Synthesis of novel thiazolylpyrazoline derivatives and evaluation of their antimicrobial activities and cytotoxicities

Article abstract of DOI:10.3906/kim-1512-12

Several novel thiazolylpyrazoline derivatives were synthesized by reacting substituted 3,5-diaryl-1-thiocarbamoyl-2-pyrazolines with phenacylbromides. The structures of the synthesized compounds were confirmed by IR, ¹H NMR, ¹³C NMR, and MS spectral data. Their antimicrobial activities against Staphylococcus aureus (ATCC-25923), Enterococcus faecalis (ATCC-29212), Enterococcus faecalis (ATCC-51922), Listeria monocytogenes (ATCC-1911), Klebsiella pneumoniae (ATCC-700603), Pseudomonas aeruginosa (ATCC-27853), Escherichia coli (ATCC-35218), Escherichia coli (ATCC-25922), Candida albicans (ATCC-90028), Candida glabrata (ATCC-90030), Candida krusei (ATCC-6258), and Candida parapsilosis (ATCC-22019) were investigated. The compounds were also studied for their cytotoxic effects using a MTT assay. Compound 7c showed the highest antimicrobial activity, possessing the same potential as chloramphenicol against K. pneumonia, P. aeruginosa, and E. coli (ATCC-25923).

Full text of DOI:10.3906/kim-1512-12

Turk J Chem
Turkish Journal of Chemistry
(2016) 40: 641 – 654
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1512-12
Research Article
Synthesis of novel thiazolylpyrazoline derivatives and evaluation of their
antimicrobial activities and cytotoxicities
Aouatef TABBI1, Zafer Asım KAPLANCIKLI2,∗, Dahmane TEBBANI1,
2
3
4
4
¨
¨
Leyla YURTTAS¸ , Zerrin CANTURK , Ozlem ATLI , Merve BAYSAL ,
Gu¨lhan TURAN-ZITOUNI2
1Department of Chemistry, Faculty of Sciences, Mentouri University, Constantin, Algeria
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eski¸sehir, Turkey
3Department of Microbiology, Faculty of Pharmacy, Anadolu University, Eski¸sehir, Turkey
4Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey
Received: 04.12.2015
•
Accepted/Published Online: 03.02.2016
•
Final Version: 21.06.2016
Abstract: Several novel thiazolylpyrazoline derivatives were synthesized by reacting substituted 3,5-diaryl-1-thiocarba-
moyl-2-pyrazolines with phenacylbromides. The structures of the synthesized compounds were confirmed by IR, 1 H
NMR, 13 C NMR, and MS spectral data. Their antimicrobial activities against Staphylococcus aureus (ATCC-25923), En-
terococcus faecalis (ATCC-29212), Enterococcus faecalis (ATCC-51922), Listeria monocytogenes (ATCC-1911), Klebsiella
pneumoniae (ATCC-700603), Pseudomonas aeruginosa (ATCC-27853), Escherichia coli (ATCC-35218), Escherichia coli
(ATCC-25922), Candida albicans (ATCC-90028), Candida glabrata (ATCC-90030), Candida krusei (ATCC-6258), and
Candida parapsilosis (ATCC-22019) were investigated. The compounds were also studied for their cytotoxic effects using
a MTT assay. Compound 7c showed the highest antimicrobial activity, possessing the same potential as chloramphenicol
against K. pneumonia, P. aeruginosa, and E. coli (ATCC-25923).
Key words: 2-Pyrazoline, thiazole, thiazolylpyrazoline, antimicrobial activity, cytotoxicity
1. Introduction
It has been reported that the morbidity, mortality, and costs related to the treatment of infectious diseases
have been increased by antimicrobial resistance. The threat from resistance (particularly multiple resistance in
bacterial strains that have disseminated widely) has never been so great. The main dynamics driving this threat
are increased antibiotic use, bigger movement of people, and increased industrial and economic development.1
The need for novel antibacterial and antifungal agents is greater than ever because of the emergence of multidrug
resistance in common pathogens, the quick emergence of new infections, and the potential for use of multidrug-
resistant agents in bioweapons.2
Every antimicrobial development candidate is considered “novel” by those who make it, but the multiple
approaches to developing new compounds neither carry equal potential to overcome preexisting resistance
mechanisms nor are they associated with equal development risk. There is a spectrum of innovation that ranges
from developments within established classes, to completely new microbial pharmacophores and molecular
targets.3
∗Correspondence: zakaplan@anadolu.edu.tr
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TABBI et al./Turk J Chem
General sources for novel antimicrobial agents include biological sources as well as large collections of
different compounds collected from various laboratories. The recent advance of synthetic libraries represents a
major advancement in the discovery of new lead compounds for antimicrobial drug development.4
Compounds having heterocyclic ring systems continue to attract considerable interest due to their wide
range of biological activities. Amongst them, five-membered heterocyclic compounds, particularly azoles, occupy
a unique place in the realm of natural and synthetic organic chemistry.5
Pyrazolines constitute a remarkable class of heterocycles due to their actual biological activities such as
anticancer, antioxidant, antibacterial, antifungal, antidepressant, anti-inflammatory, anticonvulsant, antitumor,
and analgesic properties.6,7
In addition, a thiazole ring is found in many potent biologically active compounds, such as sulfathiazole
(antimicrobial drug), ritonavir (antiretroviral drug), abafungin (antifungal drug), bleomycine, and tiazofurin
(antineoplastic drug). It has been observed over the years that thiazole derivatives have several biological
activities such as antihypertensive, anti-inflammatory, antischizophrenia, antibacterial, anti-HIV, hypnotics,
antiallergic, analgesic, antithrombotic, fibrinogen receptor antagonist, bacterial DNA gyrase B inhibitor, anti-
tumor, and cytotoxic activities.8
Thus, the synthesis and biological activities of novel thiazolylpyrazoline derivatives activated a great
deal of research. Remarkably, thiazolylpyrazoline derivatives were reported to display a variety of significant
biological importance such as antimicrobial, antiviral, anti-inflammatory, antiamebic, and anticancer activities
and β -ketoacyl-acyl carrier protein synthase III (FabH), epidermal growth factor receptor tyrosine kinase
(EGFR TK) inhibitors, superoxidase inhibitors, and free radical scavengers.9−16
In a recent study, in silico molecular docking simulation was performed to position thiazolylpyrazoline
derivatives in the DNA topoisomerase IV receptor structure active site to determine the probable binding
model.17 This study revealed that all the molecules showed good binding energy toward the target receptor
DNA topoisomerase IV. Thiazolylpyrazoline derivatives have been tested for antimicrobial activity and some
compounds showed good activity profiles against tested microbes. In another study, some thiazolylpyrazoline
derivatives were synthesized and evaluated for their antifungal activity. According to the in silico molecular
docking study, the compounds possessed the required binding energy to dock themselves with the binding
pocket of Cytochrome P450 14α-sterol demethylase (CYP51). The synthesized compounds showed significant
antifungal activity, which has been fully supported by an in silico molecular docking study.18
Keeping in view the therapeutic importance of thiazolylpyrazoline derivatives and in continuation of our
work on the synthesis of biologically active thiazolylpyrazoline compounds, herein we describe the synthesis and
evaluation of the antimicrobial and cytotoxic activities of novel molecules.19−24
2. Results and discussion
2.1. Chemistry
The synthesis of thiazolylpyrazoline derivatives (7a–7j, 8a, 8b, 9a, 9b) was carried out according to the steps
outlined in Schemes 1 and 2. The intermediate products, 1-(4’-methoxyphenyl)-3-(5,6,7,8,-tetrahydronaphthalen-
2-yl)-2-propen-1-one (1) and 1-(4’-methoxyphenyl)-3-phenylprop-2-en-1-one (2), were synthesized via the base-
catalyzed Claisen–Schmidt condensation of 4-methoxyacetophenone with 5,6,7,8-tetrahydronaphthalene-2-car-
baldehyde and benzaldehyde, respectively. Likewise 3-phenyl-1-(5,6,7,8-tetrahydronaphthalene-2-yl)prop-2-en-
1-one (3) was obtained by the condensation of 1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethanone with benzalde-
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TABBI et al./Turk J Chem
hyde. Secondly, the cyclization of chalcones (1–3) with thiosemicarbazide in the presence of sodium hy-
droxide led to 3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1-thiocarbamoyl-2-pyrazoline (4), 3-
(4’-methoxyphenyl)-5-phenyl-1-thiocarbamoyl-2-pyrazoline (5), and 5-phenyl-3-(5,6,7,8-tetrahydronaphthalen-
2-yl)-1-thiocarbamoyl-2-pyrazoline (6), respectively. Finally, reactions of 4, 5, and 6 with phenacylbromide
derivatives gave compounds 7a–7j, 8a, 8b, 9a, and 9b (Table 1).
O
O
O
1
a
b
N
N
O
O
O
H2N
S
4
O
O
2
c
b
N
N
O
H2N
S
O
5
O
N
O
c
b
3
N
H2N
S
6
a: 5,6,7,8-Tetrahydronaphthalene-2-carbaldehyde / NaOH, b: Thiosemicarbazide / NaOH,
c: Benzaldehyde / NaOH
Scheme 1. Synthesis of intermediate compounds.
The structures of the compounds were elucidated by IR, 1 H NMR, 13 C NMR, and MS spectral data.
The spectral analysis for intermediate original compound 4 was given, but the spectral analysis for compounds
5 and 6, which were examined in previous studies, was not given.19,25 In the IR spectra of the final compounds
7a–7j, 8a, 8b, 9a, and 9b, C=N and C=C stretching vibrations were observed in the region 1630–1450 cm−1
The aromatic C–H stretching vibrations gave rise to bands at 3117–3015 cm−1
.
.
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TABBI et al./Turk J Chem
R1
O
R2
Br
R3
R4
4
5
6
O
O
N
N
N
N
N
N
N
N
R1
R1
S
N
S
S
R2
R3
R3
R4
7a-j
8a-b
9a-b
Scheme 2. Synthesis of title compounds.
Table 1. Some properties of the compounds.
Compounds R1
R2
H
H
H
H
H
H
Cl
O–CH2–O
NO2
H
H
H
R3
F
Cl
R4
H
H
H
H
H
H
H
H
H
Molecular formula Yield (%) Mp (◦C)
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
H
H
H
H
H
H
H
H
C29H26FN3OS
C29H26ClN3OS
C29H26BrN3OS
C30H29N3OS
89
91
87
82
80
91
80
85
84
88
82
80
80
78
192
193
187
178
172
221
171
194
170
164
213
212
176
185
Br
CH3
OCH3
NO2
Cl
C30H29N3O2S
C29H26N4O3S
C29H25Cl2N3OS
C30H27N3O3S
C29H26N4O3S
H
H
H
H
Cl
CH3
7j
OCH3
H
OH
H
OCH3 C31H31N3O3S
8a
8b
9a
9b
H
H
H
H
C25H21N3OS
C25H21N3O2S
C28H26ClN3S
C29H27N3S
H
H
H
In the 1 H NMR spectra, C6 and C7 protons resonated as multiplets at δ 1.77–1.83 ppm and C5 and
C8 protons at 2.70–2.80 ppm, corresponding to tetrahydronaphthalenes; the methylene of the pyrazoline ring
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TABBI et al./Turk J Chem
resonated as a pair of doublets of doublets at δ 3.30–3.40 ppm (HA) and 3.85–3.92 ppm (HB). The CH proton
(HX) at position 5 of the pyrazoline ring appeared as a doublet of doublets or a broad signal at δ 5.60–5.68 ppm
due to vicinal coupling with the two magnetically nonequivalent protons of the methylene group at position 4
of pyrazoline (Figure). All the other aromatic and aliphatic protons were observed at expected regions.
HA
Ar
HB
N
N
HX
N
Ar
S
Ar
Figure. ABX system of pyrazoline ring.
13 C NMR chemical shift values of the carbon atoms at 43.61–44.62 ppm (pyrazoline C4), 64.42–64.88
ppm (pyrazoline C5), and about 148.20–161.10 ppm (pyrazoline C3) corroborate the 2-pyrazoline character
deduced from the 1 H NMR data. All the other aromatic and aliphatic carbon atoms were observed at expected
regions.
The mass spectra (EIMS) of the compounds (7a–7j, 8a, 8b, 9a, and 9b) are also in agreement with
their molecular formula.
2.2. Biology
MICs were recorded as the minimum concentration of a compound that inhibits the growth of tested microor-
ganisms. All of the compounds tested illustrated significant antibacterial and antifungal activity when compared
with reference drugs. When compared with chloramphenicol (MIC = 200 µg/mL), all of the compounds and
chloramphenicol showed the same level of activity against K. pneumoniae (ATCC-700603) and P. aeruginosa
(ATCC-27853) (Table 2). A similar result was obtained from E. coli (ATCC-25923): compound 7c showed the
same level of activity when compared with chloramphenicol. When compared with ketoconazole (MIC = 2 and
3.125 µg/mL), all of the compounds showed low activity against the tested fungi.
The necessity when generating a chemotherapeutic agent is to show minimal or no side-effects on healthy
cells in patients receiving chemotherapy. The cytotoxic activities of these compounds were evaluated against a
normal mouse embryonic fibroblast cell line, NIH/3T3, for determining the selectivity of potential antimicrobial
agents.
When we evaluated the effects of the synthesized compounds against the NIH/3T3 cell line (healthy),
most of the compounds were found to have higher IC50 values (Table 2) than their effective doses (MIC = 200
µg/mL), which were also the same as the positive control, chloramphenicol, against K. pneumoniae and P.
aeruginosa. Thus, they may be regarded as nontoxic at their effective antibacterial doses. Only compounds 8a
and 8b exhibited antimicrobial activity with MIC values lower than their IC50 values against K. pneumonia
and P. aeruginosa as a result of cytotoxicity.
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Table 2. Antimicrobial activity and cytotoxicity of the compounds (µg/mL).
A
B
C
D
E
F
G
H
I
J
K
L
Cyt.
7a
7b
7c
7d
7e
7f
7g
7h
7i
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 200 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
400 400 400 400 200 200 400 400 200 200 100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
-
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
301.63
371.60
> 500
> 500
-
7j
8a
8b
9a
9b
Ref.1 6.25 25
Ref.2
200 25
-
200 200 200 200
-
2
-
2
-
-
-
-
-
-
-
-
3.125 3.125
-
A: S. aureus, B: E. faecalis (ATCC-29212), C: E. faecalis (ATCC-51922), D: L. monocytogenes, E: K. pneumonia,
F: P. aeruginosa, G: E. coli (ATCC-35218), H: E. coli (ATCC-25923), I: C. albicans, J: C. glabrata, K: C. krusei,
L: C. parapsilopsis,
Ref.1: Chloramphenicol, Ref.2: Ketoconazole, Cyt (Cytotoxicity): IC50 values for cell lines (NIH3T3)
3. Conclusion
All the synthesized compounds showed antibacterial activity against K. pneumoniae and P. aeruginosa, with a
MIC value of 200 µg/mL. They did not show any cytotoxicity against fibroblasts. The results mentioned above
suggest that thiazolylpyrazolines have potential as antibacterial compounds that are worth being investigated
further for the development of new drugs to treat infectious diseases.
4. Experimental
4.1. General remarks
All chemicals were purchased from commercial suppliers and used without purification. Melting point (mp)
was determined on an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK)
and are uncorrected. Spectroscopic data were recorded with the following instruments: IR, Shimadzu 8400S
spectrophotometer (Shimadzu, Tokyo, Japan); NMR, Bruker 500 MHz spectrometer in CDCl3 using TMS
internal standard; and MS, LC/MS/MS Mass Spectrometer (3200 Q TRAP, AB Sciex Instruments, USA).
4.2. Chemistry
Chalcones (1, 2, 3): All chalcone derivatives were synthesized according to the literature.19,25
4.2.1. General procedure for the synthesis of the intermediate compounds (4, 5, 6)
A mixture of chalcone (0.01 mol), thiosemicarbazide (0.012 mol), and sodium hydroxide (0.01 mol) was refluxed
in ethanol (30 mL) for 8 h. The solution was poured into crushed ice. The precipitated solid was filtered,
washed with water, and dried. The product was crystallized from ethanol.
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4.2.1.1 3-(4’-Methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1-thiocarbamoyl-2-pyrazoline
(4)
Yield: 87%, mp 230 ◦ C. IR νmax (cm−1): 3470.7, 3375.2 (N–H stretching), 1560.9, 1510.4, 1467.5 (C=N and
C=C stretching), 1210.2, 1168.0, 1095.5, 1010.0 (C–N stretching and aromatic C–H bending). 1 H NMR (500
MHz, CDCl3)δ (ppm): 1.72–1.83 (4H, m, tetrahydronaphthalene C6,7 –H), 2.70–2.78 (4H, m, tetrahydronaph-
thalene C5,8 –H), 3.20 (1H, dd, J = 17.5 Hz, J = 3.4 Hz, pyrazoline C4 –H), 3.74 (1H, dd, J = 17.41 Hz, J =
11.31 Hz, pyrazoline C4 –H), 3.88 (3H, s, OCH3), 5.99 (1H, dd, J = 11.12 Hz, J = 2.68 Hz, pyrazoline C5 –H),
6.91 (1H, s, Ar–H), 6.95 (3H, d, J = 8.86 Hz, Ar–H), 7.03 (1H, d, J = 7.86 Hz, Ar–H), 7.70 (2H, d, J = 8.82
Hz, Ar–H).
4.2.2. General procedure for compounds 7a–7j
A mixture of 3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-1-thiocarbamoyl-2-pyrazoline (4) (0.001
mol) and appropriate 2-bromoacetophenone derivative (0.001 mol) was refluxed in ethanol (20 mL) for 4 h. The
reaction mixture was cooled and filtered.
4.2.2.1. 4-(4’-Fluorophenyl)-2-[3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-
pyrazolin-1-yl]thiazole (7a)
IR νmax (cm−1): 3125.7, 3045.3 (aromatic C–H), 1620.9, 1500.3 (C=N and C=C stretching), 1220.4, 1170.1,
1055.2 (C–N stretching and aromatic C–H bending). 1 H NMR (500 MHz, CDCl3) δ (ppm): 1.75–1.86 (4H, m,
tetrahydronaphthalene C6,7 –H), 2.73–2.81 (4H, m, tetrahydronaphthalene C5,8 –H), 3.35 (1H , dd, J = 17.37
Hz, J = 6.42 Hz, pyrazoline C4 –H), 3.89 (1H, dd, J = 16.75 Hz, J = 12.90 Hz, pyrazoline C4 –H), 3.90 (3H, s,
OCH3), 5.62–5.73 (1H, br, pyrazoline C5 –H), 6.73 (1H, s, thiazole–H), 6.97 (2H, d, J = 8.83 Hz, Ar–H), 7.02
(2H, d, J = 8.71 Hz, Ar–H), 7.08 (2H, d, J = 8.37 Hz, Ar–H), 7.17 (1H, d, J = 6.52 Hz, Ar–H), 7.70–7.74
(2H, m, Ar–H), 7.75 (2H, d, J = 8.71 Hz, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 23.16 (2CH2), 29.15 (CH2), 29.48 (CH2), 43.71 (pyrazoline
C4), 55.41 (OCH3), 64.43 (pyrazoline C5), 102.46 (thiazole C5), 114.17, 115.32, 128.09, 129.44 (2CH, Ar–C),
124.14, 130.89, 136.79, 137.40, 138.65 (Ar–C), 123.80 127.61, 127.72 (Ar–CH), 157.37 (thiazole C2), 161.10
(pyrazoline C3), 161.39 (C–OCH3), 163.35 (C–F), 165.27 (S–C=N).
For C29 H26 FN3 OS calculated: (%) C 72.03, H 5.42, N 8.69; found: (%) C 72.08, H 5.38, N 8.56.
MS [M+1]+ : m/z 484
4.2.2.2. 4-(4’-Chlorophenyl)-2-[3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-
pyrazolin-1-yl]thiazole (7b)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.75–1.86 (4H, m, tetrahydronaphthalene C6,7 –H), 2.73–2.81 (4H, m,
tetrahydronaphthalene C5,8 –H), 3.35 (1H, dd, J = 17.37 Hz, J = 6.42 Hz, pyrazoline C4 –H), 3.72–3.76 (1H,
dd, J = 16.75 Hz, J = 12.90 Hz, pyrazoline C4 –H), 3.90 (3H, s, OCH3), 5.62–5.73 (1H, br, pyrazoline C5 –H),
6.79 (1H, s, thiazole–H), 6.97 (2H, d, J = 8.83 Hz, Ar–H), 7.07 (1H, d, J = 8.38 Hz, Ar–H), 7.18 (2H, br,
Ar–H), 7.32 (2H, d, J = 8.55 Hz, Ar–H), 7.68 (2H, d, J = 8.54 Hz, Ar–H), 7.74 (2H, d, J = 8.89 Hz, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 23.16 (2CH2), 29.16 (CH2), 29.49 (CH2), 43.67 (pyrazoline
C4), 55.41 (OCH3), 64.43 (pyrazoline C5), 103.35 (thiazole C5), 114.17, 127.26, 128.07, 128.52 (2CH, Ar–C),
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TABBI et al./Turk J Chem
123.82, 127.68, 129.28 (Ar–CH), 124.16, 133.07, 136.79, 137.38, 138.69 (Ar–C), 149.94 (thiazole C2), 152.15
(pyrazoline C3), 161.08 (C-OCH3), 165.27 (S–C=N).
MS [M+1]+ : m/z 500
4.2.2.3. 4-(4’-Bromophenyl)-2-[3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-
pyrazolin-1-yl]thiazole (7c)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.76–1.85 (4H, m, tetrahydronaphthalene C6,7 –H), 2.73–2.82 (4H, m,
tetrahydronaphthalene C5,8 –H), 3.36 (1H, dd, J = 17.39 Hz, J = 6.57 Hz, pyrazoline C4 –H), 3.87 (1H, dd, J
= 17.39 Hz, J = 11.89 Hz, pyrazoline C4 –H), 3.88 (3H, s, OCH3), 5.58–5.67 (1H, br, pyrazoline C5 –H), 6.80
(1H, s, thiazole–H), 6.97 (2H, d, J = 8.91 Hz, Ar–H), 7.06 (1H, d, J = 8.39 Hz, Ar–H), 7.17 (2H, m, Ar–H),
7.47 (2H, d, J = 8.60 Hz, Ar–H), 7.62 (2H, d, J = 8.54 Hz, Ar–H), 7.74 (2H, d, J = 8.87 Hz, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 23.16 (2CH2), 29.16 (CH2), 29.49 (CH2), 43.67 (pyrazoline
C4), 55.41 (OCH3), 64.44 (pyrazoline C5), 103.48 (thiazole C5), 114.17, 127.57, 128.07, 131.46 (2CH, Ar-C),
121.29, 133.76, 136.79, 137.38, 138.66 (Ar–C), 123.82,127.68, 129.45 (Ar–CH), 149.90 (thiazole C2), 152.23
(pyrazoline C3), 161.08 (C–OCH3), 165.27 (S–C=N).
MS [M+1]+ : m/z 546
4.2.2.4. 4-(4’-Methylphenyl)-2-[3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-
pyrazolin-1-yl]thiazole (7d)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.75–1.85 (4H, m, tetrahydronaphthalene C6,7 –H), 2.38 (3H, s, CH3),
2.72–2.84 (4H, m, tetrahydronaphthalene C5,8 –H), 3.35 (1H, dd, J = 17.36 Hz, J = 6.55 Hz, pyrazoline C4 –H),
3.86 (1H, dd, J = 17.35 Hz, i=11.91 Hz, pyrazoline C4 –H), 3.88 (3H, s, OCH3), 5.59–5.68 (1H, br, pyrazoline
C5 –H), 6.76 (1H, s, thiazole–H), 6.97 (2H, d, J = 8.91 Hz, Ar–H), 7.06 (1H, d, J = 8.45 Hz, Ar–H), 7.17 (2H,
d, J = 8.14 Hz, Ar–H), 7.18 (1H, s, Ar–H), 7.19 (1H, d, J = 6.52 Hz, Ar–H), 7.65 (2H, d, J = 8.11 Hz, Ar–H),
7.74 (2H, d, J = 8.88 Hz, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 21.25 (CH3), 23.18 (2CH2), 29.47 (CH2), 29.73 (CH2), 43.61
(pyrazoline C4), 55.40 (OCH3), 64.42 (pyrazoline C5), 102.19 (thiazole C5), 114.15, 123.86, 125.93, 129.41
(2CH, Ar–C), 124.27, 132.06, 136.69, 137.33, 138.76 (Ar–C), 127.72, 128.04, 129.41 (Ar–CH), 151.05 (thiazole
C2), 152.02 (pyrazoline C3), 161.01 (C-OCH3), 165.14 (S–C=N).
MS [M+1]+ : m/z 480
4.2.2.5. 4-(4’-Methoxyphenyl)-2-[3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-
pyrazolin-1-yl]thiazole (7e)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.74–1.85 (4H, m, tetrahydronaphthalene C6,7 –H), 2.70–2.84 (4H, m,
tetrahydronaphthalene C5,8 –H), 3.35 (1H, dd, J = 17.37 Hz, J = 6.36 Hz, pyrazoline C4 –H), 3.84 (3H, s,
OCH3), 3.87 (1H, dd, J = 17.50 Hz, J = 10.00 Hz, pyrazoline C4 –H), 3.8 (3H, s, OCH3), 5.60–5.83 (1H, br,
pyrazoline C5 –H), 6.67 (1H, s, thiazole–H), 6.90 (2H, d, J = 8.80 Hz, Ar–H), 6.97 (2H, d,J = 8.84 Hz, Ar–H),
7.06 (1H, d, J = 8.20 Hz, Ar–H), 7.18 (1H, s, Ar–H), 7.19 (1H, d, J = 7.00 Hz, Ar–H), 7.70 (2H, d, J = 8.79
Hz, Ar–H), 7.74 (2H, d, J = 8.84 Hz, Ar–H).
648

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13 C NMR (500 MHz, CDCl3) δ (ppm): 23.15 (2CH2), 29.16 (CH2), 29.47 (CH2), 43.76 (pyrazoline
C4), 55.30 (OCH3), 55.41 (OCH3), 64.43 (pyrazoline C5), 101.00 (thiazole C5), 113.84, 114.18, 123.84, 129.45
(2CH, Ar–C), 127.41, 127.57, 128.18 (Ar–CH), 124.06, 129.36, 136.81, 137.44, 138.55 (Ar–C), 156.41 (thiazole
C2), 159.32 (pyrazoline C3), 161.08 (C–OCH3), 161.18 (C–OCH3), 165.09 (S–C=N).
MS [M+1]+ : m/z 496
4.2.2.6. 4-(4’-Nitrophenyl)-2-[3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-pyra-
zolin-1-yl]thiazole (7f)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.77–1.84 (4H, m, tetrahydronaphthalene C6,7 –H), 2.74–2.82 (4H, m,
tetrahydronaphthalene C5,8 –H), 3.40 (1H, dd, J = 17.43 Hz, J = 6.15 Hz, pyrazoline C4 –H), 3.89 (3H, s,
OCH3), 3.93 (1H, dd, J = 17.43 Hz, J = 11.76 Hz, pyrazoline C4 –H), 5.76–5.85 (1H, br, pyrazoline C5 –H),
6.98 (2H, d, J = 8.87 Hz, Ar–H), 7.01 (1H, s, thiazole–H), 7.08 (1H, d, J = 8.35 Hz, Ar–H), 7.18 (1H, s,
Ar–H), 7.19 (1H, d, J = 6.86 Hz, Ar–H), 7.76 (2H, d, J = 8.88 Hz, Ar–H), 7.89 (2H, d, J = 8.91 Hz, Ar–H),
8.22 (2H, d, J = 8.95 Hz, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 23.11 (2CH2), 29.15 (CH2), 29.50 (CH2), 43.90 (pyrazoline
C4), 55.44 (OCH3), 64.47 (pyrazoline C5), 106.91 (thiazole C5), 114.25, 123.93, 126.55, 128.27 (2CH, Ar–C),
123.76, 127.55, 129.56 (Ar–CH), 123.40, 137.09, 137.57, 138.12, 140.04 (Ar–C), 146.86 (thiazole C2), 148.20
(pyrazoline C3), 161.38 (C–OCH3), 165.37 (S–C=N).
MS [M+1]+ : m/z 511
4.2.2.7. 4-(3’,4’-Dichlorophenyl)-2-[3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-
2-pyrazolin-1-yl]thiazole (7g)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.75–1.86 (4H, m, tetrahydronaphthalene C6,7 –H), 2.70–2.80 (4H, m,
tetrahydronaphthalene C5,8 –H), 3.36 (1H, dd, J = 17.44 Hz, J = 6.77 Hz, pyrazoline C4 –H), 3.87 (1H, dd, J
= 17.50 Hz, J = 10.00 Hz, pyrazoline C4 –H), 3.88 (3H, s, OCH3), 5.19–5.36 (1H, br, pyrazoline C5 –H), 6.79
(1H, s, thiazole–H), 6.97 (2H, d, J = 8.84 Hz, Ar–H), 7.07 (1H, d, J = 8.00 Hz, Ar–H), 7.15 (1H, dd, J = 9.5
Hz, J = 1.62 Hz, Ar–H), 7.19 (1H, s, Ar–H), 7.38 (1H, d, J = 8.40 Hz, Ar–H), 7.52 (1H, dd, J = 8.38 Hz, J
= 2.02 Hz, Ar–H), 7.73 (2H, d, J = 8.85 Hz, Ar–H), 7.81 (1H, d, J = 1.98 Hz, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 23.14 (2CH2), 29.15 (CH2), 29.45 (CH2), 43.67 (pyrazoline
C4), 55.40 (OCH3), 64.54 (pyrazoline C5), 104.31 (thiazole C5), 114.18, 128.08 (2CH, Ar–C), 123.73, 124.96,
127.89, 128.04, 129.53, 130.24 (Ar–CH), 124.06, 129.57, 131.01, 132.46, 136.89, 137.41, 138.47 (Ar–C), 148.66
(thiazole C2), 152.47 (pyrazoline C3), 161.13 (C–OCH3), 165.28 (S–C=N).
MS [M+1]+ : m/z 534
4.2.2.8. 4-(Benzo[1,3]dioxol-5-yl)-2-[3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-
2-pyrazolin-1-yl]thiazole (7h)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.71–1.81 (4H, m, tetrahydronaphthalene C6,7 –H), 2.68–2.80 (4H, m,
tetrahydronaphthalene C5,8 –H), 3.45 (1H, dd, J = 17.55 Hz, J = 3.55 Hz, pyrazoline C4 –H), 3.90 (3H, s,
OCH3), 4.01 (1H, dd, J = 17.57 Hz, J = 6.45 Hz, pyrazoline C4 –H), 5.95–6.02 (1H, br, pyrazoline C5 –H),
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6.00 (2H, s, dioxolane), 6.53 (1H, s, thiazole–H), 6.87 (1H, d, J = 8.15 Hz, Ar–H), 7.00 (2H, d, J = 8.85 Hz,
Ar–H), 7.05 (1H, d, J = 7.93 Hz, Ar–H), 7.22 (1H, s, Ar–H), 7.25 (1H, s, Ar–H), 7.41 (1H, d, J = 8.12 Hz,
Ar–H), 7.67 (1H, d, J = 8.81 Hz, Ar–H), 7.78 (2H, d, J = 9.23 Hz, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 22.99 (2CH2), 29.17 (CH2), 29.43 (CH2), 44.62 (pyrazoline
C4), 55.51 (OCH3), 64.57 (pyrazoline C5), 101.43 (thiazole C5), 107.16 (CH2 –dioxolane), 114.42, 123.74
(2CH, Ar–C), 108.62, 113.99, 123.64, 127.22, 129.18, 129.88 (Ar–CH), 121.64, 128.46, 134.37, 136.91, 138.07
(Ar–C), 144.34 (thiazole C2), 147.98, 148.73 (2C–dioxolane), 149.84 (pyrazoline C3), 162.37 (C–OCH3), 164.64
(S–C=N).
MS [M+1]+ : m/z 510
4.2.2.9. 4-(3’-Nitrophenyl)-2-[3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-pyra-
zolin-1-yl]thiazole (7i)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.75–1.85 (4H, m, tetrahydronaphthalene C6,7 –H), 2.73–2.89 (4H, m,
tetrahydronaphthalene C5,8 –H), 3.39 (1H, dd, J = 17.43 Hz, J = 6.69 Hz, pyrazoline C4 –H), 3.90 (1H, dd, J
= 17.37 Hz, J = 11.94 Hz, pyrazoline C4 –H), 3.89 (3H, s, OCH3), 5.57–5.67 (1H, br, pyrazoline C5 –H), 6.94
(1H, s, thiazole–H), 6.99 (2H, d, J = 8.94 Hz, Ar–H), 7.10 (1H, d, J = 8.37 Hz, Ar–H), 7.21 (1H, d, J = 6.76
Hz, Ar–H), 7.22 (1H, s, Ar–H), 7.50 (1H, t, J = 7.97 Hz, Ar–H), 7.75 (2H, d, J = 8.84 Hz, Ar–H), 8.03 (1H,
d, J = 7.82 Hz, Ar–H), 7.74 (1H, dd, J = 8.15 Hz, J = 1.4 Hz, Ar–H), 8.59 (1H, s, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 23.14 (2CH2), 29.17 (CH2), 29.43 (CH2), 43.75 (pyrazoline
C4), 55.42 (OCH3), 64.56 (pyrazoline C5), 105.13 (thiazole C5), 114.20, 128.10 (2CH, Ar–C), 121.05, 121.92,
123.93, 127.71, 129.21, 129.57, 131.43 (Ar–CH), 124.04, 136.49, 136.96, 137.57, 138.52 (Ar–C), 148.59 (C–NO2),
148.72 (thiazole C2), 152.53 (pyrazoline C3), 161.17 (C–OCH3), 165.44 (S–C=N).
MS [M+1]+ : m/z 511
4.2.2.10. 4-(2’,5’-Dimethoxyphenyl)-2-[3-(4’-methoxyphenyl)-5-(5,6,7,8-tetrahydronaphthalen-2-
yl)-2-pyrazolin-1-yl]thiazole (7j)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.74–1.85 (4H, m, tetrahydronaphthalene C6,7 –H), 2.69–2.82 (4H, m,
tetrahydronaphthalene C5,8 –H), 3.33 (1H, dd, J = 17.43 Hz, J = 7.06 Hz, pyrazoline C4 –H), 3.80 (3H, s,
OCH3), 3.87 (3H, s, OCH3), 3.88 (1H, dd, J = 15.00 Hz, J = 10.00 Hz, pyrazoline C4 –H), 3.89 (3H, s,
OCH3), 5.57–5.81 (1H, br, pyrazoline C5 –H), 6.79 (1H, dd, J = 8.87 Hz, J = 3.03 Hz, Ar–H), 6.85 (1H, s,
thiazole–H), 6.97 (2H, d, J = 8.94 Hz, Ar–H), 7.04 (1H, d, J = 7.83 Hz, Ar–H), 7.19 (1H, s, Ar–H), 7.21 (1H,
d, J = 8.00 Hz, Ar–H), 7.35 (1H, m, Ar–H), 7.58 (1H, d, J = 3.03 Hz, Ar–H), 7.74 (2H, d, J = 8.60 Hz,
Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 23.16 (2CH2), 29.17 (CH2), 29.42 (CH2), 43.92 (pyrazoline
C4), 55.41 (OCH3), 55.69 (OCH3), 56.03 (OCH3), 64.83 (pyrazoline C5), 108.21 (thiazole C5), 114.16, 129.35
(2CH, Ar–C), 112.42, 114.44, 123.92, 127.33, 128.06 (Ar–CH), 124.20, 136.61, 137.44, 138.95 (Ar–C), 151.33
(thiazole C2), 153.53 (pyrazoline C3), 161.06 (C–OCH3), 163.53 (S–C=N).
MS [M+1]+ : m/z 526
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TABBI et al./Turk J Chem
4.2.3. General procedure for compounds 8a, 8b
A mixture of 3-(4’-methoxyphenyl)-5-phenyl-2-pyrazolin-1-carbothioamide (5) (0.001 mol) and 2-bromoacetophe-
none (0.001 mol) in ethanol (20 mL) was refluxed for 4 h. The reaction mixture was cooled and filtered.
4.2.3.1. 2-[3-(4’-Methoxyphenyl)-5-phenyl-2-pyrazolin-1-yl]-4-phenylthiazole (8a)
1 H NMR (500 MHz, CDCl3) δ (ppm): 3.36 (1H, dd, J = 17.34 Hz, J = 6.36 Hz, pyrazoline C4 –H), 3.89 (3H,
s, OCH3), 3.93 (1H, dd, J = 17.34 Hz, J = 11.87 Hz, pyrazoline C4 –H), 5.78–5.89 (1H, br, pyrazoline C5 –H),
6.81 (1H, s, thiazole–H), 6.98 (2H, d, J = 8.91 Hz, Ar–H), 7.27–7.42 (6H, m, Ar–H), 7.48 (2H, d, J = 8.37 Hz,
Ar–H), 7.71 (2H, d, J = 8.47 Hz, Ar–H), 7.75 (2H, d, J = 8.89 Hz, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 43.80 (pyrazoline C4), 55.42 (OCH3), 64.62 (pyrazoline C5),
103.02 (thiazole C5), 114.20, 126.01, 126.67, 128.13, 128.43, 128.73 (2CH, Ar–C), 126.55, 127.78 (CH), 124.00,
127.66, 141.54 (C), 145 (thiazole C2), 150.05 (pyrazoline C3), 161.18 (C–OCH3), 165.12 (S–C=N).
MS [M+1]+ : m/z 412
4.2.3.2. 2-[2-(3-(4’-Methoxyphenyl)-5-phenyl-2-pyrazolin-1-yl]thiazol-4-yl]phenol (8b)
1 H NMR (500 MHz, CDCl3) δ (ppm): 3.33 (1H, dd, J = 17.47 Hz, J = 7.06 Hz, pyrazoline C4 –H), 3.88 (3H,
s, OCH3), 3.94 (1H, dd, J = 17.47 Hz, J = 11.83 Hz, pyrazoline C4 –H), 5.56 (1H, dd, J = 11.79 Hz, J =
7.00 Hz, pyrazoline C5 –H), 6.80 (1H, two d, J = 7.10 Hz, J = 1.18 Hz, Ar–H), 6.81 (1H, s, thiazole–H), 6.91
(1H, d, J = 8.17 Hz, Ar–H), 6.98 (2H, d, J = 8.89 Hz, Ar–H), 7.15 (1H, two d, J = 7.23 Hz, J = 1.61 Hz,
Ar–H), 7.33 (2H, d, J = 8.88 Hz, Ar–H), 7.35 (1H, m, Ar–H), 7.42 (4H, m, Ar–H), 7.49 (1H, dd, J = 7.84 Hz,
J = 1.56 Hz, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 44.44 (pyrazoline C4), 55.43 (OCH3), 64.88 (pyrazoline C5),
101.66 (thiazole C5), 114.27, 126.10, 128.24, 129.31 (2CH, Ar–C), 117.71, 119.12, 125.80, 128.32, 129.54 (CH),
123.59, 125.93, 140.67 (C), 148.78 (thiazole C2), 153.19 (pyrazoline C3), 155.73 (C–OH), 161.40 (C–OCH3),
164.82 (S–C=N).
MS [M+1]+ : m/z 428
4.2.4. General procedure for compounds 9a, 9b
A mixture 5-phenyl-3-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-pyrazolin-1-carbothioamide (6) and 2-bromoacetophe-
none (0.001 mol) in ethanol (20 mL) was refluxed for 4 h. The reaction mixture was cooled and filtered.
4.2.4.1. 2-[5-Phenyl-3-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-pyrazolin-1-yl]-4-(4’-chlorophenyl)]
thiazole (9a)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.80–1.91 (4H, m, tetrahydronaphthalene C6,7 –H), 2.79–2.90 (4H, m,
tetrahydronaphthalene C5,8 –H), 3.36 (1H, dd, J = 17.42 Hz, J = 6.42 Hz, pyrazoline C4 –H), 3.92 (1H, dd, J
= 17.42 Hz, J = 11.92 Hz, pyrazoline C4 –H), 5.72–5.84 (1H, br, pyrazoline C5 –H), 6.80 (1H, s, thiazole–H),
7.15 (1H, d, J = 7.97 Hz, Ar–H), 7.29 (1H, m, Ar–H), 7.30 (2H, d, J = 8.59 Hz, Ar–H), 7.38 (2H, t, J = 7.36
Hz, Ar–H), 7.45 (2H, d, J = 7.32 Hz, Ar–H), 7.48 (1H, s, Ar–H), 7.53 (1H, dd, J = 7.85 Hz, J = 1.18 Hz,
Ar–H), 7.62 (2H, d, J = 8.52 Hz, Ar–H).
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13 C NMR (500 MHz, CDCl3) δ (ppm): 23.02–23.07 (2CH2 – tetrahydronaphthalene), 29.43–29.49
(2CH2 –tetrahydronaphthalene), 43.76 (pyrazoline C4), 64.59 (pyrazoline C5), 103.47 (thiazole C5), 126.60,
127.22, 128.56, 128.72 (2CH, Ar–C), 123.67, 127.18, 127.78, 129.54 (CH), 128.47, 129.23, 133.18, 137.61, 139.77,
141.61 (C), 149.81 (thiazole C2), 152.81 (pyrazoline C3), 165.14 (S–C=N).
MS [M+1]+ : m/z 470
4.2.4.2. 2-[5-Phenyl-3-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-pyrazolin-1-yl]-4-(4’-methylphenyl)]
thiazole (9b)
1 H NMR (500 MHz, CDCl3) δ (ppm): 1.80–1.89 (4H, m, tetrahydronaphthalene C6,7 –H), 2.36 (3H, s, CH3),
2.79–2.88 (4H, m, tetrahydronaphthalene C5,8 –H), 3.36 (1H, dd, J = 17.42 Hz, J = 6.27 Hz, pyrazoline C4 –H),
3.92 (1H, dd, J = 17.40 Hz, J = 11.91 Hz, pyrazoline C4 –H), 5.76–5.92 (1H, br, pyrazoline C5 –H), 6.75 (1H,
s, thiazole–H), 7.13 (1H, s, Ar–H) 7.15 (2H, d, J = 8.00 Hz, Ar–H), 7.29 (1H, m, Ar–H), 7.37 (2H, t, J = 7.83
Hz, Ar–H), 7.47 (2H, dd, J = 8.50 Hz, J = 1.15 Hz, Ar–H), 7.48 (1H, s, Ar–H), 7.54 (1H, dd, J = 7.91 Hz,
J = 1.6 Hz, Ar–H), 7.60 (2H, d,J = 8.12 Hz, Ar–H).
13 C NMR (500 MHz, CDCl3) δ (ppm): 21.24 (1H, s, CH3), 23.03–23.07 (2CH2 – tetrahydronaphthalene),
29.42–29.49 (2CH2 –tetrahydronaphthalene), 43.71 (pyrazoline C4), 64.58 (pyrazoline C5), 102.25 (thiazole C5),
125.92, 126.67, 128.69, 129.11 (2CH, Ar–C), 123.68, 127.19, 127.73, 129.52 (CH), 128.53, 137.39, 137.59, 139.73,
141,70 (C), 149.65 (thiazole C2), 152.65 (pyrazoline C3), 164.98 (S–C=N).
MS [M+1]+ : m/z 450
4.3. Microbiology
The microbiological assay was carried out according to the CLSI reference M7-A7 broth microdilution method.26
Chloramphenicol and ketoconazole were used as reference drugs. In the current work, thiazolylpyrazoline deriva-
tives (7a–7j, 8a, 8b, 9a, and 9b) were tested for their in vitro antimicrobial activity against Staphylococcus
aureus (ATCC-25923), E. faecalis (ATCC-29212), E. faecalis (ATCC-51922), L. monocytogenes (ATCC-1911),
K. pneumoniae (ATCC-700603), P. aeruginosa (ATCC-27853), E. coli (ATCC-35218), E. coli (ATCC-25922),
C. albicans (ATCC-90028), C. glabrata (ATCC-90030), C. krusei (ATCC-6258), and C. parapsilosis (ATCC-
22019) (Table 2).
4.4. Cytotoxicity
Cytotoxicity tests were performed using the MTT assay. The tetrazolium salt MTT [3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide] is used to measure the metabolic activity of viable cells. Tetrazolium
salts are reduced to formazan by mitochondrial succinate dehydrogenase, an enzyme that is only active in cells
with an intact metabolism. The formazan can be quantified photometrically and it is in correlation with the
number of viable cells.27 Cytotoxicity was tested using NIH3T3 (mouse embryonic fibroblast cell line) cells.
NIH3T3 cells were incubated in RPMI medium (Hyclone, Thermo Scientific, USA) supplemented with fetal
calf serum (Hyclone, Thermo Scientific, USA), 100 IU/mL penicillin and 100 mg/mL streptomycin (Hyclone,
Thermo Scientific, USA) at 37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 . NIH3T3 cells were
seeded at 10,000 cells into each well of 96-well plates. After 24 h of incubation, the culture media were removed
and compounds were added to culture medium in the range between 3.9 and 500 µg mL−1 concentrations
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with a dilution factor of 2. After 24 h of incubation, 20 µL of MTT solution (5 mg mL−1 MTT powder in
PBS) was added to each well. After 3 h of incubation at 37 ◦ C, 5% CO2 , contents of the wells were removed
and 100 µL of dimethyl sulfoxide (DMSO) was added to each well. Then OD of the plate was read at 540
nm. Inhibition% was calculated for each concentration of the compounds and IC50 values were estimated by
nonlinear regression analysis. Stock solutions of compounds were prepared in dimethyl sulfoxide (DMSO) and
further dilutions were made with fresh culture medium. The final DMSO concentration was under 0.1%. All
experiments were performed in triplicate (Table 2).27
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