Synthesis, characterization, and preliminary in vitro cytotoxic evaluation of a series of 2-substituted benzo [d] [1,3] azoles

Article abstract of DOI:10.3390/molecules26092780

A series of benzo [d] [1,3] azoles 2-substituted with benzyl-and allyl-sulfanyl groups were synthesized, and their cytotoxic activities were in vitro evaluated against a panel of six human cancer cell lines. The results showed that compounds BTA-1 and BMZ

Full text of DOI:10.3390/molecules26092780

molecules
Article
Synthesis, Characterization, and Preliminary In Vitro Cytotoxic
Evaluation of a Series of 2-Substituted Benzo [d] [1,3] Azoles
1
1,
1
, Luis Ángel Gil-Ruiz 1,2,
Ozvaldo Linares-Anaya , Alcives Avila-Sorrosa * , Francisco Díaz-Cedillo
José Correa-Basurto 2 , Domingo Salazar-Mendoza , Adrian L. Orjuela , Jorge Alí-Torres
3
4
4
,
María Teresa Ramírez-Apan 5 and David Morales-Morales 5
1
Instituto Politécnico Nacional, Departamento de Química Orgánica, Carpio y Plan de Ayala S/N,
Escuela Nacional de Ciencias Biológicas, Colonia Santo Tomás, Ciudad de México 11340, Mexico;
ozvaldo55@hotmail.com (O.L.-A.); fdiazc@ipn.mx (F.D.-C.); angeluzzy@hotmail.com (L.Á.G.-R.)
Laboratorio de Diseño y Desarrollo de Nuevos Fármacos e Innovación Biotecnológica, Instituto Politécnico
Nacional, Escuela Superior de Medicina, Ciudad de México 11340, Mexico; corrjose@gmail.com
Carretera a Acatlima, Huajuapan de León, Universidad Tecnológica de la Mixteca, Oaxaca 69000, Mexico;
dsalazar@mixteco.utm.mx
Departamento de Química, Universidad Nacional de Colombia-Sede, Bogotá 111321, Colombia;
alorjuelar@unal.edu.co (A.L.O.); jialit@unal.edu.co (J.A.-T.)
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria,
Ciudad de México 04510, Mexico; mtrapan@yahoo.com.mx (M.T.R.-A.); damor@unam.mx (D.M.-M.)
Correspondence: aavilas@ipn.mx; Tel.: +52-555-729-6000
2
3
4
5
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
*
Citation: Linares-Anaya, O.;
Avila-Sorrosa, A.; Díaz-Cedillo, F.;
Gil-Ruiz, L.Á.; Correa-Basurto, J.;
Salazar-Mendoza, D.; Orjuela, A.L.;
Alí-Torres, J.; Ramírez-Apan, M.T.;
Morales-Morales, D. Synthesis,
Characterization, and Preliminary
In Vitro Cytotoxic Evaluation of a
Series of 2-Substituted Benzo [d] [1,3]
Azoles. Molecules 2021, 26, 2780.
https://doi.org/10.3390/molecules
Abstract: A series of benzo [d] [1,3] azoles 2-substituted with benzyl- and allyl-sulfanyl groups were
synthesized, and their cytotoxic activities were in vitro evaluated against a panel of six human cancer
cell lines. The results showed that compounds BTA-1 and BMZ-2 have the best inhibitory effects,
compound BMZ-2 being comparable in some cases with the reference drug tamoxifen and exhibiting
a low cytotoxic effect against healthy cells. In silico molecular coupling studies at the tamoxifen
binding site of ERα and GPER receptors revealed affinity and the possible mode of interaction of
both compounds BTA-1 and BMZ-2.
Keywords: 2-substituted benzo [d] [1,3] azoles; in vitro cytotoxicity; breast cancer; ERα and GPER;
2
6092780
molecular docking
Academic Editors: Maria Emília de
Sousa and Maria-João R. P. Queiroz
1
. Introduction
Received: 16 February 2021
Accepted: 13 April 2021
Published: 8 May 2021
Heterocyclic structures are widely distributed in nature and occur as key fragments of
biomolecules, e.g., amino acids, nucleic acids, vitamins, natural products, enzymes, bio-
logical targets, etc. Besides, they constitute numerous synthetic compounds with relevant
applications in different branches of chemistry and technology [1–4].
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
The structures of benzo [d] [1,3] azoles, such as benzimidazole (BZM), benzothiazole
(
BTA), and benzoxazole (BOX), are highly valued heterocyclic scaffolds in the design and
synthesis of new drugs and bioactive agents. These heterocyclic nuclei are privileged
pharmacophores that exhibit an inherent affinity for various types of receptors involved
in different biological signaling pathways, whose derivatives have shown multiple phar-
macological functions, including anti-inflammatory, diuretic, antiviral, anti-insomnia,
antiparasitic, anticancer, among others (Scheme 1) [5–15].
On the other hand, cancer is considered a complex group of diseases that can origi-
nate anywhere in the body, characterized by uncontrolled cell proliferation and abnormal
growth of the affected tissue (neoplasm), being able to migrate causing metastasis. Cancer
is the second leading cause of death worldwide; only in 2018 9.6 million deaths were esti-
mated, with low- and middle-income countries being the most affected with approximately
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
7
0% of deaths. Lung, colorectal, stomach, liver, and breast cancers are metastatic diseases
with the highest mortality rates [16]. Globally, breast cancer affects around 2.1 million
4
.0/).
Molecules 2021, 26, 2780. https://doi.org/10.3390/molecules26092780
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2780
2 of 16
women per year, accounting for approximately 15% of all cancer deaths in women [16,17].
Breast cancer is linked to estrogen receptors (ERs) called hormone receptor-positive (ER-
positive) cancers. ERs are present in cells and are nuclear transcription factors with both
dependent and independent transcriptional activity of the 17β-estradiol ligand, related
to the physiology and pathophysiology of different tissues. The activated receptor is
transported to the nucleus and binds to DNA to modulate the activities of different genes.
Steroid hormones, including the ligand 7
ment and physiology of the mammary glands, as well as being an important risk factor
in breast cancer. These hormonal actions are mainly mediated by ER and ER- 18 21],
which are strongly expressed in human breast cancer, ER being predominant in benign
breast tumors, while ER is dominant in malignant tumors [22 24], in such a way that ER
is associated with growth and cell proliferation in breast cancer [25 26]. Likewise, multiple
β-estradiol, play an important role in the develop-
α
β [ –
β
α
–
α
,
studies have shown that G-protein coupled estrogen receptor 1 (GPER) is also involved in
the transduction of estrogenic signals for the regulation of cell growth and proliferation in
different types of solid tumors, such as colon cancers, lung, cervix, and breast cancer [27].
In fact, in approximately 50% of human breast cancers, the GPER has been found at high
levels [28–31]. The genomic action mechanism of estrogens occurs: (1) when the ligand
interacts with ERs, inducing a conformational change that activates them, or (2) a dimer is
generated that is the active form, it translocates to the nucleus interacting with the estrogen
response elements regulating the transcription of specific genes. In the case of GPER1, it is
a component of the family of G-protein-coupled receptors, it is responsible for regulating
cell growth (HER), including proliferation and apoptosis through a non-genomic response
activating the epidermal growth factor [21]. Estrogen signaling is of great interest due
to the high levels of ERs and GPER protein in breast cancers, for which these proteins
are vital targets in the therapy of ER-linked breast cancer. Therefore, ERs signaling can
be blocked, thus reducing proliferation. Tamoxifen (TAM) and other estrogen receptor
selective drugs are used to treat or prevent breast cancer by inhibiting the proliferative
effects of estrogen, mediated through ERs. The active metabolite of TAM, 4-hydroxy ta-
moxifen, exhibits a high binding affinity for ERs [32]. Among the main cancer treatments,
chemotherapy using multiple antineoplastic drugs remains one of the most common basic
alternatives. However, very often, these pharmacological treatments are accompanied by
several serious side effects frequently not tolerated by the patients [33–35]. In this context,
and considering the alarming statistics projected by the WHO for the next decade, there is a
pressing urgency in the search for new targeted and effective chemotherapeutic agents for
safer treatments. For this purpose, ERs are considered potential targets for the development
of new anticancer agents.
A quick look at the literature shows several works on derivatives of BZM, BTA, and
BOX, especially those substituted at the 2 position, which have shown important biological
activity against different types of neoplasms, including breast cancer [36
pointed out that the mechanisms of action of these benzo [d] [1,3] azole are different,
highlighting in an important way the signaling pathways modulated by ERs [41 44]. Thus,
–40]. It has been
–
following our continuous interest in the synthesis of benzofused heterocyclic derivatives
and the study of their potential biological activities, in this opportunity, we report here the
synthesis and characterization of a series of benzo [d] [1,3] azoles substituted at position 2
with benzyl and allyl-sulfanyl groups (BTA-1, BZM-2, BOX-3, BTA-4, BZM-5 and BOX-6)
and their preliminary in vitro cytotoxic assays against different human cancer cell lines.
Additionally, in silico molecular docking between the different heterocyclic derivatives
(BTA-1, BZM-2, BOX-3, BTA-4, BZM-5 and BOX-6) and ERα and GPER receptors where
performed, seeking to explore the affinity and possible binding modes of these compounds
with ERs. These molecular couplings were performed at the reference drug TAM binding
site of both estrogen receptors.

Molecules 2021, 26, 2780
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Scheme 1. Drugs containing heterocyclic cores of BZM, BTA, and BOX.
2. Results and Discussion
2.1. Synthesis and Characterization
The series of 2-substituted benzo [d] [1,3] azoles (BTA-1, BZM-2, BOX-3, BTA-4,
BZM-5 and BOX-6) were easily synthesized by a procedure previously reported by our
research group [45], involving the substitution reactions between 2-mercaptobenzimidazole
and their analogs, 2-mercaptobenzothiazole and 2-mercaptobenzoxazole, with the corre-
sponding 1-(bromomethyl)-4-vinylbenzene or allyl bromide. Most of the benzo[d]azoles
heterocycle derivatives were obtained as pure microcrystalline solids in good yields (70 to
◦
8
3%) with melting points between 49 and 140 C (BTA-4 and BOX-6 were liquids) (Table 1).
1
13
1
The series of compounds were fully characterized by IR and NMR ( H, C{ H})
spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-
MS afforded spectra that display peaks with the molecular ions plus one mass unit (M+
+
1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while
those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for
1
the proposed structures. Additionally, analyses by H NMR of compounds BZM-2 and
BZM-5 produced spectra exhibiting signals around
signals due to the heteroaromatic cores were observed between
signals due to the hydrogens H4 and H7 are displaced to lower field at
δ
11 ppm due to the -NH- proton, while
8.0 and 7.0 ppm, where
7.91–7.42 ppm.
6.68–5.01 ppm, with
δ
δ
Besides, signals due to the vinyl hydrogens appear in the region of
δ
0
multiplicities and couplings constants (J s) typical for cis, trans, and geminal couplings.
The methine signal (H13) of the benzyl-substituted derivatives BTA-1, BZM-2, and BOX-3

Molecules 2021, 26, 2780
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is observed as a doublet of doublets (dd) with J = 17 and 10 Hz corresponding to the
couplings with protons H14a and H14b, respectively. Conversely, in the allyl-substituted
compounds BTA-4
the additional allylic couplings with proton H8. The latter signal for the benzyl-substituted
compounds BTA-1 BZM-2, and BOX-3 appear as singlets at 4.38–4.58 ppm, and for the
allyl-substituted compounds BTA-4 BZM-5, and BOX-6 as doublets (J = 6.9 Hz) at about
.0 ppm. These spectroscopic data are summarized in Table 2.
, BZM-5, and BOX-6, these signals were observed as multiplets due to
,
δ
,
4
Table 1. Synthesis of 2-substituted benzo [d] [1,3] azoles derivatives (BTA-1, BZM-2, BOX-3, BTA-4, BZM-5, and BOX-6).
Entry
2-Mercaptobenzo [d] Azole
Br-R
Product
Yield (%) a
N
Br
Br
Br
N
S
1
SH
S
70
S
BTA -1
N
N
SH
2
3
S
83
80
N
H
N
H
BZM-2
BOX-3
N
N
O
SH
O
S
N
N
4
5
6
SH
S
S
75
70
77
Br
S
BTA -4
N
N
SH
S
Br
N
H
N
H
BZM -5
N
N
O
S
SH
O
Br
BOX-6
a
Isolated yields based on 2-mercaptobenzo [d] azoles.
Further analysis by 13C{ H} NMR yielded spectra displaying all the expected signals
consistent with the proposed structures for all the compounds. In general, the signal
due to the quaternary carbons attached to heteroatoms appears at lower field. While
1
the signals due to the carbon C2 binding the three heteroatoms is located between
δ
166.26 and 149.87 ppm, showing further deprotection for the BTA derivatives, and signals
due to carbons C3a and C7a at the bicyclic linking bonds were observed in the range
δ
153.22–139.27 ppm (see experimental section).

Molecules 2021, 26, 2780
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1
Table 2. H NMR data for 2-substituted benzo [d] [1,3] azoles derivatives (BTA-1, BZM-2, BOX-3, BTA-4, BZM-5, and BOX-6).
Comp.
H4
H5
H6
H7
H8
H10
H11
H13
H15a
H15b
BTA-1
BZM-2
BOX-3
BTA-4
BZM-5
BOX-6
7.89
7.43–7.40
7.62
7.68
7.47–7.37
7.60
7.44–7.25a
7.12–7.08
7.30–7.20
7.36–7.31
7.12–7.04
7.29–7.21
7.44–7.25a
7.12–7.08
7.30–7.20
7.24–7.19
7.12–7.04
7.29–7.21
7.73
7.43–7.40
7.44–7.43
7.67
7.47–7.37
7.43
4.58
4.38
4.54
3.92
3.87
3.95
7.40–7.38
7.12–7.08
7.41–7.40
7.35–7.36
7.12–7.08
7.37–7.36
6.68
6.51
6.68
6.01–5.88
5.97–5.83
6.08–6.00
5.72
5.57
5.73
5.30
5.18
5.39
5.23
5.10
5.24
5.13
5.01
5.21
−
−
−
−
−
−
Chemical shifts (
3 4
δ) are reported in ppm down of Si(CH ) (δ = 0.0). Compounds BZM-2 and BZM-5 showed the H1 signals (NH) at δ 10.26
and δ 11.20 ppm, respectively.
2.2. Preliminary In Vitro Antiproliferative Screening
With the compounds on hand, preliminary in vitro cytotoxicity studies were per-
formed to explore their potential anticancer activity against a panel of six human cancer
cell lines, i.e., CNS glia (U-251), prostate (PC-3), leukemia (K-562), colon (HTC-15), lung
(SKLU-1), breast (MCF-7), and non-cancerous human gingival fibroblast cells (FGH) using
TAM as reference drug. As shown in Table 3, the allyl-substituted compounds BTA-4
,
BZM-5, and BOX-6, did not cause cytotoxic effects against U-251, PC-3, and K-562 cancer
cell lines, and they were also inactive against gingival fibroblast cells (FGH), while for the
cell lines HTC-15, MCF-7, and SKLU-1, inhibition values ranging from 2 to 12% were ob-
served. Besides, the benzyl-substituted compounds BTA-1, BZM-2, and BOX-3 exhibited
better growth inhibitory activity compared with those of TAM, being the best BZM-2 and
the worst BOX-3, being active only on three cell lines, i.e., HTC-15 (1.1%), MCF-7 (16.7%),
and SKLU-1 (10.4%).
Interestingly, derivatives BTA-1 and BZM-2 displayed the highest cytotoxic activities
of all heterocyclic derivatives against the six cancer cell lines explored. Thus, compound
BTA-1 showed moderate inhibition percentages of about 15% against U-251, PC-3, K-
562, and HTC-15, being better than the allyl-substituted compounds BTA-4, BZM-5, and
BOX-6. For breast cancer (MCF-7), derivative BTA-1 exhibited moderate inhibition (21.4%)
compared to TAM (71.3%), while the cytotoxic effect was better against SKLU-1 with
38.4% compared to 43.3% of inhibition for TAM. However, BZM-2 was the one exhibiting
the highest inhibitory profile among the series of benzo [d] [1,3] azole derivatives tested
against the different cancer cell lines, being at least ten times more cytotoxic than the BTA-1
derivative against the different types of carcinomas, thus indicating that the BZM core
increases the cytotoxic activity compared with the BTA and BOX moieties. Hence, BZM-2
exhibited the best cytotoxic effects against PC-3 (47.0%), K-562 (37.2%), MCF-7 (46.8%),
and SKLU-1 (41%) cell lines. Notably, the inhibitory effect of compound BZM-2 against
PC-3 was 10% greater than the reference drug, and for SKLU-1, the inhibitory percentage
is very similar, more notably the fact that it exhibited a very low inhibitory effect of only
7.7% in FGH non-cancerous cells, contrasted with the 100% observed for TAM. Regarding
cytotoxicity in non-cancer cells, it can be observed that most of the heterocyclic derivatives
have practically null or very low cytotoxicity, with BZM-5 being the one with the greatest
inhibitory effect (12%), still very low compared to TAM. In the case of the compounds with
the best cytotoxic profiles against cancer cells from the in vitro studies, BTA-1 and BZM-2
the cytotoxic effect on FGH is even lower compared to the reference drug, so that BTA-1 is
not active, and in the case of BZM-2, the cytotoxicity in healthy cells is very low, with a
percentage of inhibition of about 8%. These values are positive and of great relevance in
the search for more selective anticancer agents with low side effects. Finally, IC50 values
,

Molecules 2021, 26, 2780
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for compound BZM-2 were determined on PC3 (34.79
32.21 ± 0.78), and SKLU (27.93 ± 1.8).
±
0.13), K562 (22.79
±
3.0), MCF7
(
Table 3. % growth inhibitor of benzo [d] [1,3] azole derivatives (BTA-1, BZM-2, BOX-3, BTA-4, BZM-5, and BOX-6) against
six human cancer cell lines (25 µM) *.
Compound
U-251
PC-3
K-562
HTC-15
MCF-7
SKLU-1
FGH
N
S
1
2
5.7
7.5
12.7
14.6
14.0
21.4
38.4
NA
S
BTA -1
N
47.0
37.2
31.2
46.8
41
7.7
S
N
H
BZM-2
N
O
NA
NA
NA
2.5
NA
NA
1.1
5.8
16.7
4.0
10.4
12.4
NA
NA
S
BOX-3
N
S
S
BTA-4
N
S
NA
NA
NA
NA
NA
NA
NA
NA
10.3
2.5
12.1
3.2
12.0
NA
N
BZM-5
H
N
O
S
BOX-6
O
(
H C) N
3 2
CH3
80.6
36.3
54.3
58.8
71.3
43.3
100
Tamoxifen (TAM)
*
The results are the average of three runs. NA: not active.
Attempts to get IC50 values in SKLU with compound BTA-1 revealed that the response
was not concentration-dependent at M concentrations of 100, 75, 50, and 25 where the r
µ
value for cytotoxicity was practically linear, and thus, if a value could be reported for this
particular species, it would be >100 µM.
2.3. Molecular Docking Studies
In order to validate the performance of the molecular docking protocol, we used the
crystalized X-ray ligand of the receptors with three docking programs. The receptors used
for the molecular dynamic simulations were selected, since they are important targets
in the development of several types of cancer. Thus, mammalian target of rapamycin
receptor (mTOR) was selected, because it regulates relevant cellular pathways such as cell

Molecules 2021, 26, 2780
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proliferation, autophagy, and apoptosis [46]. Additionally, the progesterone receptor (Pr),
besides being a marker of the Er activity, its overexpression is observed in the development
of breast cancer [47]. These facts fully justify the use of multitarget docking to evaluate the
biological activity of the series of benzo [d] [1,3] azoles derivatives. The binding energy of
these receptors were used as a reference to the in silico evaluation of the biological activity
for the derivatives of benzo [d] [1,3] azole. To calculate the precision of the binding mode of
the ligand, we calculated the root-mean-square deviation (RMSD) comparing the position
of the ligand in the best poses obtained from the three programs and the crystalized ligand
◦
(
(
Supplementary Materials Figure S19). We found a RMSD for all receptors lesser than 1 A
Table 4), which is a good performance for docking simulations. In addition, the binding
energy for the redocking process results in a strong biding energy with the three programs.
Table 4. Docking validation for the four receptors using three different molecular docking programs. The calculated
root-mean-square deviation (RMSD) respect to the crystalized ligand is also shown.
Binding Energy
Binding Energy
(kcal/mol) Vina
Binding Energy
(kcal/mol) Smina
◦
Compound
Receptor
Mean RMSD (A )
(kcal/mol) AD4
AEE788
TAM
EGFR
Erα
−6.7
−8.1
−9.4
−15.0
−8.3
−6.2
−8.5
−9.1
−6.7
−7.9
−10.6
−6.9
0.845
0.568
0.352
0.158
Ulipristal acetate
RAP
Pr
mTOR
To select the best poses among all the conformations explored by the three programs,
we used an exponential consensus rank approach. These results are shown in Table 5
in which it can be observed a good correlation between the experimental data and the
molecular docking results. Since tamoxifen (TAM) was the crystal ligand in Er
α; this was
used as reference. As shown in Table 5, TAM has a strong interaction with all the receptors
that could be related with an inhibition of the metabolic pathway. Compounds BTA-4
,
BZM-5, and BOX-6 establish weak interaction with all the receptors, while compound
BZM-2 prefers the interactions with Er
α and mTOR receptors. In addition, compound BTA-
1
interacts with the EGRF pathway and the ligand BOX-3 with Pr. In spite of the molecular
similarity, the series of compounds interact in different targets of cancer pathways.
Table 5. Exponential rank consensus between receptors and derivatives of benzo [d] [1,3] azole. * The
0
tamoxifen (TAM) was the crystal ligand of Er
α
and LogP calculated by Crippen s fragmentation
method [48].
Ligand
EGFR
Erα
Pr
mTOR
LogP ± 0.47
BTA-1
BZM-2
BOX-3
BTA-4
BZM-5
BOX-6
TAM
3
4
5
6
7
8
1
2
4
2
3
5
5
6
1 *
1
5
4
3
6
7
7
2
1
4
3
5
6
7
7
2
1
6.04
4.60
4.67
4.25
2.82
2.86
6.07
Reference ligand (crystal structure)
To evaluate the ability of the derivatives of benzo [d] [1,3] azole to enter into the
cellular membrane, we calculated the LogP for all the derivates. The results are presented
in Table 5. As can be seen from this table, TAM and compound BTA-1 presented the
higher values of LogP compared with the other derivates. The sulfur atom in the azole
group increases the hydrophobicity of the derivates BTA-1 and BTA-4 lowering the LogP
values. From the whole group of ligands, the less hydrophobics were derivates BZM-5
and BOX-6. These low values suggest a bigger difficulty to enter into cell and generate a
pharmacological activity.

Molecules 2021, 26, 2780
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The derivatives of benzo [d] [1,3] azole exhibit the same pose and interactions with the
receptor EGFR, since the size and charge in the atoms were similar, but the presence of an
heteroatom changes the electronic structure of the azole derivate. All azole derivates form
pi–alkyl interactions with Leu 718 and hydrogen bonds with the nitrogen atom from the
azole group and the backbone of Met 793. The vinylbenzene group of the molecules BTA-1
,
BZM-2, and BOX-3 exhibit a pi–sigma interaction with Thr 790 and pi–sulfur interaction
with Met 766 as shown in Figure 1. The derivates BTA-4, BZM-5, and BOX-6 establish
weak interactions with the receptor, since the substituent group only forms a pi–alkyl
interaction with Leu 844.
Figure 1. Interaction maps between receptor EGFR and molecules (
A) BTA-1, BZM-2, and BOX-3 (B) BTA-4, BZM-5, and
BOX-6. Cyan dashed lines are hydrogen bonds, magenta are pi–alkyl interactions, and yellow are pi–sulfur interactions.
The Er
between the nitrogen atom from the benzo [d] [1,3] azole scaffold and Lys 449. Sulphur
and oxo derivates BTA-1 BOX-3 BTA-4, and BOX-6 form hydrogen bonds with Arg 394,
α receptor presents interactions with all derivates through hydrogen bonds
,
,
meanwhile nitrogen atoms from derivates BZM-2 and BZM-5 form hydrogen bonds with
Glu 353. All derivates establish pi–alkyl interactions with Pro 324. The vinylbenzene
derivates BTA-1
electronic structure compared with the compounds BTA-4
,
BZM-2, and BOX-3 exhibit pi–alkyl interactions due to their different
BZM-5, and BOX-6 (Figure 2).
,
Figure 2. Interactions maps between receptor Er
α and molecules (A) BTA-1, BZM-2, and BOX-3 (B) BTA-4, BZM-5, and
BOX-6. Cyan dashed lines are hydrogen bonds, magenta are pi–alkyl interactions, and yellow are pi–sulfur interactions.

Molecules 2021, 26, 2780
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All benzo [d] [1,3] azole derivatives present a pi–cation interaction with Arg 766
and hydrogen bond interactions between the nitrogen from the azole and Gln 725. The
benzimidazole derivates BZM-2 and BZM-5 form hydrogen bonds with Asp 697. While
the vinylbenzene derivates BTA-1, BZM-2, and BOX-3 exhibit pi–sigma interactions with
valine. The vinyl group in the derivates BTA-4, BZM-5, and BOX-6 gives place to pi–alkyl
interactions, as shown in Figure 3.
Figure 3. Interactions maps between receptor Pr and molecules (
A) BTA-1, BZM-2, and BOX-3 (B) BTA-4, BZM-5, and
BOX-6. Cyan dashed lines are hydrogen bonds, magenta are pi–alkyl interactions, and yellow are pi–sulfur interactions.
Compounds BTA-1, BZM-2, BOX-3, BTA-4, and BOX-6 establish pi–pi stacking inter-
action with the benzo [d] [1,3] azole center and Trp 90, Tyr 57, and Phe 130. In addition, the
nitrogen atom from the azole ring forms hydrogen bonds with the backbone of Tyr 113.
While the nitrogen atom from compound BZM-2 forms a hydrogen bond with Tyr 57, the
benzo vinyl ligand exhibits a pi–pi interaction with Pro 120. Additionally, the vinyl group
in derivates BTA-4 and BOX-6 does not interact with the receptor. The derivate BZM-5
exhibits a different binding mode with two hydrogen bonds in the benzo [d] [1,3] azole
ring (Figure 4).
Figure 4. Interactions maps between receptor mTOR and molecules (
A) BTA-1, BZM-2, and BOX-3; (B) BTA-4 and BOX-6;
(
C) BZM-5 cyan dashed lines represent hydrogen bonds, and pink lines represent pi–pi stacking interactions.
As it can be observed, the molecular docking simulations described the stabilizing
interactions between the derivatives and four proteins related to the development of
various types of cancer. The in silico evaluation is in good agreement with the preliminary
in vitro cytotoxicity bioassays. That is, compounds BTA-1, BZM-2, and BOX-3 exhibit
better interactions with the proteins due to the presence of the benzyl moiety in their

Molecules 2021, 26, 2780
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structures thus facilitating the formation of stabilizing pi–sigma interactions. In contrast,
the vinyl group in derivatives BTA-4
interaction with the proteins. Compounds BTA-1
,
BZM-5, and BOX-6 hampers the formation of any
BZM-2, and BOX-3 also exhibited the
,
larger values of LogP this being in favor of a better membrane transports of these species
that in turn may also favor their cytotoxic activities, as was the case for compounds BZM-2
and BTA-1.
3. Materials and Methods
3.1. Reagents and Apparatus
All reagents used were purchased commercially from Aldrich Chemical. Co. Inc.
(St Louis, MO, USA), and used without further purification. Solvents were supplied by J.T.
Baker (Phillipsburg, NJ, USA), which were dried and distilled prior to use, using standard
procedures established under dinitrogen atmosphere. The melting points were determined
(
without corrections) using a MELT-TEMP II Laboratory Devices, vibrational spectroscopy
−
1
IR were performed in the range of
υ
4000 to 350 cm in a NICOLETMAGNA spectrometer
MS were carried out using a JEOL JMS-SX102A
and JEOL JMS-T100LC spectrometer, respectively. NMR spectra were recorded in CDCl
7
50 FT-IR in KBr tablet. EI-MS and DART
±
3
at room temperature on a JEOL spectrometer GX300 ECLIPSE at 300 MHz frequency for
1
13
1
1
13
H and 75 MHz for C{ H}. The chemical shifts (
δ) for H and C are reported in ppm
down field of Si (Me)4 ( = 0.0). The abbreviations used in the description of the NMR data
δ
are the following: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet of
doublets; td, triplet of doublets; and m, multiplet. Coupling constants are reported as J in
Hz. The number of protons (n) for a given resonance is indicated by nH. NMR spectra,
mass spectra and docking results are available in supporting material.
3
.2. Synthesis of 2-Substituted Benzo[d] [1,3] Azole Heterocycle Derivatives (BTA-1, BZM-2,
BOX-3, BTA-4, BZM-5, and BOX-6)
The series of 2-substituted benzo[d] [1,3]azoles derivatives were obtained by the
following general procedure: To a suspension of 2-mercaptobenzothiazole (3.53 mmol) and
K CO (0.553 g, 4 mmol) in THF (30 mL), the corresponding 1-chloromethyl-4-vinylbenzene
2
3
(or allyl bromide) (0.5 mL, 3.8 mmol) was added dropwise. The reaction mixture was stirred
for 48 h at room temperature under N atmosphere. The reaction was monitored by thin-
2
layer chromatography to completion. Then, the reaction was filtered and washed with
CH Cl (3
×
5 mL). The organic filtrate was dried over anhydrous Na SO and evaporated.
2
2
2 4
Solid compounds were recrystallized from hexane/AcOEt and dried under vacuum to
afford the desired product.
2
-(4-vinylbenzylthio) benzo [d] thiazole (BTA-1)
◦
−1
υ (cm ): 3083, 3053,
White solid (0.703 g, 2.48 mmol, 70%), mp 63–65 C. IR (KBr),
3
9
026, 2999, 2925, 2853, 1687, 1820, 1605, 1506, 1307, 1421, 1200, 1278, 1235, 1127, 1101, 1076,
+
92, 935, 905, 843, 750, 725, 673, 484, 434. EI-MS, m/z: 283 (70, [M] ), 250 (30), 166 (10),
1
1
17 (100), 91 (15). H NMR (300 MHz, CDCl ),
δ
(ppm): 7.89 (ddd, J = 8.1, 1.2, 0.6 Hz, 1H,
3
Ar), 7.73 (ddd, J = 7.9, 1.3, 0.6 Hz, 1H, Ar), 7.44–7.24 (m, 6H, Ar), 6.68 (dd, J = 17.6, 10.9 Hz,
1
2
H, CH), 5.72 (dd, J = 17.6, 0.9 Hz, 1H, CH), 5.23 (dd, J = 11.0, 0.9 Hz, 1H, CH), 4.58 (s,
H, CH2). 13C{ H} NMR (75 MHz, CDCl ),
1
δ
(ppm): 166.26, 153.14, 137.11, 136.27, 135.71,
3
1
35.33, 129.31, 126.49, 126.03, 124.27, 121.54, 120.98, 114.14, 37.40. Anal. Cal. for C H NS
16 13 2
(
283.41 g/mol): C, 68.08; H, 4.68; N, 4.94; S, 22.63. Found: C, 68.12; H, 4.71; N, 4.99; S, 22.54.
2
-(4-vinylbenzylthio)-1H-benzo[d]imidazole (BZM-2)
◦
−1
υ (cm ): 3064, 3053,
White solid (0.832 g, 3.12 mmol, 83%), mp 136–138 C. IR (KBr),
3
020, 2957, 2924, 2853, 2789, 2697, 2609, 1623, 1586, 1503, 1436, 1404, 1343, 1295, 1268, 1230,
+
1194, 1115, 1012, 982, 909, 875, 844, 812, 741, 615, 596, 477, 430. DART -MS, m/z: 267 (100,
+
1
[M] + 1), 151 (35), 117 (15). H NMR (300 MHz, CDCl ),
δ
(ppm): 7.42 (dd, J = 5.7, 3.0 Hz,
3
2
H, Ar), 7.12–7.08 (m, 6H, Ar), 6.51 (dd, J = 17.6, 10.9 Hz, 1H, CH), 5.57 (d, J = 17.6 Hz, 1H,
13
1
CH), 5.10 (d, J = 10.9 Hz, 1H, CH), 4.38 (s, 2H, CH2). C{ H} NMR (75 MHz, CDCl ),
δ
3
(ppm): 150.05, 138.27, 137.03, 136.30, 136.11, 129.17, 126.57, 122.58, 114.34, 114.22, 137.36.

Molecules 2021, 26, 2780
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Anal. Cal. for C H N S (266.36 g/mol): C, 72.15; H, 5.30; N, 10.52; S, 12.04. Found: C,
16
14
2
71.96; H, 5.34; N, 10.43; S, 12.01.
2
-(4-vinylbenzylthio) benzo [d] oxazole (BOX-3)
◦
−1
υ (cm ): 3044, 2928,
White solid (0.798 g, 2.98 mmol, 80%), mp 49–54 C. IR (KBr),
822, 1626, 1599, 1541, 1497, 1451, 1404, 1343, 1286, 1241, 1214, 1132, 1096, 990, 928, 908, 844,
1
8
+
1
03, 742, 623, 488, 425. EI-MS, m/z: 267 (45, [M] ), 234 (10), 117 (100). H NMR (300 MHz,
(ppm): 7.62 (d, J = 7.4 Hz, 1H, Ar), 7.43–734 (m, 5H, Ar), 7.30–7.20 (m, 2H, Ar),
.68 (dd, J = 17.6, 10.9 Hz, 1H, CH), 5.73 (d, J = 17.6 Hz, 1H, CH), 5.24 (d, J = 10.9 Hz, 1H,
CDCl3),
δ
6
13
1
CH), 4.54 (s, 2H, CH2). C{ H} NMR (75 MHz, CDCl ),
δ
(ppm): 164.54, 151.94, 141.96,
37.34, 136.30, 135.41, 129.40, 126.65, 124.42, 124.05, 118.55, 114.42, 110.01, 36.43. Anal. Cal.
for C H NOS (267.35 g/mol): C, 71.88; H, 4.90; N, 5.24; S, 11.99. Found: C, 71.94; H, 4.94;
3
1
16
13
N, 5.18; S, 11.95.
2
-(allylthio) benzo [d] thiazole (BTA-4) [49].
Amber liquid (0.753 g, 3.63 mmol, 75%). IR (KBr),
(cm 1): 3060, 2978, 2920, 1941,
−
υ
1
7
902, 1860, 1636, 1560, 1456, 1423, 1307, 1274, 1234, 1158, 1125, 1075, 990, 921, 859, 752,
24, 669, 591, 548, 525, 427. DART -MS, m/z: 208 (100, [M] + 1). H NMR (300 MHz,
+
+
1
CDCl ), δ (ppm): 7.80 (d, J = 8.1 Hz, 1H, Ar), 7.67 (d, J = 7.9 Hz, 1H, Ar), 7.36–7.31 (m, 1H,
3
Ar), 7.24–7.18 (m, 1H, Ar), 6.01–5.58 (m, 1H, CH), 5.30 (dd, J = 16.9, 1.1 Hz, 1H, CH), 5.13
d, J = 10.0 Hz, 1H, CH), 3.92 (d, J = 6.9 Hz, 2H, CH2). 13C{ H} NMR (75 MHz, CDCl ),
1
δ
(
(
3
ppm): 166.20, 153.22, 135.34, 132.33, 126.06, 124.29, 121.60, 120.98, 119.19, 36.26. Anal. Cal.
for C H NS (207.32 g/mol): C, 57.93; H, 4.38; N, 6.76; S, 30.93. Found: C, 57.85; H, 4.35;
10
9
2
N, 6.74; S, 30.87.
2
-(allylthio)-1H-benzo [d] imidazole (BZM-5) [49].
◦
−1
υ (cm ): 3079, 3051,
White solid (0.701 g, 3.68 mmol, 70%), mp 138–140 C. IR (KBr),
2
925, 2867, 2729, 1911, 1818, 1789, 1686, 1624, 1604, 1504, 1452, 1420, 1307, 1277, 1234, 1196,
+
1
198, 1126, 1101, 1076, 991, 935, 905, 843, 750, 725, 673, 599, 484, 435. DART -MS, m/z: 191
+
1
(100, [M] ). H NMR (300 MHz, CDCl ), δ (ppm): 7.42 (d, J = 2.8 Hz, 2H, Ar), 7.08 (dd,
3
J = 5.7, 2.9 Hz, 1H, Ar), 5.90 (td, J = 16.8, 8.1 Hz, 1H, CH), 5.18 (d, J = 16.9 Hz, 1H, CH),
.01 (d, J = 9.9 Hz, 1H, CH), 3.87 (d, J = 6.8 Hz, 1H, CH2). 13C{ H} NMR (75 MHz, CDCl ),
1
5
3
δ
(ppm): 149.87, 139.59, 132.98, 121.87, 118.46, 114.14, 35.36. Anal. Cal. for C H N S
10 10 2
(
190.26 g/mol): C, 63.13; H, 5.30; N, 14.72; S, 16.85. Found: C, 62.97; H, 5.32; N, 14.68;
S, 16.76.
2
-(allylthio) benzo [d] oxazole (BOX-6) [50].
-1
Amber liquid (0.768 g, 4.01 mmol, 77%). IR (KBr),
υ (cm ): 3082, 2979, 2928, 1638,
1
7
599, 1498, 1451, 1424, 1402, 1337, 1281, 1236, 1213, 1118, 1129, 1094, 987, 923, 888, 860, 805,
+
+
1
40, 622, 598, 426. DART -MS, m/z: 192 (100, [M] ), 117 (32), 89 (18). H NMR (300 MHz,
(ppm): 7.60 (d, J = 7.6 Hz, 1H, Ar), 7.43 (d, J = 7.9 Hz, 1H, Ar), 7.29–7.21 (m,
H, Ar), 6.08–6.00 (m, 2H, Ar), 5.39 (d, J = 16.9 Hz, 1H, CH), 5.21 (d, J = 10.0 Hz, 1H, CH),
CDCl3),
2
3
δ
.95 (d, J = 6.9 Hz, 2H, CH2). 13C{ H} NMR (75 MHz, CDCl ),
1
δ (ppm): 164.39, 151.93,
3
1
41.97, 132.18, 124.37, 123.99, 119.38, 119.83, 118.54, 109.96, 34.96. Anal. Cal. for C H NOS
10 9
(
191.25 g/mol): C, 62.80; H, 4.74; N, 7.32; S, 16.77. Found: C, 62.79; H, 4.68; N, 7.31; S, 16.72.
3.3. Biological Evaluation
The carcinogenic cell lines were supplied by the National Cancer Institute (USA).
Antiproliferative assays against different human cancer cell lines were determined using
the protein-binding dye sulphorhodamine B (SRB) in a microculture assay to measure cell
growth, as described in the protocols established by NCI1 [51]. Cell lines were cultured
in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine,
10,000 units/mL of sodium penicillin G, 10,000 lg/mL of streptomycin sulfate, 25 µg/mL
of amphotericin B (Gibco), and 1% non-essential amino acids (Gibco). They were kept
◦
at 37 C in a humidified atmosphere with 5% CO . The viability of the cells used in the
2
experiments exceeds 95%, as determined with trypan blue.
The cytotoxic activity of benzo [d] [1,3] azoles derivatives (BTA-1, BZM-2, BOX-3,
BTA-4, BZM-5, and BOX-6) against human cancer cell lines: glioblastoma (U-251), prostatic

Molecules 2021, 26, 2780
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adenocarcinoma (PC-3), chronic myelogenous leukemia (K-562), colorectal adenocarcinoma
(
HTC-15), lung (SKLU-1), mammary adenocarcinoma (MCF-7), and non-cancer gingival
fibroblast cells (FGH). The different carcinogenic cell lines were removed from tissue culture
flasks by trypsin treatment and diluted with fresh medium. From these cell suspensions,
1
00
µ
L containing 5000–10,000 cells per well were pipetted into 96-well microtiter plates
◦
(Costar), and the material was incubated at 37 C for 24 h in a 5% CO atmosphere.
2
Subsequently, 100
µL of a solution of each well was added the compound obtained by
diluting the reserves. The cultures were exposed for 48 h to the compound at 25
µM
concentrations. After the incubation period, cells were fixed to the plastic substrate by
◦
adding 50
µ
L of cold 50% acidic aqueous trichloroacetic. The plates were incubated at 4 C
for 1 h, washed with tap water, and air-dried. Cells fixed with trichloroacetic acid were
stained with the addition of 0.4% SRB. Free SRB solution was removed by washing with
1
% aqueous acetic acid. The plates were then air-dried, and the bound dye was solubilized
by the addition of tris without 10 mM base buffer (100 L). The plates were placed and
µ
shaken for 10 min, and the absorption was determined at 515 nm using an ELISA plate
reader (Bio-Tex Instruments).
3.4. Methodology and Computational Details
Electronic structure calculations were carried out by using the B3LYP density func-
tional. The initial structure of ligands was drawn with Avogadro Software [52]. For all
atoms, we used the Pople 6 – 31 + g basis set. All ligands were fully optimized in their
geometries, and all stationary points were characterized as minima through frequency
calculations. Solvent effects (water) were included with the SMD continuum method [53].
Atomic charges to be used in the molecular docking simulations were calculated with the
NPA scheme. All calculations were carried out with gaussian16 suite of programs [54].
To improve the Docking outcomes and reduce the effects of force fields and sys-
tem dependence, we used an exponential rack consensus (ECR) proposed by Palacio-
Rodriguez et al. [55]. The ECR of each molecule was described using a consensus score
P(i) corresponding to the sum of the exponential rank of j programs, by following the
following equation:
!
j
1
σ
exp −ri
P(i) =
(1)
∑
σ
j
where
σ
is the expected value of the exponential distribution respect to the number of
j
molecules i, and r is the rank of molecule i predicted with the program j.
i
We used this consensus for three molecular docking programs. AutoDock 4 (AD4) [56],
AutoDock Vina (Vina) [57], and Smina (vinardo scoring function) [58]. Current cancer
therapies seek to target the hormone receptors. The receptors considered in this work were
Er
ERT [59], 4OAR [60], 2J6M [61], and 4DRH [62]. These structures were used as a target in
previous computational works related with anticancer activity [63 65]. The receptors files
α, PR, EGFR, and mTOR. These tridimensional structures were taken from PDB codes
3
–
were prepared in the graphic interface Auto-Dock Tools 1.4.5 (ADT) [56] where the water
molecules were removed, and all hydrogens atoms were properly added, and nonpolar
hydrogen were merged to carbon atoms. The Gasteiger charges were added in the receptors
to prepare the pdbqt files. The charges of the ligands were taken from the NPA population
analysis based on density functional theory calculations. In all docking experiments, a grid
◦
3
◦
box of size 60
×
60
×
60 A in X, Y, and Z coordinates with a spacing of 0.375 A was used.
The ligand–receptor complexes were analyzed with LigPlot + [66] PyMol [67] and Maestro
Schrodinger programs [68].
4
. Conclusions
We have presented a small library of 2-substituted benzo[d] [1,3]azoles derivatives;
these species can be synthesized in a simple and good yield manner. Preliminary in vitro
cytotoxicity bioassays with the series of compounds BTA-1 BZM-2 BOX-3 BTA-4 BZM-
,
,
,
,

Molecules 2021, 26, 2780
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5, and BOX-6 has been complemented with in silico molecular docking studies, these
results suggesting that molecules containing the aromatic vinyl moieties are anchoring
better to ER and GPER than those including the allylic fragments. While the higher affinity
α
of BTA-1 and BZM-2 may be due to the atoms with a higher electron density that they
present (S and N), compared to BOX-3, which includes a harder, more electronegative O in
its structure. The major bioactivity in vitro of BZM-2 and its affinity for ERs may be due
to the additional interaction by hydrogen bonds that it establishes with the nitrogenous
unit -NH-. The latter compound BMZ-2 is especially interesting for future studies and
structural modifications, since it was not only the most active compound of the series, but
also the one with the lower activity against healthy cells. Hence, further studies may allow
us to shed further light in the design of other species with enhanced activities aimed to
develop new, specific, yet safer antineoplastic agents, related to the breast cancer estrogen
receptors (ERα and GPER).
Supplementary Materials: The following are available online, Figure S1: 1H NMR spectrum for
1
3
1
BTA-1; Figure S2: C{ H} NMR spectrum for BTA-1; Figure S3: Mass spectrometry (EI) for BTA-1
;
1
13
1
Figure S4: H NMR (300 MHz, CDCl ) spectrum for BZM-2; Figure S5: C{ H} NMR spectrum
3
+
1
for BZM-2; Figure S6: Mass spectrometry (DART ) for BZM-2; Figure S7: H NMR spectrum for
1
3
1
BOX-3; Figure S8: C{ H} NMR spectrum for BOX-3; Figure S9: Mass spectrometry (EI) for BOX-3
;
:
:
1
13
1
Figure S10: H NMR spectrum for BTA-4; Figure S11: C{ H} NMR spectrum for BTA-4
Mass spectrometry (DART ) for BTA-4; Figure S13: H NMR spectrum for BZM-5
C{ H} NMR spectrum for BZM-5; Figure S15: Mass spectrometry (DART ) for BZM-5; Figure S16:
H NMR spectrum for BOX-6; Figure S17: C{ H} NMR spectrum for BOX-6; Figure S18: Mass
;
Figure S12
+
1
; Figure S14
13
1
+
1
13
1
+
spectrometry (DART ) for BOX-6. Table S1. Docking results of receptor EGFR with crystalized
ligand as reference and tamoxifen. Table S2. Docking result of receptor Erα with crystalized ligand as
reference and tamoxifen. Table S3. Docking result of receptor Pr with crystalized ligand as reference
and tamoxifen. Table S4. Docking result of receptor mTOR with crystalized ligand as reference and
tamoxifen. Figure S19. Redocking of crystalized ligand in (A) EGFR receptor (B) Er
mTOR receptor (D) Pr Receptor.
α receptor (C)
Author Contributions: Conceptualization, A.A.-S.; methodology, M.T.R.-A., O.L.-A., J.C.-B., D.M.-M.
and A.A.-S.; synthesis, O.L.-A. and A.A.-S.; software and in silico analysis, J.C.-B., L.Á.G.-R.,
A.L.O., and J.A.-T.; validation, J.C.-B., D.M.-M. and A.A.-S.; formal analysis, J.C.-B., D.M.-M. and
A.A.-S.; investigation, F.D.-C. and D.S.-M.; resources, A.A.-S., J.C.-B. and D.M.-M.; writing—original
draft preparation, A.A.-S.; writing—review and editing, A.A.-S., J.C.-B. and D.M.-M.; supervision,
A.A.-S.; project administration, A.A.-S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by CONACYT 62267, SIP-IPN (grants 2018500, 20190027,
20200028). D.M.-M. would like to thank PAPIIT-DGAPA-UNAM (PAPIIT IN210520) and CONA-
CYT A1-S-33933 and FORDECYT-PRONACES FON.INST 22/2020 (FOINS 307152) for generous
financial support.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.
Acknowledgments: O.L.-A and L.Á.G.-R. would like to thank Programa de Beca de Estímulo
Institucional de Formación de Investigadores (BEIFI-IPN), (Oficios: SIP/DI/DAI/PIFI/42-0697/2017;
SIP/DI/DAI/PIFI/2-0500/2018).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.

Molecules 2021, 26, 2780
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