Studies on non-steroidal inhibitors of aromatase enzyme; 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives

Article abstract of DOI:10.1016/j.bmc.2018.02.048

Steroidal and non-steroidal aromatase inhibitors target the suppression of estrogen biosynthesis in the treatment of breast cancer. Researchers have increasingly focused on developing non-steroidal derivatives for their potential clinical use avoiding steroidal side-effects. Non-steroidal derivatives generally have planar aromatic structures attached to the azole ring system. One part of this ring system comprises functional groups that inhibit aromatization through the coordination of the haem group of the aromatase enzyme. Replacement of the triazole ring system and development of aromatic/cyclic structures of the side chain can increase selectivity over aromatase enzyme inhibition. In this study, 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives were synthesized and physical analyses and structural determination studies were performed. The IC₅₀ values were determined by a fluorescence-based aromatase inhibition assay and compound 1 (4-(2-hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole) were found potent inhibitor of enzyme (IC₅₀:0.42 nM). Then, their antiproliferative activity over MCF-7 and HEK-293 cell lines was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds 1, 7, 8, 13, 15, 18, 21 were active against MCF-7 breast cancer cells. Lastly, a series of docking experiments were undertaken to analyze the crystal structure of human placental aromatase and identify the possible interactions between the most active structure and the active site.

Full text of DOI:10.1016/j.bmc.2018.02.048

Accepted Manuscript
Studies on Non-Steroidal Inhibitors of Aromatase Enzyme; 4-(aryl/heteroar-
yl)-2-(pyrimidin-2-yl)thiazole Derivatives
Zafer Şahin, Merve Ertaş, Barkın Berk, Sevde Nur Biltekin, Leyla Yurttaş,
Şeref Demirayak
PII:
S0968-0896(18)30024-5
https://doi.org/10.1016/j.bmc.2018.02.048
BMC 14234
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
8 January 2018
22 February 2018
26 February 2018
Please cite this article as: Şahin, Z., Ertaş, M., Berk, B., Nur Biltekin, S., Yurttaş, L., Demirayak, S., Studies on
Non-Steroidal Inhibitors of Aromatase Enzyme; 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole Derivatives,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.048
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Studies on Non-Steroidal Inhibitors of Aromatase Enzyme; 4-(aryl/heteroaryl)-2-
pyrimidin-2-yl)thiazole Derivatives
(
The authors whose names are listed immediately below certify that they have NO affi liations with or
involvement in any organization or entity with any fi nancial interest (such as honoraria; educational
grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership,
or other equity interest; and expert testimony or patent-licensing arrangements), or non-fi nancial
interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject
matter or materials discussed in this manuscript.
1*
1
1
2
3
Zafer Şahin , Merve Ertaş , Barkın Berk , Sevde Nur Biltekin , Leyla Yurttaş , Şeref
1
Demirayak
1
İstanbul Medipol University, School of Pharmacy, Department of Pharmaceutical
Chemistry, 34083 İstanbul, Turkey.
2
İstanbul Medipol University, School of Pharmacy, Department of Pharmaceutical
Microbiology, 34083 İstanbul, Turkey.
3
Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470
Eskişehir, Turkey.
*Corresponding author: Zafer Şahin (E-mail address: zsahin@medipol.edu.tr)

Abstract
Steroidal and non-steroidal aromatase inhibitors target the suppression of estrogen
biosynthesis in the treatment of breast cancer. Researchers have increasingly focused on
developing non-steroidal derivatives for their potential clinical use avoiding steroidal side-
effects.
Non-steroidal derivatives generally have planar aromatic structures attached to the azole ring
system. One part of this ring system comprises functional groups that inhibit aromatization
through the coordination of the haem group of the aromatase enzyme. Replacement of the
triazole ring system and development of aromatic/cyclic structures of the side chain can
increase selectivity over aromatase enzyme inhibition.
In this study, 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives were synthesized and
physical analyses and structural determination studies were performed. The IC50 values were
determined by a fluorescence-based aromatase inhibition assay and compound 1 (4-(2-
hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole) were found potent inhibitor of enzyme
(IC50:0.42nM). Then, their antiproliferative activity over MCF-7 and HEK cell lines was
evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Compounds 1, 7, 8, 13, 15, 18, 21 were active against MCF-7 breast cancer cells.
Lastly, a series of docking experiments were undertaken to analyze the crystal structure of
human placental aromatase and identify the possible interactions between the most active
structure and the active site.
Key Words: Anticancer, molecular modelling, thiazoles, aromatase, non-steroidal

1. Introduction
Breast cancer is one of the most common cancer types. Surgical methods, radiotherapy, and
reversible inhibition of estrogen synthesis or its interaction with drugs constitute the basis of
1
treatment for breast cancer . The investigated history of breast cancer suggests that many
2
tumors occur and develop through estrogen-dependent mechanisms . Accordingly, it can be
assumed that estrogen deprivation may prevent the formation of these cancers or may result in
3,4
the regression of occurred tumors . The target of this suppression is the inhibition of the
aromatase enzyme responsible for the cyclization and structuring of estrogen. These processes
are mechanically constructed by inhibiting the last step, in which the aromatic ring in the
steroid structure is formed during biosynthesis. In this step, the ketone group at position 3 is
5
converted to 3-hydroxyl by dihydronicotinamide-adenine dinucleotide phosphate (NADPH) .
The methyl group at position 19 is eliminated and aromatization of the A-ring of estrogen
takes place. These processes are responsible for aromatase, a cytochrome P450 derivative,
6,7
and related flavoproteins .
The key role of aromatase in estrogen biosynthesis makes this enzyme a promising candidate
8
,9
for the development of drugs to be used in the treatment of endocrine-derived tumors . Due
to the serious side effects of steroid-derived inhibitors involved in the treatment regimen,
research in recent years has mostly focused on the development of non-steroidal derivatives
10,11
and undertaking clinical trials
.
1
2
Non-steroidal compounds usually contain triazole structures attached to the aromatic ring .
In addition, it forms part of the ring structure in functions that are coordinated with the iron in
the haem group of the aromatase enzyme and effectively inhibit hydroxylation reactions for
13
aromatization . Linked to the heme group and the active site, this combination is responsible
for a high effect and target specificity. By changing the triazole system with bioisosteres and
improving the aromatic / cyclic structures in the side chain selectively inhibits the aromatase
1
4,15
enzyme . Studies on pyrimidinyl thiazole derivatives have shown varying pharmacological
properties through anti-inflammatory, histamine H receptor antagonistic, and antibacterial
2
16,17
activities on Helicobacter pylori . In a study investigating the aromatase activity of the
thiazole ring system, nitrogen containing heterocyclic structures were attached using
diarylmethane and diarylmethanol intermediate chain structures, and the compounds showed
1
8
enzyme inhibition at the nanomolar level in in vitro experiments . In another study, 2-bromo
or non-substituted thiazole structures linked to the free nitrogen group of the 4-amino-1,2,4-
triazole structures by the methyl bridge at position 5 were found to have an inhibitory activity
1
9
on the aromatase enzyme . A similar situation was observed in the work of Shibata et al., in
which the activity of aromatase enzyme was obtained by adding diazole, triazole and 4-
2
0
cyanophenyl structures to a carbon bridge as well as thiazole and benzothiazole rings . In
2008, Eriksson et al. found that combining different thiazole derivatives / analogs with

tamoxifen showed a high aromatase inhibitor activity and a regression in the proliferation of
2
1
breast cancer cells at the micromolar level .
Thiazole and phenol groups in resveratrol, thiazofurin, and bleomycin are common
functionals exhibiting anticancer activity in the structure of natural compounds and drug
molecules. The resveratrol structure is able to modulate many steps in carcinogenesis;
however, despite its high efficiency, this structure also has certain disadvantages. In the
literature, it has been found that the effect of aromatase inhibition increases at a high rate
when the ethylenic structure of resveratrol is replaced with thiadiazole, and phenolic groups
2
2,23
are replaced with pyridines . Furthermore, replacing the central ring system of the
resveratrol analogues by 1,3-thiazole ring (IV) results in an aromatase inhibitor activity of
6,000 times greater than the original structure (I). In particular when it is nitrogen
nd
th
heterocyclic ring (III,IV), the aromatic side chains at 2 and 4 positions of central ring
2
4
systems seems to be significantly responsible for the enhanced activity (Figure 1).
Figure 1. Resveratrol and derived structures similar to synthesized compounds
Molecular docking, quantitative structure–activity relationship (QSAR) and high-throughput
virtual screening (HTVS) studies have a very widespread use in computer-assisted drug
2
5-28
design . Suvannang et al. used the published x-ray crystallographic data to examine known
aromatase inhibitors in the docking process and found that the enzyme polar (ASP309,
THR310, SER478, and MET374), aromatic (PHE134, PHE221, and TRP224), and non-polar
(ALA306, ALA307, VAL370, LEU372, and LEU477) have played an important role in the
29
interaction . Ferlin et al. determined that the synthesized pyrrolo [2,3-h] quinoline and
pyrrolo [3,2-f] quinoline derivatives inhibited the aromatase CYP19 enzyme but did not
inhibit the CYP11B1 and CYP17 enzymes against the letrozole standard. In the same study,
the interactions of the compounds with enzymes were examined using a docking method to
3
0
identify the amino acids that played an active role in the active region . There are also other
studies involving the use of different QSAR-linked docking analyses to determine such
31-36
interactions and activities
.

In the present study, we prepared 21 novel non-steroidal 4-(aryl/heteroaryl)-2-(pyrimidin-2-
yl)thiazole derivatives to inhibit the active site of the aromatase cytochrome P450. The
aromatase inhibitory activities of the molecules were measured by a fluorescence-based assay
using ketoconazole as the standard. Then, antiproliferative activities of the compounds on the
human breast adenocarcinoma (MCF7) and human embryonic kidney (HEK-293) cell lines
were evaluated with an MTT assay. Further interactions between the active molecule and the
active site of the enzyme were analyzed through docking experiments using the “crystal
structure of human placental aromatase cytochrome P450 in complex with androstenedione”
37
(
3EQM) . In addition, absorption, distribution, metabolism, and excretion (ADME), drug-
likeness, and molecular properties of the molecules were predicted by in silico techniques.
2
2
. Results
.1. Chemistry
The synthesis of all 21 compounds was carried out at a yield of 66-91% using the proposed
method (Scheme 1). The compounds were purified by crystallization twice with ethanol
before undertaking biological activity tests. The purity of the compounds was determined to
be above 95% by high-pressure liquid chromatography. In the IR spectrum of the compounds,
-1
aromatic C-H stretching bands were observed in the range of 2800-3000 cm and O-H
stretching bands of Compounds 1-6 containing OH functional groups were recorded to be
-1
1
around 3100 cm . The results of the H-NMR spectrums of the compounds were as follows:
Thiazole hydrogen was observed to coincide with pyridine hydrogens in the 3 and 4 pyridine
derivatives. For the remaining 19 compounds, thiazole hydrogens were present as a singlet
and one hydrogen in the range of 8.04-8.77 ppm while two hydrogen atoms in the position
closest to the nitrogen atoms of the pyrimidine ring system were observed as a doublet in two
hydrogen sites in the range of 9 ± 0.34 ppm. The other hydrogen atoms were observed as a
triplet or overlapping with aryl groups at approximately 7.6 ppm. In the hydroxyl group-
containing compounds, hydrogen belonging to this functional group was observed as a singlet
in the range of 9.57-11 ppm and in the methoxy group-containing compounds (Compounds 5,
7
-9), methyl groups were present in the 3H range and in the range of 3.77-3.95 ppm. In
addition, the numbers of hydrogens on the aryl groups showed d, t, dd and m breaks in the
field. For all compounds, the H number in the spectra was consistent with the calculated
1
3
number. In the C NMR spectra, the observed carbon numbers were consistent with the
calculated numbers. The carbon atom at position 2 of the thiazole and carbon atoms adjacent
to the nitrogen in the pyrimidine were observed separately from the other carbons in the range
+1
of 155-160. The M peaks in the mass spectra calculated with the ESI method were parallel
to the molecular weights of the compounds.

Scheme 1. Synthesized 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivative compounds
and general synthesis
2
2
.2. Biological evaluation
.2.1. Enzymatic tests on aromatase inhibition
Measurement of aromatase enzyme inhibition activity of 21 compounds and standard
ketoconazole (IC50 = 0.95 μM) was performed at a concentration range of 15 μM to 4.5 nM.
The obtained values are listed in Table 1.
Compound
IC50 (µM)
0.00042
3.04568
5.71579
3.59114
Compound
IC50 (µM)
3.40925
1.09268
3.32179
0.93546
1
2
3
4
12
13
14
15

5
6
7
8
9
3.00088
3.56519
1.18136
0.17682
3.18768
1.70322
3.25328
16
2.60071
3.48215
3.34523
1.80456
1.98440
0.99974
0.95148
17
18
19
20
21
10
11
Ketoconazole
Table 1. The IC50 values obtained after the aromatase inhibition activity test of the
synthesized substances (at 9 different concentrations from 15 μM to 4.5 nM)
Figure 2 shows the dose-response curves of the ketoconazole standard and Compounds 1, 7,
8, 13, 15 and 21.
AROMATASE INHIBITORY
ACTIVITY
Figure 2. The dose-response curves of Compounds 1, 7, 8, 13, 15 and 21 and ketoconazole

Aromatase inhibition activity of 21 compounds and the standard ketoconazole were
determined based on the principle of measuring the amount of fluorescein formed in the
medium. The IC50 values obtained from all compounds were promising. Compound 1 was
2000 times and Compound 8 was 9 times more active than ketoconazole. Furthermore, the
activities of Compounds 15 and 21 were equivalent to ketoconazole. Other IC50 values that
were close to that of ketoconazole belonged to Compounds 7 (1.18 μM), 8 (0.17 μM), 1 (0.41
nM), 13 (1.09 μM), 15 (IC50 = 0.93 μM), and 21 (IC50 = 0.99 μM).
2.2.2. MTT proliferation assay
-4
To measure the effects of the substances on cell viability, 10 M concentrations of the
substances were studied on HEK as breast cancer cell and MCF-7 as control (healthy) cell
groups (Figure 3).
-
4
Figure 3. Effects of compounds on the viability of MCF-7 and HEK cells at the 10 M
concentration
-4
1
0 M concentrations of all compounds were added to the breast cancer cell MCF-7 and
healthy cell group HEK to examine their effects on the life cycles of the cells. Percent
viability of the compounds on these cell lines was measured. Compounds 13, 14, 15, 16 and
21 were more effective on the MCF7 cell line in terms of viability. The inhibitory effect of
the most active compound (Compound 1) was very similar on the HEK and MCF7 cell lines.
In the MTT viability test, Compound 21 exhibited viability effect as high as 40% on the
MCF7 cell line, but it did not have any toxic effect on the HEK cell line (Table 2).

MCF7
HEK293
Compound
Compound
% Viability
129,23
111,91
227,15
195,07
208,17
161,79
129,89
136,94
127,44
125,40
86,88
% Viability
131,18
113,31
94,02
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
91,84
81,26
78,00
86,95
94,98
100,52
89,41
1
1
1
1
1
1
1
1
1
1
2
2
0
1
2
3
4
5
6
7
8
9
0
1
106,73
100,98
101,28
89,05
146,08
74,18
75,45
76,62
76,05
101,20
58,96
135,74
131,23
119,92
127,87
76,99
107,72
83,51
101,52
6
0,72
117,34
Table 2. MTT % control viability values of all 21 compounds comparingly HEK 293 versus
MCF7
Different concentrations of compounds were tested on both cell lines but IC50 values could not
be given because of high concentrations.
2
2
.3. Molecular modeling studies
.3.1. Docking studies
Compound 1 had the lowest IC50 value during aromatase inhibition; thus, this compound was
used in the docking experiment to determine 3EQM and identify its interaction pattern with
the active site. The experiment was first performed utilizing the XP algorithm of Glide
simulating the receptor as rigid and the ligands as flexible. For the docking protocol, the same
constraints were used as described by 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
3
8,39
subsequent oxidation reaction of the substrate . A van der Waals radius scaling factor of
.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
0

heme residue, and default sizes were used for both the enclosing and bounding boxes. The
ligand poses in which an aromatic nitrogen atom interacts with the heme iron were
o
determined with a metal constraint. The ligand nitrogen must be within 2.4 A of the metal to
satisfy the constraint. To validate the experiment, anastrozole, a non-steroidal aromatase
inhibitor interacting 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 1 (Figure 4). Then,
the experiment was improved using the induced-fit protocol of Glide, in which both receptor
and ligand were treated as flexible. The overall settings of this protocol were the same as
38
described by Caporuscio et al. except for the initial use of the XP protocol rather than SP
(Figure 5).
Figure 4. The anastrozole structure and position of Compound 1 in the active site (ligand C in
the crystal structure: dark gray, anastrozole C: light green, H: white, N: blue, O: red, S:
yellow)
A

B
C
Figure 5. The poses (A, B.) and interactions (C) of Compound 1 obtained through the
induced fit protocol (C: dark gray, N: blue S: yellow O: red)
The final step of our work consisted of molecular modeling studies for examining the pattern
of activity. In this step, 3EQM published in the protein database and different docking
37
protocols was used as a guide . The iron atom located in the center of the heme group in the
active site of the aromatase enzyme was present in + 3 charge during attaching. In particular,
a charge-based interaction between the unpaired electrons of the nitrogen structures found in

non-steroidal compounds and the charged atom was observed. While the nitrogens of the
pyrimidine ring in the structure of Compound 1 promoted this interaction, they also pushed
the molecule towards more hydrophobic portions of the active site relative to the anastrozole
structure. The H bond was formed by the OH structure of the ortho position of Compound 1
and Leu 477 presented in the active region by this process. The presence of an OH group in
the meta or para position of the OH structure did not support this interaction.
There was an H bond between the pyrimidine nitrogen atom and the active site Thr310. The
interconnection of the three aromatic rings ensured that they were in harmony with the
surrounding hydrophobic domain. At the same time, the addition of voluminous hydrophobic
groups and the pyrimidine ring to the thiazole ring did not support this interaction. The
resulting pose was compatible with the poses obtained from similar compounds containing
22,23
the pyrimidine ring in the literature
. Discussion
.
3
If the enzyme activity results taken together, excluding 8 and 15, compounds with electron-
nd
rich groups at 2 position of Ar group are also more active as explained in modelling (Figure
6). Compound 1 (0.00042 µM) has hydroxyl group at second position which is considered to
bind Leu 477 residue. Similarly 2-methoxyphenyl (7) substition (1.18136 µM) may have
effect on enzyme. Although the compounds 4, 5 and 6 contains the 2-hydroxy group, other
substituents in the ring may have affected this positioning. 2-pyridine (13) (1.09268 µM) and
2
-benzothiazole (21) (0.99974 µM) substitions may show its effect by nitrogen at second
position. 2-chlorophenyl (10) (1.70322 µM) substition also effective against enzyme.
Benzimidazole substition were found less effective possibly due to steric effect of methyl
group. According to this positioning, 4-pyridile group of 15 may be interacted with Met374.
Ar
Y
X
N
N
N
S
Figure 6. Active compounds according to enzymatic results where Ar is 2-hydroxyphenyl (1), 2-
methoxyphenyl (7), 2-chlorophenyl (10), 2-pyridyle (13) and benzothiazole (21). X or Y is a
heteroatom (N, O or Cl).
According to MTT results, expected property is to inhibit proliferation by saving the
reproduction of HEK293. Compounds 11 (%13), 13 (%26), 14 (%24), 15 (%23), 16 (%24)

and 21 % (40) inhibited the proliferation of MCF7 breast cancer cells. Compounds 14 and 16
also inhibited the reproduction of HEK293 cells undesirably. Besides, compounds 11, 13, 15
and 21 were selective for inhibition of MCF7 cells. They did not show toxicity on HEK293
cells. Remarkably, selective compounds 13, 15 and 21 also showed good activity in
enzymatic assay.
Aromatase enzyme structure complexed with androstanedione is reported in prominent
publications in this area. According to that docking results, androstanedione 3-oxo group
interacts with Asp309 in active site, where 17-oxo group interacts with Met374. Methyl group
th
at 19 position in a contact with haem group. In this active site, Thr310 is coordinated with
haem group but not in interaction with androstandione. In these studies, even if they do not
interact with androstanedione, Leu477, Ser478 and Arg115 were represented as key
aminoacids in active site.
nd
According to compund 1 docking results, hydroxyl group at 2 position of phenyl interacts
with Leu477 at active site. Nitrogen in pyrimidine ring coordinated to Thr310 which is an
interaction with haem group for featured efficiency on enzyme.
4
. Conclusion
In this study, 21 non-steroidal compounds inhibiting the catalytic site of the aromatase
enzyme and showing anticancer effects were synthesized and purity and structural analyses
were performed on the synthesized compounds by various methods. The activities of the
compounds at the enzyme specific and cellular levels were determined, and the interaction
between the active site and the most active compound was further evaluated using molecular
modeling techniques.
The biological activity of all the compounds was measured by two different tests. The IC50
value obtained from the enzyme inhibition assay of Compound 1 (0.42 nM) with the structure
of 4-(2-Hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole was higher than that of anastrozole (IC50
40
=
1.5 nM) . In addition, cytotoxicity studies on the HEK and MCF7 cell lines showed that
Compound 1 had a promising activity as non-steroidal aromatase inhibitors. Thus, Compound
can be considered for further development as a potential and valuable non-steroidal
aromatase inhibitor.
1
5
5
. Experimental section
.1. General
All the chemicals used in the synthesis and biological activity analysis were purchased from
Sigma Aldrich Chemical Corp. (St. Louis, Missouri, USA) and Merck KGaA (Merck,
Darmstadt, Germany). During the reactions, all the compounds were checked for their purities
using an Alliance HPLC System (Waters Inc., Milford, Massachusetts, USA) as well as Thin

Layer Chromatography (TLC) with a Silica Gel 60 F254 TLC plate (Merck KGaA,
Darmstadt, Germany). All the melting points were measured by a Stuart Melting Point
Apparatus SMP30 (Staffordshire, UK). The analysis of the C, H and N fractions of the
crystallized compounds was performed using a Leco TruSpec Micro CHN/CHNS elemental
analyzer (Saint Joseph, Michigan, USA). Spectroscopic data were determined with the
following instruments: IR, Perkin Elmer Spectrum Two FTIR Spectrometer (Perkin Elmer,
1
Inc., Waltham, Massachusetts, USA); H NMR, Bruker 500 MHz UltraShield NMR
13
Spectrophotometer (Bruker Corp., Billerica, Massachusetts, USA); and C NMR, Bruker 125
MHz UltraShield NMR Spectrophotometer (Bruker Corp., Billerica, Massachusetts, USA) in
DMSO-d using standard TMS. The mass spectra of the compounds were determined on an
6
Agilent 1100 series mass spectrometer (Agilent Tech., Santa Clara, California, USA) by the
electron spray method (ESI). The IC50 values of the compounds were recorded using
GraphPad Prism software Version 7.02 (La Jolla, California, USA).
5
.2. General procedure for the synthesis of 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole
(1-21)
In the synthesis process, different 1-(aryl/heteroaryl)ethanone (50 mmol) derivatives were
brominated in the presence of copper (II) bromide (100 mmol) followed by a reaction with
equimolar pyrimidine-2-carbothioamide (50 mmol). In the course of the reaction, the
components were mixed in ethyl acetate for approximately 1 hour in room temperature and
o
then refluxed at 77-78 C for 15-20 minutes. After cooling, the precipitate was filtered,
neutralized with sodium acetate, and recovered from the HBr salt. All the synthesized
compounds were purified twice by crystallization from ethanol. Pyrimidine-2-carbonitrile and
phosphorus pentasulfide were mixed in dioxane for 48 hours to obtain pyrimidine-2-
carbothioamide as the starting material. This is the first time in the literature that synthesis of
pyrimidine thioamides has been undertaken with this method.
5
.2.1. 4-(2-Hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole (1)
o
-1
Yield 75%, mp 202.5 C. IR (KBr) ῡmax (cm ): 3113 (OH), 3034 (C-H, aromatic), 1247 (C-
1
O), 750 (1,2-disubstituted benzene). H-NMR (300 MHz, DMSO-d6, δ, ppm): 7.55 (d, J=8.55
Hz, 2H, Ph), 7.61 (t, J=4.89 Hz, 1H, Pyr), 8.08 (d, J=8.58 Hz, 2H, Ph), 8.45 (s, 1H, Th), 8.97
13
(
d, J=4.89 Hz, 2H, Pyr), 9.67 (s, 1H, OH). C-NMR (75 MHz, DMSO-d6, δ, ppm): 116.02,
1
1
16.37, 122.02, 128.07, 158.22, 158.61. For C13
H
9
N OS calculated: 61.16% C, 3.55% H,
3
6.46% N; found: 61.20% C, 3.56% H, 16.44% N. Mass (ESI), m/z: 256.05 (M+1).
5
.2.2. 4-(3-Hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole (2)
o
-1
Yield 80%, mp 268.7 C. IR (KBr) ῡmax (cm ): 3113 (OH), 1235 (C-O), 778 (1,3-

1
disubstituted benzene). H-NMR (300 MHz, DMSO-d6, δ, ppm): 6.78 (d, J=8.90 Hz, 1H, Ph),
7.30 (t, J=7.92 Hz, 1H, Ph), 7.45-7.62 (m, 3H, Ph and Pyr), 8.30 (s, 1H, Th), 8.97 (d, J=4.89
13
Hz, 2H, Pyr), 9.57 (s, 1H, OH). C-NMR (75 MHz, DMSO-d6, δ, ppm): 113.53, 115.88,
17.35, 119.01, 122.12, 130.36, 135.59, 156.92, 158.29, 158.65, 159.07. For C13 OS
calculated: 61.16% C, 3.55% H, 16.46% N; found: 61.13 % C, 3.54% H, 16.49% N. Mass
ESI), m/z: 256.05 (M+1).
1
H N
9 3
(
5
.2.3. 4-(4-Hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole (3)
o
-1
Yield 68%, mp 231.1 C. IR (KBr) ῡmax (cm ): 3106 (OH), 2801 (C-H, aromatic), 1241 (C-
1
O), 830 (1,4-disubstituted benzene). H-NMR (300 MHz, DMSO-d6, δ, ppm): 6.93-7.02 (m,
2H, Ph), 7.21-7.27 (m, 1H, Ph), 7.61 (t, J=7.62 Hz, 1H, Pyr), 8.11 (dd, J1,2=7.95, 1.98, 1H,
13
Ph), 8.45 (s, 1H, Th), 8.99 (d, J=3.15 Hz, 2H, Pyr), 10.99 (s,1H, OH). C-NMR (75 MHz,
DMSO-d6, δ, ppm): 117.20, 119.48, 119.79, 120.65, 122.22, 128.69, 130.07, 154.15, 155.73,
1
58.71, 165.22. For C13
H
9
N OS calculated: 61.16% C, 3.55% H, 16.46% N; found: 61.15%
3
C, 3.54% H, 16.48% N. Mass (ESI), m/z: 256.05 (M+1).
5
.2.4. 4-(2-Hydroxy-5-methylphenyl)-2-(pyrimidine-2-yl)thiazole (4)
o
-1
1
Yield 75%, mp 226.6 C. IR (KBr) ῡmax (cm ): 3103 (OH), 3021 (C-H, aromatic). H-NMR
300 MHz, DMSO-d6, δ, ppm): 2.30 (s, 3H, Ph-CH ), 6.89 (d, J=8.22 Hz, 1H, Ph), 7.04 (d,
J=8.28 Hz, 1H, Ph), 7.62 (t, J=7.62 Hz, 1H, Pyr), 7.92 (s, 1H, Ph), 8.42 (s, 1H, Th), 8.99 (d,
(
3
13
J=4.86 Hz, 2H, Pyr), 10.99 (s,1H, OH). C-NMR (75 MHz, DMSO-d6, δ, ppm): 20.68,
17.04, 119.15, 120.56, 122.21, 128.19, 128.82, 130.61, 153.56, 154.26, 158.71. For
OS calculated: 62.43% C, 4.12% H, 15.60% N; found: 62.39% C, 4.11% H, 15.63%
Mass (ESI), m/z: 270.07 (M+1).
1
C
14 11
H N
3
5
.2.5. 4-(2-Hydroxy-5-methoxyphenyl)-2(pyrimidine-2-yl)thiazole (5)
o
-1
1
Yield 73%, mp 212.4 C. IR (KBr) ῡmax (cm ): 3100 (OH), 3047 (C-H, aromatic). H-NMR
(
300 MHz, DMSO-d6, δ, ppm): 3.77 (s, 3H, O-CH ), 6.83-6.93 (m, 2H, Ph), 7.60-7.65 (m, 2H,
3
13
Ph, Pyr), 8.50 (s, 1H, Th), 8.99 (d, J=4.86 Hz, 2H, Pyr), 10.54 (s,1H, OH). C-NMR (75
MHz, DMSO-d6, δ, ppm): 56.06, 112.88, 116.34, 116.97, 119.63, 121.07, 122.24, 149.78,
1
5
5
52.68, 154.08, 158.74. For C14
H
11
N
3
O S calculated: 58.93% C, 3.89% H, 14.73% N; found:
2
8.90% C, 3.88% H, 14.75% N. Mass (ESI), m/z: 286.06 (M+1).
.2.6. 4-(2-Hydroxy-5-chlorophenyl)-2-(pyrimidine-2-yl)thiazole (6)
o
-1
Yield 68%, mp 265 C. IR (KBr) ῡmax (cm ): 3096 (OH), 3044 (C-H, aromatic), 1247 (C-
1
O). H-NMR (300 MHz, DMSO-d6, δ, ppm): 7.02 (d, J=8.70 Hz, 1H, Ph), 7.26 (dd, J=11.55,
2
8
.73 Hz, 1H, Ph), 7.62 (t, J=4.89 Hz, 1H, Pyr), 8.17 (d, J=2.70 Hz, 1H, Ph), 8.52 (s, 1H, Th),
13
.99 (d, J=4.89 Hz, 2H, Pyr), 11.07 (s,1H, OH). C-NMR (75 MHz, DMSO-d6, δ, ppm):

1
18.81, 121.55, 122.31, 123.37, 128.09, 129.37, 152.35, 154.56, 158.73. For C13
calculated: 53.89% C, 2.78% H, 14.50% N; found: 53.92% C, 2.79% H, 14.48% N. Mass
ESI), m/z: 290.01 (M+1).
H
8
ClN OS
3
(
5
.2.7. 4-(2-Methoxyphenyl)-2-(pyrimidine-2-yl)thiazole (7)
o
-1
Yield 78%, mp 131.9 C. IR (KBr) ῡmax cm ): 3061 (C-H, aromatic), 1558 (C=N), 1506
1
(
C=C), 1256 (C-O), 731 (1,2-disubstituted benzene). H-NMR (300 MHz, DMSO-d6, δ, ppm):
3
3.95 (s, 3H, O-CH ), 7.10 (t, J=7.80 Hz, 1H, Ph), 7.18 (d, 1H, J=8.90 Hz, Ph), 7.38 (td,
J=7.80, 1.05 Hz, 1H, Ph), 7.60 (t, J=4.89 Hz, 1H, Pyr), 8.26 (dd, J=7.80, 1.77 Hz, 1H, Ph),
1
3
8
1
.32 (s, 1H, Th), 8.97 (d, J=4.89 Hz, 2H, Pyr). C-NMR (75 MHz, DMSO-d6, δ, ppm): 56.05,
12.29, 121.09, 121.08, 122.52, 129.87, 129.98, 158.64. For C14 OS calculated: 62.43%
11
H N
3
2
C, 4.12% H, 15.60% N; found: 62.42% C, 4.13% H, 15.62% N. Mass (ESI), m/z: 270.07
M+1).
(
5
.2.8. 4-(3-Methoxyphenyl)-2-(pyrimidine-2-yl)thiazole (8)
o
-1
Yield 74%, mp 115.4 C. IR (KBr) ῡmax (cm ): 3010 (C-H, aromatic), 1506 (C=N), 1403
1
(
C=C), 1227 (C-O), 737 (1,3-disubstituted benzen). H-NMR (300 MHz, DMSO-d6, δ, ppm):
3
7
.85 (s, 3H, O-CH
3
), 6.95 (dd, J=8.10, 2.52 Hz, 1H, Ph), 7.40 (t, J=8.01 Hz, 1H, Ph), 7.59-
13
.66 (m, 3H, Ph ve Pyr), 8.42 (s, 1H, Th), 8.98 (d, J=4.89 Hz, 2H, Pyr). C-NMR (75 MHz,
DMSO-d6, δ, ppm): 55.71, 111.86, 114.60, 119.02, 122.16, 130.48, 135.72, 156.69, 158.66,
59.03, 160.20. For C14 OS calculated: 62.43% C, 4.12% H, 15.60% N; found: 62.39%
C, 4.11% H, 15.62% N. Mass (ESI), m/z: 270.07 (M+1).
1
11
H N
3
2
5
.2.9. 4-(4-Methoxyphenyl)-2-(pyrimidine-2-yl)thiazole (9)
o
-1
Yield 82%, mp 179.2 C. IR (KBr) ῡmax (cm ): 3119 (C-H, aromatic), 1557 (C=N), 1404
1
(
C=C), 1244 (C-O), 837 (1,4-disubstituted benzene). H-NMR (300 MHz, DMSO-d6, δ, ppm):
3
.82 (s, 3H, O-CH ), 7.05 (d, J=8.91 Hz, 2H, Ph), 7.60 (t, J=4.89 Hz, 1H, Pyr), 7.99 (d,
3
13
J=8.91 Hz, 2H, Ph), 8.23 (s, 1H, Th), 8.96 (d, J=3.99 Hz, 2H, Pyr). C-NMR (75 MHz,
DMSO-d6, δ, ppm): 55.69, 114.69, 117.21, 122.07, 127.17, 128.00, 156.82, 158.63, 159.11,
1
59.90, 166.36. For C14
11
H N
3
OS calculated: 62.43% C, 4.12% H, 15.60% N; found: 62.45%
2
C, 4.11% H, 15.57% N. Mass (ESI), m/z: 270.07 (M+1).
5
.2.10. 4-(2-Chlorophenyl)-2-(pyrimidine-2-yl)thiazole (10)
o
-1
Yield 78%, mp 166 C. IR (KBr) ῡmax (cm ): 3067 (C-H, aromatic), 1556 (C=N), 1396
1
(
C=C), 703 (1,2-disubstituted benzene). H-NMR (300 MHz, DMSO-d6, δ, ppm): 7.15-7.18
(
m, 1H, Ph), 7.58-7.68 (m, 4H, Ph and Pyr), 8.23 (s, 1H, Th), 8.96 (d, J=4.89 Hz, 2H, Pyr).
13
C-NMR (75 MHz, DMSO-d6, δ, ppm): 117.53, 122.22, 125.32, 126.95, 128.63, 138.11,

1
5
51.63, 158.66, 158.83. For C13
H
8
ClN S calculated: 57.04% C, 2.95% H, 15.35% N; found:
3
7.08% C, 2.94% H, 15.38% N. Mass (ESI), m/z: 274.02 (M+1).
5
.2.11. 4-(3-Chlorophenyl)-2-(pyrimidine-2-yl)thiazole (11)
o
-1
Yield 80%, mp 128.5 C. IR (KBr) ῡmax (cm ): 3037 (C-H, aromatic), 1556 (C=N), 1411
1
(
(
(
C=C), 732 (1,3-disubstituted benzene). H-NMR (300 MHz, DMSO-d6, δ, ppm): 7.44-7.63
m, 3H, Ph and Pyr), 8.03 (dt, J1,2=7.68, 1.95 Hz, 1H, Ph), 8.12 (t, J=1.80 Hz, 1H, Ph), 8.53
13
s, 1H, Th), 8.98 (d, J=4.89 Hz, 2H, Pyr). C-NMR (75 MHz, DMSO-d6, δ, ppm): 120.54,
22.28, 125.18, 126.22, 128.61, 131.32, 134.22, 136.34, 155.12, 158.69, 158.90, 166.89. For
ClN S calculated: 57.04% C, 2.95% H, 15.35% N; found: 57.01% C, 2.94% H, 15.39%
N. Mass (ESI), m/z: 274.02 (M+1).
1
C
13
H
8
3
5
.2.12. 4-(4-Chlorophenyl)-2-(pyrimidine-2-yl)thiazole (12)
o
-1
Yield 75%, mp 148 C. IR (KBr) ῡmax (cm ): 3027 (C-H, aromatic), 1553 (C=N), 1454
1
(
C=C), 807 (1,4-disubstituted benzen). H-NMR (300 MHz, DMSO-d6, δ, ppm): 7.55 (d,
J=8.55 Hz, 2H, Ph), 7.61 (t, J=4.89 Hz, 1H, Pyr), 8.08 (d, J=8.58 Hz, 2H, Ph), 8.45 (s, 1H,
13
Th), 8.97 (d, J=4.89 Hz, 2H, Pyr). C-NMR (75 MHz, DMSO-d6, δ, ppm): 119.83, 122.24,
28.35, 129.40, 133.21, 133.37, 155.52, 158.68, 158.94. For C13 ClN S calculated: 58.27%
C, 4.03% H, 23.98% N; found: 58.29% C, 4.02% H, 23.89% N. Mass (ESI), m/z: 274.02
M+1).
1
H
8
3
(
5
.2.13. 4-(Pyridine-2-yl)-2-(pyrimidine-2-yl)thiazole (13)
o
-1
1
Yield 80%, mp 212.2 C. IR (KBr) ῡmax (cm ): 3001 (C-H, aromatic). H-NMR (300 MHz,
DMSO-d6, δ, ppm): 7.39-7.44 (m, 1H, Py), 7.62 (t, J=4.89 Hz, 1H, Pyr), 7.95 (td, J=7.86, 1.08
Hz, 1H, Py), 8.19 (dt, J=7.89, 1.05 Hz, 1H, Py), 8.53 (s, 1H, Th), 8.65-8.68 (m, 1H, Py), 8.99
13
(
d, J=4.89 Hz, 2H, Pyr). C-NMR (75 MHz, DMSO-d6, δ, ppm): 121.09, 122.26, 122.55,
1
2
23.94, 138.02, 150.13, 152.15, 158.69. For C12
H
8
N S calculated: 59.98% C, 3.36% H,
4
3.32% N; found: 59.86% C, 3.35% H, 23.29% N. Mass (ESI), m/z: 241.05 (M+1).
5
.2.14. 4-(Pyridine-3-yl)-2-(pyrimidine-2-yl)thiazole (14)
o
-1
1
Yield 90%, mp 260 C. IR (KBr) ῡmax (cm ): 3054 (C-H, aromatic). H-NMR (300 MHz,
DMSO-d6, δ, ppm): 7.64 (t, J=4.89 Hz, 1H, Pyr), 7.72-7.76 (m, 1H, Py), 8.62-8.71 (m, 3H,
13
Py, Th), 9.00 (d, J=4.86 Hz, 2H, Pyr), 9.34 (d, J=2.10 Hz, 1H, Py). C-NMR (75 MHz,
DMSO-d6, δ, ppm): 121.76, 122.41, 125.54, 158.75. For C12 S calculated: 59.98% C,
.36% H, 23.32% N; found: 59.91% C, 3.35% H, 23.28% N. Mass (ESI), m/z: 241.05 (M+1).
H N
8 4
3
5
.2.15. 4-(Pyridine-4-yl)-2-(pyrimidine-2-yl)thiazole (15)

o
-1
1
Yield 85%, mp 208 C. IR (KBr) ῡmax (cm ): 3034 (C-H, aromatic). H-NMR (300 MHz,
DMSO-d6, δ, ppm): 7.63 (t, J=4.89 Hz, 1H, Pyr), 8.01 (d, J=6.15 Hz, 2H, Py), 8.68-8.72 (m,
13
3
1
3
H, Py, Th), 9.00 (d, J=4.89 Hz, 2H, Pyr). C-NMR (75 MHz, DMSO-d6, δ, ppm): 120.83,
22.38, 123.09, 141.07, 150.87, 154.22, 158.72, 167.38. For C12 S calculated: 59.98% C,
.36% H, 23.32% N; found: 59.95% C, 3.34,% H, 23.28% N. Mass (ESI), m/z: 241.05
8
H N
4
(M+1).
5
.2.16. 4-(Furan-2-yl)-2-(pyrimidine-2-yl)thiazole (16)
o
-1
1
Yield 66%, mp 138.4 C. IR (KBr) ῡmax (cm ): 3037 (C-H, aromatic). H-NMR (300 MHz,
DMSO-d6, δ, ppm): 6.62-6.64 (m, 1H, Fur), 6.93 (d, J=3.18 Hz, 1H, Fur), 7.59 (t, J=4.86 Hz,
13
1
H, Pyr), 7.79 (d, J=1.02 Hz, 1H, Fur), 8.04 (s, 1H, Th), 8.96 (d, J=4.89 Hz, 2H, Pyr). C-
NMR (75 MHz, DMSO-d6, δ, ppm): 107.99, 112.37, 117.81, 122.25, 143.78, 148.53, 149.84,
1
5
58.62, 158.85, 167.23. For C11
H
7
N OS calculated: 57.63% C, 3.08% H, 18.33% N; found:
3
7.59% C, 3.07% H, 18.30% N. Mass (ESI), m/z: 230.04 (M+1).
5
.2.17. 4-(Thiophen-2-yl)-2-(pyrimidine-2-yl)thiazole (17)
o
-1
1
Yield 90%, mp 172.5 C. IR (KBr) ῡmax (cm ): 3086-3067 (C-H, aromatic). H-NMR (300
MHz, DMSO-d6, δ, ppm): 7.15-7.18 (m, 1H, Thip), 7.58-7.68 (m, 3H, Thip and Pyr), 8.22 (s,
1
3
1
1
H, Th), 8.97 (d, 2H, J=4.89 Hz, Pyr). C-NMR (75 MHz, DMSO-d6, δ, ppm): 117.53,
22.22, 125.31, 126.94, 128.62, 138.11, 151.62, 158.66, 158.82, 166.62. For C11
7 3 2
H N S
calculated: 57.63% C, 3.08% H, 18.33% N; found: 57.59% C, 3.07% H, 18.30% N. Mass
ESI), m/z: 246.02 (M+1).
(
5
.2.18. 4-(Benzofuran-2-yl)-2-(pyrimidine-2-yl)thiazole (18)
o
-1
1
Yield 68%, mp 186.5 C. IR (KBr) ῡmax (cm ): 2955 (C-H, aromatic). H-NMR (300 MHz,
DMSO-d6, δ, ppm): 7.28-7.74 (m, 6H, Pyr and Bzf), 8.35 (s, 1H, Th), 8.99 (d, 2H, J=4.89 Hz,
13
Pyr). C-NMR (75 MHz, DMSO-d6, δ, ppm): 104.12, 111.67, 120.87, 122.20, 122.43,
1
6
2
23.93, 125.57, 128.81, 147.95, 151.52, 154.74, 158.73, 167.78. For C15
H
9
N OS calculated:
3
4.50% C, 3.25% H, 15.04% N; found: 64.47% C, 3.24% H, 15.08% N. Mass (ESI), m/z:
80.05 (M+1).
5
.2.19. 4-(Benzothiophen-2-yl)-2-(pyrimidine-2-yl)thiazole (19)
o
-1
1
Yield 91%, mp 202.3 C. IR (KBr) ῡmax (cm ): 2985 (C-H, aromatic). H-NMR (300 MHz,
DMSO-d6, δ, ppm): 7.38-7.42 (m, 2H, Bzt), 7.62 (t, J=4.89 Hz, 1H, Pyr), 7.88-8.02 (m, 3H,
13
Bzt), 8.44 (s, 1H, Th), 8.99 (d, 2H, J=4.89 Hz, Pyr). C-NMR (75 MHz, DMSO-d6, δ, ppm):
1
1
19.91, 121.62, 122.33, 123.09, 124.43, 125.31, 125.40, 137.97, 139.46, 140.45, 151.36,
58.72. For C15 calculated: 60.99% C, 3.07% H, 14.23% N; found: 60.98% C, 3.06%
9
H N
S
3 2
H, 14.19% N. Mass (ESI), m/z: 296.03 (M+1).

5
.2.20. 4-(1-Methylbenzimidazol-2-yl)-2-(pyrimidine-2-yl)thiazole (20)
o
-1
Yield 76%, mp 195 C. IR (KBr) ῡmax (cm ): 3124 (C-H, aromatic), 2958 (C-H, aliphatic).
1
H-NMR (300 MHz, DMSO-d6, δ, ppm): 4.29 (s, 3H, N-CH
.72 (m, 3H, Bzi and Pyr), 8.70 (s, 1H, Th), 9.02 (d, 2H, J=4.89 Hz, Pyr). C-NMR (75 MHz,
DMSO-d6, δ, ppm): 111.16, 119.43, 122.43, 122.84, 123.31, 126.94, 158.80. For C15
calculated: 61.42% C, 3.78% H, 23.87% N; found: 61.38% C, 3.77% H, 23.85% N. Mass
ESI), m/z: 294.08 (M+1).
3
), 7.27-7.33 (m, 2H, Bzi), 7.63-
13
7
H
11
5
N S
(
5
.2.21. 4-(Benzothiazol-2-yl)-2-(pyrimidine-2-yl)thiazole (21)
o
-1
1
Yield 88%, mp 247 C. IR (KBr) ῡmax (cm ): 3031 (C-H, aromatic). H-NMR (300 MHz,
DMSO-d6, δ, ppm): 7.47-7.68 (m, 3H, Bt and Pyr), 8.14 (d, J=7.11 Hz, 2H, Bt), 8.77 (s, 1H,
13
Th), 9.02 (d, 2H, J=4.89 Hz, Pyr). C-NMR (75 MHz, DMSO-d6, δ, ppm): 122.58, 123.08,
1
5
2
23.34, 124.27, 126.16, 127.22, 135.24, 150.62, 158.49, 158.80. For C14
H
8
N
4
S calculated:
2
6.74% C, 2.72% H, 18.91% N; found: 56.74% C, 2.73% H, 18.87% N. Mass (ESI), m/z:
97.03 (M+1).
5
5
.3. Experimental procedure for biological activity
.3.1. Aromatase inhibitory activity assay
Aromatase inhibition is quantified by measuring the fluorescent intensity of fluorescein, the
hydrolysis product of dibenzylfluorescein, by aromatase. Experiments were carried out
following the instructions provided by the “CYP19/MFC High Throughput Inhibitor
Screening assay kit” (Corning, Catalog number: 459520) using the modified method detailed
4
1
by Stresser . Stock solutions of the compounds were prepared with DMSO (1%). In brief, the
test substance (10 μM) and the standard solution 3l HFC (7-hydroxy-4-trifluoromethyl
coumarone 0.25 M), 147 l NADPH-Cofactor Mix without acetonitrile were preincubated
+
with the NADPH regenerating system (1.3 mM NADP , 66 mM glucose 6-phosphate
dehydrogenase, 66 mM MgCl ) for 10 min at 37 °C. Then, 100 μL of the enzyme and
2
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 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 duplicate and
the average values were used to construct the dose−response curves. A minimum of eight
concentrations of each test substance were used to calculate the IC50 values (Table 1).
5
.3.2. Measurement of activity by MTT assay
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a water soluble
4
2
tetrazolium salt. The MTT assay was discovered by Mossmann (1983) . In this colorimetric

technique, the tetrazolium ring is converted to an insoluble purple formazan by cleavage of
that ring through the increased activity of succinate dehydrogenase on live cells within the
mitochondria. Experiments were carried out following the instructions given in the “Cell
Proliferation Kit I (MTT)” (Roche, Catalog Number: 11 465 007 001). The effects of the
tested compounds on the cell viability of the MCF7 and HEK-293 cell lines were determined
using this colorimetric technique. The 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 heat
inactivated fetal bovine serum (GIBCO, UK), 1% penicillin-streptomycin (GIBCO, UK), 1%
4
L-glutamine (GIBCO, UK) per well at a cell density of 5 × 10 cell / wells. Experiment sets of
different concentrations were prepared. Control wells were formed by preparing culture
media without compounds. The cells were incubated for 24 hours (37 °C and 5% CO ).
2
Following this step, each medium in wells was replaced with a fresh medium and an
appropriate amount of compounds was added and incubated for 6 hours. After incubation, 10
µl of MTT (final concentration 0.5 mg/ml) was added and left for 4 hours. 100 µl of a
solubilization solution containing 10% SDS in 0.01 M HCl was added into each well.
4
3,44
Absorbance at 550 nm was recorded with an ELISA plate reader
were performed in triplicate.
. Each set of experiments
4
5
54. Molecular modeling studies
.4.1. Preparation of the ligand set
The 3D structures of all the compounds were drawn using a 3D builder implemented in the
Maestro Suite (Schrödinger LCC, Oregon, USA) and minimized by means of the OPLS_2005
force field. Then, 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.
5
.4.2. Preparation of the target
The preparation of the protein structure and ligand, GRID files, docking, and scoring were
performed using algorithms available in the Maestro modules (Schrodinger Inc, USA). The
X-ray crystallographic structure of human placental aromatase (PDB entry code: 3EQM) was
3
7
obtained from the Protein Data Bank (RCSB) . The 3EQM PDB file was edited using the
protein preparation wizard in Maestro for hydrogen insertion and rotamer adjustment, and H-
bond optimization using OPLS 2005 as the energy parameters. During the process, the wizard
identified both the heme group and the iron atom in protein content with the appropriate
formal charges. It was also determined that the amide functions of Asn / Gln and the thiolate
group on Cys437 were negatively configurated both in the axial position to the iron atom
present in the heme group.
5
.4.3. Docking Experiments
The compound that had the highest aromatase inhibition activity was Compound 1 with the
structure of 4-(2-hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole. series of docking
A

experiments were performed on this compound to identify the probable interactions between
the ligand and the aromatase active site. 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. for docking
38
studies .
Acknowledgments
The authors acknowledge The Scientific and Technological Research Council of Turkey
TUBİTAK) for their financial support for the project SBAG-115S168.

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-
-
-
Synthesis of pyrimidine 2-carbothioamide were carried out by a new original method
New resveratrol analogues as aromatase inhibitors defined
Compound 1 was 2000 times and Compound 8 was 9 times more active than
ketoconazole. Compounds 15 and 21 were equivalent to ketoconazole.
Compounds 13, 15, and 21 showed significant biological effect in both tests
aromatase inhibitory activity and cytotoxicity.
-

HO
N
N
N
S
IC50 1 = 0.42 (nM)
KTZ = 0.95148 (µM)
N
N
N
S
S
N
IC50 21 = 0.99974 (µM)
Active against MCF7 versus HEK 293
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