Potent ribonucleotide reductase inhibitors: Thiazole-containing thiosemicarbazone derivatives

Article abstract of DOI:10.1002/ardp.201900033

The antioxidant, antimalarial, antibacterial, and antitumor activities of thiosemicarbazones have made this class of compounds important for medicinal chemists. In addition, thiosemicarbazones are among the most potent and well-known ribonucleotide reductase inhibitors. In this study, 24 new thiosemicarbazone derivatives were synthesized, and the structures and purity of the compounds were determined by IR, ¹H NMR, ¹³C NMR, mass spectroscopy, and elemental analysis. The IC₅₀ values of these 24 compounds were determined with an assay for ribonucleotide reductase inhibition. Compounds 19, 20, and 24 inhibited ribonucleotide reductase enzyme activity at a higher level than metisazone as standard. The cytotoxic effects of these compounds were measured on the MCF7 (human breast adenocarcinoma) and HEK293 (human embryonic kidney) cell lines. Similarly, compounds 19, 20, and 24 had a selective effect on the MCF7 and HEK293 cell lines, killing more cancer cells than cisplatin as standard. The compounds (especially 19, 20, and 24 as the most active ones) were then subjected to docking experiments to identify the probable interactions between the ligands and the enzyme active site. The complex formation was shown qualitatively. The ADME (absorption, distribution, metabolism, and excretion) properties of the compounds were analyzed using in-silico techniques.

Full text of DOI:10.1002/ardp.201900033

Received: 30 January 2019
Revised: 30 July 2019
Accepted: 1 August 2019
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DOI: 10.1002/ardp.201900033
F U L L P A P E R
Potent ribonucleotide reductase inhibitors:
Thiazole‐containing thiosemicarbazone derivatives
Merve Ertas1
Barkin Berk1
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Zafer Sahin1
Leyla Yurttas3
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Emre F. Bulbul1
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Ceysu Bender1
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Sevde N. Biltekin2
Şeref Demirayak1
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Aysu M. Nalbur4 Hayati Celik4
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1Department of Pharmaceutical Chemistry,
School of Pharmacy, Istanbul Medipol
University, Istanbul, Turkey
Abstract
The antioxidant, antimalarial, antibacterial, and antitumor activities of thiosemicar-
bazones have made this class of compounds important for medicinal chemists. In
addition, thiosemicarbazones are among the most potent and well‐known ribonucleo-
tide reductase inhibitors. In this study, 24 new thiosemicarbazone derivatives were
synthesized, and the structures and purity of the compounds were determined by IR,
1H NMR, 13C NMR, mass spectroscopy, and elemental analysis. The IC50 values of
these 24 compounds were determined with an assay for ribonucleotide reductase
inhibition. Compounds 19, 20, and 24 inhibited ribonucleotide reductase enzyme
activity at a higher level than metisazone as standard. The cytotoxic effects of these
compounds were measured on the MCF7 (human breast adenocarcinoma) and
HEK293 (human embryonic kidney) cell lines. Similarly, compounds 19, 20, and 24
had a selective effect on the MCF7 and HEK293 cell lines, killing more cancer cells
than cisplatin as standard. The compounds (especially 19, 20, and 24 as the most
active ones) were then subjected to docking experiments to identify the probable
interactions between the ligands and the enzyme active site. The complex formation
was shown qualitatively. The ADME (absorption, distribution, metabolism, and
excretion) properties of the compounds were analyzed using in‐silico techniques.
2Department of Pharmaceutical Microbiology,
School of Pharmacy, Istanbul Medipol
University, Istanbul, Turkey
3Department of Pharmaceutical Chemistry,
School of Pharmacy, Anadolu University,
Eskişehir, Turkey
4Department of Analytical Chemistry, School
of Pharmacy, Yeditepe University, Istanbul,
Turkey
Correspondence
Merve Ertas, M.Sc., Department of
Pharmaceutical Chemistry, School of
Pharmacy, Istanbul Medipol University,
34810 İstanbul, Turkey.
Email: mertas@medipol.edu.tr
Funding information
Anadolu University Scientific Research
Projects Commission, Grant/Award Number:
1609S624
K E Y W O R D S
antitumor agents, ribonucleotide reductase, thiazole, thiosemicarbazone
transferrin.[3–5] In biological systems, iron chelators are responsible
for capturing the free iron produced from transferrin and limit the
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1
INTRODUCTION
As current chemotherapies are not effective against common and
aggressive tumors, such as breast, prostate, lung, and brain cancers,
research on new compounds focusing on the proliferation of
cancerous cells remains crucial. In such research, two main
approaches are adopted; either developing new chemotherapeutics
targeting DNA or developing the components of cell metabolism and
replication.[1] Iron is a key metal that catalyzes important reactions in
metabolism, respiration, and oxygen transport as well as DNA
synthesis.[2] In cancer cells, the need for iron causes an increase in
transferrin receptor 1 that provides intracellular uptake of iron from
activity of iron‐dependent proteins, such as ribonucleotide reductase.
The amount of ribonucleotide reductase determines the rate of
cell replication.[6,7] According to Cory and Chiba,[8] ribonucleoside
diphosphate reductase inhibitors are better inhibitors of DNA
synthesis than DNA polymerase inhibitors.
The antioxidant, antimalarial, antibacterial, and antitumor activities
of thiosemicarbazones have made these compounds more interesting
for medicinal chemists.[8–32] In addition, with their capability to
produce metal complexes, thiosemicarbazones are known to be one
of the most potent ribonucleotide reductase enzyme inhibitors.[33]
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Arch Pharm Chem Life Sci. 2019;e1900033.
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201900033

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(a)
(b)
FIGURE 1 (a) Examples of chelators of hydrazones, thiosemicarbazones, and thiohydrazones. Electron‐donor atoms are shown in bold.
(b) Gemcitabine, MDL 101.731, 3‐AP, hydroxyurea structures
Some chelate‐producing derivatives of thiosemicarbazones (I),
hydrazones (II, III), and thiohydrazones (IV) are listed in Figure 1a.
These structures create the Fe/ligand complex. Oxidation of metal is
primarily dependent on the presence of electron‐donating atoms. O,
N, O are FeIII chelators of hydrazones, whereas N, N, O are specific to
FeII.[34,35] S (sulfur) acts as an electron‐donating precursor specific to
iron II and III in thiosemicarbazones and thiohydrazones.
tyrosyl radical and is the main moiety required for reductase activity.
There are also two classes of ribonucleotide reductase inhibitors;
nucleoside analogs that bind to the R1 subunit, such as gemcitabine
(2′,2′‐difluoro‐2′‐deoxycytidine; V) and MDL 101.731 (2′‐fluoro-
methylene‐2′‐deoxydidine; VI), and those that bind to either the
non‐heme iron or the free radicals in the R2 subunit; for example,
heterocyclic carboxaldehyde thiosemicarbazone 3‐AP (VII), 3‐amino-
pyridine‐2‐carboxyaldehyde thiosemicarbazone, and N‐hydroxyurea
(VIII). Heterocyclic carboxaldehyde thiosemicarbazones have been
found to be more effective inhibitors compared to N‐hydroxyurea,
which has a relatively short half‐life (Figure 1b).[38–43]
The antitumor activity of thiosemicarbazone derivatives can be
explained through two separate mechanisms: In the first, thiosemi-
carbazides bind to free iron in the cytoplasm and form a redox‐active
Fe complex by sulfur and two nitrogen atoms, N–N–S.[6,36] The
structure–activity relationships of this pattern were examined by
Logan et al.[37] in detail, and the –HN–(C═S)–NH– group was shown
to be essential for antitumor activity.[37]
In this study, 24 novel thiosemicarbazone analogs were
synthesized, and their cytotoxic activities were assessed on the
human embryonic kidney (HEK293) and human breast cancer
(MCF7) cell lines. Ribonucleotide reductase enzyme inhibition
activities were evaluated at 10 μM concentration compared to
metisazone. As the active compounds gave similar results in both
tests, a docking study was also performed to clarify the possible
interactions between the active site and the molecules.
In the second mechanism, the target is ribonucleotide reductase
enzyme. It binds to iron at high levels and can also be targeted by
iron chelators. The ribonucleotide reductase enzyme in mammals
consists of two subunits responsible for antitumor activity; part R1
contains polythiols and part R2 contains non‐heme iron and the free
a1–a8
b1–b8
1–24
SCHEME 1 Synthesis pathway of compounds 1–24

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TABLE 1 Structures of compounds 1–24
2
RESULTS AND DISCUSSION
Chemistry
Compound
R
Rʹ
S
R″
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2.1
1
4‐Cl phenyl
4‐OCH3 phenyl
2‐Pyridyl
H
2
S
H
The synthetic pathway and structures of the final thiazolylmethyl-
ketone‐(thio)semicarbazones (compounds 1–24) are detailed in
3
S
H
Scheme
1 and Table 1, respectively. Initially, aryl‐substituted
4
3‐Pyridyl
S
H
carbonitriles were reacted with phosphorus pentasulfide in 1,4‐
dioxane to produce aryl‐substituted thioamide derivatives (a1–a8).
Then, 1‐(2‐aryl‐4‐methylthiazol‐5‐yl)ethanone derivatives (b1–b8)
were obtained by the synthesis of aryl thioamides and 3‐chloro‐
2,4‐pentanedione. Finally, 1‐(2‐aryl‐4‐methylthiazol‐5‐yl)ethanones
and (thio)semi‐carbazide derivatives were reacted together using
an acetic acid catalyst to acquire compounds 1–24.[20,44] The
structures and purity of these novel compounds were elucidated by
the spectroscopic methods of infrared (IR), 1H NMR, 13C NMR, and
mass and elemental analysis.
5
4‐Pyridyl
S
H
6
2‐Pyrazinyl
2‐Pyrimidinyl
Phenyl
S
H
7
S
H
8
S
H
9
4‐Cl phenyl
4‐OCH3 phenyl
2‐Pyridyl
O
O
O
O
O
O
O
O
S
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
3‐Pyridyl
4‐Pyridyl
2‐Pyrazinyl
2‐Pyrimidinyl
Phenyl
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2.2
Anticancer activity assay
4‐Cl phenyl
4‐OCH3 phenyl
2‐Pyridyl
To assess the cytotoxic activity of compounds on the HEK293 and
MCF7 cell lines, various concentrations (5 × 10−4–10−6 M) were
applied to the medium. The IC50 cytotoxicity values were considered
to be the compound concentrations that reduced the absorbance to
50% compared with the level in untreated control wells and were
derived from at least six separate experiments. The control group
cells that contained nothing incubated at 37°C were accepted as
having 100% viability. The effect of the compounds’ IC50 values is
presented in Table 2. Statistical comparisons were made using one‐
way analysis of variance and a module of the GraphPad Prism
software version 7.02 (La Jolla, CA). Differences in the mean values
were considered to be significant when p < 0.05.
S
S
3‐Pyridyl
S
4‐Pyridyl
S
2‐Pyrazinyl
2‐Pyrimidinyl
Phenyl
S
S
S
Furthermore, stoichiometric analysis of complexes belonging to
metisazone, compounds 1, 19, 20, and 24, and Fe(III) was under-
taken. Finally, the ADME properties of the compounds were
analyzed using in‐silico techniques.
When the 48‐hr effect of the substances was assessed on the
MCF7 and HEK293 cell lines, the toxicity of most of the compounds
TABLE 2 IC50 values (μM) of the substances in the HEK293 and MCF7 cell lines
Compound
MCF7
HEK293
55.9 2.2
21 5.1
Compound
MCF7
HEK293
1
14.2 1.4
10.4 0.33
19.2 2.4
13
14
15
16
17
18
19
20
21
22
23
24
62.1 1.5
34.2 3.5
56.5 2.2
12.4 1.3
19.2 3.5
7.9 1.1
1.4 0.25
4.1 0.9
57.9 1.9
28.4 2.1
51.9 2.4
15.3 3.9
50.1 3.2
18.2 3.4
23.4 1.1
33.4 5.1
38.4 4.4
2
3
53.1 3.3
8.6 3.4
57.9 4.2
11 0.4
4
7
1.33
2
5
16.1
6
12.5 0.9
37.4 1.2
17 4.2
7
45 4.1
8
10.2 2
9
58.3 2.2
50.9 3.2
15.4 2.3
56.1 2.5
41.4 5.2
10.51 1.31
4.5
2
10
19.5
2
7.4 1.3
45.4 3
11
63.7 0.2
55.1 3.2
5.75 0.02
11.2
2
50.2 2.2
13 0.9
12
2.4 0.1
Cisplatin*
Note: Asterisk represents the standart. Bold figures mean significant values.

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FIGURE 2 The results of the enzyme inhibition assays of 10 μM of the standard inhibitor metisazone and 24 substances. Vertical bars
showing standard deviation values. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
was more selective for cancer cells than for normal cells. The IC50
values of compounds 19, 20, 21, and 24 in the MCF7 cells were lower
compared to the standard substance, cisplatin. While the IC50 values
of compounds 20 and 21 were very close to that of cisplatin, the IC50
value of compound 24 was 2.4 μM, which was almost half the value of
the standard. The IC50 value of the most active compound (19) was
1.4 μM. This compound also had a higher IC50 value in the HEK293
cell line.
2 (82.46%), 23 (80.77%), 22 (77.69%), and 19 (77.50%). Compound 8
had the least‐inhibiting effect on ribonucleotide reductase activity
(∼50% inhibition).
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2.4
Molecular docking studies
Docking experiments were undertaken with all synthesized com-
pounds and 1W68 to identify the possible interaction patterns on the
largest active site closer to Fe found with the Site Map. The
experiments were initially performed using the SP algorithm of Glide,
treating the receptor as rigid and the ligands as flexible. The
enclosing box was centered on the center of the largest binding site,
and default sizes were used for both the enclosing and bounding
boxes. All ligands seemed to fit well into the pocket, giving similar
poses (Figure 3).
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2.3
Ribonucleotide reductase enzyme inhibition
activity
The ribonucleotide reductase enzyme inhibition activity of 24
compounds and the standard metisazone (% inhibition = 77.44) was
measured at a concentration of 10 μM. For this purpose, the standard
from the kit was diluted at various dilutions. A standardized graph
was plotted based on the results (R2: 0.9809). The values obtained as
a result of the experimental studies were evaluated according to this
graph. The values obtained are given in Figure 2 and Table 3.
Compound 24 was the most potent inhibitor of ribonucleotide
reductase activity (85.85% inhibition). Other compounds with higher
activity than metisazone were compounds 20 (83.34% inhibition),
The binding site between two helical secondary structures seem
to produce two hydrophobic cavities connected with a narrow
hydrophobic tunnel. Therefore
a three compartment structure,
seemingly, can best fit horizontally to this binding site. The vertical
positioning of functional groups belong to Phe 241, Tyr 324, and Asn
346 with Phe 245 seem to lock the positioning of the central thiazole
moiety with hydrophobic interactions. The nitrogen atom of the
TABLE 3 Ten‐micromolar enzyme inhibition values of metisazone, used as a standard inhibitor, and the derived substances
Compound
% Inhibition
64.83 4.58
82.46 3.06
69.12 2.08
65.33 3.79
63.12 1.15
61.38 1.15
62.75 1.53
53.16 3.61
72.44
Compound
% Inhibition
62.49 2.08
61.98 2.16
65.04 3.51
67.65 3.46
64.97 3.46
69.96 3.61
64.80 4.51
63.84 2.65
Compound
% Inhibition
63.16 2.52
63.22 3.46
77.50 2.00
83.34 2.31
70.78 2.08
77.69 1.73
80.77 2.50
85.85 2.65
1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2
3
4
5
6
7
8
Metisazone*
Note: Asterisk represents the standart. Bold figures mean significant values.

ERTAS ET AL.
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from compounds 19, 20, and 24, therefore two‐ and three‐
dimensional (3D) interaction patterns for these compounds are given
in Figures 4 and 5 representing all the compounds.
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2.5
Absorption spectra and stoichiometry of
complexes
The complex formation between Fe(III) ions and the ligands/
metisazone was investigated. For representative purposes, the
experimental details of compound 1 are given in Figure 6a as the
same results were obtained from other ligands including metisazone.
This figure shows the absorption spectra of the Fe(III)–compound 1
complex recorded in the wavelength range of 200 to 400 nm.
The stoichiometry of the Fe(III)–compound 1 complex was
determined by continuous variation of the compound 1 concen-
tration while keeping the concentration of the Fe(III) ions
constant. The complex had a maximum absorbance at 343 nm.
The stoichiometric ratio between the metal and ligand was found
by plotting the absorbance values at 343 nm versus mole ratio
(Figure 6b).
FIGURE 3 Poses of all compounds in the active site
thiosemicarbazone moiety binds with a hydrogen bond to the p‐OH
group of the Tyr 324 in all compounds. Aromatic functional groups,
pyrimidine, pyridine, and phenyl, which are directly connected to the
thiazole ring system, position themselves in the shallow cavity
surrounded by Phe 348, Lys 249, Trp 246, and Phe 245 with
hydrophobic interactions. However, the substitution on these ring
systems can produce van der Waals clashes with the side chains and
are not favored. On the contrary, the cavity produced by Phe 327,
Val 328, Arg 331, Phe 237, and Ser 238 is deeper. This pocket can
easily host freely rotating over a single‐bond ring system such as
phenyl, though the smaller hydrophobic functional groups are
capable of creating the same hydrophobic interactions. The lowest
IC50 values during ribonucleotide reductase inhibition were obtained
The plot obtained by the molar ratio method indicated that the
ligand and metal ions formed the complex at a molar ratio of 1:4. In
this study, we only showed the complex formation between ferric
ions and ligands. The determination of conditional stability constants
Compound 19
Compound 20
Compound 24
FIGURE 4 Two‐dimensional interaction diagrams for compounds 19, 20, and 24

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(a)
(b)
FIGURE 6 (a) Absorption spectra of Fe(III)–compound 1 complex
in the presence of 10% ethanol in a buffer solution at pH 7.40. Mole
ratios (nL/nM): (a) 0.5, (b) 1.0, (c) 1.5, (d) 2.0, (e) 2.5, (f) 3.0, (g) 3.5,
(h) 4.0, (i) 4.5, and (j) 5.0. (b) Molar ratio method: [Fe3+] = 0.002 mM
and [L] = 0.001–0.010 mM in phosphate buffer at pH 7.40. A on the
y‐axis means absorption
early as possible in the drug development process. Studies conducted
in recent years have shown a significant increase in the number of
compounds required for early delivery of ADME.[45] For this reason,
we analyzed the ADME properties of compounds 1–24 using the
online Molinspiration property calculator, which allows easy inter-
active determination of molecular properties.[46] Lipinski’s rule of five
was also employed using this software. This set of rules require that
an orally active drug should not violate more than one criterion.[47]
The ADME parameters of molecular weight, topological polar surface
area (tPSA), number of hydrogen donors, number of hydrogen
acceptors, number of rotatable bonds, and volume were calculated
theoretically and evaluated in accordance with Lipinski’s rule
(Table 4). According to the results, none of the compounds (1–24)
violated more than one criterion. Thus, this analysis confirmed the
biological significance of compounds 19, 20, and 24 having a good
pharmacological profile.
FIGURE 5 Three‐dimensional interaction diagrams for
compounds 19, 20, and 24
and formation of complexes with other metals ions, including nickel,
copper, cobalt, and lead is currently under investigation.
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2.6
Theoretical calculation of the absorption,
distribution, metabolism, and excretion (ADME)
parameters
Poor pharmacokinetics is one of the major causes of failure in the
costly late‐stage drug development process. Therefore, the pharma-
cokinetic profiles of potential novel drugs should be evaluated as

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TABLE 4 Physicochemical properties of compounds 1–24 used in
the prediction of ADME profiles
HEK293 cell lines, killing cancer cells more than the cisplatin standard.
The ribonucleotide reductase inhibition effect of the compounds
targeted with the RRM2 ELISA kit was examined using metisazone as
standard. Compounds 19, 20, and 24 inhibited ribonucleotide reductase
enzyme activity at a higher level than the standard. Molecular docking
studies on the selected most active compounds (19, 20, and 24)
revealed good docking poses. The complex formation between the
metal ion and these compounds (including metisazone) was shown
quantitatively. Furthermore, the ADME parameters were calculated and
these molecules were found to have a good pharmacokinetic profile.
The results indicated that (thio)semicarbazone containing sulfur and
small hydrophobic moieties inhibit ribonucleotide reductase activity
effectively, as observed in compounds 19, 20, and 24 in this study.
Compound MW
tPSA nON nOHNH Vol
nrotb Vio
1
324.86 63.31
4
5
5
5
5
6
6
4
5
6
6
6
6
7
7
5
4
5
5
5
5
6
6
4
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
263.48
275.49
245.79
245.79
245.79
241.63
241.63
249.94
327.12
339.13
309.43
309.43
309.43
305.27
305.27
313.59
281.15
293.16
263.46
263.46
263.46
259.30
259.30
267.62
4
5
4
4
4
4
4
4
4
5
4
4
4
4
4
4
5
6
5
5
5
5
5
5
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
320.44 72.54
291.40 76.20
291.40 76.20
291.40 76.20
292.39 89.09
292.39 89.09
290.42 63.31
384.89 66.38
380.47 75.61
351.44 79.27
351.44 79.27
351.44 79.27
352.42 92.17
352.42 92.17
350.45 66.38
338.89 49.31
334.47 58.54
305.43 62.20
305.43 62.20
305.43 62.20
306.42 75.09
306.42 75.09
304.44 49.31
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
|
4
EXPERIMENTAL
Chemistry
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4.1
|
4.1.1
General
All chemicals used 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), and 1H‐NMR spectra were obtained with a Bruker
DPX‐500, 300 MHz High‐Performance Digital FT‐NMR. 13C‐NMR
spectra were measured using a DPX‐125, 75 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) and were found to be
within 0.4% of the theoretical values for C, H, and N.
Abbreviations: ADME, absorption, distribution, metabolism, and excre-
tion; MW, molecular weight; nOHNH, number of hydrogen donors; nON,
number of hydrogen acceptors; nrotb, number of rotatable bonds; tPSA,
total polar surface area; Vio, Violation; Vol, molecular volume.
For evaluating the drug‐likeness properties of the compounds,
the QikProp module of Maestro was used, and the results are
presented in Table 5.
The original IR and NMR spectra are provided as Supporting
Information, as are the InChI codes of the investigated compounds
together with some biological activity data.
The results obtained from this study indicate that the compounds
may have an effect on CNS as well as showing K+ channel inhibitor
activity and binding to human serum albumin. The brain/blood
partition coefficient and high oral absorption values were within the
acceptable range of drug‐likeness.
|
4.1.2
General procedure for the synthesis of
thioamide derivatives (a1–8
)
Carbonitrile derivatives (10 mmol) were stirred in 1,4‐dioxane (40 ml)
with P2S5 (10 mmol) for 1 week at room temperature. Ice was added
to the resulting product. The solution was neutralized with ammonia
and the crude product was obtained by filtration.
|
3
CONCLUSION
In this study, novel thiazolylmethylketone(thio)semicarbazone deriva-
tives were synthesized by reacting acetylthiazole derivatives with
thiosemicarbazides. The structure and purity of the obtained com-
pounds was determined by IR, 1H NMR, 13C NMR, mass spectroscopy,
and elemental analysis. The effect of the compounds on cellular
proliferation was monitored using the Cell Proliferation Reagent Kit I.
Compounds 19, 20, and 24 had a selective effect on the MCF7 and
|
4.1.3
General procedure for the synthesis of
thiazole derivatives (b1–8
)
The thioamide derivatives (5 mmol) were refluxed with 3‐chloro‐2,4‐
pentanedione (5 mmol) in ethanol. The obtained product was cooled,
filtered, and washed with ethanol.

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TABLE 5 Predicted drug‐likeness properties of the compounds 1–24
Compound
CNS
0
logPo/w
3.651
2.949
2.245
1.928
1.925
1.399
1.647
2.823
4.88
logHERG
−5.785
−5.214
−5.283
−5.145
−5.142
−5.057
−5.153
−5.276
−5.51
PCaco
logBB
0.033
PMDCK
logKHSA
0.16
HOA
3
%HOA
100
1
1,762.828
1,764.07
998.017
754.08
10,000
2
−1
−1
−1
−1
−1
−1
−1
0
−0.197
−0.367
−0.49
3,503.967
2,042.255
1,432.696
1,431.647
1,014.433
1,210.466
3,500.883
3,613.102
1,465.214
1,079.678
751.664
0.002
−0.182
−0.254
−0.255
−0.406
−0.339
−0.029
0.64
3
100
3
3
93.767
89.736
89.719
83.801
86.491
100
4
3
5
754.042
523.723
614.148
1,765.753
1,348.86
1,347.696
983.171
726.401
724.81
−0.49
3
6
−0.646
−0.584
−0.114
−0.233
−0.466
−0.54
3
7
3
8
3
9
1
100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0
4.448
3.826
3.485
3.485
2.921
3.192
4.384
3.985
3.583
2.99
−5.435
−5.511
−5.411
−5.41
0.529
0.324
0.228
0.228
0.032
0.124
0.522
0.279
0.189
−0.017
−0.081
−0.083
−0.287
−0.184
0.193
3
100
0
3
100
0
−0.69
3
100
0
−0.691
−0.846
−0.775
−0.393
0.281
750.423
3
100
−1
−1
0
−5.308
−5.389
−5.609
−5.336
−5.266
−5.317
−5.193
−5.187
−5.115
−5.171
−5.399
519.647
609.176
1,347.695
3,205.332
3,203.695
2,302.331
1,746.449
1,750.214
1,197.232
1,449.935
3,257.18
541.789
3
92.655
95.479
100
643.512
3
1,465.214
10,000
3
1
3
100
1
0.046
6,005.222
4,299.548
2,679.481
2,697.81
2,135.208
2,290.109
5,275.57
3
100
0
−0.028
−0.177
−0.175
−0.316
−0.257
0.098
3
100
0
2.626
2.625
2.043
2.325
3.515
3
100
0
3
100
0
3
94.003
100
0
3
1
3
100
Note: Predicted central nervous system (CNS) activity on a scale of −2 (inactive) to +2 (active); PlogPo/w, predicted octanol/water partition coefficient
(−2.0 to 6.5); logHERG, predicted IC50 value for blockage of HERG K+ channels (below −5); logBB, predicted brain/blood partition coefficient (−3.0 to 1.2);
logKHSA, prediction of binding to human serum albumin (−1.5 to 1.5); Human Oral Absorption (HOA), predicted qualitative human oral absorption
measured as 1, 2, or 3 for low; Percent HOA, high if >80% and poor if <25%.
|
4.1.4
General procedure for the synthesis of the
2‐(1‐(2‐(4‐Methoxyphenyl)‐4‐methylthiazole‐5‐yl)ethylidene)hydra-
zinecarbothioamide (
thiazolylmethylketone(thio)semicarbazones 1–24
2
)
Yield, 66–68%; m.p. 224.8°C. IR Vmax (cm−1): 3,266,5 (NH), 3,132
(arom.), 2,958–2,833.4 (aliph.), 1,605.23–1,499.96 (C═N, C═C),
1,171.87 (C–O), 1,087.41 (C═S), and 706.53 (C–S). 1H‐NMR
(300 MHz, DMSO‐d6) δ (ppm): 2.37 (3H, s, CH3), 2.58 (3H, s, thiazole
CH3), 3.82 (3H, s, OCH3), 7.03–7.06 (2H, m, phenyl C3,5–H), 7.40 (1H,
4‐Acetyl‐5‐methylthiazole derivatives (3 mmol) were refluxed with
(thio)semicarbazide derivatives in ethanol by an acetic acid catalyst.
The resulting product was filtered and washed with ethanol.[20,44]
s, NH), 7.84–7.88 (2H, m, phenyl C2,6–H), and 8.38 (1H, brs, NH). 13
C
2‐(1‐(2‐(4‐Chlorophenyl)‐4‐methylthiazole‐5‐yl)ethylidene)hydrazi-
necarbothioamide (1)
NMR (75 MHz, DMSO‐d6) δ (ppm): 17.90, 18.73, 55.90, 115.07,
75.98, 128.11, 131.11, 144.16, 152.07, 161.56, 164.87, and 179.21.
Mass (ESI), m/z: 321.0836 (M+). Anal. calcd. for C, 52,48; H, 5.03; N,
17.48; O, 4.99; S, 20.01. Found: C, 52.49; H, 5.02; N, 17.49; O, 5.00; S,
20.00.
Yield, 70–74%; m.p. 220.9°C. IR Vmax (cm−1): 3,411 (NH),
3,283–3,151.7 (arom.), 2,968–2,912.1 (aliph.), 1,582.30–1,493.57
(C═N, C═C), 1,087.95 (C═S), 692.81 (C–S), and 531.23 (C–Cl). 1H‐
NMR (300 MHz, DMSO‐d6) δ (ppm): 2.39 (3H, s, CH3), 2.60 (3H, s,
thiazole CH3), 7.44 (1H, brs, NH), 7.54–7.58 (2H, m, phenyl C3,5–H),
7.91–7.95 (2H, m, phenyl C2,6–H), and 8.41 (1H, brs, NH). 13C‐NMR
(75 MHz, DMSO‐d6) δ (ppm): 17.87, 18.67, 128.12, 129.79, 131.99,
132.79, 135.46, 143.77, 152.34, 163.42, and 179.31. Mass (ESI), m/z:
325.0345 (M+). Anal. calcd. for C, 48.06; H, 4.03; Cl, 10.91; N, 17.25;
S, 19.74. Found: C, 48.08; H, 4.01; Cl, 10.93; N, 17.24; S, 19.75.
2‐(1‐(4‐Methyl‐2‐(pyridine‐2‐yl)thiazole‐5‐yl)ethylidene)hydrazine
carbothioamide (3)
Yield, 72–74%; m.p. 218.2°C. IR Vmax (cm−1): 3,233.7 (NH),
3,115.6–3,053.2 (arom.), 2,912.1–2,767.7 (aliph.), 1,688–1,619.1
(C═N, C═C), 1,088.35 (C═S), and 781.18 (C–S). 1H NMR (300 MHz,

ERTAS ET AL.
9 of 13
|
DMSO‐d6) δ (ppm): 2.40 (3H, s, CH3), 2.62 (3H, s, thiazole CH3),
7.47–7.52 (2H, m, pyridine C4–H, NH), 7.95 (1H, td, J = 7.50, 1.68 Hz,
pyridine C5–H), 8.10 (1H, d, J = 7.82 Hz, pyridine C3–H), 8.39 (1H, s,
NH), and 8.61 (1H, d, J = 4.85 Hz, pyridine C6–H). 13C NMR (75 MHz,
DMSO‐d6) δ (ppm): 18.15, 18.90, 119.46, 75.71, 134.24, 138.20,
143.90, 150.22, 150.70, 152.58, 165.89, and 179.35. Mass (ESI), m/z:
292.0679 (M+). Anal. calcd. for C, 49.46; H, 4.50; N, 24.03; S, 22.01.
Found: C, 49.49; H, 4.51; N, 24.05; S, 22.00.
DMSO‐d6) δ (ppm): 2.41 (3H, s, CH3), 2.64 (3H, s, thiazole CH3), 7.52
(1H, s, NH), 7.57 (1H, t, J = 4.76 Hz, pyrimidine C5–H), 8.41 (1H, brs,
NH), and 8.92 (2H, d, J = 4.76 Hz, pyrimidine C4,6–H). 13C NMR
(75 MHz, DMSO‐d6) δ (ppm): 18.18, 18.92, 122.04, 135.86, 143.57,
153.25, 158.57, 158.91, 163.33, and 179.42. Mass (ESI), m/z:
293.0648 (M+). Anal. calcd. for C, 45.19; H, 4.14; N, 28.74; S,
21.93. Found: C, 45.16; H, 4.16; N, 28.70; S, 21.95.
2‐(1‐(4‐Methyl‐2‐(pyridine‐3‐yl)thiazole‐5‐yl)ethylidene)hydrazine-
carbothioamide (4)
2‐(1‐(4‐Methyl‐2‐phenylthiazole‐5‐yl)ethylidene)hydrazinecar-
bothioamide (8)
Yield, 74–78%; m.p. 202°C. IR Vmax (cm−1): 3,240.3 (NH), 3,151.7
(arom.), 2,981 (aliph.), 1,489.62–1,476.66 (C═N, C═C), 1,084.2 (C═S),
and 703.90 (C–S). 1H NMR (300 MHz, DMSO‐d6) δ (ppm): 2.40 (3H, s,
CH3), 2.62 (3H, s, thiazole CH3), 7.45–7.55 (2H, m, pyridine C4–H,
NH), 8.27 (1H, d, J = 7.73 Hz, pyridine C5–H), 8.44 (1H, brs, NH), 8.66
(1H, d, J = 5.05 Hz, pyridine C6–H), and 9.10 (1H, s, pyridine C2–H).
13C NMR (75 MHz, DMSO‐d6) δ (ppm): 17.87, 18.63, 124.72, 129.13,
133.21, 133.81, 143.66, 147.19, 151.48, 152.43, 161.78, and 179.35.
Mass (ESI), m/z: 292.0688 (M+). Anal. calcd. for C, 49.46; H, 4.50; N,
24.03; S, 22.01. Found: C, 49.49; H, 4.51; N, 24.05; S, 22.00.
Yield, 68–70%; m.p. 221.7°C. IR Vmax (cm−1): 3,447 (NH),
3,322.3–3,197.6 (arom.), 2,977.8–2,902.3 (aliph.), 1,573.2–1,487.58
(C═N, C═C), 1,080.16 (C═S), and 753.51 (C–S). 1H‐NMR (300 MHz,
DMSO‐d6) δ (ppm): 2.39 (3H, s, CH3), 2.61 (3H, s, thiazole CH3), 7.44
(1H, s, NH), 7.47–7.52 (3H, m, phenyl C3,4,5–H), 7.91–7.94 (2H, m,
phenyl C2,6–H), and 8.41 (1H, s, NH). 13C NMR (75 MHz, DMSO‐d6) δ
(ppm): 17.93, 18.73, 126.45, 129.72, 130.95, 132.25, 133.17, 143.94,
152.25, 164.83, and 179.29. Mass (ESI), m/z: 291.0729 (M+). Anal.
calcd. for C, 53.77; H, 4.86; N, 19.29; S, 22.08. Found: C, 53.75; H,
4.88; N, 19.30; S, 22.10.
2‐(1‐(4‐Methyl‐2‐(pyridine‐4‐yl)thiazole‐5‐yl)ethylidene)hydrazine-
carbothioamide (
5
)
2‐(1‐(2‐(4‐Chlorophenyl)‐4‐methylthiazole‐5‐yl)ethylidine)‐N‐phenyl-
Yield, 68–72%; m.p. 221.1°C. IR Vmax (cm−1): 3,417.5 (NH),
3,250.1–3,138.6 (arom.), 2,977.8 (aliph.), 1,691.3–1,595.84 (C═N,
C═C), 1,078.84 (C═S), and 715.72 (C–S). 1H NMR (300 MHz, DMSO‐
d6) δ (ppm): 2.40 (3H, s, CH3), 2.63 (3H, s, thiazole CH3), 7.47 (1H, brs,
NH), 7.84 (2H, d, J = 5.57 Hz, pyridine C3,5–H), 8.44 (1H, brs, NH), and
8.70 (2H, d, J = 6.12 Hz, pyridine C2,6–H). 13C NMR (75 MHz, DMSO‐
d6) δ (ppm): 17.90, 18.67, 120.21, 134.42, 139.62, 143.42, 151.23,
152.78, 161.95, and 179.41. Mass (ESI), m/z: 292.0683 (M+). Anal.
calcd. for C, 49.46; H, 4.50; N, 24.03; S, 22.01. Found: C, 49.49;
H, 4.51; N, 24.05; S, 22.00.
9
hydrazinecarboxamide ( )
Yield, 76–78%; m.p. 191.6°C. IR Vmax (cm−1): 3,368.3 (NH),
3,263.3–3,175.92 (arom.), 2,977.8 (aliph.), 1,644.07 (C═O),
1,620.23–1,506.62 (C═N, C═C), 716.68 (C–S), and 565.95 (C–Cl).
1H‐NMR (300 MHz, DMSO‐d6) δ (ppm): 2.39 (3H, s, CH3), 2.60 (3H, s,
CH3), 7.18 (2H, s, HAr), 7.55–7.58 (3H, d, J = 9 Hz, HAr), 7.92–7.95 (2H,
d, J = 9 Hz, HAr), 8.41 (1H, s, HAr), 8.63 (2H, s, HAr), and 10.51
(15, s, NH). 13C‐NMR (75 MHz, DMSO‐d6) δ (ppm): 17.88, 18.68, 39.15,
39.43, 39.71, 39.98, 40.26, 40.54, 40.82, 128.13, 129.80, 131.99,
135.47, 143.78, 152.35, 179.31, and 181.66. Mass (ESI), m/z: 386.0813
(M+). Anal. calcd. for C, 59.29; H, 4.45; Cl, 9.21; N, 14.56;
O, 4.16; S, 8.33. Found: C, 59.30; H, 4.47; Cl, 9.22; N, 14.53; O, 4.18;
S, 8.34.
2‐(1‐(4‐Methyl‐2‐(pyrazine‐2‐yl)thiazole‐5‐yl)ethylidene)hydrazine-
carbothioamide (6)
Yield, 68–72%; m.p. 232.6°C. IR Vmax (cm−1): 3,414.2 (NH),
3,194.3–3,135.3 (arom.), 3,046.7–2,987.6 (aliph.), 1,588.02–1,501.61
(C═N, C═C), 1,084.72 (C═S), and 719.96 (C–S). 1H NMR (300 MHz,
DMSO‐d6) δ (ppm): 2.40 (3H, s, CH3), 2.64 (3H, s, thiazole CH3), 7.51
(1H, s, NH), 8.41 (1H, s, NH), 8.69 (1H, d, J = 2.78 Hz, pyrazine C5–H),
8.73 (1H, d, J = 2.64 Hz, pyrazine C6–H), and 9.27 (1H, s, pyrazine
C3–H). 13C NMR (75 MHz, DMSO‐d6) δ (ppm): 18.10, 18.83, 135.32,
140.79, 143.53, 145.03, 146.10, 146.31, 153.01, 163.16, and 179.41.
Mass (ESI), m/z: 293.0633 (M+). Anal. calcd. for C, 45.19; H, 4.14; N,
28.74; S, 21.93. Found: C, 45.16; H, 4.15; N, 28.77; S, 21.96.
2‐(1‐(2‐(4‐Methoxyphenyl)‐4‐methylthiazole‐5‐yl)ethylidene)‐N‐
methylhydrazinecarboxamide (10)
Yield, 66–70%; m.p. 204.5°C. IR Vmax (cm−1): 3,361.7 (NH),
3,184.5–3,073 (arom.), 2,994.2–2,836.6 (aliph.), 1,677.31 (C═O),
1,536.16–1,518.39 (C═N, C═C), and 750.87 (C–S). 1H‐NMR
(300 MHz, DMSO‐d6) δ (ppm): 2.27 (3H, s, CH3), 2.62 (3H, s, CH3),
3.83 (3H, s, OCH3), 7.00–7.09 (3H, m, HAr), 7.27–7.33 (2H, m, HAr),
7.57–7.60 (2H, m, HAr), 7.86–7.91 (2H, m, HAr), 8.61 (1H, s, NH), and
9.97 (1H, s, NH). 13C‐NMR (75 MHz, DMSO‐d6) δ (ppm): 17.60, 18.54,
39.16, 39.44, 39.71, 39.99, 40.27, 40.55, 40.83, 55.89, 115.06,
119.14, 119.53, 122.96, 126.08, 128.07, 129.18, 139.32, 141.69,
151.06, 153.46, 161.47, and 164.24. Mass (ESI), m/z: 381.1378 (M+).
Anal. calcd. for C, 63.14; H, 5.30; N, 14.73; O, 8.41; S, 8.43. Found: C,
63.15; H, 5.33; N, 14.71; O, 8.40; S, 8.45.
2‐(1‐(4‐Methyl‐2‐(pyrimidine‐4‐yl)thiazole‐5‐yl)ethylidene)hydrazi-
necarbothioamide (7)
Yield, 72–76%; m.p. 252.5°C. IR Vmax (cm−1): 3,427.3 (NH),
3,112.3–3,033.5 (arom.), 2,971.2 (aliph.), 1,668.4–1,600.48
(C═N, C═C), 1,078.84 (C═S), and 806.38 (C–S). 1H NMR (300 MHz,

10 of 13
ERTAS ET AL.
|
2‐(1‐(4‐Methyl‐2‐(pyridine‐2‐yl)thiazole‐5‐yl)ethylidene)‐N‐phenyl-
hydrazinecarboxamide (11)
J = 9 Hz, HAr), 8.67–8.75 (3H, m, HAr), 9.29 (1H, s, HAr), and 10.05 (1H,
s, NH). 13C NMR (75 MHz, DMSO‐d6) δ (ppm): 17.78, 18.67, 18.94,
119.65, 123.02, 125.12, 125.68, 128.71, 129.17, 139.30, 140.77,
141.14, 145.05, 146.21, 146.37, 152.18, 153.42, and 162.52. Mass
(ESI), m/z: 353.1164 (M+). Anal. calcd. for C, 57.94; H, 4.58; N, 23.85;
O, 4.54, S, 9.10. Found: C, 57.96; H, 4.60; N, 23.83; O, 4.52, S, 9.09.
Yield, 68–70%; m.p. 250.1°C. IR Vmax (cm−1): 3,358.4 (NH),
3,184.5–3,086 (arom.), 2,928.5 (aliph.), 1,678.99 (C═O), 1,533.06
(C═N, C═C), and 742.22 (C–S). 1H‐NMR (300 MHz, DMSO‐d6) δ
(ppm): 2.35 (H, s, CH3), 2.68 (3H, s, CH3), 7.03 (1H, t, J = 7.5 Hz, HAr),
7.31 (2H, t, J = 6 Hz, HAr), 7.47–7.51 (1H, m, HAr), 7.59 (2H, d, J = 9 Hz,
HAr), 7.96 (1H, td, J = 9 Hz, HAr), 8.12 (1H, d, J = 9 Hz, HAr), 8.62 (1H,
2‐(1‐(4‐Methyl‐2‐(pyrimidine‐2‐yl)thiazole‐5‐yl)ethylidene)‐N‐phe-
d, J = 9 Hz, HAr), 8.66 (1H, s, HAr), and 10.02 (1H, s, NH). 13C‐NMR
(75 MHz, DMSO‐d6) δ (ppm): 17.86, 18.73, 39.16, 39.44, 39.72, 40.00,
40.27, 40.55, 40.83, 119.40, 119.61, 122.99, 125.60, 129.17, 134.39,
138.16, 139.32, 141.45, 150.22, 150.80, 151.71, 153.49, and 165.24.
Mass (ESI), m/z: 352.1212 (M+). Anal. calcd. for C, 61.52; H, 4.88; N,
19.93; O, 4.55; S, 9.12. Found: C, 61.54; H, 4.90; N, 19.91; O, 4.57; S,
9.15.
nylhydrazinecarboxamide (15)
Yield, 72–76%; m.p. 258.7°C. IR Vmax (cm−1): 3,358.4 (NH),
3,187.8–3,059.8 (arom.), 2,990.9–2,902.3 (aliph.), 1,678.90 (C═O),
1,554.89–1,531.72 (C═N, C═C), and 754.65 (C–S). 1H NMR
(300 MHz, DMSO‐d6) δ (ppm): 2.36 (3H, s, CH3), 2.70 (3H, s, CH3),
7.02 (1H, t, J = 6 Hz, HAr), 7.31 (2H, t, J = 6 Hz, HAr), 7.55–7.59 (3H, m,
HAr), 8.69 (1H, m, HAr), 8.93 (2H, d, J = 3 Hz, HAr), 10.05 (1H, s, NH).
13C NMR (75 MHz, DMSO‐d6) δ (ppm): 17.88, 18.79, 119.63, 121.97,
123.01, 129.17, 139.31, 141.15, 153.45, and 158.58. Mass (ESI), m/z:
353.1165 (M+). Anal. calcd. for C, 57.94; H, 4.58; N, 23.85; O, 4.54, S,
9.10. Found: C, 57.96; H, 4.60; N, 23.83; O, 4.52, S, 9.09.
2‐(1‐(4‐Methyl‐2‐(pyridine‐3‐yl)thiazole‐5‐yl)ethylidene)‐N‐phenyl-
hydrazinecarboxamide (12
)
Yield, 70–72%; m.p. 225.4°C. IR Vmax (cm−1): 3,368.3 (NH),
3,191–3,053.2 (arom.), 2,984.3–2,882.6 (aliph.), 1,692.73 (C═O),
1,523.86–1,499.71 (C═N, C═C), and 752.77 (C–S). 1H‐NMR
(300 MHz, DMSO‐d6) δ (ppm): 2.35 (3H, s, CH3), 2.67 (3H, s, CH3),
7.03 (1H, t, J = 7.5Hz, HAr), 7.31 (2H, t, J = 6 Hz, HAr), 7.52–7.60 (3H,
m, HAr), 8.30 (1H, dt, J = 9 Hz, HAr), 8.63 (1H, s, HAr), 8.68 (1H, dd,
J = 6 Hz, HAr), 9.12 (1H, s, NH), and 10.04 (1H, s, NH). 13C‐NMR
(75 MHz, DMSO‐d6) δ (ppm): 17.53, 18.46, 119.58, 123.01, 124.73,
129.18, 133.38, 133.81, 139.29, 141.28, 147.20, 151.41, 151.51,
153.38, and 161.17. Mass (ESI), m/z: 352.1219 (M+). Anal. calcd. for
C, 61.52; H, 4.88; N, 19.93; O, 4.55; S, 9.12. Found: C, 61.54; H, 4.90;
N, 19.91; O, 4.57; S, 9.15.
2‐(1‐(4‐Methyl‐2‐phenylthiazole‐5‐yl)ethylidene)‐N‐phenylhydrazine-
carboxamide (16
)
Yield, 66–68%; m.p. 190.4°C. IR Vmax (cm−1): 33.61.7 (NH),
3,273.1–3,194.3 (arom.), 2,987.48–2,902.3 (aliph.), 1,684.8 (C═O),
1,637.40–1,489.28 (C═N, C═C), and 757.91 (C–S). 1H‐NMR
(300 MHz, DMSO‐d6) δ (ppm): 2.34 (3H, s, CH3), 2.65 (3H, s, CH3),
7.02 (1H, t, J = 6 Hz, HAr), 7.30 (2H, t, J = 6 Hz, HAr), 7.51 (3H, m, HAr),
7.60 (2H, s, HAr), 7.91–7.99 (2H, m, HAr), 8.63 (1H, s, HAr), and 10.00
(1H, s, NH). 13C NMR (75 MHz, DMSO‐d6) δ (ppm): 17.61, 18.55,
19.20, 119.56, 122.98, 126.43, 126.48, 126.65, 129.18, 129.72,
130.84, 131.10, 132.40, 133.10, 133.25, 139.31, 141.50, 153.44,
and 166.19. Mass (ESI), m/z: 351.1258 (M+). Anal. calcd. for C, 65.12;
H, 5.18; N, 15.99; O, 4.57; S, 9.15. Found: C, 65.14; H, 5.20; N, 16.00;
O 4.55; S, 9.12.
2‐(1‐(4‐Methyl‐2‐(pyridine‐4‐yl)thiazole‐5‐yl)ethylidene)‐N‐phenyl-
hydrazinecarboxamide (13)
Yield, 66–70%; m.p. 240.6°C. IR Vmax (cm−1): 3,378.1 (NH),
3,194.3–3,079.5 (arom.), 2,984.3–2,885.9 (aliph.), 1,703.32 (C═O),
1,599.46–1,526.91 (C═N, C═C), 754.50 (C–S). 1H‐NMR (300 MHz,
DMSO‐d6) δ (ppm): 2.36 (3H, s, CH3), 2.68 (3H, s, CH3), 7.03 (1H, t,
J = 7.5 Hz, HAr), 7.31 (2 H, t, J = 6 Hz, HAr), 7.59 (2H, d, J = 9 Hz, HAr),
7.87 (2H, dd, J = 6 Hz, HAr), 8.65 (1H, s, HAr), 8.71 (2H, dd, J = 6 Hz,
2‐(1‐(2‐(4‐Chlorophenyl)‐4‐methylthiazole‐5‐yl)ethylidene)‐N‐
methylhydrazinecarbothioamide (17)
Yield, 72–76%; m.p. 117.9°C. IR Vmax (cm−1): 3,299.4 (NH), 3,092.6
(arom.), 2,990.9–2,925.3 (aliph.), 1,661.44 (C═N, C═C), 1,087.16
(C═S), and 649.80 (C–S). 1H‐NMR (300 MHz, DMSO‐d6) δ (ppm): 2.38
(3H, s, CH3), 2.60 (3H, s, CH3), 3.04 (3H, s, CH3), 7.58 (2H, d, J = 8 Hz,
HAr), 7.93 (2H, d, J = 8 Hz, HAr), 8.11 (1H, s, HAr), and 10.52 (1H, s,
NH). 13C NMR (75 MHz, DMSO‐d6) δ (ppm): 17.86, 18.56, 30.64,
31.65, 128.08, 128.74, 129.82, 131.98, 132.84, 135.44, 143.56,
152.25, 163.36, and 178.95. Mass (ESI), m/z: 340.0425 (M+). Anal.
calcd. for C, 49.62; H, 4.46; Cl, 10.46; N, 16.53; S, 18.92. Found: C,
49.64; H, 4.47; Cl, 10.43; N, 16.56; S, 18.94.
H
Ar), and 10.07 (1H, s, NH). 13C‐NMR (75 MHz, DMSO‐d6) δ (ppm):
17.54, 18.51, 119.27, 119.60, 120.21, 123.03, 129.18, 134.58,
139.28, 139.64, 139.70, 141.06, 151.22, 151.36, 151.89, 153.35,
and 161.33. Mass (ESI), m/z: 352.1216 (M+). Anal. calcd. for C, 61.52;
H, 4.88; N, 19.93; O, 4.55; S, 9.12. Found: C, 61.54; H, 4.90; N, 19.91;
O, 4.57; S, 9.15.
2‐(1‐(4‐Methyl‐2‐(pyrazine‐2‐yl)thiazole‐5‐yl)ethylidene)‐N‐phenyl-
hydrazinecarboxamide (14)
Yield, 72–74%; m.p. 210°C. IR Vmax (cm−1): 3,381.4 (NH),
3,197.6–3,092.6 (arom.), 2,984.3–2,902.3 (aliph.), 1,680.57 (C═O),
1,539.55–1,520.38 (C═N, C═C), and 760.19 (C–S). 1H NMR
(300 MHz, DMSO‐d6) δ (ppm): 2.36 (3H, s, CH3), 2.70 (3H, s, CH3),
7.03 (1H, t, J = 6 Hz, HAr), 7.31 (2H, t, J = 6 Hz, HAr), 7.58 (1H, d,
2‐(1‐(2‐(4‐Methoxyphenyl)‐4‐methylthiazole‐5‐yl)ethylidene)‐N‐
methylhydrazinecarbothioamide (18)
Yield, 66–70%; m.p. 214.3°C. IR Vmax (cm−1): 3,371.6 (NH),
3,217.3–3,004 (arom.), 2,928.5–2,836.6 (aliph.), 1,609.3 (C═N,
C═C), 1,054.37 (C═S), and 827.03 (C–S). 1H‐NMR (300 MHz,

ERTAS ET AL.
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|
DMSO‐d6) δ (ppm): 2.36 (3H, s, CH3), 2.71 (3H, s, thiazole CH3), 3.04
(3H, s, NCH3), 3.82 (3H, s, OCH3), 7.04 (2H, d, J = 8.53 Hz, phenyl
N‐Methyl‐2‐(1‐(4‐methyl‐2‐(pyrazine‐2‐yl)thiazole‐5‐yl)ethylidene)‐
hydrazinecarbothioamide (22
)
C
3,5–H), and 7.84–7.87 (2H, d, J = 7.68 Hz, phenyl C2,6–H). 13C NMR
Yield, 74–78%; m.p. 210°C. IR Vmax (cm−1): 3,361.7 (NH),
3,227.2–3,043.4 (arom.), 2,987.6–2,931.8 (aliph.), 1,680.57 (C═O),
1,655.2–1,496.61 (C═N, C═C), 1,049.08 (C═S), and 755.03 (C–S). 1H
NMR (300 MHz, DMSO‐d6) δ (ppm): 2.40 (3H, s, CH3), 2.64 (3H, s,
thiazole CH3), 3.03 (3H, s, NCH3), 8.15 (1H, brs, NH), 8.69–8.71 (1H,
m, piperazine C5–H), 8.91 (1H, d, J = 2.45 Hz, piperazine C6–H), 8.92
(1H, d, J = 1.60 Hz, pyrazine C3–H), and 10.49 (1H, s, NH). 13C NMR
(75 MHz, DMSO‐d6) δ (ppm): 17.93, 18.67, 31.68, 135.49, 140.80,
143.30, 145.01, 146.13, 146.29, 152.90, 163.2, and 179.06. Mass
(ESI), m/z: 307.0791 (M+). Anal. calcd. for C, 47.04; H, 4.61; N, 27.43;
S, 20.93. Found: C, 47.01; H, 4.62; N, 27.45; O, 4.52, S, 20.94.
(75 MHz, DMSO‐d6) δ (ppm): 17.88, 18.61, 31.61, 55.89, 115.06,
126.01, 128.07, 131.15, 143.94, 151.95, 161.53, 164.82, and 178.98.
Mass (ESI), m/z: 335.0994 (M+). Anal. calcd. for C, 53.87; H, 5.42; N,
16.75; O, 4.78; S, 19.17. Found: C, 53.88; H, 5.44; N, 16.72; O, 4.80; S,
19.19.
N‐Methyl‐2‐(1‐(4‐methyl‐2‐(pyridine‐2‐yl)thiazole‐5‐yl)ethylidene)‐
hydrazinecarbothioamide (19
)
Yield, 74–76%; m.p. 232.7°C. IR Vmax (cm−1): 3,358.4 (NH), 3,151.7
(arom.), 2,977.8–2,931.8 (aliph.), 1,544.81–1,502.98 (C═N, C═C),
1,049.43 (C═S), 782.89 (C–S). 1H NMR (300 MHz, DMSO‐d6) δ (ppm):
2.39 (3H, s, CH3), 2.62 (3H, s, thiazole CH3), 3.04 (3H, s, NCH3),
7.47–7.51 (1H, m, pyridine C4–H), 7.95 (1H, t, J = 7.95 Hz, pyridine
C5–H), 8.11 (1H, d, J = 8.40 Hz, pyridine C3–H), 8.13 (1H, brs, NH),
and 8.61 (1H, d, pyridine C6–H). 13C NMR (75 MHz, DMSO‐d6) δ
(ppm): 17.99, 18.74, 31.65, 19.46, 75.68, 134.40, 138.18, 143.69,
150.18, 150.73, 152.46, 165.94, and 179.04. Mass (ESI), m/z:
306.0837 (M+). Anal. calcd. for C, 51.12; H, 4.95; N, 22.93; S,
21.00. Found: C, 51.10; H, 4.96; N, 22.10; S, 21.01.
N‐Methyl‐2‐(1‐(4‐methyl‐2‐(pyridine‐2‐yl)thiazole‐5‐yl)ethylidene)‐
hydrazinecarbothioamide (23)
Yield, 68–70%; m.p. 216.3°C. IR Vmax (cm−1): 3,365 (NH), 3,155
(arom.), 2,997.4–2,908.8 (aliph.), 1,523.30–1,496.72 (C═N, C═C),
1,124.02 (C═S), and 797.92 (C–S). 1H NMR (300 MHz, DMSO‐d6) δ
(ppm): 2.40 (3H, s, CH3), 2.64 (3H, s, thiazole CH3), 3.04 (3H, s,
NCH3), 8.15 (1H, brs, NH), 8.69–8.71 (1H, m, pyrimidine C5–H), 8.92
(2H, d, J = 4.85 Hz, pyrimidine C4.6–H), 10.49 (1H, s, NH). 13C NMR
(125 MHz, DMSO‐d6) δ (ppm): 18.03, 18.76, 31.68, 122.03, 136.01,
143.33, 153.14, 158.56, 158.93, 163.38, and 179.07. Mass (ESI), m/z:
307.0795 (M+). Anal. calcd. for C, 47.04; H, 4.61; N, 27.43; S, 20.93.
Found: C, 47.01; H, 4.60; N, 27.44; S, 20.91.
N‐Methyl‐2‐(1‐(4‐methyl‐2‐(pyridine‐3‐yl)thiazole‐5‐yl)ethylidene)‐
hydrazinecarbothioamide (20)
Yield, 66–70%; m.p. 209.8°C. IR Vmax (cm−1): 3,371.6 (NH), 3,148.4
(arom.), 2,971.2–2,905.6 (aliph.), 1,583–1,500.27 (C═N, C═C),
1,053.67 (C═S), and 705.01 (C–S). 1H NMR (300 MHz, DMSO‐d6) δ
(ppm): 2.39 (3H, s, CH3), 2.62 (3H, s, thiazole CH3), 3.04 (3H, s,
NCH3), 7.52–7.56 (1H, m, pyridine C5–H), 8.09 (1H, s, NH), 8.26 (1H,
d, J = 8.64 Hz, pyridine C4–H), 8.66 (1H, d, J = 4.94 Hz, pyridine
C6–H), and 9.09 (1H, s, pyridine C2–H). 13C NMR (75 MHz, DMSO‐d6)
δ (ppm): 17.90, 18.50, 31.64, 124.76, 129.16, 133.25, 133.80, 143.42,
147.17, 151.50, 152.35, 161.72, and 179.06. Mass (ESI), m/z:
306.0837 (M+). Anal. calcd. for C, 51.12; H, 4.95; N, 22.93; S,
21.00. Found: C, 51.10; H, 4.96; N, 22.90; S, 21.01.
N‐Methyl‐2‐(1‐(4‐methyl‐2‐phenylthiazole‐5‐yl)ethylidene)hydrazi-
necarbothioamide (24
)
Yield, 64–66%; m.p. 219.4°C. IR Vmax (cm−1): 3,358.4 (NH), 3,171.4
(arom.), 2,974.5–2,902.3 (aliph.), 1,520.04–1,491.79 (C═N, C═C),
1,052.52 (C═S), and 761.54 (C–S). 1H NMR (300 MHz, DMSO‐d6) δ
(ppm): 2.38 (3H, s, CH3), 2.60 (3H, s, thiazole CH3), 3.03 (3H, s,
NCH3), 7.48–7.52 (3H, m, phenyl C3,4,5–H), 7.91–7.94 (2H, m, phenyl
C
2.6–H), 8.07 (1H, brs, NH), and 10.46 (1H, s, NH). 13C NMR (75 MHz,
DMSO‐d6) δ (ppm): 17.93, 18.59, 31.63, 126.42, 129.73, 130.93,
132.29, 133.19, 143.71, 152.15, 164.79, and 179.03. Mass (ESI), m/z:
305.0879 (M+). Anal. calcd. for C, 55.23; H, 5.30; N, 18.40; S, 21.07.
Found: C, 55.21; H, 5.32; N, 18.39; S, 21.04.
N‐Methyl‐2‐(1‐(4‐methyl‐2‐(pyridine‐4‐yl)thiazole‐5‐yl)ethylidene)‐
|
4.2
Anticancer activity
hydrazinecarbothioamide (21
)
Yield, 72–74%; m.p. 257.2°C. IR Vmax (cm−1): 3,368.3 (NH),
3,145.1–3,059.8 (arom.), 2,941.7–2,902.3 (aliph.), 1,599.5–1,515.61
(C═N, C═C), 1,057.63 (C═S), and 702.44 (C–S). 1H NMR (300 MHz,
DMSO‐d6) δ (ppm): 2.40 (3H, s, CH3), 2.63 (3H, s, thiazole CH3),
3.05 (3H, s, NCH3), 7.84 (2H, d, J = 4.52 Hz, pyridine C3,5–H), 8.13
(1H, brs, NH), 8.69 (2H, d, J = 4.52 Hz, pyridine C2,6–H), and 10.53
(1H, s, NH). 13C NMR (75 MHz, DMSO‐d6) δ (ppm): 17.90, 18.53,
31.66, 120.19, 134.45, 139.65, 143.17, 151.26, 152.69, 161.89,
and 179.09. Mass (ESI), m/z: 306.0837 (M+). Anal. calcd. for C,
51.12; H, 4.95; N, 22.93; S, 21.00. Found: C, 51.10; H, 4.96; N,
22.90; S, 21.01.
The effect of the compounds on cellular proliferation was monitored
using the cell proliferation reagent kit I (MTT, 3‐(4,5‐dimethylthiazol‐
2‐yl)‐2,5‐diphenyltetrazolium bromide; Roche Applied Science, Cat-
alog Number: 11465007001).[48,49] The HEK293 (American Type
Culture Collection, Manassas, VA) and MCF7 (American Type
Culture Collection) cell lines were seeded in 100 μl of growth
medium at a density of 5 × 103 cells/well in 96‐well plates and
allowed to adhere for 24 hr. Then, serially diluted compounds were
added to an additional 100 μl of growth medium. The cells were
incubated for 48 hr at 37°C in humidified 5% CO2. After incubation,
10 μl of MTT stock solution was added to each well and further

12 of 13
ERTAS ET AL.
|
incubated at 37°C for 4 hr. Then, 100 μl of solubilizing solution was
added, and the plates were read at 550–600 nm using a scanning
multiwell spectrophotometer. The results were expressed as a
percentage of the control based on the means of three replicates.
The IC50 cytotoxicity values were determined as the compound
concentration that reduced the absorbance to 50% of that in the
untreated control wells.
using the mole ratio technique.[50] Blanks were prepared by
mixing 1 ml of ethanol and 9 ml of buffer. In the mole ratio
method, ligand–metal solutions were buffered at pH 7.4. The ratio
(moles of ligand/moles of metal) ranged from 0.5 to 5.0, in which
the amount of metal was kept constant but that of the ligand
varied. The absorbance values of the complex were measured
immediately after mixing the ligand and metal at room tempera-
ture. For each solution, absorbance at λmax was plotted against the
mole ratio (nL/nM).
|
4.3
Ribonucleotide reductase enzyme assay
|
Ribonucleotide reductase enzyme inhibition was monitored using the
RRM2 ELISA Kit (MyBioSource, ABD). The synthesized compounds
were first dissolved (under 1%) in dimethyl sulfoxide. Under sterile
conditions, the HEK293 cell lines were cultured and collected
separately for each compound in the amount appropriate for the
kit content, and then transferred from the wells to microcentrifuge
tubes. The cell suspension was diluted with 1× PBS (pH 7.2–7.4) until
the cell concentration reached 100 million/ml. The cells were stored
overnight at −20°C. After two freeze‐thaw cycles to break up the cell
membranes, the cell lysates were centrifuged for 5 min at 5,000g,
2–8°C. The supernatant was collected, and the enzyme activity of all
compounds was evaluated in accordance with the manufacturer’s
instructions.
4.6
Molecular modeling studies
|
4.6.1
Ligand preparation and docking experiment
The preparation of the protein structure and ligand, binding site
mapping, GRID files, docking, and scoring were performed using
algorithms included in the Maestro modules (Schrodinger Inc.).
The X‐ray crystallographic structure of human ribonucleotide
reductase (PDB entry code: 1W68) was obtained from the Protein
Data Bank (RCSB).[51] The 1W68 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 3e as the energy parameters. The 3D
diagrams of the synthesized compounds were drawn and
inspected using the 3D builder implemented in the Maestro suite
(Schrödinger LCC, OR). These compounds were prepared and
minimized by means of the OPLS 3e force field and the partial
atomic charges, ionization and tautomerization states were
computed at a pH range of 6–8 by Epik using the LigPrep module
|
4.4
Theoretical calculation of the ADME
parameters
The physicochemical parameters of molecular weight, tPSA, number
of hydrogen donors, number of hydrogen acceptors, number of
rotatable bonds and volume were analyzed for all compounds using
the online Molinspiration property calculator.[46] Drug‐likeness
properties of the compounds were calculated using the QikProp
module of Maestro (Schrodinger Inc.).
of the same suite. Then they were subjected to
a docking
experiments to identify the probable interactions between the
ligands and the enzyme‐active site by using the Glide module in
Schrodinger. The standard precision protocol included in Glide
was employed with the default parameter settings to the most
probable site near iron atoms found by the site map function of
the same software. The highest e‐model scoring poses of the
compounds were selected for further evaluation to explain the
probable ligand–enzyme interactions.
|
4.5
Spectrophotometric determination of
complex formation
Absorption measurements were carried out using an Agilent 8453
model UV‐Vis spectrophotometer with a 1.00‐cm quartz cell. The
experiments were performed at a wavelength of 190 to 800 nm.
An Oakton pH 2100 series pH‐meter was used in combination
with a glass electrode for pH measurements. All the ligands were
used without further purification. All chemicals and buffer used
were of reagent or spectral grade purity. NaH2PO4·2H2O and
Na2HPO4 were supplied by Sigma‐Aldrich, and ethanol and
acetonitrile were purchased from Merck. Ferric nitrate nonahy-
drate was supplied by Riedel‐de Haen. 0.1 mM standard solutions
of various ligands and ferric nitrate were dissolved in ethanol and
metisazone was dissolved in acetonitrile. All these solutions were
used as stock solutions throughout the study. The acidity of the
solution was kept constant at pH 7.40 within a buffer solution
containing NaH2PO4/Na2HPO4 having 0.1 M ionic strength. The
stoichiometric ratio of the ligand–metal complex was determined
ACKNOWLEDGMENT
This study was supported by the Anadolu University Scientific
Research Projects Commission, Eskisehir, Turkey (project number:
1609S624).
CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
ORCID
Merve Ertas
Barkin Berk
Leyla Yurttas
http://orcid.org/0000-0002-2289-7950
http://orcid.org/0000-0001-6047-2796
http://orcid.org/0000-0002-0957-6044

ERTAS ET AL.
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