Novel amine-functionalized benzimidazolium salts: Synthesis, characterization, bioactivity, and molecular docking studies

Article abstract of DOI:10.1016/j.molstruc.2020.127802

A series of amine-tethered benzimidazolium salts were synthesized by the reactions between 1-(1-methyl-2-dimethylaminoethyl)benzimidazole and various alkyl halides. The characterization of the newly synthesized salts was done by spectroscopic methods. Also, 2e, 2f, and 2h have been docked into the catalytic active site hCA I, hCA II, AChE, BChE, and α-glycosidase enzymes. We have identified high binding affinity and explained inhibition mechanism of the compounds against the enzymes. These novel amine-functionalized benzimidazolium salts derivatives were good inhibitor compounds of the α-glycosidase, hCA I and II isoforms, and both cholinesterase enzymes with K_i values in the range of 0.63 ± 0.05–3.63 ± 0.83 nM for α-glycosidase, 8.42 ± 1.03–27.04 ± 3.74 nM for hCA I, 7.94 ± 0.74–21.82 ± 5.81 nM for hCA II, 136.38 ± 19.55–247.34 ± 34.06 nM for BChE, and 124.24 ± 13.94–283.55 ± 54.06 nM for AChE, respectively. Among the inhibitors, 2e, 2e, 2f, 2f, and 2h were obtained to be the excellent inhibitors with Ki values of 8.42 ± 1.03, 7.94 ± 0.74, 124.24 ± 13.94, 136.38 ± 19.55, and 0.63 ± 0.05 nM for hCA I, hCA II, AChE, BChE, α-glycosidase enzymes, respectively. The ability to model some metabolic enzymes receptors and theirs inhibitors in silico are important because they can save valuable resources and help to rationalize the mode of binding, and to design better inhibitors.

Full text of DOI:10.1016/j.molstruc.2020.127802

Journal Pre-proof
Novel amine-functionalized benzimidazolium salts: Synthesis, characterization,
bioactivity, and molecular docking studies
Murat Yiğit, Beyhan Yiğit, Parham Taslimi, İsmail Özdemir, Muhammet Karaman,
İlhami Gulçin
PII:
S0022-2860(20)30126-5
DOI:
https://doi.org/10.1016/j.molstruc.2020.127802
MOLSTR 127802
Reference:
To appear in: Journal of Molecular Structure
Received Date: 15 December 2019
Revised Date: 13 January 2020
Accepted Date: 25 January 2020
Please cite this article as: M. Yiğit, B. Yiğit, P. Taslimi, İ. Özdemir, M. Karaman, İ. Gulçin, Novel amine-
functionalized benzimidazolium salts: Synthesis, characterization, bioactivity, and molecular docking
studies, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.127802.
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Author Contributions
The compounds were synthesised by M. Yiğit, B. Yiğit and İ. Özdemir. The inhibition
properties of compounds were investigated by P. Taslimi and İ. Gulçin. The docking studies
were performed by M. Karaman. The manuscript was written and corrected by M. Yiğit and
P. Taslimi. All the authors have given approval to the final version of the manuscript.

Novel amine-functionalized benzimidazolium salts: Synthesis, characterization, bioactivity, and
molecular docking studies
Murat Yiğit, Beyhan Yiğit, Parham Taslimi, İsmail Özdemir, Muhammet Karaman, İlhami
Gulçin
N
N
X-
+)
N
Rn

Novel amine-functionalized benzimidazolium salts: Synthesis, characterization,
bioactivity, and molecular docking studies
Murat Yiğit,1 Beyhan Yiğit,1 Parham Taslimi2, İsmail Özdemir3, Muhammet Karaman4,
İlhami Gulçin5
1Department of Chemistry, Faculty of Science and Art, Adıyaman University, 02040
Adıyaman, Turkey
2Department of Biotechnology, Faculty of Science, Bartin University, 74100 - Bartin, Turkey
3Department of Chemistry, Faculty of Science and Art, İnönü University, 44280 Malatya,
Turkey
4Department of Molecular Biology and Genetics, Faculty of Arts and Science, Kilis 7 Aralik
University, 79000 Kilis, Turkey
5Department of Chemistry, Faculty of Science, Atatürk University, 25240 - Erzurum, Turkey
Corresponding author e-mail address: myigit@adiyaman.edu.tr
Abstract
A series of amine-tethered benzimidazolium salts were synthesized by the reactions between
1-(1-methyl-2-dimethylaminoethyl)benzimidazole and various alkyl halides. The
characterization of the newly synthesized salts was done by spectroscopic methods. Also, 2e,
2f, and 2h have been docked into the catalytic active site hCA I, hCA II, AChE, BChE, and α-
glycosidase enzymes. We have identified high binding affinity and explained inhibition
mechanism of the compounds against the enzymes. These novel amine-functionalized
benzimidazolium salts derivatives were good inhibitor compounds of the α-glycosidase, hCA
I and II isoforms, and both cholinesterase enzymes with Ki values in the range of 0.63±0.05–
3.63±0.83 nM for α-glycosidase, 8.42±1.03-27.04±3.74 nM for hCA I, 7.94±0.74-21.82±5.81
nM for hCA II, 136.38±19.55-247.34±34.06 nM for BChE, and 124.24±13.94-283.55±54.06
nM for AChE, respectively. Among the inhibitors, 2e, 2e, 2f, 2f, and 2h were obtained to be
the excellent inhibitors with Ki values of 8.42±1.03, 7.94±0.74, 124.24±13.94, 136.38±19.55,
and 0.63±0.05 nM for hCA I, hCA II, AChE, BChE, α-glycosidase enzymes, respectively.
The ability to model some metabolic enzymes receptors and theirs inhibitors in silico are
important because they can save valuable resources and help to rationalize the mode of
binding, and to design better inhibitors.

Keywords: N-Heterocyclic carbene; benzimidazolium salt; enzyme inhibition; molecular
docking
1. Introduction
The benzimidazolium salts are the quaternary ammonium compounds, which have attracted
much attention in recent years owing to their applications in different fields such as
coordination, material and medicinal chemistry [1-3]. These salts are important precursors for
N-heterocyclic carbenes (NHCs), which are popular as ligands in organometallic chemistry
due to their strong σ-donating and weak π-accepting features, and facile synthesis [4]. NHC
ligands can form stable complexes with almost all transition metals and the complexes are
generally used in catalytic, biomedical, luminescent and functional materials applications [5-
7]. A variety of metal complex of N-heterocyclic carbenes has been studied for medical
applications. Especially, Ag and Au complexes of NHC ligands have been extensively
explored as efficient antibacterial and anticancer agents against numerous bacterial species
and various cancer cell lines [8-12]. Also, the benzimidazolium salts have a variety of
numerous applications such as ionic liquids, fluorescence sensors, batteries and solar cells
[13-16]. In addition, these salts have shown a broad range of biological activity including
antibacterial, antitumor and antimicrobial activities [17-19]. Recently, benzimidazole,
imidazole and imidazoline salts have been explored as enzyme inhibitors by different research
groups, and studies showed that these salts possess inhibitory potential against metabolic
enzymes [20-24].
Carbonic anhydrases (CAs) play a crucial role in different physiological and
biochemical processes such as ureagenesis, photosynthesis, carboxylation, respiration,
calcification, bone resorption, and pH regulation, which are indirectly or directly associated
with CO2 hydration reaction [25,26]. Besides the important role of CA isoenzymes in carrying
out several biochemical activities, the deregulated activity of CA isoenzymes is also
recognized to be associated with diverse CA related pathologies; like overexpression of
ubiquitous isoenzyme hCA I is associated with retinal edema and cerebral; the deregulated
activity of hCA II is associated with glaucoma, epilepsy, and edema; membrane-bound
isoform hCA IV is associated with glaucoma, retinitis pigmentosa, stroke; whereas
transmembrane isoform hCA IX is associated with abnormal growth of tumor cells [27,28].
Indeed, CA isoforms offer a significant drug aim and the synthesis of isoenzyme-selective
inhibitors is a great approach for obtaining a drug with the least side effects. The CA
inhibitors (CAIs) are utilized as anti-glaucoma, anti-epileptics, and diuretics agents but new

applications in the management of neuropathic pain, cancer, migraine, sleep apnea, lowering
intracranial pressure, and cerebral ischemia were recently reported [29,30].
Cholinesterase inhibitor (ChEI) drugs are approved for the symptomatic treatment of
Alzheimer's disease (AD) dementia and these agents (donepezil; rivastigmine; galantamine)
have consistently demonstrated modest improvement in cognitive outcomes [31]. AChE
enzyme has a significant aim for rational drug design and the discovery of mechanism-based
inhibitor compounds for the therapy of peripheral and central neural system diseases, like
glaucoma, schizophrenia, myasthenia gravis, and AD. Thus, the utilize of AChE inhibitors
(AChEIs) is considered a therapeutically related strategy for these diseases and justifies the
search for new leads [32,33]. The effects of antidiabetic drugs happen via different
mechanisms of action. Antidiabetic drugs can lower blood glucose levels by affecting insulin
sensitivity, carbohydrate metabolism, insulin secretion, and lipid metabolism. The α-
glycosidase enzyme plays a significant role in the absorption of glucose units in the
gastrointestinal tract. Inhibition of α-glycosidase enzyme can decrease blood glucose levels
and prevents postprandial hyperglycemia in patients with type 2 diabetes mellitus [34,35].
We have previously reported glycosidase inhibitory properties of imidazolinium salts
[23, 24]. These salts demonstrated the good inhibition effects against metabolic enzymes. In
this work, we wish to report the synthesis, molecular docking, and metabolic enzymes
inhibitory properties of benzimidazolium salts bearing both amino and benzyl groups (2a-i).
2. Experimental
2.1. Materials and methods
All reactions for the preparation of the benzimidazolium salts were carried out under air. All
commerical chemicals were purchased from Sigma-Aldrich and used as received. The
benzimidazole derivatives (1, 2a and 2f) were synthesized according to reported procedures
1
[36,17]. H-NMR and 13C-NMR spectra were recorded with a Varian AS 400 Merkur
spectrometer operating at 400 MHz (1H), 100 MHz (13C) in CDCl3 with tetramethylsilane as
an internal reference. Coupling constants (J values) are given in Hertz. NMR multiplicities are
abbreviated as follows: s = singlet, bs = broad singlet, d = doublet, t = triplet and m = multiplet
signal. FT-IR spectra were recorded on ATR unit in the range 400-4000 cm-1 on Perkin Elmer
Spectrum 100. Melting points were measured in open capillary tubes with Stuart SMP 40
melting point apparatus and uncorrected. Elemental analyses were performed at Inonu
University research center.

2.2. Synthesis of 1-(1-methyl-2-dimethylaminoethyl)benzimidazole, 1
To a solution of benzimidazole (2.36 g, 20 mmol) in ethanol (30 mL), potassium hydroxide
(0.56 g, 10 mmol) was added and stirred at room temperature for 1 h. After this time, 2-
chloro-N,N-dimethylpropylamine (2.43 g, 20 mmol) was added slowly and the resulting
mixture was refluxed for 6 h. After reaction completed, the reaction mixture was cooled to
room temperature. The formed inorganic salt was filtered through filter paper and the solvent
was removed under reduced pressure to give the product 1. The crude product was
recrystallized from ethanol/diethyl ether.
2.3. General procedure for the preparation of the benzimidazolium salts, 2
To a solution of 1-(1-methyl-2-dimethylaminoethyl)benzimidazole (9.85 mmol) in N,N-
dimethylformamide (DMF) (5 mL), alkyl chloride (9.90 mmol) was added. The reaction
mixture was stirred at room temperature for 2 h and heated at 80 oC for 18 h. After reaction
completed, the reaction mixture was cooled to room temperature. Diethyl ether (10 mL) was
added to obtain a white crystalline solid, which was filtered off. The solid was washed with
diethyl ether and dried under vacuum. The crude product was recrystallized from
ethanol/diethyl ether.
2.3.1. 1-(1-Methyl-2-dimethylaminoethyl)-3-benzylbenzimidazolium chloride, 2a
This compound was prepared according to the literature [17].
2.3.2. 1-(1-Methyl-2-dimethylaminoethyl)-3-(2-methylbenzyl)benzimidazolium chloride, 2b
This compound was prepared from 1-(1-methyl-2-dimethylaminoethyl)benzimidazole (2 g,
9.85 mmol) and 2-methylbenzyl chloride (1.3 g, 9.90 mmol) in DMF (5 mL).
Yield: 95%, m.p.: 179-180 oC, IR: ν(NCN) = 1562 cm-1. 1H NMR (CDCl3) δ: 1.14 (d, 3H, J =
8.0 Hz, CH(CH3)CH2N(CH3)2), 2.28 (s, 6H, CH(CH3)CH2N(CH3)2), 3.22-3.28 (m, 1H,
CH(CH3)CH2N(CH3)2), 4.48 and 4.71 (d, 2H, J = 12.0 Hz, CH(CH3)CH2N(CH3)2), 5.81 and
5.99 (d, 2H, J = 16.0 Hz, CH2C6H4CH3-2), 2.40 (s, 3H, CH2C6H4CH3-2), 7.11-7.73 (m, 8H,
Ar–H), 11.41 (s, 1H, NCHN). 13C NMR (CDCl3) δ: 9.5, 40.3, 49.6, 50.1
(CH(CH3)CH2N(CH3)2), 19.4 (CH2C6H4CH3-2), 57.9 (CH2C6H4CH3-2), 112.9, 113.8, 126.6,
126.7, 126.8, 128.3, 129.3, 130.9, 131.2, 131.3, 131.6, 136.8 (Ar-C), 145.2 (NCHN). Anal.
Calcd for C20H26N3Cl (%): C, 69.87; H, 7.57; N, 12.23. Found: C, 69.84; H, 7.54; N, 12.26.
2.3.3. 1-(1-Methyl-2-dimethylaminoethyl)-3-(3-methylbenzyl)benzimidazolium chloride (2c).

This compound was prepared from 1-(1-methyl-2-dimethylaminoethyl)benzimidazole (2 g,
9.85 mmol) and 3-methylbenzyl chloride (1.3 g, 9.90 mmol) in DMF (5 mL).
o
1
Yield: 87%, m.p.: 192-193 C, IR: ν(NCN) = 1562 cm-1. H NMR (CDCl3) δ: 1.13 (d, 3H, J =
4.0 Hz, CH(CH3)CH2N(CH3)2), 2.31 (s, 6H, CH(CH3)CH2N(CH3)2), 3.21-3.30 (m, 1H,
CH(CH3)CH2N(CH3)2), 4.48, 4.52, 4.66 and 4.70 (d, 2H, J = 4.0 Hz, CH(CH3)CH2N(CH3)2),
5.77 and 5.88 (d, 2H, J = 12.0 Hz, CH2C6H4CH3-3), 2.31 (s, 3H, CH2C6H4CH3-3), 7.13-7.77
(m, 8H, Ar–H), 11.33 (s, 1H, NCHN). 13C NMR (CDCl3) δ: 9.6, 40.3, 49.6, 51.3
(CH(CH3)CH2N(CH3)2), 21.4 (CH2C6H4CH3-3), 57.9 (CH2C6H4CH3-3), 113.0, 113.7, 125.0,
126.8, 126.9, 128.6, 129.1, 129.8, 130.9, 131.7, 133.0, 139.2 (Ar-C), 144.3 (NCHN). Anal.
Calcd for C20H26N3Cl (%): C, 69.87; H, 7.57; N, 12.23. Found: C, 69.91; H, 7.54; N, 12.26.
2.3.4. 1-(1-Methyl-2-dimethylaminoethyl)-3-(4-methylbenzyl)benzimidazolium chloride, 2d
This compound was prepared from 1-(1-methyl-2-dimethylaminoethyl)benzimidazole (2 g,
9.85 mmol) and 4-methylbenzyl chloride (1.3 g, 9.90 mmol) in DMF (5 mL).
o
1
Yield: 89%, m.p.: 151-152 C, IR: ν(NCN) = 1561 cm-1. H NMR (CDCl3) δ: 1.50 (d, 3H, J =
8.0 Hz, CH(CH3)CH2N(CH3)2), 2.26 (s, 6H, CH(CH3)CH2N(CH3)2), 3.65-3.70 (m, 1H,
CH(CH3)CH2N(CH3)2), 4.93 and 5.17 (d, 2H, J = 12.0 Hz, CH(CH3)CH2N(CH3)2), 5.65 and
5.89 (d, 2H, J = 16.0 Hz, CH2C6H4CH3-4), 2.29 (s, 3H, CH2C6H4CH3-4), 7.09-7.53 (m, 8H,
Ar–H), 11.24 (s, 1H, NCHN). 13C NMR (CDCl3) δ: 13.2, 46.5, 47.9, 51.8
(CH(CH3)CH2N(CH3)2), 21.2 (CH2C6H4CH3-4), 58.1 (CH2C6H4CH3-4), 113.2, 115.6, 123.9,
127.3, 128.2, 129.4, 129.8, 130.4, 132.0, 133.5, 139.3, 140.9 (Ar-C), 143.5 (NCHN). Anal.
Calcd for C20H26N3Cl (%): C, 69.87; H, 7.57; N, 12.23. Found: C, 69.83; H, 7.59; N, 12.25.
2.3.5. 1-(1-Methyl-2-dimethylaminoethyl)-3-(3,4-dimethylbenzyl)benzimidazolium chloride,
2e
This compound was prepared from 1-(1-methyl-2-dimethylaminoethyl)benzimidazole (2 g,
9.85 mmol) and 3,4-dimethylbenzyl chloride (1.52 g, 9.90 mmol) in DMF (5 mL).
o
1
Yield: 76%, m.p.: 189-190 C, IR: ν(NCN) = 1560 cm-1. H NMR (CDCl3) δ: 1.15 (d, 3H, J =
8.0 Hz, CH(CH3)CH2N(CH3)2), 2.21 (s, 6H, CH(CH3)CH2N(CH3)2), 3.28-3.37 (m, 1H,
CH(CH3)CH2N(CH3)2), 4.53, 4.56, 4.73 and 4.76 (d, 2H, J = 8.0 Hz, CH(CH3)CH2N(CH3)2),
5.72 and 5.81 (d, 2H, J = 12.0 Hz, CH2C6H3(CH3)2-3,4), 2.34 (s, 6H, CH2C6H3(CH3)2-3,4),
13
7.08-7.78 (m, 7H, Ar–H), 11.30 (s, 1H, NCHN). C NMR (CDCl3) δ: 9.7, 40.3, 49.5, 51.2
(CH(CH3)CH2N(CH3)2), 19.5, 19.8 (CH2C6H3(CH3)2-3,4), 58.1 (CH2C6H3(CH3)2-3,4), 113.0,

113.7, 113.8, 125.4, 126.7, 126.8, 129.2, 130.3, 130.4, 131.0, 131.8, 137.7 (Ar-C), 144.2
(NCHN). Anal. Calcd for C21H28N3Cl (%): C, 70.49; H, 7.83; N, 11.75. Found: C, 70.43; H,
7.86; N, 11.71.
2.3.6. 1-(1-Methyl-2-dimethylaminoethyl)-3-(2,4,6-trimethylbenzyl)benzimidazolium chloride,
2f
This compound was prepared according to the literature [17].
2.3.7. 1-(1-Methyl-2-dimethylaminoethyl)-3-(4-tert-butylbenzyl)benzimidazolium bromide, 2g
This compound was prepared from 1-(1-methyl-2-dimethylaminoethyl)benzimidazole (2 g,
9.85 mmol) and 4-tert-butylbenzyl bromide (2.24 g, 9.90 mmol) in DMF (5 mL).
o
1
Yield: 82%, m.p.: 186-187 C, IR: ν(NCN) = 1563 cm-1. H NMR (CDCl3) δ: 1.19 (d, 3H, J =
8.0 Hz, CH(CH3)CH2N(CH3)2), 2.39 (s, 6H, CH(CH3)CH2N(CH3)2), 3.34-3.38 (m, 1H,
CH(CH3)CH2N(CH3)2), 4.62, 4.65, 4.77 and 4.80 (d, 2H, J = 8.0 Hz, CH(CH3)CH2N(CH3)2),
5.75 and 5.84 (d, 2H, J = 12.0 Hz, CH2C6H4(CH3)3C-4), 1.26 (s, 9H, CH2C6H4(CH3)3C-4),
13
7.35-7.87 (m, 8H, Ar–H), 11.00 (s, 1H, NCHN). C NMR (CDCl3) δ: 9.9, 40.4, 49.2, 51.1
(CH(CH3)CH2N(CH3)2), 31.2 (CH2C6H4(CH3)3C-4), 34.7 (CH2C6H4(CH3)3C-4), 58.2
(CH2C6H4(CH3)3C-4), 113.2, 113.7, 126.2, 126.3, 126.9, 127.1, 127.9, 128.1, 129.8, 130.9,
131.7, 143.4 (Ar-C), 152.3 (NCHN). Anal. Calcd for C23H32N3Br (%): C, 64.19; H, 7.44; N,
9.77. Found: C, 64.26; H, 7.46; N, 9.73.
2.3.8. 1-(1-Methyl-2-dimethylaminoethyl)-3-phenylethylbenzimidazolium chloride, 2h
This compound was prepared from 1-(1-methyl-2-dimethylaminoethyl)benzimidazole (2 g,
9.85 mmol) and 2-phenylethyl chloride (1.30 g, 9.90 mmol) in DMF (5 mL).
o
1
Yield: 81%, m.p.: 148-149 C, IR: ν(NCN) = 1557 cm-1. H NMR (CDCl3) δ: 1.08 (d, 3H, J =
4.0 Hz, CH(CH3)CH2N(CH3)2), 2.30 (s, 6H, CH(CH3)CH2N(CH3)2), 3.24-3.29 (m, 1H,
CH(CH3)CH2N(CH3)2), 4.46, 4.50, 4.64 and 4.67 (d, 2H, J = 4.0 Hz, CH(CH3)CH2N(CH3)2),
3.38 (t, 2H, J = 8.0 Hz, CH2CH2C6H5), 4.87-4.92 (m, 2H, CH2CH2C6H5), 7.18-7.76 (m, 9H,
Ar–H), 11.01 (s, 1H, NCHN). 13C NMR (CDCl3) δ: 9.8, 40.3, 49.4, 58.1
(CH(CH3)CH2N(CH3)2), 35.8 (CH2CH2C6H5), 48.6 (CH2CH2C6H5), 112.8, 113.1, 126.7,
126.8, 127.3, 128.8, 128.9, 131.1, 131.3, 136.1 (Ar-C), 143.9 (NCHN). Anal. Calcd for
C20H26N3Cl (%): C, 69.87; H, 7.57; N, 12.23. Found: C, 69.82; H, 7.59; N, 12.20.
2.3.9. 1-(1-Methyl-2-dimethylaminoethyl)-3-benzhydrylbenzimidazolium bromide, 2i

This compound was prepared from 1-(1-methyl-2-dimethylaminoethyl)benzimidazole (2 g,
9.85 mmol) and benzhydryl bromide (2.44 g, 9.90 mmol) in DMF (5 mL).
o
1
Yield: 88%, m.p.: 213-215 C, IR: ν(NCN) = 1533 cm-1. H NMR (CDCl3) δ: 1.11 (d, 3H, J =
4.0 Hz, CH(CH3)CH2N(CH3)2), 2.17 (s, 6H, CH(CH3)CH2N(CH3)2), 3.11 (bs, 1H,
CH(CH3)CH2N(CH3)2), 4.63-4.79 (m, 2H, CH(CH3)CH2N(CH3)2), 7.42 (s, 1H, CH(C6H5)2),
13
7.34-7.88 (m, 14H, Ar–H), 9.95 (s, 1H, NCHN). C NMR (CDCl3) δ: 9.6, 40.1, 49.5, 57.9
(CH(CH3)CH2N(CH3)2), 66.2 (CH(C6H5)2), 113.4, 114.5, 126.8, 127.0, 128.4, 128.7, 129.4,
129.5, 130.8, 132.1, 135.2, 135.3 (Ar-C), 143.5 (NCHN). Anal. Calcd for C25H28N3Br (%): C,
66.67; H, 6.22; N, 9.33. Found: C, 66.74; H, 6.25; N, 9.30.
2.4. Bioactivity Studies
Both hCA isoenzyme inhibition effects are described in Verpoorte et al. [37] and were
spectrophotometrically recorded at 348 nm using p-nitrophenyl acetate substrate (PNA) [38].
On the other hand, the AChE and BChE inhibitory effects of the complexes were determined
according to Ellman’s procedure [39]. They were recorded spectrophotometrically at 412 nm
using acetylthiocholine iodide and butyrylcholine iodide as substrates for the enzymatic
reaction according to previous study [40]. The 5,5’-dithio-bis (2-nitro-benzoic) acid
compound was used as second substrtate to measure cholinesterases activies [41]. In addition,
the inhibitory effect of these complexes on α-glycosidase enzyme activity was carried out
using the p-nitrophenyl-D-glycopyranoside (p-NPG) substrate according to the analysis of
Tao et al. [42]. First, 200 ꢀL of phosphate buffer was mixed with 40 ꢀL of homogenate
solution in phosphate buffer (0.15 U/mL, pH 7.4). Furthermore, after preincubation, 50 µL p-
NPG in phosphate buffer (5 mM, pH 7.4) was added and incubated again at 30°C.
Absorbances were measured spectrophotometrically at 405 nm according to previous studies
[43,44].
2.5. In Silico Studies
The Small Drug Discovery Suites version 2019-3 (Schrödinger, LLC, USA) was used to
perform in silico studies of most active novel compounds against hCA I, hCA II, AChE,
BChE, and α-glycosidase enzymes. Procedures for protein and ligand preparation, binding site
prediction and induced-fit docking were similar to those reported recently [45-48]. X-ray
crystal structures of hCA I, hCA II, AChE, BChE, and α-glycosidase (PDB code: 4WR7,
5AML, 4M0E, 5NN0 and 3L4Y, respectively) were used in silico studies because they had

best resolution and good percentile ranks. The crystal structures have been prepared with
steps including assigning bond order and charges, adding hydrogen atoms, filling missing side
chains, ionizing amino acids, removing water molecules and minimizing energy and
optimizing geometry, respectively. Fallowing protein preparation, binding site of all crystal
structures was predicted using the default parameter of SiteMap module. SiteScore and
Druggability Score (Dscore) of binding sites were also calculated. Most active novel
compounds have been prepared in the steps including sketching 2D structure, producing 3D
structure, and preparing correct molecular geometries and protonation state. Fallowing,
prepared most active compounds were docked in the steps including generating grid box,
trimming side chains, and refining ligands into the predicted catalytic active site of AChE,
BChE, hCA I, hCA II and α-glycosidase enzymes. In addition, the docking methodology was
tested by re-docking co-crystallized ligand of receptors.
3. Results and discussion
3.1. Synthesis of benzimidazolium salts
The unsymmetrical benzimidazolium salts are generally prepared by the stepwise N-
alkylation of benzimidazole, which is low cost starting material. In this study, the
benzimidazolium salts 2a-i were synthesized by the two-step procedure as shown in Scheme
1. Firstly, the benzimidazole reacted with potassium hydroxide in ethanol to afford the
benzimidazolide anion, which was subsequently converted to 1-(1-methyl-2-
dimethylaminoethyl)benzimidazole by alkylation with 2-chloro-N,N-dimethylpropylamine.
Then, 1-(1-methyl-2-dimethylaminoethyl)benzimidazole were reacted with variety alkyl
halides in DMF to obtain the targeted benzimidazolium salts 2a-i (Scheme 1). After
purification, unsymmetrical benzimidazolium salts 2a-i were isolated as white solids in good
yields of 76-95%. These salts are soluble in the polar solvents like water, methanol and
ethanol, and are air- and moisture stable both in the solid state and in solution. The structures
1
of these salts have been fully identified by H and 13C NMR spectroscopy, FTIR and
elemental analysis, and their melting points were determined. All results were consistent with
the proposed structure. The formation of benzimidazolium salts were confirmed by the
characteristic chemical shift values of NCHN protons as singlets between 9.55 and 11.41 ppm
1
in H NMR spectroscopy and the characteristic chemical shift values of NCHN carbons
between 143.5 and 153.3 ppm in 13C NMR spectroscopy. These downfield signals observed in
1
13
the H NMR and C NMR spectra of 2a-i indicate the formation of benzimidazolium salts
and their high acidity. The IR data for 2a-i clearly indicate the presence of the –C=N- group

with a ν(C=N) vibration between 1533 and 1563 cm-1. The NMR and IR values were similar to
those found for other benzimidazolium salts [49]. The results obtained from the elemental
analysis of the benzimidazolium salts 2a-i are in agreement with the theoretical requirements
of their structures. The other benzimidazolium salts (2a and 2f) were previously reported by
our group, however, for a comparison of the influence of substituents on phenyl group on the
enzyme inhibition activity, benzimidazolium salts 2a and 2f were also used in this study.
N
N
N
H
N
N
N
N
Cl
RX
+) X-
N
DMF, 18 h, 80 oC
N
KOH, EtOH, 6h, 76 oC
R
1
2a-i
N
N
N
N
N
N
N
N
+)
N
N
N
Cl-
+)
+) Cl-
N
Cl-
Cl-
+) Cl-
N
+)
N
N
2c
2a
2b
2d
2e
N
Cl-
N
N
N
Cl-
N
N
+)
N
N
+)
N
N
Br-
+)
+) Br-
N
N
2g
2i
2f
2h
Scheme 1. Synthesis of benzimidazolium salts
3.2. Bioactivity results
3.2.1. hCA I and II isoenzymes inhibition results
The results presented in Table 1 indicate that novel benzimidazolium salts (2a-i) derivatives
had effective inhibition profile against slow cytosolic hCA I isoform. The hCA I isoform was
inhibited by novel benzimidazolium salts in low nanomolar levels. Ki of novel
benzimidazolium salts differed between 8.42±1.03 and 27.04±3.74 nM. On the other hand,

acetazolamide (AZA), considered being a broad-specificity CA inhibitor owing to its
widespread inhibition of CAs, showed Ki value of 51.83±7.83 nM against hCA I. Among
novel
benzimidazolium
salts,
1-(1-methyl-2-dimethylaminoethyl)-3-(3,4-
dimethylbenzyl)benzimidazolium chloride, 2e and 1-(1-methyl-2-dimethylaminoethyl)-3-(4-
methylbenzyl)benzimidazolium chloride, 2d were obtained to be the excellent hCA I inhibitor
with Ki of 8.42±1.03 and 10.82±0.93 nM, respectively. The hCA I inhibition effects of novel
benzimidazolium salts (2a-i) derivatives were found to be greater than that of acetazolamide,
which was a clinically standard CA inhibitor (Table 1 and Figure 1). For hCA I, IC50 values of
AZA as positive control and some novel benzimidazolium salts (2a-i) derivatives the
following order: 2e (7.47 nM, r2: 0.9925) < 2b (9.71 nM, r2: 0.9407) < 2d (10.16 nM, r2:
0.9733) < 2c (10.53 nM, r2: 0.9889) < 2a (13.83 nM, r2: 0.9736) < AZA (57.36 nM, r2:
0.9790). Against the physiologically dominant isoform hCA II, the novel benzimidazolium
salts (2a-i) derivatives demonstrated Kis varying from 7.94±0.74 to 21.82±5.81 µM (Table 1
and Figure 1). These compounds had high inhibition effects on hCA II. On the other hand,
standard compound AZA showed Ki of 60.77±14.07 nM against hCA II. The 1-(1-methyl-2-
dimethylaminoethyl)-3-(3,4-dimethylbenzyl)benzimidazolium chloride (2e) and 1-(1-methyl-
2-dimethylaminoethyl)-3-(2-methylbenzyl)benzimidazolium chloride, (2b) had shown the
most inhibition effect with Ki values of 7.94±0.74 and 8.96±1.73 nM, respectively. For hCA
II, IC50 values of AZA as positive control and some novel benzimidazolium salts (2a-i)
derivatives the following order: 2b (6.44 nM, r2: 0.9588) < 2e (7.47 nM, r2: 0.9925) < 2a
(8.76 nM, r2: 0.9910) < 2d (8.83 nM, r2: 0.9667) < AZA (64.08 nM, r2: 0.9903). The most
investigated CAIs are those possessing a sulfonamide/sulfamate zinc binder group, of which
topiramate and acetazolamide are two clinically utilized representatives [50].
3.2.2. Cholinesterase inhibition results
All of novel benzimidazolium salts (2a-i) derivatives had significantly higher AChE
inhibitory activity than that of standard AChE inhibitors such as Tacrine. Furthermore, the Ki
values of novel benzimidazolium salts and tacrine are summarized in Table 1. As can be seen
from the results obtained in Table 1 and Figure 1, novel benzimidazolium salts effectively
inhibited AChE, with Ki values in the range of 124.24±13.94 to 283.55±54.06 nM. However,
all of novel benzimidazolium salts had almost similar inhibition profiles. Currently,
cholinesterase inhibitor compounds are the most popular therapy for AD with diverse degrees
of efficacy in patients’ memory and cognitive functions. These drugs have been recorded to
decrease systemic pro-inflammatory cytokine production via the cholinergic anti-

inflammatory pathway, to exert anti-inflammatory effects in peripheral blood mononuclear
cells of AD patients, and to have inhibitory effects on Aβ oligomer-induced inflammation in
vivo and in vitro by inhibiting microglial activation [51,52]. The most active 1-(1-methyl-2-
dimethylaminoethyl)-3-(2,4,6-trimethylbenzyl)benzimidazolium chloride, 2f showed Ki
values of 124.24±13.94 nM. For AChE, IC50 values of TAC as positive control and some
novel benzimidazolium salts (2a-i) derivatives the following order: 2f (152.84 nM, r2: 0.9937)
< 2h (198.30 nM, r2: 0.9770) < 2e (225.90 nM, r2: 0.9789) < 2g (230.77 nM, r2: 0.9485) <
TAC (438.43 µM, r2: 0.9348). For BChE, IC50 values of TAC as positive control and some
novel benzimidazolium salts (2a-i) derivatives the following order: 2f (187.45 nM, r2: 0.9581)
< 2h (190.26 nM, r2: 0.9915) < 2c (193.66 nM, r2: 0.9811) < 2d (197.36 nM, r2: 0.9731) <
TAC (382.75 µM, r2: 0.9862). Additionally, the novel benzimidazolium salts (2a-i)
derivatives effectively inhibited BChE, with Ki values in the range of 136.38±19.55 to
247.34±34.06 nM. However, all of these compounds had almost similar inhibition profiles.
The most active (2h) showed Ki values of 0.63±0.05 nM.
3.2.3. α-Glycosidase inhibition results
For enzyme glycosidase, novel benzimidazolium salts (2a-i) derivatives have IC50 values in
the range of 0.55-2.67 nM and Ki in the range of 0.63±0.05-2.95±0.43 µM (Table 1 and Fig.
1). The results have clearly documented that all of these derivatives have shown the inhibitory
effects of α-glycosidase efficient acarbose (IC50: 3.87 nM) as a standard glycosidase inhibitor.
In fact, the most effective Ki values of 1-(1-methyl-2-dimethylaminoethyl)-3-
phenylethylbenzimidazolium chloride (2h) and 1-(1-methyl-2-dimethylaminoethyl)-3-(3,4-
dimethylbenzyl)benzimidazolium chloride (2e) complexes were with Ki values of 0.63±0.05
and 0.85±0.11 nM, respectively. For α-glycosidase, IC50 values of ACR as positive control
and novel benzimidazolium salts (2a-i) derivatives the following order: 2h (0.55 nM, r2:
0.9943) < 2e (0.74 nM, r2: 0.9285) < 2c (0.83 nM, r2: 0.9784) < 2g (0.96 nM, r2: 0.9880) < 2a
(0.98 nM, r2: 0.9638) < Acarbose (3.87 nM, r2: 0.9738). Diabetes mellitus is a prevalent
degenerative disorder and characterized by high blood glucose levels. Indeed, effective
antidiabetic treatments attempt to reduce blood glucose amounts; keeping glucose under
control is very significant. Recent studies have recorded that the α-glucosidase inhibitor
improves postprandial hyperglycemia and then decreases the risk of developing type 2
diabetes in patients. Thus, the synthesis and design of high-affinity glucosidase inhibitors had
significance importance [53,54].

3.3. In Silico Studies
As defined in vitro studies, 2e, 2f, and 2h compounds have exhibited a more potent inhibition
than standard inhibitors of hCA I, hCA II, AChE, BChE, and α-glycosidase enzymes.
Therefore, we decided to investigate inhibition mechanism of the most active compound on
the enzyme using molecular docking technique. We have predicted binding site of the
enzymes before carried out the docking studies. SiteScore and Dscore are an important
parameter for evaluation of its catalytic active site characteristics and druggability. For this
reason, we have calculated SiteScore and Dscore for each binding site. The binding site with
best SiteScore was identified as catalytic active site of the enzyme. Dscores of the catalytic
active sites were founded as 1.113, 1.217, 1.061, 1.106, and 0.961 for hCA I, hCA II, AChE,
BChE, and α-glycosidase enzymes. The results showed that the catalytic active sites have to
feature of druggable site. The catalytic active sites have been shown in Figure 2. The catalytic
active sites have also used for evaluation of docking hits.
Figure 2
At the outset, we have tested the induced-fit docking technique’s reliability using co-
crystallized ligand of the enzymes. After reliability tests, the results were analysed on the
basis of similarity of binding position of co-crystallized and re-docked ligand. The binding
position of ligands has been presented in Figure 3. Moreover, we have calculated root-mean-
square deviation (RMSD) value of co-crystallized and re-docked ligand which was docked
into the catalytic active site of the enzymes. The results were also shown in Figure 3.
Reliability test’s results have revealed that the compounds might be dock with high accuracy
by the docking technique. In order to understand possible inhibition mechanism of the most
active novel compounds against hCA I, hCA II, AChE, BChE and α-glycosidase enzymes, the
compounds have been docked into the predicted catalytic active site of the enzymes using
induced-fit docking technique. In addition to that, standard inhibitor of the enzymes was
docked with the same docking technique and best-poses of most active novel compounds and
standard inhibitors have been compared with each other. Docking scores of best-pose of most
active compound and standard inhibitors of the enzyme have been presented in Table 2.
Figure 3
A zinc atom which is the catalytic active site of hCA I play an important role in its activity.
The enzyme had inhibited by molecules which have sulfonamide group because of interaction
between the molecules and the zinc atom formed coordination with His94, His96, and His119

residues and water molecule [55]. It was remarked that tetrafluorobenzene sulfonamides
which was a quite potent inhibitor against CAs tightly interacted with Leu198, Thr199,
His200, and Pro201 residues [56]. Best-pose of compound 2e which has -6,206 kcal/mol
docking score was located to a similar position with position of AZA in the catalytic active
sites of hCA I. But there were no direct interactions between 2e and catalytic site residues. 2D
and 3D diagrams showed that the compound formed mostly hydrophobic interaction the
catalytic active sites residues especially Leu198 and Pro 201. Even it couldn't any hydrophilic
interactions with the catalytic active sites residue, 2e was embedded into the catalytic active
sites through its phenyl rings. The hydrophobic interaction between 2e and the residues which
are responsible for the enzyme activity caused to the enzyme inhibition (Figure 4a and Figure
5a). Compound 2e also was most active compound against hCA II in vitro studies. Therefore,
we have docked the compound into the catalytic active site of hCA II enzyme too. The
docking result shown that it formed favourable interaction with some residues compared to
AZA and 2D interaction diagram has been presented in Figure 4b. 2e formed π-π interaction
with HID94 (histidine protonated the delta nitrogen) residue. Additionally, it interacted with
Pro201 residue through H-bond via water molecule. Previously reported study has revealed
that some sulfanamide derivatives inhibited hCA II enzyme by interacting with Asn67 and
Gln92 residues through H-bound and Phe131, Val135, Leu198, Leu204 and Pro202 residues
through hydrophobically [57]. The compound was surrounded by similar hydrophobic
residues of the enzyme. Moreover, it formed an aromatic hydrogen bond with Thr200 residues
as seen in Figure 5b.
Figure 4
The catalytic active site of AChE contains Ser203, Glu334, and His447 residues.
Apart from the active site, the peripheral site is also used for the enzyme inhibition. The
peripheral site is very close to the active site and contains many aromatic side chain [58].
According to our docking results, compound 2f located into the peripheral site and formed π-π
interaction with Tyr124, Tyr337, and Tyr341 residues. Besides the interaction, it formed π-
cation with Tyr337 residue. The compound also interacted with Trp86 residues in active site
through π-cation interaction (Figure 4c). After the visual analyses, it has determined that
nitrogen atoms, as well as benzimidazolium and trimethylbenzoyl moieties, were responsible
for inhibition of AChE enzyme. The result is quite similar to Territrem B which is more
potent inhibitor of AChE [59]. Compound 2f also most actively inhibited BChE enzyme in
vitro studies. its docking score indicated that it was potent inhibitor for the enzyme because

the compound formed many close interactions with catalytic active site residues as seen in
Figure 5d. Nitrogen atom of 2-dimethylaminoethyl moity interacted through two π-cation
with Asp304 and Trp522 residues.
Figure 5
The interactions were crucial for the enzyme inhibition as reported by Košak et. al.
[60]. It was also formed hydrogen bond with Phe227 and salt bridges with Asp 304 residues
(Figure 4d). Sim et. al. have determined that active site residues including Asp203, Asp327,
Asp366, Asp443, Arg526, Trp536, Asp542, Asp571, and His600 have played a key role for
the enzyme inhibition, after X-ray crystallographic studies of α-glycosidase complexed with
acarbose, ACR [61]. When 2D diagram presented in Figure 3e of compound 2h was
examined, it has been seen that the compound exhibited very similar interactions with ACR.
Besides the interaction, it also formed π-cation with Tyr299 and π-π interaction with Try406
and Phe575 residues of the catalytic active site. Moreover, 2h formed an aromatic hydrogen
bond with Asp203 residue as seen in Figure 4e. According to the visual analyses, 1-Methyl-2-
dimethylaminoethyl and benzimidazolium moieties play an important role possible inhibition
mechanism of 2h. We have shown that the most active compounds were surrounded by which
type catalytic active site residues of the enzyme in Figure 2. According to the Figure, except
α-glycosidase enzyme, the others mostly have hydrophobic residues in the catalytic active
sites (Figures 2a-2d). For this reason, their inhibitors exhibited hydrophobic wander walls
interaction. However, α-glycosidase enzyme mostly has charged residue in the catalytic active
sites. In the enzyme inhibition, electrostatic interactions have played an important role (Figure
2e).
4. Conclusion
In summary, the benzimidazolium salts bearing both amine and benzyl moieties were
succesfully synthesized and characterized using spectroscopic methods. On the other hand,
AD can be controlled by inhibition of AChE and BChE; however, BChE has limited quantity
and is localized in the peripheral tissues. Thus, the discovery of new selective AChE
inhibitors with lesser peripheral inhibition is therapeutically suitable for the treatment of AD.
The novel benzimidazolium salts (2a-i) derivatives were identified as good inhibitors. On the
other hand, novel benzimidazolium salts (2a-i) derivatives were observed to have antidiabetic,
antiepileptic and anticholinergic properties; therefore, they are suitable for future important

drug studies. Further studies on the enzyme inhibitory properties of benzimidazole derived
metal complexes are in progress.
Conflicts of interest
The authors declare that they have no conflict of interest.
References
[1] F. E. Hahn, M. C. Jahnke, V. Gomez-Benitez, D. Morales-Morales, T. Pape,
Organometallics 24 (2005) 6458.
[2] R. Dang, Y. Wang, J. Zeng, Z. Huang, Z. Fei, P. J. Dyson, J. Mater. Chem. A 5 (2017)
13526-13534.
[3] J. Grave, A. R. Schmitzer, Org. Biomol. Chem. 15 (2017) 1051-1071.
[4] E. Peris, Chem. Rev. 118 (2018) 9988-10031.
[5] S. Demir, M. Yiğit, İ. Özdemir, J. Organomet. Chem. 732 (2013) 21-26.
[6] W. Liu, R. Gust, Chem. Soc. Rev. 42 (2013) 755-773.
[7] L. Mercs, M. Albrecht, Chem. Soc. Rev. 39 (2010) 1903-1912.
[8] S. Günal, N. Kaloğlu, İ. Özdemir, S. Demir, İ. Özdemir, Inorg. Chem. Commun. 21
(2012) 142-146.
[9] C. R. Shahini, G. Achar, S. Budagumpi, H. Müller-Bunz, M. Tacke, S. A. Patil, J.
Organomet. Chem. 868 (2018) 1-13.
[10] Ş. Yaşar, T. K. Köprülü, Ş. Tekin, S. Yaşar, Inorg. Chim. Acta. 479 (2018) 17-23.
[11] H. Z. Zulikha, R. A. Haque, S. Budagumpi, AMSA Majid, Inorg. Chim. Acta. 411
(2014) 40-47.
[12] R. Rubbiani, S. Can, I. Kitanovic, H. Alborzinia, M. Stefanopoulou, M. Kokoschka, S.
Mönchgesang, W. S. Sheldrick, S. Wölfl, I. Ott, J. Met. Chem. 54 (2011) 8646-8657.
[13] P. N. Muskawar, S. S. Kumar, P. R. Bhagat, J. Mol. Catal. A: Chem. 380 (2013) 112-
117.
[14] A. K. Lal, M. D. Milton, Sens. Actuators B 202 (2014) 257-262.
[15] J. B. Goodenough, Y. Kim, Chem. Mater. 22 (2010) 587-603.
[16] R. Dang, Y. Wang, J. Zeng, Z. Huang, Z. Fei, P. J. Dyson, J. Mater. Chem. A 5 (2017)
13526-13534.
[17] B. Yiğit, Y. Gök, İ. Özdemir, S. Günal, J. Coord. Chem. 65 (2012) 371.
[18] S. Akkoç, V. Kayser, İ. Ö. İlhan, D. E. Hibbs, Y. Gök, P. A. Williams, B. Hawkins, F.
Lai, J. Organomet. Chem. 839 (2017) 98.

[19] Y. Gök, S. Akkoç, H. Erdoğan, S. Albayrak, J. Enzyme Inhib. Med. Chem. 31 (2016)
1322-1327.
[20] Y. Sarı, A. Aktaş, P. Taslimi, Y. Gök, İ. Gülçin, J. Biochem. Mol. Toxicol. 32 (2018)
e22009.
[21] M. O. Karataş, H. Uslu, B. Alıcı, B. Gökçe, N. Gencer, O. Arslan, N. B. Arslan, N.
Özdemir, Bioorg. Med. Chem. 24 (2016) 1392-1401.
[22] M. O. Karataş, H. Uslu, S. Sarı, M. A. Alagöz, A. Karakurt, B. Alıcı, C. Bilen, E. Yavuz,
N. Gencer, O. Arslan, J. Enzyme Inhib. Med. Chem. 31 (2016) 760-772.
[23] B. Yiğit, M. Yiğit, D. Barut Celepci, Y. Gök, A. Aktaş, M. Aygün, P. Taslimi, İ. Gülçin,
ChemistrySelect 3 (2018) 7976-7982.
[24] B. Yiğit, R. Kaya, P. Taslimi, Y. Işık, M. Karaman, M. Yiğit, İ. Özdemir, İ. Gülçin, J.
Mol. Struct. 1179 (2019) 709-718.
[25] F. Erdemir, D. Barut Celepci, A. Aktaş, Y. Gök, R. Kaya, P. Taslimi, Y. Demir, İ.
Gülçin, Bioorg. Chem. 91(2019) 103134.
[26] S. Bayındır, C. Çağlayan, M. Karaman, İ. Gülçin, Bioorg. Chem. 90 (2019) 103096.
[27] H. İ. Gül, M. Tuğrak, M. Gül, S. Mazlumoğlu, H. Sakagami, İ. Gülçin, C. T. Supuran
Bioorg. Chem. 90 (2019) 103057.
[28] P. Taslimi, F. M. Kandemir, Y. Demir, M. İleritürk, Y. Temel, C. Cağlayan, İ. Gülçin, J.
Biochem. Mol. Toxicol. 33 (2019) e22313.
[29] M. Boztas, P. Taslimi, M. A. Yavari, İ. Gülçin, E. Sahin, A. Menzek, Bioorg. Chem. 89
(2019) 103017.
[30] U. Atmaca, R. Kaya, H. S. Kahraman, M. Çelik, İ. Gülçin, Bioorg. Chem. 88 (2019)
102980.
[31] H. Genc Bilgicli, A. Kestane, P. Taslimi, O. Karabay, A. Bytyqi-Damoni, M. Zengin, İ.
Gülçin, Bioorg. Chem. 88 (2019) 102931.
[32] U. M. Koçyiğit, Y. Budak, M. B. Gürdere, N. Dürü, P. Taslimi, İ. Gülçin, M. Ceylan,
Monatsh. Chem. 150 (2019) 721-731.
[33 F. Türkan, Z. Huyut, P. Taslimi, İ. Gülçin, Arch. Physiol. Biochem. 125 (2019) 12-18.
[34] E. Bursal, A. Aras, Ö. Kılıç, P. Taslimi, A. C. Gören, İ. Gülçin, J. Food Biochem. 43
(2019) e12776.
[35] C. Çağlayan, Y. Demir, S. Küçükler, P. Taslimi, F. M. Kandemir, İ. Gülçin, J. Food
Biochem. 43 (2019), e12720.
[36] İ. Özdemir, N. Şahin, Y. Gök, S. Demir, B. Çetinkaya, J. Mol. Catal. A: Chem. 234
(2005) 181-185.

[37] J. A. Verpoorte, S. Mehta, J. T. Edsall, J. Biol. Chem, 242 (1967) 4221-4229.
[38] N. Öztaskin, R. Kaya, A. Maraş, E. Sahin, İ. Gülçin, S. Göksu, Bioorg. Chem. 87 (2019)
91-102.
[39] G.L. Ellman, K. D. Courtney, V. Andres, R. M. Featherston, Biochem. Pharmacol. 7
(1961) 88-95.
[40] B. Kuzu, M. Tan, P. Taslimi, İ. Gülçin, M. Taspınar, N. Menges, Bioorg. Chem. 86
(2019) 187-196.
[41] N. Eruygur, M. Ataş, M. Tekin, P. Taslimi, U. M. Koçyiğit, İ. Gülçin, S. Afr. J. Bot. 120
(2019) 141-145.
[42] Y. Tao, Y. Zhang, Y. Cheng, Y. Wang, Biomed. Chromatogr. 27 (2013) 148-155.
[43] A. Aktaş, D. Barut Celepci, R. Kaya, P. Taslimi, Y. Gök, M. Aygün, İ. Gülçin,
Polyhedron 159 (2019) 345-354.
[44] S. Okten, M. Ekiz, U. M. Koçyiğit, A. Tutar, İ. Çelik, M. Akkurt, M. Gökalp, P. Taslimi,
İ. Gülçin, J. Mol. Struct. 1175 (2019) 906-915.
[45] Ç. Bayrak, P. Taslimi, H. S. Kahraman, İ. Gülçin, A. Menzek, Bioorg. Chem. 85 (2019)
128-139.
[46] F. Türkan, A. Çetin, P. Taslimi, H. S. Karaman, İ. Gülçin, Arch. Pharm. 352 (2019)
e1800359.
[47] U. Atmaca, R. Kaya, H. S. Kahraman, M. Çelik, İ. Gülçin, Bioorg. Chem. 88 (2019)
102980.
[48] P. Taslimi, F. Türkan, A. Çetin, H. Burhan, M. Karaman, I. Bildirici, İ. Gülçin, F. Şen,
Bioorg. Chem. 92 (2019) 103213.
[49] B. Yiğit, M. Yiğit, İ. Özdemir, E. Çetinkaya, Heterocycles 81 (2010) 943-953.
[50] A. Biçer, P. Taslimi, G. Yakali, İ. Gülçin, M. S. Gültekin, G. Turgut Cin, Bioorg. Chem.
82 (2019) 393-404.
[51] M. Tuğrak, H. İ. Gül, H. Sakagami, İ. Gülçin, C. T. Supuran, Bioorg. Chem. 81 (2018)
433-439.
[52] M. Huseynova, P. Taslimi, A. Medjidov, V. Farzaliyev, M. Aliyeva, G. Gondolova, O.
Şahin, B. Yalçın, A. Sujayev, E. B. Orman, A. R. Özkaya, İ. Gülçin, Polyhedron 155
(2018) 25-33.
[53] M. Zengin, H. Genç, P. Taslimi, A. Kestane, E. Güçlü, A. Ögütlü, O. Karabay, İ. Gülçin,
Bioorg. Chem. 81 (2018) 119-126.
[54] P. Taslimi, H. E. Aslan, Y. Demir, N. Öztaşkın, A. Maraş, İ. Gülçin, Ş. Beydemir, Ş.
Göksu, Int. J. Biol. Macromol. 119 (2018) 857-863.

[55] V. M. Krishnamurthy, G. K. Kaufman, A. R. Urbach, I. Gitlin, K. L. Gudiksen, D. B.
Weibel, G. M. Whitesides, Chem. Rev. 108 (2008) 946-1051.
[56] A. Zubrienė, J. Smirnovienė, A. Smirnov, V. Morkūnaitė, V. Michailovienė, J. Jachno,
V. Juozapaitienė, P. Norvaišas, E. Manakova, S. Gražulis, D. Matulis, Biophys. Chem.
205 (2015) 51-65.
[57] J. Ivanova, J. Leitans, M. Tanc, A. Kazaks, R. Zalubovskis, C. T. Supuran, K. Tars,
Chem. Commun. 51 (2015) 7108–7111.
[58] M. Harel, D. M. Quinn, H. K. Nair, I. Silman, J. L. Sussman, J. Am. Chem. Soc. 118
(1996) 2340–2346.
[59] J. Cheung, E. N. Gary, K. Shiomi, T. L. Rosenberry, ACS Med. Chem. Lett. 4 (2013)
1091–1096.
[60] U. Košak, B. Brus, D. Knez, S. Žakelj, J. Trontelj, A. Pišlar, R. Šink, M. Jukič, M. Živin,
A. Podkowa, F. Nachon, X. Brazzolotto, J. Stojan, J. Kos, N. Coquelle, K. Sałat, J. P.
Colletier, S. Gobec, J. Med. Chem. 61 (2018) 119-139.
[61] L. Sim, K. Jayakanthan, S. Mohan, R. Nasi, B. D. Johnston, B. M. Pinto, D. R. Rose,
Biochemistry 49 (2010) 443–451.

Table 1. The enzyme inhibition results of novel benzimidazolium salts (2a-i) against human carbonic anhydrase isoenzymes I and II (hCA I and
II), butyrylcholinesterase (BChE), achethylcholinesterase (AChE) and α-glycosidase (α-Gly) enzymes
IC50 (nM)
r2
Ki (nM)
AChE
Compounds
hCA I
r2
hCA II
r2
AChE
BChE
r2
α-Gly
0.98
1.63
0.83
2.67
0.74
1.43
0.96
0.55
2.64
r2
hCA I
hCA II
BChE
α-Gly
0.9638
0.9273
0.9783
0.9598
0.9285
0.9591
0.9880
0.9943
0.9737
1.25±0.12
2.16±0.23
1.43±0.16
3.63±0.83
0.85±0.11
1.68±0.36
1.15±0.08
0.63±0.05
2.95±0.43
2a
2b
13.83
9.71
10.53
10.16
7.47
16.04
25.93
14.87
22.73
57.36
-
0.9736
0.9407
0.9889
0.9733
0.9925
0.9784
0.9638
0.9844
0.9658
0.9790
-
8.76
6.44
9.26
8.83
7.05
13.64
23.17
10.28
18.37
64.08
-
0.9910
0.9588
0.9943
0.9667
0.9599
0.9842
0.9395
0.9681
0.9814
0.9903
-
274.03
294.38
305.82
248.08
225.90
152.84
230.77
198.30
347.17
-
0.9683
205.83
254.90
193.66
197.36
265.84
187.45
211.48
190.26
278.38
-
0.9837
0.9773
0.9811
0.9731
0.9847
0.9581
0.9796
0.9915
0.9732
-
18.54±1.76
11.26±2.87
13.08±1.84
10.82±0.93
8.42±1.03
20.04±2.88
22.62±4.43
17.23±2.16
27.04±3.74
51.83±7.83
-
10.45±0.94
8.96±1.73
11.85±1.08
10.32±1.16
7.94±0.74
15.84±3.55
19.27±2.78
12.48±1.30
21.82±5.81
60.77±14.07
-
231.54±25.94
248.06±18.04
232.88±35.82
198.43±45.93
183.72±24.85
124.24±13.94
205.92±20.15
157.48±31.66
283.55±54.06
-
156.32±22.65
211.05±35.73
166.83±27.48
145.22±48.11
225.37±44.14
136.38±19.55
173.64±15.27
144.76±37.85
247.34±34.06
-
0.9505
0.9274
0.9418
0.9789
0.9937
0.9485
0.9770
0.9842
-
2c
2d
2e
2f
2g
2h
2i
AZA*
TAC**
ACR***
-
-
-
-
-
-
438.43
-
0.9348
-
382.75
-
0.9862
-
372.33±26.55
-
266.12±22.54
-
3.87
0.9738
4.07±0.38
-
-
-
-
-
-
*Acetazolamide (AZA) was used as a control for hCA I, and II.
**Tacrine (TAC) was used as a control for AChE and BChE enzymes.
***Acarbose (ACR) was used as a control for α-glycosidase enzyme.

Table 2. Binding affinity scores (kcal/mol) of most active compounds in the catalytic sites of
the enzymes.
Docking Score
Compounds
hCA I
hCA II
AChE
BChE α- glycosidase
-6.206
-6.715
-
-
-
2e
-
-
-11.461
-6.016
-
2f
-
-
-
-
-8.488
2h
-8.171
-8.121
-
-9.579
-
-
-8.829
-
-
-
AZA*
TAC**
ACR***
-
-
-
-
-16.245
* Acetazolamide (AZA) was used as a standard inhibitor for hCA I and II enzymes.
** Tacrine (TAC) was used as a standard inhibitor for the AChE and BChE enzymes.
***Acarbose (ACR) was used as a standard inhibitor for the α-glycosidase enzyme.

75
60
45
30
15
0
hCA I
hCA II
α-Gly
AZA, 2a
ACR
2b
2c
2d
2e
2f
2g
2h
2i
Compounds
400
300
200
100
0
AChE
BChE
TAC 2a
2b
2c
2d
2e
2f
2g
2h
2i
Compounds
Figure 1. Compare the results of the study (Ki values)

Figure 2. The predicted catalytic active sites of the enzymes. (a) 2e-hCA I, (b) 2e-hCA II, (c) 2f-AChE, (d) 2f-
BChE, and (e) 2h-α-glycosidase. Most active compounds have been represented as grey ball-stick. The catalytic
active site has been represented as grey mash surface, the hydrophobic site has been represented as yellow
bubble, hydrophilic site has been represented as blue bubble and metal-binding site has been represented as
purple bubble.

Figure 3. Induced-fit docking reliability test. Co-crystalized ligands were re-docked into predicted catalytic
active sites of the enzyme and RMSD value between co-crystalized and re-docked ligands was calculated as (a)
1.053 Å for 3TV-hCA I, (b) 0.337 for 51J-hCA II, (c) 0.092 for 1YL-AChE, (d) 2.777 for 93H-BChe, and (e)
1.011 for NR4-α-glycosidase. Co-crystalized ligands were represented with black ball-stick and re-docked
ligands were represented with grey ball-stick.

Figure 4. 2D diagram of interaction between most active compounds and the enzymes. (a) 2e-hCA I, (b) 2e-hCA
II, (c) 2f-AChE, (d) 2f-BChE, (e) 2h-α-glycosidase, (f) AZA-hCA I, (g) AZA-hCA II, (h) TAC-AChE, (i) TAC-
BChE, and (j) ACR-α-glycosidase.

Figure 5. 2D diagram of the interaction between most active compounds and the enzymes. (a) 2e-hCA I, (b) 2e-
hCA II, (c) 2f-AChE, (d) 2f-BChE, (e) 2h-α-glycosidase, (f) AZA-hCA I, (g) AZA-hCA II, (h) TAC-AChE, (i)
TAC-BChE, and (j) ACR-α-glycosidase. Receptor structure was represented as coloured cartoon model, amino
acid residues were represented as grey thick tube model, compound 2e was represented as pink ball-stick model,
compound 2f was represented as orange ball-stick model and compound 2h was represented as green ball-stick
model.

•
Novel amine-functionalized benzimidazolium salts were synthesized via stepwise N-
alkylation of benzimidazole.
•
•
These salts were characterized by elemental analysis, IR, 1H, 13C NMR spectroscopy.
Their inhibition effects on α-glycosidase, hCA I, hCA II, BChE and AChE enzymes
were investigated.
•
•
The these salts were identified as good inhibitors.
The molecular docking studies of the some inhibitors were performed.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: