Pyridine-substituted thiazolylphenol derivatives: Synthesis, modeling studies, aromatase inhibition, and antiproliferative activity evaluation

Article abstract of DOI:10.1002/ardp.201700272

Drugs used in breast cancer treatments target the suppression of estrogen biosynthesis. During this suppression, the main goal is to inhibit the aromatase enzyme that is responsible for the cyclization and structuring of estrogens either with steroid or non-steroidal-type inhibitors. Non-steroidal derivatives generally have a planar aromatic structure attached to the triazole ring system in their structures, which inhibits hydroxylation reactions during aromatization by coordinating the heme group. Bioisosteric replacement of the triazole ring system and development of aromatic/cyclic structures of the side chain can increase the selectivity for aromatase enzyme inhibition. In this study, pyridine-substituted thiazolylphenol derivatives, which are non-steroidal triazole bioisosteres, were synthesized using the Hantzsch method, and physical analysis and structural determination studies were performed. The IC₅₀ values of the compounds were determined by a fluorescence-based aromatase inhibition assay. Then, their antiproliferative activities on the MCF7 and HEK 293 cell lines were evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, the crystal structure of human placental aromatase was subjected to a series of docking experiments to identify the possible interactions between the most active structure and the active site. Lastly, an in silico technique was performed to analyze and predict the drug-likeness, molecular and ADME properties of the synthesized molecules.

Full text of DOI:10.1002/ardp.201700272

Received: 24 August 2017
Revised: 12 February 2018
Accepted: 13 February 2018
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DOI: 10.1002/ardp.201700272
FULL PAPER
Pyridine-substituted thiazolylphenol derivatives: Synthesis,
modeling studies, aromatase inhibition, and antiproliferative
activity evaluation
Merve Ertas1
Sevde N. Biltekin3
Zafer Sahin1
Barkin Berk1
Leyla Yurttas2
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Seref Demirayak1
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1 Department of Pharmaceutical Chemistry,
School of Pharmacy, İstanbul Medipol
University, İstanbul, Turkey
Abstract
Drugs used in breast cancer treatments target the suppression of estrogen
biosynthesis. During this suppression, the main goal is to inhibit the aromatase
enzyme that is responsible for the cyclization and structuring of estrogens either with
steroid or non-steroidal-type inhibitors. Non-steroidal derivatives generally have a
planar aromatic structure attached to the triazole ring system in their structures, which
inhibits hydroxylation reactions during aromatization by coordinating the heme group.
Bioisosteric replacement of the triazole ring system and development of aromatic/
cyclic structures of the side chain can increase the selectivity for aromatase enzyme
inhibition. In this study, pyridine-substituted thiazolylphenol derivatives, which are
non-steroidal triazole bioisosteres, were synthesized using the Hantzsch method, and
physical analysis and structural determination studies were performed. The IC50 values
of the compounds were determined by a fluorescence-based aromatase inhibition
assay. Then, their antiproliferative activities on the MCF7 and HEK 293 cell lines were
evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. Furthermore, the crystal structure of human placental aromatase was
subjected to a series of docking experiments to identify the possible interactions
between the most active structure and the active site. Lastly, an in silico technique was
performed to analyze and predict the drug-likeness, molecular and ADME properties of
the synthesized molecules.
2 Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Anadolu
University, Eskisehir, Turkey
3 Department of Pharmaceutical Microbiology,
School of Pharmacy, İstanbul Medipol
University, İstanbul, Turkey
Correspondence
Merve Ertas, Department of Pharmaceutical
Chemistry, School of Pharmacy, İstanbul
Medipol University, Kavacık street no. 19,
34810 İstanbul, Turkey.
Email: mertas@medipol.edu.tr
Funding information
Anadolu University Scientific Research
Projects Commission, Grant number:
1410S422
K E Y W O R D S
anticancer activity, aromatase, breast cancer, MCF-7, non-steroidal inhibitor
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1
INTRODUCTION
idea that estrogens stimulate the growth of residual cancer cells or
contribute to the initiation of a new primary cancer over time. Selective
Breast cancer is the most common cancer among women worldwide.[1]
Most breast cancers in postmenopausal women are estrogen receptor
positive (ER+)[2,3] and adjuvant endocrine therapy has an important role
in treating this type. Efficacy of suppression treatment is based on the
estrogen-receptor modulators (SERMs) and aromatase inhibitors (AIs)
are two basic regimes used in clinics.
During the final step of estrogen biosynthesis, the aromatase
enzyme plays a crucial role by both enhancing the aromatic feature of
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Arch Pharm Chem Life Sci. 2018;e1700272.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201700272

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FIGURE 1 Key role of aromatase in the estrogen pathway
androstenedione/testosterone and eliminating a methyl group from
the ring system.[4,5] Therefore, inhibition of aromatase with AIs is a
promising method of developing new solutions (Figure 1).[6,7]
systems and changing phenolic rings with the pyridine ring within this
structure has resulted in improvement of aromatase inhibitory activity
(Figure 2).[16–19]
In the last few decades, three generations of AIs have been
developed. Those that are most preferred in clinical therapy are third-
generation AIs since they have more favorable tolerability profiles
and they are more selective and/or potent compared with first- and
second-generation agents.[8,9] In terms of chemistry and pharmacol-
In the current study, we prepared 18 non-steroidal pyridine-
substituted thiazolylphenol derivatives for the purpose of inhibiting
the active site of aromatase cytochrome P450. Aromatase inhibitory
activities of the molecules were measured by a fluorescence-based
assay using ketoconazole as standard. Then, antiproliferative and
anticancer activities of the compounds were evaluated with an MTT
assay over the human breast adenocarcinoma (MCF7) and human
embryonic kidney (HEK 293) cell lines. Further interactions between
the active molecule and the active site of the enzyme was analyzed
through docking experiments using the crystal structure of human
placental aromatase cytochrome P450 in complex with androstenedi-
one (3EQM).[7] Furthermore, absorption, distribution, metabolism, and
excretion (ADME), drug-likeness, and molecular properties of all the
molecules were predicted by in silico techniques. Although compounds
2 and 4–8 had been previously synthesized using different methods
and investigated in other subjects such as chemiluminescence,[20–26]
we resynthesized them and performed enzymatic and cellular experi-
ments to test their activities.
ogy, there are two broad categories of third-generation AIs[10]
;
reversible non-steroidal agents consist of anastrozole and letrozole
(triazole derivatives), and irreversible steroidal inhibitors comprise
exemestane and formestane.[11] Recent studies on breast cancer
treatment involve non-steroidal inhibition of the aromatase enzyme
rather than steroidal therapy[12–14] due to the prolonged inactivating
effect of irreversible steroidal agents on the enzyme even after the
drug is cleared from circulation. In this case, continuation of estrogen
production depends on the synthesis of new aromatase molecules.[15]
Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a stilbenoid, which
can modulate several steps in carcinogenesis; however, it has several
negative effects and disadvantages in clinical use.[16] Research
involving the addition of central 1,3-thiazole or 1,3,5-thiadiazole
FIGURE 2 Aromatase inhibition of resveratrol and analogs[17–19]

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TABLE 1 The IC50 valuesa of aromatase-active compounds (10 μM–
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2
RESULTS AND DISCUSSION
4.5 nM)
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2.1 Chemistry
A synthetic pathway consists of a two-step reaction. In the first step,
1-(substituted phenyl)ethanone derivatives were brominated using
copper(II) bromide in ethyl acetate resulting in 2-bromo-1-(substituted
phenyl)ethanones. For these types of bromination reactions, acetic-
acid medium is generally used[27]; however, in the current study,
copper(II) bromide was preferred since in acetic medium, structures
containing phenolic parts can direct the bromine to be substituted over
the ring system. In the second step, through the Hantzsch thiazole[28]
synthesis, 2-bromo-1-(substituted phenyl)ethanones and various
pyridinethioamides were reacted in ethanol to obtain either 2/3/4-
[2-(pyridin-2/3/4-yl)thiazol-4-yl]phenol or 4-substituted-2-[2-(pyri-
din-2/3/4-yl)thiazol-4-yl]phenol structured compounds with yields
ranging from 65 to 82% (Figure 3).
Compound
R1
R2
IC50 (μM)
1
2-OH Phenyl
2-Pyridinyl
3-Pyridinyl
4-Pyridinyl
2-Pyridinyl
3-Pyridinyl
4-Pyridinyl
2-Pyridinyl
3-Pyridinyl
4-Pyridinyl
2-Pyridinyl
3-Pyridinyl
4-Pyridinyl
2-Pyridinyl
3-Pyridinyl
4-Pyridinyl
2-Pyridinyl
3-Pyridinyl
4-Pyridinyl
12.68 0.23
9.63 2.13
1.84 0.20
2.86 0.44
8.87 1.33
0.9 1.12b
7.92 3.06
1.51 0.35
0.96 0.44
1.25 0.62
15.49 0.44
1.62 0.11
1.74 0.18
11.63 4.70
10.35 1.18
1.38 0.31
7.39 3.13
9.48 3.82
0.95 0.09
2
2-OH Phenyl
3
2-OH Phenyl
4
3-OH Phenyl
5
3-OH Phenyl
6
3-OH Phenyl
7
4-OH Phenyl
8
4-OH Phenyl
9
4-OH Phenyl
In the IR spectra, all the compounds had a strong C─O stretching
band for the phenol group at 1200 cm−1. The 1H NMR spectra of the
compounds showed that the protons belonging to the hydroxyl group
differed between 9.55 and 11.4 ppm according to their substitution
position. The only hydrogen of the central thiazole was found to be a
singlet between 8.0 and 8.75 ppm. The other aromatic hydrogens of
both phenol and pyridine rings ranged from 6.9 to 8.9 ppm. All the
remaining protons were observed according to the expected chemical
shift and integral values. In the 13C NMR spectra, the carbons of the
phenol, thiazole, and pyridine ring systems were observed at 116–129,
162–191, and 150–155 ppm, respectively. The molecular ion peaks
(M+) of the compounds confirmed their molecular weights.
10
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12
13
14
15
16
17
18
KTZc
2-OH-5-CH3 Phenyl
2-OH-5-CH3 Phenyl
2-OH-5-CH3 Phenyl
2-OH-5-OCH3 Phenyl
2-OH-5-OCH3 Phenyl
2-OH-5-OCH3 Phenyl
2-OH-5-Cl Phenyl
2-OH-5-Cl Phenyl
2-OH-5-Cl Phenyl
aThe values represent mean SD of triplicate determinations.
bThe IC50 value of compound 6 and its SD were both in nM units.
cKetoconazole (KTZ) was used as standard for aromatase.
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2.2 Biological activity
The bold values displays meaningful results according to the standard.
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2.2.1 Aromatase enzyme inhibitory activity
3
(IC50 = 1.84 μM), 6 (IC50 = 0.91 nM), 8 (IC50 = 1.51 μM), 9
Aromatase inhibitory activities of compounds 1–18 and ketoconazole
(IC50 = 0.95 μM) were determined within a concentration range of
10 μM–4.5 nM. Most efficient molecules were found to be compounds
(IC50 = 0.96 μM), 10 (IC50 = 1.25 μM), 12 (IC50 = 1.62 μM), 13
(IC50 = 1.74 μM), and 16 (IC50 = 1.38 μM). These results are shown in
Table 1 and Figure 4.
FIGURE 3 General synthesis and structures of the synthesized compounds

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FIGURE 4 Dose–response curves for compounds 3, 6, 8–10, 12, 13, and 16 and ketoconazole
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As shown in Table 1, compound 6 inhibited the enzyme at
1000 times lower concentration compared to ketoconazole. In
addition, the efficacy of compound 9 was very similar to that of
ketoconazole.
2.2.2 Anticancer activity against tumor and normal
cells in culture
The 10−4 M concentrations of compounds were tested on MCF7 and
HEK 293 cell lines for measuring the viability of the exposed cells.

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Synthesized compound 13 exhibited the highest cytotoxic activities
against MCF7 cells (56.44%), the lowest proliferative activities against
HEK 293 cells (10.86%). Compound 14 had the most cytotoxic activity
on MCF7 (68.32%), so this compound showed the most potent
anticancer activity. Compounds 13 (56.44%), 16 (49.58%), 8 (47.67%),
and 10 (46.09%) were more active than the others. Compound 6 that
had the highest anti-aromatase activity had a toxic effect on MCF7;
however, its activity was 10% less compared to HEK 293. Compounds
14 and 15 showed cytotoxic effects on both the HEK 293 and MCF7
cell lines. The obtained values are listed in Figure 5 and Table 2.
TABLE 2 % Viability values (10−4 M) for compounds in MCF7 and
HEK 293 cell lines
MCF7
HEK 293
Compound
% Control
Compound
% Control
1
83.39 2.64
84.40 3.60
86.12 2.51
70.81 3.05
75.24 2.00
83.44 3.05
84.71 4.93
52.33 4.35
68.96 3.05
53.01 2.00
73.22 4.72
58.02 3.05
43.56 4.04
31.68 3.05
55.03 4.00
50.42 1.52
57.51 3.00
67.28 3.05
1
80.94 3.21
86.69 2.08
66.41 2.51
63.60 3.21
67.19 2.51
72.89 4.04
85.93 3.51
90.86 2.64
74.73 2.51
87.51 2.64
79.74 3.00
90.69 2.64
110.86 1.52
63.23 3.51
43.49 2.00
80.46 2.08
83.95 3.60
76.15 3.51
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2
3
3
4
4
5
5
6
6
7
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2.3 Molecular modeling studies
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8
9
9
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2.3.1 Docking studies
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11
12
13
14
15
16
17
18
10
11
12
13
14
15
16
17
18
The lowest IC50 value during aromatase inhibition was obtained from
compound 6; therefore, a docking experiment was undertaken with
this compound and to determine 3EQM in order to identify the
interaction pattern between the active site and the compound. The
experiment was initially performed using the XP algorithm of Glide,
treating the receptor as rigid and the ligands as flexible. The docking
protocol had the same constraints as a previous study detailing high
throughput docking to find potent azole compounds as aromatic
nitrogen heterocycles that are able to inhibit P450 enzymes by
coordinating the heme iron atom, thus preventing oxygen binding and
the subsequent oxidation reaction of the substrate.[29,30] A van der
Waals radius scaling factor of 0.50 for atoms with a partial atomic
charge (absolute value) less than 0.15 was used to soften the potential
for the non-polar parts of the receptor. The enclosing box was
centered on the heme residue, and default sizes were used for both the
enclosing and bounding boxes. A metal constraint was used to
determine ligand poses where an aromatic nitrogen atom interacts
with the heme iron. The ligand nitrogen must be within 2.4 Å of the
metal in order to satisfy the constraint. To validate the experiment,
anastrozole, a non-steroidal aromatase inhibitor which interacts with
the enzyme through a triazole moiety, was first docked to check
whether the binding mode was consistent with the available data.
After achieving a successful repetition of the results, the experiment
was performed with compound 6. Then, the experiment was improved
with the induced-fit protocol of Glide, in which both receptor and
ligand were simulated as flexible. The overall settings of this protocol
were also the same as described by Caporuscio et al.[29] with the only
exception being the initial use of the XP protocol, rather than SP. The
ligand seemed to fit well into the pocket similar to anastrazole where
the pyridine ring system was coordinated with heme moiety (Figure 6).
Nitrogen atom of the pyridine system acted as an H-bond acceptor
from the Thr 310 side chain. This part of the molecule was in
hydrophobic interactions with Ala 306 and Val 370 of the active site.
Other active site amino acids that stabilized the ligand with
hydrophobic interactions were Val 373, Leu 372, Met 374, Leu 228,
Phe 116, and Trp 224. These amino acids surrounded the phenolic part
of the molecule producing a small hydrophobic cavity. Furthermore,
this phenolic part had a π–π stacking interaction with Phe 134. A
similar kind of stacking was also observed between the thiazole part
and Arg 115.
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2.3.2 Prediction of drug-likeness, molecular and
ADME properties
In this study, the drug-likeness, molecular and ADME properties of all
the compounds were calculated using the QikProp function of Maestro
(Schrödinger 2016-4). All the compounds showed promising findings
presenting with a drug-like/lead-like profile according to their #stars
rankings. The combinations of the HOA values being 3, %HOA of all
FIGURE 5 Effect of compounds 1–18 at the concentration of
10−4 M on the viability of MCF7 and HEK 293 cells

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FIGURE 6 Positioning of compound 6 docked with the induced-fit protocol in the active site and compound 6 in the active site of
aromatase
compounds being 100%, and all PCaco values being very high indicated
that these compounds are suitable for oral use. The log Kp values
varying between −1.80 (compounds 5, 6, 8, and 9) and −1.07
(compound 1) indicate that these compounds can be effectively
used in topical preparations. Furthermore, the logarithm of the
predicted binding constant to human serum albumin of the selected
compounds, i.e., the log KHSA values, varied between 0.08 (compounds
2 and 3) and 0.33 (compound 10). It is important to note that the
binding of drugs to plasma proteins such as human serum albumin,
lipoprotein, glycoprotein, α, β, and γ globulins greatly reduces the
quantity of the drug in general blood circulation. In other words, the
less bound a drug is, the more efficiently it can traverse cell membranes
or diffuse. The log KHSA values showed that these derivatives could
freely circulate and easily traverse cell membranes. The logHERG
values of the compounds predicted with the in silico method were
between −5.38 (compound 15) and −5.64 (compounds 4 and 7). The
HERG K+ channel, best known for its impact on the electrical activity of
the heart by synchronizing beating activity, appears to be the
molecular target responsible for the cardiac toxicity of a wide range
of therapeutic drugs.[31] HERG has also been found associated with
modulating the functions of certain cells of the nervous system and
with establishing and maintaining cancer-like features in leukemic
cells.[32] Thus, HERG K+ channel blockers are potentially toxic and IC50
values often provide reasonable predictions for the cardiac toxicity of
drugs in the early stages of drug discovery.[33] The results showed the
presence of a mild risk of compounds acting as HERG K+ channel
blockers. As a basic rule, polar drugs cannot easily penetrate the BBB.
The blood/brain partition coefficient (logBB) [−0.20 (compounds 5, 6,
8, and 9) to 0.29 (compound 16)], PMDCK [1216.28 (compound 8) and
7046.34 (compound 16)], and logPo/w [2.82 (compounds 5 and 6) and
3.70 (compound 16)] values are useful for determining the penetration
capacity of a compound from BBB. The values predicted for these
parameters of the synthesized compounds were within the ranges
defined for 95% of drugs. Moreover, the predicted CNS value of the
compounds with a maximum of 1 indicated mild to medium activity.
Although the predictive results must be checked with actual
experiments, the activity studies showed that compounds 3, 6, 8–10,
12, 13, and 15 exhibited very promising IC50 values during aromatase
and cellular inhibition assays. Using a thiazole ring system instead of a
double bond in resveratrol and the 1,3,5-thiadiazole systems worked
extremely well with the insertion of pyridine and phenol to both sides
of the molecule. Such improvements with these structures can lead to
more effective oral non-steroidal aromatase inhibitors with less
cytotoxicity to healthy human cells.
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3
EXPERIMENTAL
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3.1 Chemistry
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3.1.1 General
All the chemicals were purchased from Aldrich Chemical Co.
(Steinheim, Germany). Melting points were determined with a Stuart
melting point apparatus SMP30 (Staffordshire, UK). IR spectra (KBr)
were recorded on a PerkinElmer Spectrum Two FT-IR spectrometer
(Waltham, MA, USA) and 1H NMR spectra were obtained by Bruker
DPX-500, 500 MHz High Performance Digital FT-NMR. 13C NMR
spectra were measured using a DPX-125, 125 MHz High Performance
Digital FT-NMR. All the chemical shift values were recorded as δ (ppm).
Mass spectra were obtained using an Agilent 1100 MSD series mass
spectrometer. The purity of the compounds was checked by thin-layer
chromatography on silica gel-coated aluminum sheets (Merck,
1.005554, silica gel HF254–361, Type 60, 0.25 mm; Darmstadt,
Germany). Elemental analyses were performed with a Leco CHNS 932
analyzer (Leco Corp., MI, USA) and were found to be within 0.4% of
the theoretical values for C, H, and N.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
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3.1.2 General procedure for the synthesis of
2-bromo-1-(substituted phenyl)ethanone compounds
1-(Substituted phenyl)ethanone derivatives (0.1 mol) and CuBr2
(0.2 mol) were mixed in ethyl acetate and refluxed for 6–8 h until
the green color disappeared. The mixtures were cooled and kept
refrigerated overnight for the total precipitation of CuBr. The
precipitates were filtered and the ethyl acetate phases were extracted
with water (2 × 100 mL) to remove all the inorganic leftovers. Then, the
ethyl acetate phase was dried with anhydrous sodium sulfate, filtered,
and concentrated in a vacuum to yield target compounds.

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3.1.3 General procedure for the synthesis of
7.48–7.50 (m, 1H, Ph), 7.50–7.53 (m, 2H, Py and Ph), 8.01 (td, 1H,
J = 1.53 Hz, J = 7.72 Hz, J = 7.72 Hz, Py), 8.17 (s, 1H, Th), 8.24 (d, 1H,
J = 7.87, Py), 8.66 (d, 1H, J = 4.78, Th), 9.57 (s, 1H, OH). 13C NMR:
113.49, 115.82, 117.27, 117.41, 119.69, 125.65, 130.31, 135.72,
138.24, 150.20, 150.91, 156.22, 158.27, 168.55. For C14H10N2OS
calcd. 66.12% C, 3.96% H, 11.02% N; found: 66.08% C, 3.96% H,
11.08% N. Mass (ESI), m/z: 255 (M+1).
2/3/4-[2-(pyridin-2/3/4-yl)thiazol-4-yl]phenol and
4-substituted-2-[2-(pyridin-2/3/4-yl)thiazol-4-yl]-
phenol compounds
Equimolar appropriate amounts of pyridinethioamides (3.6 mmol) and
2-bromo-1-(substituted phenyl)ethanone derivatives were mixed and
stirred in ethanol (50 mL) at room temperature for 30 min followed by
refluxing for an hour. The mixtures were cooled to 0°C and the
precipitates collected through filtration were dissolved in water and
treated with 1 M sodium acetate solution. New precipitates were
collected by filtration, dried, and recrystallized from ethanol or an
ethanol/water (1:1) mixture.
3-[2-(Pyridin-3-yl)thiazol-4-yl]phenol (5)
Yield: 78%, mp: 193.2°C, IR υmax (cm−1): 3173.3–2976 (aromatic
C─H), 1274.60 (C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm): 6.81
(dd, 1H, J = 2.24 Hz, J = 7.96 Hz, Ph), 7.29 (t, 1H, J = 7.80 Hz,
J = 7.80 Hz, Ph), 7.48 (s, 1H, Ph), 7.50 (d, 1H, J = 8.6 Ar C6-H), 7.59
(dd, 1H, J = 4.77 Hz, J = 7.93 Hz, Py), 8.19 (s, 1H, Th), 8.38 (dt, 1H,
J = 1.88 Hz, J = 1.88 Hz, J = 7.97 Hz, Py), 8.71 (dd, 1H, J = 1.39 Hz,
J = 4.77 Hz, Py), 9.21 (d, 1H, J = 2.20 Hz, Py), 9.56 (s, 1H, OH).
13C NMR: 113.60, 115.91, 117.50, 124.76, 129.51, 130.34, 134.11,
135.47, 147.41, 151.45, 156.11, 158.25, 164.20. For C14H10N2OS
calcd. 66.12% C, 3.96% H, 11.03% N; found: 66.10% C, 3.95% H,
11.06% N. Mass (ESI) m/z: 255 (M+1).
2-[2-(Pyridin-2-yl)thiazol-4-yl]phenol (1)
Yield: 76%, mp: 124.3°C, IR υmax (cm−1): 3104–3039 (aromatic C─H),
1247 (C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm): 6.95 (t, 1H, J:
7.50 Hz, J = 7.50 Hz, Ph), 7.01 (d, 1H, J = 8.13 Hz, Ph), 7.23 (t, 1H,
J = 7.64 Hz, Ph), 7.54 (dd, 1H, J = 7.49 Hz, J = 4.66 Hz, Py), 8.02 (t, 1H,
J = 7.72 Hz, J = 7.72 Hz, Py), 8.17 (d, 1H, J = 7.7, Ph), 8.25 (d, 1H,
J = 7.213 Hz, Py), 8.31 (s, 1H, Th), 8.68 (dd, 1H, J = 4.68 Hz, J = 0.6 Hz,
Py), 10.56 (s, 1H, OH). 13C NMR: 116.98, 119.43, 119.77, 120.121,
125.72, 129.05, 129.73, 138.33, 150.27, 150.71, 153.17, 155.53,
167.29. For C14H10N2OS calcd. 66.12% C, 3.96% H, 11.02% N; found:
66.08% C, 3.96% H, 11.08% N. Mass (ESI), m/z: 255 (M+1).
3-[2-(Pyridin-4-yl)thiazol-4-yl]phenol (6)
Yield: 80%, mp: 234°C, IR υmax (cm−1): 3556.37–2986.72 (aromatic
C─H), 1262.25 (C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm): 6.84
(dd, 1H, J = 2.24 Hz, J = 8.11 Hz, Ph), 7.31 (t, 1H, J = 8.08 Hz,
J = 8.08 Hz, Ph), 7.50–7.52 (m, 2H, Ph), 8.26 (d, 2H, Py), 8.42 (s, 1H,
Th), 8.90 (d, 2H, J = 4.42 Hz, Py), 9.62 (s, 1H, OH). 13C NMR: 113.67,
116.25, 117.59, 119.01, 121.88, 130.44, 135.07, 143.39, 147.82,
157.04, 158.33, 163.13. For C14H10N2OS calcd. 66.12% C, 3.96% H,
11.03% N; found: 66.08% C, 3.97% H, 11.03% N. Mass (ESI) m/z: 255
(M+1).
2-[2-(Pyridin-3-yl)thiazol-4-yl]phenol (2)
Yield: 67%, mp: 134.1°C, IR υmax (cm−1): 3120–3000 (aromatic C─H),
1248 (C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm): 6.95 (t, 1H,
J = 7.49 Hz, J = 7.49 Hz, Ph), 7.01 (d, 1H, J = 8.08 Hz, Ph), 7.21–7.25
(m, 1H, Ph), 7.59 (dd, 1H, J = 4.83 Hz, J = 7.99 Hz, Ph), 8.19 (d, 1H,
J = 7.79 Hz, Py), 8.32 (s, 1H, Th), 8.39–8.41 (m, 1H, Py), 8.71 (d, 1H,
J = 4.79 Hz, Py), 9.22 (d, 1H, J = 1.64 Hz, Py), 10.55 (s, 1H, OH).
13C NMR: 116.94, 119.43, 119.78, 120.13, 124.78, 129.29, 129.31,
129.81, 134.14, 147.45, 151.47, 152.97, 155.53, 162.95. For
4-[2-(Pyridin-2-yl)thiazol-4-yl]phenol (7)
Yield: 70%, mp: 148.2°C, IR υmax (cm−1): 3109.26 (aromatic C─H),
1247.79 (C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm): 6.88 (d, 2H,
J = 8.57 Hz, Ph), 7.52 (dd, 1H, J = 4.93 Hz, J = 6.86 Hz, Py), 7.89 (d, 2H,
J = 8.56 Hz, Ph), 7.99–8.02 (m, 2H, Py and Th), 8.24 (d, 1H, J = 7.87 Hz,
Py), 8.66 (d, 1H, J = 4.73 Hz, Py), 9.66 (s, 1H, OH). 13C NMR: 114.58,
116.02, 119.68, 125.57, 125.79, 127.99, 138.22, 150.18, 151.01,
156.48, 158.14, 168.33. For C14H10N2OS calcd. 66.12% C, 3.96% H,
11.02% N; found: 66.10% C, 3.95% H, 11.06% N. Mass (ESI) m/z: 255
(M+1).
C
14H10N2OS calcd. 66.12% C, 3.96% H, 11.02% N; found: 66.05%
C, 3.98% H, 11.03% N. Mass (ESI), m/z: 255 (M+1).
2-[2-(Pyridin-4-yl)thiazol-4-yl]phenol (3)
Yield: 69%, mp: 131°C, IR υmax (cm−1): 3131.3–2856.8 (aromatic
C─H), 1279.58 (C─O). 1H NMR (500 MHz), (DMSO-d6) δ (ppm): 6.96
(t, 1H, J = 7.47 Hz, J = 7.47 Hz, Ph), 7.02 (d, 1H, J = 8.07 Hz, Ph), 7.24 (t,
1H, J = 7.64 Hz, J = 7.65 Hz, Ph), 7.97 (d, 2H, J = 5.4 Hz, Py), 8.19 (dd,
1H, J = 1.35 Hz, J = 7.79 Hz, Ph), 8.38 (s, 1H, Th), 8.74 (d, 2H, Py), 10.55
(s, 1H, OH). 13C NMR: 116.94, 119.33, 119.81, 120.56, 129.36,
129.29, 129.31, 129.36, 129.92, 139.87, 151.24, 153.31, 155.55,
163.29. For C14H10N2OS calcd. 66.12% C, 3.96% H, 11.02% N; found:
66.11% C, 3.92% H, 11.01% N. Mass (ESI) m/z: 255 (M+1).
4-[2-(Pyridin-3-yl)thiazol-4-yl]phenol (8)
Yield: 65%, mp: 252.8°C, IR υmax (cm−1): 3093.77–2805.3 (aromatic
C─H), 1257.70–1241.32 (C─O). 1H NMR (500 MHz) (DMSO-d6) δ
(ppm): 6.87 (d, 2H, J = 8.68 Hz, Ph), 7.59 (dd, 1H, J = 4.69 Hz,
J = 7.80 Hz, Py), 7.90 (d, 2H, J = 8.65 Hz, Ph), 8.02 (s, 1H, Th), 8.39
(d, 1H, J = 7.99 Hz, Py) 8.72 (s, 1H, Py), 9.23 (s, 1H, Py), 9.68 (s, 1H, OH).
13C NMR: 113.13, 116.03, 124.83, 125.56, 128.14, 134.11, 147.30,
151.25, 156.38, 158.24, 163.95. For C14H10N2OS calcd. 66.12% C,
3.96% H, 11.02% N; found: 66.15% C, 3.97% H, 11.03% N. Mass (ESI)
m/z: 255 (M+1).
3-[2-(Pyridin-2-yl)thiazol-4-yl]phenol (4)
Yield: 82%, mp: 172.4°C, IR υmax (cm−1): 3133.3 (aromatic C─H),
1235.02 (C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm): 6.82 (dd, 1H,
J = 2.21 Hz, J = 7.95 Hz, Ph), 7.29 (t, 1H, J = 7.85, J = 7.85 Hz, Ph),

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4-[2-(Pyridin-4-yl)thiazol-4-yl]phenol (9)
150.24, 150.67, 152.70, 153.08, 167.28. For C15H12N2O2S calcd.
63.36% C, 4.25% H, 9.85% N; found: 62.78% C, 3.83% H, 9.76% N.
Mass (ESI), m/z: 285 (M+1).
Yield: 73%, mp: 252.8°C, IR υmax (cm−1): 3118–3006.76 (aromatic
C─H), 1241.86 (C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm): 6.88
(d, 2H, J = 8.57 Hz, Ph), 7.90 (d, 2H, J = 8.56 Hz, Ph), 7.97 (d, 2H,
J = 4.70 Hz, Py and Ph), 8.11 (s, 1H, Th), 8.78 (d, 2H, Py), 9.70 (s, 1H,
OH). 13C NMR: 114.40, 116.07, 120.60, 125.42, 128.18, 140.13,
151.18, 156.77, 158.35, 164.39. For C14H10N2OS calcd. 66.12% C,
3.96% H, 11.01% N; found: 66.09% C, 3.98% H, 11.04% N. Mass (ESI)
m/z: 255 (M+1).
4-Methoxy-2-[2-(pyridin-3-yl)thiazol-4-yl]phenol (14)
Yield: 81.3%, mp: 167°C, IR υmax (cm−1): 3063 (aromatic C─H),
2944–2906 (aliphatic C─H), 1221 (C─O). 1H NMR (500 MHz) (DMSO-
d6) δ (ppm): 3.7 (s, 3H, OCH3), 6.8 (dd, 1H, J = 3.1 Hz, J = 8.8 Hz, Ph), 6.9
(d, 1H, J = 8.8 Hz, Ph), 7.60–7.57 (m, 1H, m, Py), 7.7 (d, 1H, J = 3.11 Hz,
Ph), 8.3 (s, 1H, Th), 8.4 (d, 1H, J = 5.9 Hz, Py), 8.7 (d, 1H, J = 3.3 Hz, Py),
9.2 (s, 1H, Py), 10.1 (s, 1H, OH). 13C NMR: 55.9, 113.68, 115.89,
117.69, 118.40, 120.35, 124.77, 129.27, 134.17, 147.47, 149.57,
151.48, 152.69, 152.88, 163.17. For C15H12N2O2S calcd. 63.36% C,
4.25% H, 9.85% N; found: 63.05% C, 4.18% H, 9.70% N. Mass (ESI),
m/z: 285 (M+1).
4-Methyl-2-[2-(pyridin-2-yl)thiazol-4-yl]phenol (10)
Yield: 78.4%, mp: 167°C, IR υmax (cm−1): 3102–3048 (aromatic C─H),
2987–2914 (aliphatic C─H), 1246 (C─O). 1H NMR (500 MHz) (DMSO-
d6) δ (ppm): 2.3 (s, 3H, CH3), 6.8 (d, 1H, J = 8.1 Hz, Ph), 7.0 (dd, 1H,
J = 2.0 Hz, J = 8.2 Hz, Ph), 7.55–7.52 (m, 1H, Py), 7.9 (d, 1H, J = 1.7 Hz,
Ph), 8.0 (td, 1H, J = 1.6 Hz, J = 7.7 Hz, Py), 8.2 (d, 1H, J = 7.8 Hz, Py), 8.2
(s, 1H, Th), 8.6 (d, 1H, J = 4.1 Hz, Py), 10.2 (s, 1H, OH). 13C NMR: 20.74,
116.87, 119.2, 119.78, 119.82, 125.70, 128.17, 129.15, 130.27,
138.32, 150.26, 150.71, 153.35, 167.24. For C15H12N2OS calcd.
67.14% C, 4.51% H, 10.44% N; found: 66.12% C, 4.33% H, 9.99% N.
Mass (ESI), m/z: 269 (M+1).
4-Methoxy-2-[2-(pyridin-4-yl)thiazol-4-yl]phenol (15)
Yield: 78.6%, mp: 153.4°C, IR υmax (cm−1): 3124–3065 (aromatic
C─H), 2951 (aliphatic C─H), 1215 (C─O). 1H NMR (500 MHz) (DMSO-
d6) δ (ppm): 3.7 (s, 3H, OCH3), 6.8 (dd, 1H, J = 3.1 Hz, J = 8.8 Hz, Ph), 6.9
(d, 1H, J = 8.8 Hz, Ph), 7.7 (d, 1H, J = 3.12 Hz, Ph), 7.9 (d, 2H, J = 6.0 Hz,
Py), 8.4 (s, 1H, Th), 8.7 (d, 2H, J = 4.6 Hz, Py), 10.1 (s, 1H, OH).
13C NMR: 55.9, 113.78, 115.96, 117.70, 119.69, 120.34, 120.57,
139.81, 149.60, 151.28, 152.70, 153.20, 163.29. For
4-Methyl-2-[2-(pyridin-3-yl)thiazol-4-yl]phenol (11)
Yield: 80.2%, mp: 217°C, IR υmax (cm−1): 3095, 3044, 3021 (aromatic
C─H), 2919 (aliphatic C─H), 1251–1238 (C─O). 1H NMR (500 MHz)
(DMSO-d6) δ (ppm): 2.3 (s, 3H, CH3), 6.9 (d, 1H, J = 8.1 Hz, Ph), 7.03 (dd,
1H, J = 2.0 Hz, J = 8.1 Hz, Ph), 7.76–7.73 (m, 1H, Py), 8.0 (d, 1H,
J = 1.7 Hz, Ph), 8.3 (s, 1H, Th), 8.6 (d, 1H, J = 6.4 Hz, Py), 8.7 (d, 1H,
J = 4.4 Hz, Py), 9.3 (s, 1H, Py), 10.3 (s, 1H, OH). 13C NMR: 20.72,
116.83, 118.54, 119.68, 125.65, 128.18, 129.45, 130.12, 130.41,
136.40, 145.64, 149.30, 153.24, 153.37, 161.94. For C15H12N2OS
calcd. 67.14% C, 4.51% H, 10.44% N; found: 66.87% C, 4.13% H,
9.31% N. Mass (ESI), m/z: 269 (M+1).
C15H12N2O2S calcd. 63.36% C, 4.25% H, 9.85% N; found: 62.77%
C, 3.90% H, 9.31% N. Mass (ESI), m/z: 285 (M+1).
4-Chloro-2-[2-(pyridin-2-yl)thiazol-4-yl]phenol (16)
Yield: 79.5%, mp: 193.7°C, IR υmax (cm−1): 3105 (aromatic C─H), 1247
(C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm): 7.0 (d, 1H, J = 8.6 Hz,
Ph), 7.2 (dd, 1H, J = 2.7 Hz, J = 8.6 Hz, Ph), 7.54–7.52 (m, 1H, m, Py), 8.0
(td, 1H, J = 1.6 Hz, J = 7.6 Hz, Py), 8.2 (d, 1H, J = 2.7 Hz, Ph), 8.2 (d, 1H,
J = 7.8 Hz, Py), 8.3 (s, 1H, Th), 8.6 (d, 1H, J = 4.7 Hz, Py), 10.7 (s, 1H,
OH). 13C NMR: 118.61, 119.94, 120.91, 122.10, 123.41, 125.77,
128.30, 129.07, 138.30, 150.22, 150.64, 151.47, 154.40, 167.51. For
4-Methyl-2-[2-(pyridin-4-yl)thiazol-4-yl]phenol (12)
Yield: 79.3%, mp: 184.5°C, IR υmax (cm−1): 3106–3031 (aromatic
C─H), 2913 (aliphatic C─H), 1238 (C─O). 1H NMR (500 MHz) (DMSO-
d6) δ (ppm): 2.3 (s, 3H, CH3), 6.9 (d, 1H, J = 8.2 Hz, Ph), 7.0 (dd, 1H,
J = 1.8 Hz, J = 8.1 Hz, Ph), 7.9 (d, 2H, J = 6.0 Hz, Ph), 8.0 (d, 1H,
J = 1.6 Hz, Ph), 8.3 (s, 1H, Th), 8.7 (d, 2H, J = 6.0 Hz, Py), 10.3 (s, 1H,
OH). 13C NMR: 20.72, 116.83, 119.19, 119.71, 120.57, 128.21,
129.45, 130.45, 139.86, 151.28, 153.38, 153.46, 163.23. For
C14H9ClN2OS calcd. 58.23% C, 3.14% H, 9.70% N; found: 57.34% C,
2.90% H, 9.45% N. Mass (ESI), m/z: 289 (M+1).
4-Chloro-2-[2-(pyridin-3-yl)thiazol-4-yl]phenol (17)
Yield: 80.3%, mp: 179.3°C, IR υmax (cm−1): 3094–3041 (aromatic
C─H), 1246 (C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm): 7.0 (d, 1H,
J = 8.67 Hz, Ph), 7.2 (dd, 1H, J = 2.7 Hz, J = 8.6 Hz, Ph), 7.59–7.56
(m, 1H, Py), 8.2 (d, 1H, J = 2.6 Hz, Ph), 8.3 (s, 1H, Th), 8.4 (d, 1H,
J = 9.7 Hz, Py), 8.7 (d, 1H, J = 4.7 Hz, Py), 9.2 (s, 1H, Py), 10.7 (s, 1H,
OH). 13C NMR: 118.58, 119.50, 121.97, 123.41, 124.75, 128.51,
129.18, 129.22, 134.22, 147.53, 151.29, 151.53, 154.40, 163.17. For
C15H12N2OS calcd. 67.14% C, 4.51% H, 10.44% N; found: 66.31%
C, 3.99% H, 10.31% N. Mass (ESI), m/z: 269 (M+1).
4-Methoxy-2-[2-(pyridin-2-yl)thiazol-4-yl]phenol (13)
Yield: 79.8%, mp: 142.8°C, IR υmax (cm−1): 3076 (aromatic C─H), 2981
(aliphatic C─H), 1222 (C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm):
3.8 (s, 3H, OCH3), 6.8 (dd, 1H, J = 3.1 Hz, J = 8.8 Hz, Ph), 6.9 (d, 1H,
J = 8.8 Hz, Ph), 7.54–7.52 (m, 1H, Py), 7.7 (d, 1H, J = 3.0 Hz, Ph), 8.0 (td,
1H, J = 1.7 Hz, J = 7.7 Hz, Py), 8.2 (d, 1H, J = 7.8 Hz, Py), 8.3 (s, 1H, Th),
8.6 (d, 1H, J = 3.6 Hz, Py), 10.1 (s, 1H, OH). 13C NMR: 55.9, 113.52,
115.76, 117.71, 119.79, 119.82, 120.43, 125.72, 138.31, 149.57,
C14H9ClN2OS calcd. 58.23% C, 3.14% H, 9.70% N; found: 57.78% C,
2.99% H, 9.21% N. Mass (ESI), m/z: 289 (M+1).
4-Chloro-2-[2-(pyridin-4-yl)thiazol-4-yl]phenol (18)
Yield: 78.5%, mp: 203.6°C, IR υmax (cm−1): 3032 (aromatic C─H), 1254
(C─O). 1H NMR (500 MHz) (DMSO-d6) δ (ppm): 7.0 (d, 1H, J = 8.6 Hz,
Ph), 7.2 (dd, 1H, J = 2.7 Hz, J = 8.6 Hz, Ph), 7.9 (d, 2H, J = 6.0 Hz Py), 8.2

ERTAS ET AL.
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(d, 1H, J = 2.7 Hz, Ph), 8.4 (s, 1H, Th), 8.7 (d, 2H, J = 4.6 Hz, Py), 10.7 (s,
1H, OH). 13C NMR: 118.60, 120.66, 120.88, 121.95, 123.42, 128.55,
129.30, 139.75, 151.29, 151.60, 154.43, 163.58. For C14H9ClN2OS
calcd. 58.23% C, 3.14% H, 9.70% N; found: 57.77% C, 2.95% H, 9.55%
N. Mass (ESI), m/z: 289 (M+1).
with a stop reagent, 0.5 M Tris base. After the reaction was terminated,
fluorescence was measured at 409 nm (excitation) and 530 nm
(emission). Experiments were performed in triplicate and the average
values were used to construct the dose–response curves. GraphPad
Prism software Version 7.02 (La Jolla, California, USA) was used to
calculate the IC50 values of a minimum of eight concentrations of each
test substance (Table 3).
|
3.2 Pharmacology
|
|
3.2.1 Aromatase activity assay
3.2.2 Evaluation of anticancer activity against MCF7
and HEK 293 cells
Aromatase inhibition is quantified by measuring the fluorescent
intensity of fluorescein, the hydrolysis product of dibenzylfluorescein,
by aromatase. With slight modification to the method described by
Stresser,[34] experiments were carried out following the instructions
provided by the “CYP19/MFC High Throughput Inhibitor Screening
assay kit” (Corning, Catalog number: 459520). Stock solutions of the
compounds were prepared with DMSO (1%). In brief, the test
substance (10 µM) and the standard solution [3 μL HFC (7-hydroxy-
4-trifluoromethyl coumarone 0.25 M), 147 μL NADPH-Cofactor Mix
without acetonitrile] were preincubated with the NADPH regenerating
system (1.3 mM NADP+, 66 mM glucose 6-phosphate dehydrogenase,
66 mM MgCl2) for 10 min at 37°C. Then, 100 µL of the enzyme and
substrate mixture [1 µM enzyme (CYP19), 25 mM 7-methoxy-4-
trifloromethyl coumarone and 0.5 M phosphate buffer, pH 7.4] were
added. The reaction mixture was incubated for 30 min at 37°C to allow
aromatase to generate the product and the reaction was quenched
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
is a water soluble tetrazolium salt. The MTT assay was discovered by
Mosmann (1983).[35] In this colorimetric technique, the tetrazolium
ring is converted to an insoluble purple formazan by cleavage of that
ring by increased activity of succinate dehydrogenase on live cells
within the mitochondria. Experiments were carried out following the
instructions given in “Cell Proliferation Kit I (MTT)” (Roche, Catalog
Number: 11 465 007 001). The effects of tested compounds on cell
viability of the human breast adenocarcinoma cell line (MCF7) and
human embryonic kidney cell line (HEK 293) were determined using
this colorimetric technique. MCF7 and HEK 293 cells were cultured in
96-well plates in 100 μL of Dulbecco's Modified Eagle's Medium
(GIBCO, UK) with 10% of charcoal stripped fetal bovine serum (GIBCO,
UK), 1% penicillin-streptomycin (GIBCO, UK), 1% L-glutamine (GIBCO,
UK) per well at a cell density of 5 × 104 cell/wells. Experiment sets of
TABLE 3 Calculated drug-likeness, molecular properties, and ADME predictions for the active compounds using QikProp
Compound
#stars
0.00
0.00
0.00
0.00
0.00
0.00
1.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1.00
0.00
CNS
1.00
0.00
1.00
0.00
0.00
0.00
0.00
0.00
0.00
1.00
0.00
0.00
1.00
0.00
0.00
1.00
1.00
1.00
logPo/w
3.28
2.95
2.95
3.16
2.82
2.82
3.17
2.83
2.83
3.44
3.22
3.22
3.34
3.03
3.03
3.70
3.36
3.36
logHERG
−5.59
−5.48
−5.46
−5.64
−5.52
−5.52
−5.64
−5.53
−5.53
−5.55
−5.43
−5.43
−5.49
−5.41
−5.38
−5.55
−5.42
−5.42
PCaco
logBB
0.13
PMDCK
2876.81
1923.02
2009.79
1743.99
1221.57
1224.67
1738.77
1216.28
1220.20
2864.81
2002.83
2000.72
2848.85
1899.45
2015.03
7046.34
4927.40
4930.27
log Kp
−1.07
−1.40
−1.36
−1.50
−1.80
−1.80
−1.50
−1.80
−1.80
−1.27
−1.56
−1.56
−1.17
−1.51
−1.46
−1.24
−1.53
−1.53
log KHSA
0.18
0.08
0.08
0.19
0.09
0.09
0.19
0.09
0.09
0.33
0.23
0.23
0.19
0.10
0.09
0.29
0.19
0.19
HOAa
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
%HOA
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
1
2993.15
2149.23
2233.53
1900.06
1411.73
1413.03
1894.97
1406.67
1408.05
2980.62
2228.99
2223.69
2989.42
2115.32
2238.54
2979.05
2227.58
2225.93
2
−0.02
0.00
3
4
−0.07
−0.20
−0.20
−0.08
−0.20
−0.20
0.11
5
6
7
8
9
10
11
12
13
14
15
16
17
18
−0.02
−0.02
0.05
−0.10
−0.07
0.29
0.16
0.16
aThe assessment uses a knowledge-based set of rules including checking for suitable values for the percent of human oral absorption, number of metabolites,
number of rotatable bonds, logP, solubility, and cell permeability.

10 of 11
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|
different concentrations were prepared. Control wells were formed by
preparing culture media without compounds. The cells were incubated
for 24 h (37°C and 5% CO2). Following this step, each medium in wells
was changed with fresh medium and different concentrations of
compounds was added and incubated for 6 h. After incubation, 10 μL
of MTT (final concentration 0.5 mg/mL) was added and left for 4 h. A
total of 100 μL of a solubilization solution containing 10% SDS in
0.01 M HCl was added into each well. Absorbance at 550 nm was
recorded with an ELISA plate reader.[36,37] Each set of experiments
was performed in triplicate.
channels, logHERG (concern <−5); Caco-2 cell membrane permeability
in nm/s, PCaco (<5 low to >100 high); the logarithm of the predicted
blood/brain barrier (BBB) partition coefficient, logBB (−3.0 to 1.0);
apparent Madin–Darby canine kidney cell permeability (PDMCK) that
mimics BBB for non-active transport in nm/s, PMDCK (<25 poor to
>500 great); skin permeability, log Kp (−8.0 to −1.0); the logarithm of
binding constant to human serum albumin, log KHSA (−1.5 to 1.2);
qualitative human oral absorption (HOA) (1: low, 2: medium, 3: high);
and percent of HOA (>80%: high, <25%: poor).
ACKNOWLEDGMENT
|
3.3 Molecular modeling studies
This study was supported by Anadolu University Scientific Research
Projects Commission (Project No: 1410S422).
|
3.3.1 Ligand preparation and docking experiment
The 3D diagrams of the synthesized compounds were drawn using the
3D builder implemented in the Maestro suite (Schrödinger LCC,
Oregon, USA). The ligands were prepared and minimized by means of
the OPLS_2005 force field and the partial atomic charges, ionization,
and tautomerization states were computed at a pH range of 6–8 by
Epik v1620716 using the LigPrep module of the same suite.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ORCID
The most active compound found using the methods of the
aromatase inhibition assay, 3-[2-(pyridin-4-yl)thiazol-4-yl]phenol
(compound 6), was subjected to a series of docking experiments to
identify the probable interactions between the ligand and the
aromatase active site. The preparation of the protein structure and
ligand, GRID files, docking, and scoring were performed using
algorithms included in the Maestro modules (Schrodinger Inc, USA).
The X-ray crystallographic structure of human placental aromatase
(PDB entry code: 3EQM) was obtained from the Protein Data Bank
(RCSB).[7] The 3EQM PDB file was edited using the protein preparation
wizard from the Workflows menu (Maestro) for hydrogen insertion
and rotamer adjustment, and H-bond optimization using OPLS 2005 as
the energy parameters. Ligand docking was performed with the Glide
module in Schrodinger. Extra Precision (XP) and induced-fit protocols
included in Glide were employed with the parameter settings
recommended by Caporuscio et al.[29] for docking studies.
Merve Ertas
Leyla Yurttas
http://orcid.org/0000-0002-2289-7950
http://orcid.org/0000-0002-0957-6044
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How to cite this article: Ertas M, Sahin Z, Berk B, Yurttas L,
Biltekin SN, Demirayak S. Pyridine-substituted
thiazolylphenol derivatives: Synthesis, modeling studies,
aromatase inhibition, and antiproliferative activity evaluation.
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