A step-by-step synthesis of triazole-benzimidazole-chalcone hybrids: Anticancer activity in human cells+

Article abstract of DOI:10.1016/j.molstruc.2019.127487

Novel series of triazole-benzimidazole-chalcone hybrid compounds have been synthesized via click chemistry, between different azide derivatives and substituted benzimidazole terminal alkynes bearing a chalcone moiety. The starting alkynes are prepared via base-catalysed nitrogen alkylation of pre-synthetized benzimidazole-chalcone substrates. All the intermediates as well as the final products are fully characterized by 1D and 2D NMR and mass spectrometry techniques. HMBC correlations permits the identification of a unique 1,4-disubstitued triazole-benzimidazole-chalcone isomer. Evaluation of the anti-proliferative potential in breast and prostate cancer cell lines showed that the presence of chloro substituents at the chalcone ring of the triazole-benzimidazole-chalcone skeleton enhanced the cytotoxic effects. The benzyl group linked to the 1,2,3-triazole moiety provides more antiproliferative potential.

Full text of DOI:10.1016/j.molstruc.2019.127487

Journal Pre-proof
A step-by-step synthesis of triazole-benzimidazole-chalcone hybrids: Anticancer
+
activity in human cells
Amar Djemoui, Abdelkader Naouri, Mohammed Ridha Ouahrani, Djamila Djemoui,
Souli Lahecen, Mokhtar Boualem Lahrech, Leila Boukenna, Hélio M.T. Albuquerque,
Liza Saher, Djenisa H.A. Rocha, Fátima Liliana Monteiro, Luísa A. Helguero,
Khaldoun Bachari, Oualid Talhi, Artur M.S. Silva
PII:
S0022-2860(19)31596-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.127487
MOLSTR 127487
Reference:
To appear in: Journal of Molecular Structure
Received Date: 4 October 2019
Revised Date: 26 November 2019
Accepted Date: 27 November 2019
Please cite this article as: A. Djemoui, A. Naouri, M.R. Ouahrani, D. Djemoui, S. Lahecen, M.B. Lahrech,
L. Boukenna, Hé.M.T. Albuquerque, L. Saher, D.H.A. Rocha, Fá.Liliana. Monteiro, Luí.A. Helguero, K.
Bachari, O. Talhi, A.M.S. Silva, A step-by-step synthesis of triazole-benzimidazole-chalcone hybrids:
+
Anticancer activity in human cells , Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.127487.
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A step-by-step synthesis of Triazole-Benzimidazole-Chalcone
hybrids: Anticancer activity in human cells+
Amar Djemoui,*[a,b,c] Abdelkader Naouri,[a,d] Mohammed Ridha Ouahrani,[a] Djamila Djemoui,[b] Souli
Lahecen,[c] Mokhtar Boualem Lahrech,[c] Leila Boukenna,[d] Hélio M. T. Albuquerque,[e] Liza Saher,[e,f]
Djenisa H. A. Rocha,[f,g] Fátima Liliana Monteiro,[f] Luísa A. Helguero,[f] Khaldoun Bachari,[d] Oualid
Talhi,*[d,e] and Artur M. S. Silva*[e]
[a]
Department of Chemistry, Faculty of Exact Sciences and Informatics
ZIANE Achour University. Djelfa (Algeria)
E-mail: djamarchimie@yahoo.fr
[b]
Department of Chemistry, Faculty of Exact Sciences
Echahid Hamma Lakhdar University of El Oued (Algeria)
Laboratory of Organic Chemistry and Natural Substance
Faculty of Exact Sciences and informatics, ZIANE Achour University. Djelfa (Algeria)
Centre de Recherche Scientifique et Technique en Analyses Physico-Chimiques
CRAPC, BP384, Bou-Ismail, 42004 Tipaza (Algeria)
E-mail: oualid.talhi@crapc.dz
[c]
[d]
[e]
QOPNA and LAQV-REQUIMTE, Department of Chemistry
University of Aveiro, 3810-193 Aveiro (Portugal)
E-mail: oualid.talhi@ua.pt, artur.silva@ua.pt
[f]
Institute of Biomedicine (iBiMED), Department of Medical Sciences
University of Aveiro, 3810-193 Aveiro (Portugal)
[g]
CICECO-Aveiro Institute of Material
University of Aveiro, 3810-193 Aveiro (Portugal)
+ Supporting information available
Abstract: Novel series of triazole-benzimidazole-chalcone hybrid
compounds have been synthesized via click chemistry, between
different azide derivatives and substituted benzimidazole terminal
alkynes bearing a chalcone moiety. The starting alkynes are
prepared via base-catalyzed nitrogen alkylation of pre-synthetized
benzimidazole-chalcone substrates. All the intermediates as well as
the final products are fully characterized by 1D and 2D NMR and
mass spectrometry techniques. HMBC correlations permits the
identification of a unique 1,4-disubstitued triazole-benzimidazole-
chalcone isomer. Evaluation of the anti-proliferative potential in
breast and prostate cancer cell lines showed that the presence of
chloro substituents at the chalcone ring of the triazole-
benzimidazole-chalcone skeleton enhanced the cytotoxic effects.
The benzyl group linked to the 1,2,3-triazole moiety provides more
antiproliferative potential.
enzymatic degradation, but reductive cleavage sometimes may
occur under forcing conditions leading to the formation of
triazolium salts.[11] There are many metal-free methods for the
synthesis of 1,2,3-triazoles, however, the most commonly
reported strategy for their high yield preparation is the copper(I)-
catalysed azide-alkyne cycloaddition (CuAAC) reaction
developed by Sharpless et al.,[12] which involves the reaction of
an azide with a terminal-alkyne.
Aiming at the construction of a hybrid compound,[13] we focus on
using the benzimidazole nucleus, which is another outstanding
nitrogen-containing heterocycle. In recent years, benzimidazole
nucleus has been associated to several biological properties,
namely antimicrobial,[14] antiallergic,[15] anticancer,[16] antiviral,[17]
anti-inflammatory,[18] antimalarial,[19] antitubercular,[20] and
antiparasitic.[21] The benzimidazole-pyrazole combination has
been described to show effective activity against lung cancer cell
line (A549) and epidermal growth factor receptor (EGFR)
binding affinity.[22a] More recently, Padhy et al.,[22b] have also
reported the synthesis of N-benzylbenzimidazole linked to
pyrimidine as moderate anticancer agents on breast cancer cell
line (MDA-MB-231). Further synthetic strategies have been
Introduction
1,2,3-Triazoles and their derivatives are important nitrogen
heterocycles, having relevant applications in both medicinal and
technological fields. This kind of heterocycles is well recognized
for its biological properties, such as anti-HIV,[1] antifungal,[2]
antiplasmodial,[3] antibacterial,[4] anticancer,[5] antileishmanial,[6]
anti-inflammatory,[7] antitubercular,[8] antiviral,[9] among others.
From a medicinal chemistry point of view, 1,2,3-triazoles have
gained relevance due to their implication in binding interactions
designed
towards
benzothiazole-piperazine-1,2,3-triazole
hybrids, being considered as promising active candidates
against selected human cancer cell lines.[23a] Besides, the use of
click chemistry to construct the flavone-triazole-benzimidazole
hybrid was successful and compounds exhibited good
antiproliferative and anti-mycobacterial activities.[23b] In relation to
the topic of hybrid organic compounds, the chalcone-
benzimidazole combination have been studied showing that the
N-substituted benzimidazole derivatives by an alkyl chain and
nitrogen-containing 5- or 6-membered rings enhanced the
cytotoxic effects on human breast adenocarcinoma (MCF-7) and
human ovarian carcinoma (OVCAR-3) cell lines with IC50 around
9-11 µM.[23c] The cytotoxicity of some synthesized chalcone
linked-1,2,3-triazoles was tested against four human cancer cell
with different biological targets, maintaining
a
good
pharmacokinetic profile.[10] They are effective amide surrogates
in bioactive molecules because of their strong dipole moments
which can even be used as linkers, presenting bio-isosteric
effects on peptide linkage, aromatic ring, double bonds and
imidazole ring. Moreover, the 1,2,3-triazoles are remarkably
stable towards hydrolysis, oxidative/reductive conditions, and
1

lines (MCF-7, MIA-PA-Ca-2, A549, HepG2). The most active
compound presented IC50 of 4-11 µM with better or comparable
activity to the reference drug tested. These hybrid compounds
have also induced apoptosis, G2/S arrest and triggers
mitochondrial potential loss in pancreatic cancer MIA-PA-Ca-
2.[23d]
Following similar synthetic pathways towards the elaboration of
novel heterocyclic hybrids,[23-26] in this paper we intend to
combine, in one molecule, two different nitrogen heterocycles,
1,2,3-triazole and benzimidazole incorporating the functional
chalcone moiety, as a Michael acceptor, expecting that this
series of 1,2,3-triazole-benzimidazole-chalcone hybrids may
present improved and selective biological activities comparing to
the isolated parental structures.
Taking as a representative example, compound 10a, where it
was apparently possible to distinguish between the two
methylene groups N(1’)-CH2 and N(1)-CH2 appearing as singlets
at δH 5.52 and 5.99 ppm, respectively. The N(1)-CH2 represents
the linking bridge between the triazole and the benzimidazole
heterocycles. These protons have shown important HMBC
cross-peak correlations with the surrounding quaternary carbons
related to both nitrogen-containing rings. Therefore, N(1)-CH2
3
could evidently entertain JH/C HMBC correlations with C-2 (δC
146.9 ppm) and C-7a (δC 136.7 ppm) of the benzimidazole part.
2
While, the same one is showing interesting JH/C with C-4’ (δC
3
143.1 ppm) and JH/C with the protonated triazolic carbon C-5’
(δH 8.12 ppm and δC 124.0 ppm determined via HSQC 2D-NMR)
(Figure 1, See supporting information).
Results and Discussion
Synthesis
Our synthetic strategy begins by the preparation of the key
intermediates
of
N-propargyl-substituted
benzimidazole-
chalcones 7a-h, which have been obtained in a four-step
reaction sequence. The condensation of o-phenylenediamine 1
with lactic acid 2 in hydrochloric acid (HCl, 4N) affords the
intermediate 3, which after the oxidation of its hydroxyl group
using potassium dichromate, the desired 2-acetylbenzimidazole
4 is furnished in appreciable yields (83%) (Scheme 1). The
synthesis of the benzimidazole-chalcone derivatives 6a-h was
accomplished through the base-catalysed (NaOH) aldol-
condensation of 2-acetylbenzimidazole 4 with a series of
substituted aromatic aldehydes 5a-h at room temperature (yield
up to 93%, Scheme 1). The final step of the reaction sequence
involved the N-alkylation of benzimidazole-chalcones 6a-h with
propargyl bromide, affording N-propargylated compounds 7a-h
in excellent yields (89-92%) (Scheme 1). 1H-NMR analysis of
compounds 6a-h, showed an NH group displayed at δH 11.50-
13.50 ppm. On the other hand, in the 1H NMR spectra of N-
propargylated compounds 7a-h, we observed the disappearance
of the signal corresponding to NH, and the appearance of two
new signals at δH 1.20 and 5.00 ppm, being attributed to terminal
alkyne and the N-CH2, respectively.
In parallel, important azide intermediates 8a-b and 9a-b are
required for our “step-by-step” synthetic strategy and their
classical preparation was performed via substitution reactions
using sodium azide NaN3, which also occurred in good yields
(67-74%) (See supporting information).
The combination of all the parts to construct the target hybrid
was achieved via click chemistry, which is the key step towards
generating the 1,2,3-triazole-benzimidazole-chalcone structure
(Scheme 1). Hence, the reaction of propargyl-compounds 7a-h
with the pre-synthetized azide derivatives (8a-b or 9a-b) was
conducted in dichloromethane/water (1:1) as a solvent system
catalyzed by CuSO4.5H2O and sodium ascorbate at room
temperature to deliver a structural variety of 1,2,3-triazole-
benzimidazole-chalcone hybrids 10a-h and 11a-f in good-to-
excellent yield (79-91%) and high purity after simple
recrystallisation step.
Scheme 1. Synthetic route for the preparation of benzimidazole-chalcone
intermediates 7a-h and triazole-benzimidazole-chalcone hybrids 10a-h and
11a-f.
A deep examination of the HMBC spectrum of compound 10a,
sharing a benzyl group on the triazole nucleus, can inform us
about the regioselective formation of a 1,4-disubstitued triazole
isomer. This is proved when referring to the methylene group
N(1’)-CH2 of the 1’-N-benzyl substituent, which does not
1D and 2D NMR analysis are used to unequivocally establish
the exact structure of the triazole-benzimidazole-chalcone hybrid
compounds 10a-h and 11a-f (See supporting information).
3
establish any noticeable JH/C HMBC connectivity with C-4’ (or
carbon C-5’ in case of 1,5-triazole), thus, the 1,5-disubstituted
2

triazole isomer is not expected as demonstrated in figure 1.
Furthermore, the chalcone moiety can also be observed through
the presence of signals corresponding to the protons of the
ketone α,β-unsaturated system HβC=CHα–C=O represented as
two doublets at δH 8.06 and 7.80 ppm, respectively with coupling
MDA-MB-231 (Table 1). The most active compound 10d, is
about 4-fold less active in MDA-MB-231, 48-fold less active in
T47-D and 15-fold less active in PC3 than doxorubicin. However,
the obtained results do not take into consideration the in vivo
bioavailability aspect of the triazole-benzimidazole-chalcone
hybrid. Further experiences have revealed that the presence of
1-N-benzyl-1,2,3-triazole at the triazole-benzimidazole-chalcone
scaffold (compounds 10) favors the cytotoxicity on the tested cell
lines, where a chloro-substitution (10d and 10h) at the chalcone
ring enhanced the cytotoxic effects. Regarding the structure-
activity-relationship (SAR) aspect, the reported chloro-
substituted benzimidazole-triazole hybrids have already shown
good cytotoxic effects against mouse embryonic fibroblast cell
lines NIH/3T3 (IC50 1.63 µM).[23e] In the other hand, benzylated
1,2,3-triazole chalcone hybrid are proved as excellent
antiproliferative agents against SK-N-SH cancer cells (IC50 1.53
ꢀM).[23f] In our case, we have noticed that, in all the cancer cell
lines tested, derivatives 11a-b have stimulated the cell viability
by 5%. The unique structural difference between compounds 10
and 11 settles in the presence of the benzyl group linked to the
1,2,3-triazole moiety of the whole triazole-benzimidazole-
chalcone scaffold. It could be underlined that the absence of the
methylene group has provided a counter-effect of the molecules
favoring cell proliferation. Such a structure-activity-relationship
should be considered for future design and synthesis of novel
heterocyclic hybrids bearing a triazole moiety, hence, an
adequate choice of the linking groups of a hybrid molecule is
primordial.[23]
constant
J =
16 Hz evidencing the (E)-configuration.[24e]
Additional HMBC connectivities have been considered for
compound 10a, showing the proximity of Hα to the carbonyl C=O
at δC 182.0 ppm. Nearby, Hβ shows HMBC correlations with the
phenyl substituent as illustrated in figure 1. In a similar way, the
novel library of triazole-benzimidazole-chalcone hybrid
compounds 10a-h and 11a-f have been finely characterized
(See supporting information).
Figure 1. Main HMBC cross-peak correlations observed in the 2D-NMR
spectra of the compound 10a.
Table 1. In vitro cytotoxicity assessment of compounds 10a, 10d, 10e and 10h
in breast cancer (T47-D and MDA-MB-231) and prostate cancer (PC3) cell
lines.
Biological assays
The triazole-benzimidazole-chalcone hybrid compounds (10a,
10d, 10e, 10h, 11a-b) were selected to test their cytotoxic
effects in vitro using two human breast cancer cell lines (T47-D
and MDA-MB-231) and one prostate cancer cell line (PC3). The
selection was based on compounds’ solubility in DMSO and we
started the cytotoxic studies by applying a 10 nM–100 ꢀM
concentration range with a dilution factor of 10. Thereafter, the
1–100 ꢀM concentration range was expanded to allow
calculation of the IC50 values for the compounds that exhibited
nearly total cytotoxicity at 100 ꢀM concentrations, but no effects
at concentrations lower than 1 ꢀM (Table 1). Compound 10h has
no significant effect on MDA-MB-231 but stimulate the cell
viability of T47-D cell lines. It is important to observe that
compound 10h showed excellent cytotoxic effect in PC3 cell line
being the most active one compared to compounds 10a, 10d
and 10e (Table 1). Further results showed that in breast cancer
cell lines (MDA-MB-231 and T47-D), compounds 10d has
obviously displayed activity with IC50 value ≤ 10 µM.
The preliminary evaluation of in vitro antiproliferative activity of
compounds 10a, 10d, 10e, 10h and 11a-b has allowed
identifying the triazole-benzimidazole-chalcones 10a, 10d, 10e
as active heterocyclic hybrids. Among them, compound 10d with
a chloro substituent (2-Cl) on the aromatic rings displayed the
highest cytotoxicity effects on all cancer cell lines tested. In this
group of compounds, 10h derivative sharing a chloro substituent
(4-Cl) on the aromatic rings exerted the highest effects in PC3
cells, with IC50 value of 5.64 µM (Table 1).
IC50 (ꢀM)[a]
Compounds
T47-D
MDA-MB-231
PC3
10a
36.7±4,62
6.23±1,03
38.8±2,58
>100
51.2±5.17
5.89±1.35
58.4±0.18
>100
87.6 ±4,81
10.7±1,25
53.2±6,75
5.64 ±1,33
0.73±0.14
10d
10e
10h
Doxorubicin
0.13±0.003
1.51±0.97
[a] IC50 determined using a resazurin-based method after 72 h of
incubation. Each value is the mean (IC50±SD) of two independent
experiments performed in quadruplicate
Conclusions
In summary, we designed a step-by-step synthetic strategy to
build the triazole-benzimidazole-chalcone heterocyclic hybrid
bearing varied aromatic substitutions. The novel synthesized
compounds are obtained in excellent yields and purities, by
using a combination of classical aldol-condensation and click
chemistry. 2D NMR techniques were used to unveil the exact
molecular structure and prove the regioselective formation of
1,4-disubsituted triazole via HMBC correlations. Further
synthetic work on similar nitrogen containing heterocyclic
hybrids is undertaken aiming the construction of new libraries for
biological screenings. The preliminary anticancer screenings on
human cancer cell lines demonstrate that the triazole-
benzimidazole-chalcone hybrid sharing a chloro substituent and
1-N-benzyl-1,2,3-triazole moiety show the best cytotoxic effect
The positive control used in this study, doxorubicin, yielded IC50
values less than 1 µM for PC3 and T47-D and less than 2 µM for
3

α, H-4, H-7), 8.06 (d, 1H, H-β, J 16.0 Hz), 8.12 (s, 1H, H-5’) ppm. 13C-
NMR (100 MHz, [D6]DMSO): δ = 40.7 [N(1)-CH2], 53.2 [N(1’)-CH2], 55.9
(4’’’-OCH3), 112.5 (C-7), 115.2 (C-3’’’,5’’’), 120.9 (C-β), 121.7 (C-4),
124.0 (C-5’), 124.2 (C-5), 126.3 (C-6), 127.4 (C-1’’’), 128.3 (C-2’’,6’’),
128.5 (C-4’’), 129.2 (C-3’’,5’’), 131.4 (C-2’’’,6’’’), 136.4 and 136.7 (C-1’’
and C-7a), 141.7 (C-3a), 143.7 (C-4’), 144.7 (C-α), 146.9 (C-2), 162.3 (C-
4’’’), 182.5 (C=O) ppm. ESI-HRMS: calcd. for C27H24N5O2 [M+H]+
450.1930; found 450.1917.
on all the selected PC-3, MDA-MB-231 and T47-D cell lines.
Based on this preliminary cytotoxicity screening, further studies
are required to provide more insights about the combination of
heterocyclic pharmacophores in a hybrid molecule and their
structure-activity-relationship in cancer therapy.
Experimental Section
(E)-1-{1-[(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-
benzo[d]imidazol-2-yl}-3-(4-methylphenyl)prop-2-en-1-one
(11a):
Chemistry
Yellow Cristal (80 mg, 89%); mp 201-202 °C; 1H-NMR (400 MHz,
[D6]DMSO): δ = 2.37 (s, 3H, 4’’’-CH3), 3.80 (s, 3H, 4’’-OCH3), 6.10 [s, 2H,
N(1)-CH2], 7.08 (d, 2H, H-3’’,5’’, J 9.0 Hz), 7.31 (d, 2H, H-3’’’,5’’’, J 7.8
Hz), 7.40 (t, 1H, H-5, J 7.7 Hz), 7.50 (t, 1H, H-6, J 7.7 Hz), 7.73-7.75 (m,
4H, H-2’’,6’’ and H-2’’’,6’’’), 7.85-7.92 (m, 3H, H-α, H-7 and H-4), 8.18 (d,
1H, H-β, J 16.0 Hz), 8.63 (s, 1H, H-5’) ppm. 13C-NMR (100 MHz,
[D6]DMSO): δ = 21.6 (4’’’-CH3), 40.7 [N(1)-CH2], 56.0 (4’’-OCH3), 112.5
(C-7), 115.2 (C-3’’,5’’), 121.8 (C-4), 122.0 (C-5’), 122.3 (C-2’’.6’’), 122.4
(C-β), 124.3 (C-5), 126.4 (C-6), 129.4 (C-2’’’,6’’’), 130.3 (C-3’’’,5’’’), 130.4
(C-1’’), 132.1 (C-1’’’), 136.8 (C-7a), 141.8 (C-3a and C-4’’’), 144.3 (C-4’),
144.6 (C-α), 146.8 (C-2), 159.7 (C-4’’), 182.6 (C=O) ppm. ESI-HRMS:
calcd. for C27H24N5O2 [M+H]+ 450.1930; found 450.1916.
General: Melting points were measured on a Buchi melting point B-545
apparatus and are uncorrected. 1H and 13C NMR spectroscopy data were
recorded on a Bruker NMR 400 MHz (for 1H) and 100 MHz (for 13C)
spectrometer in [D6]DMSO as a solvent. Chemical shifts (δ) are reported
in ppm and coupling constants (J values) are given in Hz. High-resolution
mass spectra were recorded on a QTOF THERMO spectrometer. All
reactions were monitored by means of TLC on silica or alumina gel
plates and the spots were detected with UV light (l=254 nm). Chemicals
were purchased from Merck or Sigma–Aldrich and used without prior
purification.
General procedure for the synthesis of benzimidazole chalcones
derivatives (6a-h): A solution of 2-acetyl-benzimidazole 4 (3.2 g, 20
mmol) and the diversely substituted aromatic aldehydes 5a-h (24 mmol)
in 40 ml of absolute methanol were taken in a flask. Potassium hydroxide
(8.0 g, 142.6 mmol) was added and the reaction mixture was stirred for
24 hours at room temperature under TLC monitoring. The mixture was
then poured into crushed ice water. The product obtained is filtered and
washed with distilled water and finally recrystallized from methanol.
Biological assays.
Cell culture: MDA-MB-231 metastatic breast cancer cell line was grown
in DMEM culture medium supplemented with 10% fetal bovine serum
(FBS) (GibcoTM by Life Technology) and 1% penicillin/streptomycin
solution (PEST; GibcoTM). T47-D non-metastatic luminal breast cancer
and PC3 metastatic prostate cancer cell lines were grown in RPMI
culture medium supplemented with 10% FBS and 1% PEST. Cell
cultures were maintained at 37 ˚C in a 5% CO2 humidified atmosphere.
Stock solutions of the test compounds were prepared in sterile DMSO to
100 mM final concentration. Thereafter, serial dilutions were prepared in
growth medium. Maximum DMSO concentration applied to the cells was
1/1000 to avoid toxic effects associated to higher concentrations of this
solvent.
General procedure for the synthesis of terminal alkyne
benzimidazole chalcones derivatives (7a-h): To a stirred solution of
benzimidazole chalcones derivatives 6a-h (1 mmol) and propargyl
bromide (0.143 g, 1.2 mmol) in acetone (15 mL), potassium carbonate
(0.346 g, 2.5 mmol) was added and the mixture is allowed to reflux for 5
hours. After the reaction is completed as indicted by TLC, the solvent
was removed, and the crude was recrystallized from ethanol to provide
the desired compound as yellow crystals.
Reagents and test compounds: Compounds 10a, 10d, 10e, 10h, 11a-b
were dissolved in 100% DMSO to obtain a stock concentration of 100
mm. Thereafter, serial dilutions were prepared in growth medium.
General procedure for the synthesis of Triazole-benzimidazole-
chalcone hybrid compounds 10a-h and 11a-f: Synthetic phenyl azide
derivatives 8a-b or benzyl azide derivatives 9a-b (0.21 mmol) and
terminal alkyne of benzimidazole chalcones derivatives 7a-h (0.2 mmol)
were taken in CH2Cl2 and H2O (10 mL, 1:1) under continuous stirring for
5 min. After that, CuSO4.5H2O (0.035 g, 0.22 mmol) and sodium
ascorbate (0.055 g, 0.28 mmol) were added to the reaction mixture,
which was allowed to stirring at room temperature for further 24 hours
under TLC monitoring. After completion of the reaction, the crude mixture
was partitioned between H2O and CH2Cl2. The aqueous layer was
extracted with ethyl acetate (3×15 mL). The combined organic phases
were dried over magnesium sulfate (MgSO4) and concentrated under
reduced pressure. The compounds obtained were recrystallized from
absolute ethanol.
Cell viability assays: Serial dilutions ranged 10 nM-100 µM to cover a
wide scale for generation of dose-response curves. In all experiments the
solvent DMSO (1/1000) alone was used as negative control. Seven
thousand cells/wells were seeded in 96-well plates and allowed to adhere
for 24h. Cells were then exposed to the test compounds diluted in culture
medium for 48 h (200 µL), after this time 100 µl of the culture medium
was replaced by test solution for additional 24 h. Thereafter, cell viability
was assessed using 100 µL/well of the rezasurin-based PrestoBlueTM
reagent (Life Technologies) according to the manufacturer´s instructions.
Values considered were within the linear range of the reading which was
after 3h incubation.
The IC50 values from at least two independent experiments performed in
quadruplicate were calculated using GraphPad Prism software (version
6.00), using the log (inhibitor) vs. response (variable slope-four
parameters) function. For simplification and comparison among different
experiments the figure shows the viability index which was calculated by
normalizing the mean absorbance values of each treatment by the mean
absorbance of the DMSO control.
Fourteen (14) new triazole-benzimidazole-chalcone hybrid compounds
10a-h and 11a-f have been generated; their NMR data and spectra are
reported in the supporting information section. In the following, only NMR
data of compound 10a and 11a are given as representative examples.
(E)-1-{1-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]-1H-benzo[d]imidazol-
2-yl}-3-(4-methoxyphenyl)prop-2-en-1-one (10a): Yellow solid (72.9
mg, 81%); mp 188-190 °C; 1H-NMR (400 MHz, [D6]DMSO): δ = 3.84 (s,
3H, 4’’’-OCH3), 5.53 [s, 2H, N(1’)-CH2], 5.99 [s, 2H, N(1)-CH2], 7.06 (d,
2H, H-3’’’,5’’’, J 8.8 Hz), 7.23-7.34 (m, 5H, H-2’’,3’’,4’’,5’’,6’’), 7.38 (t, 1H,
H-5, J 7.6 Hz), 7.47 (t, 1H, H-6, J 7.6 Hz), 7.81-7.89 (m, 5H, H-2’’’,6’’’, H-
Acknowledgements
4

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A. Massarotti, S. Aprile, V. Mercalli, E. Del Grosso, G. Grosa, G.
Sorba, G. C. Tron, Chem. Med. Chem. 2014, 9, 2497.
Thanks are due to University of Aveiro and FCT/MEC for the
financial support to the QOPNA research project (FCT
UID/QUI/00062/2013) and to the CICECO-Aveiro Institute of
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Dheer, D.; Singh, V.; Shankar, R. Bioorg. Chem. 2017, 71, 30.
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Materials
(POCI-01-0145-FEDER-007679;FCT
UID/CTM/50011/2013), financed by national funds and when
appropriate co-financed by FEDER under the PT2020
Partnership Agreement, and to the Portuguese NMR Network.
We would like also to thank FCT/MEC and the General
Directorate for Scientific Research and Technological
Development – DGRSDT of Algeria and Agence Thématique de
Recherche en Sciences et Technologie ATRST for approving
the co-financed bilateral project PT-DZ/0005. We further wish to
thank Pr. Farid Messelmi the Dean of Faculty of Exact
Sciences and informatics in the University of Djelfa for providing
necessary laboratory facilities to carry out the research work
smoothly. The biological assays were carried out at iBiMED´s
cell culture facility which is supported by national funds through
FCT project UID/BIM/04501/2019
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Keywords: 1,2,3-triazole • benzimidazole • chalcones • click
chemistry • Anticancer activity
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Highlights:
•
•
•
•
Triazole-benzimidazole-chalcone heterocyclic hybrids were prepared via click
chemistry with variable substitutions
1D and 2D NMR (HMBC correlations) have identified the 1,4-disubstitued
triazole-benzimidazole-chalcone as a unique isomer.
Anti-proliferative potential in breast and prostate cancer cell lines have been
evidenced for the triazole-benzimidazole-chalcone hybrid.
The synthetized triazole-benzimidazole-chalcone hybrid sharing a chloro
substituent and 1-N-benzyl-1,2,3-triazole moiety enhanced the cytotoxic
effects.

Author Contributions Section:
Djemoui A., Naouri A; and TALHI O. conceptualized the work and co-wrote the
manuscript.
Ouahrani M.R., Djemoui D. and Lahecen S. performed the synthetic experimental
work and wrote the original draft preparation
Lahrech M.B., Boukenna L. and Albuquerque H.M.T. performed the 2D NMR analysis
and interpretation
Saher L., Rocha D.H.A., Monteiro F.L. and Helguero L.A. were responsible for the
biological screening work
Bachari K. and Silva A.M.S. conceptualized the work and co-wrote the manuscript.

Declaration of interests
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.