Antiproliferative activities of 2-hydroxyethyl substituted benzimidazolium salts and their palladium complexes against human cancerous cell lines

Article abstract of DOI:10.1080/00397911.2019.1650187

Benzimidazolium salts (1a and 1b) and respective palladium complexes (2a and 2b) were prepared and characterized with ¹H and ¹³C NMR, IR, elemental analysis as well as HRMS (for 2a). All target compounds were screened as potential an

Full text of DOI:10.1080/00397911.2019.1650187

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
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Antiproliferative activities of 2-hydroxyethyl
substituted benzimidazolium salts and their
palladium complexes against human cancerous
cell lines
Senem Akkoç
To cite this article: Senem Akkoç (2019): Antiproliferative activities of 2-hydroxyethyl substituted
benzimidazolium salts and their palladium complexes against human cancerous cell lines, Synthetic
Communications, DOI: 10.1080/00397911.2019.1650187
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https://doi.org/10.1080/00397911.2019.1650187
Antiproliferative activities of 2-hydroxyethyl substituted
benzimidazolium salts and their palladium complexes
against human cancerous cell lines
Senem Akkoc¸
Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, Suleyman Demirel University,
Isparta 32260, Turkey
ABSTRACT
ARTICLE HISTORY
Benzimidazolium salts (1a and 1b) and respective palladium com-
Received 2 July 2019
1
13
plexes (2a and 2b) were prepared and characterized with H and
C
KEYWORDS
NMR, IR, elemental analysis as well as HRMS (for 2a). All target com-
pounds were screened as potential anticancer agents against human
cell lines for assessing their cytotoxicity. Heterocyclic organic com-
pounds (1a and 1b) showed more cytotoxic activity than their com-
plexes (2a and 2b) in the tested two cell lines. Particularly, a
benzimidazolium salt including a 4-methylbenzyl group had a high
cytotoxic potency towards MDA-MB-231 and DLD-1 cell lines with
IC50 values comparable to a well-known anticancer drug cisplatin,
which is generally used in clinical studies. Furthermore, a compound
namely 1-(2-hydroxyethyl)-3-(2,3,4,5,6-pentamethylbenzyl)-1H-benzo[-
d]imidazol-3-ium bromide was found to be more cytotoxic activity in
MDA-MB-231 cell line compared to cisplatin with following IC50 value
of 7.59 ± 0.68 lM.
Anticancer; benzimidazo-
lium salts; cytotoxic activity;
N-heterocyclic car-
bene; PEPPSI
GRAPHICAL ABSTRACT
CONTACT Senem Akkoc¸
senemakkoc@sdu.edu.tr, senemakkoc44@gmail.com
Faculty of Pharmacy, Department
of Basic Pharmaceutical Sciences, Suleyman Demirel University, Isparta 32260, Turkey.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsyc.
Supplemental data for this article is available online at on the publisher’s website
ß 2019 Taylor & Francis Group, LLC

2
S. AKKOC¸
Introduction
Benzimidazole and its derivatives are a class of very versatile chemicals that display high
desirable properties including pharmacological activities. Several products containing
benzimidazole nucleus are already been commercialized and are being employed in
treating different cancers and other ailments (Scheme 1). For example; bendamustine
(Treanda) is a chemotherapy drug which is used in the treatment of non-hodgkin’s
lymphoma and chronic lymphocytic leukemia. On the other hand, fenbendazole is a
veterinary anthelmintic drug and it is used against gastrointestinal parasites such as
roundworms, giardia, whipworms, hookworms, pinworms and paragonimiasis. The 5,6-
dimethyl-1-(a-D-ribofuranosyl)benzimidazole ring system is an integral part of the
N
N
Cl
O
NH
O
N
OCH3
N
N
N
N
N
H
O
O
Cl
N
H
O
Bendamustine (Anticancer)
Carbendazim (Fungicide)
Bezitramide (Analgesic)
H
N
H
N
O
H
N
O
S
O
CH PhCl
N
O
N
H
O
2
H
N
H
N
N
Fenbendazole (Veterinary anthelmintic)
Oxibendazole (Veterinary anthelmintic)
Diabazole (Vasodilator
spasmolytic hypotensive)
O
O
NH
H
N
N
N
N
OCH3
O
N
N
N
H
F
Flubendazole (Antineoplastic agent
Microtubule inhibitor)
N
Cl
Clemizole penicillin
(Bactericide)
F
F
Pimozide
Pyschopharmacological agent)
(
Scheme 1. A few examples of commercial products containing benzimidazole nucleus.

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SYNTHETIC COMMUNICATIONS
3
vitamin B12 structure, which was discovered by Bonnett in 1948, also possesses a benzi-
[
1]
midazole nucleus.
Additional applications of benzimidazole and its derivatives involve preparations such
[
2]
as polymer dyes and optical laser in optoelectronics, fluorescent tags and organic
luminophores particularly for the detection of DNA, RNA, or other biological signifi-
[
3–5]
cant molecules including enzymes and proteins.
Furthermore, compounds having
benzimidazole nucleus also exhibit catalytic activity, as a result, much attention has
been paid to those compounds because they can be used as catalysts in different
[
6,7]
[8]
[9–15]
reactions like arylation,
Negishi, Suzuki-Miyaura cross-coupling,
Mizoroki-
[
16,17]
[18]
Heck cross-coupling,
Sonogashira cross-coupling,
and Hiyama coupling
[
19,20]
reactions.
It is well-known that cancer is a big health problem in the worldwide. Although there
are many different developed anticancer drugs such as cisplatin, doxorubicin, oxalipla-
tin, melphalan, irinotecan hydrochloride trihydrate, 5-fluorouracil, yet people still lose
their lives because of cancer in every year. For instance, according to global cancer sta-
tistics data of 2018, 18.1 million new cancer cases and more than half of these cases
[
21]
approximately 9.6 million estimated cancer deaths will be in 2018.
Therefore,
[
22–28]
researchers are studying to come out with the most effective anticancer drugs.
For
reported a series of bis-NHC Ag(I) and Pd complexes that
demonstrate promising anticancer activity against breast cancer cell line (MCF-7). Yang
[
29]
example, Haque et al.
[
30]
et al.
have reported that 10-fluorocamptothecin derivatives showed important potent
anticancer effect against MDA-MB-231, A-549, KB-VIN, MCF-7 and KB cell lines.
In this study, two benzimidazolium salts were synthesized and they were used as
starting material for preparing palladium complexes. All the prepared compounds were
tested as anticancer agents towards human cancerous cell types containing cervix,
breast, liver and colon. Benzimidazolium salts had in vitro cytotoxic activity in tested
cell lines. Especially, compounds namely 1-(2-hydroxyethyl)-3-(4-methylbenzyl)-1H-ben-
zo[d]imidazol-3-ium bromide and 1-(2-hydroxyethyl)-3-(2,3,4,5,6-pentamethylbenzyl)-
1H-benzo[d]imidazol-3-ium bromide appear to be anticancer drugs with lower inhibi-
tory concentration (IC ) values against MDA-MB-231 cell line.
5
0
Results and discussion
-Hydroxyethyl substituted benzimidazolium salts and their palladium complexes
2
Two benzimidazolium salts (1a and 1b) and their metal complexes (2a and 2b) includ-
ing 2-hydroxyethyl substituent were synthesized in yields ranging from 21 to 58% for
1
a, 1b, 2a and 2b (Scheme 2). The structural characterizations of these compounds
OH
OH
Cl
Cl
X
OH
N
N
N
N
N
N
N
N
Br
Pd
Br
R
Br
8
Br-
+
PdCl2 + K CO + KBr
N
2
3
80 oC, 16 h
Ethyl alcohol
reflux, 6 h
DMF
0 oC, 24 h
N
H
R
R
R
1
a, 1b
R : -C H (CH )- 4
(for 1a and 2a)
(for 1b and 2b)
2a, 2b
6
4
3
-
C
6
3 5
(CH ) - 2,3,4,5,6
Scheme 2. Synthesis of benzimidazolium salts (1a and 1b) and their Pd complexes (2a and 2b).

4
S. AKKOC¸
Table 1. A few selected properties of compounds.
Melting
ꢁ
Entry Compounds Yield (%) point ( C)
Color
Elemental analysis found (calc.)
IR(C¼N)
1
2
3
4
1a
1b
2a
2b
58
57
49
21
239–240
194–196
215–217
174–179
white
yellow
yellow
C: 58.61 (58.80); H: 5.80 (5.52); N: 8.03 (8.07) 1556.8
C: 62.37 (62.53); H: 6.99 (6.75); N: 6.92 (6.95) 1550.7
C: 40.81 (40.90); H: 3.55 (3.43); N: 6.47 (6.50) 1402.2
light yellow C: 44.40 (44.47); H: 4.46 (4.31); N: 5.99 (5.98) 1456.2
1
13
were done with elemental analysis, IR, H and C NMR. Some properties of synthe-
sized compounds were done in Table 1.
1
The H NMR spectra confirm the structures of 1a, 1b and 2a and 2b. The sharp sin-
gle signal belonging to the NCHN proton at the C2 position of the benzimidazole ring
1
was obtained in quite a downfield region in the H NMR spectra for 1a and 1b. For
example, these signals were observed at d 11.54 and 9.77 ppm for 1a and 1b, respect-
ively. However, as a result of deprotonation of this acidic proton in each ligand (from
1
NCHN to NCN) when complexing, these single signals disappeared in H NMR spectra
of 2a and 2b. This indicates the synthesis of 2a and 2b. The corresponding to the two
protons of methylene group (NCH C ) as a singlet signal was observed at d 5.82 and
2
6
1
1
5
.70 ppm for 1a and 1b in H NMR spectra, respectively. On the other hand, in H
NMR spectra of 2b, these proton signals belonging to NCH C group shifted further
2
down area and observed at d 6.04 ppm.
1
3
In the C NMR spectra, the carbon signal of NCHN group was observed at d 142.72
and 141.34 ppm for 1a and 1b, respectively. Carbene carbon signal (NCN) was obtained
13
at lower chemical shift at d 162.25 and 161.13 ppm for 2a and 2b, respectively.
C
NMR spectra shows the methyl carbon signal belonging to NCH C H (CH )-4 at d
2
6
4
3
21.17 and 21.20 ppm for 1a and 2a, respectively.
The formation of the compounds was also made evident through IR spectra, which
ꢀ
1
showed the v(C¼N) band at 1556.8, 1550.7, 1402.2 and 1456.2 cm for 1a, 1b, 2a and
2
b, respectively. The v(C–O) bands of compounds (1a, 1b, 2a and 2b) appeared at
ꢀ
1
1189.5, 1193.8, 1201.6 and 1255.6 cm in the IR spectra, respectively. Furthermore, the
HRMS of 2a was taken and it demonstrated molecular ion peak m/z 433.07378. The cal-
culated and found values were obtained very similar to each other as 433.08 and
433.07378, respectively. This result also confirms the proposed structure of 2a.
Cytotoxic activity results of the synthesized compounds
Using MTT assay method, the antiproliferative activities of all synthesized compounds
(1a, 1b, 2a and 2b) were screened towards four different cell lines, namely; HeLa,
MDA-MB-231, HepG2 and DLD-1. Furthermore, the obtained cytotoxic activity results
were compared with cisplatin, which is a well-known anticancer drug and it was used
as a positive control drug under the same experimental conditions with the tested drug
candidates in this study. The standard deviation values of the compounds tested at least
twice and at different times were calculated and the all cytotoxic activity results are
given in Table 2.
It is noteworthy that compounds 1b showed measurable cytotoxic activity in HeLa
cell line with the following IC50 values of 80.64 ± 1.25 mM. However, Pd complexes (2a

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SYNTHETIC COMMUNICATIONS
5
Table 2. IC50 results for 1a, 1b, 2a and 2b against HeLa, MDA-MB-231, HepG2 and DLD-1 cell lines.
IC50 (mM)
Compounds
HeLa
MDA-MB-231
HepG2
DLD-1
1
1
2
2
a
b
a
b
N.T.
80.64 ± 1.25
>200
>200
2.14 ± 0.063
11.53 ± 1.21
7.59 ± 0.68
>200
>200
8.25 ± 1.56
123.7
175.7
>200
>200
9.82
9.19 ± 1.51
39.51
>200
>200
5.71 ± 0.98
Cisplatin
N.T. not tested.
1
1
20
00
80
60
40
20
0
1
2
b
a
2b
Cisplaꢀn
2
00 100 50 20 10
5
2
1
0.5
Concentraꢀon (mM)
Figure 1. Viability ratios of HeLa cells as dependent on drug concentrations after completion of
the process.
1
1
20
00
80
60
40
20
0
1
1
a
b
2a
2
b
2
00 100
50
20
10
5
2
1
0.5
Concentraꢀon (mM)
Figure 2. Viability ratios of MDA-MB-231 cells as dependent on drug concentrations after completion
of the process.
and 2b) of these organic compounds (1a and 1b) did not show anticancer activity in
the same cell line.
All developed compounds (1a, 1b, 2a and 2b) were tested in MDA-MB-231 cell line.
Compound 1a including 4-methylbenzyl substituent and compound 1b containing
2,3,4,5,6-pentamethylbenzyl group exhibited higher cytotoxic activities as compared to

6
S. AKKOC¸
1
1
20
00
80
60
40
20
0
1
a
1b
2
2
a
b
Cisplaꢀn
2
00 100 50 20 10
5
2
1
0.5
Concentraꢀon (mM)
Figure 3. Viability ratios of HepG2 cells as dependent on drug concentrations after completion of
the process.
2a and 2b with IC50 values of 11.53 ± 1.21 and 7.59 ± 0.68 mM, respectively. Particularly,
compound 1b with an IC50 value of 7.59 ± 0.68 mM exhibited the highest potency for
killing breast cancerous cells. Furthermore, it was found more active than cisplatin
against MDA-MB-231 cell line. The cytotoxicity effects of 2a and 2b were not observed
towards breast cancerous cell line for the tested nine different concentrations
(0.5–200 mM) after 72 h of incubation. Also, according to the activity results obtained,
compounds 1a and 1b screened against breast cell line was found to be dose-dependent
(Fig. 2).
All compounds were further screened in liver cancer cell line (HepG2). Compounds
1a and 1b) had cytotoxic activity in HepG2 cell line and demonstrated IC50 values of
(
123.7 and 175.7 mM, respectively. However, compounds (2a and 2b) were found to have
no activity toward HepG2 cell line, too.
Compounds (1a, 1b, 2a and 2b) were tested in DLD-1. Compound 1a particularly
showed lowest IC50 value (IC : 9.19 ± 1.51 mM) compared to compounds 1b, 2a, 2b
50
towards DLD-1 cell line. This value is very close to the positive control drug cisplatin
value (IC : 5.71 ± 0.98 mM).
5
0
From the results obtained, the percentages of live cells were observed to increase with
decreasing concentrations of drug candidates (Figs. 1–3). This condition strongly
showed that concentrations of the tested compounds are very significant for obtaining
measurable cytotoxic activity results.
As can be seen in Figures 1–3, the complexes (2a and 2b) were almost seen to have
equal inactive cytotoxicity to all the tested cell lines. However, benzimidazolium salts
(1a and 1b) exhibited toxic activity and cell viabilities changed depending on the tested
drug concentrations. For example, in high concentrations like 200 or 100 mM, cell viabil-
ities of cancerous cells were obtained in lower percentages compared to cell viabilities
treated with low concentrations of drug candidates (Fig. 1 for 1b; Figs. 2 and 3 for 1a
and 1b). In Figure 2, compound 1a can be seen as the most effective drug candidate
against MDA-MB-231 cell line because of it exhibited lowest cell viabilities compared to
compounds (1b, 2a and 2b).

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7
Conclusion
In this study, four compounds were successfully synthesized and screened against HeLa,
MDA-MB-231, HepG2 and DLD-1 cell lines. Benzimidazolium salts (1a and 1b) exhib-
ited in vitro cytotoxic activity towards the above-mentioned cell lines with IC50 values
ranging from 7.59 lM to >200 lM. Compound 1a showed significant cytotoxicity
against MDA-MB-231 and DLD-1 cell lines with the following lowest IC50 values of
11.53 and 9.19 lM, respectively. Furthermore, the results of antiproliferative activity
evaluation towards breast cancerous cell line demonstrated that a compound namely 1-
(
(
2-hydroxyethyl)-3-(2,3,4,5,6-pentamethylbenzyl)-1H-benzo[d]imidazol-3-ium bromide
1b) had remarkable cytotoxicity with IC50 value of 7.59 ± 0.68 lM, which is lower than
IC50 value of cisplatin. This compound may be used as a potential anticancer drug
towards MDA-MB-231 cell line. However, the developed palladium complexes (2a and
2
b) were found to be inactive in screened all cell lines with IC50 values greater than
200 lM. In conclusion, synthesized organic compounds (1a and 1b) gave better anti-
cancer activity results than their metal complexes (2a and 2b).
Experimental section
Materials and instrumentations
All solvents and reagents for the synthesis of compounds (1a, 1b, 2a and 2b) were
bought from Merck, Sigma-Aldrich or Scharlau.
1
13
H and C NMR spectra were taken using a Bruker 400 or 600 MHz Ultra Shield
NMR in CDCl solvent. The IR spectra of compounds were taken with a Shimadzu FT-
3
IR 8400 spectrophotometer.
Human cervix epithelial adenocarcinoma (HeLa), human breast epithelial adenocar-
cinoma (MDA-MB-231), human liver epithelial hepatocellular carcinoma (HepG2) and
human colorectal adenocarcinoma (DLD-1) cell lines were bought from the American
Type Culture Collection (ATCC, USA). Reagents and materials like GlutaMAX, penicil-
lin streptomycin, Dulbecco’s Modified Eagle’s Medium (DMEM), DMEM:F-12, Fetal
Bovine Serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(
MTT), cisplatin, ninety six well plates and cryofreezing containers were bought from
Medicago (Astral scientific, Sweden), Gibco (Life technologies, USA) or Sigma-Aldrich
USA) for cell culture studies.
(
General procedure for the synthesis of benzimidazolium salts, 1a-b
The synthesis of the compounds (1a and 1b) were conducted according to literature
[
5,31,32]
protocols.
Benzimidazole (1 mmol) was dissolved in ethyl alcohol (15 mL) and
potassium hydroxide (KOH) (1 mmol) was added to this solution. The resulting mixture
ꢁ
was stirred for 1 h at 25 C. Then, aryl halide such as 1-(chloromethyl)-4-methylbenzene
(for 1a), 1-(chloromethyl)-2,3,4,5,6-pentamethylbenzene (for 1b) was slowly incorpo-
rated into this reaction mixture. While refluxing, the reaction was maintained for 6 h.
So, two different N-alkyl benzimidazole, which are 1-(4-methylbenzyl)-1H-benzo[d]imi-
dazole (for 1a) and 1-(2,3,4,5,6-pentamethylbenzyl)-1H-benzo[d]imidazole (for 1b),

8
S. AKKOC¸
were prepared. The purification of these two N-alkyl benzimidazole were done with
crystallization. 2-Bromoethanol (1 mmol) was slowly put to the solution of N-alkyl ben-
zimidazole (1 mmol) in N,N-dimethylformamide (DMF) (4 mL) and the reaction mix-
ꢁ
ture was stirred at 80 C for 24 h. Finally, DMF was aspirated under vacuum after the
completion of the reaction and the obtained targeted crude products (1a and 1b) were
purified by crystallization.
1
-(2-Hydroxyethyl)-3-(4-methylbenzyl)-1H-benzo[d]imidazol-3-ium bromide, 1a
A similar compound namely 1-(2-hydroxyethyl)-3-(4-methylbenzyl)benzimidazolium
[
33]
iodide was prepared by Erdemir et al.
However, in here, compound 1a was synthe-
sized from 1-(4-methylbenzyl)-1H-benzo[d]imidazole (1.00 g, 1 mmol) and 2-bromoe-
ꢁ
1
thanol (0.56 g, 1 mmol) in shorter time. Yield: 58%, m.p.: 239-240 C, color: white. IR:
ꢀ
1
1
189.5 (C–O); 1556.8 (C¼N); 2773.5 and 2967.4 cm
(C–H). H NMR (400 MHz,
CDCl , 298 K), d: 2.18 (s, 1H, NCH CH OH); 2.27 (s, 3H, NCH C H (CH )-4); 4.20 (t,
3
2
2
2
6
4
3
2
7
2
1
H, NCH CH OH); 4.55 (t, 2H, NCH CH OH); 5.82 (s, 2H, NCH C H (CH )-4);
2 2 2 2 2 6 4 3
13
.14–7.59 (m, 8 H, Ar–H); 11.54 (s, 1H, NCHN). C NMR (100 MHz, CDCl , 298 K), d:
3
1.17 (NCH C H CH ); 34.56 and 51.48 (NCH CH OH and NCH C H CH ); 113.83,
2
6
4
3
2
2
2
6
4
3
27.09, 128.30, 129.53, 130.04, 131.37 and 139.28 (Ar–C); 142.72 (NCHN). Elemental
analysis for C H N OBr (347.25 g/mol) %: Found C: 58.61; H: 5.80; N: 8.03. Anal.
1
7
19 2
Calc. C: 58.80; H: 5.52; N: 8.07.
Synthesis of palladium complexes, 2a-b
The synthesis of Pyridine Enhanced Precatalyst Preparation Stabilization and Initiation
(
PEPPSI) palladium N-heterocyclic carbene complexes were conducted according to lit-
[
5,7]
erature protocols.
Complexes (2a and 2b) were prepared using compounds 1a and
1
b (1 mmol), PdCl2 (1 mmol), 3-chloropyridine (3 mL), KBr (1 mmol) and K CO3
2
(
2 mmol) as a base. The reaction was conducted in a Schlenk tube and the reaction mix-
ꢁ
ture was stirred at 80 C for 16 h. After completion of the reaction, dichloromethane
was added to the reaction medium and filtered through a column over celite-silicagel.
Then, the solvent in the medium was aspirated under vacuum. The resulting crude
product was initially washed several times with diethyl ether. Then, it was purified by
crystallization.
Dibromo-[1-(2-hydroxyethyl)-3-(4-methylbenzyl)benzimidazol-2-ylidene]-N-(3-chloro-
pyridine) palladium (II) complex, 2a
Compound 2a was prepared from 1-(2-hydroxyethyl)-3-(4-methylbenzyl)-1H-benzo[d]i-
midazol-3-ium bromide 1a (1 mmol), PdCl (1 mmol), 3-chloropyridine (3 mL) and
2
ꢁ
K CO (5 mmol) as a base. Yield: 49%, m.p.: 215–217 C, color: yellow. IR: 1201.6
C–O); 1402.2 (C¼N); 2854.4, 2933.5 and 3006.8 cm (C–H). H NMR (400 MHz,
CDCl , 298 K), d: 2.30–2.35 [m, 6 H, NCH CH OH; NCH C H (CH )-4]; 6.00–6.24 [m,
2
3
ꢀ
1
1
(
3
2
2
2
6
4
3
13
4
H, NCH CH OH; NCH C H (CH )-4]; 6.96–9.07 (m, 12 H, Ar–H).
C NMR
2
2
2
6
4
3
(
100 MHz, CDCl , 298 K), d: 21.20 [NCH C H (CH )-4]; 53.02, 53.30 and 53.56
3
2
6
4
3

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SYNTHETIC COMMUNICATIONS
9
[
NCH CH OH and NCH C H (CH )-4]; 111.53, 123.11, 123.37, 124.87, 127.97, 128.02,
2 2 2 6 4 3
1
29.30, 129.56, 131.77, 131.93, 132.63, 134.76, 137.97, 138.06, 149.25, 150.03 and 151.69
þ
(Ar–C); 162.25 (NCN). HRMS [M-OHClBr2] calcd for C H N Pd: 433.08, found
22
21 3
m/z: 433.07378. Elemental analysis for C H Br ClN OPd (646.12 g/mol) %: Found
2
2
22
2
3
C: 40.81; H: 3.59; N: 6.47. Anal. Calc. C: 40.90; H: 3.43; N: 6.50.
Cytotoxic activity of new compounds
For in vitro cytotoxic activity studies, the stock solutions of 1a and 1b were prepared in
sterile PBS. However, the stock solutions of 2a and 2b were made ready in 0.5% DMSO
due to the fact that they are not soluble in PBS only. So, in negative control tests of the
metal complexes in the cell culture studies, 0.5% DMSO was used as co-solvent.
[
5,32,34,35]
The cytotoxic activity procedures in the literatures were applied to this study.
The HeLa, MDA-MB-231 and DLD-1 cells were cultured in DMEM supplemented with
% GlutaMAX, 1% penicillin streptomycin and 10% FBS. DMEM:F-12 instead of
1
DMEM was used in HepG2 cells as medium.
Firstly, the cancerous cells were seeded into sterile 96-well plates at a density of 3 x
3
1
0 cells/well. Plates were incubated for 24 h in an incubator. Secondly, medium in the
plates were removed with vacuum pump and the cells were treated with the synthesized
compounds (1a, 1b, 2a and 2b), which were prepared in nine different concentrations
as follows; 0.5, 1, 2, 5, 10, 20, 50, 100 and 200 mM. The 96-well plates were again incu-
bated for 72 h. Thirdly, the medium was aspirated after 72 h and MTT stock solution
(50 mL, 5 mg/mL) was added to each well. After 4 h incubation, DMSO (200 mL) was put
to each well and the plates were mixed on a rocker for 1/2 h. ELISA micro plate reader
device was used for measuring the absorbance values at 595 nm. The GraphPad Prism
software program was used for calculating IC50 values.
Spectra and full characterization data of compounds can be found via the
“Supplementary Content”.
Acknowledgments
I would like to express my heartfelt gratitude to Erciyes University, Genom and Stem Cell Center
(ERU GENKOK) for giving me study places for cell culture. I would also like to thank Yahuza
Nantomah Abdulai for reading the manuscript and correcting its grammatical mistakes.
Disclosure statement
The author declares that no conflict of interest is associated with this work.
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