Design, synthesis, in vitro and in silico studies of some novel triazoles as anticancer agents for breast cancer

Article abstract of DOI:10.1016/j.molstruc.2021.131198

Against the increasing incidence of breast cancer in postmenopausal women in recent years, a few clinically approved inhibitors and their side-effect profiles indicate the need for the development of new aromatase inhibitors. In this study, carried out to develop a new aromatase inhibitor, the triazole ring system was preferred because of its known activity in the field. The triazole ring, which is in the structure of the most commonly used aromatase inhibitors such as anastrazole and letrazole, was synthesized from the thiourea residue. Inhibitor structures were elucidated using the ¹H-NMR, ¹³C-NMR, 2D-NMR, and HRMS spectroscopic methods. A cytotoxicity (MTT) test was performed to determine the anticancer activity of the compounds on breast (MCF7) carcinoma cell type. In addition, to determine selectivity of their action, the final compounds were screened against a healthy NIH3T3 cell line (mouse embryonic fibroblast cells). In terms of the MTT assay, it was observed that the calculated IC₅₀ values of compound 5e for the NIH3T3 cell line were found to be higher than for the MCF7 cell lines. Considering the viability results, it was found that the selected compound 5e showed a favorable safety profile and that it has anticancer activities. It was determined by in vitro studies that compound 5e showed inhibition potential on the aromatase enzyme with an IC₅₀ = 0.028 μM value. The docking study of compound 5e revealed that there is a strong interaction between the active sites of the human aromatase enzyme and the analyzed compound.

Full text of DOI:10.1016/j.molstruc.2021.131198

Journal of Molecular Structure 1246 (2021) 131198
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Design, synthesis, in vitro and in silico studies of some novel triazoles
as anticancer agents for breast cancer
Derya Osmaniyea,b,∗, Begüm Nurpelin Sag˘lıka,b, Serkan Leventa,b, Sinem Ilgınc,
Yusuf Özkaya,b, Zafer Asım Kaplancıklıa
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir, Turkey
b Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir, Turkey
c Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Against the increasing incidence of breast cancer in postmenopausal women in recent years, a few clin-
ically approved inhibitors and their side-effect profiles indicate the need for the development of new
aromatase inhibitors. In this study, carried out to develop a new aromatase inhibitor, the triazole ring
system was preferred because of its known activity in the field. The triazole ring, which is in the struc-
ture of the most commonly used aromatase inhibitors such as anastrazole and letrazole, was synthesized
from the thiourea residue. Inhibitor structures were elucidated using the 1H-NMR, 13 C-NMR, 2D-NMR,
and HRMS spectroscopic methods. A cytotoxicity (MTT) test was performed to determine the anticancer
activity of the compounds on breast (MCF7) carcinoma cell type. In addition, to determine selectivity of
their action, the final compounds were screened against a healthy NIH3T3 cell line (mouse embryonic
fibroblast cells). In terms of the MTT assay, it was observed that the calculated IC50 values of compound
5e for the NIH3T3 cell line were found to be higher than for the MCF7 cell lines. Considering the viability
results, it was found that the selected compound 5e showed a favorable safety profile and that it has an-
ticancer activities. It was determined by in vitro studies that compound 5e showed inhibition potential on
the aromatase enzyme with an IC50 = 0.028 μM value. The docking study of compound 5e revealed that
there is a strong interaction between the active sites of the human aromatase enzyme and the analyzed
compound.
Received 21 May 2021
Revised 13 July 2021
Accepted 26 July 2021
Available online 28 July 2021
Keywords:
Triazole
Anticancer
Aromatase
2D-NMR
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
ways: Tamoxifen, which inhibits estrogen receptors or aromatase
inhibitors (AI), which inhibit estrogen biosynthesis [1].
It is known that an increasing number of patients all over the
world are affected by breast cancer. GLOBOCAN 2012, published
by the International Agency for Research on Cancer (AIRC), stated
that breast cancer is the most common type of cancer affecting
women worldwide, both in terms of new cases and deaths [1].
The World Health Organization (WHO) reported that breast can-
cer caused more than half a million deaths in 2015 [2]. In addi-
tion, it has been reported that approximately 70-80% of the causes
of breast tumors in postmenopausal women are estrogen or pro-
gesterone dependent [3]. Pharmaceutical treatment of breast can-
cer, including chemotherapy, Her2 antibody therapy, and endocrine
therapy, are currently used clinically. Endocrine therapy, based on
the ability to prevent the effect of estrogen, can be applied in 2
The aromatase enzyme, which contains heme (hemoglobine),
is a microsomal enzyme complex responsible for the conversion
of androgens to estrogens in peripheral tissues by aromatization
of the steroid A ring [3–6]. Aromatase inhibitors, categorized as
steroidal and non-steroidal blockers, have a significant role in the
treatment of breast cancer in women and gynecomastia in men
[7,8].
Steroidal-type agents (Type I), sometimes called “suicide in-
hibitors”, are androstenedione analogs that act by binding to the
active site of aromatase through a competitive or irreversible pro-
cess, based on a mechanism or through covalent interactions.
Type II inhibitors or nonsteroidal inhibitors contain an azole
structure and bind reversibly to the cytochrome P450 portion of
the aromatase molecule, thus causing a reversible inhibition. The
first generation of nonsteroidal aromatase inhibitors is aminog-
lutethimide. Problems associated with aminoglutethimide side ef-
fects and selectivity have led to the development of a second gen-
∗
Corresponding author at: Anadolu University, Faculty of Pharmacy, Department
of Pharmaceutical Chemistry, 26470 Eskis¸ ehir, Turkey.
E-mail address: dosmaniye@anadolu.edu.tr (D. Osmaniye).
https://doi.org/10.1016/j.molstruc.2021.131198
0022-2860/© 2021 Elsevier B.V. All rights reserved.

D. Osmaniye, B.N. Sag˘lık, S. Levent et al.
Journal of Molecular Structure 1246 (2021) 131198
Scheme 1. Synthesis pathway for obtained compounds.
eration nonsteroidal aromatase inhibitor (fadrazol-bearing imida-
zole structure). However, this compound still exhibits some non-
selective inhibitory activity regarding progesterone, corticosterone,
and aldosterone biosynthesis [9,10]. Among the third generation
type II aromatase inhibitors, anastrozole, letrozole, and vorozol are
the strongest, most selective, and least toxic AIs. It is known that
they can reduce serum estradiol by more than 95% [11,12].
the steroid ring is the bulky aryl part [11]. Moreover, imidazole or
triazole-containing compounds have been reported as novel aro-
matase inhibitors [11,14–28].
In addition to all these, in many studies conducted in recent
years, new compounds containing triazole have been synthesized
and their anticancer profiles have been examined. As a result of
activity studies, activity profiles ranging from 9.02 nM to 1.9 μM
were obtained. It was noted that letrazole was used as the precur-
sor compound in the design of its compounds and that there were
2 aromatic structures attached to the triazole ring [15,20,26,28].
Five-year long-term therapy that may intensify side effects
(bone loss, joint pain, and cardiac events) and acquired resistance
are among the reasons limiting the clinical use of third AIs [29].
Thus, the development of new non-steroidal AIs is a popular area
for researchers for purposes such as limiting side effects and in-
While non-steroidal aromatase inhibitors (NSAIs) do not exhibit
any fatal androgenic side effects, long-term use of SAIs can result
in decreased estrogen levels, which can result in infertility and os-
teoporosis, as well as cancer. [13]. The structure of nonsteroidal
aromatase inhibitors, which consists of 2 main parts, can be ex-
plained as follows: The part in which the cytochrome P450 of the
aromatase must interact with the heme-iron atom is the azole part
with a nitrogen atom; the other part of the substrate that mimics
2

D. Osmaniye, B.N. Sag˘lık, S. Levent et al.
Journal of Molecular Structure 1246 (2021) 131198
phenol derivative was first reacted with NaH so that the pheno-
lic OH could be converted into sodium salt, facilitating the pro-
gression of the reaction. Since NaH is a flammable substance with
water, solvents with 99% purity and above were used in this step.
In addition, the dryness of the glass materials used is of great
importance. Using microwaves, compared to normal reflux pro-
cedures, shortened the experiment time by 4 times. Secondly, 2-
(2,4-dichlorophenoxy)acetohydrazide (2) was synthesized using hy-
drazine hydrate. An excess of hydrazine hydrate was used in the
reaction. Thirdly, the resulting hydrazide (2) was reacted with
ethyl isothiocyanate to obtain 2-(2-(2,4-dichlorophenoxy)acetyl)-
N-ethylhydrazine-1-carbothioamide (3). The ring closure reaction
was then performed using 2-(2-(2,4-dichlorophenoxy)acetyl)-N-
ethylhydrazine-1-carbothioamide (3) and carbondisulfide in pres-
ence of sodium hydroxide. HCl was carefully added dropwise dur-
ing the finishing step. The pH was also constantly checked. It
should be noted that if excess HCl is added, it is removed from
obtaining the molecule in solid form. Finally, the resulting triazole
compound (4) and the appropriate 2-bromoacetophenone were re-
acted to synthesize the target compounds. The obtained products
were crystallized from ethanol using activated charcoal. In order to
completely remove the activated charcoal from the environment,
filtering was performed with 3 layers of filter paper. The structures
of the gained compounds were demonstrated by means of spec-
troscopic methods, namely 1H-NMR, 13C-NMR, and HRMS (Supple-
mentary Data).
Fig 1. Design of target compounds.
In the final compounds, the triazole-linked ethyl group was
recorded as 3H triplet at 1.33 ppm and as 2H quartet at 4.09 ppm
in 1H-NMR and 15.51 ppm and 30.31 ppm in 13C-NMR. Hydroxy
groups for compound 5e were observed as br.s. at 5.11 ppm in 1H-
NMR. Carbonyl carbons were recorded between 190.07 ppm and
194.90 ppm in 13C-NMR. All remaining protons and carbons were
observed at expected values. 2D-NMR studies, namely HSQC and
HMBC were performed for compound 5e (Fig. 1), which had the
most active compound of all ones obtained. Using HMBC data, it
was found that the triazole carbons coded 3, 5 were recorded at
151.38 ppm and 151.03 ppm, respectively. In light of the infor-
mation obtained from HMBC and HSQC, dichlorophenyl carbons
(1,2,3,4,5,6) were found to be 152.38 ppm, 125.85 ppm, 129.90
ppm, 122.86 ppm, 128.58 ppm, and 116.13 ppm, respectively. Using
the information obtained from HMBC and HSQC, dihydroxyphenyl
carbons coded (1,2,3,4,5,6) were found to be 122.85 ppm, 158.87
ppm, 112.94 ppm, 147.43 ppm, 123.60 ppm, and 115.04 ppm, re-
spectively. HRMS results were also well-suited with theoretical m/z
values.
Fig 2. The 3D interacting mode of compound 5e in the active region of human aro-
matase enzyme (PDB ID: 3EQM). The inhibitor, colored with maroon, the important
residues, colored with gray, and the HEME(hemoglobine), colored with green in the
active site of the enzyme are presented by tube model.
2.2. Cytotoxicity test
The MTT test, one of the most preferred cytotoxicity tests, is
based on the principle that metabolically active cells convert the
pale yellow MTT color to a spectrophotometrically measured blue
formazan salt [32]. The antitumor potential of the novel triazole
derivatives against the MCF-7 cell line at various concentrations
(1000 μM, 316 μM, 100 μM, 31.6 μM, 10 μM, 3.16 μM, 1 μM, 0.316
μM) was evaluated. In addition, the NIH3T3 cells were applied as
a healthy cell line reference. IC50 values and the selectivity indices
(SI) of compounds against the MCF7 cell line are shown in Table 1.
According to the obtained MTT test results, compounds 5b, 5c, and
creasing clinical efficacy [30,31]. For this purpose, the aromatase
activities of newly designed triazole compounds were investigated.
While designing the compounds, the third generation inhibitors
anastrazole and letrazole were chosen as precursors (Fig. 1). The
azole group is therefore provided with a triazole ring system, and
a bulky aryl part is placed on both sides of this ring system.
2. Result and discussion
5d showed activity with IC50 = 11.003
0.112 μM, 15.235
0.470
2.1. Chemistry
μM and 13.587 0.297 μM values against the MCF7 cell line, re-
spectively. The efficacy of these compounds on the MCF7 cell line
is very valuable, but the selectivity indices calculated according to
the MTT test result performed with the healthy cell line (NIH3T3)
are 3.443, 1.528, 10.007, respectively. On the other hand, compound
5e was the most active derivative in the series with IC50 = 7.501
The compounds 5a-5h were obtained as presented in Scheme 1.
Firstly, Ethyl 2-(2,4-dichlorophenoxy)acetate (1) was obtained by
initiating
a reaction between 2,4-dichlorophenol and ethyl 2-
chloroacetate using NaH using of microwave (mw) irradiation. The
3

D. Osmaniye, B.N. Sag˘lık, S. Levent et al.
Journal of Molecular Structure 1246 (2021) 131198
Fig 3. The 2D interacting mode of compound 5e in the active region of human aromatase enzyme (PDB ID: 3EQM).
Table 1
IC50 values (μM) of compounds (5a-5h) against MCF7 and
NIH3T3 cell lines.
Compounds
MCF7
NIH3T3
SI∗
5a
5b
5c
20.411 0.497
11.003 0.112
15.235 0.470
13.587 0.297
7.501 0.305
33.762 0.936
39.395 1.797
37.159 0.837
16.385 0.658
187.685 2.322
37.885 0.991
23.286 1.879
135.965 2.811
387.685 1.325
34.782 1.091
40.896 0.541
53.662 2.073
1141.688 5.268
9.195
3.443
1.528
10.007
51.684
1.030
1.038
1.444
69.678
5d
5e
5f
5g
5h
Dox
∗
Selectivity index (SI)=[IC50(NIH3T3)/IC50(MCF7)], n=4
Table 2
IC50 values (μM) of compound 5e and le-
Fig 4. The 3D interacting mode of letrazole in the active region of human aro-
matase enzyme (PDB ID: 3EQM). The inhibitor, colored with orange, the important
residues, colored with blue in the active site of the enzyme are presented by tube
model.
trazole.
Compounds
Aromatase inhibition
5e
0.028 0002
0.024 0.001
Letrazole
0.305 μM. In addition, compound 5e resulted in a safe profile with
a selectivity index value of 51.684.
letrazole showed activity with a value of 0.024 μM. When com-
pared with the reference drug letrazole, it was observed that com-
pound 5e has a similar level of aromatase inhibitor activity with
letrazole.
2.3. Aromatase inhibition assay
The in vitro aromatase inhibition activity of the most active
compound, compound 5e, the results of which are presented in
Table 2, was assessed by the commercial fluorometric assay kit
(Aromatase-CYP19A Inhibitor Screening kit, Bio Vision). Letrozole
was used as the reference drug. According to the activity result,
compound 5e shows activity with an IC50 value of 0.028 μM, while
2.4. Molecular docking
Molecular docking studies were achieved to find the binding
modes of compound 5e on the human aromatase enzyme active
site. Therefore, the crystal structure of the human aromatase en-
4

D. Osmaniye, B.N. Sag˘lık, S. Levent et al.
Journal of Molecular Structure 1246 (2021) 131198
Fig 5. The 2D interacting mode of letrazole in the active region of human aromatase enzyme (PDB ID: 3EQM).
zyme (PDB ID: 3EQM) [33] was recovered from the Protein Data
Bank server (www.pdb.org). The 2D and 3D docking poses of com-
pound 5e can be seen in Figs 2–3. The 2D and 3D docking poses
of letrazole are shown in Figs. 4–5.
pounds, the dihydroxy phenyl ring in compound 5e creates the π-
π interaction itself, while one of the OH groups interacted with the
hemoglobine molecule, which is very important for the aromatase
enzyme active site.
As seen in the docking poses, compound 5e properly settled
down in the enzyme active site. According to the molecular dock-
ing results, this compound displayed many interactions. The first
of these belonged to triazole. This ring formed a π-π interaction
with the phenyl of Phe134. The other interaction belonged to the
dihydroxyphenyl ring. There was a π-π interaction between the
phenyl and the indole of Trp224. When the 3D pose of compound
5e (Fig. 2) is examined, it can be observed that the Cl atom in
the 2nd position of the phenyl forms a halogen bond with the hy-
droxy group of the Leu372. Another interaction took place between
the hydroxy group in the 2nd position of the phenyl ring and the
hemoglobine in the enzyme active site. A salt bridge formed be-
tween these 2 structures.
3. Conclusion
Aromatase inhibitors are frequently used in hormone-
dependent breast cancer cases, especially in postmenopausal
women. The most distinctive feature of this group is that they
contain azole groups in their structures and that the azole group
is bound to 2 aromatic structures. For this purpose, the aromatase
activities of newly designed triazole compounds were investigated.
Compound 5e showed activity against the MCF7 cell line with
an IC50 = 7.501
0.305 μM. In addition, this compound showed
similar efficacy to letrazole by inhibiting the aromatase enzyme
with a value of IC50 = 0.028 μM. When compound 5e was ana-
lyzed in terms of its chemical properties, it was observed that it
had a 2,4-dihydroxyphenyl ring that was different from the other
derivatives in the series. This is thought to have been caused by
the fact that the substituent of the hydroxy group of the phenyl
ring activated the compounds biologically. The superiority of this
compound could be explained by the molecular modeling studies.
According to the results of docking studies, compound 5e bounded
to the active site by forming additional interaction (salt bridge)
via its substituent of the hydroxy atom at the 2nd position with
hemoglobine. In addition, the arrangement that occurred in the
conformation of the compound originating from this substituent
enabled the chlorine atom of the compound to form a halogen
bond.
As seen in the docking poses, letrozole is settled down in
the enzyme active site properly. It interacted with the active site
in a similar position to compound 5e. For letrozole, it is seen
that benzonitrile ring makes π–π interaction with hemoglobin.
In compound 5e, this interaction was observed as a salt bridge.
Another π–π interaction is detected between 1,2,4-triazole and
Arg115. Likewise, this 1,2,4-triazole ring forms two hydrogen bonds
through its second and fourth nitrogen atoms with amino groups
of Ala438 and Arg115, respectively.
When looking at the results obtained, the triazole ring inter-
acted with the enzyme active site. In addition, the dichlorophenyl
ring contributed significantly to the activity by making a halogen
bond in the 2-Cl substituent in the structure. Unlike other com-
5

D. Osmaniye, B.N. Sag˘lık, S. Levent et al.
Journal of Molecular Structure 1246 (2021) 131198
When looking at the data obtained, the fact that the com-
pounds show anticancer activity in relation to breast cancer is ob-
servable. Compound 5e is very important for both its anticancer
activity and aromatase inhibitory property. In summary, structural
modifications can be further made on the basis of the new tria-
zoles to look for compounds with higher inhibitory activity against
the human aromatase enzyme.
4.1.5. Synthesis of target compounds (5a-5h)
5-((2,4-Dichlorophenoxy)methyl)-4-ethyl-4H-1,2,4-triazole-
3-thiol (4) (0.485 gr, 1.6 mmol) and the appropriate 2-
bromoacetophenone (1.6 mmol) were reacted in the presence
of potassium carbonate in acetone (≥99.5%) at room temperature.
After completion of the reaction, the solvent was removed under
reduced pressure. The residue was washed with water to remove
potassium carbonate, then dried and recrystallized from EtOH.
4. Experimental
4.1.5.1. 2-((5-((2,4-Dichlorophenoxy)methyl)-4-ethyl-4H-1,2,4-triazol-
3-yl)thio)-1-phenylethan-1-one (5a). Yield: 85 %, M.P.: 167.8-169.2
°C. 1H-NMR (300 MHz, DMSO-d6): δ = 1.33 (3H, t, J=7.2 Hz,
-CH2CH3), 4.09 (2H, q, J=7.2 Hz, -CH2CH3), 5.00 (2H, s, -CH2-),
5.41 (2H, s, -CH2-), 7.42-7.43 (2H, m, 1,2,4-Trisubstituebenzene),
7.56 (2H, t, J=7.5 Hz, Monosubstituebenzene), 7.61 (1H, dd, J1=0.8
Hz, J2=1.9 Hz, 1,2,4-Trisubstituebenzene), 7.69 (1H, t, J=7.4 Hz,
Monosubstituebenzene), 8.03 (2H, d, J=7.2 Hz, Monosubstitueben-
zene). 13C-NMR (75 MHz, DMSO-d6): δ = 15.51, 33.03, 40.99,
61.19, 116.14, 122.83, 125.87, 128.59, 129.30, 129.31, 129.93, 134.25,
135.72, 151.17, 152.36, 193.14. HRMS (m/z): [M+H]+ calcd for
C19 H17 N3O2SCl2: 422.0491; found: 422.0489.
4.1. Chemistry
All reagents purchased from commercial suppliers were used
without additional purification. (M.p.), and the melting points de-
termined on the Mettler Toledo-MP90 Melting Point System are
uncorrected. For 1H-NMR (nuclear magnetic resonance), a Bruker
DPX 300 FT-NMR spectrometer was used and for 13C-NMR, a
Bruker DPX 75 MHz spectrometer (Bruker Bioscience, Billerica, MA,
USA). The coupling constants (J) were expressed in Hertz (Hz).
Mass spectra were recorded on an LCMS-IT-TOF (Shimadzu, Kyoto,
Japan) using the ESI method.
4.1.5.2. 1-(4-Chlorophenyl)-2-((5-((2,4-dichlorophenoxy)methyl)-
4-ethyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one (5b). Yield: 79 %,
M.P.: 199.4-201.3 °C. 1H-NMR (300 MHz, DMSO-d6): δ = 1.33
(3H, t, J=7.2 Hz, -CH2CH3), 4.09 (2H, q, J=7.2 Hz, -CH2CH3),
4.98 (2H, s, -CH2-), 5.41 (2H, s, -CH2-), 7.42-7.43 (2H, m, 1,2,4-
Trisubstituebenzene), 7.61 (1H, br.s., 1,2,4-Trisubstituebenzene),
7.63 (2H, d, J=8.7 Hz, 1,4-Disubstituebenzene), 8.04 (2H, d,
J=8.6 Hz, 1,4-Disubstituebenzene). 13C-NMR (75 MHz, DMSO-d6):
δ = 15.50, 30.45, 40.85, 61.18, 116.14, 122.83, 125.88, 128.59,
129.42, 129.93, 130.81, 134.45, 139.15, 150.73, 151.21, 152.36,
192.71. HRMS (m/z): [M+H]+ calcd for C19 H16 N3O2SCl3: 456.0102;
found: 456.0096.
4.1.1. Synthesis of ethyl 2-(2,4-dichlorophenoxy)acetate (1)
2,4-Dichlorophenol (4.859 gr, 0.030 mol) was dissolved in ace-
tone (ACS reagent) (≥99.5%). Sodium hydride (0.864 gr, 0.036 mol)
was added in reaction mixture as 4 portions. Ethyl 2-chloroacetate
(3.69 mL, 0.036 mol) was added to the reaction medium; then,
the reaction was continued in the microwave reactor for 20 m. At
the end of the reaction, the solvent was removed. The filtrate was
washed with water, dried, and recrystallized from EtOH. The prod-
uct was obtained in 80% yield.
4.1.2. Synthesis of 2-(2,4-dichlorophenoxy)acetohydrazide (2)
Ethyl 2-(2,4-dichlorophenoxy)acetate (1) (5.952 gr, 0.024 mol)
was dissolved in ethanol (absolute) (50 mL). A solution of hy-
drazine hydrate in ethanol was added dropwise to the reaction
medium. After the dropping was completed, the reaction was
stirred for 5 h at room temperature. The precipitated product was
filtered. The hydrazide (2) was washed 3 times with cold ethanol
(95%) to remove the excess of hydrazine hydrate. The product was
obtained in 75% yield.
4.1.5.3. 2-((5-((2,4-Dichlorophenoxy)methyl)-4-ethyl-4H-1,2,4-triazol-
3-yl)thio)-1-(4-fluorophenyl)ethan-1-one (5c). Yield: 82 %, M.P.:
191.7-192.9 °C. 1H-NMR (300 MHz, DMSO-d6): δ = 1.33 (3H,
t, J=7.2 Hz, -CH2CH3), 4.09 (2H, q, J=7.2 Hz, -CH2CH3), 4.99
(2H, s, -CH2-), 5.41 (2H, s, -CH2-), 7.38 (2H, d, J=8.8 Hz, 1,4-
Disubstituebenzene), 7.42-7.43 (2H, m, 1,2,4-Trisubstituebenzene),
7.61 (1H, dd, J1=0.8 Hz, J2=1.9 Hz, 1,2,4-Trisubstituebenzene), 8.11
(2H, dd, J1=4.8 Hz, J2=8.8 Hz, 1,4-Disubstituebenzene). 13C-NMR
(75 MHz, DMSO-d6): δ = 15.51, 29.80, 40.86, 61.18, 116.14, 116.36
(d, J=21.9 Hz), 122.83, 125.88, 128.59, 129.92, 131.98 (d, J=9.6 Hz),
132.52 (d, J=2.5 Hz), 150.79, 151.19, 152.36, 165.77 (d, J=252.2
Hz), 192.24. HRMS (m/z): [M+H]+ calcd for C19 H16 N3O2FSCl2:
440.0397; found: 440.0389.
4.1.3. Synthesis of
2-(2-(2,4-dichlorophenoxy)acetyl)-N-ethylhydrazine-1-carbothioamide
(3)
2-(2,4-Dichlorophenoxy)acetohydrazide (2) (4.914 gr, 0.021 mol)
and isothiocyanatoethane (1.827 gr, 0.021 mol) were dissolved in
ethanol (absolute) (40 mL). The reaction mixture was refluxed for
10 h. After completion of the reaction, the mixture was cooled and
the precipitated compound was filtered and recrystallized from
ethanol. The product was obtained in 83% yield.
4.1.5.4. 1-(4-Bromophenyl)-2-((5-((2,4-dichlorophenoxy)methyl)-
4-ethyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one
%, M.P.: 207.9-209.0 °C. 1H-NMR (300 MHz, DMSO-d6):
1.33 (3H, t, J=7.2 Hz, -CH2CH3), 4.09 (2H, q, J=7.2
(5d). Yield:
88
δ
=
Hz, -CH2CH3), 4.98 (2H, s, -CH2-), 5.41 (2H, s, -CH2-
), 7.42-7.43 (2H, m, 1,2,4-Trisubstituebenzene), 7.61 (1H,
d, J=1.7 Hz, 1,2,4-Trisubstituebenzene), 7.77 (2H, d, J=8.5
Hz, 1,4-Disubstituebenzene), 7.96 (2H, d, J=8.6 Hz, 1,4-
Disubstituebenzene). 13C-NMR (75 MHz, DMSO-d6): δ = 15.51,
30.31, 40.83, 61.18, 116.13, 122.83, 125.88, 128.38, 128.58, 129.93,
130.88, 132.37, 134.77, 150.73, 151.21, 152.36, 192.93. HRMS (m/z):
[M+H]+ calcd for C19 H16 N3O2SCl2Br: 499.9596; found: 499.9583.
4.1.4. Synthesis of
5-((2,4-dichlorophenoxy)methyl)-4-ethyl-4H-1,2,4-triazole-3-thiol (4)
2-(2-(2,4-dichlorophenoxy)acetyl)-N-ethylhydrazine-1-
carbothioamide (3) (5.778 gr, 0.018 mol) was dissolved in ethanol
(absolute) (40 mL). Sodium hydroxide solution (0.840 gr, 0.021
mol) in ethanol and carbon disulfide (1.270 mL, 0.021 mol) were
added to the reaction vial. The reaction mixture was refluxed for
5 h. After completion of the reaction, the mixture was poured in
ice water. The reaction medium is acidified with 20% HCl to pH 2.
The precipitated product was filtered, then dried and recrystallized
from EtOH. The product was obtained in 80% yield.
4.1.5.5. 2-((5-((2,4-Dichlorophenoxy)methyl)-4-ethyl-4H-1,2,4-triazol-
3-yl)thio)-1-(2,4-dihydroxyphenyl)ethan-1-one (5e). Yield: 77 %,
M.P.: 203.7-205.8 °C. 1H-NMR (300 MHz, DMSO-d6): δ = 1.33
(3H, t, J=7.2 Hz, -CH2CH3), 4.09 (2H, q, J=7.2 Hz, -CH2CH3),
6

D. Osmaniye, B.N. Sag˘lık, S. Levent et al.
Journal of Molecular Structure 1246 (2021) 131198
4.79 (2H, s, -CH2-), 5.11 (2H, br.s., -OH), 5.41 (2H, s, -CH2-),
6.56 (1H, d, J=8.3 Hz, 1,2,4-Trisubstituebenzene), 7.22 (1H, d,
J=1.6 Hz, 1,2,4-Trisubstituebenzene), 7.34 (1H, d, J=10.0 Hz, 1,2,4-
Trisubstituebenzene), 7.42 (2H, br.s., 1,2,4-Trisubstituebenzene),
7.61 (1H, br.s., 1,2,4-Trisubstituebenzene). 13C-NMR (75 MHz,
DMSO-d6): δ = 15.55, 39.40, 40.95, 61.24, 112.90, 115.04, 116.13,
122.84, 122.85, 123.60, 125.85, 128.58, 129.90, 147.43, 151.03,
151.38, 152.38, 158.87, 190.06. HRMS (m/z): [M+H]+ calcd for
C19 H17 N3O4SCl2: 454.0390; found: 454.0378.
4.3. Aromatase inhibition assay
In vitro aromatase inhibition tests were performed using a
kit procedure (Bio Vision, Aromatase (CYP19A) Inhibitor Screening
Kit (Fluorometric)), in accordance with the method previously re-
ported by our study group [39,40]. The compounds were dissolved
in dimethyl sulfoxide (DMSO) and added to the assay in at least 7
concentrations ranging from 10−3-10−9 M. Letrozole was used as a
positive inhibition control.
4.4. Molecular docking
4.1.5.6. 2-((5-((2,4-Dichlorophenoxy)methyl)-4-ethyl-4H-1,2,4-
triazol-3-yl)thio)-1-(2,4-dichlorophenyl)ethan-1-one
80 %, M.P.: 175.7-177.7 °C. 1H-NMR (300 MHz, DMSO-d6):
1.33 (3H, t, J=7.2 Hz, -CH2CH3), 4.09 (2H, q, J=7.2
(5f). Yield:
In this study, the structure based in the silico docking method,
including protein-ligand interaction analysis, was applied to deter-
mine the binding and interaction points of compound 5e to the
human aromatase enzyme active site. The crystal structure of hu-
man aromatase (PDB ID: 3EQM) [33] was regained from the Protein
Data Bank server (www.pdb.org).
δ
=
Hz, -CH2CH3), 4.82 (2H, s, -CH2-), 5.41 (2H, s, -CH2-),
7.42-7.43 (2H, m, 1,2,4-Trisubstituebenzene), 7.56 (1H, dd,
J1=2.0 Hz, J2=8.4 Hz, 1,2,4-Trisubstituebenzene), 7.61 (1H,
d, J=1.9 Hz, 1,2,4-Trisubstituebenzene), 7.76 (1H, d, J=2.0
Hz, 1,2,4-Trisubstituebenzene), 7.83 (1H, d, J=8.4 Hz, 1,2,4-
Trisubstituebenzene). 13C-NMR (75 MHz, DMSO-d6): δ = 15.37,
32.31, 42.72, 61.12, 116.12, 122.82, 125.88, 128.01, 128.59, 129.93,
130.54, 131.90, 132.03, 135.77, 137.24, 150.50, 151.28, 152.33,
194.90. HRMS (m/z): [M+H]+ calcd for C19 H15N3O2SCl4: 489.9712;
found: 489.9697.
The structures of the ligands were constructed by means of the
Schrödinger Maestro [41] interface and were then acquiesced to the
Protein Preparation Wizard protocol of the Schrödinger Suite 2016
Update 2 [42]. The ligands were set by LigPrep 3.8 [43] to assign
the protonation states at pH 7.4 1.0 and the atom types appropri-
ately, as well. Bond instructions were assigned and hydrogen atoms
were added to the structures. The grid generation was designed us-
ing Glide 7.1 [44]. Flexible docking runs were achieved with single
precision docking mode (SP).
4.1.5.7. 2-((5-((2,4-Dichlorophenoxy)methyl)-4-ethyl-4H-1,2,4-triazol-
3-yl)thio)-1-(2,4-difluorophenyl)ethan-1-one (5g). Yield: 82 %,
M.P.: 192.7-194.6 °C. 1H-NMR (300 MHz, DMSO-d6): δ = 1.33
(3H, t, J=7.2 Hz, -CH2CH3), 4.09 (2H, q, J=7.2 Hz, -CH2CH3),
4.87 (2H, d, J=2.5 Hz, -CH2-), 5.41 (2H, s, -CH2-), 7.27 (1H, td,
J1=2.2 Hz, J2=8.4 Hz, 1,2,4-Trisubstituebenzene), 7.42-7.43 (2H,
m, 1,2,4-Trisubstituebenzene), 7.48 (1H, td, J1=2.5 Hz, J2=9.4
Hz, 1,2,4-Trisubstituebenzene), 7.61 (1H, d, J=1.3 Hz, 1,2,4-
Trisubstituebenzene), 7.97 (1H, td, J1=6.7 Hz, J2=8.7 Hz, 1,2,4-
Trisubstituebenzene). 13C-NMR (75 MHz, DMSO-d6): δ = 15.46,
30.88, 44.01 (d, J=7.9 Hz), 61.17, 105.79 (t, J=26.8 Hz), 112.99,
113.15, 116.14, 122.82, 125.88, 128.59, 129.92, 133.31 (q, J=5.1 Hz),
150.71, 151.22, 152.35, 165.76 (d, J=252.8 Hz), 165.94 (d, J=251.8
Hz), 190.13. HRMS (m/z): [M+H]+ calcd for C19 H15N3O2F2SCl2:
458.0303; found: 458.0290.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.131198.
CRediT authorship contribution statement
Derya Osmaniye: Conceptualization, Methodology, Software,
Formal analysis, Investigation, Writing – original draft, Writing –
review & editing, Visualization. Begüm Nurpelin Sag˘lık: Method-
ology. Serkan Levent: Formal analysis. Sinem Ilgın: Methodology.
Yusuf Özkay: Supervision, Conceptualization, Investigation, Visu-
alization. Zafer Asım Kaplancıklı: Supervision, Conceptualization,
Investigation, Writing – original draft, Writing – review & editing.
4.1.5.8. 2-((5-((2,4-Dichlorophenoxy)methyl)-4-ethyl-4H-1,2,4-
triazol-3-yl)thio)-1-(3,4-dichlorophenyl)ethan-1-one
76 %, M.P.: 165.8-166.9 °C. 1H-NMR (300 MHz, DMSO-
d6):
1.33 (3H, t, J=7.2 Hz, -CH2CH3), 4.09 (2H, q,
(5h). Yield:
δ
=
J=7.2 Hz, -CH2CH3), 4.99 (2H, s, -CH2-), 5.41 (2H, s, -CH2-
), 7.42-7.43 (2H, m, 1,2,4-Trisubstituebenzene), 7.61 (1H, d,
J=1.7 Hz, 1,2,4-Trisubstituebenzene), 7.84 (1H, d, J=8.4 Hz,
1,2,4-Trisubstituebenzene), 7.97 (1H, dd, J1=2.0 Hz, J2=8.4
Hz, 1,2,4-Trisubstituebenzene), 8.25 (1H, d, J=2.0 Hz, 1,2,4-
Trisubstituebenzene). 13C-NMR (75 MHz, DMSO-d6): δ = 15.51,
30.49, 40.79, 61.17, 116.13, 122.82, 125.88, 128.59, 128.89, 129.92,
130.85, 131.65, 132.34, 135.94, 137.01, 150.58, 151.25, 152.35,
192.08. HRMS (m/z): [M+H]+ calcd for C19 H15N3O2SCl4: 489.9712;
found: 489.9692.
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8