Synthesis and in silico studies of Novel Ru(II) complexes of Schiff base derivatives of 3-[(4-amino-5-thioxo-1,2,4-triazole-3-yl)methyl]-2(3H)-benzoxazolone compounds as potent Glutathione S-transferase and Cholinesterases Inhibitor

Article abstract of DOI:10.1016/j.molstruc.2021.129943

Novel Ru(II) complexes of Shiff base derivatives of 3-[(4-amino-5-thioxo-1,2,4-triazole-3-yl)methyl]-2(3H)-benzoxazolone were synthesized. The ligands (1a-e) were confirmed by IR, ¹H NMR, and ¹³C NMR spectra (only 1b and 1c). Structures of the synthesized Ru(II) complexes (2a-e) were illuminated by elemental analysis, IR, ¹H NMR, ¹³C NMR, and mass spectra. As the biological studies, the inhibitory potency of the ligands and the novel synthesized complexes were evaluated against the glutathione S-transferase (GST), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes in vitro conditions. Ki values in the range of 26.87-47.63 μM for AChE, 23.51-42.81 μM for BChE, and 33.14-51.73 μM for GST, respectively. The free binding energy of most active inhibitors against AChE, BChE, and GST enzymes were detected as -10.183 kcal/mol, -9.111 kcal/mol, and -6.097 kcal/mol, respectively. All compounds docked were observed to bind in the active site of the enzymes with similar binding orientation and binding interactions with the surrounding amino acids.

Full text of DOI:10.1016/j.molstruc.2021.129943

Journal of Molecular Structure 1231 (2021) 129943
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis and in silico studies of Novel Ru(II) complexes of Schiff base
derivatives of 3-[(4-amino-5-thioxo-1,2,4-triazole-3-yl)methyl]-2(3H)-
benzoxazolone compounds as potent Glutathione S-transferase and
Cholinesterases Inhibitor
Ragip Adiguzela,b,c, Fikret Türkand,∗, Ümit Yildikoe, Abdülmelik Arasf, Enes Evreng,
Tijen Onkolh
a Department of Chemistry and Chemical Process Technologies/Laboratory Technology Program, Vocational School of Tunceli, Munzur University,
62000 Tunceli, Turkey
b Head of Department of Chemical Technologies in Institute of Graduate Studies, Munzur University, 62000 Tunceli, Turkey
c Rare Earth Elements Research and Application Center, Munzur University, 62000 Tunceli, Turkey
d Health ServicesVocational School, Igdır University, Igdır, Turkey
e Department of Biochemistry, Faculty of Science and Letters Igdir University, 76000 Igdır, Turkey
f Architecture and Engineering Faculty, Department of Bioengineering, Kafkas University, Kars, Turkey
g Catalysis Research and Application Center, Inonu University, 44280Malatya, Turkey
h Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey,
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel Ru(II) complexes of Shiff base derivatives of 3-[(4-amino-5-thioxo-1,2,4-triazole-3-yl)methyl]-2(3H)-
benzoxazolone were synthesized. The ligands (1a-e) were confirmed by IR, 1H NMR, and 13 C NMR spectra
(only 1b and 1c). Structures of the synthesized Ru(II) complexes (2a-e) were illuminated by elemental
analysis, IR, 1H NMR, 13 C NMR, and mass spectra. As the biological studies, the inhibitory potency of the
ligands and the novel synthesized complexes were evaluated against the glutathione S-transferase (GST),
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes in vitro conditions. Ki values in
the range of 26.87-47.63 μM for AChE, 23.51-42.81 μM for BChE, and 33.14-51.73 μM for GST, respectively.
The free binding energy of most active inhibitors against AChE, BChE, and GST enzymes were detected as
-10.183 kcal/mol, -9.111 kcal/mol, and -6.097 kcal/mol, respectively. All compounds docked were observed
to bind in the active site of the enzymes with similar binding orientation and binding interactions with
the surrounding amino acids.
Received 20 October 2020
Revised 7 January 2021
Accepted 10 January 2021
Available online 13 January 2021
Keywords:
2(3H)-benzoxazolone
4-amino-5-thioxo-1,2,4-triazole
Schiff base
Ruthenium complexesy
Enzyme inhibition
Docking
© 2021 Published by Elsevier B.V.
1. Introduction
their effects on functional materials, agrochemicals, and pharma-
ceuticals is becoming more important [9].
Schiff bases are organic compounds that have an important po-
sition as ligands in metal coordination chemistry since their dis-
covery. They can form stable complexes with some transition met-
als including rare-earth metals [1-3]. Schiff bases containing nitro-
gen, oxygen, and sulfur donor atoms have great importance for bi-
ological systems due to their ability to illuminate the mechanism
of reactions such as transformation [4-6]. There are various biolog-
ical activities of the 1,2,4-triazole Schiff bases such as antioxidant,
antitumor, antimicrobial, antitubercular [7], anti-inflammatory, an-
tipyretic, antifungal, and antibacterial [8]. Hence, the application of
The importance of sulphonamide Schiff bases for the de-
sign of enzyme inhibitors has been demonstrated in the lit-
erature [10]. Therefore, herein Glutathione S-transferases (GST),
acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) en-
zymes were examined regarding the inhibitory activity. Acetyl-
cholinesterase enzymes belong to the hydrolase class and are en-
coded by chromosomes 7 [11]. They are used as the target of drugs
in the treatment of some diseases, including Alzheimer’s disease
(AD) [9]. The biological responsibility of the AChE is to swiftly ter-
minate the neural impulse transmission in the synaptic cleft by hy-
drolysis of the ACh to acetate and choline [12-14].
Butyrylcholinesterase (BChE) accepted as a standard approach
synthesized in the liver is used in the symptomatic treatment
∗
Corresponding author.
E-mail address: fikret.turkan@igdir.edu.tr (F. Türkan).
https://doi.org/10.1016/j.molstruc.2021.129943
0022-2860/© 2021 Published by Elsevier B.V.

R. Adiguzel, F. Türkan, Ü. Yildiko et al.
Journal of Molecular Structure 1231 (2021) 129943
of neurodegenerative diseases [15]. BChE with three-dimensional-
structures is a serine hydrolase related to AChE. Their catalytic
mechanisms are close, however, substrate specificity and inhibitor
sensitivity are different. Unlike AChE, which enables acetylcholine
to terminate the effect of acetylcholine in the cholinergic system,
studies have not demonstrated direct participation of BChE in the
cholinergic system [16,17].
2.2.1. Spectral results for ligands
1a: FT-IR ν (cm-1), 3230, 1776, 1484, 1236; 1H NMR (DMSO-d6)
δ 14.10 (1H, s, NH), 10.01 (1H, s, N=CH), 7.16-7.85 (8H, m, Ar-H),
5.30 (2H, s, CH2);
1b: FT-IR ν (cm-1), 3248, 1744, 1480, 1269; 1H NMR (400 MHz
DMSO-d6) δ(ppm): 14.17 (1H, s, NH), 10.82 (1H, s, N=CH),7.14-
8.14 (8H, m, Ar-H), 5.34 (2H, s, CH2); 13C NMR 13C{1H} NMR (100
MHz, DMSO-d6) δ (ppm):162.6 (C=S), 159.5 (C=O), 146.5 (NCH(o-
BrC6H4)), 110.2, 110.3, 123.2, 124.5, 126.1, 128.7, 131.1, 131.8, 134.0,
134.7, 142.4 (Ar-C) of NCH(o-BrC6H4) and CCH2NC(C6H4) in either
benzene ring, 153.8 (NCCH2), 37.4 (NCCH2).
Glutathione S-transferase (GST) enzyme family located in the
liver plays an important role in detoxification and takes part in the
process of adding glutathione to oxidative stress products [18,19].
However, to maintain their survival and obtain drug resistance this
feature is similarly used by cancer cells. Therefore, various mem-
bers of the GST enzyme were determined overexpressed in some
types of cancers [20]. It has been determined that GST enzyme
inhibitors eliminate drug resistance by sensitizing tumor cells to
different anticancer drugs [21], and therefore inhibitory studies of
this enzyme have gained more importance.
1c: FT-IR ν (cm−1), 3258, 1759, 1462, 1265.; 1H NMR (400 MHz,
DMSO-d6) δ (ppm): 14.12 (1H, s, NH), 10.06 (1H, s, N=CH), 7.13-
7.81 (8H, m, Ar-H), 5.30 (2H, s, CH2); 13C{1H} NMR (100 MHz,
DMSO-d6) δ (ppm): 162.6 (C=S), 162.4 (C=O),
146.1 (NCH(p-BrC6H4)), 110.1, 110.3, 123.2, 124.5, 126.9,
130.9, 131.0, 131.6, 132.7, 142.3 (Ar-C) of NCH(o-BrC6H4) and
CCH2N(C6H4) in either benzene ring, 153.8 (NCCH2), 37.4 (NCCH2).
1d: FT-IR ν (cm−1), 3238, 1748, 1482, 1242; 1H NMR (DMSO-d6)
δ (ppm): 14.10 (1H, s, NH), 10.01 (1H, s, N=CH), 7.16-7.85 (8H, m,
Ar-H), 5.30 (2H, s, CH2);
In this study, starting from the known fact that Shiff base
derivatives of 3-[(4-amino-5-thioxo-1,2,4-triazole-3-yl)methyl]-
2(3H)-benzoxazolone have various biological activities [7,8],
we synthesized new Ruthenium(II) complexes of Shiff base
derivatives of 3-[(4-amino-5-thioxo-1,2,4-triazole-3-yl)methyl]-
2(3H)benzoxazolone. We determined some metabolic enzyme
inhibitory effects of these compounds.Besides, the molecular dock-
ing study was carried out to observe enzyme inhibitor interaction.
1e: 3298, 1744, 1484, 1241; 1H NMR (DMSO-d6) δ (ppm): 14.06
(1H, s, NH), 10.06 (1H, s, N=CH), 7.17-7.88 (8H, m, Ar-H), 5.30 (2H,
s, CH2)
2.3. Design and synthesis of the Ru(II) complexes (2a-e)
2. Experimental section
All the Ru(II) complexes benzoxazolone-based ligands (1a-e)
were prepared under the argon gas atmosphere.
2.1. Materials and methods
2.3.1. Synthesis of novel Ru(II) complexes (2a-e)
All chemicals were acquired from commercial firms that are
concerning chemicals, without further purification. The starting
materials necessary for the synthesis of ligands were available in
our laboratory. From the solvents, only toluene that was used dur-
ing the synthesis of Ru(II) complexes (2a-e) was purified by distil-
lation over the drying agents indicated and was transferred to the
reaction media under argon. [Ru(p-cymene)Cl2]2 compounds were
purchased from Sigma Aldrich Co. (Dorset, UK). Melting points
were determined using the Electrothermal 9100 melting point de-
tection apparatus in capillary tubes and the melting points are
reported as uncorrected values. Elemental analyses were carried
out with a Leco CHNS–O model 932 elemental analyzer at Inonu
University, Malatya, Turkey. FT-IRspectra of the Ru(II) complexes
(2a-e) were recorded using a JASCO 6700 spectrophotometer in
the wavenumber range of 4000–400 cm−1 at Munzur University,
Tunceli, Turkey. All spectra represented 32 scans and resolution
The ligands 1a-e (0.122 mmol), [Ru(p-cymene)Cl2]2 (0.122
mmol) and dry PhMe solvent (7 mL), was added in a schlenk flask
(50 mL) The mixture was stirred for 2 h at 70 °C and then the
brick-colored mixture was stirred for 20 h at 95 °C. After the re-
action was finished, PhMe was removed under vacuum. The crude
product was crystallized in CH2Cl2/C2H5)2O (1:3 v/v) and yellow-
brown RCl2L(η6-p-cymene) 2a, 2c-e were obtained. Since enough
precipitate formed, only 2b complex was directly washed with
C2H5)2O and dried in a vacuum.The crude precipitate was crys-
tallized in a CH2Cl2/C2H5)2O, because in the complexes (2a, 2c-e)
weren’t formed sufficient precipitate.
2a: Yield: 78%, mp >225°C. Elemental Analysis (EA): Calcd
for C27H27Cl2N5O2SRu (656.97): C, 49.31%; H, 4.11%; N, 10.65%;
S, 4.87%. Found: C, 48.87%; H, 4.25%; N, 10.32%; S, 5.17%. FT-
IR ν ATR (cm−1) 3197 N-H, 3050 C-H arom., 3028 H-C=N, 2965
asym. C-H, 2934 sym. C-H, 1779 C=O, 1609,1589 C=N, 1482 C=C,
1361 C-H(bend),1235 C=S, 586 Ru-N. 1H NMR (400 MHz, CDCl3)
δ (ppm): 13.12 (1H, s, NH), 9.62 (1H, s, N=CH), 7.02-7.91 (9H
m, Ar-H of NCH(C6H5) and CCH2N(C6H4)), 5.26-5.45 (4H d, Ru-
C6H4), 5.20 (2H, s, CH2); 3.02(1H h, Ru-C6H4CH(CH3)2; 2.26 (3H
s, Ru-C6H4CH3); 1.33 (6H d, Ru-C6H4CH(CH3)2. 13C NMR 13C{1H}
NMR (100 MHz, CDCl3) δ (ppm): 164.2 (C=S), 160.3 (C=O); 145.6
(NCH(C6H5)); 109.3, 110.1, 123.3, 124.4, 128.2, 129.2, 129.5, 129.7
131.1, 133.5, 134.3 142.3 (Ar-C) of NCH(C6H5) and CCH2N(C6H4)
in either benzene ring; 153.6 (NCCH2); 82.1, 83.5, 98.8, 103.7
Ru-C6H4CH(CH3)2; 36.5 CCH2N;30.6 Ru-C6H4CH(CH3)2; 22.2 Ru-
C6H4CH(CH3)2; 18.4 Ru-C6H4CH3. ESI-MS (positive mode, m/z):
4 cm−1 1H NMR and 13C NMR data were obtained in DMSO-
.
d6 solvent on a Bruker AVANCE 400 spectrometer at room tem-
perature, at Inonu University, Malatya, Turkey. The high-resolution
mass spectra (HRMS) were obtained on a Waters LCT Premier XE
Mass Spectrometer also coupled to an EQUITY Ultra Performance
Liquid Chromatography System at Faculty of Pharmacy, Gazi Uni-
versity, Ankara, Turkey.
2.2. Design and synthesis of the ligands (1a-e)
3-[(o/p-substitutedphenylmethylidene]amino-5-thioxo-1,2,4-
triazol-3-yl)methyl]-2(3H)-benzoxazolone(1a-e)
which
are
calcd 586.07, found 586.08 for [M-2Cl]2+
.
Shiff base derivatives of 3-[(4-amino-5-thioxo-1,2,4-triazole-
3-yl)methyl]-2(3H)-benzoxazolone were synthesized by using
microwave technique as shown in Scheme 1, according to given
method in the literature. The structures of the synthesized ligands
(1a-e) were checked out by IR, 1H NMR, and 13C NMR spectra. 13C
NMR spectra data of the ligands (1b and 1c) were obtained firstly
in our current study.
2b: Yield: 84%, mp >235°C. Elemental Analysis (EA): Calcd for
C27H26BrCl2N5O2SRu (735.97): C, 44.02%; H, 3.53%; N, 9.51%; S,
4.34%. Found: C, 43.55%; H, 3.69%; N, 9.23%; S, 4.48%. FT-IR ν ATR
(cm−1) 3176 N-H, 3058 C-H arom., 3029 H-C=N, 2959 asym. C-H,
2936 sym. C-H, 1785 C=O, 1609, 1584 C=N, 1480 C=C, 1336 C-
H(bend), 1237 C=S, 749 C-Br, 590 Ru-N. 1H NMR (400 MHz, CDCl3)
δ (ppm): 13.09 (1H, s, NH), 10.38 (1H, s, N=CH), 7.06-8.36 (8H m,
2

R. Adiguzel, F. Türkan, Ü. Yildiko et al.
Journal of Molecular Structure 1231 (2021) 129943
Schema 1. Synthesis steps of the ligands 3-[(o/p-substitutedphenylmethylidene]amino-5-thioxo-1,2,4-triazol-3-yl)methyl]-2(3H)-benzoxazolone (1a-e).
Ar-H of NCH(o-BrC6H4) and CCH2N(C6H4)), 5.26-5.47 (4H d, Ru-
C6H4), 5.23 (2H, s, CH2); 3.05(1H h, Ru-C6H4CH(CH3)2; 2.28 (3H
s, Ru-C6H4CH3); 1.34 (6H d, Ru-C6H4CH(CH3)2. 13C{1H} NMR (100
MHz, CDCl3) δ (ppm): 164.7 (C=S), 160.3 (C=O); 145.8 (NCH(o-
BrC6H4)), 109.5, 110.1, 123.3, 124.5, 126.8, 128.4, 129.2, 129.7,130.7
133.4, 134.4, 142.3 (Ar-C) of NCH(o-BrC6H4) and CCH2N(C6H4)
in either benzene ring; 153.6 (NCCH2); 82.1, 83.9, 98.9, 103.8
Ru-C6H4CH(CH3)2; 36.5 CCH2N;30.6 Ru-C6H4CH(CH3)2; 22.3 Ru-
C6H4CH(CH3)2; 18.4 Ru-C6H4CH3. ESI-MS (positive mode, m/z):
C6H4CH(CH3)2; 18.4 Ru-C6H4CH3. ESI-MS (positive mode, m/z):
calcd 709.42, found 709.02 for [M+NH4]+.
2e: Yield: 80%, mp >300°C. Elemental Analysis (EA): Calcd
for C27H26Cl3N5O2SRu (691.42): C, 46.86%; H, 3.76%; N, 10.12%; S,
4.62%. Found: C, 46.39%; H, 4.02%; N, 9.69%; S, 4.71%. FT-IR ν ATR
(cm−1) 3220 N-H, 3051 arom. C-H,3021 H-C=N, 2965 asym. C-
H, 2927 sym. C-H, 1772 C=O, 1609, 1592 C=N, 1483 C=C, 1361
C-H(bend), 1235 C=S, 744 C-Cl, 586 Ru-N. 1H NMR (400 MHz,
CDCl3) δ (ppm): 13.16 (1H, s, NH), 9.65 (1H, s, N=CH), 7.03-7.86
(8H m, Ar-H of NCH(p-ClC6H4) and CCH2N(C6H4)), 5.26-5.45 (4H
d, Ru-C6H4), 5.20 (2H, s, CH2); 3.01 (1H h, Ru-C6H4CH(CH3)2;
2.26 (3H s, Ru-C6H4CH3); 1.33 (6H d, Ru-C6H4CH(CH3)2. 13C{1H}
NMR (100 MHz, CDCl3) δ (ppm): 162.4 (C=S), 160.3 (C=O); 145.6
(NCH(p-ClC6H4)), 109.4, 110.1, 123.3, 124.5, 128.4,129.6, 130.7, 131.8,
132.6, 138.3,139.9 142.3 (Ar-C)of NCH(p-ClC6H4) and CCH2N(C6H4)
in either benzene ring; 153.6 (NCCH2); 82.1, 83.6, 98.9, 103.8
Ru-C6H4CH(CH3)2; 36.6 CCH2N;30.6 Ru-C6H4CH(CH3)2; 22.3 Ru-
C6H4CH(CH3)2; 18.4 Ru-C6H4CH3. ESI-MS (positive mode, m/z):
calcd 664.97, found 664.49 for [(M-2Cl]2+
.
2c: Yield: 81%, mp >300°C. Elemental Analysis (EA): Calcd for
C27H26BrCl2N5O2SRu (735.97): C, 44.02%; H, 3.53%; N, 9.51%; S,
4.34%. Found: C, 43.54%; H, 3.64%; N, 9.33%; S, 4.45%. FT-IR ν ATR
(cm−1) 3172 N-H, 3051 C-H arom., 3021 H-C=N, 2967 asym. C-H,
2929 sym. C-H, 1775 C=O, 1609, 1584 C=N, 1480 C=C, 1361 C-
H(bend), 1235 C=S, 747 C-Br, 588 Ru-N. 1H NMR (400 MHz, CDCl3)
δ (ppm): 13.16 (1H, s, NH), 9.65 (1H, s, N=CH), 7.03-8.7.79 (8H m,
Ar-H of NCH(p-BrC6H4) and CCH2N(C6H4)), 5.26-5.45 (4H d, Ru-
C6H4), 5.19 (2H, s, CH2); 3.01 (1H h, Ru-C6H4CH(CH3)2; 2.31 (3H
s, Ru-C6H4CH3); 1.33 (6H d, Ru-C6H4CH(CH3)2. 13C{1H} NMR (100
MHz, CDCl3) δ (ppm):162.4 (C=S), 160.4 (C=O); 145.6 (NCH(p-
BrC6H4)), 109.4, 110.1, 123.3, 124.4, 125.3, 128.2, 128.6 129.0, 129.6,
130.0 130.8, 132.6 (Ar-C) of NCH(p-BrC6H4) and CCH2N(C6H4)
in either benzene ring; 153.6 (NCCH2); 82.1, 83.9, 98.9, 103.8
Ru-C6H4CH(CH3)2; 36.5 CCH2N;30.6 Ru-C6H4CH(CH3)2; 22.2 Ru-
C6H4CH(CH3)2; 18.4 Ru-C6H4CH3. ESI-MS (positive mode, m/z):
calcd 620.52, found 620.05 for [M-2Cl]2+
.
2.4. Enzyme inhibitory activity
2.4.1. Determination of AChE and BChE inhibitory activity
The determination of cholinesterase activity was done by the
Ellman method with some change [22,23]. For BChE and AChE in-
hibitions test butyrylthiocholine iodide and acetylthiocholine io-
dide were used as substrates. To determine enzyme activities three
calcd 664.97 found 664.99 for [M-2Cl]2+
.
ꢀ
2d: Yield: 83%, mp >233°C. Elemental Analysis (EA): Calcd
for C27H26Cl3N5O2SRu (691.42): C, 46.86%; H, 3.76%; N, 10.12%; S,
4.62%. Found: C, 46.37%; H, 3.62%; N, 9.66%; S, 4.18%. FT-IR ν ATR
(cm−1) 3169 N-H, 3049 arom. C-H, 3019 H-C=N, 2967 asym. C-H,
2924 sym. C-H, 1775 C=O, 1610, 1586 C=N, 1482 C=C, 1359 C-
H(bend), 1235 C=S, 744 C-Cl, 586 Ru-N. 1H NMR (400 MHz, CDCl3)
δ (ppm): 13.16 (1H, s, NH), 9.63 (1H, s, N=CH), 7.03-7.79 (8H m,
Ar-H of NCH(o-ClC6H4) and CCH2N(C6H4)), 5.26-5.45 (4H d, Ru-
C6H4), 5.20 (2H, s, CH2); 2.99 (1H h, Ru-C6H4CH(CH3)2; 2.26 (3H
s, Ru-C6H4CH3); 1.33 (6H d, Ru-C6H4CH(CH3)2. 13C{1H} NMR (100
MHz, CDCl3) δ (ppm): 162.3 (C=S), 160.4 (C=O); 145.6 (NCH(o-
ClC6H4)), 109.4, 110.1, 123.3, 124.4, 125.3, 128.6, 130.4, 130.7, 131.9,
132.6, 134.5, 142.3 (Ar-C) of NCH(o-ClC6H4) and CCH2N(C6H4)
in either benzene ring; 153.6 (NCCH2); 82.1, 83.5, 98.9, 103.8
Ru-C6H4CH(CH3)2; 36.5 CCH2N;30.6 Ru-C6H4CH(CH3)2; 22.2 Ru-
solutions of 50 μL BChE-AChE, 5,5 -Dithiobis(2-nitrobenzoic acid)
(DTNB), 100 μL tris/HCl buffer solution (1 mM, pH 8.0), and drug
solution (10 mL) was stirred and then left in the dark at room
temperature for 10 minutes. Finally, 0.5 mM, 50 μL of 2-nitro-
benzoic (5,5-Dithiobis acid) was added to the prepared mixture,
and the reaction was initiated. By the formation of DTNB color
change was calculated at 412 nm by UV–Vis spectrophotometer
[24].
2.4.2. Determination of GST inhibitory activity
Novel Ru(II) complexes of Shiff base derivatives of 3-[(4-amino-
5-thioxo-1,2,4-triazole-3-yl)methyl]-2(3H)-benzoxazolone inhabita-
tion effects were determined in the range of 20-100 μM
concentration on GST enzyme activity according to the to
(Habig et al. 1974) previous publications [25,26]. Their Ki
3

R. Adiguzel, F. Türkan, Ü. Yildiko et al.
Journal of Molecular Structure 1231 (2021) 129943
Fig. 1. Optimized 3D structures of the Shiff base derivatives and Ru(II) complexes ligands.
and IC50 values were calculated and results were given in
Table 1.
3. Results and discussion
3.1. Experimental
Characteristic peaks of the ligands in the IR spectrum (1a-
e) were confirmed with values are given in literature The struc-
tures of the synthesized Ru(II) complexes (2a-e) were illuminated
by elemental analysis, IR, 1H NMR, 13C NMR, and mass spectra
(See Fig. 2). According to mass spectra and elemental analyses,
the general formula of all complexes was determined as [Ru(η6-
p-cymene)LCl2].
2.5. Molecular docking studies
In the molecular docking study, acetylcholinesterase (PDB ID:
6O4W) [27], butyrylcholinesterase (PDB ID: 6SAM) [28], and glu-
tathione s-transferase (PDB ID: 5JCU) [29]. enzymes were used
as receptors. Crystallographic structures of enzymes obtained
from the protein database (PDB) at the Research Collaboratory
for Structural Bioinformatics (RCSB) (see http//www.rcsb.org/pdb).
The Maestro Molecular Modeling platform (version 11.8) of the
Schrödinger, LLC model was used as the docking program [30].
The structures of the ligands that were papered with ChemBio3D
is part of the ChemBioOffice 2019 Suite in SDF file format as 3D
structures (Fig. 1). The ionization is created by removing or adding
protons from the ligand. Ionization work can be quite useful for
generating a varied range of ionization filled with a chosen pH
range. In the Ligand optimization, the force field geometry is opti-
mized in the protein, and partial atomic loads are perfectly pre-
sented using the force field. During the enzymatic process, it is
very important to evaluate the changes in the protonation status of
functional groups at the right time and to elucidate the proton ad-
dition mechanism. The Missing residues or atoms fixes by the pro-
gram. Water molecules play an important role in ligand binding.
It should be classified according to their interactions with neigh-
boring protein atoms and water molecules. When the right water
molecules are targeted and removed, significant gains in affinity
and selectivity are obtained [31] Lig prep, Protein Preparation Wiz-
ard, and Receptor Grid Generation modules of Maestro software,
at this stage all water molecules were removed and polar hydro-
gen atoms were added. A grid box was formed around the active
site of the proteins containing natural ligands. These studies were
carried out according to the methods used in the previous stud-
ies [32,33]. The Glide score and energy were analyzed for receptor
binding affinity and the interactive nature of the Ligand - proteins.
Molecular docking studies were performed with the Glide dock-
ing module under Maestro. The resulting receptor model, 2D, and
3D interactions were visualized with Maestro and Discovery Studio
2017 version [34].
3.1.1. IR spectra
The N-H stretching bands in the IR spectra of all ligands
(1a-e) were observed at 3230, 3248, 3258, 3238, and 3298
cm−1respectively[7]. These N-H stretching bands for Ru(II) com-
plexes (2a-e) shifted to the low-frequency region and were ob-
served at 3197, 3176, 3172, 3169, and 3220 cm−1 respectively (2a-
e). The shift to low wave number is compatible with decreasing
electron density in the N-H bond [35,36], In other vibration fre-
quencies of all complexes didn’t appear a considerable change ex-
cept the C=O stretching bands. In general, it is reported in the lit-
erature when the metal ion is coordinated by the C=O group, in
contrast to results the C=O band shifts to the lower frequency re-
gion [37]. The reason for the shifting to the high frequency of the
CO band (from 1744 to 1785 cm−1) can be explained by the in-
crease in electron density of the CO bond due to the coordination
of nitrogen of triazole ring with the metal. On the other side, Ru-N
vibration frequency has been reported that observed at about 700-
416 cm−1region in the literature [35,38,39]. As for in Ru(II) com-
plexes synthesized by our research group Ru-N peak appeared be-
tween 586-590 cm−1 as a mild intense band.
3.1.2. NMR spectra
While in the 1H- NMR spectrum of ligands (1a-e), N-H proton
over 1,2,4-triazole ring was observed at δ=14.10, 14.17, 14.12, 14.10,
and 14.06 ppm as a singlet peak respectively, this peak for Ru(II)
complexes (2a-e) shifted to a lower frequency and was observed
at observed between at δ=13.09-13.16 ppm [7,40]. This shifting to
lower frequency shows that around N-H proton over 1,2,4-triazole
ring change. In this case, it can be explained over N atom in the
1,2,4-triazole ring with the coordination of metal. So all ligands
acted as a monodentate ligand.
4

R. Adiguzel, F. Türkan, Ü. Yildiko et al.
Journal of Molecular Structure 1231 (2021) 129943
Table 1
The inhibition results of the Shiff base derivatives ligand (1a-e) (a) and Ru(II) complex (2a, e) (b) against glutathione S-transferase (GST), acetylcholinesterase (AChE), and
butyrylcholinesterase (BChE) enzymes.
(a) Shiff base derivatives ligand (1a-e)
IC50 (µM)
AChE
Ki (µM)
Ligand
Structures
R2
BChE
R2
GST
R2
AChE
BChE
GST
41.70
32.03
43.42
0.9491
30.14
0.9555
48.12
0.8821
37.88
30.25
38.96
23.51
40.07
0.9603
0.9606
40.10
36.79
0.9748
0.9455
58.23
58.72
0.8627
0.815
35.78
31.58
47.54
51.73
33.62
44.41
0.9712
0.9432
43.63
45.05
0.9739
0.9814
46.2
0.931
30.18
41.13
33.45
40.09
38.75
38.76
48.98
0.9582
Tacrine
7.80
0.9443
0.9628
8.77
19.73
22.36
(b) Ru(II) complex (2a, e)
∗
Used as a positive control for AChE and BChE enzymes.
5

R. Adiguzel, F. Türkan, Ü. Yildiko et al.
Journal of Molecular Structure 1231 (2021) 129943
˚
˚
˚
Fig. 2. High resulition 3D crystal structures of proteins with the interactive regions. a) AChE (Resolution: 2.35 A), b) BChE (Resolution: 2.50 A) c) GST (Resolution: 1.93 A).
On the other hand, it can be shown the observed peaks that for
p-cymene group protons between 5.26-5.47 ppm (4H d, Ru-C6H4),
2.99-3.02 ppm (1H h, Ru-C6H4CH(CH3)2, 2.26-2.31 ppm (3H s, Ru-
C6H4CH3), and 1.33-1.34 ppm (6H d, Ru-C6H4CH(CH3)2as evidence
[41]. Also, the four peaks corresponding to p-cymene carbons ap-
peared between at 83.1-103.8 ppm in 13C NMR spectra of Ru(II)
complexes [42].
42.0 μM IC50.1c compound showed very week activity against the
GST with 51.73 μM Ki and 58.72 μM IC50 values.
3.3. Computational analysis
Molecular docking is useful for studying the binding mecha-
nism between the ligand-receptor and understanding the interac-
tion of possible binding modes at the molecular level. Here, molec-
ular docking was performed to obtain the preferred binding sites
of the ligands with the receptor and to substantially confirm the
experimental observations [44-46]. In the study consisting of 5 dif-
ferent Shiff Base Derivatives and 4 enzyme sets, 20 docking results
were obtained (Table 1). These ligands were placed in the catalytic
active region of the enzyme and the docking results were analyzed
based on binding affinity and interaction mode. The halogenated
structures at the ortho-position achieved a good binding score with
all enzymes relative to the para position. However, as a molecu-
lar structure, the best binding affinity score was observed in AChE
and BChE enzymes. The interactions of molecules with enzymes
are shown in Figs. 3 and 4. These figures show the interactions
of ligands (1a-e) with enzymes. The interaction of the Ru(II) com-
plexes (2a-e) could not be obtained due to the very high molec-
ular volume [15,47]. Molecular structural similarity to the natural
ligand in protein structure increases this value. Fig. 2 shows the
regions where proteins can interact with 3D crystal structures and
small molecules (ligands). The dynamics of the protein play an im-
portant role in how proteins interact with Shiff base derivatives to
form complexes, which can increase or inhibit its biological func-
tion. Since AChE is in the binding inner regions of proteins, higher
affinity was found compared to other enzymes.
3.1.3. Mass spectra
The mass spectrum of [Ru(η6p-cymene)LCl2] complexes (2a-
c and 2e) showed as the ion peak of the cationic complexes
at m/z=586.08, 664.49, 664.99, 620.05 respectively for [M-2Cl]2+
.
Only for Ru(II) complex (2d) found at m/z=709.02 [M+NH4]+ be-
cause ammonium ion was bounded to the molecule during the dis-
solution of the complex [43].
3.2. Determination of enzyme inhibition study
The synthesized ligands (1a-e) and their Ru(II) complex (2a-e)
compounds were investigated for their inhibitory activity on AChE,
BChE, and GST. Ki and the half-maximal inhibitory concentration
(IC50) values of the ligand and its complexes were calculated and
evaluated for three metabolic enzymes and the molecular docking
study was performed. The results derived for the ligand (1a-e) and
their complexes (2a-e) against the AChE, BChE, and GST are given
in Table 1. 1a compound was found to be the best inhibitor of
BChE and its Ki and IC50 values were 23.51 μM and 30.14 μM. BChE
inhibited by 2d complex with lower Ki (42.81 μM) and IC50 (50.46
μM) values when compared with other compounds. For AChE 2c
compound was the best inhibitor with 26.87 μM Ki and 35.76 μM
IC50 values. 2a compound was the weakest inhibitor for the AChE
with 47.63 μM Ki and 53.23 μM IC50 values. While2a demonstrated
very good inhibitory activity against the GST with 33.14 μM Ki and
Ligand-receptor docking binding and interaction parameters are
listed in Table 2. In the ligands compared to other enzymes, the
highest glide score was calculated as -10.183 kcal/mol between
compound 1d and AChE. According to the results Glide scores, 1c
6

R. Adiguzel, F. Türkan, Ü. Yildiko et al.
Journal of Molecular Structure 1231 (2021) 129943
Fig. 3. 2D View of the interaction modes between Shiff base derivatives and enzymes, a) 1c-AChE, b)1e-AChE, c) 1d-BChE, d)1a-GST.
Table 2
covalent bond with an energy of more than 47.8 kcal, non-covalent
interactions were assumed to be much weaker. While it has an en-
ergy of between 5.97 and 9.55 kcal(6) for a hydrogen bond, the
energies of non-covalent interactions in Table 1 are less than 2.39
kcal. However, these energies are sufficient for binding [48,49]. In-
hibitor activity was powerful due to the π-donor hydrogen bond
(PHE 338) and conventional hydrogen bond (TYR 124). Other bind-
ing parameters are given in Fig. 3 a-b. Binding scores also provided
well results in the interaction of BChE with Schiff bases deriva-
tives and herein the highest score was found to be -9.111 kcal/mol.
π-alkyl is well docked with interactions such as ALA 328, HIS
438, LEU 286, carbon-hydrogen bond TRP 82, DMS 601 (Fig. 3c).
Docking of compound 1a with GST gave the best score with 6.097
kcal/mol. Interactions are present such as protein residues, LEU108,
LEU 107, and ALA 216 π-alkyl, ARG 15, VAL 55, conventional hy-
drogen bond that contributes to binding affinity (Table 3). Other
binding parameters are given in Fig. 3d.
Best-binding affinity scores (kcal/mol) of Shiff base derivatives in the
catalytic sites of the enzymes.
Shiff base
derivatives
Glide Score
AChE
BChE (PDB:
GST
(PDB:6O4W)
6SAM)
(PDB:5JCU)
1a
1b
1c
1d
1e
-9.933
-10.004
-9.518
-10.183
-9.659
-8.513
-8.384
-7.558
-9.111
-7.288
-6.097
-5.667
-5.182
-5.936
-5.725
and 1e exhibited excellent binding affinities to AChE enzymes, as
seen in Table 1. The high binding affinity here maybe the reason
for the catalytic site selectivity of AChE.In addition to conventional
hydrogen bonds, it is another type of hydrogen bond in which π-
systems (especially aromatic rings) play the role of proton accep-
tors. This type of hydrogen bonding is important in understanding
biochemical processes such as hydrophobic interactions in protein,
ligand-protein interactions [48,49]. π -π stacking, unlike a single
After the best pose selection in all ligand-enzyme docking,
binding modes were analyzed to understand the inhibition mech-
anisms. When Fig. 4a is examined, blue-colored surfaces for aro-
matic ring edges, an orange-colored surface represents the inter-
action formed on the aromatic ring faces. In Fig. 4 b, d, the ma-
7

R. Adiguzel, F. Türkan, Ü. Yildiko et al.
Journal of Molecular Structure 1231 (2021) 129943
Fig. 4. The interaction mode between Shiff base derivatives - the enzymes, 3D View of a) the solvent accessibility interaction surface on 1c-AChEb) the hydrogen bonds
donor/acceptor surface on 1e-AChE, c) the aromatic interaction surface on 1d-BChE, d) the hydrogen bonds donor/acceptor surface on 1a-GST.
Table 3
The parameters of the interaction modes between Shiff base derivatives and enzymes, a) 1c-AChE, b)1e-AChE, c)1d-BChE, d)1a-GST.
AChE - 1c
Bond
AChE - 1e
Bond
BChE-1d
GST-1a
Bond
Important
Residues
Residues
Residues
Bond
Length (A)
Residues
˚
˚
˚
˚
interactions
Length (A)
Length (A)
Length (A)
Conventional
TYR 124-N
PHE 295-O
2.17
2.52
TYR 124-NPHE
295-O
2.22
2.50
ARG 15-O
VAL 55-S VAL
55-H
1.87-2.16,
2.72
Hydrogen Bond
2.62
Pi-Donor Hydrogen
Bond
PHE 338-S
3.78
Pi- Sulfur
TYR 337-S
TRP 286,
PHE 338
3.55
TYR 337-S
TRP 286
3.55
3.73,
4.78
5.76
5.07
4.93
5.38
MET 208
PHE 220
5.15
4.25
Pi-Pi stacked
3.73, 4.70
5.76
DMS 601-Cl
2.88
PHE 338 TRP
86 –Cl TRP 86
-Ar
Pi- Alkyl
LEU 130
5.05
LEU 130-Cl
ALA 328,
HIS 438,
LEU 286,
PHE 329
4.94
4.38
4.83
4.78
LEU 108 LEU
107 ALA 216
5.50
5.44
4.75
Pi-Donor Hydrogen
Bond
PHE 338 –S
3.78
Pi-Pi T-Shaped
TRP 231
PHE 329
TRP 82 DMS
601
4.88,4.96,
4.78,5.28
3.06
CarbonHydrogen
Bond
2.59,3.10
4.67,5.16
Pi – Lone Pair
GLY 116
8

R. Adiguzel, F. Türkan, Ü. Yildiko et al.
Journal of Molecular Structure 1231 (2021) 129943
genta surfaces in hydrogen bonds represent the donor group, and
in green the acceptors. In Fig. 4c, the solvent accessibility of the re-
ceptor residues creates a colored surface that decreases from blue
to green.
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Herein, [Ru(η6-p-cymene)LCl2] complexes (2a-e) of Shiff base
derivatives of 3-[(4-amino-5-thioxo-1,2,4-triazole-3-yl)methyl]-
2(3H)-benzoxazolone ligands (1a-e) were synthesized in very
short reaction times and very good yields. The novel synthesized
complex structures of ligands were illuminated by spectroscopic
methods and elemental analysis. In vitro and in silico study was
used to evaluate the inhibition mechanism of the compounds
against the AChE, BChE, and GST enzyme. When compared to
standard tacrine it is possible to say the synthesized molecules
have significant inhibition effects against the enzymes used.. 1a,
2a, 2c compounds were observed to be the best inhibitors rela-
tively. Both 1a and 2a showed good inhibition effects against the
BChE. Inhibition properties of the compounds could be attributed
to the contribution of donor atoms of the 1,2,4-triazole group,
2(3H)-benzoxazolone group, and (-CH=N) imine group of the
ligands. However, further in vitro and in vivo pharmacological
researches need to illuminate the biological activities of these
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Declaration of Competing Interest
[19] N. Verma, A. Yadav, S. Bal, R. Gupta, N. Aggarwal, In vitro studies on amelio-
rative effects of limonene on cadmium-induced genotoxicity in cultured hu-
man peripheral blood lymphocytes, Appl. Biochem. Biotechnol. 187 (4) (2019)
1384–1397.
There are no conflicts to declare.
[20] N. Allocati, M. Masulli, C. Di Ilio, L. Federici, Glutathione transferases: sub-
strates, inihibitors and pro-drugs in cancer and neurodegenerative diseases,
Oncogenesis 7 (1) (2018) 1–15.
Acknowledgments
[21] S. Boy, F. Türkan, M. Beytur, A. Aras, O. Akyıldırım, H.S. Karaman, H. Yüksek,
Synthesis, design, and assessment of novel morpholine-derived Mannich bases
as multifunctional agents for the potential enzyme inhibitory properties in-
cluding docking study, Bioorg. Chem. (2020) 104524.
[22] F. Turkan, Z. Huyut, P. Taslimi, I. Gulcin, The effects of some antibiotics from
cephalosporin groups on the acetylcholinesterase and butyrylcholinesterase
enzymes activities in different tissues of rats, Arch. Physiol. Biochem. 125 (1)
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A. Sujayev, N. Sadeghian, S.H. Alwasel, The biological activities, molecular
docking studies, and anticancer effects of 1-arylsuphonylpyrazole derivatives,
J. Biomol. Struct. Dyn. (just-accepted) (2020) 1–20.
We appreciate the Catalysis Research and Application Center In-
onu University (for analyses of 1H NMR and 13C NMR spectra), Fac-
ulty of Pharmacy Department of Pharmaceutical Chemistry Gazi
University (for mass spectra), and Tunceli Vocational School, De-
partment of Chemistry and Chemical Process Technologies Mun-
zur University (for FT-IR spectra). The enzyme inhibition study was
carried out in Ig˘dır University Research Laboratory Application and
Research Center (ALUM) in Turkey.
[24] P. Taslimi, A. Sujayev, F. Turkan, E. Garibov, Z. Huyut, V. Farzaliyev, S. Mame-
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