Imidazolinium chloride salts bearing wingtip groups: Synthesis, molecular docking and metabolic enzymes inhibition

Article abstract of DOI:10.1016/j.molstruc.2018.11.038

A series of symmetrical imidazolinium chloride salts bearing secondary N-alkyl substituents were synthesized in good yield by the reaction of N,N′-dialkylethane-1,2-diamines and HC(OEt)₃ in the presence of NH₄Cl. These salts were characterized by spectroscopic methods. All compounds were tested as enzyme inhibitory agents. These novel symmetrical imidazolinium chloride salts derivatives (3a-h) effectively inhibited the cytosolic hCA I and hCA II, BChE, α-glycosidase and AChE with K_i values in the range of 18.41–121.73 nM for hCA I, 12.50–63.12 nM for hCA II, 3.72–34.58 nM for AChE, 5.50–32.36 nM for BChE, and 94.72–364.51 nM for α-glycosidase, respectively. CA isoenzymes play a crucial roles including acid-base balance homeostasis by excreting and secreting protons (H⁺) due to the CO₂ hydration, HCO₃ ^? reabsorption mechanisms, and renal NH₄ ⁺ output. Also, the molecular modeling is an implementation for estimation of the binding proximity of symmetrical imidazolinium chloride salts bearing secondary wingtip groups and their inhibition mechanisms and kinetics in atomic levels at the catalytic domains.

Full text of DOI:10.1016/j.molstruc.2018.11.038

Accepted Manuscript
Imidazolinium chloride salts bearing wing tip groups: Synthesis, molecular docking
and metabolic enzymes inhibition
Beyhan Yiğit, Ruya Kaya, Parham Taslimi, Yılmaz Işık, Muhammet Karaman, Murat
Yiğit, İsmail Özdemir, İlhami Gulçin
PII:
S0022-2860(18)31348-6
DOI:
https://doi.org/10.1016/j.molstruc.2018.11.038
MOLSTR 25867
Reference:
To appear in: Journal of Molecular Structure
Received Date: 5 October 2018
Revised Date: 9 November 2018
Accepted Date: 10 November 2018
Please cite this article as: B. Yiğit, R. Kaya, P. Taslimi, Yı. Işık, M. Karaman, M. Yiğit, İ. Özdemir,
İ. Gulçin, Imidazolinium chloride salts bearing wing tip groups: Synthesis, molecular docking and
metabolic enzymes inhibition, Journal of Molecular Structure (2018), doi: https://doi.org/10.1016/
j.molstruc.2018.11.038.
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ACCEPTED MANUSCRIPT
Imidazolinium Chloride Salts Bearing Wing Tip Groups: Synthesis, Molecular Docking
and Metabolic Enzymes Inhibition
Beyhan Yiğit, Ruya Kaya, Parham Taslimi, Yılmaz Işık, Muhammet Karaman,
Murat Yiğit, İsmail Özdemir, İlhami Gulçin*
b
a
d
c
e
In this study, a serie of novel imidazolinium chloride salts bearing wing tip groups
synthesised and characterized. Also, their inhibition effects were tested against carbonic
anhydrase I and II isoenzymes and acetylcholinesterase; butyrylcholinesterase and α-
glycosidase enzymes.

ACCEPTED MANUSCRIPT
Imidazolinium Chloride Salts Bearing Wing Tip Groups: Synthesis,
Molecular Docking and Metabolic Enzymes Inhibition
Beyhan Yiğit1, Ruya Kaya2,3, Parham Taslimi2, Yılmaz Işık1, Muhammet Karaman4,5,
Murat Yiğit1, İsmail Özdemir6, İlhami Gulçin2,*
1Department of Chemistry, Faculty of Science and Art, Adiyaman University, 02040-
Adıyaman, Turkey
2Department of Chemistry, Faculty of Sciences, Ataturk University, 25240-Erzurum, Turkey
3Agri Ibrahim Cecen University, Central Research and Application Laboratory, 04100-Agri,
Turkey
4Department of Molecular Biology and Genetics, Faculty of Arts and Science, Kilis 7 Aralik
University, 79000-Kilis, Turkey
5Advanced Technology Application and Research Center, Kilis 7 Aralik University, 79000-
Kilis, Turkey
6Department of Chemistry, Faculty of Art and Science, Inonu University, 44260-Malatya,
Turkey
Address for Correspondence:
Prof. Dr. İlhami GÜLÇİN
Atatürk University
Faculty of Sciences
Department of Chemistry
TR-25240-Erzurum, Turkey
Phone : +90 442 2314375
Fax : +90 442 2314109
E-mails: igulcin@atauni.edu.tr
igulcin@yahoo.com

ACCEPTED MANUSCRIPT
ABSTRACT
A series of symmetrical imidazolinium chloride salts bearing secondary N-alkyl substituents
were synthesized in good yield by the reaction of N,N’-dialkylethane-1,2-diamines and
HC(OEt)3 in the presence of NH4Cl. These salts were characterized by spectroscopic
methods. All compounds were tested as enzyme inhibitory agents. These novel symmetrical
imidazolinium chloride salts derivatives (3a-h) effectively inhibited the cytosolic hCA I and
hCA II, BChE, α-glycosidase and AChE with Ki values in the range of 18.41-121.73 nM for
hCA I, 12.50-63.12 nM for hCA II, 3.72-34.58 nM for AChE, 5.50-32.36 nM for BChE, and
94.72-364.51 nM for α-glycosidase, respectively. CA II isoenzyme plays an crucial roles
including acid-base balance homeostasis by excreting and secreting protons (H+) due to the
-
+
CO2 hydration, HCO3 reabsorption mechanisms, and renal NH4 output. Also, the molecular
modeling are implementation for estimation of the binding proximity of symmetrical
imidazolinium chloride salts bearing secondary wing tip groups and their inhibition
mechanisms and kinetics in atomic levels at the catalytic domains.
Keywords: Carbonic anhydrase; butyrylcholinesterase; acetylcholinesterase; α-glycosidase;
molecular modeling
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1.INTRODUCTION
Azolium salts are the quaternary ammonium compounds, which had considerable attention in
recent years because of their different applications [1,2]. These salts are an important class of
carbene precursors, which have played an important role in the development of N-
heterocyclic carbene (NHC) chemistry. The synthesis of N-heterocyclic carbenes is usually
made by the deprotonation form of the azolium salts. Removal of the C2-proton of azolium
salts in approtic solvents using a suitable strong base generates the N-heterocyclic carbenes.
NHCs are neutral, two-electron donor ligands, which have strong σ-donating and weak π-
accepting properties, they form stable N-heterocyclic carbene complexes with a range of
transition metals [3,4]. NHC complexes have a variety of numerous applications such as
catalysis, material and medicinal sciences [5,6]. Also, azolium salts are known to exhibit a
broad range of biological activities including antibacterial, antitumor and antimicrobial
activities [7,8]. Recently, studies on enzyme inhibitory properties of imidazole and
benzimidazole salts have been reported [9-11]. Therefore, the synthesis of these salts is
important. In literature, various methods have been reported for the synthesis of the azolium
salts [12,13]. The most widely used methods for the synthesis of saturated imidazolium salts
are (i) the cyclization reaction of the corresponding secondary diamine with an orthoformate
in the presence of HX, (ii) N-alkylation of an imidazoline with an appropriate alkyl halide,
and (iii) reactions of a formamidine with ethyl dication equivalent [14-17]. A great number of
symmetrical and unsymmetrical imidazolinium salts have been prepared using these methods.
Although aromatic aldehydes have been generally used for the synthesis of five-, six- and
seven-membered NHC precursors as starting materials. The usge of inexpensive aromatic
ketones was very limited due to probably poor reactivity toward nucleophiles [18].
The α-carbonic anhydrase (CA, E.C.4.2.1.1) isozymes efficiently perform the
interconversion among water (H2O) and carbon dioxide (CO2) to protons (H+) and
-
bicarbonate (HCO3 ), with a wide number of isozymes being explained in mammals: sixteen
number in human cells and primates, and sixteen number in other vertebrate’s groups [19-21].
These catalysts are motivated by CA reaction and very slow at physiologic pH levels for the
hydration and dehydration processes [22,23]. Additionally, the large CO2 quantity occurs in
electron transport chain and oxidative metabolism [24-26]. Indeed, CA isozymes are key
enzymes in most living organisms. Their role is connected with tightly controlled processes
including pH regulation [27,28].
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Two enzymes rated among cholinesterases are known as AChE (E.C.3.1.1.7.) and
BChE (E.C.3.1.1.8.) and presented in humans [29-31]. AChE is expressed in multiple cells. It
is present even on erythrocytes surface. However, the majority of AChE activity is present in
cholinergic nervous system where it terminates cholinergic neurotransmission via fast
hydrolysis of neurotransmitter acetylcholine [32-34]. Due to presence of AChE on
erythrocytes, it was formerly called as a blood cholinesterase in some sources. Compared with
AChE, BChE is not expressed in situ whereas it is produced by livers and distributed through
plasma into the body [35,36]. For the reason, BChE was in some sources called plasmatic,
serum cholinesterase or pseudocholinesterase. The both enzymes are markers of different
pathologies including poisoning with cholinesterases inhibitors for the both cholinesterases
and liver parenchyma degradation for BChE [37-39]. α-Glycosidase is available in the small
intestine and catalyses the final phase in the digestive mechanism of carbohydrate molecules
to release absorbable monosaccharide molecules resulting in increased blood glucose levels.
Inhibition action of this enzyme regulates the liberation of D-glucose from the complex
carbohydrates [40-42].
In this work, a series of novel symmetrical imidazolinium chloride salts derivatives
(3a-h), which synthesized from aromatic ketones and ethylenediamine as low-cost starting
materials. Also their inhibition properties were investigated against some metabolic enzymes.
2.MATERIALS AND METHODS
All preparative reactions for the imidazolinium salts (3a-h) were performed under Ar in
flame-dried glassware using standard Schlenk-type flasks. All reagents were purchased from
1
Sigma Aldrich and Fluka. All H- and 13C-NMR spectra were recorded in CDCl3 with
tetramethylsilane as an internal reference using a Varian AS 400 Merkur spectrometer
operating at 400 MHz (1H), 100 MHz (13C). Chemical shifts (
δ) are given in ppm relative to
TMS, coupling constants (J) in hertz. NMR multiplicities are abbreviated as follows: quint =
quintet, m = multiplied, s = singlet, d = doublet, t = triplet signal. FT-IR spectra were
recorded as KBr pellets in the range 400-4000 cm-1 on Perkin Elmer Spectrum 100. Melting
points (m.p.) were determined in open capillary tubes with an Electrothermal-9200 melting
point apparatus and uncorrected. Elemental analyses were carried out at Inonu University
Research Center.
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2.1.General preparation of diimines, 1
Ethylenediamine (5 mmol) was slowly added to a solution of the appropriate aromatic ketone
(10 mmol), which was previously dissolved in toluene (30 mL) over a period of 2 min. The
resulting mixture was refluxed for 12 h. After reaction completed, the solvent was removed
under vacuum to dryness. The residue was recrystallized in dichloromethane or ethyl alcohol /
diethyl ether.
2.2. General preparation of N,N’-dialkylethane-1,2-diamines, 2
NaBH4 (15 mmol) was added slowly to a solution of the appropriate diimine (10 mmol) in
methyl alcohol (30 mL) at room temperature over a period of 30 min. The reaction mixture
was stirred during 12 h and then refluxed for 1 h. After cooling, the mixture was treated with
1.0 N HCl, and organic phase was extracted with CH2Cl2 (3 × 30 mL). After drying over
MgSO4 and evaporation, the residue was recrystallized from toluene / n-hexane.
2.3. General preparation of symmetrical 1,3-dialkylimidazolinium salts, 3
A mixture of N,N’-dialkylethylenediamines (6.2 mmol), NH4Cl (6.2 mmol) and HC(OEt)3 (10
mL) was heated for 12 h at 110 oC. After cooling, colorless crystals were formed. The crystals
were filtered, washed with diethyl ether (3 × 15 mL) and dried under vacuum. The obtained
salt was recrystallized from ethyl alcohol / diethyl ether.
2.4.1. 1,3-Di(1-phenylethyl)imidazolinium chloride, 3a
This compound was prepared according to published procedures [18].
2.4.2. 1,3-Di[1-(4-methylphenyl)ethyl]imidazolinium chloride, 3b
o
Yield: 1.86 g, 88%, m.p. 185-186 C. IR: ν(NCN) = 1634 cm-1. Anal. Calc. for C21H27N2Cl: C,
73.58 H, 7.88; N, 8.18. Found: C, 73.60; H, 7.85; N, 8.20%. 1H NMR (CDCl3) δ: 1.79 (d, 6H,
J = 7.0 Hz, CH(CH3)C6H4-CH3), 5.24 - 5.32 (m, 2H, CH(CH3)C6H4-CH3), 2.33 (s, 6H,
CH(CH3)C6H4-CH3), 3.50 - 3.76 (m, 4H, NCH2CH2N), 7.18 (d, 4H, J = 7.8 Hz, Ar-H) and
7.30 (d, 4H, J = 8.1 Hz, Ar-H), 10.57 (s, 1H, NCHN). 13C NMR (CDCl3) δ: 18.9
(CH(CH3)C6H4-CH3), 21.1 (CH(CH3)C6H4-CH3), 45.5 (NCH2CH2N), 57.4 (CH(CH3)C6H4-
CH3), 127.0, 129.8, 134.4 and 138.8 (Ar-C), 156.8 (NCHN).
2.4.3. 1,3-Di[1-(3,4-dimethylphenyl)ethyl]imidazolinium chloride, 3c
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o
Yield: 1.85 g, 81%, m.p. 191-193 C. IR: ν(NCN) = 1629 cm-1. Anal. Calc. for C23H31N2Cl: C,
1
74.49; H, 8.37; N, 7.56. Found: C, 74.51; H, 8.35; N, 7.54%. H NMR (CDCl3) δ: 1.79 (d,
6H, J = 6.9 Hz, CH(CH3)C6H3-(CH3)-3,4), 5.19 - 5.26 (m, 2H, CH(CH3)C6H3-(CH3)2-3,4),
2.24 (s, 6H, CH(CH3)C6H3-(CH3)2-3,4), 2.26 (s, 6H, CH(CH3)C6H3-(CH3)2-3,4), 3.51-3.74
(m, 4H, NCH2CH2N), 7.13 - 7.15 (m, 6H, Ar-H), 10.41 (s, 1H, NCHN). 13C NMR (CDCl3) δ:
19.1 (CH(CH3)C6H3-(CH3)2-3,4), 19.4 (CH(CH3)C6H3-(CH3)2-3,4), 19.8 (CH(CH3)C6H3-
(CH3)2-3,4), 45.3 (NCH2CH2N), 57.5 (CH(CH3)C6H3-(CH3)2-3,4), 124.4, 128.2, 128.3, 130.3,
134.8, 134.9, 137.4, 137.5 and 137.6 (Ar-C), 156.6 (NCHN).
2.4.4. 1,3-Di[1-(3,4-dimethoxyphenyl)ethyl]imidazolinium chloride, 3d
Yield: 2.04 g, 76%, m.p. 161-163oC. IR: ν(NCN) = 1628 cm-1. Anal. Calc. for C23H31N2O4Cl:
C, 63.52; H, 7.13; N, 6.44. Found: C, 63.54; H, 7.12; N, 6.45%. 1H NMR (CDCl3) δ: 1.78 (d,
6H, J = 6.9 Hz, CH(CH3)C6H3(OMe)2-3,4), 3.49 - 3,69 (m, 4H, NCH2CH2N), 3.83 (s, 6H,
CH(CH3)C6H3(OMe)2-3,4), 3.91 (s, 6H, CH(CH3)C6H3(OMe)2-3,4), 5.18 (quart, 2H, J = 6.9
13
Hz, CH(CH3)C6H3(OMe)2-3,4), 6.77 - 7.19 (m, 6H, Ar-H), 10.68 (s, 1H, NCHN). C NMR
(CDCl3) δ: 18.8 and 18.9 (CH(CH3)C6H3(OMe)2-3,4), 45.1 and 45.2 (NCH2CH2N), 55.9
CH(CH3)C6H3(OMe)2-3,4), 56.6 CH(CH3)C6H3(OMe)2-3,4), 57.6 (CH(CH3)C6H3(OMe)2-
3,4), 110.7, 110.8, 110.9, 111.0, 119.0, 129.8, 129.9, 149.4, 149.6 and 149.7 (Ar-C), 156.8
(NCHN).
2.4.5. 1,3-Di[1-(3,4,5-trimethoxyphenyl)ethyl]imidazolinium chloride, 3e
This compound was prepared according to previous procedures [18].
2.4.6. 1,3-Dibenzhydrylimidazolinium chloride, 3f
This compound was prepared according to previous procedures [18].
2.4.7. 1,3-Di[1-phenyl-1-(4-methylphenyl)methyl]imidazolinium chloride, 3g
o
Yield: 2.51 g, 87%, m.p. 86-88 C. IR: ν(NCN) = 1638 cm-1. Anal. Calc. for C31H31N2Cl: C,
79.74; H, 6.65; N, 6.00. Found: C, 79.75; H, 6.64; N, 6.01%. 1H NMR (CDCl3) δ: 4.04 (s, 4H,
NCH2CH2N), 6.37 (s, 2H, CH-Ar), 7.15 and 7.25 (d, 8H, J = 8.0 Hz, C6H4-CH3), 7.30 - 7.37
(m, 10H, C6H5), 2.31 (s, 6H, C6H4-CH3), 8.11 (s, 1H, NCHN). 13C NMR (CDCl3) δ: 48.7
(NCH2CH2N), 66.0 (CH-Ar), 21.1 (C6H4-CH3), 128.3, 128.4, 128.5, 128.8, 129.2, 129.8,
132.6, 135.8 and 138.9 (Ar-C), 158.4 (NCHN).
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2.4.8. 1,3-Di[1,1-di(4-methylphenyl)methyl]imidazolinium chloride, 3h
o
Yield: 2.41 g, 79%, m.p. 219-221 C. IR: ν(NCN) = 1658 cm-1. Anal. Calc. for C33H35N2Cl: C,
80.08; H, 7.08; N, 5.66. Found: C, 80.10; H, 7.06; N, 5.67%. 1H NMR (CDCl3) δ: 4.03 (s, 4H,
NCH2CH2N), 6.32 (s, 2H, CH-Ar), 7.15 and 7.24 (d, 16H, J = 8.0 Hz, C6H4-CH3), 2.31 (s,
13
12H, C6H4-CH3), 8.15 (s, 1H, NCHN). C NMR (CDCl3) δ: 48.5 (NCH2CH2N), 65.8 (CH-
Ar), 21.1 (C6H4-CH3), 128.3, 129.8, 132.8 and 138.7 (Ar-C), 158.4 (NCHN).
2.5. Biochemical studies
CA inhibitory effects of novel symmetrical imidazolinium chloride salts derivatives (3a-h)
were measured according to Verpoorte et al. [43] conforming to previous studies [44,45] and
measured at 348 nm spectrophotometrically using p-nitrophenylacetate (PNA) substrate.
AChE and BChE inhibitory effects of novel symmetrical imidazolinium chloride salts
derivatives (3a-h) were determined according to Ellman et al. [46] conforming to previous
studies [47,48] and measured at 412 nm spectrophotometrically using acetylthiocholine iodide
as a substrate. 5,5’-Dithio-bis(2-nitrobenzoic) acid compound was used for activities
measurements of both cholinergic enzymes. Inhibitory effect of novel symmetrical
imidazolinium chloride salts derivatives (3a-h) against α-glycosidase activity was realized by
p-Nitrophenyl-D-glycopyranoside (p-NPG) substrate [49]. For this purpose, an aliquot of
phosphate buffer (200 µL, pH 7.4) was mixed with 40 µL of the homogenate solution, which
prepared in same buffer. Then, an aliquot of p-NPG (50 µL) in phosphate buffer (pH 7.45,
mM) was added to mixture and incubated at 30ºC again. α-Glycosidase activity was
spectrophotometrically recorded at 405 nm according to previous studies [50,51].
2.6. Docking studies
x-Ray crystal structures of AChE, BChE, hCA I, hCA II and α-glycosidase (PDB code:
4WR7, 5AML, 4M0E, 5NN0 and 3L4U, respectively) were taken from RCSB Protein Data
Bank with resolution of 1.5 Å, 1.36 Å, 2 Å, 2.1 Å and 1.9 Å, respectively. Molecular docking
studies were performed using Small Drug Discovery Suites package (Schrödinger 2017-2,
LLC, USA). The 3D crystal structures were repaired and prepared via protein preparation
wizard in Maestro 11.4. Bond order and charges were assigned and then all missing hydrogen
atoms were added to protein structure. Missing side chains were filled using Prime module of
the program. Amino acids were ionized by setting physiological pH with the help of Propka
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software. Water molecules that were formed less than 3 contacts with the protein or ligand
were removed. Finally, energy minimization has also been performed using OPLC force field.
After protein preparation, top-ranked potential protein binding sites and single binding site
were identified using SiteMap module in Maestro 11.4 [52].
3D structures of synthesized compounds were produced with Maestro 11.4. by
sketching 2D structures. 3D structure of ligands was created using LigPrep module of
Schrodinger. In order to obtain correct molecular geometries and protonation state at pH 7.0
± 2.0, Epik module and OPLS-2005 force field were used [52].
Molecular docking was performed to identify binding affinity and interactions between
synthesized compounds and receptors. Briefly, grid box was generated by selecting
crystallized inhibitors at the binding site using the Receptor Grid Generation platform before
the docking process. Docking calculations were set as Extra Precision (XP) by keeping the
ligand flexible. After the docking process, the types of interactions and interacted residues for
best-scored compound results were analyzed. Docking validation was performed with re-
docking procedure by extracting inhibitor complexed in the crystal structure of receptor [52].
3.RESULTS AND DISCUSSION
3.1.Synthesis of imidazolinium salts
The symmetrical 1,3-dialkylimidazolinium chloride salts (3a-h) were prepared according to
literature procedure (Scheme 1). Ethylenediamine was reacted with the appropriate aromatic
ketones (2 eq.) in ethanol to give the corresponding diimines 1. Subsequent reduction of 1
with NaBH4 in methyl alcohol at room temperature, followed by their cyclization with triethyl
ortoformate in the presence of NH4Cl led to the expected imidazolinium salts 3a-h. These
salts were isolated as colourless solids in 76-88% yields and completely characterized using
1
FTIR, elemental analysis, H and 13C NMR spectroscopies. Their melting points were
determined. All results support the proposed structure. The 1H NMR spectra of imidazolinium
salts 3 show the characteristic resonance signals of NCHN protons as singleds at 10.57 for 3b,
10.41 for 3c, 10.68 for 3d, 8.11 for 3g and 8.15 ppm for 3h, respectively. The 13C NMR
spectra of imidazolinium salts 3 show characteristic chemical resonances at 156.8 for 3b and
3d, 156.6 for 3c and 158.4 ppm for 3g and 3h, corresponding to the NCHN carbons. The IR
spectra of 3 also exhibit characteristic υ(NCN) band of imidazolinium ring at 1634, 1629, 1628,
1638 and 1658 cm-1 for 3b, 3c, 3d, 3g and 3h, respectively. The elemental analysis data of
imidazolinium salts are in agreement with the theoretical requirements of their structures.
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Scheme 1
3.2.Metabolic enzymes inhibition results
The activation or inhibition properties of many CA isozymes in tissues including brain and
kidney are crucial for biochemical applications. The diuretic effect of the CAIs is well
recorded in recent. They lead to the progress of diuretics belonging to multiple classes
including thiazides and acetazolamide compound [53]. Many drugs had strong CA inhibition
effects resulting in diuretic effects against several mammalian CA isoenzymes are extensively
utilized clinically. Among them, furosemide, thiazides, metolazone, quinethazone,
indapamide and chlorthalidone countable. Their applications were dealt with widely in a
previous study [54]. Most of the recent work relevant to the renal applications of the CA
isoforms and their inhibition effects deal predominantly with aspects relevant to the decrease
of drug toxicity effect by suing CAIs [55]. Novel symmetrical imidazolinium chloride salts
derivatives (3a-h) were tested for evaluation of inhibition effects towards the hCA I and hCA
II isoenzymes, AChE, BChE and α-glycosidase enzymes. The chemical structure of novel
symmetrical imidazolinium chloride salts derivatives (3a-h) was shown in Scheme 1. Their
inhibition data related to AChE, BChE, α-glycosidase, and hCA I, and hCA II are given in
Table 1. The following results are concluded in this Table. The hCA I isoenzyme was
inhibited by these compounds with Ki values between 18.41±3.76 and 121.73±52.55 µM. In
addition, compounds 3a, and 3g demonstrated as the most effective hCA I inhibition profile
with Ki values of 18.41±3.76 and 28.39±10.22 µM, respectively. Acetazolamide (AZA),
which is positive control and used a clinical drug, demonstrated a Ki value of 127.63±14.77
µM. The results clearly showed that the investigated molecules shown better inhibitory effects
when compared to AZA (Figure 1 and Table 1). The results demonstrated that hCA II
isoenzyme was impressively inhibited by the novel symmetrical imidazolinium chloride salts
derivatives (3a-h). These compounds had strong hCA II inhibition with Ki values in ranging
of 12.50±6.50 - 63.12 ± 26.98 nM. Ki values of novel molecules are better than that of AZA
(Ki: 91.37 ± 10.62 µM). All the evaluated novel molecules showed effective inhibition
properties against hCA II. However, the compounds 3f, and 3a showed the best inhibition
profiles against hCA II with Ki values of 12.50±6.50 and 25.68±10.40 µM, respecticelly
(Table 1).
Table 1
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Both AChE and BChE are a structurally similar proteins belonging to the esterase-lipase
family. Their inhibitors are promising for the therapy as they are activating cholinergic system
as well [56]. The current drugs are donepezil, galantamine, rivastigmine and memantine and
used in most countries for therapy of Alzheimer’s disease (AD). Among them, memantine is a
non-competitive antagonist for N-methyl-D-aspartic acid receptors [57,58]. The other drugs,
(donepezil, galantamine and rivastigmine) are used as inhibitors of cholinesterases. Donepezil
is marketed under different trade [59]. It is a reversible inhibitor for both cholinergic
enzymes. The inhibitory effects of novel synthesized compounds on AChE enzyme are shown
in Table 1. In this work, AChE was also extremely inhibited by novel symmetrical
imidazolinium chloride salts derivatives (3a-h) at the low nanomolar inhibition (Kis:
3.72±0.70-34.58±4.79 nM, Table 1). However, the most effective AChE inhibition was
recorded by novel compound of 3e (Ki: 3.72 ± 0.70 nM, Figure 1). Also, the other compounds
had highly efficient inhibition effects against AChE. Additionally, 1,2,3,4-tetrahydroacridin-
9-amine (Tacrine) that acts as cholinesterase inhibitor used for AD therapy showed Ki value
of 118.93 ± 9.07 nM against AChE. Finally, novel symmetrical imidazolinium chloride salts
derivatives (3a-h) inhibited another cholinergic enzyme (BChE). Ki values were found in
range of 5.50 ± 1.25-32.36 ± 7.70 nM obtained from Lineweaver-Burk plots (Table 1).
Whereas, tacrine, as the first cholinesterase inhibitor and used for the management of AD
symptoms since 1993, had Ki value of 97.26±16.48 nM against BChE. Also, donepezil
showed lower AChE inhibition activity (IC50: 55.0 nM). As seen in Table 2, IC50 values of
new synthesized compounds are between 48.34-290.04 nM against AChE with range of
47.91-108.37 nM for BChE (Figure 1 and Table 1).
The modern antidiabetic drugs are available in both oral and injectable forms. Among
them, α-glycosidase inhibitors (AGIs) are the main therapeutic agents to treat type II diabetes
[60,61]. In diabetic patients, AGIs help to retard the metabolism of carbohydrates and regulate
the elevation of high postprandial blood glucose level. Acarbose, miglitol and voglibose are
some of the currently used AGI drugs. Although AGIs prevent the hypoglycemic condition
and also control the micro- and macrovascular complications, side effects like gastrointestinal
irritation, diarrhea, flatulence, and abdominal discomfort occur with them [62,63]. For this
metabolic enzyme, the novel symmetrical imidazolinium chloride salts derivatives (3a-h) had
IC50 values of 100.37-303.63 µM. Ki values were found in the range of 94.72±17.53-
364.51±104.23 µM for novel synthesized compounds (Figure 1 and Table 1). It is obvious
that all novel symmetrical imidazolinium chloride salts derivatives (3a-h) had efficient α-
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glycosidase inhibition profile when compared to acarbose (IC50: 22.80 ꢀM) as a positive
control α-glycosidase [59,60]. On the other hand, the most effective Ki value was obtained by
1,3-di[1-(3,4-dimethoxyphenyl)ethyl]imidazolinium chloride, 3d and 1,3-di[1-(3,4-
dimethylphenyl)ethyl]imidazolinium chloride, 3c, with Ki values of 94.72 ± 17.53 and 109.38
± 24.71 µM, respectively.
Figure 1
3.3.Docking studies
We performed docking studies to identify the best pose of the compounds, which interacts
with the active site residues of the receptors and possible inhibition mechanism after in vitro
experimental results. Besides, AZA was used as a reference inhibitor compound for hCA I
and hCA II receptors, TAC was used as a reference inhibitor compound for cholinergic
receptors and ACR was used as a reference inhibitor compound for α-glycosidase receptor.
The reference inhibitors were similarly docked into catalytic active site of receptors. In order
to analyse the interactions between best-scored compounds and receptors, pose viewer
module was used. Molecular docking studies showed that all synthesised compounds fit well
into the catalytic active sites of BChE, AChE, α-glycosidase, hCA I, and hCA II enzymes
along with high binding affinity towards the enzyme. The results of Glide docking are
summarized in Table 2. We detected that in vitro end in silico result be compatible on a large
scale. In present study, we focus on explanation for interactions and binding mechanisms
between ligands and receptors and we considered glide score, H-bonds and non-bonded
interactions for docking results analysis. The docking process was confirmed with re-docking
procedure. In the validation, the docking accuracy showed that co-crystallized and docked
ligands are well docked into the receptors as seen in Figure 2.
Table 2 and Figure 2
Docking studies exhibited that all compounds could easily fit into the catalytic active
site of all the receptors. However, we have illustrated only best pose of compounds exhibited
best inhibition effect in results of in vitro study for each receptor. According to results of in
vitro study, compound 3a was most active inhibitor and was also one of compounds with best
docking score towards hCA I isoenzyme. The compound formed π-π interaction with Hie94
residue and also interacted through π-cation with Zn301, which is cofactor of the enzyme into
the catalytic active site of hCA I isoenzyme. Additionally, the compound formed salt bridge
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with Act304, which is ion at protein crystal structure. We detected similarity between
inhibition mechanisms of compound 3a and AZA, a standard inhibitor for hCAs. Two
compounds interacted with Zn301 and Act304 into the catalytic active site of hCA I
isoenzyme. We also detected that very similar hydrophobic and polar amino acid residues as
seen Figure 3a and 5f surrounded best-poses of compounds 3a and AZA. Compound 3a
exhibited very similar interactions as formerly reported inhibitors [63,64].
Although compound 3g was identified as best scored compound towards hCA II with
docking study. We observed that compound 3f was the most active inhibitor of hCA II in vitro
experiment. Therefore, interaction and binding mechanism of compound 3f were analyzed.
The compound formed π-π interaction with His94 residue. In previously study, it was
revealed that the residue is located into the catalytic active site of hCA II and plays a critical
role inhibiting the enzyme [65-67]. AZA formed hydrogen bond with Asn67 and Thr199
amino acid residues and moreover formed a salt bridge with Zn265 atom into the catalytic
active site of hCA II (Figures 3b and 3g).
According to results of docking study, all of synthesised compounds exhibited good
binding affinity ranges from -7.414 to - 12.435 kcal/mol the catalytic active site of AChE
receptor in comparison with binding affinity of TAC standard inhibitor. Compound 3e was
identified as most active compound against AChE enzyme. Docking score of the compound
was more negative than docking score of TAC. Therefore, we analyzed interaction and
binding mechanism of 3e with active site residues of AChE. The compound formed hydrogen
bond with Tyr124, Trp286, and Tyr337 residues. Furthermore, the compound formed salt
bridge with Asp74 residue. 3e was surrounded by a number of hydrophobic amino acid
residues and binding pocket of 3e and TAC is very similar due to hydrophobic amino acid
residues including Try72, Trp286, Phe295, Phe297, Tyr341, Tyr337 and Phe338. 3e exhibited
very similar interaction likes previously reported inhibitors [68-70].
Compound 3c has both most active compound and highest binding affinity against
BChE enzyme. The compound also exhibited excellent binding affinity against BChE enzyme
in comparison with binding affinity of TAC, standard inhibitor. The compound formed π-π
interaction with Tpr82 residue and π-cation interaction Try332 residue into the catalytic active
site of BChE (Figure 3d). It has observed that the interactions play a critical role in the
enzyme activity and inhibition as previously studies [71]. Besides, TAC formed π-π
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interaction with Tpr82 residue and also was surrounded with greatly similar hydrophobic
residues as seen Figure 3i.
We detected that compound 3b was best-scored compound against α-Gly with docking
study. According result of in vitro study, compound 3d was determined most active
compound for the enzyme. Therefore, we analysed best pose of compound 3d to understand
inhibiting mechanism of the compound. The compound formed hydrogen bond with Gly603
residue and salt bridge with Asp542 residue (Figure 3e). Asp542 residue was one of residues
interacted of ACR. Moreover, compound 3d and ACR located similar binding pocket and
were surrounded with similar residues as seen Figure 3e and 4j. Our finding is similar with the
result, which given in literature [72]. They have detected that (R)-2-ethylhexyl 2H-1,2,3-
triazole-4-carboxylate and (E)-2-(prop-1-enyl)-N-methylquinolinium-4-olate extracted from
the stems of Paramignya trimera inhibited human α-glycosidase by interacting through
hydrogen bond with Asp542 residue of the enzyme [72]. The results show that Asp542 is a
key residue inhibiting the enzyme.
Figure 3
Superimposed pose of compounds, which are most active inhibitor, showed that the
compounds well placed into the catalytic active site of the receptors as seen in Figure 4. In
this figure, the detailed binding mode of the compounds has also shown. The binding mode of
compounds revealed that this compounds interacted with the key residues into the catalytic
active site of the receptors.
Figure 4
4.CONCLUSION
In summary, the symmetrical imidazolinium chloride salts were successfully prepared in good
yields by the treatment of N,N’-dialkylethane-1,2-diamines with HC(OEt)3 in the presence of
NH4Cl and characterized by spectroscopic methods, and microanalysis techniques. The hCA I
inhibitors are clinically utilized predominantly as anti-epileptics and diuretics. However, the
novel synthesized compounds can be used in clinical applications in the management of some
disease after some further evaluations because of their inhibition effects against some
metabolic enzymes including carbonic anhydrase I and II isoenzymes, acetylcholinesterase;
butyrylcholinesterase and α-glycosidase. Also, the molecular modeling are implementation
for estimation of the binding proximity of symmetrical imidazolinium chloride salts bearing
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secondary wing tip groups and their inhibition mechanisms and kinetics in atomic levels at the
catalytic domains.
Acknowledgments
We thank the Adiyaman University Research Fund (FEFYL/2017-0004) for financial support
of this work. The authors are grateful to Dr. Halide Sedef Karaman for giving her technical
guidance while processing docking study of this article and for supporting small drug
discovery suite software.
REFERENCES
[1] M. Xia, C. Shi, M. Chai, Z. Liu, Solvent-free synthesis of alkylbenzimidazolium iodides
and their applications in dye-sensitized solar cells. Solar Energy, 84 (2010) 1828-1832.
[2] Y. Shimakawa, T. Morikawa, S. Sakaguchi, Facile route to benzils from aldehydes via
NHC-catalyzed benzoin condensation under metal-free conditions. Tetrahedron Lett. 51
(2010) 1786-1798.
[3] S. Diez-Gonzalez, N. Marison, S.P. Nolan, N-Heterocyclic carbenes in late transition
metal catalysis. Chem. Rev. 109 (2009) 3612-3676.
[4] P. De Fremont, N. Marion, S.P. Nolan, Carbenes: Synthesis, properties, and
organometallic chemistry. Coord. Chem. Rev. 253 (2009) 862-892.
[5] M. Yiğit, B. Yiğit, İ. Özdemir, E. Çetinkaya, B. Çetinkaya, Active ruthenium-(N-
heterocyclic carbene) complexes for hydrogenation of ketones. Appl. Organometal.
Chem. 20 (2006) 322-327.
[6] İ. Özdemir, Y. Gök, Ö. Özeroğlu, M. Kaloğlu, H. Doucet, C. Bruneau, N Heterocyclic
carbenes: Useful ligands for the palladium-catalysed direct c5 arylation of
heteroaromatics with aryl bromides or electron-deficient aryl chlorides. Eur. J. Inorg.
Chem. 12 (2010) 1798-1805.
[7] B. Yiğit, Y. Gök, İ. Özdemir, S. Günal, Synthesis and antimicrobial studies of 1-methyl-2-
dimethylaminoethyl-substituted benzimidazolium salts and N-heterocyclic carbene-silver
complexes. J. Coord. Chem. 65 (2012) 371-379.
[8] S. Akkoç, V. Kayser, İ.Ö. İlhan, D. E. Hibbs, Y. Gök, P.A. Williams, B. Hawkins, F. Lai,
New compounds based on a benzimidazole nucleus: synthesis, characterization and
14

ACCEPTED MANUSCRIPT
cytotoxic activity against breast and colon cancer cell lines. J. Organomet. Chem. 839
(2017) 98-107.
[9] M.O. Karataş, H. Uslu, B. Alıcı, B. Gökçe, N. Gencer, O. Arslan, N.B. Arslan, N.
Özdemir, Functionalized imidazolium and benzimidazolium salts as paraoxonase 1
inhibitors: Synthesis, characterization and molecular docking studies. Bioorg. Med.
Chem. 24 (2016) 1392-1401.
[10] M.O. Karataş, H. Uslu, S. Sarı, M.A. Alagöz, A. Karakurt, B. Alıcı, C. Bilen, E. Yavuz,
N. Gencer, O. Arslan, Coumarin or benzoxazinone based novel carbonic anhydrase
inhibitors: synthesis, molecular docking and anticonvulsant studies. J. Enzyme Inhib.
Med. Chem. 31 (2016) 760-772.
[11] A. Aktaş, P. Taslimi, İ. Gülçin, Y. Gök, Novel NHC precursors: Synthesis,
characterization and carbonic anhydrase and acetylcholinesterase inhibitory properties.
Arch. Pharm. 350 (2017) e1700045.
[12] L. Benhamou, E. Chardon, G. Lavigne, S. Bellemin-Laponnaz, V. Cesar, Synthetic
routes to N-heterocyclic carbene precursors. Chem. Rev. 111 (2011) 2705-2733.
[13] A. Albright, D. Eddings, R. Black, C.J. Welch, N.N. Gerasimchuk, R.E. Gawley, Design
and synthesis of C2-symmetric N-heterocyclic carbene precursors and metal carbenoids.
J. Org. Chem. 2011, 76, 7341-7351.
[
14] A.J. Arduengo, R. Krafczyk, R. Schmutzler, Imidazolylidenes, imidazolinylidenes and
imidazolidines. Tetrahedron 55 (1999) 14523-14533.
[
15] S. P. Roche, M.L. Teyssot, A. Gautier, Synthesis of 1,2 diamines under environmentally benign
conditions: application for the preparation of imidazolidiniums. Tetrahedron Lett. 51 (2010) 1265-
1268.
[
[
16] A. Paczal, A.C. Benyei, A. Kotschy, Modular synthesis of heterocyclic carbene precursors. J.
Org. Chem. 71 (2006) 5969-5979.
17] E.M. MgGarrigle, S.P. Fritz, L. Favereau, M. Yar, V.K. Aggarwal, An efficient synthesis of
imidazolinium salts using vinyl sulfonium salts. Org. Lett. 13 (2011) 3060-3063.
[18] B. Yiğit, The synthesis and catalytic properties of imidazolinium salts and their
palladium (II) complexes. Transition Metal Chem. 37 (2012) 183-188.
[19] Şentürk, M., Gülçin, İ., Daştan, A., Küfrevioğlu, Ö.İ., Supuran, C.T. Carbonic anhydrase
inhibitors. Inhibition of human erythrocyte isozymes I and II with a series of antioxidant
phenols. Bioorg. Med. Chem. 17(8) (2009) 3207-3211.
15

ACCEPTED MANUSCRIPT
[20] Coban, T.A., Beydemir, S., Gücin, İ., Ekinci, D., Innocenti, A., Vullo, D., Supuran, C.T.
Sildenafil is a strong activator of mammalian carbonic anhydrase isoforms I-XIV. Bioorg.
Med. Chem. 17(16) (2009) 5791-5795.
[21] 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, Synthesis,
characterization, crystal structure, electrochemical studies and biological evaluation of
metal complexes with thiosemicarbazone of glyoxylic acid. Polyhedron 155 (2018) 25-
33.
[22]M. Tuğrak, H.İ. Gül, H. Sakagami, İ. Gülçin, C.T. Supuran, New azafluorenones with
cytotoxic and carbonic anhydrase inhibitory properties: 2-Aryl-4-(4-hydroxyphenyl)-5H-
indeno[1,2-b]pyridin-5-ones. Bioorg. Chem. 81 (2018) 433-439.
[23]K. Aksu, H. Akıncıoğlu, A. Akıncıoğlu, S. Göksu, F. Tümer, İ. Gulçin, Synthesis of
novel sulfamides ıncorporating phenethylamines and determination of their ınhibition
profiles against some metabolic enzymes. Arch. Pharm. 351(9) (2018) e1800150.
[24] A. Innocenti, S.B. Öztürk Sarıkaya, İ. Gülçin, C.T. Supuran, Carbonic anhydrase
inhibitors. Inhibition of mammalian isoforms I-XIV with a series of natural product
polyphenols and phenolic acids. Bioorg. Med. Chem. 18(6) (2010) 2159–2164.
[25]P. Taslimi, S. Osmanova, C. Çağlayan, F. Turkan, S. Sardarova, F. Farzaliyev, E.
Sujayev, N. Sadeghian, İ. Gulçin, Novel amides of 1,1-bis-(carboxymethylthio)-1-
arylethanes: Synthesis, characterization, and acetylcholinesterase, butyrylcholinesterase,
and carbonic anhydrase ınhibitory properties. J. Biochem. Mol. Toxicol. 32(9) (2018)
e22191.
[26] A. Innocenti, İ. Gülçin, A. Scozzafava, C.T. Supuran Carbonic anhydrase inhibitors.
Antioxidant polyphenol natural products effectively inhibit mammalian isoforms I-XV.
Bioorg. Med. Chem. 20 (18) (2010) 5050-5053.
[27] S.B. Öztürk Sarıkaya, F. Topal, M. Şentürk, İ. Gülçin, C.T. Supuran, In vitro inhibition
of α-carbonic anhydrase isozymes by some phenolic compounds. Bioorg. Med. Chem.
Lett. 21 (14) (2011) 4259-4262.
[28] B. Yiğit, M. Yiğit, P. Taslimi, Y. Gök, İ. Gulçin, Schiff bases and their amines:
synthesis, and discovery of carbonic anhydrase and acetylcholinesterase enzymes
ınhibitors. Arch. Pharm. 351(9) (2018) e1800146.
[29] N. Öztaşkın, Y. Çetinkaya, P. Taslimi, S. Göksu, İ. Gülçin, Antioxidant and
acetylcholinesterase inhibition properties of novel bromophenol derivatives. Bioorg.
Chem. 60 (2015) 49-57.
16

ACCEPTED MANUSCRIPT
[30]M. Zengin, H. Genç, P. Taslimi, A. Kestane, E. Güçlü, A. Ögütlü, O. Karabay, İ. Gülçin,
Novel thymol bearing oxypropanolamine derivatives as potent some metabolic enzyme
inhibitors-their antidiabetic, anticholinergic and antibacterial potentials. Bioorg. Chem.
81 (2018) 119-126.
[31]U.M. Koçyiğit, Y. Budak, M.B. Gürdere, F. Ertürk, B. Yencilek, P. Taslimi, İ. Gulçin, M.
Ceylan, Synthesis of chalcone-imide derivatives and investigation of their anticancer and
antimicrobial activities, carbonic anhydrase and acetylcholinesterase enzymes inhibition
profiles. Arch. Physiol. Biochem. 124(1) (2018) 61-68.
[32] H. Gocer, F. Topal, M. Topal, M. Küçük, D. Teke, İ. Gülçin, S.H. Alwasel, C.T.
Supuran, Acetylcholinesterase and carbonic anhydrase isoenzymes I and II inhibition
profiles of taxifolin. J. Enzyme Inhib. Med. Chem. 31(3) (2016) 441-447.
[33]Y. Sarı, A. Aktaş, P. Taslimi, Y. Gök, C. Caglayan, İ. Gulçin, Novel N-
propylphthalimide and 4-vinylbenzyl substituted benzimidazole salts: synthesis,
characterization and determination of their metal chelating effects and inhibition profiles
against acetylcholinesterase, and carbonic anhydrase enzymes. J. Biochem. Mol. Toxicol.
32(1) (2018) e22009.
[34]U.M. Koçyiğit, Y. Budak, M.B. Gürdere, F. Ertürk, B. Yencilek, P. Taslimi, İ. Gulçin, M.
Ceylan, Synthesis and investigation of anti-bacterial activities and carbonic anhydrase
and acetyl cholinesterase inhibition profiles of novel 4,5-dihydropyrazol and pyrazolyl-
thiazole derivatives containing methanoisoindol-1,3-dion unit. Synt. Commun. 47(24)
(2017) 2313-2323.
[35]P. Taslimi, H. Akıncıoğlu, İ. Gulçin, Synephrine and phenylephrine act as α-amylase, α-
glycosidase, acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase enzymes
inhibitors. J. Biochem. Mol. Toxicol. 31(11) (2017) e21973.
[36] B. Özgeriş, S. Göksu, L. Köse Polat, İ. Gülçin, R.E. Salmas, S. Durdagi, F. Tümer, C.T.
Supuran, Acetylcholinesterase and carbonic anhydrase inhibitory properties of novel urea
and sulfamide derivatives incorporating dopaminergic 2-aminotetralin scaffolds. Bioorg.
Med. Chem. 24(10) (2016) 2318-2329.
[37] B. Turan, K. Sendil, E. Sengul, M.S. Gultekin, P. Taslimi, İ. Gulcin, C.T. Supuran, The
synthesis of some β-lactams and investigation of their metal chelating activity, carbonic
anhydrase and achetylcholinesterase inhibition profiles. J. Enzyme Inhib. Med. Chem.
31(S1) (2016) 79-88.
[38]İ. Gulçin, M. Abbasova, P. Taslimi, Z. Huyut, L. Safarova, A. Sujayev, V. Farzaliyev, S.
Beydemir, S.H. Alwasel, C.T. Supuran, Synthesis and biological evaluation of
17

ACCEPTED MANUSCRIPT
aminomethyl and alkoxymethyl derivatives as carbonic anhydrase, acetylcholinesterase
and butyrylcholinesterase inhibitors. J. Enzyme Inhib. Med. Chem. 32 (2017) 1174-1182.
[39] U.M. Koçyiğit, P. Taslimi, H. Gezegen, İ. Gulçin, M. Ceylan, Evaluation of
acetylcholinesterase, and carbonic anhydrase inhibition profiles of 1,2,3,4,6-
pentasubstituted-4-hydroxy-cyclohexanes. J. Biochem. Mol. Toxicol. 31(9) (2017).
e21938.
[40]İ. Gulçin, P. Taslimi, A. Aygün, N. Sadeghian, E. Bastem, Ö.İ. Küfrevioğlu, F. Turkan,
F. Şen, Antidiabetic and antiparasitic potentials: Inhibition effects of some natural
antioxidant compounds on α-glycosidase, α-amylase and human glutathione S-transferase
enzymes. Int. J. Biol. Macromol. 119 (2018) 741-746.
[41]P. Taslimi, H.E. Aslan, Y. Demir, N. Öztaşkın, A. Maraş, İ. Gulçin, Ş. Beydemir, Ş.
Göksu, Diarilmethanon, bromophenols and diarilmetan compounds: discovery of potent
aldose reductase, α-amylase and α-glycosidase inhibitors as new therapeutic approach in
diabetes and functional hyperglycemia. Int. J. Biological. Macromol. 119 (2018) 857-
863.
[42]G. Gondolova, P. Taslimi, A. Medjidov, F. Farzaliyev, A. Sujayev, M. Huseuinova, O.
Şahin, B. Yalçın, F. Turkan, İ. Gulçin, Synthesis, crystal structure and biological
evaluation of spectroscopic characterization of Ni(II) and Co(II) complexes with N-
salicyloil-N’-maleoil-hydrazine as anticholinergic and antidiabetic agents. J. Biochem.
Mol. Toxicol. 32(9) (2018) e22197.
[43] J.A. Verpoorte, S. Mehta, J.T. Edsall, Esterase activities of human carbonic anhydrases B
and C. J. Biol. Chem. 242 (1967) 4221-4229.
[44] M. Boztaş, Y. Çetinkaya, M. Topal, İ. Gülçin, A. Menzek, E. Şahin, M. Tanc, C.T.
Supuran, Synthesis and carbonic anhydrase isoenzymes I, II, IX, and XII inhibitory
effects of dimethoxy-bromophenol derivatives incorporating cyclopropane moieties. J.
Med. Chem. 58 (2015) 640-650.
[45] A. Yıldırım, U. Atmaca, A. Keskin, M. Topal, M. Çelik, İ. Gülçin, C.T. Supuran, N-
Acylsulfonamides strongly inhibit human carbonic anhydrase isoenzymes I and II.
Bioorg. Med. Chem. 23 (2015) 2598-2605.
[46] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherston, A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochem Pharmacol. 7 (1961) 88-95.
[47] B. Yiğit, M. Yiğit, D. Barut Celepci, Y. Gök, A. Aktaş, M. Aygün, P. Taslimi, İ. Gulçin,
Novel
benzylic
substituted
imidazolinium,
tetrahydropyrimidinium
and
18

ACCEPTED MANUSCRIPT
tetrahydrodiazepinium salts-potent carbonic anhydrase and acetylcholinesterase
inhibitors. ChemistrySelect 3(27) (2018) 7976-7982.
[48] İ. Gulçin, P. Taslimi, Sulfonamide inhibitors: A patent review 2013-present. Exp. Opin.
Therap. Pat. 28(7) (2018) 541-549.
[49] Y. Tao, Y. Zhang, Y. Cheng, Y. Wang, Rapid screening and identification of α-
glucosidase inhibitors from mulberry leaves using enzyme-immobilized magnetic beads
coupled with HPLC/MS and NMR. Biomed. Chrom. 27 (2013)148-155.
[50] P. Taslimi, C. Caglayan, İ. Gulçin, The impact of some natural phenolic compounds on
carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase, and α-glycosidase
enzymes: an antidiabetic, anticholinergic, and antiepileptic study. J. Biochem. Mol.
Toxicol. 31(12) (2017) e21995.
[51] F. Erdemir, D. Barut Celepci, A. Aktaş, P. Taslimi, Y. Gök, H. Karabıyık, İ. Gulçin, 2-
Hydroxyethyl substituted NHC precursors: Synthesis, characterization, crystal structure
and carbonic anhydrase, α-glycosidase, butyrylcholinesterase, and acetylcholinesterase
inhibitory properties. J. Mol. Struct. 1155 (2018)) 797-806.
[52] L. Schrödinger, Drug Discov. Suite. (2017).
[53] P. Taslimi, İ. Gulçin, Antioxidant and anticholinergic properties of olivetol. J. Food
Biochem. 42(3) (2018) e12516.
[54] M. Rezai, Ç. Bayrak, P. Taslimi, İ. Gulçin, A. Menzek, The first synthesis, antioxidant
and anticholinergic activities of 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative
bromophenols including natural products. Turk. J. Chem. 42(3) (2018) 808-825.
[55] A. Behcet, T. Çağlılar, D. Barut Celepci, A. Aktaş, P. Taslimi, Y. Gök, M. Aygün, R.
Kaya, İ. Gulçin, Synthesis, characterization and crystal structure of 2-(4-
hydroxyphenyl)ethyl and 2-(4-nitrophenyl)ethyl substituted benzimidazolium bromide
salts: their inhibitory properties against carbonic anhydrase and acetylcholinesterase. J.
Mol. Struct. 1170 (2018) 160-169.
[56] C. Yamali, H.İ. Gül, A. Ece, P. Taslimi, İ. Gulçin, Synthesis, molecular modeling and
biological
evaluation
of
4-[5-aryl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-
yl]benzenesulfonamides towards acetylcholinesterase, carbonic anhydrase I and II
enzymes. Chem. Biol. Drug Des. 91(4) (2018) 854-866.
[57] A. Akıncıoğlu, E. Kocaman, H. Akıncıoğlu, R.E. Salmas, S. Durdağı, İ. Gülçin, C.T.
Supuran, S. Göksu, The synthesis of novel sulfamides derived from β-
benzylphenethylamines as acetylcholinesterase, butyrylcholinesterase and carbonic
anhydrase enzymes inhibitors. Bioorg. Chem. 74 (2017) 238-250.
19

ACCEPTED MANUSCRIPT
[58] N. Öztaşkın, P. Taslimi, A. Maraş, S. Göksu, İ. Gülçin, Novel antioxidant bromophenols
with acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase inhibitory
actions. Bioorg. Chem.74 (2017) 104-114.
[59] Ç. Bayrak, P. Taslimi, İ. Gülçin, A. Menzek, The first synthesis of 4-phenylbutenone
derivative bromophenols including natural products and their inhibition profiles for
carbonic anhydrase, acetylcholinesterase and butyrylcholinesterase enzymes. Bioorg.
Chem. 72 (2017) 359-366.
[60] P. Taslimi, İ. Gulçin, Antidiabetic potential: in vitro inhibition effects of some natural
phenolic compounds on α-glycosidase and α-amylase enzymes. J. Biochem. Mol.
Toxicol. 31(10) (2017) e21956.
[61] P. Taslimi, H. Akıncıoğlu, İ. Gulçin, Synephrine and phenylephrine act as α-amylase, α-
glycosidase, acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase enzymes
inhibitors. J. Biochem. Mol. Toxicol. 31(11) (2017) e21973.
[61] M. Torres-Naranjo, A. Suarez, G. Gilardoni, L. Cartuche, P. Flores, V. Morocho,
Chemical constituents of Muehlenbeckia tamnifolia (Kunth) Meisn (Polygonaceae) and
its in vitro α-amilase and α-glucosidase inhibitory activities. Molcules, 21 (2016) 1461-
1465.
[62] H. Teng, L. Chen, T. Fang, B. Yuan, Q. Lin, Rb2 inhibits α-glucosidase and regulates
glucose metabolism by activating AMPK pathways in HepG2 cells. J. Funct. Foods 28
(2017) 306-313.
[63] R. Ekhteiari Salmas, M. Mestanoglu, S. Durdagi, M. Sentürk, A.A. Kaya, E.Ç. Kaya,
Kinetic and in silico studies of hydroxy-based inhibitors of carbonic anhydrase isoforms I
and II. J. Enzyme Inhib. Med. Chem. 31 (2016) 31-37.
[64] U.M. Kocyigit, Y. Budak, F. Eliguzel, P. Taslimi, D. Kilic, I. Gulcin, M. Ceylan,
Synthesis and carbonic anhydrase inhibition of tetrabromo chalcone derivatives. Arch.
Pharm. 350(12) (2017) e1700198.
[65]L. Polat Köse, İ. Gulcin, Inhibition effects of some lignans on carbonic anhydrase,
acetylcholinesterase and butyrylcholinesterase enzymes. Rec. Nat. Prod. 11(6) (2017)
558-561.
[66] A. Akıncıoğlu, E. Kocaman, H. Akıncıoğlu, R.E. Salmas, S. Durdagi, I. Gülçin, CT.
Supurane, S. Göksu, The synthesis of novel sulfamides derived from β-
benzylphenethylamines as acetylcholinesterase, butyrylcholinesterase and carbonic
anhydrase enzymes inhibitors. Bioorg. Chem. 74 (2017) 238-225.
20

ACCEPTED MANUSCRIPT
[66] S. Burmaoglu, A.O. Yilmaz, P. Taslimi, O. Algul, D. Kilic, I. Gulcin, Synthesis and
biological evaluation of phloroglucinol derivatives possessing α-glycosidase,
acetylcholinesterase, butyrylcholinesterase, carbonic anhydrase inhibitory activity. Arch.
Pharm. 351 (2018) e1700314.
[68] M. Mladenović, B.B. Arsić, N. Stanković, N. Mihović, R. Ragno, A. Regan, J.S.
Milićević, T.M. Trtić-Petrović, R. Micić, The targeted pesticides as acetylcholinesterase
inhibitors: Comprehensive cross-organism molecular modelling studies performed to
anticipate the pharmacology of harmfulness to humans in vitro. Molecules 23(9) (2018)
2192.
[69] N. Chufarova, K. Czarnecka, R. Skibiński, M. Cuchra, I. Majsterek, P. Szymański, New
tacrine-acridine hybrids as promising multifunctional drugs for potential treatment of
Alzheimer's disease. Arch. Pharm. 351 (2018) e1800050.
[70] A. Kulshreshtha, P. Piplani, Design, synthesis and pharmacological evaluation of
carboxamide and carbothioamide derivatives of 1,3,4-thiadiazole as the inhibitors of
acethylycholinesterase and oxipiperazine)ative stress for the management of cognitive
debility. Med. Chem. Res. 27 (2018) 1800-1821.
[71]I. Daouda, N. Melkemic, T. Salahc, S. Ghalem, Combined QSAR, molecular docking and
molecular dynamics study on new acetylcholinesterase and butyrylcholinesterase
inhibitors. Comp. Biol. Chem. 74 (2018) 304-326.
[72]N.T. Nguyen, P.H. Dang, N.X.T. Vu, T.H. Le, M.T.T. Nguyen, Quinoliniumolate and
2H-1,2,3-triazole derivatives from the stems of Paramignya trimera and their α-
glucosidase inhibitory activities: in vitro and in silico studies. J. Nat. Prod. 80(7) (2017)
2151-2155.
[73]K. Tadera, Y. Minami, K. Takamatsu, T.J. Matsuoka, Inhibition of alpha-glucosidase and
alpha-amylase by flavonoids. J. Nutr. Sci. Vitaminol. 52 (2006) 149153.
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Table 1. Human carbonic anhydrase isoenzymes I, and II, α-Glycosidase, AChE and BChE enzymes inhibition values of novel symmetrical
imidazolinium chloride salts derivatives (3a-h)
IC50 (nM)
Ki (nM)
hCA II
Compounds
3a
hCA I
r2
hCA II
r2
AChE
r2
BChE
r2
α-Gly
r2
hCA I
AChE
BChE
α-Gly
248.12
0.9614
267.13±63.12
11.43
0.9844
13.54
0.9692
113.56
0.9832
49.78
0.9922
18.41±3.76
25.68±10.40
23.64±10.23
26.50±4.10
293.05
0.9889
303.73±50.23
3b
3c
17.02
15.15
16.16
19.19
18.47
30.59
20.49
134.14
-
0.9631
0.9693
0.9702
0.9291
0.9862
0.9841
0.9872
0.9811
-
24.10
20.34
28.72
31.17
24.38
20.56
28.43
103.11
-
0.9402
0.9671
0.9631
0.9321
0.9441
0.9501
0.9333
0.9709
-
210.68
255.41
226.55
174.60
118.47
290.04
48.34
-
0.9812
0.9931
0.9871
0.9781
0.9651
0.9761
0.9842
-
61.61
86.58
63.63
59.45
108.37
49.06
47.91
-
0.9881
0.9723
0.9851
0.9753
0.9641
0.9681
0.9772
-
40.84±10.20
54.13±17.02
39.97±5.57
48.90±4.50
121.73±52.55
28.39±10.22
52.67±6.34
127.63±14.77
-
62.44±25.34
40.70±14.93
53.67±14.83
63.12 ±26.98
12.50±6.50
40.50±10.90
43.56±11.60
91.37±10.62
-
7.65±0.88
12.60±0.60
7.60±0.92
3.72±0.70
21.42±3.48
7.02±1.47
34.58±4.79
-
24.03±11.80
5.50±1.25
17.10±2.49
12.33 ±0.98
7.05±2.39
32.36±7.70
14.29±0.95
-
123.74
100.37
303.63
347.71
265.72
217.54
-
0.9720
0.9924
0.9691
0.9377
0.9633
0.9521
-
109.38±24.71
94.72±17.53
348.20±73.51
364.51±104.23
239.76±94.32
263.98±66.14
-
3d
3e
3f
3g
3h
AZA*
TAC**
ACR***
-
-
-
156.82
-
0.9691
-
128.02
-
0.9508
-
118.93±9.07
-
97.26±16.48
-
2800
0.9634
12600±780
-
-
-
-
-
-
*Acetazolamide (AZA) was used as a standard inhibitor for both hCA I, and II isoenzymes.
** Tacrine (TAC) was used as a standard inhibitor for AChE and BChE enzymes.
***Acarbose (ACR) was used as positive control for α-glycosidase enzyme, which obtained from reference 73.
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Table 2. Glide and IFD scores (kcal/mol) of the pyrazoline derivatives in the catalytic sites of hCA I,
hCA II, AChE, BChE, and α-Gly enzymes
XP GScore
Compounds
hCA I
hCA II
AChE
BChE
α-Gly
-4.777
-4.570
-3.386
-4.438
-4.374
-4.408
-4.946
-3.980
-8.331
-
-4.377
-3.807
-3.914
-3.803
-3.601
-4.631
-6.213
-1.920
-8.172
-
-10.190
-10.452
-10.343
-11.139
-9.560
-7.414
-12.435
-7.892
-
-9.703
-10.282
-11.501
-9.664
-10.524
-6.587
-10.182
-8.928
-
-6.000
-6.233
-5.929
-5.147
-4.269
-3.136
-4.411
-3.719
-
3a
3b
3c
3d
3e
3f
3g
3h
AZA*
TAC**
ACR***
-8.978
-
-8.440
-
-
-
-
-16.933
*Acetazolamide (AZA) was used as a standard inhibitor for both hCA I, and II isoenzymes.
** Tacrine (TAC) was used as a standard inhibitor for AChE and BChE enzymes.
***Acarbose (ACR) was used as positive control for α-glycosidase enzyme, which taken of reference 73.
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FIGURE LEGENDS
Figure 1. Ki values of novel compounds on metabolic enzymes
Figure 2. Docking validation. Receptors are depicted in the ribbon model which hCA I with
grey color, hCA II with orange color, AChE with blue color, BChE with faded red,
and α-Gly with yellow color. The poses of co-crystallized ligands are represented in
grey color ball and stick modeling while that of docked ligands is represented in
magenta color ball and stick modeling.
Figure 3. 2D receptor–ligand interaction profile; (a) 3a-hCA I, (b) 3f-hCA II, (c) 3e-AChE,
(d) 3c-BChE, (e) 3d-α-Gly, (f) AZA-hCA I, (g) AZA-hCA II, (h) TAC-AChE, (i)
TAC-BChE, and (j) ACR-α-glycosidase.
Figure 4. The lowest energy conformation of compounds into hCA I, hCA II, AChE, BChE,
and α-glycosidase receptors. (a) Best-pose of 3a into catalytic active site of hCA I, (b)
Best-pose of 3f into catalytic active site of hCA II, (c) Best-pose of 3e into catalytic
active site of AChE, (d) Best-pose of 3c into catalytic active site of BChE, and (e)
Best-pose of 3d into catalytic active site of α-glycosidase. Receptors are depicted in
the ribbon model which hCA I with grey color, hCA II with orange color, AChE with
blue color, BChE with faded red, and α-glycosidase with yellow color. Catalytic active
sites are represented as solid surface. Compounds are represented in ball and stick
modeling which magenta color carbon and residues are represented in ball and stick
modeling which grey color carbon.
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Figure 1

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Figure 2

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a
b
c
d
e
g
h
i
j
f
Figure 3

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Figure 4