New thiazoline-tetralin derivatives and biological activity evaluation

Article abstract of DOI:10.3390/molecules23010135

In this study, novel N’-(3-cyclohexyl/phenyl-4-(substituted phenyl)thiazole-2(3H)-ylidene)-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetohydrazide (4a–4k) derivatives were synthesized and their anticancer potency were evaluated on human breast adenocarcinoma cell line (MCF-7), human lung carcinoma cell line (A549) and mouse embryoblast cell line (NIH/3T3) using the MTT method, DNA synthesis inhibition and flow cytometric analysis. Compound 4e bearing 4-methoxyphenyl moiety exhibited the highest antitumor efficiency against MCF-7 cell line with higher DNA synthesis inhibition and apoptotic cell percentages (ealy+late apoptotic cell). On the other hand, compounds 4f, 4g, and 4h bearing 4-bromo, 4-chloro and 4-florophenyl moieties, respectively caused excellent apoptosis levels against A549 cell line when treated with lower concentration even than cisplatin. Anticholinesterase activity of the compounds were also tested, compound 4h showed 49.92% inhibition of acetylcholinesterase (AChE).

Full text of DOI:10.3390/molecules23010135

molecules
Article
New Thiazoline-Tetralin Derivatives and Biological
Activity Evaluation
1
Gülhan Turan-Zitouni 1,*, Leyla Yurttas¸ , Aouatef Tabbi 2, Güls¸en Akalın Çiftçi 3,
Halide Edip Temel 3 and Zafer Asım Kaplancıklı 1
ID
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskisehir,
Turkey; lyurttas@anadolu.edu.tr (L.Y.); zakaplan@anadolu.edu.tr (Z.A.K.)
Department of Chemistry, Faculty of Sciences, Mentouri University, 25000 Constantin, Algeria;
2
awateftabbi@yahoo.fr
3
Department of Biochemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskisehir, Turkey;
gakalin@anadolu.edu.tr (G.A.Ç.); heincedal@anadolu.edu.tr (H.E.T.)
*
Correspondence: gturan@anadolu.edu.tr; Tel.: +90-222-335-05-80 (ext. 3777)
Received: 27 December 2017; Accepted: 9 January 2018; Published: 10 January 2018
Abstract: In this study, novel N’-(3-cyclohexyl/phenyl-4-(substituted phenyl)thiazole-2(3H)-ylidene)-
2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetohydrazide (4a–4k) derivatives were synthesized and
their anticancer potency were evaluated on human breast adenocarcinoma cell line (MCF-7), human
lung carcinoma cell line (A549) and mouse embryoblast cell line (NIH/3T3) using the MTT method,
DNA synthesis inhibition and flow cytometric analysis. Compound 4e bearing 4-methoxyphenyl
moiety exhibited the highest antitumor efficiency against MCF-7 cell line with higher DNA synthesis
inhibition and apoptotic cell percentages (ealy+late apoptotic cell). On the other hand, compounds
4f, 4g, and 4h bearing 4-bromo, 4-chloro and 4-florophenyl moieties, respectively caused excellent
apoptosis levels against A549 cell line when treated with lower concentration even than cisplatin.
Anticholinesterase activity of the compounds were also tested, compound 4h showed 49.92%
inhibition of acetylcholinesterase (AChE).
Keywords: thiazoline; tetralin (tetrahydronaftalene); cytotoxicity; DNA synthesis inhibition;
apoptosis; anticholinesterase activity
1. Introduction
Thiazole ring is an important pharmacologically active heterocylic ring whose mono-, di-,
three-substituted, condensed derivatives and reduced analogs (thiazoline, thiazolidine) have been
widely studied in medicinal chemistry [
derivatives have attracted attention due to existing in many biochemical reactions in organisms [
Consequently, various thiazoline derivatives were reported with a broad spectrum of activity
attributed to this property [ ]. When considering molecular aspects and biological diversity,
1
–6
]. Among these molecules with common origin, thiazoline
7].
8,9
a wide variety of thiazoline derivatives were encountered with anticancer activity. From natural
sources, oligothiazoline includes some marine products such as tantazole B, mirabazole and
thiangazole, and were demonstrated to have high selective cytotoxicity on murine solid tumor [10,11].
Furthermore, bacterial isolates ulbactin F, G, aerugine and pyochelin, which possess tandemly
connected thiazoline and thiazolidine ring systems, were declared with tumor cell migration inhibitory
activity (Figure 1) [12]. Therewithal, synthetic thiazoline derivatives exhibiting antiproliferative activity
were widely studied and well documented in literature [13–22]. Apoptosis inducing [23] and cell
division inhibiting [24] abilities were reported as anticancer activity mechanism.
Molecules 2018, 23, 135; doi:10.3390/molecules23010135
www.mdpi.com/journal/molecules

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N
N
O
H
O
H
S
S
S
S
N
N
N
N
S
S
OH
OH
Ulbactin F
Ulbactin G
OH
S
OH
S
O
H
N
H
N
N
OH
H
OH
S
Aerugine
Pyochelin
Figure 1. Some thiazoline containing anticancer molecules.
On the other hand, tetrahydronaftalene (tetralin) is an efficient ring, which is found in
structures of anthracycline antibiotics doxorubicin, daunorubicin, epirubicin, and idarubicin (Figure 2).
These clinically used anticancer drugs have been known to act as DNA intercalators [25].
Another naturally occurring plant-derived podophyllotoxin glycosides have been established with
high cytotoxicity since 1950s [26]. Their semisynthetic derivatives etoposide and teniposide are
topoisomerase II inhibitor anticancer drugs in clinical use. Due to high toxicity, low water solubility,
acquired drug resistance and gastrointestinal discomfort, numerous structural modifications were
performed, consequently NK611, NPF, GL-311 and TOP53 molecules have been discovered which are
being tested in clinical trials, presently [27–29]. Besides, many studies have been carried out to achieve
appropriate tetralin derivatives in terms of all aspects to be an efficacious anticancer drug [30–40].
Figure 2. Tetralin including anticancer drugs.
Acetylcholinesterase is an enzyme responsible for the breakdown of cholinergic neurotransmitter
acetylcholine. The ability of inhibiting acetylcholinesterase activity is closely associated with
myasthenia gravis, Alzheimer, post-operative ileus, bladder distention, glaucoma diseases and
Parkinson’s disease dementia [41]. Since the 1980s, regulation of acetylcholinesterase during tumor
formation process including adhesion, differentiation, and proliferation stages have been investigated
and it was found to show an overexpression of some cholinesterase activities on some tumor varieties.

Molecules 2018, 23, 135
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Accordingly, some researches showed that the importance of cholinergic signal system in carcinogenic
tissues and reported acetylcholinesterase (AChE) inhibition-dependent reduction of cell proliferation
in primary cell lines derived from lung tumor then suggested that main antitumoral effects may be
responsible for AChE activity inhibition [42,43].
Considering the above data, we have combined thiazoline and tetralin rings using a
hydrazine bridge. We have synthesized novel N’-[3-cyclohexyl/phenyl-4-(substituted phenyl)thiazole-
2(3H)-ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetohydrazides (4a–4k) and evaluate their
anticancer effects on MCF-7 and A549 cell lines. The anticholinesterase activity of the compounds was
also studied on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes.
2. Results and Discussion
2.1. Chemistry
Eleven new compounds were synthesized in a three step synthetic procedure and this is outlined
in Scheme 1. Starting material 5,6,7,8-tetrahydro-2-naphthol was reacted with ethyl 2-chloroethyl
acetate to give ethyl 2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetate (
hydrazinated with hydrazine hydrate to give ethyl 2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]
acetohydrazide ( ) and these intermediates were reported, previously [44]. The resulting hydrazide
was boiled with phenyl and cyclohexyl isothiocyanate in ethanol to give N-phenyl/cyclohexyl-
2-[2-((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)acetyl]hydrazine carbothioamide (3a 3b) according
1), this intermediate was
2
,
to the literature [45]. In the last step these compounds were reacted with phenacyl bromide
derivatives to give N’-[3-phenyl/cyclohexyl-4-(substituted phenyl)thiazol-2(3H)-ylidene]-2-[(5,6,7,8-
tetrahydronaphthalen-2-yl)oxy]acetohydrazides (4a–4k). The substituents were showed in Table 1.
The structures of final compounds were confirmed by using spectral and elemental analysis. In the
IR spectra of the compounds (see Supplementary materials), characteristic bands belong to N–H and
C=O bonds of acetamide moiety were observed at 3109–3199 cm−1 and 1712–1745 cm−1 whereas the
protons of acetamide moiety were observed at 4.75–4.61 ppm as singlets belong to methylene and at
11.79–11.98 ppm belong to amino protons in the 1H-NMR spectra of the compounds. –CH2 Protons of
tetrahydronaftalene ring were detected at 1.67–1.98 ppm and 2.48–2.69 ppm region. In aromatic field,
proton of thiazole ring, C5–H was seen at about 7.0–7.19 ppm as singlet peak, mostly. In the 13C-NMR
spectra of the compounds, methylene carbons of acetamide group were resonated at 56.21–66.15 ppm,
whereas methylene carbons of tetrahydronaftalene ring were observed at 20.91–31.17 ppm. In the MS,
M + 1 peaks were detected for all final compounds. Elemental analysis data was also determined
within the accurate range of ±0.4%.
Table 1. The structures of the compounds (4a–4k).
Compounds
R1
R2
R3
R4
4a
4b
4c
4d
4e
4f
4g
4h
4i
Cyclohexyl
Cyclohexyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
NO2
H
H
OCH3
H
CH3
OCH3
Br
Cl
F
H
NO2
H
4j
4k
Phenyl
Phenyl
Cl

Molecules 2018, 23, 135
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Scheme 1. The synthesis of the compounds (4a–4k). Reactants and reagents: i: ClCH2COOEt, 3–5 h,
reflux; ii: NH2NH2 H2O, EtOH, 3 h, reflux; iii: C6H5NCS/C6H11NCS, EtOH, 2 h, reflux; iv: phenacyl
·
bromide derivative, EtOH, 5 h, reflux.
2.2. Biological Results
2.2.1. Cytotocity
In order to determine anticancer efficiency of the synthesized compounds, three-step activity
studies were carried out. Accordingly, cytotoxicity and DNA synthesis inhibition experiments and
tests to determine apoptosis/necrosis ratios were performed. Tested tumor cells were human breast
adenocarcinoma cell line (MCF-7), human lung carcinoma cell line (A549) and healthy cell mouse
embryoblast cell line (NIH/3T3) were used to detect cytotoxicity of the compounds according to
classical MTT method. The calculated inhibitory concentrations (IC50) and selectivity index (SI) are
represented in Table 2.
Table 2. IC50 values (µM) of the compounds and SI values against MCF-7, A549 and NIH/3T3 cell lines.
SIMCF-7
SIA549
Compounds
MCF-7
A549
NIH/3T3
4a
4b
4c
4d
4e
4f
4g
4h
>1000
157.6 ± 14.8
137.5 ± 7.2
>1000
206.1 ± 15.3
55.7 ± 1.2
>1000
>1000
>1000
146.7 ± 5.4
224.5 ± 20.5
246.7 ± 12.2
>1000
0.2
0.8
3.4
13.9
10.9
0.5
0.5
1.0
. . .
1.3
0.4
. . .
69.2 ± 5.8
296.7 ± 46.6
71.8 ± 12.5
91.4 ± 9.8
280.9 ± 56.2
483.0 ± 31.2
243.1 ± 14.9
>1000
>1000
. . .
707.9 ± 83.3
54.3 ± 6.8
28.6 ± 4.1
48.6 ± 6.0
>1000
1.4
2.7
7.8
5.1
. . .
20.4
. . .
. . .
4i
4j
4k
>1000
49.0 ± 1.41
>1000
. . .
1.1
. . .
152.7 ± 27.5
>1000
165.4 ± 11.0
Cisplatin
100.0 ± 3.3
108.3 ± 11.8
-: not tested; . . . :not calculated.
-
Compounds 4b (IC50 = 69.2 µM) and 4d (IC50 = 71.8 µM) showed the highest cytotoxicity against
MCF-7 cell line. However, compounds 4e and 4k also exhibited considerable cytotoxic activity. None of
these compounds were found as active as standard drug cisplatin. Among these four compounds 4b
and 4k showed toxicity to normal cells. When we compare, antiproliferative effects of the compounds,
their potency against A549 was found to be greater than against MCF-7 cells. It has been determined
that compounds 4f, 4g and 4h act at a lower concentration than the inhibition concentration of cisplatin

Molecules 2018, 23, 135
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which possessed 4-bromo, 4-chloro and 4-fluoro substituent moieties respectively on A549 cell line.
Besides, these compounds exhibited selectivity due to their low cytotoxicity on healthy cells with
selectivity index in between 2.7 and 7.8. However, the IC50 values of compounds 4c, 4d, 4i and 4k
were not determined at the highest concentration tested against A549. Additionally, compounds 4a
4b and 4j exhibited moderate antiproliferative activity.
,
2.2.2. DNA Synthesis Inhibition
DNA synthesis inhibition study, which was considered as the second stage in anticancer activity
studies, have been conducted on compounds with high cytotoxicity. Compounds 4b 4d 4e, and 4k
,
,
were determined to possess more cytotoxic activity on the MCF cell line. The graph generated
according to the percent inhibitions of the selected compounds at a: IC50/2 and b: IC50 concentrations
is shown in Figure 3. Compound 4b inhibited DNA synthesis with ratio of 31.39% at 34.6
48.50% at 69.2 M concentration on MCF-7 cell line. It was seen this ratio was very close to the
inhibition exhibited by cisplatin. The inhibition rates for compounds 4d 4e and 4k were calculated
higher than the rate of standard, ranging from 47.75% to 72.15%. Among these three, compound 4d
µM and
µ
,
exhibited the best inhibition potential of 47.75% at 36.9
MCF-7 cells.
µM concentration and 72.15% at 71.8 µM on
DNA synthesis inhibition studies have also been conducted on those compounds that exhibit
high cytotoxic activity on A549 cells. Compounds 4a 4b 4f 4g and 4j were selected for this study.
,
,
,
The graph based on the obtained results is shown in Figure 4. DNA synthesis inhibition on A549
revealed that these six compounds showed close inhibition values of 20.13–50.33%. The compound
4h bearing 4-fluoro substitution on phenyl ring showed the highest inhibitory effect. The inhibition
percentages were specified as 8.97% at 24.3 µM and 50.78% at 48.6 µM whereas these values were
established 32.86% at 54.2 µM and 50.33% at 108.3 µM for standard drug cisplatin.
MCF-7
80
60
40
20
0
4b1 4b2 4d1 4d2 4e 1 4e 2 4k1 4k2 cisp1 cisp2
Compounds and doses
Figure 3. DNA synthesis inhibitory activity of compounds 4b
cells. Mean percent absorbance of untreated control cells were assumed 0%. In addition, two different
concentrations (1: IC50/2; 2: IC50 M) of test compounds and cisplatin were given. Data points
, 4d, 4e, 4k and cisplatin on MCF-7
µ
represent means for three independent wells ± SD. p < 0.05.
2.2.3. Apoptosis/Necrosis Detection
To determine the death mechanism of the most active compounds, flow cytometric analysis was
done according to the instructions of the Annexin V-FITC Apoptosis Detection Kit. The results were
calculated in percentage of which the ratios were demonstrated as early apoptotic, late apoptotic, viable
and necrotic cells. The numerical values were represented in Table 3 and diagrams in Figures 5 and 6.
Compounds 4b, 4d, 4e and 4k against MCF-7 cell line and compounds 4a, 4b, 4f, 4g, 4h and 4j against
A549 cells were tested in flow cytometry at IC50 concentrations of the compounds. Against MCF-7 cell
line, compounds 4b and 4e exhibited 29.8% and 32.5% apoptosis (ealy + late apoptotic cell percentage),

Molecules 2018, 23, 135
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respectively whereas cisplatin caused 29.9% apoptosis of the cells. Other two compounds did not
showed prominent activity. All selected compounds for flow cytometric analysis on A549 cell line
displayed marvelous activity that apoptotic cell ratios were found even higher than cisplatin caused
(40.2%). Among them, compound 4b showed the highest apoptosis level of 73.6% which was a great
value for such a study. Besides, compounds 4a and 4h caused apoptosis over 50% and compounds 4f
,
4g and 4j caused apoptosis over 41.5%. Among these compounds, the results of compounds 4f 4g and
,
4h bearing p-halogenated phenyl moiety are more meaningful, which, is because they have lower
IC50 values than cisplatin. Consequently, the dose administered to this test is lesser, but apoptotic cell
percentage found is higher. In addition, these three compounds have exhibited non-toxic profile which
is a required property for druglikeness of a molecule.
A549
60
50
40
30
20
10
0
4a1 4a2 4b1 4b2 4f1 4f2 4g1 4g2 4h1 4h2 4j1 4j2 cisp1 cisp2
Compounds and doses
Figure 4. DNA synthesis inhibitory activity of compounds 4a
cells. Mean percent absorbance of untreated control cells were assumed 0%. In addition, two different
concentrations (1: IC50/2; 2: IC50 M) of test compounds and cisplatin were given. Data points
, 4b, 4f, 4g, 4h, 4j and cisplatin on A549
µ
represent means for three independent wells ± SD. p < 0.05.
Table 3. Percents of typical quadrant analysis of Annexin V FITC/Propidium Iodide flow cytometry of
MCF-7 and A549 cells treated with compounds and Cisplatin.
Groups
Control
Compound 4b
Compound 4d
Compound 4e
Compound 4k
Cisplatin
% Early Apoptotic
% Late Apoptotic
% Viable
% Necrotic
1.7
0.7
1.1
0.8
2.4
2.8
4.3
29.1
12.1
31.7
8.9
84.9
6.3
50.7
24.8
54.3
42.0
9.1
63.8
36.1
39.1
34.4
28.1
MCF-7 cell
line
27.1
Control
3.1
7.4
6.6
16.5
18.7
23.7
11.2
14.9
6.1
89.0
31.5
10.9
47.7
49.4
45.9
47.0
53.1
1.8
12.8
15.6
6.5
5.8
4.0
Compound 4a
Compound 4b
Compound 4f
Compound 4g
Compound 4h
Compound 4j
Cisplatin
48.3
67.0
29.3
26.0
26.4
30.3
25.3
A549 cell line
11.4
6.7

Molecules 2018, 23, 135
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Figure 5. Flow cytometric diagrams of MCF-7 cells treated with compounds 4a, 4d, 4e, 4k, and cisplatin.
2.2.4. Anticholinesterase Activity
The inhibitory effects of the compounds on the acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE) enzymes were investigated. The efficacy of the compounds is compared to the anticholinesterase
effective donepezil drug molecule used in Alzheimer's treatment. The results obtained are shown in
Table 4.
Table 4. % AChE and BuChE inhibitions of the compounds at 80 µg/mL concentration.
AChE
BuChE
% Inhibition (80 µg/mL)
Compounds
% Inhibition (80 µg/mL)
4a
4b
4c
4d
4e
4f
4g
4h
38.26 ± 2.18
11.08 ± 1.48
19.30 ± 1.87
43.22 ± 1.56
10.81 ± 0.94
42.87 ± 2.84
24.37 ± 1.20
49.92 ± 1.44
41.85 ± 0.23
44.96 ± 1.60
15.19 ± 1.99
28 ± 1.88
15.89 ± 1.72
16.48 ± 0.08
33.19 ± 2.38
—
—
—
—
—
—
—
4i
4j
4k
−3
Donepezil IC50 (µM)
4.85 ± 0.55
(11.8 ± 0.7) × 10
—: no inhibition.

Molecules 2018, 23, 135
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Figure 6. Flow cytometric diagrams of A549 cells treated with compounds 4a
, 4b, 4f, 4g, 4h, 4j
and cisplatin.
Based on the enzyme inhibition studies, AChE and BuChE inhibitions were determined
at 80 g/mL (~160 M) concentrations of the compounds. Compound 4h showed the highest
µ
µ
acetylcholinesterase inhibition with 49.92%. It was followed by compounds 4j with 44.96%, 4d with
43.22%, 4f with 42.87% and 4i with 41.85%. Compounds 4b and 4e exhibited the lowest inhibition
among remaining others. Since the compounds did not provide over 50% enzyme inhibition, they were
not studied at lower doses to detect inhibition concentrations (IC50). The IC50 value of the standard
drug, donepezil, was determined to be (11.8
enzyme at concentration of 80 g/mL was found in between 15.89% and 33.19% for compounds 4a
4b 4c and 4d while other compounds did not inhibit this enzyme. Compounds 4f 4g 4h and 4j are
±
0.7)
×
10−3 µM. The inhibition of butyrylcholinesterase
µ
,
,
,
,
the most active compounds on A549 cancer cells. The AChE inhibition effect of these substances may
enhance antiproliferative activity of them.
3. Materials and Methods
3.1. Chemistry
All chemicals were purchased from Sigma-Aldrich Chemical Co., (Sigma-Aldrich Corp., St. Louis,
MO, USA) and Merck (Merck, Darmstadt, Germany). All melting points (m.p.) were determined by
Electrothermal 9300 digital melting point apparatus (Electrothermal, Essex, UK) and are uncorrected.
All the reactions were monitored by thin-layer chromatography (TLC) using Silica gel 60 F254
TLC plates (Merck, Darmstadt, Germany). Spectroscopic data were recorded with the following
instruments: IR, Shimadzu 8400S spectrophotometer (Shimadzu, Tokyo, Japan); 1H-NMR, Agilent 400

Molecules 2018, 23, 135
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MHz NMR spectrometer (Agilent Technologies, Palo Alto, CA, USA); 13C-NMR, Agilent 100 MHz
NMR spectrometer (Agilent Technologies, Palo Alto, CA, USA); Shimadzu 8040 LC/MS/MS system
(Shimadzu, Tokyo, Japan), and elemental analyses were performed on a Perkin Elmer EAL 240
elemental analyser (Perkin Elmer, Norwalk, CT, USA).
General procedure for the synthesis of N0-[3-cyclohexyl/phenyl-4-(substituted phenyl)thiazole-2(3H)-
ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetohydrazide (4a–4k). The synthesized intermediates
N-phenyl/cyclohexyl-2-[2-((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)acetyl]hydrazine carbothioamide
(3a, 3b) were refluxed with phenacyl bromide derivatives for 5 h in ethanol. After overnight standing
in a cool place, crystals of final thiazole compounds were filtered off.
N0-(3-Cyclohexyl-4-phenylthiazole-2(3H)-ylidene)-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetohydrazide
◦
(
4a): Yield: % 68. m.p. 277 C. IR
C–H), 1745 (amide C=O), 1595–1442 (C=C and C=N), 1215–1004 (C–N and C–O). 1H-NMR (400 MHz,
DMSO-d6, ppm): 1.13–2.60 (19H, m, CH2), 4.61 (2H, s, OCH2), 6.46–7.51 (9H, m, Ar–H), 11.85 (1H, s,
NH). 13C-NMR (100 MHz, DMSO-d6, ppm)
22.5, 22.8, 24.3, 24.5, 27.9, 28.9, 31.2, 55.2, 58.5, 66.0, 100.9,
ν
(cm−1): 3199 (N–H), 3070 (aromatic C–H), 2854 (aliphatic
max
δ
δ
112.1, 114.1, 114.3, 118.9, 129.5, 129.5, 130.1, 137.6, 140.8, 155.1, 160.6, 167.3. HRMS (m/z): [M + H]+ for
C27H31N3O2S calculated: 461.62; found: 462.2195. For C27H31N3O2S calculated: 70.25% C, 6.77% H,
9.10% N; found: 70.23% C, 6.76% H, 9.12% N.
N0-[3-Cyclohexyl-4-(4-methoxyphenyl)thiazole-2(3H)-ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]
◦
acetohydrazide (4b): Yield: % 71. m.p. 266 C. IR
2972 (aliphatic C–H), 1714 (amide C=O), 1602–1502 (C=C and C=N), 1247–1037 (C–N and C–O).
1H-NMR (400 MHz, DMSO-d6, ppm):
1.12–2.61 (19H, m, CH2), 4.62 (2H, s, OCH2), 6.45–6.49 (2H, m,
Ar–H), 6.85 (1H, d, J = 8.4 Hz, Ar–H), 6.95 (2H, d, J = 8.8 Hz), 7.12 (1H, s, Ar–H), 7.43 (2H, d, J = 8.8 Hz,
Ar–H), 11.79 (1H, s, NH). 13C-NMR (100 MHz, DMSO-d6, ppm)
22.5, 22.7, 27.8, 28.9, 66.1, 112.0,
ν
(cm−1): 3182 (N–H), 3073 (aromatic C–H),
max
δ
δ
114.3, 122.6, 128.1, 129.4, 129.5, 129.9, 130.0, 130.7, 131.4, 132.6, 137.5, 137.7, 155.1, 167.8. HRMS (m/z):
[M + H]+ for C28H33N3O3S calculated: 491.65; found: 492.2296. For C28H33N3O3S calculated: 68.40%
C, 6.77% H, 8.55% N; found: 68.42% C, 6.76% H, 8.56% N.
0
N -[3,4-Diphenylthiazole-2(3H)-ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy] acetohydrazide (4c): Yield:
◦
% 73. m.p. 253 C. IR
C=O), 1600–1444 (C=C and C=N), 1217–1055 (C–N and C–O). 1H-NMR (400 MHz, DMSO-d6, ppm):
1.98 (4H, br, CH2), 2.56–2.61 (4H, m, CH2), 4.65 (2H, s, OCH2), 6.49–7.58 (13H, m, Ar–H), 7.07 (1H, s,
thiazole–H), 11.98 (1H, br, NH). 13C-NMR (100 MHz, DMSO-d6, ppm)
22.6, 22.8, 22.9, 24.5, 25.2, 27.9,
ν
max (cm−1): 3120 (N–H), 3053 (aromatic C–H), 2987 (aliphatic C–H), 1714 (amide
δ
δ
29.0, 30.7, 56.2, 60.2, 112.6, 112.6, 114.5, 123.8, 129.6, 129.8, 129.8, 137.7, 140.2, 149.1, 150.1, 151.7, 155.1,
192.6. HRMS (m/z): [M + H]+ for C27H25N3O2S calculated: 455.58; found: 456.1719. For C27H25N3O2S
calculated: 71.18% C, 5.53% H, 9.22% N; found: 71.20% C, 5.54% H, 9.23% N.
N0-[3-Phenyl-4-(4-methylphenyl)thiazole-2(3H)-ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]
◦
acetohydrazide (4d): Yield: % 72. m.p. 258 C. IR
2860 (aliphatic C–H), 1745 (amide C=O), 1599–1496 (C=C and C=N), 1199–1074 (C–N and C–O).
1H-NMR (400 MHz, DMSO-d6, ppm):
1.67 (4H, br, CH2), 2.32 (3H, s, CH3), 2.56–2.59 (4H, m, CH2),
4.65 (2H, s, OCH2), 6.48–7.56 (12H, m, Ar–H), 7.00 (1H, s, thiazole–H), 11.98 (1H, br, NH). 13C-NMR
ν
(cm−1): 3109 (N–H), 3047 (aromatic C–H),
max
δ
(100 MHz, DMSO-d6, ppm)
δ 22.4, 22.7, 27.8, 28.9, 66.0, 112.1, 114.1, 122.7, 123.6, 129.3, 129.4, 129.5,
130.1, 133.7, 137.4, 138.1, 147.6, 155.1, 168.0. HRMS (m/z): [M + H]+ for C28H27N3O2S calculated:
469.60; found: 470.1885. For C28H27N3O2S calculated: 71.62% C, 5.80% H, 8.95% N; found: 71.61% C,
5.79% H, 8.94% N.
N0-[3-Phenyl-4-(4-methoxyphenyl)thiazole-2(3H)-ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]
◦
acetohydrazide (4e): Yield: % 67. m.p. 240 C. IR
2860 (aliphatic C–H), 1745 (amide C=O), 1599–1496 (C=C and C=N), 1199–1074 (C–N and C–O).
1H-NMR (400 MHz, DMSO-d6, ppm):
1.68 (4H, br, CH2), 2.48–2.59 (4H, m, CH2), 3.77 (3H, s, OCH3),
4.65 (2H, s, OCH2), 6.47–7.57 (13H, m, Ar–H), 11.98 (1H, br, NH). 13C-NMR (100 MHz, DMSO-d6,
ν
(cm−1): 3109 (N–H), 3047 (aromatic C–H),
max
δ

Molecules 2018, 23, 135
10 of 15
ppm)
δ 22.6, 22.9, 27.9, 29.0, 66.0, 112.2, 114.4, 123.5, 127.3, 128.4, 128.7, 129.5, 129.7, 129.7, 131.1, 130.3,
137.6, 140.6, 155.2, 167.7. HRMS (m/z): [M + H]+ for C28H27N3O3S calculated: 485.60; found: 486.1834.
For C28H27N3O3S calculated: 69.26% C, 5.60% H, 8.65% N; found: 69.24% C, 5.61% H, 8.66% N.
N0-[3-Phenyl-4-(4-bromophenyl)thiazole-2(3H)-ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]
◦
acetohydrazide (4f): Yield: % 74. m.p. 272 C. IR
2935 (aliphatic C–H), 1712 (amide C=O), 1598–1454 (C=C and C=N), 1313–1006 (C–N and C–O).
1H-NMR (400 MHz, DMSO-d6, ppm):
1.67 (4H, br, CH2), 2.50–2.63 (4H, m, CH2), 4.67 (2H, d,
ν
(cm−1): 3122 (N–H), 3039 (aromatic C–H),
max
δ
J = 4.4 Hz, OCH2), 6.48–6.51 (2H, m, Ar–H), 6.87 (1H, d, J =7.6 Hz, Ar–H), 7.04 (1H, s, thiazole–H),
7.33–7.38 (3H, m, Ar–H), 7.45 (2H, d, J = 8.4 Hz), 7.52–7.56 (2H, m, Ar–H), 7.60 (2H, d, J = 8.4 Hz,
Ar–H), 11.89 (1H, br, NH). 13C-NMR (100 MHz, DMSO-d6, ppm)
δ 22.6, 22.9, 28.0, 29.0, 66.0, 112.2,
114.4, 123.2, 126.3, 128.8, 129.5, 129.6, 130.1, 130.3, 134.9, 137.6, 139.26, 155.2, 167.9. HRMS (m/z):
[M + H]+ for C27H24N3O2SBr calculated: 534.47; found: 536.0798. For C27H24N3O2SBr calculated:
60.68% C, 4.53% H, 7.86% N; found: 60.67% C, 4.52% H, 7.87% N.
N0-[3-Phenyl-4-(4-chlorophenyl)thiazole-2(3H)-ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]
◦
acetohydrazide (4g): Yield: % 67. m.p. 268 C. IR
2972 (aliphatic C–H), 1712 (amide C=O), 1593–1456 (C=C and C=N), 1313–1055 (C–N and C–O).
1H-NMR (400 MHz, DMSO-d6, ppm):
1.68 (4H, br, CH2), 2.50–2.62 (4H, m, CH2), 4.64 and 4.70 (2H,
d, J = 15.6 Hz, OCH2), 6.49–6.51 (2H, m, Ar–H), 6.87 (1H, d, J = 8.8 Hz, Ar–H), 7.05 (1H, s, thiazole–H),
7.35–7.56 (9H, m, Ar–H), 11.93 (1H, br, NH). 13C-NMR (100 MHz, DMSO-d6, ppm)
20.9, 22.6, 22.9,
ν
(cm−1): 3122 (N–H), 3032 (aromatic C–H),
max
δ
δ
29.0, 66.0, 112.2, 114.4, 123.5, 124.4, 128.8, 129.3, 129.5, 129.6, 130.4, 137.6, 139.9, 140.7, 155.2, 167.7.
HRMS (m/z): [M + H]+ for C27H24N3O2SCl calculated: 490.02; found: 490.1328. For C27H24N3O2SCl
calculated: 66.18% C, 4.94% H, 7.58% N; found: 66.16% C, 4.93% H, 8.57% N.
N0-[3-Phenyl-4-(4-florophenyl)thiazole-2(3H)-y◦lidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]
(cm−1): 3116 (N–H), 3043 (aromatic C–H),
max
acetohydrazide (4h): Yield: % 72. m.p. 257 C. IR
ν
2916 (aliphatic C–H), 1714 (amide C=O), 1600–1494 (C=C and C=N), 1313–1035 (C–N and C–O).
1H-NMR (400 MHz, DMSO-d6, ppm):
δ
1.67 (4H, br, CH2), 2.57–2.62 (4H, m, CH2), 4.62 and 4.69
(2H, dd, J = 15.6 Hz, OCH2), 6.50–6.53 ( 2H, m, Ar–H), 6.86 (1H, d, J = 8.0 Hz, Ar–H), 7.03 (1H, s,
thiazole–H), 7.21–7.56 (9H, m, Ar–H), 11.93 (1H, br, NH). 13C-NMR (100 MHz, DMSO-d6, ppm)
δ
22.6, 22.8, 27.9, 29.0, 66.0, 112.1, 114.4, 123.1, 123.6, 126.6, 129.5, 129.6, 130.2, 130.2, 131.6, 137.6,
139.3, 155.2, 167.8. HRMS (m/z): [M + H]+ for C27H24FN3O2S calculated: 473.57; found: 474.1629.
For C27H24FN3O2S calculated: 68.48% C, 5.11% H, 8.87% N; found: 68.49% C, 5.12% H, 8.88% N.
N0-[3-Phenyl-4-(3-nitrophenyl)thiazole-2(3H)-ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]
◦
acetohydrazide (4i): Yield: % 69. m.p. 252 C. IR
2949 (aliphatic C–H), 1710 (amide C=O), 1604–1344 (C=C and C=N), 1217–1062 (C–N and C–O).
1H-NMR (400 MHz, DMSO-d6, ppm):
1.64 (4H, br, CH2), 2.47–2.57 (4H, m, CH2), 4.52 and 6.72 (2H,
dd, J = 15.6 Hz, OCH2), 6.41–6.43 (2H, m, Ar–H), 6.74 (1H, d, J = 8.0 Hz, Ar–H), 7.16 (1H, s, thiazole–H),
7.30–8.29 (9H, m, Ar–H), 11.95 (1H, br, NH). 13C-NMR (100 MHz, DMSO-d6, ppm)
22.6, 22.8, 27.9,
ν
(cm−1): 3118 (N–H), 3045 (aromatic C–H),
max
δ
δ
29.0, 55.3, 66.1, 112.2, 114.1, 114.4, 119.4, 123.6, 129.5, 129.6, 130.0, 130.6, 137.6, 140.6, 155.2, 160.5, 167.7.
HRMS (m/z): [M + H]+ for C27H24N4O4S calculated: 500.57; found: 501.1567. For C27H24N4O4S
calculated: 64.79% C, 4.83% H, 11.19% N; found: 64.78% C, 4.82% H, 11.18% N.
N0-[3-Phenyl-4-(4-nitrophenyl)thiazole-2(3H)-ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]
◦
acetohydrazide (4j): Yield: % 67. m.p. 268 C. IR
2931 (aliphatic C–H), 1714 (amide C=O), 1602–1338 (C=C and C=N), 1288–1035 (C–N and C–O).
1H-NMR (400 MHz, DMSO-d6, ppm):
1.65 (4H, br, CH2), 2.50–2.57 (4H, m, CH2), 4.61 and 4.75
(2H, dd, J = 15.6 Hz, OCH2), 6.48–6.54 ( 2H, m, Ar–H), 6.81 (1H, d, J = 8.0 Hz, Ar–H), 7.19 (1H, s,
thiazole–H), 7.30–8.20 (9H, m, Ar–H), 11.94 (1H, br, NH). 13C-NMR (100 MHz, DMSO-d6, ppm)
22.6,
ν
(cm−1): 3126 (N–H), 3037 (aromatic C–H),
max
δ
δ
22.8, 27.9, 29.0, 66.03, 112.2, 114.3, 115.7, 115.9, 123.3, 123.8, 127.4, 129.6, 129.6, 130.3, 130.8, 130.9, 137.6,
139.5, 155.1, 161.7, 164.2, 167.8. HRMS (m/z): [M + H]+ C27H24N4O4S için calculated: 500.57; found:

Molecules 2018, 23, 135
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501.1572. For C27H24N4O4S calculated: 64.79% C, 4.83% H, 11.19% N; found: 64.80% C, 4.84% H,
11.20% N.
N0-[3-Phenyl-4-(2,3-dichlorophenyl)thiazole-2(3H)-ylidene]-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]
◦
acetohydrazide (4k): Yield: % 75. m.p. 272 C. IR
2916 (aliphatic C–H), 1732 (amide C=O), 1610–1305 (C=C and C=N), 1219–1028 (C–N and C–O).
1H-NMR (400 MHz, DMSO-d6, ppm):
1.68 (4H, br, CH2), 2.49–2.62 (4H, m, CH2), 4.63 and 4.72 (2H,
dd, J = 15.6 Hz, OCH2), 6.48–6.50 (2H, m, Ar–H), 6.84 (1H, d, J = 8.0 Hz, Ar–H), 7.04 (1H, s, thiazole–H),
7.27–7.82 (8H, m, Ar–H), 11.92 (1H, br, NH). 13C-NMR (100 MHz, DMSO-d6, ppm)
22.4, 22.7, 27.8,
ν
(cm−1): 3109 (N–H), 3047 (aromatic C–H),
max
δ
δ
28.8, 66.2, 111.9, 114.1, 122.9, 124.4, 128.8, 129.4, 129.5, 130.6, 130.2, 134.7, 137.4, 138.0, 147.4, 155.0, 168.0.
HRMS (m/z): [M + H]+ for C27H23Cl2N3O2S calculated: 524.46; found: 524.0938. For C27H23Cl2N3O2S
calculated: 61.83% C, 4.42% H, 8.01% N; found: 61.82% C, 4.43% H, 8.02%N.
3.2. Biochemistry
3.2.1. MTT Assay (Mitochondrial Activity)
This is a colorimetric assay that measures the reduction of 3-(4,5-dimethylthiazol-2-yl)-
2,4,diphenyltetrazolium bromide (MTT) by mitochondria [46]. All final compounds were tested against
human breast adenocarcinoma cell line (MCF-7), human lung carcinoma cell line (A549) and mouse
embryooblast cell line (NIH/3T3) to determine their cytotoxic profile. Cancer cells were cultured in
◦
RPMI medium (Hyclone, Thermo Scientific, Waltham, MA, USA) at 37 C in a humidified atmosphere
of 95% air and 5% CO2. The studied cells were situated at 2
×
104 cells into each well of 96-well
microtiter tissue culture plates (Nunc, Denmark) and incubated for 24 h. After that, test compounds
were dissolved in DMSO and added to culture wells at varying concentrations (85–1000 µM). After a
24 h incubating period, 20 mL MTT solution (5 mg/mL MTT powder in PBS) was added to each well
and the cells were incubated for 4 h at 37 ◦C. The purple formazan crystals produced were dissolved
in DMSO and the absorbance was read by ELISA reader (OD570 nm). The percent values of cell
proliferations were calculated relative to controls, whose cell proliferations were accepted as 100% [47].
3.2.2. DNA Synthesis Inhibition
The 5-bromo-20-deoxy-uridine (BrdU) cell proliferation assay [48] was used to detect the
proliferation of A549 and C6 cells. This method was performed in the 96-well flat-bottomed microtiter
plates by using cisplatin as positive control drug. A549 and C6 cells were collected from cell cultures
by 0.25% trypsin/EDTA solution and counted in a hemocytometer. Suspensions of cell lines were
seeded into 96-well flat-bottomed microtiter plates at a density of 1
×
103 cells/mL. The tumor
cell lines were cultured in the presence of various doses of the test compounds or standard drugs.
Microtiter plates were incubated at 37 ◦C in a 5% CO2/95% air humidified atmosphere for 24 h and
48 h. At the end of each day, the cells were labelled with 10 mL BrdU solution for 2 h and then
fixed. Anti-BrdU-POD (100 mL) was added and incubated for 90 min. Finally, microtiter plates were
washed with phosphate buffered saline (PBS) three times and the cells were incubated with substrate
solution until the color was sufficient for photometric detection. Absorbance of the samples was
measured with an ELx808-IUBio-Tek apparatus at 492 nm. The growth percentage was evaluated
spectrophotometrically versus untreated controls with used cell viability of growth assay. Results for
each spectrophotometrical measure were noticed as percent of growth inhibition. All experiments
were done in triplicates.
3.2.3. Flow Cytometric Analysis
Evaluation of apoptosis was carried out through the Annexin V-FITC Apoptosis Detection Kit
from BD, Pharmingen, according to the manufacturer’s instruction. This assay was performed on C6
and A549 cell lines with selected compounds which crossed the second step of anticancer activity. After
induced apoptosis of cancer cells by the addition of IC50 concentrations of selected compounds and

Molecules 2018, 23, 135
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positive controls including cisplatin (50 mM) (24 h incubation), cells were collected by centrifugation
for 5 min. Then cells were rinsed with cold water twice and resuspended in Annexin V-FITC binding
buffer at a concentration of 1
×
106 cells/mL. 5 mL of Annexin V-FITC was used to stain the cells.
After 15 min of incubation at 25 ◦C, the cell suspension was centrifugated for 5 min and cells were
resuspended in Annexin V-FITC binding buffer. Propidium iodide (5 mL) was added and the tubes
were situated on ice and away from light. The fluorescence was measured using a flow cytometer.
The results were analyzed by using FCSExpress software and represented as percentage of normal and
apoptotic cells at various stages [49]. The percentage of apoptotic cells was calculated from the number
of cells in sub-G1 phase, representing fragmented cell vesicles. The four areas in the diagrams stand
for necrotic cells (Q1, positive for PI and negative for annexin/FITC, left square on the top), live cells
(Q3, negative for annexin and PI, left square at the bottom), late apoptotic or necrotic cells (Q2, positive
for annexin and PI, right square on the top) and apoptotic cells (Q4, negative for PI and positive for
annexin, right square at the bottom), respectively. The experiment was repeated three times.
3.2.4. Inhibition of AChE/BuChE Enzymes
In order to determine the acetyl- and butyrylcholinesterase enzyme inhibitions, experiments
were carried out by making some changes in the method developed by Ellman [50]. Samples were
prepared by dissolving in the appropriate solvent. Adding 20
µ
L enzyme solution (AChE or BuChE,
L sample solution to 2.4 mL buffer solution and it was incubated for 15 min at
L DTNB and 75 mM ATCI solution or 20 L of 25 mM BTCI solution
1 U/mL) and 10
37 C. After incubation, 50
µ
◦
µ
µ
were added and allowed the mixture to incubate at room temperature for 30 min. For the control
group, only 10 L of solvent was added instead of the sample solution provided all protocols are the
µ
same. The absorbance values of the prepared mixture were measured spectrophotometrically using
polystyrene forceps at 37 ◦C at 412 nm. Experiments were run in triplicate. The inhibition fractions of
AChE yada BuChE enzymes were calculated using the following equation:
I(%) = 100 − (Abssample − Abscontrol) × 100
3.2.5. Statistical Analysis
All data were recorded as mean
one-way ANOVA plus Tukey’s test. p-values lower than 0.05 were considered significant (p < 0.05 *,
p < 0.01 **, p < 0.001 ***).
±
standard error of mean (S.E.M). Results were analyzed by the
4. Conclusions
In this study, we have synthesized novel eleven compounds combining two ring systems
thiazoline and tetralin. N0-[3-cyclohexyl/phenyl-4-(substituted phenyl)thiazole-2(3H)-ylidene]-2
-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetohydrazide (4a
determine their anticancer potency. Compounds 4b (IC50 = 69.2
the highest cytotoxicity against MCF-7 cells whereas compounds 4f
4-chloro and 4-fluoro substitutions act at lower concentration than cisplatin. Compound 4d inhibited
DNA synthesis had the best potential of 47.75% at 35.9 M concentration and 72.15% at 71.8 M on
MCF-7 cells. Compound 4h inhibited DNA synthesis with a percentage of 48.97% at 24.3 M and
50.78% at 48.6 M on A549 cell line. With regard to flow cytometric analysis, compounds 4b and
4e exhibited 29.8% and 32.5% apoptosis (ealy + late apoptotic cell percentage), respectively against
MCF-7 cell line, whereas cisplatin caused 29.9% apoptosis of the cells. Compounds 4a 4b 4f 4g,
–
4k) derivatives have been tested to
M) and 4d IC50 = 71.8 M showed
4g and 4h including 4-bromo,
µ
µ
,
µ
µ
µ
µ
,
,
,
4h and 4j showed significant apoptosis levels whose apoptosis induction ratios were higher than
cisplatin against A549 cell line. With regard to anticholinesterase activity of the compounds, compound
4h showed the highest acetylcholinesterase inhibition with 49.92% and compounds 4j with 44.96%,
4d with 43.22%, 4f with 42.87% and 4i with 41.85% followed it.

Molecules 2018, 23, 135
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Supplementary Materials: Supplementary materials are available online.
Acknowledgments: The authors present their thanks to Anadolu University for financial support within the
Anadolu University Scientific Research Project, Eskis¸ehir, Turkey (Project No: 1502S058). Also, we thank to
Anadolu University Plant, Drug and Scientific Research Center (BIBAM) for providing laboratory to study
anticancer activity.
Author Contributions: G.T.-Z. and Z.A.K. designed and organized study. A.T. and L.Y. performed the synthetic
work. G.A.Ç. and H.E.T. studied the biological activity assay. G.T.-Z. and L.Y. interpreted the results and wrote
the paper. All authors have approved the final version of the manuscript.
Conflicts of Interest: The authors confirm that the content of this article involves no conflict of interests.
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Sample Availability: Samples of the compounds 4a–4k are available from the authors.
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