell lines
including glioblastoma, the most malignant and invasive type of
with great importance in medicinal chemistry based on their ability to
act as pharmacophores and linkers between two or more substances of
interest in molecular hybridization approaches [12]. These compounds
exhibited an attractive wide range of targets biological potentials
including anti-proliferative and anticancer against a panel of human cell
lines [13–18]. Some of them, such as Cefatrizine and
d
Entry
Hydroximyl chlorides (%) b
Isoxazoles c
Time (min) Yield (%)
1
4
98
2
3
8
90
6
94
4
5
8
4
88
96
Scheme 1. Synthesis of dipolarophiles 2–4a and tyrosol-3,5-disubstituted isoxazoles (6a–e).
2

I. Aissa et al.
Bioorganic Chemistry 114 (2021) 105071
primary brain [20–22].
confirms the propargylation of the phenol function in 2 by the obser-
vation of an NOE between the methylenic protons (δH 5.25) of the
propargyl fragment and the aromatic protons of tyrosol (δH 6.95, 2H, d,
J = 8.7 Hz). The markedly high yield of 2 (76%) is explained by the
relatively high acidity of the phenolic proton compared to that of the
alcohol function, both of which react with the sodium hydride, to pre-
pare in situ the nucleophilic entity which substitute the bromine atom in
the proaprgyl bromide. We selected this quantitative dipolarophile 2 for
use in 1,3-dipolar cycloaddition reactions.
Interestingly, hybridization of 1,2,3-triazole / isoxazole with
phenolic compounds has provided a new anticancer molecules [15]. A
flavone/isoxazole fused heterocycles and flavone/1,2,3-triazole hybrid
heterocycles compounds were synthesized via an intramolecular cycli-
zation and Cu(I)-catalyzed click 1,3-dipolar cycloaddition and showed
an antiproliferative activity against human breast cancer cell line [23].
Other 1,2,3-triazole-coumarin/flavone hybrids were synthesized and
displayed potent anticancer activity against several human cell lines
[24,25] via induction of apoptosis in cancer cells after accretion of
reactive oxygen species (ROS) or reduction in mitochondrial membrane
potential –––[26,27]. Olive oil phenolic compounds are also an attrac-
tive moieties that could be important to drug design, given their wide
variety of biological activities [28]. Among these compounds, the
tyrosol (2-(4-hydroxyphenyl)-ethanol) (1), present also in wine [29],
Rhodiola rosea [30], and marine fungi [31]. Tyrosol (1) and some of its
derivatives have gained research interest due to their antiplatelet [32],
antioxidant, anti-stress [33], anticancer [34] and antibacterial [35]
activities. These data encourage to continue the hybridization of tyrosol
with other pharmcophores such as 1,2,3-triazole / isoxazole moieties
which may provide novel anticancer candidates.
2.1.2. Dipole synthesis
2.1.2.1. Preparation of hydroximyl chlorides (5a–e). Hydroximyl chlo-
rides (5a–e) are key precursors essential for in situ generation of reactive
nitrile oxides, which participate in 1,3-dipolar cycloaddition reactions
for the synthesis of tyrosol-3,5-disubstituted isoxazoles. The precursors
(5a–e) were already synthesized, in one of our previous works [2], from
the appropriate aldehydes using a two-step reaction according to the
general procedure described in the literature [36]. The desired aromatic
imidoyl chlorides were obtained in yields ranging from 85% to 96%
(Scheme 1). The intermediate nitrile oxide reagents were formed from in
situ dehydrohalogenation of the corresponding hydroximyl chlorides
(5a–e) using triethylamine as a base.
In this study, we used tyrosol (1) as a starting material to synthesize
new tyrosol-3,5-disubstituted isoxazoles (6a–e) via 1,3-dipolar cyclo-
addition using various aromatic hydroximyl chlorides, and access to 1,4-
disubstituted triazole derivatives (8a–e) via CuAAC. Additionally, we
developed a regiospecific, simple, and versatile Cu(I)-catalyzed, micro-
wave-assisted procedure for preparation of these series. Based on the
above cited findings and the potential anticancer activity of isoxazoles
and triazoles, the newly synthesized compounds have been evaluated for
their antitumor effect against human glioblastoma cells (U87) as well as
for their hemolytic activity.
2.1.2.2. Preparation of aromatic azides (7a–e). The precursors (7a–e)
were synthesized from the appropriate anilines using a two-step pro-
cedure involving diazotization with sodium nitrite under acidic condi-
tions, followed by displacement with sodium azide [13]. The desired
aromatic azides (7a–e) were obtained in high yields ranging from 90 to
98% (Scheme 2).
2.1.3. Synthesis of tyrosol-3,5-disubstituted isoxazoles (6a–e)
2. Results and discussion
A regiospecific approach was applied using dipolarophile 2 and
various aromatic hydroximyl chlorides (5a–e) in the presence of CuI and
Et3N, resulting in the formation of regiospecific tyrosol-3,5-
disubstituted isoxazoles derivatives (6a–e) in excellent yields. All re-
actions were performed under microwave irradiation (200 W) and
completed within 4 to 8 min. The new compounds (6a–e) were obtained
in good yields (88–98%) (Scheme 1). This method has the advantage of
being simple, regiospecific, fast (shorter times than those of similar
previous reactions [37]), and leading to high yields.
2.1. Chemistry
2.1.1. Synthesis of tyrosol-alkyne derivatives 2–4 as dipolarophiles
Tyrosol (1) was subjected to a propargylation in dry DMF at room
temperature in the presence of NaH and propargyl bromide for 3 h. The
reaction was monitored by TLC and yielded a mixture of differently
propargylated compounds in a 97% global yield (Scheme 1). Chro-
matographic separation of this mixture over a silica-gel column afforded
compounds 2 (76%), 3 (trace), and 4 (21%). The high yield of compound
2 relative to its analogues (3 and 4) was explained by the higher reac-
tivity of the phenol function compared with alcohol.
The synthesized tyrosol-3,5-disubstituted isoxazoles (6a–e) were
characterized by 1H, 13C NMR and ESI-HRMS analysis. The absence of
the 1H NMR signal relative to the terminal alkyne group in dipolarophile
2 at δH 2.51 (1H, t, J = 2.4 Hz) and the observation of a new signal at δH
The structures of the propargylated compounds 2–4 were unambig-
uously confirmed by their NMR and ESI-HRMS spectral data. The mo-
lecular formula of compound 2 (C11H12O2) as determined by ESI-HRMS
(m/z 177. 0925 [M + H]+) agreed with a monopropargylated structure
of tyrosol (1). Additionally, the molecular formula of compound 4
(C14H13O2) as determined by ESI-HRMS (m/z 215.1080 [M + H]+)
agreed with the dipropargylation of tyrosol (1). The position of the
alkylation in the obtained derivatives was deduced by simple compari-
son of their NMR spectral data (1H and 13C) with those of the starting
substrate 1. In addition to signals corresponding to the protons and
carbons of tyrosol (1), new signals related to the alkyl group (methylene
and methylidyne) were observed in NMR spectra (1H and 13C).
–
~ 6.58–6.65 (1H, s) corresponding to the C H proton of the isoxazole
ring supported the progress of the cycloaddition reaction to yield the
desired 3,5-disubstituted isoxazoles 6a–e. Their structures were also
supported by their 13C NMR spectra, showing the disappearance of the
terminal carbon signal of the alkyne group at δC ~ 75–78, and the
appearance of a new signal relating to the tertiary carbon in the iso-
xazole ring at δC ~ 100 , 1–100.8.
2.1.4. CuAAC for the synthesis of tyrosol-1,4-disubstituted triazoles (8a–e)
A regiospecific approach using a Huisgen 1,3-dipolar cycloaddition
reaction (CuAAC) of alkyne 2 with various aromatic azides (7a–e) in the
presence of CuI and Et3N resulted in formation of regiospecific 1,4-
disubstituted triazoles derivatives (8a–e) in excellent yields (85–96%)
(Scheme 2). All the reactions were performed under microwave irradi-
ation (250 W) and achieved within 2 to 6 min under solvent-free con-
ditions. The formed products were easily obtained from the reaction
mixture by simple extraction [38].
The distinction between the structure of the propargylated derivative
2 compared to its analogue 3 was easily deduced by the relatively high
deshielding of methylenic protons (δH 4.65, Fig. S1) of the propargyl
moiety in 2 attached to the phenol function of 1 compared to the same
protons (δH 4.15) in 3 (trace) where the propargyl moiety is attached to
the primary alcohol function (Fig. S3). This deshielding is undoubtedly
explained by the donor mesomeric effect + M exerted only by the non-
binding doublets oxygen atom of the phenol function. Moreover, the
NOESY spectrum of the cycloadduct 8c (Fig. S21), taken as an example,
The structures of the tyrosol-1,4-disubstituted triazoles regioisomers
were established according to their spectral data. The 1H NMR spectra of
compounds 8a–e showed a singlet at δH ~ 8.00–8.08 attributable to the
3

I. Aissa et al.
Bioorganic Chemistry 114 (2021) 105071
c
Entry
Aromatic azides (%)b
1,4-disubstituted triazoles
Time (min) Yield (%)
1
3
92
2
3
3
94
5
85
4
5
3
5
96
92
Scheme 2. Tyrosol-1,4-disubstituted triazoles (8a–e) prepared by the CuAACa.
H-5 proton of the triazole ring, and signals were observed in the aro-
tyrosol (1) and its derivatives (6a–e) and (8a–e) for 24 and 72 h.
Compared with natural tyrosol (1), all of the synthesized compounds
decreased U87 cell viability in a dose and time-dependent manners
(Figs. 1, 2). Interestingly, tyrosol-coupled 3,5-disubstituted isoxazole
derivatives (6a–e) (IC50 = 52–80 µg/mL) (Fig. 1A) were more active
towards U87 cells than tyrosol-coupled 1,4-disubstituted triazoles
matic region (δH 6.90–7.80 attributable to the new aromatic protons
introduced by the azides. These structures were further supported by 13
C
NMR and DEPT spectra, which showed all of the expected carbon signals
corresponding to the tyrosol-triazole derivatives, including the new ar-
omatic carbons resonating at δC ~ 114.0–160.0 introduced by the
azides. The regiospecificity of this reaction leading exclusively to 1,4-
regioisomers was supported by the dipolar interactions (NOE)
observed between H-5triazole/Hmethylene and H-5triazole/Harom, and the
absence of any NOE between Hmethylene/Harom [39,40]. HRMS data of all
of the prepared derivatives were also in agreement with the proposed
structures.
(8a–e) (IC50 > 100 μg/mL, except for 8e) after 24 h treatment (Fig. 1B).
These findings suggested that changing substitutions in the aromatic
system and differences in triazole or isoxazole rings influenced the
cytotoxic activity against glioblastoma cells.
When the treatment was extended for 72 h, all derivatives at
increasing concentrations (0–100 µg/ mL) exerted potent anti-
proliferative activity against U87 cells (Fig. 2). Moreover, the results
showed that the junction of tyrosol (1) with isoxasoles (6a–e) (Fig. 2A)
and triazoles (8a–e) (Fig. 2B) through the linker methylene improved its
antiproliferative activity at lower concentrations. Additionally, tyrosol-
coupled 3,5-disubstituted isoxazole derivatives (6a–e) (IC50 = 15–31
µg/mL) exhibited a higher degree of growth inhibition against U87 cells
2.2. Pharmacological screening and/or biological evaluation
2.2.1. Antitumor activity
2.2.1.1. Antiproliferative activity. The antitumor potential of the newly
synthesized compounds was evaluated against the human glioblastoma
cell line (U87). Cells were incubated with different concentrations of
than tyrosol-coupled 1,4-disubstituted triazoles (8a–e) (IC50 > 30
mL, except for 8e) (Table 1).
μg/
4

I. Aissa et al.
Bioorganic Chemistry 114 (2021) 105071
Fig. 1. Tyrosol derivatives affect U87 cell viability. (A) U87 cells treated with tyrosol (1) and the synthesized isoxazoles (6a–e). (B) Cells treated with tyrosol (1),
tyrosol-alkyne 2, and the synthesized triazoles (8a–e). Cells were treated with increasing concentrations for 24 h, and their viability was determined by metabolic
rate using the MTT assay. Absorbance values were measured at 560 nm and normalized against untreated cells. Data represent the mean ± SEM of three independent
experiments performed in triplicate. All the data were statistically significant (p < 0.05) except tyrosol (1) (at 12.5 and 25 µg/ mL) and 6b, 6c, 6d and 8a at 12.5 µg/
mL. MTT, 3,4,5-dimethylthiazol-2-yl, 2,5 diphenyltetrazolium bromide; SEM, standard error of the mean.
Fig. 2. Effect of tyrosol (1) and its derivatives on U87 cell proliferation. (A) U87 cells treated with tyrosol (1) and the synthesized isoxazoles (6a–e) for 72 h. (B) U87
cells treated with tyrosol (1), tyrosol-alkyne 2, and the synthesized triazoles (8a–e) at various concentrations for 72 h. Data represent the mean ± SEM of three
independent experiments performed in triplicate. All the data were statistically significant p < 0.05.SEM, standard error of the mean.
Table 1
IC50 Values (µg/mL and µM) of compounds 1, (6a-e) and (8a-e) against U87 tumor cell proliferation.
Compounds
1
6a
6b
6c
6d
6e
3
8a
8b
8c
8d
8e
Temozolomide
IC50 (µg/
mL)
>100
22.1 ±
0.5
31.0 ±
0.9
22.5 ±
0.8
15.2 ±
1.0
20.3 ±
0.4
72.1 ±
0.8
78.0 ±
1.2
30.0 ±
0.5
72.3 ±
0.2
34.2 ±
0.8
21.0 ±
0.9
–
IC50 (µM)
>400
67.6 ±
1.5
104.5 ±
3.0
72.4 ±
2.5
42.8 ±
2.8
61.4 ±
1.2
406.4 ±
4.5
263.4 ±
4.0
90.8 ±
1.5
233.1 ±
0.6
104.8 ±
2.3
52.7 ±
2.2
53.85[42]
For the 3,5-disubstituted isoxazole (6a–e) derivatives, compounds
6a (4-OCH3), 6d (4-t-Bu), and 6e (4-Cl) displayed the highest anti-
proliferative activity towards U87 cells, with IC50 values of 22.1 µg/mL
(67.6 µM), 15.2 µg/mL (42.8 µM) and 20.3 µg/mL (61.4 µM), respec-
tively. Previous studies reported the antiproliferative activity of iso-
xazole derivatives against human glioblastoma cell lines [22,41]. In the
series of the 1,4-disubstituted triazole derivatives (8a–e), compounds 8b
(4-Cl) and 8e (2,4,5-trichlo) exhibited the most potent antiproliferative
activity against U87 cells, with IC50 values of 30.0 µg/mL (90.8 µM) and
21.0 µg/mL (52.7 µM), respectively. In agreement with our results, it has
been shown that 1,4-disubstituted-1,2,3-triazole derivatives exhibited
antitumor potential against U87, GBM02 and GBM95 cell lines with IC50
values ranging from ~20 to 190 μM for 72 h treatments [20].
Interestingly, all these isoxazole and triazole derivatives exhibits
higher antiproliferative activity than Temozolomide (Table 1), the most
widely used chemotherapy for patients with glioblastoma [42].
Given this activity profile, we generated a structure–activity rela-
tionship showing that compounds with methyl, methoxy, or chlorine
substitution at R groups in isoxazole derivatives and compounds with a
chlorine substitution at R groups in triazole derivatives were more
effective against U87 cells. Furthermore, we observed that the presence
of more than one methyl group or chlorine atom (three in these cases)
5

I. Aissa et al.
Bioorganic Chemistry 114 (2021) 105071
played a significant role in enhancing the antiproliferative activity. In
the present study, a benzene ring substituted at three positions (8e)
resulted in higher activity than the derivative with a benzene ring
substituted only at a para-position (8b).
2.2.1.2. Analysis of cell morphology. To investigate the effect of tyrosol
derivatives on U87 cells, we examined changes in cell morphology
(Fig. 3). After 72 h and compared with controls, treated U87 cells with
isoxazole (Fig. 3A) and triazole (Fig. 3B) derivatives exhibited decreased
cell density and morphological changes, such acquisition of around
shape, shrinkage and spherical cellular protrusions. These abnormal
morphological characteristics provided insight into the anticancer effect
of these compounds.
2.2.1.3. Pro-apoptotic activity on U87 cells. Based on the promising
antiproliferative effect against U87 cells, compounds 6a, 6d, 8b, and 8e
were chosen for deeper evaluations aimed at better understanding its
mechanism of action. These compounds were evaluated for a possible
pro-apoptotic effect determined by Annexin V / PI assay. U87 cells were
treated with 6a, 6d, 8b (4-Cl), or 8e (2,4,5-trichlo) at their respective
IC50 values (22.1, 15.2, 30.0, and 21.0 µg/mL) for 72 h. Among the four
tested compounds, all were found to possess the ability to induce
apoptosis on U87 cells (percentage of apoptotic cells = 56%, 20%, 47%,
and 53%, respectively) (Fig. 4). These results suggested that the newly
synthesized compounds exerted its anticancer activity through the in-
duction of apoptosis in U87 cells. As it has been shown previously,
several triazole/ isoxazole derivatives trigger antiproliferative effects
through the induction of apoptosis. For example, anticancer isoxazole
derivatives have been shown to activate apoptotic pathways in U251-
MG and T98G glioma cell lines [22]. In addition to this, a pyrazolo
[3,4-d]pyrimidin-4(5H)-ones tethered to 1,2,3-triazoles compound
exhibited significant anti-proliferative and pro apoptotic effect on gli-
oma cells (U87) by the cleavage of Caspase-3, PARP and up regulation of
p53 [43]. Further investigation should be conducted in order to fully
elucidate the pro-apoptotic activity.
Fig. 4. Apoptosis induction in U87 cells treated with 6a, 6d, 8b, and 8e for 72
h according to Annexin-V and PI staining. Cells treated with 0.5 μM Staur-
osporine were used as positive control and untreated cells as negative control.
Data represent the mean ± SEM of two independent experiments. The values of
p < 0.05, p < 0.01 and p < 0.001 were considered as statistically significant (*),
very significant (**), highly significant difference (***) respectively, unless
otherwise mentioned. PI, propidium iodide; SEM, standard error of the mean.
was performed using hemolytic test on human erythrocytes (Fig. 5). All
of the isoxazole (Fig. 5A) and triazole (Fig. 5B) compounds induced <
10% hemolysis, even at a higher concentration of 400 µg/mL, except 6d
(this compound bearing an isopropyl group in its isoxazole moiety
induced ~ 40% hemolysis at 400 µg/mL). These data indicated that the
triazole and isoxazole derivatives did not induce red blood membrane
damage and hemoglobin release. This result reinforced the potential of
these compounds for future in vivo investigations as a new anticancer
prototype. These finding provide a new insight for designing novel
anticancer agents with low hemolytic activity.
3. Conclusion
2.2.2. Hemolytic activity
The promising anticancer activity shown by the synthetic com-
pounds demanded further safety analysis. In fact, hemolytic activity is a
major limitation on the development of chemical compounds as phar-
maceutical agents. Indeed, numerous therapeutic compounds do not
enter the clinical trials due to their high hemolytic activity [44].
Therefore, the hemocompatibility evaluation of the tyrosol derivatives
In summary, we synthesized novel 3,5-disubstitued isoxazole and
1,4-disubstitued triazole tyrosol conjugates (6a-e) and (8a-e), respec-
tively using microwave irradiation. The synthesized derivatives
exhibited higher antiproliferative activities towards human glioblas-
toma cells (U87) with slight hemolytic activity relative to the parent
tyrosol. Among the tested compounds, 6d and 8e exhibited the highest
Fig. 3. Morphological changes of U87 cells treated with (A) tyrosol (1) and its derivatives (6a–e) or (B) tyrosol (1), tyrosol-alkyne 2, and its derivatives (8a–e).
Morphological differences were observed by inverted phase-contrast microscopy (magnification: 10 × ) after treatment with chemical compounds (50 µg/ mL or 100
µg/ mL) for 72 h. Untreated cells were used as controls.
6

I. Aissa et al.
Bioorganic Chemistry 114 (2021) 105071
Fig. 5. Hemolytic activity of tyrosol (1) and its derivatives on human erythrocytes at different concentrations. (A) Hemolytic activity of tyrosol (1) and its derivatives
(6a–e). (B) Hemolytic activity of tyrosol (1), tyrosol-alkyne 2, and its derivatives (8a–e). Data are presented as a percentage of hemolysis relative to a positive control
(100%; 1% Triton X-100). Data represent the mean ± SEM of independent assays performed in triplicate. SEM, standard error of the mean.
selective antiproliferative effect. Preliminary results from the Annexin-V
assay indicate that these compounds induced apoptosis in human glio-
blastoma cancer cell line U87. Future studies are necessary to identify
and validate a potential lead compound, as well as elucidate the
mechanism underlying the anticancer effect.
the alkyl derivatives 2 (76%), 3 (trace), and 4 (21%).
4.2.1.1. 2-(4-Propargyloxyphenyl)-ethanol (2). White solid; mp:
112–114 ◦C; 1H NMR (300 MHz, CDCl3): δ 7.14 (2H, d, J = 8.7 Hz), 6.91
(2H, d, J = 8.7 Hz), 4.65 (2H, d, J = 2.1 Hz), 3.77 (2H, t, J = 6.6 Hz),
2.78 (2H, t, J = 6.6 Hz), 2.51 (1H, t, J = 2.4 Hz), 2.06 (1H, s); 13C NMR
(75 MHz, CDCl3): δ 155.7, 131.1, 129.5, 114.5, 78.2, 75.0, 63.1, 55.4,
37.8; HRMS (ESI+): calcd. for (C11H13O2)+ [M + H]+ 177.0924, found
177.0925.
4. Materials and methods
4.1. General experimental procedures
Tyrosol (1) was purchased from Fluka (Bucha, Switzerland). Solvents
were distilled and dried using standard methods. Melting points were
determined on a Büchi 510 apparatus using capillary tubes. Commercial
thin-layer chromatography (TLC) plates (silica gel 60; F254) were used to
monitor the progress of the reactions. Column chromatography was
4.2.1.2. 1-(propargyloxy)-4-(propargyloxyethyl)-benzene
(4). White
solid; mp: 105–107 ◦C; 1H NMR (300 MHz, CDCl3): δ 7.16 (2H, d, J =
8.4 Hz), 6.91 (2H, d, J = 8.4 Hz), 4.66 (2H, d, J = 2.4 Hz), 4.15 (2H, d, J
= 2.4 Hz), 3.72 (2H, t, J = 7.2 Hz), 2.87 (2H, t, J = 6.9 Hz), 2.50 (1H, t, J
= 2.1 Hz), 2.41 (1H, t, J = 2.4 Hz); 13C NMR (75 MHz, CDCl3): δ 155.6,
131.2, 129.3, 114.4, 79.3, 78.2, 74.8, 73.8, 70.5, 57.6, 55.3, 34.6; HRMS
(ESI+): calcd. for (C14H15O2)+ [M + H]+ 215.1081, found 215.1080.
performed with silica gel 60 (particle size: 40–63 μm). High-resolution
mass spectrometry (HRMS) was performed using an LCT Premier XE
system [electrospray ionization (ESI) technique, positive mode; Waters,
Milford, MA, USA]. For ESI experiments, leucine-enkephaline peptide
was employed as the LockSpray lock mass. 1H (300 MHz; 16–32 scans)
and BB-decoupled 13C (75 MHz; 256–2048 scans) nuclear magnetic
resonance (NMR) spectra were recorded at room temperature on an AM-
300 Fourier transform spectrometer (Bruker Daltonik, Bremen, Ger-
many) equipped with a 10-mm probe in deuterated chloroform with all
chemical shifts (δ) and reported in ppm (non-deuterated solvent was
used as a control standard). Coupling constants were measured in Hz,
with signals denoted, as follows: s, singlet; d, doublet; t, triplet; and m,
multiplet.
4.2.2. General procedure for the synthesis of 3,5-disubstituted isoxazoles
(6a–e)
To a mixture of selected alkyne 2 (0.102 g; 0.57 mmol), triethyl-
amine (1.14 mmol), and 0.1 equiv. Cu(I) iodide (CuI) in dry DMF, the
appropriate hydroximyl chlorides 5 (2 equiv.) were added at room
temperature with stirring, after which the mixture was subjected to
microwave irradiation at 200 W for 4 to 8 min. The crude mixture was
then diluted with water, extracted with ethyl acetate (3 × 40 mL), and
the organic layer was dried over anhydrous Na2SO4. After removal of
solvent in vacuo, the resulting residue was purified by silica gel column
chromatography and eluted with petroleum ether/ethyl acetate (1:1) to
obtain the new 3,5-disubstituted isoxazoles (6a–e) in 88% to 98% yields.
Cell culture medium [minimum essential medium Eagle (MEM)],
trypsin-EDTA, phosphate buffer saline (PBS), fetal bovine serum (FBS),
penicillin and streptomycin mixture, and L-glutamine (200 mM) were
purchased from GIBCO-BRL (Paisley, UK). Dimethyl sulfoxide (DMSO)
and 3,4,5-dimethylthiazol-2-yl, 2,5 diphenyltetrazolium bromide (MTT)
were purchased from Sigma (Saint Quentin Fallavier, France). All other
reagents were of analytical grade.
4.2.2.1. 2-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)methoxy)phenyl)
ethanol (6a). Yellowish solid; mp: 101–103 ◦C; 1H NMR (300 MHz,
CDCl3): δ 7.79 (2H, d, J = 6.3 Hz), 7.75 (2H, d, J = 6.6 Hz), 7.21 (2H, d,
J = 6.6 Hz), 6.94 (2H, d, J = 6.6 Hz), 6.58 (1H, s), 5.17 (2H, s), 3.94 (3H,
s), 3.85 (2H, t, J = 6.9 Hz), 2.84 (2H, t, J = 6.9 Hz); 13C NMR (75 MHz,
CDCl3): δ 168.3, 161.6, 160.6, 156.0, 131.5, 131.4, 129.6, 128.2, 127.7,
126.2, 125.5, 114.6, 114.5, 100.5, 63.1, 61.1, 55.7, 37.7; HRMS (ESI+):
calcd. for (C19H20NO4)+ [M + H]+ 326. 1401, found 326.1392.
4.2. Chemistry
4.2.1. General procedure for the synthesis of tyrosol-alkyne derivatives 2–4
To a solution of tyrosol (1) (2 g; 14.5 mmol) in dry N,N-dime-
thylformamide (DMF; 4 mL), sodium hydride (29 mmol) and propargyl
bromide (43.5 mmol) were added, and the reaction mixture was stirred
at room temperature for 3 h, with the reaction monitored by TLC. After
reaction completion, the residue was diluted with water (200 mL), and
the mixture was extracted with ethyl acetate (3 × 100 mL). The com-
bined organic layer was dried over anhydrous sodium sulfate and
evaporated to dryness, followed by purification over a silica gel column
and elution with petroleum ether/ethyl acetate (6:4 then 1:1) to obtain
4.2.2.2. 2-(4-((3-phenylisoxazol-5-yl)methoxy)phenyl)ethanol
(6b).
White solid; mp: 100–102 ◦C; 1H NMR (300 MHz, CDCl3): δ 7.83 (2H,
m), 7.47 (3H, m), 7.18 (2H, d, J = 6.6 Hz), 6.97 (2H, d, J = 6.6 Hz), 6.65
(1H, s), 5.20 (2H, s), 3.85 (2H, t, J = 6.6 Hz), 2.84 (2H, t, J = 6.6 Hz); 13
C
NMR (75 MHz, CDCl3): δ 168.1, 162.0, 156.1, 131.5, 129.6, 129.5,
128.4, 128.3, 126.3, 114.5, 100.1, 63.1, 61.1, 37.8; HRMS (ESI+): calcd.
for (C18H18NO3)+ [M + H]+ 296. 1295, found 296.1299.
7

I. Aissa et al.
Bioorganic Chemistry 114 (2021) 105071
4.2.2.3. 2-(4-((3-(p-tolyl)isoxazol-5-yl)methoxy)phenyl)ethanol (6c).
White solid; mp: 107–109 ◦C; 1H NMR (300 MHz, CDCl3): δ 7.71 (2H, d,
J = 6.9 Hz), 7.27 (2H, d, J = 6.6 Hz), 7.19 (2H, d, J = 6.6 Hz), 6.96 (2H,
d, J = 6.6 Hz), 6.62 (1H, s), 5.20 (2H, s), 3.85 (2H, t, J = 6.9 Hz), 2.84
(2H, t, J = 6.9 Hz), 2.42 (3H, s); 13C NMR (75 MHz, CDCl3): δ 167.9,
161.9, 156.1, 139.7, 131.4, 129.6, 129.4, 129.1, 126.2, 125.5, 114.6,
114.5, 100.7, 63.1, 61.1, 37.7, 20.8; HRMS (ESI+): calcd. for
(C19H20NO3)+ [M + H]+ 310. 1452, found 310.1435.
4.2.3.4. 2-(4-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)ethanol (8d). White solid; mp: 117–119 ◦C; 1H NMR (300 MHz,
CDCl3): δ 8.00 (1H, s), 7.63 (2H, d, J = 8.9 Hz), 7.17 (2H, d, J = 8.5 Hz),
7.02 (2H, d, J = 8.9 Hz), 6.97 (2H, d, J = 8.5 Hz), 5.26 (2H, s), 3.87 (3H,
s), 3.83 (2H, t, J = 6.6 Hz), 2.82 (2H, t, J = 6.6 Hz); 13C NMR (75 MHz,
CDCl3): δ 159.9, 156.9, 144.8, 131.4, 130.4, 130.1, 122.2, 121.1, 114.9,
114.8, 63.7, 62.1, 55.6, 38.3; HRMS (ESI+): calcd. for (C18H20N3O3)+
[M + H]+ 326. 1513, found 326.1523.
4.2.2.4. 2-(4-((3-(4-(tert-butyl)phenyl)isoxazol-5-yl)methoxy)phenyl)
ethanol (6d). White solid; mp: 98–100 ◦C; 1H NMR (300 MHz, CDCl3): δ
7.72 (2H, d, J = 8.7 Hz), 7.46 (2H, d, J = 8.4 Hz), 7.15 (2H, d, J = 5.4
Hz), 6.93 (2H, d, J = 6.6 Hz), 6.60 (1H, s), 5.16 (2H, s), 3.81 (2H, t, J =
6.3 Hz), 2.80 (2H, t, J = 6.3 Hz), 1.34 (9H, s); 13C NMR (75 MHz, CDCl3):
δ 167.9, 161.9, 156.1, 152.9, 131.4, 131.0, 129.6, 129.4, 129.1, 126.2,
125.4, 114.6, 114.5, 100.8, 63.1, 61.1, 37.8, 34.3, 30.6; HRMS (ESI+):
calcd. for (C22H26NO3)+ [M + H]+ 352. 1921, found 352.1922.
4.2.3.5. 2-(4-((1-(2,4,5-trichlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)ethanol (8e). White solid; mp: 111–113 ◦C; 1H NMR (300 MHz,
CDCl3): δ 8.05 (1H, s), 7.76 (1H, s), 7.68 (1H, s), 7.13 (2H, d, J = 8.4 Hz),
6.93 (2H, d, J = 8.4 Hz), 5.25 (2H, s), 3.80 (2H, t, J = 6.3 Hz), 2.79 (2H,
t, J = 6.6 Hz); 13C NMR (75 MHz, CDCl3): δ 156.2, 144.1, 139.5, 134.3,
133.3, 131.9, 131.0, 129.5, 129.4, 128.3, 126.6, 124.0, 115.0, 114.5,
63.2, 61.5, 37.7; HRMS (ESI+): calcd. for (C17H15Cl3N3O2)+ [M + H]+
398. 0239, found 398.0229.
4.2.2.5. 2-(4-((3-(4-chlorophenyl)isoxazol-5-yl)methoxy)phenyl)ethanol
(6e). Yellow paste; mp: 112–114 ◦C; 1H NMR (300 MHz, CDCl3): δ 7.72
(2H, dd, J = 6.9; 2.1 Hz), 7.41 (2H, dd, J = 6.9; 2.1 Hz), 7.15 (2H, dd, J
= 6.6; 2.1 Hz), 6.91 (2H, dd, J = 6.6; 2.1 Hz), 6.59 (1H, s), 5.16 (2H, s),
3.81 (2H, t, J = 6.3 Hz), 2.80 (2H, t, J = 6.3 Hz); 13C NMR (75 MHz,
CDCl3): δ 168.5, 161.0, 156.0, 135.7, 131.6, 129.6, 129.4, 129.0, 127.6,
126.8, 114.6, 114.5, 100.6, 63.1, 61.1, 37.7; HRMS (ESI+): calcd. for
(C18H17ClNO3)+ [M + H]+ 330. 0906, found 330.0886.
4.3. Biological assay
4.3.1. Anticancer activity
4.3.1.1. Cell line and cell culture conditions. Human glioblastoma cell
line (U87 cells) was cultured in MEM medium supplemented with 10%
fetal bovine serum, L-glutamine and 100 IU of penicillin/ streptomycin
in a humidified environment with 5% CO2 at 37 ◦C.
4.2.3. General procedure for synthesizing tyrosol-1,4-disubstituted triazoles
(8a–e): CuAAC
4.3.1.2. Cytotoxic and antiproliferative assays. The anticancer activity of
tyrosol (1) and its novel derivatives on U87 cell viability and prolifer-
ation was evaluated using an MTT assay [45]. The MTT assay evaluates
cell metabolism based on the ability of mitochondrial succinatedehy-
drogenase to convert the yellow compound MTT to a blue formazan dye.
The amount of dye produced is proportional to the number of live
metabolically active cells. U87 cells at optimal density were seeded into
96-well microplates (Nunc microplates; Thermo Fisher Scientific, Wal-
tham, MA, USA) and incubated overnight to allow attachment. Tyrosol
(1) and its derivatives were serially diluted and added to the wells to
their final respective concentrations and incubated for 24 h or 72 h.
Additionally, morphological changes in the treated cells were examined
and recorded under an inverted phase-contrast microscope (Leica,
Mannheim, Germany). After incubation with different concentrations of
compounds, MTT solution (500 µg/ mL) was added and the cells were
incubated for another 4 h. DMSO (100 µL) was then added to dissolve
the formed violet formazan crystals within metabolically viable cells.
Absorbance was determined by a microplate reader at 560 nm, and the
results were expressed as a percentage of cell viability. Cells incubated
with only medium were used as controls representing 100% viability or
proliferation. Temozolomide was employed as a positive control [42],
and all assays were performed in triplicate.
Under solvent-free conditions, 0.102 g (0.57 mmol) of dipolarophile
(2), CuI (0.5 equiv.), and triethylamine (1 equiv.) were mixed at room
temperature, followed by the addition of aryl-azide 7 (1.14 mmol) and
exposure to microwave irradiation at 250 W for 2 to 6 min. The crude
mixture was then extracted with ethyl acetate (3 × 35 mL), and the
combined organic layer was dried over anhydrous sodium sulfate,
concentrated under reduced pressure, and purified by column chroma-
tography using a petroleum ether and ethyl acetate mixture as eluents to
obtain pure (8a–e) in 85% to 98% yields.
4.2.3.1. 2-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)ethanol
(8a). White solid; mp: 120–122 ◦C; 1H NMR (300 MHz, CDCl3): δ 8.06
(1H, s), 7.73 (2H, d, J = 7.5 Hz), 7.49 (3H, m), 7.16 (2H, d, J = 8.4 Hz),
6.98 (2H, d, J = 8.4 Hz), 5.28 (2H, s), 3.83 (2H, t, J = 6.6 Hz), 2.82 (2H,
t, J = 6.6 Hz); 13C NMR (75 MHz, CDCl3): δ 156.4, 144.5, 136.4, 130.8,
129.6, 129.2, 128.3, 120.4, 120.1, 114.4, 63.2, 61.6, 37.8; HRMS (ESI+):
calcd. for (C17H18N3O2)+ [M + H]+ 296. 1408, found 296.1381.
4.2.3.2. 2-(4-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)ethanol (8b). Dark red solid; mp: 116–118 ◦C; 1H NMR (300
MHz, CDCl3): δ 8.01 (1H, s), 7.67 (2H, d, J = 7.2 Hz), 7.48 (2H, d, J =
7.5 Hz), 7.16 (2H, d, J = 8.7 Hz), 6.98 (2H, d, J = 8.7 Hz), 5.26 (2H, s),
3.82 (2H, t, J = 6.3 Hz), 2.81 (2H, t, J = 6.6 Hz); 13C NMR (75 MHz,
CDCl3): δ 156.3, 144.9, 134.9, 134.2, 130.9, 129.6, 129.4, 121.2, 120.2,
114.5, 63.1, 61.6, 37.8; HRMS (ESI+): calcd. for (C17H17ClN3O2)+ [M +
H]+ 330. 1018, found 330.1018.
4.3.1.3. Flow cytometric analysis of apoptosis. Briefly, U87 cells were
treated with 6a, 6d, 8b and 8e at their respective 50% inhibitory con-
centration (IC50) values. After 72 h, all cell populations (suspended and
attached) were collected and washed twice with PBS. The cells were
then stained with Annexin-V-fluorescein isothiocyanate-conjugated/
propidium iodide (PI) reagents (Invitrogen, Carlsbad, CA, USA) to detect
phosphatidylserine (PS) externalization for 15 min. Fluorescence-
activated cell sorting was performed on a FACScalibur flow cytometer
(Becton–Dickinson, Franklin Park, NJ, USA) to discriminate viable cells
(Annexin-Vꢀ /PIꢀ ) from cells in early apoptosis (Annexin-V+/PIꢀ ), late
apoptosis (Annexin-V+/PI+), or undergoing necrosis (Annexin-Vꢀ /PI+).
Data analyses were performed with Cell Quest software (Bec-
ton–Dickinson). Non-treated culture cells were used as a negative con-
4.2.3.3. 2-(4-((1-(m-tolyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)ethanol
(8c).. White solid; mp: 112–114 ◦C; 1H NMR (300 MHz, CDCl3): δ 8.01
(1H, s), 7.55 (1H, s), 7.49 (1H, d, J = 8.1 Hz), 7.37 (1H, t, J = 7.8 Hz),
7.23 (1H, d, J = 7.8 Hz), 7.15 (2H, d, J = 8.7 Hz), 6.95 (2H, d, J = 8.7
Hz), 5.25 (2H, s), 3.81 (2H, t, J = 6.6 Hz), 2.80 (2H, t, J = 6.6 Hz), 2.43
(3H, s); 13C NMR (75 MHz, CDCl3): δ 156.4, 144.4, 139.5, 136.4, 130.8,
129.5, 129.1, 129.0, 120.7, 120.4, 117.1, 114.4, 63.1, 61.7, 37.8, 20.8;
HRMS (ESI+): calcd. for (C18H20N3O2)+ [M + H]+ 310. 1564, found
310.1550.
trol. U87 cells treated with 0.5
positive control.
μM staurosporine (STS) were used as
8

I. Aissa et al.
Bioorganic Chemistry 114 (2021) 105071
[6] https://clinicaltrials.gov/ct2/results?cond=&term=NVP-AUY922&cntry, (n.d.).
[7] M. Abbasi, H. Sadeghi-Aliabadi, M. Amanlou, Prediction of new Hsp90 inhibitors
based on 3,4-isoxazolediamide scaffold using QSAR study, molecular docking and
molecular dynamic simulation, DARU J. Pharm. Sci. 25 (2017) 17.
[8] H. Wang, Y. Ma, Y. Lin, J. Liu, R. Chen, B. Xu, Y. Liang, An Isoxazole Derivative
SHU00238 Suppresses Colorectal Cancer Growth through miRNAs Regulation,
Molecules 24 (2019) 2335.
4.3.1.4. Hemolysis assay. The hemolytic activity of tyrosol (1) and its
derivatives was tested using human erythrocytes from healthy volun-
teers who had not taken any medication for at least 2 weeks prior to
sampling. Freshly collected blood samples were immediately mixed with
heparin. To obtain a pure suspension of erythrocytes, 1 mL of whole
blood transferred to 20 mL PBS (pH 7.4) and centrifuged at 250g for 5
min at 4 ◦C. The supernatant and buffy coats were removed by gentle
aspiration and the transfer/centrifugation process was repeated two
more times. Erythrocytes were finally re-suspended in PBS to make a 1%
solution for hemolytic assay. Various concentrations of tyrosol (1) and
its novel derivatives were then added to the suspension of red blood cells
and incubated at 37 ◦C for 1 h in a water bath, followed by centrifuga-
tion at 250g for 5 min at 4 ◦C. The absorbance of the supernatants was
measured at 545 nm to determine the extent of red blood cell lysis.
Positive (100% hemolysis) and negative (0% hemolysis) controls were
generated by incubating erythrocytes in PBS containing 1% Triton X-
100 and PBS alone, respectively [46]. All tests were performed in
triplicate.
[9] X.-L. Wang, K. Wan, C.-H. Zhou, Synthesis of novel sulfanilamide-derived 1,2,3-
triazoles and their evaluation for antibacterial and antifungal activities, Eur. J.
Med. Chem. 45 (2010) 4631–4639.
[10] P. Suman, T.R. Murthy, K. Rajkumar, D. Srikanth, Ch. Dayakar, C. Kishor,
A. Addlagatta, S.V. Kalivendi, B.C. Raju, Synthesis and structure–activity
relationships of pyridinyl-1H-1,2,3-triazolyldihydroisoxazoles as potent inhibitors
of tubulin polymerization, Eur. J. Med. Chem. 90 (2015) 603–619.
[11] E.T. Warda, I.A. Shehata, M.B. El-Ashmawy, N.S. El-Gohary, New series of
isoxazole derivatives targeting EGFR-TK: Synthesis, molecular modeling and
antitumor evaluation, Bioorg. Med. Chem. 28 (2020), 115674.
[12] D. Dheer, V. Singh, R. Shankar, Medicinal attributes of 1,2,3-triazoles: Current
developments, Bioorg. Chem. 71 (2017) 30–54.
[13] K. Chouaïb, S. Delemasure, P. Dutartre, H.B. Jannet, Microwave-assisted synthesis,
anti-inflammatory and anti-proliferative activities of new maslinic acid derivatives
bearing 1,5- and 1,4-disubstituted triazoles, J. Enzyme Inhib. Med. Chem. 31
(2016) 130–147.
[14] J. Mareddy, N. Suresh, C.G. Kumar, R. Kapavarapu, A. Jayasree, S. Pal, 1,2,3-
Triazole-nimesulide hybrid: Their design, synthesis and evaluation as potential
anticancer agents, Bioorg. Med. Chem. Lett. 27 (2017) 518–523.
4.3.1.5. Statistical analysis. Statistical analysis was performed using
Graph Pad Prism 7.0 (Graph Pad Software Inc., CA, and USA). All the
data are presented as the mean ± standard error of the mean (SEM). The
difference between two groups was evaluated using Student’s t test.
Significant difference among three or more groups was determined by
one-way ANOVA with a post hoc analysis (Turkey test). The values of p
< 0.05, p < 0.01 and p < 0.001 were considered as statistically signif-
icant (*), very significant (**), highly significant difference (***)
respectively, unless otherwise mentioned.
[15] Z. Xu, S.-J. Zhao, Y. Liu, 1,2,3-Triazole-containing hybrids as potential anticancer
agents: Current developments, action mechanisms and structure-activity
relationships, Eur. J. Med. Chem. 183 (2019), 111700.
[16] S. Balabadra, M. Kotni, V. Manga, A.D. Allanki, R. Prasad, P.S. Sijwali, Synthesis
and evaluation of naphthyl bearing 1,2,3-triazole analogs as antiplasmodial agents,
cytotoxicity and docking studies, Bioorg. Med. Chem. 25 (2017) 221–232.
[17] K. Lal, P. Yadav, A. Kumar, A. Kumar, A.K. Paul, Design, synthesis,
characterization, antimicrobial evaluation and molecular modeling studies of some
dehydroacetic acid-chalcone-1,2,3-triazole hybrids, Bioorg. Chem. 77 (2018)
236–244.
[18] P. Sooknual, R. Pingaew, K. Phopin, W. Ruankham, S. Prachayasittikul,
S. Ruchirawat, V. Prachayasittikul, Synthesis and neuroprotective effects of novel
chalcone-triazole hybrids, Bioorg. Chem. 105 (2020), 104384.
Declaration of Competing Interest
[19] J. Akhtar, A.A. Khan, Z. Ali, R. Haider, M. Shahar Yar, Structure-activity
relationship (SAR) study and design strategies of nitrogen-containing heterocyclic
moieties for their anticancer activities, Eur. J. Med. Chem. 125 (2017) 143–189.
[20] V.D. da Silva, B.M. de Faria, E. Colombo, L. Ascari, G.P.A. Freitas, L.S. Flores,
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.
˜
Y. Cordeiro, L. Romao, C.D. Buarque, Design, synthesis, structural characterization
and in vitro evaluation of new 1,4-disubstituted-1,2,3-triazole derivatives against
glioblastoma cells, Bioorg. Chem. 83 (2019) 87–97.
[21] A. Ogino, E. Sano, Y. Ochiai, S. Yamamuro, S. Tashiro, K. Yachi, T. Ohta,
T. Fukushima, Y. Okamoto, K. Tsumoto, T. Ueda, A. Yoshino, Y. Katayama, Efficacy
of ribavirin against malignant glioma cell lines, Oncol. Lett. 8 (2014) 2469–2474.
[22] I. Lampronti, D. Simoni, R. Rondanin, R. Baruchello, C. Scapoli, A. Finotti,
M. Borgatti, C. Tupini, R. Gambari, Pro–apoptotic activity of novel synthetic
isoxazole derivatives exhibiting inhibitory activity against tumor cell growth in
vitro, Oncol. Lett. 20 (2020) 151.
Acknowledgments
´
We thank Pr. Jose Luis from Laboratory of Cell Biology, Faculty of
Pharmacy, Marseille, France, for the kind gift of the human glioblastoma
cells (U87) cells. Also, the authors are grateful to Researchers Support-
ing Project number (RSP-2021/17) at King Saud University, Riyadh,
Saudi Arabia.
[23] Y.J. Rao, T. Sowjanya, G. Thirupathi, N.Y.S. Murthy, S.S. Kotapalli, Synthesis and
biological evaluation of novel flavone/triazole/benzimidazole hybrids and
flavone/isoxazole-annulated heterocycles as antiproliferative and
antimycobacterial agents, Mol. Divers. 22 (2018) 803–814.
[24] T. Sowjanya, Y. Jayaprakash Rao, N.Y.S. Murthy, Synthesis and antiproliferative
activity of new 1,2,3-triazole/flavone hybrid heterocycles against human cancer
cell lines, Russ. J. Gen. Chem. 87 (2017) 1864–1871.
Funding
The authors are grateful to the Ministry of Higher Education and
Scientific Research of Tunisia for financial support (LR11ES39).
`
´
[25] S. Chekir, M. Debbabi, A. Regazzetti, D. Dargere, O. Laprevote, H. Ben Jannet,
R. Gharbi, Design, synthesis and biological evaluation of novel 1,2,3-triazole linked
coumarinopyrazole conjugates as potent anticholinesterase, anti-5-lipoxygenase,
anti-tyrosinase and anti-cancer agents, Bioorg. Chem. 80 (2018) 189–194.
Appendix A. Supplementary data
´
ˇ
ˇ
´
ˇ
[26] A. Bistrovic, N. Stipanicev, T. Opacak-Bernardi, M. Jukic, S. Martinez, Lj. Glavas-
´
´
Obrovac, S. Raic-Malic, Synthesis of 4-aryl-1,2,3-triazolyl appended natural
coumarin-related compounds with antiproliferative and radical scavenging
activities and intracellular ROS production modification, New J. Chem. 41 (2017)
7531–7543.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105071.
[27] Y. Qi, Z. Ding, Y. Yao, D. Ma, F. Ren, H. Yang, A. Chen, Novel triazole analogs of
apigenin–7–methyl ether exhibit potent antitumor activity against ovarian
carcinoma cells via the induction of mitochondrial–mediated apoptosis, Exp. Ther.
Med. (2018).
References
[1] H.B. Jannet, Z. Mighri, L. Serani, O. Laprevote, J.-C. Jullian, F. Roblot, Isolation
and Structure Elucidation of Three New Diglyceride Compounds from Ajuga Iva
Leaves, Nat. Prod. Lett. 10 (1997) 157–164.
´
´
´
´
ˇ ´
[28] A. Karkovic Markovic, J. Toric, M. Barbaric, C. Jakobusic Brala, Hydroxytyrosol,
Tyrosol and Derivatives and Their Potential Effects on Human Health, Molecules
24 (2019) 2001.
[2] K. Chouaïb, A. Romdhane, S. Delemasure, P. Dutartre, N. Elie, D. Touboul, H. Ben
jannet, M. Ali Hamza, Regiospecific synthesis, anti-inflammatory and anticancer
evaluation of novel 3,5-disubstituted isoxazoles from the natural maslinic and
oleanolic acids, Ind. Crops Prod. 85 (2016) 287–299.
´
[29] M.I. Fernandez-Mar, R. Mateos, M.C. García-Parrilla, B. Puertas, E. Cantos-Villar,
Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A
review, Food Chem. 130 (2012) 797–813.
[30] Y. Zhang, Z. Zhou, L. Zou, R. Chi, Imidazolium-based ionic liquids with inorganic
anions in the extraction of salidroside and tyrosol from Rhodiola: The role of
cations and anions on the extraction mechanism, J. Mol. Liq. 275 (2019) 136–145.
[31] A. Chang, S. Sun, L. Li, X. Dai, H. Li, Q. He, H. Zhu, Tyrosol from marine Fungi, a
novel Quorum sensing inhibitor against Chromobacterium violaceum and
Pseudomonas aeruginosa, Bioorg. Chem. 91 (2019), 103140.
[3] K. Bozorov, J. Zhao, H.A. Aisa, 1,2,3-Triazole-containing hybrids as leads in
medicinal chemistry: A recent overview, Bioorg. Med. Chem. 27 (2019)
3511–3531.
[4] J. Zhu, J. Mo, H. Lin, Y. Chen, H. Sun, The recent progress of isoxazole in medicinal
chemistry, Bioorg. Med. Chem. 26 (2018) 3065–3075.
[5] N. Agrawal, P. Mishra, The synthetic and therapeutic expedition of isoxazole and
its analogs, Med. Chem. Res. 27 (2018) 1309–1344.
9

I. Aissa et al.
Bioorganic Chemistry 114 (2021) 105071
[32] M.B. Plotnikov, O.I. Aliev, A.V. Sidekhmenova, A.Y. Shamanaev, A.
M. Anishchenko, T.I. Fomina, T.M. Plotnikova, A.M. Arkhipov, Effect of p- tyrosol
on hemorheological parameters and cerebral capillary network in young
spontaneously hypertensive rats, Microvasc. Res. 119 (2018) 91–97.
[33] I. Aissa, M. Bouaziz, F. Frikha, R.B. Mansour, Y. Gargouri, Synthesized tyrosyl
hydroxyphenylacetate, a novel antioxidant, anti-stress and antibacterial
compound, Process Biochem. 47 (2012) 2356–2364.
[39] D. Kumar, V.B. Reddy, R.S. Varma, A facile and regioselective synthesis of 1,4-
disubstituted 1,2,3-triazoles using click chemistry, Tetrahedron Lett. 50 (2009)
2065–2068.
[40] L.-J. Li, Y.-Q. Zhang, Y. Zhang, A.-L. Zhu, G.-S. Zhang, Synthesis of 5-functional-
ized-1,2,3-triazoles via a one-pot aerobic oxidative coupling reaction of alkynes
and azides, Chin. Chem. Lett. 25 (2014) 1161–1164.
[41] P. Das, M.H. Hasan, D. Mitra, R. Bollavarapu, E.J. Valente, R. Tandon, D. Raucher,
A.T. Hamme, Design, Synthesis, and Preliminary Studies of Spiro-isoxazoline-
peroxides against Human Cytomegalovirus and Glioblastoma, J. Org. Chem. 84
(2019) 6992–7006.
[34] E.-Y. Ahn, Y. Jiang, Y. Zhang, E. Son, S. You, S.-W. Kang, J.-S. Park, J. Jung, B.-
J. Lee, D.-K. Kim, Cytotoxicity of p-tyrosol and its derivatives may correlate with
the inhibition of DNA replication initiation, Oncol. Rep. 19 (2008) 527–534.
[35] I. Aissa, R. Sghair, M. Bouaziz, D. Laouini, S. Sayadi, Y. Gargouri, Synthesis of
lipophilic tyrosyl esters derivatives and assessment of their antimicrobial and
antileishmania activities, Lipids Health Dis. 11 (2012) 13.
[42] M. Khazaei, M. Pazhouhi, S. Khazaei, Temozolomide and tranilast synergistic
antiproliferative effect on human glioblastoma multiforme cell line (U87MG), Med.
J. Islam. Republ. Iran 33 (2019) 39.
[36] F. Himo, T. Lovell, R. Hilgraf, V.V. Rostovtsev, L. Noodleman, K.B. Sharpless, V.
V. Fokin, Copper(I)-Catalyzed Synthesis of Azoles. DFT Study Predicts
Unprecedented Reactivity and Intermediates, J. Am. Chem. Soc. 127 (2005)
210–216.
[43] M. Allam, A.K.D. Bhavani, A. Mudiraj, N. Ranjan, M. Thippana, P.P. Babu,
Synthesis of pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to 1,2,3-triazoles and
their evaluation as potential anticancer agents, Eur. J. Med. Chem. 156 (2018)
43–52.
[37] R. Naresh Kumar, G. Jitender Dev, N. Ravikumar, D. Krishna Swaroop,
B. Debanjan, G. Bharath, B. Narsaiah, S. Nishant Jain, A. Gangagni Rao, Synthesis
of novel triazole/isoxazole functionalized 7-(trifluoromethyl)pyrido[2,3- d]
pyrimidine derivatives as promising anticancer and antibacterial agents, Bioorg.
Med. Chem. Lett. 26 (2016) 2927–2930.
[44] G. Jeswani, A. Alexander, S. Saraf, S. Saraf, A. Qureshi, Ajazuddin, Recent
approaches for reducing hemolytic activity of chemotherapeutic agents,
J. Controlled Release 211 (2015) 10–21.
[45] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63.
[46] M. Malagoli, A full-length protocol to test hemolytic activity of palytoxin on human
erythrocytes, Invertebr. Surviv. J. 4 (2007) 92–94.
[38] G. Wei, W. Luan, S. Wang, S. Cui, F. Li, Y. Liu, Y. Liu, M. Cheng, A library of 1,2,3-
triazole-substituted oleanolic acid derivatives as anticancer agents: design,
synthesis, and biological evaluation, Org. Biomol. Chem. 13 (2015) 1507–1514.
10