Cytotoxic effects of coumarin substituted benzimidazolium salts against human prostate and ovarian cancer cells

Article abstract of DOI:10.1007/s12039-019-1647-0

Abstract: Coumarin and benzimidazole derivatives have individual biological activities including anticancer. In this study, we aimed to synthesize coumarin-benzimidazole hybrids in order to investigate their anticancer properties. For this purpose, six 6-substituted-4-chloromethylene coumarin derivatives were synthesized. Sixteen coumarin substituted benzimidazolium chlorides were synthesized by the reaction of 4-chloromethylene coumarin and N-benzylbenzimidazole derivatives. All of the synthesized compounds were characterized by ¹H and ¹³C NMR, IR spectroscopic techniques and elemental analyses. Cytotoxicities of all compounds were tested by [3-(4,5-dimethylthiazole)-2-yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay against human prostate (PC-3) and ovarian (A2780) cancer cells. All compounds performed significant cytotoxicities at 100μM against both cancer cell lines. Moreover, some compounds performed significant activities at 1μM against both cancer cell lines and the obtained results suggest that this type of compounds are promising candidates for the treatment of human prostate and ovarian cancers. Graphic abstract: Six 4-chloromethylenecoumarin derivatives and sixteen coumarin substituted benzimidazolium chlorides were synthesized and characterized. Cytotoxic properties of all compounds were tested against human prostate and ovarian cancer cell lines.[Figure not available: see fulltext.].

Full text of DOI:10.1007/s12039-019-1647-0

J. Chem. Sci.
(2019) 131:69
© Indian Academy of Sciences
https://doi.org/10.1007/s12039-019-1647-0
REGULAR ARTICLE
Cytotoxic effects of coumarin substituted benzimidazolium salts
against human prostate and ovarian cancer cells
MERT OLGUN KARATAS¸a,∗ , SUAT TEKINb, BÜLENT ALICIa and SÜLEYMAN SANDALb
a
˙
Department of Chemistry, Faculty of Science, Inönü University, 9044280 Malatya, Turkey
b
˙
Department of Physiology, Faculty of Medicine, Inönü University, 9044280 Malatya, Turkey
E-mail: mert.karatas@inonu.edu.tr
MS received 19 November 2018; revised 15 May 2019; accepted 17 May 2019
Abstract. Coumarin and benzimidazole derivatives have individual biological activities including anticancer.
In this study, we aimed to synthesize coumarin-benzimidazole hybrids in order to investigate their anticancer
properties. For this purpose, six 6-substituted-4-chloromethylene coumarin derivatives were synthesized.
Sixteen coumarin substituted benzimidazolium chlorides were synthesized by the reaction of 4-chloromethylene
coumarin and N-benzylbenzimidazole derivatives. All of the synthesized compounds were characterized by 1H
and 13C NMR, IR spectroscopic techniques and elemental analyses. Cytotoxicities of all compounds were tested
by [3-(4,5-dimethylthiazole)-2-yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay against human prostate
(PC-3) and ovarian (A2780) cancer cells. All compounds performed significant cytotoxicities at 100 μM against
both cancer cell lines. Moreover, some compounds performed significant activities at 1 μM against both cancer
cell lines and the obtained results suggest that this type of compounds are promising candidates for the treatment
of human prostate and ovarian cancers.
Keywords. Benzimidazole; benzimidazolium salt; coumarin; cytotoxicity.
1. Introduction
Coumarin is a bicyclic compound which consists of
the fusion of α-pyron and benzene rings. The most
known coumarin derivative is Warfarin which has a
significant anticoagulant effect.1 In addition, Khel-
lactone and Calanolide derivatives are well known
coumarin-based anti-HIV agents.2 Coumarin deriva-
tives were also reported as carbonic anhydrase inhibitors
by Supuran and co-workers.3 Novobiocin is another
coumarin-based compound and was marketed as an
antibacterial drug. In a study, Zhao et al., optimized
the structure of Novobiocin as a highly active anti-
proliferative agent.4 Additionally, simple coumarin, 7-
hydroxyoumarin, Esculetin (6,7-dihydroxycoumarin),
and Scopoletin (6-methoxy-7-hydroxycoumarin) per-
formed strong cytotoxic effects against cancer cell
lines.5 Coumarin derived compounds have also a wide
spectrum of biological activities which were reviewed
by expert researchers.6,7
Cancer is one of the biggest health problems that
threaten humanity and the fight against cancer continues
with different methods across the world. Chemotherapy
is a method that uses chemical compounds includ-
ing organic, inorganic, and organometallic monomers
or polymers and nanoparticles to kill cancer cells.
However, drug resistance of cancer cells and side
effects of drugs are compelling problems encountered
in chemotherapy. Therefore, the development of novel
compounds for the treatment of cancer with low toxicity
and high selectivity is an important challenge for scien-
tists. Hybridization of some biologically active scaffolds
in a structure is one of the newest strategies for drug
design. This strategy aims to achieve higher activity and
lower toxicity due to synergistic effects. In this study,
we aimed to combine the coumarin and benzimidazole
groups which have individual biological activities in an
ionic structure in order to investigate their cytotoxicity
properties.
Benzimidazole is an important member of nitrogen
heterocycles and consist of the fusion of the imidazole
and benzene rings via 4- and 5- positions of the imida-
zole ring. The most known benzimidazole derivative is
*
For correspondence
Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12039-019-1647-0) contains
supplementary material, which is available to authorized users.
0123456789().: V,-vol

69 Page 2 of 12
J. Chem. Sci.
(2019) 131:69
N-ribosyl-5,6-dimethylbenzimidazole which serves as 2. Experimental
an axial ligand to cobalt in the structure of vitamin B12.8
2.1 Reagents and equipment
The first used benzimidazole derivatives are Thiaben-
dazole and Albendazole which have anti-helminthic
properties in agriculture.9 Due to the cytotoxic prop-
erties of some benzimidazole-based compounds, com-
mercially available anticancer drugs were developed.10
Another important usage of benzimidazole derivatives
is in the treatment of ulcer and some benzimidazole-
based anti-ulcer drugs were marketed.9 In addition,
antimicrobial, anti-coagulant, anti-oxidant, and other
Benzylchloride, 2,3,4,5,6-pentamethylbenzylchloride, 3,4,5-
trimethoxybenzyl chloride, 4-methylphenol, 4-ethylphenol,
4-ethoxyphenol, 4-iso-propylphenol, 4-tert-butylphenol, 4-
benzylphenol, ethyl-4-chloroacetoacetate and solvents were
purchased from Aldrich Chemical Co and Alfa Aesar (Istan-
bul, Turkey) and used as received. The C, H and N elemental
analysis were determined by LECO CHNS-932 elemental
analyser. Melting points were determined in open capillary
biological properties of benzimidazole derivatives were tubes by Electrothermal-9200 melting point apparatus. IR
spectra in the range of 4000–400 cm−1 were obtained in ATR
Sampling Accessory with Perkin Elmer UATR Two Spec-
trophotometer. 1H NMR and 13C NMR spectra were recorded
usingaBrukerUltraShield300operatingat300.13MHz(1H),
75.47 MHz (13C) using DMSO-d6 as a solvent. Chemical
shifts are given in ppm relative to tetramethylsilane (TMS).
NMR multiplicities are abbreviated as follows: s = singlet, d
= doublet, t = triplet, q = quartet, sep = septet, m = multiplet
signal. Coupling constants, J, are given in Hz.
reported.11
Benzimidazolium salts are 1,3-disubstituted ionic
benzimidazole derivatives that contain acidic hydrogen
at 2-position. Although they were mainly used as N-
heterocyclic carbene (NHC) precursors, in recent years,
their biological activities attracted much attention.12
In 2015, Elie et al., reported that benzimidazolium
salts perform antibacterial effects by the impairment
of membrane permeability.13 In another study, Liu et
al., reported that carbazole substituted benzimidazolium
salts perform strong cytotoxicity against various cancer
cell lines.14 Additionally, enzyme inhibitory properties
of some benzimidazolium salts were reported.15–18
In recent years, many research groups focused on
the synthesis and anti-cancer properties of hybrid
compounds of coumarin with other bio-active hetero-
2.2 Synthesis and characterization data of
compounds
2.2a Synthesis of 4-chloromethyl coumarin derivatives and
characterization data (1a–f). These compounds were synthe-
sized by the previously described procedure by Frasinyuk36
with a minor modification. The crude products were re-
cyclic moieties such as pyrimidine,19 chalcone,20 β- crystallized from ethanol instead of dioxane. The mixture of
carboline,21 indole-triazole,22 artemisinin,23 thiazole,24
18 mmol (3 g) of ethyl-4-chloroacetoacetate and 18 mmol of
and isooxazilones.25In the meantime, hybrid com-
corresponding phenol derivative was stirred in 40 mL 70%
H2SO4 for 24 h at room temperature. Then the mixture was
pounds of benzimidazole and their anticancer properties
slowly poured into an ice bath and the precipitate was col-
were reported with various heterocyclic moieties such
lected by filtration. The crude product was re-crystallized
as triazine,26 ellipticine,27 tetrazine,28 deoxynucleo-
from ethanol. Among the 1a–f, only 1a was previously
sides,29 thiazoles,30 and chrysin.31However, accord-
reported by Frasinyuk and co-workers.22
ing to our literature survey, coumarin-benzimidazole
4-Chloromethyl-6-methyl-2H-chromene-2-one, 1a. White
solid, yield: 2.8 g (75%), M.p.: 145–146◦C. Elemental anal-
ysis: Calculated for C11H9ClO2; C, 63.32; H, 4.35; Found: C,
63.16; H, 4.24. IR (cm−1): 3088, 2922, 1726 (-C=O), 1609,
hybrids are very rare. Hwu and co-workers reported
the synthesis and anti-viral properties of coumarin-
benzimidazole hybrids.32,33 Paul and co-workers syn-
thesized coumarin-benzimidazole hybrids and showed
that these compounds are highly active and selec-
tive anticancer agents.34 Most recently, Holiyachi and
co-workers reported the synthesis, antimicrobial, and
anticancer properties of coumarin-benzimidazole-
sulphonamide hybrids.35 Based on the above informa-
tion, herein, we report the synthesis and anticancer prop-
erties of coumarin substituted benzimidazolium salts.
For this purpose, six 6-substituted-4-chloromethylene
coumarinderivativesandsixteenbenzimidazoliumchlo-
rides were synthesized and characterized. Cytotoxic
properties of all compounds were tested against human
prostate (PC-3) and ovarian (A2780) cancer cell
lines.
1
1570, 1494, 1442. H NMR (300 MHz, DMSO-d6): δ 7.66
(m, 1H, ArH), 7.47 (m, 1H, ArH), 7.33 (m, 1H, ArH), 6.66 (s,
1H, -CH=C-), 5.02(s, 2H, -CH2Cl), 2.39(s, 3H, ArCH3). 13
C
NMR (75 MHz, DMSO-d6): δ 159.7 (-C=O), 151.4, 150.5,
133.7, 133.2, 124.9, 116.7, 116.5, 115.3 (-CH=C-), 41.2 (-
CH2Cl), 20.4 (ArCH3).
4-Chloromethyl-6-ethyl-2H-chromene-2-one, 1b. Yellow
solid, yield: 1.6 g (40%), M.p.: 141–142◦C. Elemental anal-
ysis; Calculated for C12H11ClO2; C, 64.73; H, 4.98; Found:
C, 64.66; H, 4.91. IR (cm−1): 3090, 2982, 2934, 1726 (-
1
C=O), 1625, 1609, 1570, 1493, 1442. H NMR (300 MHz,
DMSO-d6): δ 7.67 (m, 1H, ArH), 7.51 (m, 1H, ArH),
7.35 (m, 1H, ArH), 6.66 (s, 1H, -CH=C-), 5.04 (s, 2H, -
CH2Cl), 2.69 (q, 2H, ArCH2CH3, J = 7.6 Hz), 1.22 (t, 3H,

J. Chem. Sci.
(2019) 131:69
Page 3 of 12
69
ArCH2CH3, J = 7.6 Hz). 13C NMR (75 MHz, DMSO-d6): 1-Benzyl-3-((6-methyl-2H-chromene-4-yl)methyl)benzi-
δ 159.7 (-C=O), 151.6, 150.6, 140.0, 132.1, 123.8, 116.7, midazolium chloride, 2a. White solid, yield: 0.78 g (78%),
116.6, 115.3 (-CH=C-), 41.2 (-CH2Cl), 27.6 (ArCH2CH3), M.p.: 248–249◦C. Elemental analysis; Calculated for C25H21
15.6 (ArCH2CH3).
ClN2O2; C, 72.02; H, 5.08; N, 6.72; Found: C, 71.85; H, 4.92;
N, 6.60. IR (cm−1): 3118, 3030, 1702 (-C=O), 1687, 1617,
1576, 1557, 1482, 1452. 1H NMR (300 MHz, DMSO-d6): δ
10.18 (s, 1H, -NCHN-), 8.09–8.03 (m, 2H, ArH), 7.76–7.66
(m, 3H, ArH), 7.61–7.53 (m, 3H, ArH), 7.48–7.38 (m, 4H,
ArH), 6.23 (s, 2H, -NCH2coumarin), 6.03 (s, 1H, -CH=C-
), 5.84 (s, 2H, -NCH2Ph), 2.43 (s, 3H, ArCH3). 13C NMR
(75 MHz, DMSO-d6): δ 159.5 (-C=O), 151.1, 148.5, 143.6
(-NCHN-), 134.0, 133.7, 133.5, 131.4, 131.2, 129.0, 128.8,
128.5, 127.1, 127.0, 124.4, 116.63, 116.58, 114.1, 113.9,
112.9 (-CH=C-), 50.2 (-NCH2Ph), 46.6 (-NCH2coumarin),
20.4 (ArCH3).
4-Chloromethyl-6-ethoxy-2H-chromene-2-one, 1c. Green
solid, yield: 1.8 g (42%), M.p.: 149–150◦C. Elemental anal-
ysis; Calculated for C12H11ClO3; C, 60.39, H, 4.65; Found: C,
60.22; H, 4.53. IR (cm−1): 3090, 2982, 2876, 1729 (-C=O),
1
1624, 1568, 1499, 1477. H NMR (300 MHz, DMSO-d6):
δ 7.38 (m, 1H, ArH), 7.29 (m, 1H, ArH), 7.24 (m, 1H,
ArH), 6.67 (s, 1H, -CH=C-), 5.04 (s, 2H, -CH2Cl), 4.10 (q,
2H, ArOCH2CH3, J = 6.9 Hz), 1.36 (t, 3H, ArOCH2CH3,
J = 6.9 Hz). 13C NMR (75 MHz, DMSO-d6): δ 159.7
(-C=O), 154.7, 150.4, 147.4, 119.7, 117.8, 117.5, 115.7 (-
CH=C-), 108.8, 63.8 (ArOCH2CH3), 41.3 (-CH2Cl), 14.5
(ArOCH2CH3).
1-Benzyl-3-((6-ethyl-2H-chromene-4-yl)methyl)benzimi-
dazolium chloride, 2b. White solid, yield: 0.37 g (36%),
M.p.: 181–183◦C. Elemental analysis; Calculated for C26H23
ClN2O2; C, 72.47; H, 5.38; N, 6.50; Found: C, 72.31; H, 5.30;
N, 6.38. IR (cm−1): 3119, 3029, 2969, 1704 (-C=O), 1632,
4-Chloromethyl-6-isopropyl-2H-chromene-2-one, 1d. Be-
ige solid, yield: 2.3 g (54%), M.p.: 106–107◦C. Elemen-
tal analysis; Calculated for C13H13ClO2; C, 65.97; H, 5.54;
Found: C, 65.81; H, 5.44. IR (cm−1): 3089, 3033, 2922,
1726 (-C=O), 1625, 1609, 1570, 1493, 1442. 1H NMR (300
MHz, DMSO-d6): δ 7.69 (m, 1H, ArH), 7.56 (m, 1H, ArH),
7.37 (m, 1H, ArH), 6.67 (s, 1H, -CH=C-), 5.07 (s, 2H, -
CH2Cl), 3.00 (sep, 1H, ArCH(CH3)2, J = 6.8 Hz), 1.25 (d,
6H, ArCH(CH3)2,J = 6.8 Hz). 13C NMR (75 MHz, DMSO-
d6): δ 159.7 (-C=O), 151.6, 150.7, 144.6, 130.6, 122.6, 116.7,
116.6, 115.2 (-CH=C-), 41.3 (-CH2Cl), 33.0 (ArCH(CH3)2),
23.8 (ArCH(CH3)2).
1
1613, 1576, 1558, 1492, 1482, 1457. H NMR (300 MHz,
DMSO-d6): δ 10.18 (s, 1H, -NCHN-), 8.11–8.04 (m, 2H,
ArH), 7.74–7.67 (m, 3H, ArH), 7.62–7.54 (m, 3H, ArH),
7.47–7.39 (m, 4H, ArH), 6.26 (s, 2H, -NCH2coumarin),
6.04 (s, 1H, -CH=C-), 5.84 (s, 2H, -NCH2Ph), 2.72 (q,
2H, ArCH2CH3, J = 7.6 Hz), 1.22 (t, 3H, ArCH2CH3,
J = 7.6 Hz). 13C NMR (75 MHz, DMSO-d6): δ 159.5 (-
C=O), 151.3, 148.6, 143.6 (-NCHN-), 140.3, 133.6, 132.5,
131.3, 131.1, 129.0, 128.8, 128.5, 127.1, 127.0, 123.3, 116.7,
116.6, 114.1, 113.9, 112.9 (-CH=C-), 50.2 (-NCH2Ph), 46.6
(-NCH2coumarin), 27.6 (ArCH2CH3), 15.6 (ArCH2CH3).
4-Chloromethyl-6-tert-butyl-2H-chromene-2-one, 1e. This
compound was ready from our previous study.17
4-Chloromethyl-6-benzyl-2H-chromene-2-one, 1f. White 1-Benzyl-3-((6-ethoxy-2H-chromene-4-yl)methyl)benzi-
solid, yield: 1.9 g (37%), M.p.: 116–117◦C, Elemental midazolium chloride, 2c. Yellow solid, yield: 0.33 g (31%),
analysis; Calculated for C17H13ClO2; C, 71.71; H, 4.60; M.p.: 245–247◦C. Elemental analysis; Calculated for C26H23
Found: C, 71.56; H, 4.53. IR (cm−1): 3091, 2945, 1717 ClN2O3; C, 69.87; H, 5.19; N, 6.27; Found: C, 69.66; H,
(-C=O), 1626, 1610, 1572, 1493, 1454. 1H NMR (300 5.11; N, 6.12. IR (cm−1): 2882, 2774, 1729 (-C=O),1613,
MHz, DMSO-d6): δ 7.79 (m, 1H, ArH), 7.49 (m, 1H, 1577, 1561, 1497, 1483, 1454. 1H NMR (300 MHz, DMSO-
ArH), 7.36 (m, 1H, ArH), 7.33-7.16 (m, 5H, ArH), 6.68 d6): δ 10.17 (s, 1H, -NCHN-), 8.11–8.03 (m, 2H, ArH),
(s, 1H, -CH=C-), 5.02 (s, 2H, -CH2Cl), 4.04 (s, 2H, 7.74–7.65 (m, 2H, ArH), 7.61–7.54 (m, 2H, ArH), 7.49–
ArCH2Ar). 13C NMR (75 MHz, DMSO-d6): δ 159.7 (- 7.38 (m, 4H, ArH), 7.36–7.29 (m, 2H, ArH), 6.23 (s, 2H,
C=O), 151.8, 150.5, 140.8, 137.6, 132.9, 128.6, 128.5, 126.1, -NCH2coumarin), 6.08 (s, 1H, -CH=C-), 5.83 (s, 2H, -
124.9, 116.9, 116.8, 115.4 (-CH=C-), 41.2 (-CH2Cl), 40.3 NCH2Ph), 4.10 (q, 2H, ArOCH2CH3, J = 6.9 Hz), 1.34 (t,
(ArCH2Ar).
3H, ArOCH2CH3, J = 6.9 Hz). 13C NMR (75 MHz, DMSO-
d6): δ 159.5 (-C=O), 154.9, 148.3, 147.2, 143.6 (-NCHN-),
2.2b General procedure for the synthesis of benzimidazolium 133.7, 131.4, 131.1, 129.0, 128.8, 128.5, 127.1, 127.0, 120.1,
salts and characterization data (2a–f, 3a–e, 4a–e). The mix- 117.9, 117.3, 114.2, 113.9, 113.5 (-CH=C-), 108.3, 63.9
ture of 2.4 mmol of 1-benzylbenzimidazole derivative and (ArOCH2CH3), 50.2 (-NCH2Ph), 46.6 (-NCH2coumarin),
2.4 mmol of 4-chloromethylne coumarin derivative in 10 14.5 (ArOCH2CH3).
mL of DMF was stirred at 80◦C for 24 h. Later, DMF was
removed under reduced pressure. The crude product was
1-Benzyl-3-((6-isopropyl-2H-chromene-4-yl)methyl)ben-
zimidazolium chloride, 2d. White solid, yield: 0.55 g
dissolved in 20 mL of hot ethanol and then ethanol was evap-
(52%), M.p.: 219–221◦C. Elemental analysis; Calculated for
orated under reduced pressure to half of initial volume. Twice
C27H25ClN2O2; C, 72.88; H, 5.66; N, 6.30; Found: C, 72.71;
of the last volume of diethyl ether was added to mixture.
Obtained crystals were collected, washed three times with
H, 5.60; N, 6.17. IR (cm−1): 3033, 2966, 1720 (-C=O),
1610, 1562, 1491, 1457. 1H NMR (300 MHz, DMSO-
diethyl ether (3 × 10 mL) and dried under reduced pres-
d6): δ 10.13 (s, 1H, -NCHN-), 8.14–8.04 (m, 2H, ArH),
sure.

69 Page 4 of 12
J. Chem. Sci.
(2019) 131:69
7.75–7.52 (m, 6H, ArH), 7.48–7.38 (m, 4H, ArH), 6.28 (-CH=C-), 46.7 (-NCH2Ph(CH3)5), 46.6 (-NCH2coumarin),
(s, 2H, -NCH2coumarin), 6.05 (s, 1H, -CH=C-), 5.83 (s, 20.4 (ArCH3-coumarin), 17.0 (ArCH3), 16.7(ArCH3), 16.4
2H, -NCH2Ph), 3.02 (sep, 1H, ArCH(CH3)2, J = 6.9 Hz), (ArCH3).
1.23 (d, 2H, ArCH(CH3)2, J = 6.9 Hz). 13C NMR (75
1-(2,3,4,5,6-Pentamethylbenzyl)-3-((6-ethyl-2H-chrome-
ne-4-yl)methyl)benzimidazolium chloride, 3b. Yellow
MHz, DMSO-d6): δ 159.5 (-C=O), 151.3, 148.7, 144.9,
143.6 (-NCHN-), 133.6, 131.5, 131.11, 131.06, 129.0, 128.8,
solid, yield: 0.55 g (46%), M.p.: 249–250◦C. Elemental anal-
128.5, 127.1, 127.0, 121.8, 116.8, 116.6, 114.1, 114.0, 112.9
ysis; Calculated for C31H33ClN2O2; C, 74.31; H, 6.64; N,
(-CH=C-), 50.2 (-NCH2Ph), 46.6 (-NCH2coumarin), 33.0
5.59; Found: C, 74.47; H, 6.71; N, 5.66. IR (cm−1): 2971,
(ArCH(CH3)2), 23.7 (ArCH(CH3)2).
1714 (-C=O), 1571, 1430. 1H NMR (300 MHz, DMSO-d6):
1-Benzyl-3-((6-tert-butyl-2H-chromene-4-yl)methyl)ben-
δ 9.26 (s, 1H, -NCHN-), 8.33 (d, 1H, ArH, J = 8.1 Hz),
zimidazolium chloride, 2e. White solid, yield: 0.87 g 8.05 (d, 1H, ArH, J = 8.1 Hz), 7.86–7.66 (m, 3H, ArH),
(79%), M.p.: 220–222◦C. Elemental analysis; Calculated 7.60–7.54 (m, 1H, ArH), 7.43–7.38 (m, 1H, ArH), 6.14 (s,
for C28H27ClN2O2; C, 73.27; H, 5.93; N, 6.10; Found: C, 2H, -NCH2coumarin), 5.87 (s, 1H, -CH=C-), 5.77 (s, 2H,
73.11; H, 5.80; N, 6.02. IR (cm−1): 3032, 2965, 1721 (- -NCH2Ph(CH3)5), 2.71 (q, 2H, ArCH2CH3, J = 7.6 Hz),
1
C=O), 1612, 1563, 1490, 1458, 1449. H NMR (300 MHz, 2.24 (s, 3H, ArCH3 − p), 2.23 (s, 6H, ArCH3 − o), 2.22 (s,
DMSO-d6): δ 10.10 (s, 1H, -NCHN-), 8.17–8.03 (m, 2H, 6H, ArCH3 − m), 1.22 (t, 3H, ArCH2CH3,J = 7.6 Hz).
ArH), 7.82–7.67 (m, 4H, ArH), 7.58–7.37 (m, 6H, ArH), 13C NMR (75 MHz, DMSO-d6): δ 159.5 (-C=O), 151.1,
6.32 (s, 2H, -NCH2coumarin), 6.07 (s, 1H, -CH=C-), 5.83 149.2, 142.2 (-NCHN-), 140.3, 136.3, 133.8, 133.0, 132.4,
(s, 2H, -NCH2Ph), 1.30 (s, 9H, ArC(CH3)3). 13C NMR 131.8, 131.7, 127.3, 127.0, 125.5, 123.1, 116.7, 116.6, 114.2,
(75 MHz, DMSO-d6): δ 159.5 (-C=O), 151.1, 148.8, 147.1, 113.9, 111.8 (-CH=C-), 46.7 (-NCH2Ph(CH3)5), 46.6 (-
143.6 (-NCHN-), 133.7, 131.5, 131.1, 130.2, 129.0, 128.8, NCH2coumarin), 27.6 (ArCH2CH3), 16.9 (ArCH3), 16.7
128.4, 127.1, 127.0, 120.5, 116.5, 116.1, 114.2, 114.0, 113.0 (ArCH3), 16.4 (ArCH3), 15.6 (ArCH2CH3).
(-CH=C-), 50.2 (-NCH2Ph), 46.7 (-NCH2coumarin), 34.5
1-(2,3,4,5,6-Pentamethylbenzyl)-3-((6-ethoxy-2H-chrom-
(ArC(CH3)3), 30.9 (ArC(CH3)3).
ene-4-yl)methyl)benzimidazolium chloride, 3c. Yellow
1-Benzyl-3-((6-benzyl-2H-chromene-4-yl)methyl)benzi-
solid, yield: 0.52 g (42%), M.p.: 175–177◦C. Elemental anal-
midazolium chloride, 2f. White solid, yield: 0.53 g (45%), ysis; Calculated for; C31H33ClN2O3; C, 72.01, H, 6.43; N,
M.p.: 156–158◦C. Elemental analysis; Calculated for C31H25 5.42; Found: C, 72.24; H, 6.53; N, 5.44. IR (cm−1): 2967,
ClN2O2; C, 75.53; H, 5.11; N, 5.68; Found: C, 75.34; H, 1724 (-C=O), 1611, 15771, 1448. 1H NMR (300 MHz,
5.03; N, 5.56. IR (cm−1): 2925, 1725 (-C=O), 1666, 1613, DMSO-d6): δ 9.33 (s, 1H, -NCHN-), 8.34 (d, 1H, ArH,
1571, 1492, 1455. 1H NMR (300 MHz, DMSO-d6): δ 10.13
J = 8.1 Hz), 8.05 (d, 1H, ArH, J = 8.1 Hz), 7.85–7.70 (m,
(s, 1H, -NCHN-), 8.12–8.03 (m, 2H, ArH), 7.82 (m, 1H, 2H, ArH), 7.46–7.40 (m, 1H, ArH), 7.35–7.29 (m, 2H, ArH),
ArH), 7.75–7.65 (m, 2H, ArH), 7.63–7.53 (m, 3H, ArH), 6.18 (s, 2H, -NCH2oumarin), 5.94 (s, 1H, -CH=C-), 5.79
7.48–7.38 (m, 4H, ArH), 7.32–7.15 (m, 5H, ArH), 6.21 (s, (s, 2H, -NCH2Ph(CH3)5), 4.12 (q, 2H, ArOCH2CH3, J =
2H, -NCH2coumarin), 6.06 (s, 1H, -CH=C-), 5.83 (s, 2H, 6.9 Hz), 2.25 (s, 3H, ArCH3 − p), 2.24 (s, 6H, ArCH3 − o),
-NCH2Ph), 4.07 (s, 2H, ArCH2Ar). 13C NMR (75 MHz, 2.22 (s, 6H, ArCH3 − m), 1.36 (t, 3H, ArOCH2CH3, J =
DMSO-d6): δ 159.4 (-C=O), 151.5, 148.5, 143.6 (-NCHN- 6.9 Hz). 13C NMR (75 MHz, DMSO-d6): δ 159.5 (-C=O),
), 140.7, 137.9, 133.6, 133.3, 131.5, 131.1, 129.0, 128.8, 154.9, 149.0, 147.1, 142.2 (-NCHN-), 136.3, 133.9, 133.0,
128.6, 128.51, 128.49, 127.1, 127.0, 126.2, 124.2, 117.0, 131.9, 131.7, 127.2, 126.9, 125.6, 120.1, 117.9, 117.3, 114.2,
116.7, 114.1, 113.9, 113.1 (-CH=C-), 50.2 (-NCH2Ph), 46.6 113.9, 112.4 (-CH=C-), 108.2, 64.0 (ArOCH2CH3), 46.8
(-NCH2coumarin), 40.3 (ArCH2Ar).
(-NCH2Ph(CH3)5), 46.6 (-NCH2coumarin), 17.0 (ArCH3),
16.7 (ArCH3), 16.4 (ArCH3), 14.5 (ArOCH2CH3).
1-(2,3,4,5,6-Pentamethylbenzyl)-3-((6-methyl-2H-chrom-
ene-4-yl)methyl)benzimidazolium chloride, 3a. White 1-(2,3,4,5,6-Pentamethylbenzyl)-3-((6-isopropyl-2H-chr-
solid, yield: 0.71 g (61%), M.p.: 217–219◦C. Elemental anal- omene-4-yl)methyl)benzimidazolium chloride, 3d. White
ysis; Calculated for C30H31ClN2O2; C, 73.98; H, 6.42; N, solid, yield: 0.63 g (51%), M.p.: 237–241◦C. Elemental
5.75; Found: C, 74.18; H, 6.54; N, 5.67. IR (cm−1): 2902, analysis; Calculated for; C32H35ClN2O2; C, 74.62; H, 6.85;
1723 (-C=O), 1611, 1561, 1446. 1H NMR (300 MHz, DMSO- N, 5.44; Found: C, 74.93; H, 7.01; N, 5.31. IR (cm−1):
d6): δ 9.33 (s, 1H, -NCHN-), 8.37 (d, 1H, ArH, J = 8.1 Hz), 2957, 1717 (-C=O), 1610, 1564, 1471, 1434. 1H NMR (300
8.05 (d, 1H, ArH, J = 8.1 Hz), 7.85–7.67 (m, 3H, ArH), MHz, DMSO-d6): δ 9.29 (s, 1H, -NCHN-), 8.34 (d, 1H,
7.57–7.51 (m, 1H, ArH), 7.41–7.36 (m, 1H, ArH), 6.15 (s, ArH, J = 8.1 Hz), 8.09 (d, 1H, ArH, J = 7.9 Hz), 7.87–
2H, -NCH2coumarin), 5.91 (s, 1H, -CH=C-), 5.79 (s, 2H, 7.57 (m, 4H, ArH), 7.45–7.38 (m, 1H, ArH), 6.20 (s, 2H,
-NCH2Ph(CH3)5), 2.41 (s, 3H, ArCH3-coumarin), 2.24 (s, -NCH2coumarin), 5.86 (s, 1H, -CH=C-), 5.79 (s, 2H, -
9H, ArCH3 − o and p), 2.22 (s, 6H, ArCH3-m). 13C NMR NCH2Ph(CH3)5), 3.02 (sep, 1H, ArCH(CH3)2, J = 6.6 Hz),
(75 MHz, DMSO-d6): δ 159.5 (-C=O), 151.0, 149.1, 142.2 2.24 (m, 15H, ArCH3), 1.25 (d, 6H, ArCH(CH3)2, J = 6.6
(-NCHN-), 136.9, 133.94, 133.87, 133.5, 133.0, 131.8, 131.7, Hz). 13C NMR (75 MHz, DMSO-d6): δ 159.5 (-C=O), 151.2,
127.2, 126.9, 125.6, 124.2, 116.6, 116.5, 114.2, 113.9, 111.9 149.5, 144.8, 142.2 (-NCHN-), 136.3, 133.8, 132.9, 131.84,

J. Chem. Sci.
(2019) 131:69
Page 5 of 12
69
131.79, 131.0, 127.2, 126.9, 125.6, 121.7, 116.7, 116.6, 114.2, d6): δ 159.5 (-C=O), 153.2, 151.3, 148.8, 143.5 (-NCHN-),
113.9, 111.5 (-CH=C-), 46.8 (-NCH2Ph(CH3)5), 46.6 (- 140.3, 137.7, 132.5, 131.4, 131.2, 128.8, 127.0, 126.9, 123.3,
NCH2coumarin), 33.1 (ArCH(CH3)2), 23.8 (ArCH(CH3)2), 116.7, 116.6, 114.3, 113.8, 112.6 (-CH=C-), 106.6, 59.9
17.0 (ArCH3), 16.7 (ArCH3), 16.4 (ArCH3).
(ArOCH3), 56.1 (ArOCH3), 50.5 (-NCH2Ph(OCH3)3), 46.5
(-NCH2coumarin), 27.6 (ArCH2CH3), 15.6 (ArCH2CH3).
1-(2,3,4,5,6-Pentamethylbenzyl)-3-((6-tert-butyl-2H-chr-
omene-4-yl)methyl)benzimidazolium chloride, 3e. White 1-(3,4,5-Trimethoxybenzyl)-3-((6-ethoxy-2H-chromene-
◦
solid, yield: 0.95 g (75%), M.p.: 261–262 C. Elemental 4-yl)methyl)benzimidazolium chloride, 4c. Yellow solid,
analysis; Calculated for C33H37ClN2O2; C, 74.91; H, 7.05; yield: 0.67 g (52%), M.p.: 130–131◦C. Elemental analysis;
N, 5.29; Found: C, 75.06; H, 7.13; N, 5.14. IR (cm−1): Calculated for C29H29ClN2O6; C, 64.86; H, 5.44; N, 5.22;
2963, 1720 (-C=O), 1611, 1562, 1475, 1429. 1H NMR Found: 64.62; H, 5.30; N, 5.08. IR (cm−1): 2966, 1718 (-
1
(300 MHz, DMSO-d6): δ 9.27 (s, 1H, -NCHN-), 8.34 C=O), 1595, 1574, 1508, 1468, 1444. H NMR (300 MHz,
(d, 1H, ArH, J = 8.1 Hz), 8.09 (d, 1H, ArH, J= 8.0 DMSO-d6): δ 10.16 (s, 1H, -NCHN-), 8.25–8.19 (m, 1H,
Hz), 7.85–7.70 (m, 4H, ArH), 7.45–7.40 (m, 1H, ArH), ArH), 8.12–8.05 (m, 1H, ArH), 7.79–7.66 (m, 2H, ArH),
6.25 (s, 2H, -NCH2 coumarin), 5.85 (s, 1H, -CH=C-), 7.49–7.42 (m, 1H, ArH), 7.38–7.29 (m, 2H, ArH), 7.00
5.79 (s, 2H, -NCH2Ph(CH3)5), 2.24 (s, 3H, ArCH3 − p), (s, 2H, ArH), 6.26 (s, 2H, -NCH2 coumarin), 6.03 (s, 1H,
2.23 (s, 6H, ArCH3 − o), 2.22 (s, 6H, ArCH3 − m), -CH=C-), 5.70 (s, 2H, -NCH2Ph(OCH3)3), 4.11 (q, 2H,
1.33 (s, 9H, ArC(CH3)3). 13C NMR (75 MHz, DMSO- ArOCH2CH3, J = 7.0 Hz), 3.77 (s, 6H, ArOCH3 − m),
d6): δ 159.5 (-C=O), 150.9, 149.6, 147.1, 142.2 (-NCHN-), 3.65 (s, 3H, ArOCH3 − p), 1.34 (t, 3H, ArOCH2CH3,
136.3, 133.8, 132.9, 131.82, 131.79, 130.2, 127.2, 126.9,
J = 7.0 Hz). 13C NMR (75 MHz, DMSO-d6): δ 159.4 (-
125.6, 120.4, 116.4, 116.2, 114.2, 114.0, 111.5 (-CH=C- C=O), 154.9, 153.2, 148.5, 147.2, 143.4 (-NCHN-), 137.7,
), 46.8 (-NCH2Ph(CH3)5), 46.6 (-NCH2coumarin), 34.5 131.4, 131.2, 128.8, 127.1, 126.9, 120.1, 117.9, 117.3, 114.3,
(ArC(CH3)3), 31.0 (ArC(CH3)3), 17.0 (ArCH3), 16.7 113.8, 113.3 (-CH=C-), 108.4, 106.6, 63.9 (ArOCH2CH3),
(ArCH3), 16.4 (ArCH3).
59.9 (ArOCH3), 56.0 (ArOCH3), 50.5 (-NCH2Ph(OCH3)3),
46.5 (-NCH2coumarin), 14.5 (ArOCH2CH3).
1-(3,4,5-Trimethoxybenzyl)-3-((6-methyl-2H-chromene-
4-yl)methyl)benzimidazolium chloride, 4a. White solid, 1-(3,4,5-Trimethoxybenzyl)-3-((6-isopropyl-2H-chrome-
yield: 0.81 g (67%), M.p.: 176–177◦C. Elemental analy- ne-4-yl)methyl)benzimidazolium chloride, 4d. Yellow
sis; Calculated for C28H27ClN2O5; C, 66.34; H, 5.37; N, solid, yield: 0.91 g (71%), M.p.: 191–193◦C. Elemental
5.53; Found: C, 66.23; H, 5.26; N, 5.40. IR (cm−1): 2964, analysis, Calculated for; C30H31ClN2O5; C, 67.35; H, 5.84;
1724 (-C=O), 1603, 1593, 1574, 1560, 1508, 1449. 1H NMR N, 5.24; Found: C, 67.18; H, 5.77; N, 5.09. . IR (cm−1):
(300 MHz, DMSO-d6): δ 10.26 (s, 1H, -NCHN-), 8.26– 2968, 1721 (-C=O), 1629, 1593, 1575, 1560, 1508, 1481,
1
8.20 (m, 1H, ArH), 8.10–8.04 (m, 1H, ArH), 7.79–7.65 1444. H NMR (300 MHz, DMSO-d6): δ 10.19 (s, 1H,
(m, 3H, ArH), 7.60–7.52 (m, 1H, ArH), 7.45–7.37 (m, 1H, -NCHN-), 8.26–8.20 (m, 1H, ArH), 8.15–8.10 (m, 1H,
ArH), 7.03 (s, 2H, ArH), 6.26 (s, 2H, -NCH2coumarin), ArH), 7.78–7.67 (m, 3H, ArH), 7.66–7.60 (m, 1H, ArH),
5.98 (s, 1H, -CH=C-), 5.72 (s, 2H, -NCH2Ph(OCH3)3), 7.47–7.41 (m, 1H, ArH), 7.02 (s, 2H, ArH), 6.30 (s, 2H,
3.78 (s, 6H, ArOCH3 − m), 3.65 (s, 3H, ArOCH3 − p), -NCH2coumarin), 5.99 (s, 1H, -CH=C-), 5.71 (s, 2H, -
2.43 (s, 3H, ArCH3). 13C NMR (75 MHz, DMSO-d6): NCH2Ph(OCH3)3), 3.76 (s, 6H, ArOCH3-m), 3.64 (s, 3H,
δ 159.4 (-C=O), 153.2, 151.1, 148.7, 143.5 (-NCHN-), ArOCH3 − p), 3.02 (sep, 1H, ArCH(CH3)2, J = 6.9 Hz),
137.7, 134.0, 133.5, 131.4, 131.2, 128.8, 127.0, 126.9, 1.22 (d, 6H, ArCH(CH3)2, J = 6.9 Hz). 13C NMR (75 MHz,
124.4, 116.6, 114.3, 113.8, 112.6 (-CH=C-), 106.6, 59.9 DMSO-d6):δ 159.5(-C=O), 153.2, 151.3, 148.9, 144.9, 143.4
(ArOCH3), 56.1 (ArOCH3), 50.5 (-NCH2Ph(OCH3)3), 46.5 (-NCHN-), 137.7, 131.4, 131.2, 131.1, 128.8, 127.1, 126.9,
(-NCH2coumarin), 20.4 (ArCH3).
121.8, 116.7, 116.6, 114.3, 113.9, 112.6 (-CH=C-), 106.6,
59.9 (ArOCH3), 56.0 (ArOCH3), 50.5 (-NCH2Ph(OCH3)3),
46.6 (-NCH2coumarin), 33.0 (ArCH(CH3)2), 23.7 (ArCH
(CH3)2).
1-(3,4,5-Trimethoxybenzyl)-3-((6-ethyl-2H-chromene-4-
yl)methyl)benzimidazolium chloride, 4b. Yellow solid,
yield: 0.74 g (59%), M.p.: 212–213◦C. Elemental analy-
sis; Calculated for C29H29ClN2O5; C, 66.85; H, 5.61; N, 1-(3,4,5-Trimethoxybenzyl)-3-((6-tert-butyl-2H-chrome-
5.28; Found: C, 66.70; H, 5.52; N, 5.31. IR (cm−1): 2968, ne-4-yl)methyl)benzimidazolium chloride, 4e. Yellow
1724 (-C=O), 1630, 1593, 1575, 1558, 1507, 1479, 1445. solid, yield: 1.10 g (84%), M.p.: 230–231 ◦C. Elemen-
1H NMR (300 MHz, DMSO-d6): δ 10.23 (s, 1H, -NCHN-), tal analysis; Calculated for C31H33ClN2O5; C, 67.81; H,
8.27–8.20 (m, 1H, ArH), 8.13–8.06 (m, 1H, ArH), 7.78-7.66 6.06; N, 5.10; Found: C, 67.64; H, 6.01; N, 5.02. IR
(m, 3H, ArH), 7.63–7.55 (m, 1H, ArH), 7.47–7.40 (m, 1H, (cm−1): 2966, 1724 (-C=O), 1603, 1593, 1560, 1574, 1508,
1
ArH), 7.03 (s, 2H, ArH), 6.28 (s, 2H, -NCH2coumarin), 1449. H NMR (300 MHz, DMSO-d6): δ 10.15 (s, 1H,
5.98 (s, 1H, -CH=C-), 5.71 (s, 2H, -NCH2Ph(OCH3)3), -NCHN-), 8.26–8.18 (m, 1H, ArH), 8.18-8.10 (m, 1H,
3.77 (s, 6H, ArOCH3 − m), 3.65 (s, 3H, ArOCH3
−
ArH), 7.81–7.68 (m, 4H, ArH), 7.48–7.41 (m, 1H, ArH),
p), 2.72 (q, 2H, ArCH2CH3, J = 7.5 Hz), 1.22 (t, 3H, 6.99 (s, 2H, ArH), 6.34 (s, 2H, -NCH2 coumarin), 6.03
ArCH2CH3, J = 7.5 Hz). 13C NMR (75 MHz, DMSO- (s, 1H, -CH=C-), 5.70 (s, 2H, -NCH2Ph(OCH3)3), 3.75

69 Page 6 of 12
J. Chem. Sci.
(2019) 131:69
(s, 6H, ArOCH3 − m), 3.64 (s, 3H, ArOCH3 − p), 1.28
All P values < 0.05 were considered statistically signifi-
(s, 9H, ArC(CH3)3). 13C NMR (75 MHz, DMSO-d6): δ cant. All analyses were done by IBM SPSS Statistics 22.0 for
159.5 (-C=O), 153.2, 151.1, 148.9, 147.1, 143.4 (-NCHN- Windows. The LogIC50 values were determined by using %
), 137.7, 131.4, 131.1, 130.2, 128.8, 127.1, 127.0, 120.6, cell viability values of compounds by the GraphPad Prism 6
116.5, 116.2, 114.3, 113.9, 112.9 (-CH=C-), 106.5, 59.9 program.
(ArOCH3), 56.0 (ArOCH3), 50.5 (-NCH2Ph(OCH3)3), 46.6
(-NCH2coumarin), 34.5 (ArC(CH3)3), 30.9 (ArC(CH3)3).
3. Results and Discussion
2.3 Cytotoxicity studies
3.1 Synthesis and characterization
Cell cultures. A2780 and PC-3 cell lines were preserved in
RPMI-1640 culture medium supplemented with L-glutamine
(10% heat-inactivated fetal bovine serum, 100 μ/mL pen-
icillin-streptomycin), withtheadditionof10mMnonessential
Firstly, 6-substituted-4-chloromethylene coumarin
derivatives (1a–f) were synthesized by the previously
reported procedure.36 The structures of 1a–f are given
amino acids for the culture of prostate cancer cells. The cell
lines were kept at 37◦C in a 5% CO2 humidified incubator
(Panasonic, Japan).
in Scheme 1. Compound 1e was available from our
previous study.17 Other coumarin derivatives were
MTT assay. The synthesized compounds were screened for characterized by H and 13C NMR, IR spectroscopic
1
techniques and elemental analyses. In the 1H NMR spec-
their antitumor activities against different type cancer cell
lines (PC-3 and A2780) by MTT assay. The pale-yellow
tra of compounds, olefinic hydrogens were observed
tetrazolium salt, MTT, was transformed by active mitochon-
in the range of 6.66–6.68 ppm. Methylene protons
dria to form a dark blue formazan that was determined by a
were observed as singlets in the range of 5.02–5.07
microplate reader.37
ppm. In the 13C NMR spectra of compounds, signals
The MTT method provides a simple way to detect liv-
of olefinic carbons (-CH=C-) were observed in the
ing and growing cells without using radioactivity. For all
compounds, firstly, 100 μM and 50 mL solutions were pre-
range of 115.3–115.7 ppm. Signals of carbonyl car-
bons were observed at 159.7 ppm for all compounds.
pared by the dissolving 0.005 mmol of the compound in
1
13
Other signals in the H and C NMR spectra of com-
pounds were observed in agreement with the expected
integrities and coupling patterns (See Supplementary
100 μL of DMSO and (less than 1% DMSO) and diluting
the final volume to 50 mL with RPMI-1640. The other con-
centrations of compounds were prepared by the diluting of
this solution. Shortly, the prostate and ovarian cancer cells Information for spectra). In the IR spectra of com-
were seeded into 96-well plates at 15 × 103 cells/well in a
pounds, sharp peaks of carbonyl group were observed
in the range of 1717–1729 cm−1. Elemental analyses
final volume of 100 μL and treated with different concen-
trations of compounds (1, 10 and 100◦μM) in RPMI-1640.
results are also supportive for structures and purities of
compounds.
Then, the cells were incubated at 37 C for 24 h in a 5%
CO2 humidified incubator. After 24 h, MTT (0.005 g/mL
After the synthesis of 1a–f, benzimidazolium salts
in phosphate buffer saline) was added to the cell culture
were synthesized by the reaction of 1a–f and N-
and incubated for 3 h. The formazan crystals formed during
benzylbenzimidazole derivatives at 80◦C during 24
the reaction of active mitochondria with MTT were dis-
hours. Synthetic route and structures of compounds are
outlined in Scheme 1. Sixteen benzimidazolium salts
solved in 0.04 N (100 mL) isopropanol and readings were
recorded on a microplate reader (BioTek, Synergy HTX,
USA) using a 570 nm filter.38 By having the control wells
read, the average of the obtained absorbance values was
(2a–f, 3a–e, 4a–e) were synthesized and characterized
1
by H and 13C NMR (see Supplementary Information
taken, and this value was accepted as 100% live cell. The for spectra), IR spectroscopic techniques and elemental
absorbance values obtained from the solvent (the group into
which only DMSO was added), as well as agent-applied
wells, were proportioned to the control absorbance values,
in addition to which the percentage (%) viability values were
calculated. Each value represented an average of 10 measure-
ments. All cellular results were determined against control
cells.39,40
analyses. All salts are stable against oxygen and mois-
ture of air, and daylight. Unfortunately, the products
of the reaction of 1f with 3 and 4 could not be puri-
fied and therefore, these compounds were not included
1
in cytotoxicity assay. In the H NMR spectra of ben-
zimidazolium salts, the resonances of acidic -NCHN-
hydrogens were observed in the range of 10.10–10.18
ppm for 2a–f, 9.26–9.33 ppm for 3a–e, and 10.15–10.26
ppm for 4a–e. The appearance of these signals clearly
proves the formation of benzimidazolium chlorides.
Statistical analyses. Quantitative data were presented as
mean
standard deviation (SD). Normal distribution was
confirmedbytheKolmogorov-Smirnovtest. Quantitativedata
were analysed using the Kruskal-Wallis H test following the
Mann-Whitney U test with Bonferroni adjustment as a post-
hoc test.
1
In the H NMR spectra of pentamethylbenzyl substi-
tuted benzimidazolium chlorides (3a–e), acidic signals

J. Chem. Sci.
(2019) 131:69
Page 7 of 12
69
O
O
R2
OH
O
O
R1
70% H2SO4
rt, 24 h
N
N
O
O
R2
+
Cl
R1
R1
O
N
N
DMF, 80 oC, 24 h
Cl
Cl
1a-f
2a-f, 3a-e, 4a-e
O
O
O
O
O
O
O
O
O
R1
R1
R1
N
N
N
N
N
N
Cl
Cl
Cl
2a-f
3a-e
4a-e
1
1
1
1
1
1
a
b
c
d
e
; f
, R = -CH3 ; , R = -CH2CH3 ; , R = -OCH2CH3 ; , R = -CH(CH3)2 ; , R = -C(CH3)3 , R = -CH2C6H5
H
N
R3
N
N
Cl
R3
KOH, EtOH
+
N
5,6
N
N
H
N
Cl-
R3
Cl
N
KOH, EtOH
+
I
CH3
R3
DMF, 80 oC, 24 h
N
N
5',6'
3
5
5'
for and , R = H ;
3
6
6'
for and , R = 2,3,4,5,6-(CH3)5
Scheme 1. Synthesis and structures of compounds.
were observed at high field compared to other benz- 3.2 Cytotoxicity studies
imidazolium chlorides due to strong electron donating
In our previous studies, we had reported the human
carbonic anhydrase I and II16 and human paraoxonase
I17 inhibitory properties, and antimicrobial activities41
of some coumarin substituted benzimidazolium chlo-
rides. We had shown that these compounds have enzyme
inhibitory properties but antimicrobial properties are
dependent on their lipophilicity. In this study, we
decided to synthesize a series of 6-substituted coumarin
derivatives and their benzimidazolium salts in order to
investigate their anticancer properties. The cytotoxic
properties of methyl groups. The resonances of imino
carbons (-NCHN-) were observed at 143.6 ppm for 2a–
f, 142.2 ppm for 3a–e and 143.4 and 143.5 ppm for 4a–e.
In the 1H NMR spectra, the signals of olefinic hydrogens
were observed at the high field in the range of 5.85–
6.08 ppm, compared with 1a–f. Conversely, the signals
of methylene hydrogens shifted to downfield compared
with 1a–f. Other NMR signals, IR spectra and elemental
analyses results are also in agreement with the expected
structures.

69 Page 8 of 12
J. Chem. Sci.
(2019) 131:69
Table 1. Dose dependent cell viability results of PC-3 cells after 24 h treatment of coumarin substituted benzimidazolium
salts. Each data point is an average of 10 viability measurements.
Compound
General Structure
R1
1 µM
10 µM
65.91 7.23*
60.93 10.68* 53.99 11.91*
100 µM
42.64 10.22*
1a
1b
1c
1d
1e
1f
-CH3
95.26 8.47
90.77 8.22
101.87 12.08
60.79 6.89*
53.51 6.21*
63.25 7.16*
64.62 8.01*
63.28 8.27*
96.21 12.68
54.60 5.23*
60.42 7.29*
94.49 10.23
95.63 5.36
108.47 15.34
100.99 11.02
103.26 15.82
108.05 13.95
91.56 11.03
67.94 7.81*
60.13 9.28*
66.36 5.96*
-CH2CH3
-OCH2CH3
-CH(CH3)2
-C(CH3)3
-CH2C6H5
-CH3
92.37 10.93
35.62 5.62*
30.75 5.56*
32.49 10.93*
42.72 9.95*
50.22 8.76*
55.16 7.81*
52.61 5.26*
50.72 10.14*
102.56 18.45
105.09 12.74
72.11 8.81*
84.89 10.99
65.26 10.87*
61.65 5.17*
63.93 9.21*
45.72 5.26*
42.18 6.89*
53.29 7.09*
66.87 8.29*
20.86 4.11*
30.15 6.90*
25.70 5.85*
18.26 5.23*
32.97 4.41*
30.91 5.76*
32.54 7.29*
18.50 3.51*
36.64 5.52*
46.06 5.29*
64.17 8.91*
59.08 8.23*
62.67 9.35*
46.67 5.33*
23.67 2.84*
31.99 4.81*
30.49 5.23*
41.27 7.37*
O
O
R1
Cl
2a
2b
2c
2d
2e
2f
O
O
-CH2CH3
-OCH2CH3
-CH(CH3)2
-C(CH3)3
-CH2C6H5
-CH3
R1
N
N
Cl
3a
3b
3c
3d
3e
4a
4b
4c
4d
O
O
-CH2CH3
-OCH2CH3
-CH(CH3)2
-C(CH3)3
-CH3
R1
N
N
Cl
O
O
O
O
-CH2CH3
-OCH2CH3
-CH(CH3)2
-C(CH3)3
O
R1
N
N
Cl
4e
44.53 5.36*
44.44 6.98*
34.17 8.03*
101.96 14.83
103.31 11.67
101.82 15.99
101.65 10.02
31.45 7.89*
65.39 10.21*
106.52 14.77
99.23 16.61
100.49 13.94
20.25 3.92*
15.51 3.42*
61.61 9.99*
30.89 6.69*
84.82 13.47
2.12 0.68*
N-benzylbenzimidazole (5)
N-(2,3,4,5,6-pentamethyl)benzylbenzimidazole (6)
N-benzyl-N-methylbenzimidazolium chloride (5’)
N-(2,3,4,5,6-pentamethyl)benzyl-N-methylbenzimidazolium chloride (6’)
Docetaxel
Control value is 96.73 9.13, *p < 0.05.
effects of all compounds were tested against human difficult to make precise generalizations, but we must
prostate (PC-3) and ovarian (A2780) cancer cell lines point out that all compounds performed significant
by MTT assay at three different concentration (1, 10 cytotoxicity against both cancer cells at 100 μM. The
and 100 μM). The % cell viabilities after 24 h treat- compounds 1d, 1e, 1f, 2a, 2b, 2d, 2e, 4b, 4c, 4d
ment of compound solutions are presented in Tables 1 and 4e performed significant cytotoxicity against PC-
and 2. Docetaxel was used as a standard drug for 3 cells at 1 μM and 4e was found out as the most
comparison. All compounds reported in this study per- active among all compounds. Pentamethylbenzyl sub-
formed lower cytotoxicity than Docetaxel which has stituted salts, 3 performed lower activities than other
some adverse effects in clinical use.42 In fact, it is compounds against PC-3 cell lines. The compounds,

J. Chem. Sci.
(2019) 131:69
Page 9 of 12
69
Table 2. Dose dependent cell viability results of A2780 cells after 24 h treatment of coumarin substituted benzimidazolium
salts. Each data point is an average of 10 viability measurements.
Compound
General Structure
R1
1 µM
10 µM
100 µM
1a
1b
1c
1d
1e
1f
-CH3
106.41 9.76
98.87 16.31
95.10 9.41
82.49 18.21
95.21 9.13
72.57 4.26*
91.60 8.22
60.26 6.06*
92.33 7.06
66.28 6.35*
90.14 12.69
92.26 10.33
101.39 10.13
102.04 15.25
106.63 8.14
82.41 7.77
103.05 14.25
108.13 14.44
86.83 8.99
50.73 8.36*
101.20 18.14
85.70 20.81
82.13 8.29
55.41 8.96*
50.47 5.98*
69.88 8.14*
60.17 9.41*
57.72 6.28*
58.26 7.29*
58.07 10.02*
60.14 8.91*
58.16 19.85*
63.64 9.95*
104.83 14.75
21.55 5.06*
60.23 8.29*
34.23 9.06*
31.08 6.21*
30.22 5.21*
25.29 8.28*
31.94 6.11*
48.24 3.14*
45.75 3.94*
35.78 5.67*
36.43 7.96*
43.23 9.09*
54.51 7.26*
-CH2CH3
-OCH2CH3
-CH(CH3)2
-C(CH3)3
-CH2C6H5
-CH3
O
O
R1
Cl
2a
2b
2c
2d
2e
2f
O
O
-CH2CH3
-OCH2CH3
-CH(CH3)2
-C(CH3)3
-CH2C6H5
-CH3
R1
N
N
Cl
3a
3b
3c
3d
3e
4a
4b
4c
4d
O
O
-CH2CH3
-OCH2CH3
-CH(CH3)2
-C(CH3)3
-CH3
79.98 10.26* 60.13 10.21*
R1
75.28 10.22*
39.41 7.13*
55.98 12.81*
58.14 17.63*
90.46 15.62
49.64 8.39*
100.25 19.88
50.67 7.61*
36.89 4.09*
43.04 8.12*
27.26 5.99*
46.48 3.38*
25.32 5.02*
53.53 8.96*
N
N
Cl
O
O
O
O
-CH2CH3
-OCH2CH3
-CH(CH3)2
-C(CH3)3
O
R1
N
N
Cl
4e
95.09 16.92
65.55 13.44*
54.51 9.23*
N-benzylbenzimidazole (5)
95.80 10.13
108.24 8.42
93.73 14.23
99.85 12.42
28.65 6.19
60.15 7.41*
96.79 11.34
92.44 15.21
92.28 10.86
16.79 4.14
23.66 2.34*
66.52 12.43*
43.41 5.34*
61.16 7.12*
0.68 0.04
N-(2,3,4,5,6-pentamethyl)benzylbenzimidazole (6)
N-benzyl-N-methylbenzimidazolium chloride (5’)
N-(2,3,4,5,6-pentamethyl)benzyl-N-methylbenzimidazolium chloride (6’)
Docetaxel
Control value is 91.34 11.24, *p < 0.05.
1f, 2b, 2d, 4c performed significant cytotoxicity against of 5, 5’, 6, and 6’ were also given in Tables 1 and 2.
A2780 cells at 1 μM and 4c was found out as the most When the results compared for PC-3, 5, 5’, 6, and 6’
active among all compounds. The simple benzyl sub- performed comparable activities only at 100 μM. On
stituted salts, 2 performed stronger cytotoxicity than the other hand, compound 5 performed stronger activ-
other compounds against A2780 cell lines. Addition- ity than all coumarin substituted salts at 100 μM against
ally, methyl containing derivatives (5’ and 6’) of 5 and A2780. 5’, 6, and 5’ performed weaker cytotoxicity than
6 were synthesized according to literature43 in order to coumarin substituted salts at all concentrations. After
compare with coumarin substituted salts (Scheme 1). the completion of 24 h tests, we investigated cell via-
The cell viabilities of PC-3 and A2780 after treatment bilities of both cell lines after the treatment of selected

69 Page 10 of 12
J. Chem. Sci.
(2019) 131:69
1 µM
10 µM
100 µM
1 µM
10 µM
100 µM
120
100
35
30
25
20
15
10
5
10
9
8
7
6
5
4
3
2
1
0
1 µM
10 µM
100 µM
*
*
*
*
*
*
*
*
*
80
*
*
*
*
*
*
*
*
60
40
20
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
0
0
1e
2e
4a
4c
4e
1e
2e
4a
4c
4e
1e
2e
4a
4c
4e
1 µM
10 µM
100 µM
70
40
35
30
25
20
15
10
5
25
20
15
10
5
1 µM
10 µM
100 µM
1 µM
10 µM
100 µM
*
*
*
60
50
40
30
20
10
0
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
0
0
1e
2e
4a
4c
4e
1e
2e
4a
4c
4e
1e
2e
4a
4c
4e
Figure 1. Cell viabilities of PC-3 (top) and A2780 (bottom) after 48 h (left), 72 h (middle), and 96 h (right) treatment of
selected compounds (1e, 2e, 4a, 4c, 4e).
compounds 1e, 2e, 4a, 4c, 4e during 48, 72, and 96 4. Conclusions
hours. The results were given in Figure 1. As seen
In summary, we synthesized six 6-substituted-4-chloro-
methylene coumarin derivatives and their sixteen benz-
imidazolium chlorides. All compounds were character-
ized by 1H and 13C NMR, IR spectroscopic techniques
and elemental analyses. Cytotoxic properties of all com-
pounds were tested against PC-3 and A2780 cancer
cells and all compounds performed significant activi-
ties at different concentrations. Although these results
are preliminary, some compounds performed promising
anticancer effects at low concentrations and in future
we are planning to carry out mechanistic studies for
reported compounds.
from Figure 1, the cell viabilities significantly decreased
after 72 and 96 hours of treatment of selected com-
pounds.
The mechanisms of action of imidazolium or benzim-
idazolium salts have been investigated by some research
groups. In 2009, Minematsu and co-workers reported
the cellular uptake of 14C-labelled 1-(2-methoxyethyl)-
2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-
dihydro-1H-naphtho[2,3-d]imidazolium
bromide
(YM155 monobromide) into PC-3 cell lines.44 The
authors showed that YM155 suppress the Survivin
which acts as anti-apoptotic protein in tumor cells.
In a similar study, Lambrecht and co-workers showed
that 131I-labeled imidazolium bromide salt performs
significant uptake efficiency in MCF-7 and PC-3 cell
lines.45 In 2015, Wright and co-workers showed that
naphthalene-substituted lipophilic imidazolium salts
induce PARP-1 cleavage and reduction in procaspase-
3 so that causes the apoptotic pathway in NCI-H460
(human lung) cancer cells.46 Yang and co-workers
synthesized carbazole14 and tetrahydrobenzodifuran47
imidazolium and benzimidazolium salts and showed
that these salts induce cell cycle arrest and apopto-
sis in SMMC-7721 cancer cell lines. All mechanistic
studies clearly show that imidazolium and benzim-
idazolium salts can enter into the cancer cells. In
this study, it is possible that the synthesized ben-
zimidazolium salts performed their anticancer activ-
ity with different ways than impairment of mem-
brane integrity. Additionally, fluorescence properties of
coumarin derivatives are well known and we think that
this is an advantage in the future studies for intracellular
imaging.
Supplementary Information (SI)
1H and 13C NMR spectra of all compounds can be found at
www.ias.ac.in/chemsci.
Acknowledgements
The authors report no conflicts of interest. The authors are
alone responsible for the content and writing of the paper.
References
1. Daly A K 2013 Optimal dosing of warfarin and other
coumarin anticoagulants: the role of genetic polymor-
phisms Arch. Toxicol. 87 407
2. Yu D, Suzuki M, Xie L, Morris-Natschke S L and Lee K
H 2003 Recent progress in the development of coumarin
derivatives as potent anti-HIV agents Med. Res. Rev. 23
322
3. Maresca A, Temperini C, Vu H, Pham N B, Poulsen S A,
ScozzafavaA, QuinnRJandSupuranCT2009Non-zinc
mediated inhibition of carbonic anhydrases: coumarins

J. Chem. Sci.
(2019) 131:69
Page 11 of 12
69
are a new class of suicide inhibitors J. Am. Chem. Soc.
131 3057
4. Zhao H, Donelly A C, Kusuma B R, Brandt G E L, Brown
D, Rajewski R A, Vielhauer G, Holzbeierlein J, Cohen
M S and Blagg B S J 2011 Engineering an antibiotic to
substituted NHC precursors: synthesis, character-
ization, crystal structure and carbonic anhydrase,
α-glycosidase, butyrylcholinesterase, and acetyl-
cholinesterase inhibitory properties J. Mol. Struct. 1155
797
fight cancer: optimization of the Novobiocin scaffold to 19. Hosamani K M, Reddy D S and Devarajegowda H
produce anti-proliferative agents J. Med. Chem. 54 3839
5. Emami S and Dadashpour S 2015 Current develop-
ments of coumarin-based anti-cancer agents in medicinal
chemistry Eur. J. Med. Chem. 102 611
C 2015 Microwave-assisted synthesis of new fluori-
nated coumarin-pyrimidine hybrids as potent anticancer
agents, their DNA cleavage and X-ray crystal studies
RSC Adv. 5 11261
6. Sandhu S, Bansal Y, Silakari O and Bansal G 2014 20. Wei H, Ruan J and Zhang X 2016 Coumarin-chalcone
Coumarin hybrids as novel therapeutic agents Bioorg.
Med. Chem. 22 3806
hybrids: promising agents with diverse pharmacological
properties RSC Adv. 6 10846
7. Barot K P, Jain S V, Kremer L, Singh S and Ghate 21. Samundeeswari S, Kulkarni M V, Joshi S D, Dixit S
M D 2015 Recent advances and therapeutic journey of
coumarins: current status and perspectives Med. Chem.
Res. 24 2771
R Jayakumar S and Ezhilarasi R M 2016 Synthesis
and human anticancer cell line studies of coumarin-b-
carboline hybrids as possible antimitotic agents Chem-
istrySelect 1 5019
8. Barker H A, Smyth R D, Weissbach H, Toohey J I, Ladd
J N and Volcani B E 1960 Isolation and properties of 22. Pathoor R and Bahulayan D 2018 MCR-click synthesis,
crystalline cobamide coenzymes containing benzimida-
zole or 5,6-dimethylbenzimidazole J. Biol. Chem. 253
480
molecular docking and cytotoxicity evaluation of a new
series of indole-triazole-coumarin hybrid peptidomimet-
ics New J. Chem. 42 1810
9. Gaba M and Mohan C 2016 Development of drug-based 23. Yu H, Hou Z, Tian Y, Mou Y and Guo C 2018
imidazole and benzimidazole bioactive heterocycles:
recent advances and future directions Med. Chem. Res.
25 173
Design, synthesis, cytotoxicity and mechanism of novel
dihydroartemisinin-coumarin hybrids as potential anti-
cancer agents Eur. J. Med. Chem. 151 434
10. Shrivastava N, Naim M J, Alan M J, Nawaz F, Ahmed S 24. Ayati A, Bakhshaiesh T D, Moghimi S, Esmaeili R,
and Alam O 2017 Benzimidazole scaffold as anticancer
agent: synthetic approaches and structure–activity rela-
tionship Arch. Pharm. Chem. Life Sci. 350 e1700040
11. Yadav G and Ganguly S 2015 Structure activity relation-
Majidzadeh-A K, Safavi K, Firoozpour L, Emami S
and Foroumadi A 2018 Synthesis and biological eval-
uation of new coumarins bearing 2,4-diaminothiazole-
5-carbonyl moiety Eur. J. Med. Chem. 155 483
ship (SAR) study of benzimidazole scaffold for different 25. Lingaraju G S, Balaji K S, Jayarama S, Anil S M,
activities: a mini-review Eur. J. Med. Chem. 97 419
12. Gravel J and Schmitzer A R 2017 Imidazolium and
benzimidazolium-containing compounds: from simple
Kiran K R and Sadashiva M R 2018 Synthesis of new
coumarin tethered isoxazolines as potential anticancer
agents Bioorg. Med. Chem. Lett. 28 3606
toxic salts to highly bioactive drugs Org. Biomol. Chem. 26. Singla P, Luxami V and Paul K 2015 Triazine-
15 1051
benzimidazole hybrids: anticancer activity, DNA inter-
action and dihydrofolate reductase inhibitors Bioorg.
Med. Chem. 23 1691
13. Elie C R, David G and Schmitzer A R 2015 Strong
antibacterial properties of anion transporters: a result of
depolarization and weakening of the bacterial membrane 27. Reddy N B, Burra V R, Ravindransth L K, Kumar V N,
J. Med. Chem. 58 2358
14. Liu L X, Wang X Q, Zhou B, Yang L J, Li Y, Zhang H B
and Yang X D 2015 Synthesis and antitumor activity of
Sreenivasulu R and Sadanandan P 2016 Synthesis and
biological evaluation of benzimidazole fused ellipticine
derivatives as anticancer agents Monatsh. Chem. 147 599
novel N-substituted carbazole imidazolium salt deriva- 28. Lukowska-Chojnacka E, Winska P, Wielechowska M
tives Sci. Rep. 5 13101
and Bretner M 2016 Synthesis of polybrominated ben-
zimidazole and benzotriazole derivatives containing a
tetrazole ring and their cytotoxic activity Monatsh.
Chem. 147 1789
15. Karatas¸ M O, Alıcı B, Çetinkaya E, Bilen Ç, Gençer
N and Arslan O 2014 Synthesis, characterization and
tyrosinase inhibitory properties of benzimidazole deriva-
tives Russ. J. Bioorg. Chem. 40 46
16. Karatas¸ M O, Uslu H, Sarı S, Alagöz M A, Karakurt
A, Alıcı B, Bilen Ç, Yavuz E, Gençer N and Arslan O
2016 Coumarin or benzoxazinone based novel carbonic
29. Koronkiewicz M, Chilmonczyk Z, Kazimerczuk Z and
Orzesko A 2018 Deoxynucleosides with benzimidazoles
as a plycome moiety are potent anticancer activity Eur.
J. Pharmacol. 820 146
anhydrase inhibitors: synthesis, molecular docking and 30. Baig M F, Nayak V L, Budaganaboyina P, Mul-
anticonvulsant studies J. Enzyme Inhib. Med. Chem. 31
760
17. Karatas¸ M O, Uslu H, Alıcı B, Gökçe B, Gençer N,
Arslan O, Arslan N B and Özdemir N 2016 Func-
lagiri K, Sunkari S, Gour J and Kamal A 2018
Synthesis and biological evaluation of imidazo[2,1-
b]thiazole-benzimidazole conjıgates as microtubule-
targeting agents Bioorg. Chem. 77 515
tionalized imidazolium and benzimidazolium salts as 31. Wang Z, Deng X, Xiong S, Xiong R, Liu J, Zou L, Lei X,
paraoxonase 1 inhibitors: synthesis, characterization and
molecular docking studies Bioorg. Med. Chem. 24 1392
18. Erdemir F, Celepci D B, Aktas¸ A, Taslimi P, Gök
Cao X, Xie Z, Chen Y, Liu Y, Zhang X and Tang G 2018
Desing, synthesis and biological evaluation of chrysin
benzimidazole derivatives as potential anticancer agents
Nat. Prod. Res. 32 2900
˙
Y, Karabıyık H and Gülçin I 2018 2-Hydroxyethyl

69 Page 12 of 12
J. Chem. Sci.
(2019) 131:69
˙
32. Hwu J R, Singha R, Hung S C, Chang Y H, Das A R, 41. Karatas¸ M O, Olgundeniz B, Günal S, Özdemir I, Alıcı
Vliegen I, Clercq E D and Neyts J 2008 Synthesis of new
benzimidazole-coumarin conjugates as anti-hepatitis C
virus agents Antivir. Res. 77 157
B and Çetinkaya E 2016 Synthesis, characterization
and antimicrobial activities of novel silver(I) complexes
with coumarin substituted N-heterocyclic carbene lig-
ands Bioorg. Med. Chem. 24 643
33. Neyts J, Clercq E D, Singha R, Chang Y H, Das A R,
Chakraborty S K, Hong S C, Tsay S C, Hsu M H and 42. Sato A, Itcho N, Ishiguro H, Okamoto D, Kobayashi N,
Hwu J R 2009 Structure-activity relationship of new
anti-hepatitis C virus agents: heterobicycle-coumarin
conjugates J. Med. Chem. 52 1486
Kawai K, Kasai H, Kurioka D, Uemura H, Kubota Y
and Watanabe M 2013 Magnetic nanoparticles of Fe3O4
enhance docetaxel-induced prostate cancer cell death Int.
J. Nanomed. 8 3151
34. Paul K, Bindal S and Luxami V 2013 Synthesis of new
˙
conjugated coumarin-benzimidazole hybrids and their 43. Çekirdek S, Yas¸ar S and Özdemir I 2014 Palladium(II)-
anticancer activity Bioorg. Med. Chem. Lett. 23 3667
35. Holiyachi M, Shastri S D, Chougala B M, Shastri L A,
Joshi S D, Dixit S R, Nagarajaiah H and Sunagar V A
N-heterocyclic carbene complexes: synthesis, charac-
terization and catalytic application Appl. Organomet.
Chem. 28 423
2016 Design, synthesis and structure-activity relation- 44. Minematsu T, Iwai M, Sugimoto K, Shirai N, Nakahara
ship study of coumarin benzimidazole hybrid as potent
antibacterial and anticancer agents ChemistrySelect 1
4638
T, Usui T and Kamimura H 2009 Carrier-mediated
uptake of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-
(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-
d]imidazolium bromide (YM155 monobromide), a
novel small-molecule Survivin suppressant, into human
solid tumor and Lymphoma cells Drug Metab. Dispos.
37 619
36. Frasinyuk M S, Vinogradova V I, Bondarenko S P and
Khilya V P 2007 Synthesis of cytisine derivatives of
coumarins Chem. Nat. Compd. 43 590
37. Kolocouris N, Foscolos G B, Kolocouris A, Marakos P,
Pouli N, Fytas G, Ikeda S and Clercq E D 1994 Synthesis 45. Lambrecht F Y, Ocakog˘lu K, Colak S G, Ersoz O A
and antiviral activity evaluation of some aminoadaman-
tane derivatives J. Med. Chem. 37 2896
38. Mosmann T 1983 Rapid colorimetric assay for cellu-
and Er O 2017 Synthesis and investigation of anticancer
potential of radiolabeled naphthalane monoamide bear-
ing imidazolium salt Chem. Biol. Drug Des. 90 141
lar growth and survival: application to proliferation and 46. Wright B D, Deblock M C, Wagers P O, Duah E,
cytotoxicity assay J. Immunol. Methods 65 55
39. Görgülü A O, Koran K, Özen F, Tekin S and San-
dal S 2015 Synthesis, structural characterization and
anti-carcinogenic activity of new cyclotriphosphazenes
containing dioxybiphenyl and chalcone J. Mol. Struct.
1087 1
40. Mosmann T R, Cherwinski H, Bond M W, Giedlin M A
and Coffman R L 1986 Two types of murine helper T cell
clone. I. Definition according to profiles of lymphokine
activities and secreted proteins J. Immunol. 136 2348
Robishaw N K, Shelton K L, Southerland M R, DeBord
M A, Kersten K M, McDonald L J, Stiel J A, Panzner M
J, Tessier C A, Paruchuri S and Youngs W J 2015 Anti-
tumor activity of lipophilic imidazolium salts on select
NSCLC cell lines Med. Chem. Res. 24 2838
47. Zhang C B, Liu Y, Liu Z F, Duan S Z, Li M Y, Chen
W, Li Y, Zhang H B and Yang X D 2017 Synthesis and
cytotoxic activity of novel tetrahydrobenzodifuran imi-
dazolium salt derivatives Bioorg. Med. Chem. Lett. 27
1808