Synthesis and In Vitro Anticancer Evaluation of Some Benzimidazolium Salts

Full text of DOI:10.1002/jhet.3687

Month 2019
Synthesis and In Vitro Anticancer Evaluation of Some Benzimidazolium
Salts
a
b
Senem Akkoç,a,b,c
*
*
Veysel Kayser, and İlhan Özer İlhan
aSchool of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia
bFaculty of Sciences, Department of Chemistry, Erciyes University, Kayseri 38039, Turkey
cFaculty of Pharmacy, Department of Basic Pharmaceutical Sciences, Suleyman Demirel University, Isparta 32260,
Turkey
*E-mail: senemakkoc@erciyes.edu.tr; senemakkoc44@gmail.com; veysel.kayser@sydney.edu.au
Received March 25, 2019
DOI 10.1002/jhet.3687
Published online 00 Month 2019 in Wiley Online Library (wileyonlinelibrary.com).
A series of benzimidazolium salts having 2-cyanobenzyl (2–6), 2-(4-nitrophenyl)ethyl (7–10),
(1,3-dioxoisoindolin-2-yl)butyl (11–17) or N-methylphthalimide (18–22) substitution were synthesized
and characterized by spectroscopic and analytical techniques. The in vitro cytotoxic activity of these salts
was tested against human colon cancer (DLD-1), human breast cancer (MDA-MB-231), and normal human
noncancerous embryonic kidney (HEK-293T) cell lines using the MTT assay method. The viability rates of
the cells were determined by confocal microscopy. The majority of the newly synthesized compounds, with
the exception of compounds 2, 3, 8, 11, 12, and 17, displayed high cytotoxic activity against cancerous cells.
In fact, compounds 6 and 7 exhibited better results than the positive control drugs, cisplatin and busulfan,
against the MDA-MB-231 cells. Therefore, compounds 6 and 7 could be considered as anticancer drug can-
didates for further development.
J. Heterocyclic Chem., 00, 00 (2019).
INTRODUCTION
cervix, lymphocyte, prostate, colon, breast, lung, liver,
bone marrow, and peripheral blood [2,10–24]. Due to
their anticancer activity, many research groups have been
involved in developing new heterocyclic compounds. For
example, Shelton’s group improved a series of N,N′-
bis(arylmethyl)benzimidazolium salts as antitumor agents
against NCl-H460, NCl-H1975, HCC827, and A549 cell
lines [25]. In 2015, Haque et al. [11] reported
benzimidazole-based N-heterocyclic carbene (NHC)
proligands and their Ag complexes in human derived
colorectal adenocarcinoma (HT29) and colorectal cancer
(HCT 116) cell lines. 3,3′-(1,4-phenylenebis(methylene))
bis(1-alkyl-benzimidazolium) salts and their silver
complexes were developed by Iqbal et al. and tested their
cytotoxicity against HCT 116. The IC50 values of
compounds for this cell line ranged between 0.03 and
65.9 μM. However, dinuclear Ag(I)-NHC complexes
including benzimidazole nucleus was found to be more
active against HCT 116 than benzimidazolium salts and
5-fluorouracil, which is standard positive control drug [26].
With the aim of developing effective, safe, and
affordable anticancer drug candidates against cancerous
colon and breast cell lines, benzimidazolium salts (2–22)
were designed, synthesized, and structurally characterized
in this study.
Colon and breast cancers were two of the four major
cancers by age and sex group in America for 2016 [1].
The number of deaths from colon cancer is higher than
that from breast cancer, and deaths from colon and rec-
tum cancers are almost the same for women (23,170)
and men (26,020). Due to the high mortality rates from
colon, breast, and other cancer types, there is an ever-
increasing need to develop more effective drug therapies,
and many different types compounds have been synthe-
sized for this purpose. For instance, Asif et al. [2] pre-
pared two Ag-NHC complexes, which include
a
heterocyclic group, and screened them against HCT
116, HT-29, MCF-7, MDA-MB-231, PANC-1,
SHSY5Y, U-937 human cancer cells, and 3T3 L-1
mouse cancer cells.
Heterocyclic compounds including an imidazole
or benzimidazole nucleus have shown different
types of biological activity such as antimicrobial,
antifungal, anthelmintic, anti-allergic, antihistamine,
analgesic, antineoplastic, anti-inflammatory, hypotensive,
vasodilator, antipyretic, and antinematodal [3–9]. They
also exhibit cytotoxic effects against various cancerous
cells including the brain, pancreas, leukemia, ovaries,
© 2019 Wiley Periodicals, Inc.

S. Akkoç, V. Kayser, and İ. Ö. İlhan
Vol 000
Scheme 1. Synthesis and chemical structures of the benzimidazolium salts.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and In Vitro Anticancer Evaluation
RESULTS AND DISCUSSION 143.01, 142.77, 142.99, 143.59, and 143.16 ppm for
2–17, respectively. Furthermore, the aromatic carbon
peaks were observed in the range δ 98.98–147.15 ppm.
The formation of the desired compounds was confirmed
by IR, which showed the presence of stretching bands for
the C═N vibrations on the benzimidazole ring at 1559.8,
1566.8, 1557.8, 1552.5, 1553.9, 1510.7, 1514.9, 1514.6,
1519.7, 1553.4, 1562.9, 1557.7, 1544.9, 1557.6, 1560.4,
and 1564.8 cmÀ1, for compounds 2–17, respectively. The
structure of compound 8 was further characterized by
HRMS. The calculated value (383.42 m/z) and the found
value (383.15 m/z) are quite close to each other.
Compounds 2 and 9 were also verified by elemental
analysis.
Cytotoxic activity of synthesized new compounds. The
cytotoxic activity of compounds 2–22 was evaluated at
different concentrations ranging from 0.5 to 200 μM
against DLD-1 and MDA-MB-231 cells and at
concentrations ranging from 0.001 to 100 μM against
HEK-293T cells by using the MTT assay method. In this
work, cisplatin and busulfan were used as positive
control drugs for comparison purposes with the drug
candidates (2–22) under the same experimental
conditions. After the cells were exposed to compounds
2–22 for an incubation time of 72 h (for DLD-1 and
MDA-MB-231) or 24 h (for HEK-293T), cytotoxic
activity was expressed as IC50 values and are given in
Table 2.
Synthesis
benzimidazolium salts.
and
spectral
characterization
of
Benzimidazolium salts 2–22
were prepared in yields ranging from 35 to 93% by the
successive reaction of N-alkylbenzimidazole with various
alkyl or aryl halides in DMF (Scheme 1). After the
recrystallization of products 2–22 in suitable solvents,
pure forms of the salts were acquired and thoroughly
characterized by NMR, Fourier transform infrared, high
resolution mass spectrometry (HRMS) (for 8), and
elemental analysis (for 2 and 9). The NMR (1H and 13C)
spectra of the salts were recorded in chloroform (CDCl3)
or dimethyl sulfoxide (DMSO-d6) solvents at room
temperature. The compounds were easily soluble in polar
organic solvent DMSO; however, they were insoluble in
the nonpolar solvent diethyl ether.
1
In the H-NMR spectra, the resonance of the NCHN
proton signals belonging to the benzimidazolium salts
were found as singlets in the downfield region compared
to the other proton signals at δ 9.95, 11.61, 11.80, 10.08,
9.17, 11.23, 11.56, 9.84, 10.23, 11.69, 11.56, 11.54,
11.56, 11.55, 11.50, and 11.10 ppm for 2–17,
respectively. The methylene proton resonances belonging
to NCH2C6H4(CN)-2, which is a linker between the
phenyl groups and the nitrogen atoms of the
benzimidazole ring, were observed as singlets at δ 6.04,
6.20, 6.17, 5.82, 5.86, 5.99, and 6.17 ppm for 2–6, 8,
and 12, respectively. Moreover, the resonances of
the aromatic protons were determined as multiplets in the
low region at δ 7.02–8.92 ppm for 2–17. The important
features of 2–17 are given in Table 1.
The variety in the IC50 values showed that the
substituents at the N1 and/or N3 position have a direct
impact on the in vitro cytotoxic activity. All the
synthesized compounds showed evidence of enhanced
cytotoxic activity against DLD-1 (except 2, 3, 8, 12,
and 17) and MDA-MB-231 (except 3, 11, 12, and 17)
cells. Also, compounds 5, 6, and 10 showed moderate
activity against both cancerous cell lines. Compound 5,
In the 13C-NMR spectra, the characteristic resonance
peaks belonging to the C2 (NCHN) atom were found in
the downfield region at δ 143.60, 143.81, 144.26, 143.46,
142.93, 143.42, 143.03, 142.78, 142.23, 143.15, 143.44,
Table 1
Characterization data of the benzimidazolium salts obtained via 1H-NMR, 13C-NMR, and IR.
À1
Benzimidazolium salt
Solvent
δH (NHC═N) (ppm)
δC (NHC═N) (ppm)
IRC═
N) (cm
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
DMSO-d6
CDCl3
CDCl3
DMSO-d6
DMSO-d6
CDCl3
CDCl3
DMSO-d6
CDCl3
CDCl3
CDCl3
9.95
11.61
11.80
10.08
9.17
11.23
11.56
9.84
10.23
11.69
11.56
11.54
11.56
11.55
11.50
11.10
143.60
143.81
144.26
143.46
142.93
143.42
143.03
142.78
142.23
143.15
143.44
143.01
142.77
142.99
143.59
143.16
1559.8
1566.8
1557.8
1552.5
1553.9
1510.7
1514.9
1514.6
1519.7
1553.4
1562.9
1557.7
1544.9
1557.6
1560.4
1564.8
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
Journal of Heterocyclic Chemistry
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S. Akkoç, V. Kayser, and İ. Ö. İlhan
Vol 000
Table 2
Compounds 11–17 which all include
dioxoisoindolin-2-yl)butyl group exhibited
a
4-(1,3-
lower
IC50 results for the benzimidazolium salts against the MDA-MB-231 and
DLD-1 cancerous cell lines and HEK-293T noncancerous cell line.
cytotoxic activity compared to compounds 4–7 and 10.
Particularly, compounds 12 (including 2-cyanobenzyl
group) and 17 (including 2-morpholinoethyl group)
showed no activity against either cancerous cell lines.
Ghdhayeb et al. [27] have reported that the cytotoxic
activity of n-butyl and ally substituted benzimidazolium
salts and their silver and palladium complexes against
human cancerous colon cells (HCT 116). According to
their study, benzimidazolium salts exhibited no activity
while their silver and palladium complexes showed
anticancer activity [27]. However, in this study, almost
all the benzimidazolium salts (except 2, 3, 8, 12, and 17
against DLD-1) had cytotoxic activity against the
cancerous cell lines. A plot of drug’s effectiveness as a
function of concentration on the MDA-MB-231 and
DLD-1 cells after 72 h is depicted in Figures1 and 2,
respectively.
The cytotoxicity of the new compounds on the
MDA-MB-231 (Fig. 1) and DLD-1 cell lines (Fig. 2) was
seen to change depending on the dose. For example,
MDA-MB-231 cell viability was shown to be around 5%
when using 200 μM of compound 7 while it was around
75% when using 0.5 μM of salt 7. The big differences in
cell viability due to concentration were also valid when
other sample doses were used.
IC50 (μM)
Compound
DLD-1
>200
196.50 3.48
52.47 6.66
31.01 5.42
10.98 2.33
54.40 4.25
>200
35.08 0.49
24.98 5.44
142.25 4.94
>200
74.45 3.59
100.31 2.74
59.37 3.61
53.53 0.43
>200
MDA-MB-231
HEK-293T
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Cisplatin
Busulfan
120.96 5.69
>200
>100
>100
>100
92.71 3.59
16.22 4.20
N.T.
>100
N.T.
N.T.
>100
>100
48.48 4.25
N.T.
N.T.
>100
>100
N.T.
44.46 5.73
27.37 1.62
1.26 0.85
2.01 0.96
130.16 6.09
82.17 5.43
31.69 3.69
>200
>200
53.35 2.05
37.43 4.38
105.95 4.84
70.60 4.17
>200
180.05 6.21
133.70 2.48
173.4 3.8
135.02 2.28
29.83 6.61
5.77 0.4
>200
140.05 3.79
155.60 4.61
41.96 0.35
95.02 5.06
41.05 1.39
4.38 0.05
173.20 5.4
N.T.
>100
N.T.
87.81
33.97 2.44
>100
N.T., not tested.
which two aromatic groups, showed an IC50 value of
31.01 5.42 μM against DLD-1 and 27.37 1.62 μM
Apohan et al. have evaluated the cytotoxic activity of 14
zinc (II) and cobalt (II) complexes including those that
contain a benzimidazole nucleus against a lung cancer
cell line (A549) and a BEAS-2B cell line over various
time periods (24, 48, and 72 h). They reported the IC50
values for three complexes ranging between 1.87 and
1.97 μM at 72 h against A549 cell line [28]. In other
words, these benzimidazole-based compounds exhibited
better cytotoxic activity than cisplatin. However, they
were less cytotoxic against BEAS-2B cells than cisplatin.
In another study, Cheong et al. [22] evaluated the
anticancer activity of water soluble benzimidazole-based
organic compounds against the following three cancer
cell lines: PC3MLN4, PC3, and A549. They reported that
nearly all of the compounds exhibited low cytotoxicity
against the cell lines.
Confocal microscopy images. The photographs of the
DLD-1 and MDA-MB-231 cells, and cells treated with
test samples and positive control drugs (cisplatin and
busulfan), are depicted in Figure 3A and 3B, respectively.
The control group images of the DLD-1 and MDA-
MB-231 cells demonstrate completely confluent growth.
However, the cellular images show that the normal
morphology of the cells treated with benzimidazolium
salts changed, and the cells lost their viability compared
to the control. Therefore, the benzimidazolium salts have
a noteworthy effect on the viability of cells.
against MDA-MB-231 cells. While compounds
2
(which includes one 2-cyanobenzyl and one propyl
group) and 8 (which includes one 2-(4-nitrophenyl)ethyl
and one 2-cyanobenzyl group) were inactive against
DLD-1 with IC50 values of >200, they also had a
minimal effect against MDA-MB-231 with IC50
values of 120.96
respectively.
5.69 and 130.16
6.09 μM,
It is noteworthy that 7, which includes a naphthalen-1-
ylmethyl and an 2-(4-nitrophenyl)ethyl substituent, was
more effective than the other compounds against
MDA-MB-231 with an IC50 value of 2.01 0.96 μM.
Furthermore, compound 7 showed roughly three times
more in vitro cytotoxic activity than the well-known
chemotherapeutic agent cisplatin against MDA-MB-231
cells. While cisplatin had IC50 values of 5.77 0.4 μM
(for MDA-MB-231), compound 7 exhibited IC50 values
of 2.01 0.96 μM (for MDA-MB-231). However, drug
candidate 7 was slightly less active against the colon
cancer cells compared with the breast cancer cells.
Additionally, compound 6 exhibited higher cytotoxic
activity against MDA-MB-231 cell line with low IC50
values (2.761
0.43 μM) compared to both positive
control drugs. The in vivo study of these compounds
(6 and 7) will be performed in the future.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and In Vitro Anticancer Evaluation
Figure 1. Cell viability of MDA-MB-231 cells treated with the drug candidates. [Color figure can be viewed at wileyonlinelibrary.com]
Figure 2. Cell viability of DLD-1 cells treated with the drug candidates. [Color figure can be viewed at wileyonlinelibrary.com]
Also, the positive control drug (cisplatin) affected the
population of the DLD-1 cells, and the viability of
cells treated with cisplatin decreased significantly.
However, the morphology of both kinds of cell types
treated with busulfan protected their cell viability. The
images of the DLD-1 cells treated with 10 showed that
more cells lost their viability compared with drug
candidates 4 and 5. The images of the MDA-MB-231
cells after exposure to 9 at 20 μM show that these
cells did not survive. The anticancer potential of this
compound was more potent against breast cancer than
compound 2.
MATERIALS AND METHODS
Reagents and solvents for the synthesis of
new compounds. The chemicals 1,2-phenylenediamine,
Journal of Heterocyclic Chemistry
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S. Akkoç, V. Kayser, and İ. Ö. İlhan
Vol 000
Figure 3. Confocal images of DLD-1 (A) and MDA-MB-231 (B) cells, (A) after treatment with the synthesized compounds (20 μM) for 24 h incubation,
and (B) the cells stained with Hoechst (blue) and PI (red). [Color figure can be viewed at wileyonlinelibrary.com]
1-bromopropan, 2-(bromomethyl)benzonitrile, 10-(chloro
methyl)anthracene, 2-(2-bromoethyl)-1,3-dioxane, benzyl
chloride, 3-methylbenzylchloride, 4-methylbenzylchlo
ride, 4-vinylbenzyl chloride, 1-(chloromethyl)naphtha
lene, 1-(2-bromoethyl)-4-nitrobenzene, 2,3,5,6-tetramethy
lbenzylchloride, N-(bromomethyl)phthalimide, N-(4-bro
Aldrich, USA). Cell lines were donated by Dr Fanfan Zhou
and Dr Binh Pham of the University of Sydney.
Instrumentation.
The following instrumentation was
used in this study: a Bruker 300 or 400 MHz Ultra Shield
NMR, an electrothermal-9200 melting point apparatus,
a Shimadzu Fourier transform infrared 8400 spectropho-
mobutyl)phthalimide,
N-(2-chloroethyl)-morpholinium
tometer,
a HRMS device, an Invitrogen Countess
chloride, and N,N-dimethylformamide (DMF), were
bought from chemical companies such as Sigma-Aldrich
(Interlab A.S., USA), Merck (Darmstadt, Germany), or
Scharlau (Barcelona, Spain).
automated cell counter, and an Olympus FV1000
confocal microscope [12,18].
CONCLUSION
Cell culture. For the cell culture studies Dulbecco’s
modified Eagle’s medium, fetal bovine serum,
GlutaMAX, trypsin-EDTA, phosphate-buffered saline,
busulfan, cis-diammineplatinum (II) dichloride, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT), Hoechst 33258, pentahydrate (bis-benzimide),
propidium iodide (PI), and laboratory materials such as
tissue culture flasks and 96-well plates were purchased
from Gibco (Life Technologies, USA), Medicago (Astral
Scientific, Sweden), Lonza (Arch Chemicals, Inc., North
Sydney, Australia), Thermo Fisher Scientific (Molecular
Probes by Life Technologies, Australia), Jet Biofil
(Pathtech, China), Fluka, Nalgene, or Sigma (Sigma-
Sixteen new and five known benzimidazolium salts
were synthesized and extensively characterized using
various spectroscopic techniques. The anticancer
properties of the salts 2–22 were tested against DLD-1,
MDA-MB-231, and HEK-293T cell lines by the MTT
assay method and compared with the known anticancer
drugs cisplatin and busulfan. Our results demonstrate
that benzimidazolium salts had in vitro anticancer
activity. Specifically, compounds 6 and 7 exhibited
promising results against MDA-MB-231 cell line that
warrant further investigation as a potential anticancer
agents, even for other cell lines, such as Hela, PC-3,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and In Vitro Anticancer Evaluation
BEL-7404, A549, and HL7702. Furthermore, the low
IC50 value of compound 6 indicates its potential as a
drug candidate for DLD-1. In conclusion, our results
may be useful in designing new compounds as
anticancer agents in the future.
dioxane (1 mmol) under the same conditions and
procedure as for 2. The final product was crystallized in
ethyl alcohol. Yield: 45%, color: white. IR: 1140.0
(C-O); 1566.8 (C═N); 2218.9 (C≡N); 2854.7, 2931.6 and
1
3008.5 cmÀ1 (C-H). H-NMR (400.13 MHz, CDCl3, 298
K), δ: 1.92 (p, J: 4.0 Hz, 2 H, OCH2CH2CH2O); 2.35 (q,
J: 4.0 Hz, 2 H, NCH2CH2CHO2); 3.76 and 3.95 (dd, J:
8.0 Hz, J: 12.0 Hz, 4 H, OCH2CH2CH2O); 4.78 (t, J:
EXPERIMENTAL
8.0 Hz,
2 H, NCH2CH2CHO2); 6.20 [s, 2 H,
NCH2C6H4(CN)-2]; 7.28–8.17 (m, 8 H, Ar-H); 11.61
(s, 1 H, NCHN). 13C-NMR (100.13 MHz, CDCl3, 298
K), δ: 20.40 (OCH2CH2CH2O); 25.35 (NCH2CH2CHO2);
33.83 (OCH2CH2CH2O); 42.69 (NCH2CH2CHO2); 48.40
(NCH2CH2CHO2); 66.79 [NCH2C6H4(CN)-2]; 98.98,
112.09, 113.25, 113.43, 117.26, 127.21, 127.43, 129.95,
130.93, 131.12, 131.32, 133.19, 134.36 and 136.16
(Ar-C; C≡N); 143.81 (NCHN).
Synthesis of new benzimidazolium salts. Compounds 2–
22 were prepared according to previously published
procedures [29–31]. Benzimidazole (1 mmol) was
dissolved in 20 mL of ethanol. KOH (1.5 mmol)
was added, and the mixture was stirred for 1 h at room
temperature. Alkyl halide (1 mmol) was added to this
mixture and was refluxed for 6 h. Subsequently, it was
filtered to remove the potassium chloride that was
formed, and pure N-alkylbenzimidazole was obtained.
The solvent was removed under vacuum. This
intermediate product was washed several times
with diethyl ether. Alkyl halide (1 mmol) and
N-alkylbenzimidazole (1 mmol) were then added to a
new 100 mL of Schlenk tube with 4 mL of dried DMF.
The mixture was allowed to react further for 24 h at
80°C, before the final product was purified by
crystallization with suitable solvent or solvent mixtures.
1-(2-Cyanobenzyl)-3-(4-methylbenzyl)-1H-benzo[d]imidazol-
3-ium bromide (4).
Compound 4 (418.33 g/mol)
was synthesized from 1-(4-methylbenzyl)benzimidazole
(1 g, 1 mmol) and 2-(bromomethyl)benzonitrile (0.88 g,
1 mmol) under the same conditions and procedure as for
2. The final product was crystallized in ethyl alcohol.
Yield: 74%, m.p.: 200–201°C, color: white. IR: 1557.8
(C═N); 2226.4 (C≡N); 2927.7 and 3355.2 cmÀ1 (C-H).
1H-NMR (400.13 MHz, CDCl3, 298 K), δ: 2.31 [s, 3 H,
NCH2C6H4(CH3)-4]; 5.82 [s, 2 H, NCH2C6H4(CH3)-4];
6.17 [s, 2 H, NCH2C6H4(CN)-2]; 7.16–8.21 (m, 12 H,
Ar-H); 11.80 (s, 1 H, NCHN). 13C-NMR (100.13 MHz,
CDCl3, 298 K), δ: 21.19 [NCH2C6H4(CH3)-4]; 51.78
[NCH2C6H4(CN)-2]; 58.32 [NCH2C6H4(CH3)-4]; 111.99,
113.46, 113.92, 117.23, 127.34, 127.81, 128.34, 129.24,
130.01, 130.98, 131.13, 131.22, 133.25, 133.30, 134.50,
135.57, 135.85, 139.41 and 143.59 (Ar-C; C≡N);
144.26 (NCHN).
1-(2-Cyanobenzyl)-3-propyl-1H-benzo[d]imidazol-3-ium
bromide (2). 1-Bromopropan (0.46 g, 1 mmol) was slowly
added to the DMF solution of synthesized 2-((1H-benzo[d]
imidazol-1-yl)methyl)benzonitrile (0.868 g, 1 mmol) in the
previous step 2.4 and was stirred for 24 h at 80°C. DMF was
aspirated from the reaction medium under vacuum after the
reaction was completed. With this method, compound 2 was
synthesized in a moderate yield, and the final product
was purified by crystallization in
a
mixture of
ethyl alcohol-diethyl ether (2:1). Yield: 51%, m.p.:
1-(2-Cyanobenzyl)-3-(4-vinylbenzyl)-1H-benzo[d]imidazol-3-
ium chloride (5).
Compound 5 (385.89 g/mol) was
146–147°C, color: white. IR: 1559.8 (C═N); 2967.6 and
1
3356.9 cmÀ1 (C-H). H-NMR (400.13 MHz, DMSO-d6,
synthesized from 2-((1H-benzo[d]imidazol-1-yl)methyl)
benzonitrile (1 g, 1 mmol) and 4-vinylbenzyl chloride
(0.65 g, 1 mmol) according to the same conditions and
procedure as for 2. The final product was crystallized in
ethyl alcohol. Yield: 93%, m.p.: 172–173°C, color: white.
IR: 1552.5 (C═N); 2224.1 (C≡N); 2935.4, 3069.2,
298 K), δ: 0.94 [t, J: 4.0 Hz, 3 H, NCH2CH2CH3]; 1.95
[h, J: 8.0 Hz, 2 H, NCH2CH2CH3]; 4.54 [t, J: 12.0 Hz, 2
H, NCH2CH2CH3]; 6.04 [s, 2 H, NCH2C6H4(CN)-2];
7.49–8.19 (m, 8 H, Ar-H); 9.95 (s, 1 H, NCHN).
13C-NMR (100.13 MHz, DMSO-d6, 298 K), δ: 11.1
1
3132.8, and 3357.7 cmÀ1 (C-H). H-NMR (300.13 MHz,
(NCH2CH2CH3);
22.5
(NCH2CH2CH3);
48.7
DMSO-d6, 298 K), δ: 5.82 [s, 2 H, NCH2C6H4(CN)-2];
5.89 (d, J: 8.0 Hz, 2 H, NCH2C6H4CHCH2); 6.06
(s, 2 H, NCH2C6H4CHCH2); 6.69–6.78 (m, 1 H,
NCH2C6H4CHCH2); 7.53–8.01 (m, 12 H, Ar-H); 10.08 (s,
1 H, NCHN). 13C-NMR (75.47 MHz, DMSO-d6, 298 K),
δ: 48.58 (NCH2C6H4CHCH2); 49.86 [NCH2C6H4(CN)-2];
110.83, 113.75, 114.17, 115.32, 116.92, 126.62, 126.94,
127.07, 128.72, 129.16, 129.60, 130.91, 131.31, 133.08,
133.83, 134.02, 135.86, 136.83, and 137.55 (Ar-C; C≡N;
CH═CH2); 143.46 (NCHN).
(NCH2CH2CH3); 48.9 [NCH2C6H4(CN)-2]; 111.2, 114.1,
114.4, 114.5, 117.3, 127.2, 127.4, 129.3, 129.6, 130.0,
131.5, 131.7, 134.3, 134.4, and 137.4 (Ar-C; C≡N); 143.6
(NCHN). Elemental analysis C18H18N3Br (356.26 g/mol)
(%): Found C: 60.54; H: 4.93; N: 11.37. Anal. Calcd C:
60.68; H: 5.09; N: 11.79.
1-(2-Cyanobenzyl)-3-(1,3-dioxane-2-yl)ethyl-1H-benzo[d]
imidazol-3-ium bromide (3). Compound 3 (428.32 g/mol)
was synthesized from 2-((1H-benzo[d]imidazol-1-yl)
methyl)benzonitrile (1 mmol) and 2-(2-bromoethyl)-1,3-
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

S. Akkoç, V. Kayser, and İ. Ö. İlhan
Vol 000
1-(Anthracen-10-ylmethyl)-3-(2-cyanobenzyl)-1H-benzo[d]
imidazol-3-ium chloride (6). Compound 6 (459.97 g/mol)
124.18, 127.73, 127.96, 130.07, 130.44, 130.92,
131.38, 133.42, 134.59, 135.34, 143.03, and 143.58
(Ar-C; C≡N; NCHN). HRMS [L-Br]+ calcd for
C23H19N4O2: 383.42, found m/z: 383.15.
1-(3-Methylbenzyl)-3-[2-(4-nitrophenyl)ethyl]-1H-benzo[d]
imidazol-3-ium bromide (9). Compound 9 (452.34 g/mol)
was synthesized from 1-(3-methylbenzyl)benzimidazole
(1.0 g, 1 mmol) and 1-(2-bromoethyl)-4-nitrobenzene
(1.04 g, 1 mmol) according to the same conditions
and procedure as for 2. The final product was crystallized
was synthesized from 2-((1H-benzo[d]imidazol-1-yl)
methyl)benzonitrile (0.90 g, 1 mmol) and 10-(chloro
methyl)antracene (0.88 g, 1 mmol) according to the same
conditions and procedure as for 2. The final product was
crystallized in ethyl alcohol-diethyl ether (2:1) mixture.
Yield: 35%, m.p.: 227–228°C, color: yellow. IR: 1553.9
(C═N); 2216.5 (C≡N); 2940.2 and 3133.9 cmÀ1 (C-H).
1H-NMR (400.13 MHz, DMSO-d6, 298 K), δ: 5.86 [s,
2 H, NCH2C6H4(CN)-2]; 6.79 (s, 2 H, NCH2C14H9];
7.07–8.92 (m, 17 H, Ar-H); 9.17 (s, 1 H, NCHN).
13C-NMR (100.13 MHz, DMSO-d6, 298 K), δ: 48.67
[NCH2C6H4(CN)-2]; 56.47 [NCH2C14H9]; 110.67,
114.17, 115.06, 117.17, 122.19, 123.94, 126.13, 127.49,
127.81, 127.97, 128.33, 129.62, 129.90, 131.01, 131.50,
131.61, 131.89, 132.32, 134.07, 134.25, and 137.97
(Ar-C); 142.93 (NCHN).
in
a mixture of ethyl alcohol-diethyl ether (2:1).
Yield: 60%, m.p.: 144–145°C, color: cream. IR: 1514.6
(C═N); 1597.4 (NO2); 2948.0 and 3379.0 cmÀ1 (C-H).
1H-NMR (400.13 MHz, DMSO-d6, 298 K), δ: 2.27 [s, 3
H, NCH2C6H4(CH3)-3]; 3.43 (t, J: 12.0 Hz, 2 H,
NCH2CH2C6H4NO2); 4.89 (t, J: 8.0 Hz,
2
H,
NCH2CH2C6H4NO2); 5.68 [s, 2 H, NCH2C6H4(CH3)-3];
7.11–8.16 (m, 12 H, Ar-H); 9.84 (s, 1 H, NCHN).
13C-NMR (100.13 MHz, DMSO-d6, 298 K), δ: 21.34
[NCH2C6H4(CH3)-3]; 34.50 (NCH2CH2C6H4NO2); 47.71
1-(Naphthalen-1-ylmethyl)-3-[2-(4-nitrophenyl)ethyl]-1H-
benzo[d]imidazol-3-ium bromide (7).
Compound
7
(488.38 g/mol) was synthesized from 1-(naphthalen-1-
ylmethyl)benzimidazole (1.04 g, 1 mmol) and 1-(2-
bromoethyl)-4-nitrobenzene (0.93 g, 1 mmol) according to
the same conditions and procedure as for 2. The final
product was crystallized in a mixture of ethyl alcohol-
diethyl ether (2:1). Yield: 53%, m.p.: 149–150°C, color:
cream. IR: 1510.7 (C═N); 1559.1 (NO2); 2980.4, 3350.7,
[NCH2C6H4(CH3)-3];
50.18
(NCH2CH2C6H4NO2);
114.38, 114.48, 124.08, 125.62, 127.20, 127.28, 129.20,
129.21, 129.83, 130.78, 131.17, 131.47, 134.29, 138.74,
145.64, and 146.93 (Ar-C); 142.78 (NCHN). Elemental
analysis C23H22N3O2Br (452.34 g/mol) (%): Found
C: 59.67; H: 5.24; N: 8.73. Anal. Calcd C: 61.07;
H: 4.90; N: 9.29.
1
and 3441.9 cmÀ1 (C-H). H-NMR (400.13 MHz, CDCl3,
1-[2-(4-Nitrophenyl)ethyl]-3-(2,3,5,6-tetramethylbenzyl)-1H-
298 K), δ: 3.58 [t, J: 8.0 Hz, 2 H, NCH2CH2C6H4(NO2)-
4]; 5.03 [t, J: 4.0 Hz, 2 H, NCH2CH2C6H4(NO2)-4]; 6.19
(s, 2 H, NCH2C10H7); 7.41–8.02 (m, 15 H, Ar-H); 11.23
(s, 1 H, NCHN). 13C-NMR (100.13 MHz, CDCl3, 298 K),
δ: 35.04 [NCH2CH2C6H4(NO2)-4]; 47.87 (NCH2C10H7);
49.38 [NCH2CH2C6H4(NO2)-4]; 112.75, 113.79, 122.00,
123.97, 125.39, 126.65, 127.36, 127.48, 127.76, 127.80,
129.41, 129.95, 130.40, 130.56, 131.20, 133.86, 143.24,
143.42, and 147.11 (Ar-C and NCHN).
benzo[d]imidazol-3-ium
10 (494.42 g/mol) was synthesized from 1-(2,3,5,6-
tetramethylbenzyl)benzimidazole (0.9 g, 1 mmol) and
1-(2-bromoethyl)-4-nitrobenzene (0.78 g,
bromide
(10).
Compound
1
mmol)
according to the same conditions and procedure as for 2
[32]. The final product was crystallized in ethyl alcohol.
Yield: 45%, m.p.: 212–214°C, color: white. IR: 1519.7
(C═N); 1601.1 cmÀ1 (NO2). 1H-NMR (300.13 MHz,
CDCl3, 298 K), δ: 2.06 and 2.19 [s, 12 H, NCH2C6
H(CH3)4–2,3,5,6]; 3.44 [t, J: 7.2 Hz, 2 H, NCH2CH2
C6H4(NO2)-4]; 5.09 [t, J: 7.2 Hz, 2 H, NCH2CH2C6H4
(NO2)-4]; 5.58 [s, 2 H, NCH2C6H(CH3)4–2,3,5,6]; 7.02–
7.98 (m, 9 H, Ar-H); 10.23 (s, 1 H, NCHN). 13C-NMR
(75.47 MHz, CDCl3, 298 K), δ: 16.07 and 20.53
[NCH2C6H(CH3)4–2,3,5,6]; 35.07 [NCH2CH2C6H4(NO2)
-4]; 47.21 [NCH2C6H(CH3)4–2,3,5,6]; 47.81 [NCH2CH2
C6H4(NO2)-4]; 113.09, 113.45, 123.91, 126.97, 127.47,
130.18, 131.20, 131.37, 133.87, 133.92, 135.37, 143.52,
and 147.15 (Ar-C); 142.23 (NCHN).
1-(2-Cyanobenzyl)-3-[2-(4-nitrophenyl)ethyl]-1H-benzo[d]
imidazol-3-ium bromide (8). Compound 8 (463.33 g/mol)
was synthesized from 2-((1H-benzo[d]imidazol-1-yl)
methyl)benzonitrile (1.0 g, 1 mmol) and 1-(2-bromo
ethyl)-4-nitrobenzene (0.99 g, 1 mmol) according to the
same conditions and procedure as for 2. The final
product was crystallized in dichloromethane. Yield:
87%, m.p.: 237–238°C, color: white. IR: 1514.9
(C═N); 1601.7 (NO2); 2223.9 (C≡N); 2943.2, 3025.6,
3222.7, 3385.9, and 3446.4 cmÀ1 (C-H). 1H-NMR
(400.13 MHz, CDCl3, 298 K), δ: 3.62 [t, J: 8.0 Hz, 2
H, NCH2CH2C6H4NO2]; 5.01 [t, J: 4.0 Hz, 2 H,
NCH2CH2C6H4NO2]; 5.99 [s, 2 H, NCH2C6H4(CN)-2];
7.50–8.11 (m, 12 H, Ar-H); 11.56 (s, 1 H, NCHN).
13C-NMR (100.13 MHz, CDCl3, 298 K), δ: 35.01
(NCH2CH2C6H4NO2); 48.01 [NCH2C6H4(CN)-2]; 48.85
(NCH2CH2C6H4NO2); 112.04, 112.58, 113.70, 117.07,
1-Benzyl-3-[4-(1,3-dioxoisoindolin-2-yl)butyl]-1H-benzo[d]
imidazol-3-ium bromide (11).
According to the same
conditions and procedure as for 2, compound 11 was
synthesized from 1-benzyl-1H-benzo[d]midazole (0.7 g,
1 mmol) and N-(4-bromobutyl)phthalimide (0.95 g,
1 mmol) in DMF for 24 h at 80°C. The product
(C26H24N3O2Br: 490.4 g/mol) was purified by
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and In Vitro Anticancer Evaluation
crystallization in ethyl alcohol. Yield: 51%, m.
p.:160–162°C, color: white. IR: 1553.4 (CN); 1698.7
and 1766.4 (C═O); 2920.8, 3065.5, and 3116.8 cmÀ1
(C-H). H-NMR (400.13 MHz, CDCl3, 298 K), δ: 1.90
[p, J: 8.0 Hz, 2 H, NCH2CH2CH2CH2N(C═O)2C6H4];
1H-NMR (400.13 MHz, CDCl3, 298 K), δ: 1.90 [p, J:
8.0 Hz, 2 H, NCH2CH2CH2CH2N(C═O)2C6H4]; 2.14
[p, J: 4.0 Hz, 2 H, NCH2CH2CH2CH2N(C═O)2C6H4];
2.34 [s, 3 H, NCH2C6H4(CH3)-3]; 3.79 [t, J: 8.0 Hz, 2 H,
NCH2CH2CH2CH2N(C═O)2C6H4]; 4.77 [t, J: 8.0 Hz, 2
H, NCH2CH2CH2CH2N(C═O)2C6H4]; 5.82 [s, 2 H,
NCH2C6H4(CH3)-3]; 7.15–7.83 (m, 12 H, Ar-H); 11.54
(s, 1 H, NCHN). 13C-NMR (100.13 MHz, CDCl3, 298
K), δ: 21.36 [NCH2CH2CH2CH2N(C═O)2C6H4]; 25.59
1
2.15 [p, J: 8.0 Hz,
2
H, NCH2CH2CH2CH2N
(C═O)2C6H4]; 3.81 [t, J: 8.0 Hz, 2 H, NCH2CH2CH2
CH2N(C═O)2C6H4]; 4.77 [t, J: 8.0 Hz, 2 H, NCH2CH2
CH2CH2N(C═O)2C6H4]; 5.88 [s, 2 H, NCH2C6H5];
7.27–7.85 (m, 13 H, Ar-H); 11.69 (s, 1 H, NCHN).
13C-NMR (100.13 MHz, CDCl3, 298 K), δ: 25.61
[NCH2CH2CH2CH2N(C═O)2C6H4]; 26.66 [NCH2CH2
CH2CH2N(C═O)2C6H4]; 36.65 [NCH2CH2CH2CH2N
(C═O)2C6H4]; 46.96 [NCH2CH2CH2CH2N(C═O)2C6H4];
51.59 [NCH2C6H5]; 113.11, 113.77, 123.38, 127.27,
128.37, 129.36, 129.46, 131.45, 131.92, 132.47, and
[NCH2C6H4(CH3)-3];
(C═O)2C6H4]; 36.69 [NCH2CH2CH2CH2N(C═O)2C6H4];
46.95 [NCH2CH2CH2CH2N(C═O)2C6H4]; 51.60
26.65
[NCH2CH2CH2CH2N
[NCH2C6H4(CH3)-3]; 113.12, 113.85, 123.36, 125.35,
127.22, 128.93, 129.26, 130.10, 131.22, 131.46, 131.91,
132.41, 134.13, and 139.39 (Ar-C); 143.01 (NCHN);
168.43 [NCH2CH2CH2CH2N(C═O)2C6H4].
134.14
(Ar-C);
143.15
(NCHN);
168.43
1-[4-(1,3-Dioxoisoindolin-2-yl)butyl]-3-(4-methylbenzyl)
[NCH2CH2CH2CH2N(C═O)2C6H4].
benzimidazolium bromide (14).
According to the same
conditions and procedure as for 2, compound 14 was
synthesized from 1-(4-methylbenzyl)benzimidazole (1.3 g,
1 mmol) and N-(4-bromobutyl)phthalimide (1.65 g,
1 mmol) in DMF for 24 h at 80°C. The resulting
compound (C27H26N3O2Br: 504.42 g/mol) was purified by
crystallization in isopropyl alcohol. Yield: 33%, m.p.:
263–266°C, color: white. IR: 1544.9 (CN); 1737.7 cmÀ1
1-(2-Cyanobenzyl)-3-[4-(1,3-dioxoisoindolin-2-yl)butyl]-1H-
benzo[d]imidazol-3-ium bromide (12).
According to the
same conditions and procedure as for 2, compound 12 was
synthesized from 2-((1H-benzo[d]imidazol-1-yl)methyl)
benzonitrile (1.0 g, 1 mmol) and N-(4-bromobutyl)
phthalimide (1.21 g, 1 mmol) in DMF for 24 h at 80°C.
The product (C27H23N4O2Br: 515.4 g/mol) was purified
by crystallization in a mixture of ethyl alcohol-diethyl
ether (2:1). Yield: 58%, m.p.: 186–189°C, color: white.
IR: 1562.9 (CN); 1704.4 (C═O); 2943.9 cmÀ1 (C-H).
1H-NMR (400.13 MHz, CDCl3, 298 K), δ: 1.89 [p, J:
8.0 Hz, 2 H, NCH2CH2CH2CH2N(C═O)2C6H4]; 2.16 [p,
J: 8.0 Hz, 2 H, NCH2CH2CH2CH2N(C═O)2C6H4]; 3.80
[t, J: 8.0 Hz, 2 H, NCH2CH2CH2CH2N(C═O)2C6H4];
4.78 [t, J: 8.0 Hz, 2 H, NCH2CH2CH2CH2N(C═O)2
C6H4]; 6.17 [s, 2 H, NCH2C6H4(CN)-2]; 7.31–8.18 (m, 12
H, Ar-H); 11.56 (s, 1 H, NCHN). 13C-NMR (100.13 MHz,
CDCl3, 298 K), δ: 25.57 [NCH2CH2CH2CH2N(C═O)2
C6H4]; 26.63 [NCH2CH2CH2CH2N(C═O)2C6H4]; 36.62
[NCH2CH2CH2CH2N(C═O)2C6H4]; 47.22 [NCH2CH2
CH2CH2N(C═O)2C6H4]; 48.92 [NCH2C6H4(CN)-2];
112.03, 113.18, 113.59, 117.26, 123.36, 123.38, 127.27,
127.56, 127.72, 130.09, 131.04, 131.29, 131.91,
133.34, 134.11, 134.15, 134.59, and 135.77 (Ar-C;
C≡N); 143.44 (NCHN); 168.43 [NCH2CH2CH2CH2
N(C═O)2C6H4].
1
(C═O). H-NMR (400.13 MHz, CDCl3, 298 K), δ: 1.88
[s, 3 H, NCH2C6H4(CH3)-4]; 2.16–2.40 [m, 4 H,
NCH2CH2CH2CH2N(C═O)2C6H4]; 2.89 [t, J: 8.0 Hz, 2
H, NCH2CH2CH2CH2N(C═O)2C6H4]; 5.83 (t, J: 12.0 Hz,
2
H, NCH2CH2CH2CH2N(C═O)2C6H4]; 6.01 [s, 2
H, NCH2C6H4(CH3)-4]; 7.14–7.86 (m, 12 H, Ar-H);
11.56 (s, 1 H, NCHN). 13C-NMR (100.13 MHz, CDCl3,
298 K), δ: 21.19 [NCH2C6H4(CH3)-4]; 21.20 [NCH2CH2
CH2CH2N(C═O)2C6H4]; 21.25 [NCH2CH2CH2CH2N
(C═O)2C6H4]; 34.56 [NCH2CH2CH2CH2N(C═O)2C6H4];
51.52 [NCH2CH2CH2CH2N(C═O)2C6H4]; 51.55 [NCH2
C6H4(CH3)-4[; 113.81, 127.08, 128.29, 129.50, 130.07,
131.25, 131.41, 131.97, 132.18, 134.19, and 139.32
(Ar-C); 142.77 (NCHN); 167.70 [NCH2CH2CH2CH2
N(C═O)2C6H4].
1-[4-(1,3-Dioxoisoindolin-2-yl)butyl]-3-(4-vinylbenzyl)-1H-
benzo[d]imidazol-3-ium bromide (15).
According to the
same conditions and procedure as for 2, compound 15 was
synthesized from 1-(4-vinylbenzyl)-1H-benzo[d]imidazole
(1.4 g, 1 mmol) and N-(4-bromobutyl)phthalimide (1.69 g,
1 mmol) in DMF for 24 h at 80°C. The resulting white
compound was washed with diethyl ether and then
purified by crystallization in a mixture of ethyl alcohol-
diethyl ether (2:1). Yield: 60%, m.p.: 110–112°C, color:
cream. IR: 1557.6 (CN); 1699.3 (C═O); 2931.3
1-[4-(1,3-Dioxoisoindolin-2-yl)butyl]-3-(3-methylbenzyl)-1H-
benzo[d]imidazol-3-ium bromide (13).
According to the
same conditions and procedure as for 2, compound 13
was synthesized from 1-(3-methylbenzyl)benzimidazole
(0.82 g, 1 mmol) and N-(4-bromobutyl)phthalimide
(1.04 g, 1 mmol) in DMF for 24 h at 80°C. The product
(C27H26N3O2Br: 504.42 g/mol) was purified by
crystallization in a mixture of ethyl alcohol-diethyl ether
(2:1). Yield: 78%, m.p.: 219–221°C, color: white. IR:
1557.7 (CN); 1699.5 (C═O); 2955.1 cmÀ1 (C-H).
1
and 3069.8 cmÀ1 (C-H). H-NMR (400.13 MHz, CDCl3,
298 K), δ: 1.87 [p, J: 8.0 Hz, 2 H, NCH2CH2CH2CH2
N(C═O)2C6H4]; 2.13 [p, J: 8.0 Hz, 2 H, NCH2CH2CH2
CH2N(C═O)2C6H4]; 3.78 [t, J: 8.0 Hz, 2 H, NCH2CH2
Journal of Heterocyclic Chemistry
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S. Akkoç, V. Kayser, and İ. Ö. İlhan
Vol 000
CH2CH2N(C═O)2C6H4]; 4.75 [t, J: 8.0 Hz, 2 H, NCH2
CH2CH2CH2N(C═O)2C6H4]; 5.28 (d, J: 12 Hz, 1 H,
CH═CH2); 5.75 (d, J: 12 Hz, 1 H, CH═CH2); 5.88 [s, 2
H, NCH2C6H4(CH═CH2)-4]; 6.66 (dd, J: 8 Hz, 1 H,
CH═CH2); 7.38–7.83 (m, 12 H, Ar-H); 11.55 (s, 1 H,
NCHN). 13C-NMR (100.13 MHz, CDCl3, 298 K), δ:
25.57 [NCH2CH2CH2CH2N(C═O)2C6H4]; 26.62 [NCH2
CH2CH2CH2N(C═O)2C6H4]; 36.69 [NCH2CH2CH2CH2
N(C═O)2C6H4]; 46.93 [NCH2CH2CH2CH2N(C═O)2
C6H4]; 51.27 [NCH2C6H4(CH═CH2)-4]; 113.13, 113.86,
115.24, 123.36, 127.10, 127.23, 128.66, 131.13, 131.44,
132.89, 131.93, 134.14, 135.82, and 138.48 (Ar-C;
CH═CH2); 142.99 (NCHN); 168.43 [NCH2CH2CH2CH2
CH2N(C═O)2C6H4]; 2.97 [t, J: 8.0 Hz, 4 H, NCH2CH2N
(CH2CH2)2O]; 3.37 [t, J: 8.0 Hz, 2 H, NCH2CH2N
(CH2CH2)2O]; 3.75–3.81 [m, 6 H, NCH2CH2CH2CH2
N(C═O)2C6H4; NCH2CH2N(CH2CH2)2O]; 4.72 [tt, J:
8.0 Hz, J: 8.0 Hz, 4 H, NCH2CH2N(CH2CH2)2O];
7.62–7.83 (m, 8 H, Ar-H); 11.10 (s, 1 H, NCHN). 13C-
NMR (100.13 MHz, CDCl3, 298 K), δ: 25.54 [NCH2
CH2CH2CH2N(C═O)2C6H4]; 26.47 [NCH2CH2CH2CH2
N(C═O)2C6H4]; 36.68, 43.07, 46.87, 46.97, and 52.95
[NCH2CH2CH2CH2N(C═O)2C6H4;
NCH2CH2N(CH2
CH2)2O]; 65.65 [NCH2CH2N(CH2CH2)2O]; 113.02,
113.60, 123.37, 127.24, 127.51, 131.02, 131.25, 131.29,
131.83, 131.88, 134.12, and 134.18 (Ar-C); 143.16
(NCHN); 168.42 [NCH2CH2CH2CH2N(C═O)2C6H4].
The results of spectroscopic data for compounds 18–22
were given in previously published paper [29].
N(C═O)2C6H4].
1-[4-(1,3-Dioxoisoindolin-2-yl)butyl]-3-(naphthalen-1-
ylmethyl)-1H-benzo[d]imidazol-3-ium bromide (16).
According to the same conditions and procedure as for 2,
compound 16 was synthesized from 1-(naphthalen-1-
ylmethyl)-1H-benzo[d]imidazole (1.30 g, 1 mmol) and
N-(4-bromobutyl)phthalimide (1.42 g, 1 mmol) in DMF
for 24 h at 80°C. The resulting white compound
(C30H26N3O2Br: 540.45 g/mol) was washed with diethyl
ether and then purified by crystallization in a mixture of
ethyl alcohol-diethyl ether (2:1). Yield: 90%, m.p.:
198–200°C, color: white. IR: 1560.4 (CN); 1710.1
(C═O); 2901.6 cmÀ1 (C-H). 1H-NMR (400.13 MHz,
Cytotoxic activity studies. The cytotoxicity activity of
the compounds was determined according to our
previously established procedure [12,18,33–35]. The
DLD-1, MDA-MB-231, and HEK 293T cells were
cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum and 1%
GlutaMAX. The cells were then seeded into sterile
96-well plates at a density of 1 × 103 cells/well and
maintained at 37°C for 24 h. Cancerous cells were
exposed to 2–22 at concentrations of 0.5, 1, 2, 5, 10, 20,
50, 100, and 200 μM for 72 h. The normal human cells
were exposed to 2, 4, 5, 8, 11–13, 16, 17, 20, and 22 in the
0.001–100 μM range different concentrations for 24 h.
The plates were incubated for more than 4 h after adding
MTT stock solution (5 mg/mL) to each well.
Subsequently, the sample was dried under vacuum, and
DMSO (200 μL) was added to each well. The plate was
then shaken for 30 min on a plate rocker. GraphPad Prism
software program was then used to calculate the IC50
values. Two independent studies with triplicate data points
were conducted for all tested compounds.
CDCl3, 298 K), δ: 1.88 [p,
2
H, J:
8
Hz,
NCH2CH2CH2CH2N(C═O)2C6H4]; 2.14 [p, 2 H, J:
8.0 Hz, NCH2CH2CH2CH2N(C═O)2C6H4]; 3.80 [t, 2 H,
J: 8.0 Hz, NCH2CH2CH2CH2N(C═O)2C6H4]; 4.78 [t, 2
H, J: 8.0 Hz, NCH2CH2CH2CH2N(C═O)2C6H4]; 6.34
(s, 2 H, NCH2C10H7); 7.47–8.20 (m, 15 H, Ar-H); 11.50
(s, 1 H, NCHN). 13C-NMR (100.13 MHz, CDCl3, 298
K), δ: 25.58 [NCH2CH2CH2CH2N(C═O)2C6H4]; 26.61
[NCH2CH2CH2CH2N(C═O)2C6H4]; 36.63 [NCH2CH2
CH2CH2N(C═O)2C6H4]; 47.04 [NCH2CH2CH2CH2
N(C═O)2C6H4]; 49.64 (NCH2C10H7); 113.14, 113.95,
122.40, 123.37, 125.37, 126.63, 127.28, 127.51, 127.68,
127.83, 129.25, 130.36, 130.65, 131.44, 131.93, 133.92,
and 134.12 (Ar-C); 143.59 (NCHN); 168.40 [NCH2CH2
CH2CH2N(C═O)2C6H4].
Methods of confocal microscopy.
The confocal
(Olympus FV1000 confocal microscope with an UPLAPO
20× objective [NA 0.7]) was performed according to the
published procedure [12,18].
1-[4-(1,3-Dioxoisoindolin-2-yl)butyl]-3-(2-morpholinoethyl)-
1H-benzo[d]imidazol-3-ium bromide (17). According to the
Acknowledgments. This study was financially supported by
TUBITAK (1059B141400496), Erciyes University Research
Fund (FDK-2014-5091), and the School of Pharmacy, The
University of Sydney. We would like to thank the Key Centre
for Polymers and Colloids, The University of Sydney, for
donating some of the cells. We thank Associate Professor Nial
Wheate (The University of Sydney) for critical review of the
manuscript.
same conditions and procedure as for 2, compound 17 was
synthesized from 1-(2-morpholinoethyl)-1H-benzo[d]imi
dazole (1.0 g, 1 mmol) and N-(4-bromobutyl)phthalimide
(1.22 g, 1 mmol) in DMF for 24 h at 80°C. The resulting
off-white compound (C25H29N4O3Br: 513.43 g/mol) was
first washed with diethyl ether and then purified by
crystallization from isopropyl alcohol. Yield: 79%, color:
white, m.p.: 142–144°C. IR: 1564.8 (CN); 1705.7 (C═O);
1
2942.4 cmÀ1 (C-H). H-NMR (400.13 MHz, CDCl3, 298
CONFLICT OF INTEREST
K), δ: 1.87 [p, J: 8.0 Hz, 2 H, NCH2CH2CH2CH2
N(C═O)2C6H4]; 2.13 [p, J: 8.0 Hz, 2 H, NCH2CH2CH2
The authors have no conflict of interest to declare.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and In Vitro Anticancer Evaluation
[16] Azam, M.; Khan, A. A.; Al-Resayes, S. I.; Islam, M. S.;
AUTHOR CONTRIBUTIONS
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S. Akkoç conducted all experiments and data analysis
under the supervision of V. Kayser and İ. Ö. İlhan and
wrote the paper. V. Kayser designed the study, analyzed
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet