Synthesis, docking studies and biological activity of new benzimidazole- Triazolothiadiazine derivatives as aromatase inhibitor

Article abstract of DOI:10.3390/molecules25071642

In the last step of estrogen biosynthesis, aromatase enzyme catalyzes the conversion of androgens to estrogens. Aromatase inhibition is an important way to control estrogen-related diseases and estrogen levels. In this study, sixteen of benzimidazole-triazolothiadiazine derivatives have been synthesized and studied as potent aromatase inhibitors. First, these compounds were tested for their anti-cancer properties against human breast cancer cell line (MCF-7). The most active compounds 5c, 5e, 5k, and 5m on MCF-7 cell line were subject to further in vitro aromatase enzyme inhibition assays to determine the possible mechanisms of action underlying their activity. Compound 5e showed slight less potent aromatase inhibitory activity than that of letrozole with IC₅₀ = 0.032 ± 0.042 μM, compared to IC₅₀ = 0.024 ± 0.001 μM for letrozole. Furthermore, compound 5e and reference drug letrozole were docked into human placental aromatase enzyme to predict their possible binding modes with the enzyme. Finally, ADME parameters (absorption, distribution, metabolism, and excretion) of synthesized compounds (5a–5p) were calculated by QikProp 4.8 software.

Full text of DOI:10.3390/molecules25071642

molecules
Article
Synthesis, Docking Studies and Biological Activity of
New Benzimidazole- Triazolothiadiazine Derivatives
as Aromatase Inhibitor
Ulviye Acar Çevik 1,2, Betül Kaya Çavus¸og˘lu 3, Begüm Nurpelin Sag˘lık 1,2, Derya Osmaniye 1,2
,
Serkan Levent 1,2 , Sinem Ilgın 4, Yusuf Özkay 1,2,
*
and Zafer Asım Kaplancıklı 1
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskisehir 26470,
Turkey; uacar@anadolu.edu.tr (U.A.Ç.); bnsaglik@anadolu.edu.tr (B.N.S.);
dosmaniye@anadolu.edu.tr (D.O.); serkanlevent@anadolu.edu.tr (S.L.); zakaplan@anadolu.edu.tr (Z.A.K.)
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University, Eskisehir
26470, Turkey
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Bülent Ecevit University, Zonguldak 67100,
Turkey; betulkaya@anadolu.edu.tr
2
3
4
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, Eskisehir 26470,
Turkey; silgin@anadolu.edu.tr
*
Correspondence: yozkay@anadolu.edu.tr; Tel.: +90-222-335-0580 (ext. 3603)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Filippo Minutolo
Received: 3 March 2020; Accepted: 1 April 2020; Published: 2 April 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: In the last step of estrogen biosynthesis, aromatase enzyme catalyzes the conversion of
androgens to estrogens. Aromatase inhibition is an important way to control estrogen-related diseases
and estrogen levels. In this study, sixteen of benzimidazole-triazolothiadiazine derivatives have been
synthesized and studied as potent aromatase inhibitors. First, these compounds were tested for their
anti-cancer properties against human breast cancer cell line (MCF-7). The most active compounds 5c
5e 5k, and 5m on MCF-7 cell line were subject to further in vitro aromatase enzyme inhibition assays
to determine the possible mechanisms of action underlying their activity. Compound 5e showed
slight less potent aromatase inhibitory activity than that of letrozole with IC50 = 0.032 0.042 M,
compared to IC50 = 0.024 0.001 M for letrozole. Furthermore, compound 5e and reference drug
,
,
±
µ
±
µ
letrozole were docked into human placental aromatase enzyme to predict their possible binding
modes with the enzyme. Finally, ADME parameters (absorption, distribution, metabolism, and
excretion) of synthesized compounds (5a–5p) were calculated by QikProp 4.8 software.
Keywords: aromatase; MCF-7; NIH3T3; benzimidazole; triazolothiadiazine; docking; ADME
1. Introduction
Aromatase is a member of the cytochrome P450 enzyme that catalyzes the biosynthesis of
estrogens from androgens [1,2]. Estrogen levels have been shown to be higher in breast cancer
cells. Inhibition of aromatase is one of the effective current therapeutic strategies for controlling
estrogen-dependent breast cancer. Therefore, one of the commonly used classes of drugs for the
management of estrogen-dependent cancer is aromatase inhibitors (AIs) [1,3,4].
Aromatase inhibitors that have been used clinically can be categorized as first-, second-, and third
generations based on their evolution time or steroidal or steroidal or non-steroidal aromatase inhibitors
(NSAIs) based on their structural similarity with steroids [5,6].
Aminoglutethimide is the prototype nonsteroidal inhibitor of aromatase. Problems with the
side effects and selectivity of aminoglutethimide led to the development of the second-generation
of nonsteroidal aromatase inhibitor (Fadrazole bearing imidazole structure) [7]. However, this
Molecules 2020, 25, 1642; doi:10.3390/molecules25071642
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1642
2 of 15
compound still has some nonselective inhibitory activity with respect to progesterone, corticosterone,
and aldosterone biosynthesis. Competitive nonsteroidal inhibitors can also be constructed with
a triazole ring, which is found in the third generation of aromatase inhibitors [
nonsteroidal aromatase inhibitors of therapeutic importance act covalently bind to the substrate-binding
site of aromatase by coordinate the heme iron [11 13]. Especially, the heterocyclic nitrogen atom of
triazole and imidazole plays an important role by coordinating with the heme iron of the aromatase
enzyme [12 13]. Some studies have shown that imidazole and triazole derivatives have promising
8–10]. Most of the
–
,
aromatase inhibition [14–17].
Recent clinical studies have shown that aromatase inhibitors, especially the third-generation, are
more effective than fulvestrant and tamoxifen because of their lower side effects and higher clinical
efficacy. Nevertheless, the development of acquired resistance after prolonged AIs therapy, undesirable
side effects (bone loss, cardiac events, increased rash, insomnia, headaches, and arthralgia) limits their
use in clinical practice. Thus, the search for new potent molecules that impair cancer growth, strongly
inhibit aromatase enzyme, and present fewer side effects is of major importance [18–21].
In the previous study, 3-[4-(5-methyl-1H-benzo[d]imidazol-2-yl)phenyl]-6- (substituted phenyl)
-7H-[1,2,4] triazolo [3,4-b] [1,3,4]thiadiazines derivatives have been synthesized and promising
compounds have been obtained that need further development as a new class of aromatase
inhibitors [22]. In this study, new benzimidazole-triazolothiadiazine derivatives were synthesized
and structure of these compounds was characterized by spectroscopic data. Their antiproliferative
activities against MCF-7 were evaluated. To identify the possible modes of action, aromatase inhibition
experiments were performed for the most active compounds against the MCF-7 cell line. Finally, in
silico prediction of pharmacokinetic profiles (ADME) were calculated for physicochemical properties
of these drug candidates.
2. Results and Discussion
2.1. Chemistry
Synthesis of the target compounds was accomplished according to the steps illustrated in Scheme 1.
Substituents of the synthesized compounds (5a
compound (1a 1b) was prepared by the reaction of 1,2-phenylenediamine and sodium metabisulfite
with 4-formylbenzoic acid methyl ester as described in a previous study [23]. The ester derivative
1a 1b) reacted with hydrazine hydrate producing compounds 2a 2b in a microwave synthesis reactor.
Then, the compounds-(5-substitüe-1H-benzimidazol-2-yl)benzoic acid hydrazide derivatives (2a 2b
were treated with carbon disulfide in NaOH solution affording, after acidic treatment, compounds
3a 3b. The reaction of compound 3a, 3b with hydrazine hydrate in the presence of ethanol produced
compounds 4a 4b. Then cyclization of compounds 4a 4b with appropriate phenacyl bromide in
the presence of anhydrous ethanol provided the desired final products (5a 5p). The characterization
–5p) were shown in Table 1. The starting intermediated
,
(
,
,
,
)
,
,
,
–
of these new derivatives was done by their spectroscopic (1H NMR, 13C NMR and Mass) data.
1H NMR and 13C NMR spectra of the synthesized compounds were observed at the expected region.
Mass spectra (HRMS) of all the synthesized compounds showed that the molecular ion [M + H]+ peak
is in agreement with their molecular formula (see Supplementary Materials).

Molecules 2020, 25, 1642
3 of 15
Scheme 1. The reaction sequence for the synthesis of the compounds (5a–5p).
Table 1. The synthesized compounds (5a–5p).
Comp.
R
R1
R2
5a
5b
5c
5d
5e
5f
5g
5h
5ı
5j
5k
5l
5m
5n
5o
5p
-H
-H
-H
-H
-H
-H
-H
-H
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-H
-Cl
-F
-CH3
-CN
-Br
-F
-Cl
-H
-Cl
-F
-H
-H
-H
-H
-H
-H
-F
-Cl
-H
-H
-H
-H
-H
-H
-F
-CH3
-CN
-Br
-F
-Cl
-Cl
2.2. Cytotoxicity Assay
The newly synthesized compounds were evaluated for their in vitro anticancer potential against
MCF-7 breast cancer cell lines by MTT assay [24] using cisplatin as a reference standard. The 50%
inhibitory concentration (IC50) values were determined for these compounds. In addition, the cytotoxic
activities of compounds 5c, 5e, 5k, and 5m were assessed against healthy NIH3T3 [25] cells, in order to

Molecules 2020, 25, 1642
4 of 15
express the selectivity toward carcinogenic cells. The IC50 values of test compounds were determined
as the mean IC50 of 4 independent experiments. Results are presented in Table 2, as IC50 values are
in
cisplatin against MCF-7 cell lines. Concerning MCF-7 cell line, the most potent compounds were the
4-cyano derivatives 5e and 5m with IC50 = 0.016 0.001 and 0.018 0.001 M, respectively compared
to IC50 = 0.020 0.009 M for the reference drug cisplatin. Furthermore, the most promising activity
was observed for the compounds 5c and 5k bearing 4-fluorophenyl derivatives with IC50= 0.119 0.005
and 0.110 0.005 M, respectively compared to IC50 = 0.020 0.009 M for the reference drug cisplatin.
µM and revealed that some of the tested compounds were remarkably more cytotoxic than the
±
±
µ
±
µ
±
±
µ
±
µ
Table 2. IC50 (µM) values of compounds (5a–5p) (n = 4).
Comp.
MCF-7
NIH3T3
5a
5b
5c
5d
5e
5f
5g
5h
5ı
5j
5k
5l
5m
5n
0.142 ± 0.007
0.0414 ± 0.001
0.119 ± 0.005
0.194 ± 0.006
0.016 ± 0.001
0.318 ± 0.019
0.245 ± 0.004
0.342 ± 0.017
0.205 ± 0.005
0.258 ± 0.011
0.110 ± 0.005
0.352 ± 0.019
0.018 ± 0.001
0.264 ± 0.011
0.373 ± 0.011
0.147 ± 0.001
0.020 ± 0.009
-
-
81.8 ± 2.4
-
≥1000
-
-
-
-
-
77.6 ± 2.1
-
≥1000
-
-
-
5o
5p
Cisplatin
≥1000
Benzimidazole-triazolothiadiazine derivatives led to a promising increase in the anti-proliferative
activity. The synthesized compounds can be divided into two groups as nonsubstituted benzimidazole
and 5-chlorobenzimidazole. Test compounds showed variable activities against MCF-7. In particular,
compounds 5e and 5m displayed the highest cytotoxic activity against MCF-7 cell line. The most
active compounds 5e and 5m carried a 4-cyanophenyl substituent. The chlorine substituent in the fifth
position (5m) of the benzimidazole ring did not significantly affect the activity. Compounds 5c and 5k
carrying fluorine substituents in the fourth position of the phenyl ring showed lower activity than
compounds 5e and 5m bearing cyano group in the fourth position of the phenyl ring.
2.3. Aromatase Inhibition Assay
The in vitro anti-aromatase activity of the most active compounds 5c, 5e, 5k, and 5m was valued
using commercial fluorometric assay kit (Aromatase-CYP19A Inhibitor Screening kit, Bio Vision) with
letrozole as the reference drug. Results are presented in Table 3. The IC50 values of compounds were in
the sub-micromolar range (2.276
5e with IC50 value (0.032 ± 0.001 µM).
±
0.106–0.03 2
±
0.001 µM). The best value was shown by compound
Table 3. IC50 (µM) values of compounds 5c, 5e, 5k, 5m, and letrozole.
Comp.
Aromatase Inhibition
5c
5e
5k
1.716 ± 0.042
0.032 ± 0.001
2.276 ± 0.106
1.562 ± 0.064
0.024 ± 0.001
5m
Letrozole

Molecules 2020, 25, 1642
5 of 15
2.4. Molecular Docking
After the most active derivative was selected as compound 5e according to in vitro aromatase
enzyme inhibition assay and letrozole, molecular docking studies were performed to explain its
binding modes with human aromatase enzyme active site. For this purpose, the crystal structure of
human aromatase enzyme (PDB ID: 3EQM) [26] was retrieved from the Protein Data Bank server
(www.pdb.org). The 2D and 3D docking poses of compound 5e are presented in Figures 1 and 2.
The docking poses of letrozole are presented in Figures 3 and 4.
According to docking poses, it is understood that compound 5e displays compatible settlement
with the enzyme active region. The benzimidazole ring in the structure forms two π–π interactions with
Arg115 and Phe134. Also, it can be seen from 2D docking pose, this compound is in interaction with
Hem molecules and Cys437 via its nitrogen atoms of triazolothiadiazine ring by salt bridge. There is
another π–π interaction between 4-cyanophenyl ring and HEM molecule. The nitrogen atom of cyano
group at C-4 position of phenyl ring interacts with hydroxyl of Ser314 doing hydrogen bonding. It is
thought that this interaction is important for compound 5e in terms of explaining its inhibitory activity.
It is seen that the presence of an electron withdrawing group such as cyano at this position is a positive
contribution to the activity.
As seen in the docking poses, letrozole is settled down in the enzyme active site properly.
For letrozole, it is seen that benzonitrile ring creates π–π interaction with HEM molecule. Another π–π
interaction is observed between 1,2,4-triazole ring and Arg115. Also, this 1,2,4-triazole ring forms
two hydrogen bonds via its second and fourth nitrogen atoms with amino groups of Ala438 and
Arg115, respectively.
Figure 1. Two-dimensional interaction mode of compound 5e in the enzyme active site (Human
aromatase PDB Code: 3EQM).

Molecules 2020, 25, 1642
6 of 15
Figure 2. The interacting mode of compound 5e in the active region of human aromatase. The inhibitor,
colored with maroon, and the important residues, colored with purple, in the active site of the enzyme
are presented by tube model.
Figure 3. Two-dimensional interaction mode of letrozole in the enzyme active site (Human aromatase
PDB Code: 3EQM).

Molecules 2020, 25, 1642
7 of 15
Figure 4. The interacting mode of letrozole in the active region of human aromatase. The inhibitor,
colored with orange, and the important residues, colored with turquoise, in the active site of the enzyme
are presented by tube model.
2.5. Theoretical Determination of ADME Properties
QikProp allows to provide acceptable ranges for comparing the predicted properties of compounds
with those of 95% of known drugs and estimate drug-likeness properties. The drug-likeness of a
compound was assessed according to Jorgensen’s rule of three [27], which regards PCaco (>22 nm/s),
logS (> 5.7), primary metabolites (PM) (<7), and Lipinski’s rule of five [28], which considers number
−
of hydrogen bond acceptors (
≤
10) and donors ( 5), molecular weight (<500 Da), and octanol/water
≤
partition coefficient (≤5).
Table 4. presents the predicted ADME properties of all compounds. According to Lipinski’s rule
of five and Jorgensen’s rule of three, all compounds (5a–5p) are in accordance with the rule by causing
no more than one violation. Consequently, according to predictions of ADME properties, it can be
suggested that the active compounds may have a good pharmacokinetic profile.

Molecules 2020, 25, 1642
8 of 15
Table 4. Calculated ADME parameters.
Comp.
MW
RB
DM
MV
DHB AHB
PSA
logP
logS
PCaco logBB PMDCK PM
CNS
%HOA VRF
VRT
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
408.48
442.925
426.47
422.506
433.489
487.376
444.461
477.37
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
13.539 1230.58
11.536 1274.691
11.294 1246.689
14.175 1289.517
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4.5
4.5
4.5
4.5
6
4.5
4.5
4.5
4.5
4.5
4.5
4.5
6
66.81
66.813
66.815
66.81
92.606
66.813
65.595
64.6
66.806
66.809
66.811
66.806
92.602
66.809
65.591
64.597
5.226
5.722
5.463
5.533
4.468
5.799
5.544
5.897
5.721
6.217
5.957
6.028
4.96
−7.577 1145.907 −0.361 1066.282
−8.323 1145.871 −0.207 2630.568
−7.944 1145.883 −0.255 1928.038
−8.145 1145.907 −0.388 1066.282
−8.534 236.976 −1.255 194.119
−8.439 1145.858 −0.198 2828.358
2
2
2
3
2
2
2
2
2
2
2
3
2
2
2
2
0
0
0
100
100
100
100
95.607
100
100
100
100
100
100
100
100
92.631
100
1
1
1
1
0
1
1
1
1
1
1
1
0
2
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
7.968
1297.278
−2
11.913 1283.596
12.598 1251.337
12.709 1287.743
12.609 1274.528
10.697 1318.639
10.455 1290.637
13.229 1333.465
0
−8.02
1167.271 −0.2
2431.401
0
0
0
0
0
0
−8.422 1199.257 −0.099 3927.141
−8.321 1145.207 −0.208 2625.13
−9.068 1145.171 −0.054 6476.318
−8.688 1145.183 −0.101 4746.726
−8.891 1145.207 −0.235 2625.13
−9.274 236.832 −1.117 477.911
−9.184 1145.158 −0.044 6963.267
−8.764 1166.535 −0.046 5985.589
442.925
477.37
460.915
456.951
467.934
521.821
478.906
511.815
7.409
11.046 1327.544
11.99 1295.266
12.056 1331.676
1341.226
−2
4.5
4.5
4.5
6.294
6.038
6.391
0
0
1
−9.166 1198.487 0.055
9667.877
93.553
MW: Molecular weight RB: Number of rotatable bonds (recommended value: 0-15) DM: Computed dipole moment (recommended value: 1-12.5) MV: Total solvent-accessible volume
(recommended value: 500-2000) DHB: Estimated number of hydrogen bond donors (recommended value: 0-6) AHB: Estimated number of hydrogen bond acceptors (recommended
value: 2-20) PSA: Van der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms (recommended value: 7-200) logP: Predicted octanol/water partition
coefficient (recommended value: -2-6.5) logS: Predicted aqueous solubility (recommended value: -6.5-0.5) PCaco: Predicted apparent Caco-2 cell permeability (recommended value:
<25 poor, >500 great) logBB: Predicted brain/blood partition coefficient (recommended value: -3-1.2) PMDCK: Predicted apparent MDCK cell permeability (recommended value: <25 poor,
>500 great) PM: Number of likely metabolic reactions (recommended value: 1-8) CNS: Predicted central nervous system activity (recommended value: –2 (inactive), +2 (active)) %HOA:
Predicted human oral absorption percent (recommended value: >80% is high, <25% is poor) VRF: Number of violations of Lipinski’s rule of five. The rules are: MW < 500, logP < 5,
DHB ≤ 5, AHB≤ 10, Positive PSA value. VRT: Number of violations of Jorgensen’s rule of three. The three rules are: logS > -5.7, PCaco > 22 nm/s, PM < 7.

Molecules 2020, 25, 1642
9 of 15
3. Materials and Methods
3.1. Chemistry
Whole chemicals employed in the synthetic procedure were purchased from Sigma-Aldrich
Chemicals (Sigma-Aldrich Corp., St. Louis, MO, USA) or Merck Chemicals (Merck KGaA, Darmstadt,
Germany). Melting points of the obtained compounds were determined by MP90 digital melting point
apparatus (Mettler Toledo, OH, USA) and were uncorrected. Microwave syntheses were realized by
1
using a Monowave 300 high-performance microwave reactor (Anton-Paar, Austria). H NMR and 13
C
NMR spectra of the synthesized compounds were registered by a Bruker 300 MHz and 75 MHz digital
FT-NMR spectrometer (Bruker Bioscience, Billerica, MA, USA) in DMSO-d6, respectively. Splitting
patterns were designated as follows: s: singlet; d: doublet; t: triplet; m: multiplet in the NMR spectra.
Coupling constants (J) were reported as Hertz. M+1 peaks were determined by Shimadzu LC/MS
ITTOF system (Shimadzu, Tokyo, Japan). All reactions were monitored by thin-layer chromatography
(TLC) using Silica Gel 60 F254 TLC plates (Merck KGaA, Darmstadt, Germany).
3.1.1. Synthesis of 4-(5-substitüe-1H-benz[d]imidazol-2-yl)benzoic Acid Methyl Ester (1a,1b)
Yields: 82–78%. 4-(5-substitüe-1H-benz[d]imidazol-2-yl)benzoic acid hydrazide derivatives (2a,2b)
(yields: 76–72%) and 5-[4-(5(6)-substituted-1H-benz[d]imidazol-2-yl)phenyl]-1,3,4-oxadiazole-2-thiol
derivatives (3a,3b) (yields: 78–7 %) were prepared following the reported procedures [23].
3.1.2. 4-Amino-5-(4-(5-substitüe-1H-benzo[d]imidazol-2-yl)phenyl)-4H-1,2,4-triazole-3-thiol (4a,4b)
Compound 3a or 3b (0.02 mol) was dissolved in ethanol. Hydrazine hydrate (5 mL) was added
to the mixture. The reaction mixture was heated at 240 ◦C and 10 bar for 10 min under microwave
synthesis reactor (Anton-Paar Monowave 300). After the reaction ended, the product was washed with
water, dried, and crystallized using ethanol (96%) [22]. Yields: 68–64%.
3.1.3. 3-(4-(5-substitüe-1H-benzo[d]imidazol-2-yl)phenyl)-6-(substitüephenyl)-7H-[1,2,4]triazole
[3,4-b][1,3,4]thiadiazine (5a–5p)
Compound 4a or 4b (0.003 mol) and appropriate phenacyl bromide (0.003 mol) in ethanol was
heated under reflux for 8 h. After the reaction ended, the product was filtered and dried [22].
3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-phenyl-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine (5a):
Yields: 77%. Mp 230.7–232.9 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ = 4.52 (2H, s, -CH2-), 7.52-7.55
(2H, m, aromatic CH), 7.59–7.67 (3H, m, Aromatic CH), 7.83–7.86 (2H, m, aromatic CH), 8.06–8.09
(2H, m, aromatic CH), 8.34 (2H, d, J=8.67 Hz, 1,4-disubstitutedbenzene), 8.43 (2H, d, J=8.64 Hz,
1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-d6):
δ = 23.31, 114.89, 125.84, 126.55, 128.13,
128.50, 129.13, 129.65, 132.64, 133.78, 134.17, 143.96, 149.21, 151.22, 156.82, 163.07. HRMS (m/z): [M + H]+
calcd for C23H16N6S: 409.1224; found: 409.1230.
3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-chlorophenyl)-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine
(
5b): Yields: 72%. Mp 190.9–192.6 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ = 4.50 (2H, s, -CH2-), 7.56–7.59
(2H, m, aromatic CH), 7.65–7.70 (3H, m, aromatic CH), 7.86–7.89 (2H, m, aromatic CH), 8.06–8.09
(2H, m, aromatic CH), 8.31 (2H, d, J=8.61 Hz, 1,4-disubstitutedbenzene), 8.57 (2H, d, J=8.61 Hz, 1,4-
Disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-d6):
δ=23.27, 114.67, 125.49, 126.35, 128.87, 129.11,
129.32, 129.45, 129.73, 129.92, 130.11, 130.71, 132.62, 133.01, 137.50, 144.01, 148.52, 151.17, 156.87. HRMS
(m/z): [M + H]+ calcd for C23H15N6SCl: 443.0843; found: 443.0840.
3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-fluorophenyl)-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine
(
5c): Yields: 69%. Mp 274.2–277.1 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ = 4.50 (2H, s, -CH2-), 7.12 (1H, s,
aromatic CH), 7.29 (1H, s, aromatic CH), 7.46–7.52 (4H, m, aromatic CH), 7.81–7.84 (2H, m, aromatic CH),
8.33 (2H, d, J = 8.64 Hz, 1,4-disubstitutedbenzene), 8.51 (2H, d, J=8.76 Hz, 1,4-disubstitutedbenzene).

Molecules 2020, 25, 1642
10 of 15
13C-NMR (75 MHz, DMSO-d6):
δ
= 23.27, 114.32, 114.90, 116.76 (d, J = 21.98 Hz), 126.40, 127.68, 127.96,
128.43, 128.56, 128.69, 128.87, 128.95, 130.32 (d, J = 2.77 Hz), 130.78 (d, J = 8.43 Hz), 134.60, 143.66,
151.30, 155.71, 166.36 (d, J = 249.53 Hz). HRMS (m/z): [M + H]+ calcd for C23H15N6FS: 427.1137; found:
427.1136.
3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-methylphenyl)-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine
(
5d): Yields: 70%. Mp 207.5–209.1 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ = 2.41 (3H, s, CH3), 4.49 (2H,
s, -CH2-), 7.41 (2H, d, J = 8.13 Hz, 1,4-disubstitutedbenzene), 7.52–7.55 (2H, m, benzimidazole CH),
7.83–7.86 (2H, m, benzimidazole CH), 7.97 (2H, d, J = 8.25 Hz, 1,4-disubstitutedbenzene), 8.33 (2H, d,
J = 8.58 Hz, 1,4-disubstitutedbenzene), 8.43 (2H, d, J = 8.61 Hz, 1,4-disubstitutedbenzene). 13C-NMR
(75 MHz, DMSO-d6): δ=21.55, 23.18, 114.86, 125.84, 126.42, 128.08, 128.24, 128.47, 129.02, 129.22, 129.71,
130.20, 130.90, 134.11, 142.96, 144.00, 147.35, 149.13, 151.07, 156.69. HRMS (m/z): [M + H]+ calcd for
C24H18N6S: 423.1381; found: 423.1386.
3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-cyanophenyl)-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine
◦
1
(
5e): Yields: 68%. Mp 259.3–263.1 C. H-NMR (300 MHz, DMSO-d6): δ = 4.53 (2H, s, -CH2-),
7.56–7.58 (2H, m, benzimidazole CH), 7.85–7.88 (2H, m, benzimidazole CH), 8.06 (2H, d, J = 8.37 Hz,
1,4-disubstitutedbenzene), 8.21 (2H, d, J = 8.46 Hz, 1,4-disubstitutedbenzene), 8.30 (2H, d, J = 8.49 Hz,
1,4-disubstitutedbenzene), 8.42 (2H, d, J = 8.49 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz,
DMSO-d6):
δ = 23.35, 114.58, 114.71, 118.68, 125.51, 126.39, 127.20, 128.48, 128.74, 128.87, 129.19, 129.43,
129.84, 133.03, 133.44, 137.97, 143.97, 148.72, 151.25, 155.45. HRMS (m/z): [M + H]+ calcd for C24H15N7S:
434.1186; found: 434.1182.
3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-bromophenyl)-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine
(
5f): Yields: 74%. Mp 284.8–286.2 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ = 4.50 (2H, s, -CH2-), 7.53–7.56
(3H, m, aromatic CH), 7.81–7.85 (3H, m, aromatic CH), 7.99–8.02 (2H, m, aromatic CH), 8.32 (2H, d,
J = 8.64 Hz, 1,4-disubstitutedbenzene), 8.42 (2H, d, J = 8.70 Hz, 1,4-disubstitutedbenzene). 13C-NMR
(75 MHz, DMSO-d6):
δ = 23.20, 114.91, 125.04, 125.96, 126.50, 127.03, 128.56, 129.19, 130.09, 130.90,
132.37, 132.67, 133.00, 142.94, 143.96, 147.34, 149.17, 151.22, 155.95. HRMS (m/z): [M + H]+ calcd for
C23H15N6SBr: 487.0311; found: 487.0335.
3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-(2,4-difluorohenyl)-7H-[1,2,4]triazole
thiadiazine (5g): Yields: 72%. Mp 208.5–210.7 ◦C. 1H-NMR (300 MHz, DMSO-d6):
[3,4-b][1,3,4]
= 4.42 (2H, s,
δ
-CH2-), 7.46–7.49 (4H, m, aromatic CH), 7.79–7.82 (3H, m, aromatic CH), 7.29–7.35 (2H, m, aromatic
CH), 8.45–8.51 (2H, m, aromatic CH). 13C-NMR (75 MHz, DMSO-d6):
δ
= 25.37, 113.01 (dd, J1 = 3.08 Hz,
J2 = 21.45 Hz), 106.08 (d, J = 26.01 Hz), 114.05, 114.45, 119.72 (dd, J1 = 3.02 Hz, J2 = 11.51 Hz), 126.56,
127.17, 128.25, 128.75, 129.15, 132.37, 132.67 (dd, J1 = 3.12 Hz, J2 = 10.36 Hz), 134.23, 135.55, 143.74,
148.22, 151.06, 153.70, 164.66 (d, J = 251.18 Hz), 164.83 (d, J = 250.91 Hz). HRMS (m/z): [M + H]+ calcd
for C23H14N6SF2: 445.1053; found: 445.1041.
3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-(2,4-dichlorophenyl)-7H-[1,2,4]triazole[3,4-b]
[1,3,4]
= 4.36 (2H,
◦
1
thiadiazine (5h): Yields: 77%. Mp 190.7–193.4 C. H-NMR (300 MHz, DMSO-d6):
δ
s, -CH2-), 7.34–7.37 (3H, m, aromatic CH), 7.65 (1H, dd, J1 = 2.04 Hz, J2 = 8.34 Hz, aromatic
CH), 7.70–7.73 (2H, m, aromatic CH), 7.78–7.81 (1H, m, aromatic CH), 8.20 (2H, d, J = 8.64 Hz,
1,4-disubstitutedbenzene), 8.40 (2H, d, J = 8.61 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz,
DMSO-d6):
δ = 26.23, 115.05, 115.32, 124.22, 124.72, 126.99, 127.78, 128.08, 128.29, 128.57, 128.96, 129.59,
130.38, 132.93, 133.05, 133.74, 136.86, 143.65, 149.92, 151.62, 156.53. HRMS (m/z): [M + H]+ calcd for
C23H14N6SCl2: 477.0457; found: 477.0450.
3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-6-phenyl-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine
(
5i): Yields: 80%. Mp: 247.3–249.6 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ
= 4.50 (2H, s, -CH2-), 7.49 (1H,
dd, J1 = 1.89 Hz, J2=8.67 Hz, aromatic CH), 7.59–7.62 (3H, m, aromatic CH), 7.78–7.84 (2H, m, aromatic

Molecules 2020, 25, 1642
11 of 15
CH), 8.03–8.06 (2H, m, aromatic CH), 8.27 (2H, d, J = 8.64 Hz, 1,4-disubstitutedbenzene), 8.37 (2H, d,
J = 8.64 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-d6):
= 23.32, 114.60, 116.29, 125.95,
δ
126.40, 128.11, 128.47, 128.97, 129.20, 129.63, 129.94, 132.62, 133.27, 133.71, 135.39, 143.97, 150.42, 151.04,
156.76. HRMS (m/z): [M + H]+ calcd for C23H15N6SCl: 443.0826; found: 443.0840.
3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-chlorophenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4]
thiadiazine (5j): Yields: 70%. Mp: 226.0–228.9 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ = 4.46 (2H, s,
-CH2-), 7.33–7.37 (1H, m, benzimidazole CH), 7.63 (2H, d, J = 8.61 Hz, 1,4-disubstitutedbenzene),
7.68–7.73 (2H, m, benzimidazole CH), 8.04 (2H, d, J = 8.61 Hz, 1,4-disubstitutedbenzene), 8.19 (2H, d,
J = 8.46 Hz, 1,4-disubstitutedbenzene), 8.39 (2H, d, J = 8.43 Hz, 1,4-disubstitutedbenzene). 13C-NMR
(75 MHz, DMSO-d6):
δ = 23.13, 115.14, 116.51, 124.18, 127.79, 128.20, 128.33, 128.83, 128.98, 129.19,
129.68, 129.87, 130.51, 131.57, 132.60, 137.42, 143.58, 151.40, 155.59. HRMS (m/z): [M + H]+ calcd for
C23H14N6SCl2: 477.0427; found: 477.0450.
3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-fluorophenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4]
◦
1
thiadiazine (5k): Yields: 71%. Mp: 249.1–250.9 C. H-NMR (300 MHz, DMSO-d6):
δ = 4.48 (2H,
s, -CH2-), 7.38 (1H, dd, J1 = 1.98 Hz, J2 = 8.58 Hz, aromatic CH), 7.46–7.48 (2H, m, aromatic CH),
7.71–7.74 (1H, m, aromatic CH), 7.77 (1H, s, aromatic CH), 8.12–8.14 (2H, m, aromatic CH), 8.25 (2H, d,
J = 8.64 Hz, 1,4-disubstitutedbenzene), 8.40 (2H, d, J = 8.61 Hz, 1,4-disubstitutedbenzene). 13C-NMR
(75 MHz, DMSO-d6):
δ = 23.45, 114.95, 116.45, 116.77 (d, J = 22.09 Hz), 124.83, 128.07, 128.21, 128.39,
128.84, 128.89, 130.30 (d, J = 2.99 Hz), 130.85 (d, J = 8.93 Hz), 135.26, 137.58, 143.69, 150.99, 151.27, 155.73,
164.82 (d, J = 249.42 Hz). HRMS (m/z): [M + H]+ calcd for C23H14N6FSCl: 461.0741; found: 461.0746.
3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-methylphenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4]
thiadiazine (5l): Yields: 65%. Mp: 250.7–252.3 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ = 4.46 (2H, s,
-CH2-), 7.40 (2H, d, J = 8.07 Hz, 1,4-disubstitutedbenzene), 7.45 (1H, dd, J1 = 1.95 Hz, J2 = 8.67 Hz,
benzimidazole CH), 7.76–7.79 (1H, m, benzimidazole CH), 7.82–7.83 (1H, m, benzimidazole CH), 7.95
(2H, d, J = 8.31 Hz, 1,4-disubstitutedbenzene), 8.28 (2H, d, J = 8.67 Hz, 1,4-disubstitutedbenzene), 8.37
(2H, d, J = 8.67 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-d6):
δ = 21.54, 23.15, 114.88,
116.44, 125.26, 127.74, 128.08, 128.21, 128.95, 129.24, 129.72, 130.21, 130.90, 134.62, 136.89, 142.95, 143.90,
150.99, 151.15, 156.66. HRMS (m/z): [M + H]+ calcd for C24H17N6SCl: 457.0994; found: 457.0997.
3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-cyanophenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4]
◦
1
thiadiazine (5m): Yields: 62%. Mp: 254.0–255.7 C. H-NMR (300 MHz, DMSO-d6):
δ = 4.53 (2H,
s, -CH2-), 7.50 (1H, dd, J1 = 1.95 Hz, J2 = 8.70 Hz, benzimidazole CH), 7.85–7.86 (1H, m, aromatic
CH), 8.06–8.08 (3H, m, aromatic CH), 8.21 (2H, d, J = 8.61 Hz, 1,4-disubstitutedbenzene), 8.26 (2H, d,
J = 8.64 Hz, 1,4-disubstitutedbenzene), 8.38 (2H, d, J = 8.64 Hz, 1,4-disubstitutedbenzene). 13C-NMR
(75 MHz, DMSO-d6):
δ = 23.30, 114.57, 114.71, 118.70, 125.83, 126.85, 128.48, 128.86, 129.11, 129.37,
129.81, 133.46, 133.58, 135.73, 137.98, 143.88, 150.56, 151.33, 155.41. HRMS (m/z): [M + H]+ calcd for
C24H14N7SCl: 468.0782; found: 468.0793.
3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-bromophenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4]
thiadiazine (5n): Yields: 76%. Mp: 243.4–247.3 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ = 4.48 (2H, s,
-CH2-), 7.43 (1H, dd, J1 = 1.98 Hz, J2 = 8.67 Hz, benzimidazole CH), 7.75 (1H, s, aromatic CH), 7.80–7.82
(3H, m, aromatic CH), 7.99 (2H, d, J = 8.70 Hz, 1,4-disubstitutedbenzene), 8.26 (2H, d, J = 8.58 Hz,
1,4-disubstitutedbenzene), 8.37 (2H, d, J = 8.64 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz,
DMSO-d6): δ=23.14, 115.01, 116.52, 124.92, 126.45, 128.10, 129.05, 130.07, 131.76, 132.00, 132.15, 132.67,
133.00, 141.45, 143.77, 148.89, 149.69, 151.39, 155.91. HRMS (m/z): [M + H]+ calcd for C23H14N6SClBr:
520.9930; found: 520.9945.
3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-6-(2,4-difluorohenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4]
thiadiazine (5o): Yields: 66%. Mp: 182.2–185.0 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ = 4.39 (2H, s,

Molecules 2020, 25, 1642
12 of 15
-CH2-), 7.28–7.34 (1H, m, aromatic CH), 7.42–7.48 (1H, m, aromatic CH), 7.48–7.52 (2H, m, aromatic
CH), 7.77–7.83 (2H, m, aromatic CH), 7.93–8.01 (1H, m, aromatic CH), 8.25 (2H, d, J = 8.64 Hz,
1,4-disubstitutedbenzene), 8.44 (2H, d, J = 8.58 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz,
DMSO-d6):
δ = 25.31, 105.76 (d, J = 26.01 Hz), 106.28, 113.19 (dd, J1 = 2.63 Hz, J2 = 20.86 Hz), 114.74,
116.39, 119.76 (dd, J1 = 3.73 Hz, J2 = 7.83 Hz), 125.45, 127.49, 128.32, 128.94, 129.14, 129.40, 132.71 (dd,
J1 = 3.21 Hz, J2 = 9.80 Hz), 134.22, 143.80, 151.60, 151.26, 153.78, 161.28 (d, J = 252.62 Hz), 164.46 (d,
J = 256.22 Hz). HRMS (m/z): [M + H]+ calcd for C23H13N6F2SCl: 479.0632; found: 479.0652.
3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-6-(2,4-dichlorophenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4]
thiadiazine (5p): Yields: 68%. Mp: 114.4–116.5 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ = 4.35 (2H, s,
-CH2-), 7.25 (1H, dd, J1 = 1.74 Hz, J2 = 8.52 Hz, aromatic CH), 7.65 (2H, dd, J1 = 2.07 Hz, J2 = 8.34 Hz,
aromatic CH), 7.88 (2H, d, J = 8.34 Hz, aromatic CH), 7.90 (1H, m, aromatic CH), 8.16 (2H, d, J = 8.61 Hz,
1,4-disubstitutedbenzene), 8.32 (2H, d, J = 8.58 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz,
DMSO-d6):
δ = 26.33, 106.76, 125.55, 127.29, 127.35, 128.58, 128.90, 129.64, 130.37, 131.63, 132.35, 132.96,
133.06, 133.44, 133.77, 136.56, 136.84, 143.51, 149.25, 151.77, 156.48. HRMS (m/z): [M + H]+ calcd for
C23H13N6SCl3: 511.0037; found: 511.0061.
3.2. Cytotoxicity Assay
The anticancer activity of compounds 5a–5p were screened according to the MTT assays. The MTT
assays were performed as previously described [24
the MCF7 cell line in the MTT assays.
,25]. Cisplatin was used as the reference drug for
3.3. Aromatase Inhibition Assay
This method was carried out according to the kit procedure (Bio Vision, Aromatase (CYP19A)
Inhibitor Screening Kit (Fluorometric)). The compounds were dissolved in dimethyl sulfoxide (DMSO)
and added to the assay in at least seven concentrations ranging from 10−3–10−9 M. The recombinant
human aromatase stock was prepared by reconstituting with 1 mL of aromatase assay buffer.
The contents were mixed thoroughly by vortexing to obtain a homogeneous solution and the solution
was transferred to a 15-mL conical tube. The volume was brought to 2450
µL with the aromatase
assay buffer and 50 l of NADPH production system (100X) was added for a final total volume of
µ
2.5 mL. Letrozole was used as a positive inhibition control. For solvent control, a small aliquot of
aromatase assay buffer containing the organic solvent was used to dissolve the test compounds that
were prepared. Reaction wells containing test compounds and the corresponding no inhibitor controls
(which may also serve as a solvent control), as well as a background control (containing no fluorogenic
Aromatase Substrate) were prepared. The plate was incubated for at least 10 min at 37 ◦C to allow
test ligands to interact with the aromatase. After incubation, 30 µl of the aromatase substrate/NADP+
mixture was added to each well. Immediately (within 1 min), the fluorescence at Ex/Em = 488/527 nm
was measured.
3.4. Molecular Docking
A structure-based in silico procedure was applied to discover the binding modes of compound
5e to human aromatase enzyme active site. The crystal structures of human aromatase (PDB ID:
3EQM) [26] was retrieved from the Protein Data Bank server (www.pdb.org).
The structures of ligands were built using the Schrödinger Maestro [29] interface and then were
submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2 [30].
The ligands were prepared by the LigPrep 3.8 [31] to assign the protonation states at pH 7.4
±
1.0
and the atom types, correctly. Bond orders were assigned, and hydrogen atoms were added to the
structures. The grid generation was formed using Glide 7.1 [32]. Flexible docking runs were performed
with single precision docking mode (SP).

Molecules 2020, 25, 1642
13 of 15
3.5. Theoretical Determination of ADME Properties
Physicochemical parameters of obtained compounds (5a
–5p) were evaluated by using QikProp
4.8 [33].
4. Conclusions
Inhibition of aromatase has proved to be an effective approach for the treatment of
hormone-dependent breast cancer in the postmenopausal women. Imidazole and triazole groups
are important rings for the development new potent aromatase inhibitors with high affinity for the
enzyme. A series of benzimidazole-triazolothiadiazine derivatives have been synthesized with different
substituents at benzimidazole and phenyl rings. The newly synthesized compounds were tested for
their anti-cancer properties against human breast cancer cell line (MCF-7). The synthesized compounds
were then tested in in vitro aromatase assay and two compounds (5e and 5m) exhibited activity similar
to letrozole.
Supplementary Materials: The following are available online.
Author Contributions: Z.A.K., Y.Ö., conceived and designed the experiments; D.O., U.A.Ç. performed the
synthesis; S.L. performed analysis studies; B.N.S., U.A.Ç., and S.I. performed activity tests; D.O., U.A.Ç., B.N.S.,
B.K.Ç. wrote the paper. All authors have read and agreed to the published version of the manuscript.
Acknowledgments: This study was financially supported by Anadolu University Scientific Projects Fund, Project
No: 1905S032.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
Chamduang, C.; Pingaew, R.; Prachayasittikul, V.; Prachayasittikul, S.; Ruchirawat, S.; Prachayasittikul, V.
Novel triazole-tetrahydroisoquinoline hybrids as human aromatase inhibitors. Bioorg. Chem. 2019, 93, 103327.
[CrossRef] [PubMed]
ˇ
Doiron, J.; Richard, R.; Touré, M.M.; Picot, N.; Richard, R.; Cuperlovic´-Culf, M.; Robichaud, G.A.; Touaibia, M.
Synthesis and structure–activity relationship of 1-and 2-substituted-1, 2, 3-triazole letrozole-based analogues
as aromatase inhibitors. Eur. J. Med. Chem. 2011, 46, 4010–4024. [CrossRef] [PubMed]
Henneberta, O.; Montes, M.; Favre-Reguillon, A.; Chermetted, H.; Ferroudc, C.; Mortin, R. Epimerase activity
of the human 11β-hydroxysteroid dehydrogenase type 1 on 7-hydroxylated C19-steroids. J. Steroid Biochem.
Mol. Biol. 2009, 114, 57–63. [CrossRef] [PubMed]
Leechaisit, R.; Pingaew, R.; Prachayasittikul, V.; Worachartcheewan, A.; Prachayasittikul, S.; Ruchirawat, S.;
Prachayasittikul, V. Synthesis, molecular docking, and QSAR study of bis-sulfonamide derivatives as
potential aromatase inhibitors. Bioorg. Med. Chem. 2019, 27, 115040. [CrossRef]
Brueggemeier, R.W.; Hackett, J.C.; Diaz-Cruz, E.S. Aromatase inhibitors in the treatment of breast cancer.
Endocr. Rev. 2005, 26, 331–345. [CrossRef]
Cepa, M.M.; da Silva, E.J.T.; Correia-da-Silva, G.; Roleira, F.M.; Teixeira, N.A. Synthesis and biochemical
studies of 17-substituted androst-3-enes and 3,4-epoxyandrostanes as aromatase inhibitors. Steroids 2008, 73,
1409–1415. [CrossRef]
5.
6.
7.
8.
9.
Bonfield, K.; Amato, E.; Bankemper, T.; Agard, H.; Steller, J.; Keeler, J.M.; Roy, D.; McCallum, A.; Paula, S.;
Ma, L. Development of a new class of aromatase inhibitors: Design, synthesis and inhibitory activity of
3-phenylchroman-4-one (isoflavanone) derivatives. Bioorg. Med. Chem. 2012, 20, 2603–2613. [CrossRef]
Rampogu, S.; Baek, A.; Bavi, R.S.; Son, M.; Cao, G.P.; Kumar, R.; Park, C.; Zeb, A.; Rana, R.M.; Park, S.J.; et al.
Identification of Novel Scaffolds with Dual Role as Antiepileptic and Anti-Breast Cancer. IEEE/ACM Trans.
Comput. Biol. Bioinform. 2018, 16, 1663–1674. [CrossRef]
Trunet, P.F.; Vreeland, F.; Royce, C.; Chaudri, H.A.; Cooper, J.; Bhatnagar, A.S. Clinical use of aromatase
inhibitors in the treatment of advanced breast cancer. J. Steroid. Biochem. Mol. Biol. 1997, 61, 241–245.
[CrossRef]

Molecules 2020, 25, 1642
14 of 15
10. Steele, R.E.; Mellor, L.B.; Sawyer, W.K.; Wasvary, J.M.; Browne, L.J. In vitro and in vivo studies demonstrating
potent and selective estrogen inhibition with the nonsteroidal aromatase inhibitor CGS 16949A. Steroids 1987
,
50, 147161. [CrossRef]
11. Kato, S.; Endoh, H.; Masuhiro, Y.; Kitamoto, T.; Uchiyama, S.; Sasaki, H.; Masushige, S.; Gotoh, Y.; Nishida, E.;
Kawashima, H.; et al. Activation of the estrogen receptor through phosphorylation by mitogen-activated
protein kinase. Science 1995, 270, 1491–1494. [CrossRef] [PubMed]
12. Sable, P.M.; Potey, L.C. Synthesis and antiproliferative activity of imidazole and triazole derivatives of
flavonoids. Pharm. Chem. J. 2018, 52, 438–443. [CrossRef]
13. Gilardi, G.; Di Nardo, G. Heme iron centers in cytochrome P450: Structure and catalytic activity. Rend. Lincei
2017, 28, 159–167. [CrossRef]
14. Asadi, P.; Khodarahmi, G.; Farrokhpour, H.; Hassanzadeh, F.; Saghaei, L. Quantum mechanical/molecular
mechanical and docking study of the novel analogues based on hybridization of common pharmacophores
as potential anti-breast cancer agents. Res. Pharm. Sci. 2017, 12, 233.
15. Mojaddami, A.; Sakhteman, A.; Fereidoonnezhad, M.; Faghih, Z.; Najdian, A.; Khabnadideh, S.; Rezaei, Z.
Binding mode of triazole derivatives as aromatase inhibitors based on docking, protein ligand interaction
fingerprinting, and molecular dynamics simulation studies. Res. Pharm. Sci. 2017, 12, 21. [CrossRef]
16. Song, Z.; Liu, Y.; Dai, Z.; Liu, W.; Zhao, K.; Zhang, T.; Dai, Y. Synthesis and aromatase inhibitory evaluation
of 4-N-nitrophenyl substituted amino-4H-1,2,4-triazole derivatives. Bioorg. Med. Chem. 2016, 24, 4723–4730.
[CrossRef]
17. Adhikari, N.; Amin, S.A.; Jha, T.; Gayen, S. Integrating regression and classification-based QSARs with
molecular docking analyses to explore the structure-antiaromatase activity relationships of letrozole-based
analogs. Can. J. Chem. 2017, 95, 1285–1295. [CrossRef]
18. Prachayasittikul, V.; Pingaew, R.; Worachartcheewan, A.; Sitthimonchai, S.; Nantasenamat, C.;
Prachayasittikul, S.; Ruchirawat, S.; Prachayasittikul, V. Aromatase inhibitory activity of 1, 4-naphthoquinone
derivatives and QSAR study. EXCLI J. 2017, 16, 714.
19. Augusto, T.V.; Amaral, C.; Varela, C.L.; Bernardo, F.; da Silva, E.T.; Roleira, F.F.; Costa, S.; Teixeria, N.;
Correia-da-Silva, G. Effects of new C6-substituted steroidal aromatase inhibitors in hormone-sensitive breast
cancer cells: Cell death mechanisms and modulation of estrogen and androgen receptors. J. Steroid. Biochem.
Mol. Biol. 2019, 195, 105486. [CrossRef]
20. Shoombuatong, W.; Schaduangrat, N.; Nantasenamat, C. Towards understanding aromatase inhibitory
activity via QSAR modeling. EXCLI J. 2018, 17, 688.
21. Prior, A.M.; Yu, X.; Park, E.J.; Kondratyuk, T.P.; Lin, Y.; Pezzuto, J.M.; Sun, D. Structure-activity relationships
and docking studies of synthetic 2-arylindole derivatives determined with aromatase and quinone reductase
1. Bioorg. Med. Chem. Lett. 2017, 27, 5393–5399. [CrossRef] [PubMed]
22. Acar Çevik, U.; Sag˘lık, B.N.; Osmaniye, D.; Levent, S.; Kaya Çavus¸og˘lu, B.; Karaduman, A.B.; Ozkay, Y.;
Kaplancıklı, Z.A. Synthesis and docking study of benzimidazole–triazolothiadiazine hybrids as aromatase
inhibitors. Arch. Pharm. 2020, e2000008. [CrossRef] [PubMed]
23. Çevik, U.A.; Osmaniye, D.; Çavus¸og˘lu, B.K.; Sag˘lik, B.N.; Levent, S.; Ilgin, S.; Ozkay, Y.; Kaplancikli, Z.A.
Synthesis of novel benzimidazole–oxadiazole derivatives as potent anticancer activity. Med. Chem. Res. 2019
,
28, 2252–2261. [CrossRef]
24. Evren, A.E.; Yurttas¸, L.; Eksellı, B.; Akalın-Cıftcı, G. Novel tri-substituted thiazoles bearing piperazine ring:
Synthesis and evaluation of their anticancer activity. Lett. Drug Des. Discov. 2019, 16, 547–555. [CrossRef]
25. Çevik, U.A.; Sag˘lık, B.N.; Ardıç, C.M.; Özkay, Y.; Atlı, Ö. Synthesis and evaluation of new benzimidazole
derivatives with hydrazone moiety as anticancer agents. Turk. J. Biochem. 2018, 43, 151–158. [CrossRef]
26. Ghosh, D.; Griswold, J.; Erman, M.; Pangborn, W. Structural basis for androgen specificity and oestrogen
synthesis in human aromatase. Nature 2009, 457, 219. [CrossRef]
27. Jorgensen, W.L.; Duffy, E.M. Prediction of drug solubility from structure. Adv. Drug Deliv. Rev. 2002
54, 355–366. [CrossRef]
,
28. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to
estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev.
1997, 23, 3–25. [CrossRef]
29. Maestro, version 10.6; Schrödinger, LLC: New York, NY, USA, 2016.
30. The Schrödinger Suite 2016 Update 2; Schrödinger, LLC: New York, NY, USA, 2016.

Molecules 2020, 25, 1642
15 of 15
31. LigPrep, version 3.8; Schrödinger, LLC: New York, NY, USA, 2016.
32. Glide, version 7.1; Schrödinger, LLC: New York, NY, USA, 2016.
33. QikProp, version 4.8; Schrödinger, LLC: New York, NY, USA, 2016.
Sample Availability: Samples of the compounds 5a–5p are available from the authors.
©
2020 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 (http://creativecommons.org/licenses/by/4.0/).