Synthesis, characterization, crystal structure of novel bis-thiomethylcyclohexanone derivatives and their inhibitory properties against some metabolic enzymes

Article abstract of DOI:10.1016/j.bioorg.2018.11.001

In this study, a series of novel bis-thiomethylcyclohexanone compounds (3a–3j) were synthesized by the addition of thio-Michael to the bis-chalcones under mild reaction conditions. The bis-thiomethylcyclohexanone derivatives (bis-sulfides) were characterized by ¹H NMR, ¹³C NMR, FTIR and elemental analysis techniques. Furthermore, the molecular and crystal structures of 3h, 3i and 3j compounds were determined by single crystal X-ray diffraction studies. In this study, X-ray crystallography provided an alternative and often-complementary means for elucidating functional groups at the enzyme inhibitory site. Acetylcholinesterase (AChE) is a member of the hydrolase protein super family and has a significant role in acetylcholine-mediated neurotransmission. Here, we report the synthesis and determining of novel bis-thiomethylcyclohexanone compounds based hybrid scaffold of AChE inhibitors. The newly synthesized bis-thiomethylcyclohexanone compounds showed K_i values of in range of 39.14–183.23 nM against human carbonic anhydrase I isoenzyme (hCA I), 46.03–194.02 nM against human carbonic anhydrase II isoenzyme (hCA II), 4.55–32.64 nM against AChE and 12.77–37.38 nM against butyrylcholinesterase (BChE). As a result, novel bis-thiomethylcyclohexanone compounds can have promising anti Alzheimer drug potential and record novel hCA I, and hCA II enzymes inhibitor.

Full text of DOI:10.1016/j.bioorg.2018.11.001

Accepted Manuscript
Synthesis, Characterization, Crystal Structure of Novel Bis-Thiomethylcyclo-
hexanone Derivatives and Their Inhibitory Properties Against Some Metabolic
Enzymes
Abdullah Biçer, Parham Taslimi, Gül Yakalı, Ilhami Gülçin, Mehmet Serdar
Gültekin, Günseli Turgut Cin
PII:
S0045-2068(18)30994-5
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.001
YBIOO 2609
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 September 2018
3 October 2018
1 November 2018
Please cite this article as: A. Biçer, P. Taslimi, G. Yakalı, I. Gülçin, M. Serdar Gültekin, G. Turgut Cin, Synthesis,
Characterization, Crystal Structure of Novel Bis-Thiomethylcyclohexanone Derivatives and Their Inhibitory
Properties Against Some Metabolic Enzymes, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.11.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, Characterization, Crystal Structure of Novel Bis-
Thiomethylcyclohexanone Derivatives and Their Inhibitory Properties
Against Some Metabolic Enzymes
Abdullah Biçera, Parham Taslimib, Gül Yakalıc,d, Ilhami Gülçinb,*,
Mehmet Serdar Gültekinb,e and Günseli Turgut Cina,*
aDepartment of Chemistry, Faculty of Science, Akdeniz University, 07058-Antalya, Turkey
bDepartment of Chemistry, Faculty of Science, Atatürk University, 25240-Erzurum, Turkey
cSerik Gülsün Süleyman Sural Vocational School of Higher Education, Department of Opticianry
Program, Akdeniz University, 07058-Antalya, Turkey
dCentral Research Laboratory, İzmir Katip Çelebi University, 35620-İzmir, Turkey
eFaculty of Pharmacy, Ağrı İbrahim Çeçen University, 04100-Agrı, Turkey
*Corresponding authors
E-mail address: gturgut@akdeniz.edu.tr (G. Turgut Cin).
E-mail address: igulcin@atauni.edu.tr, Phone: +904422314375, Fax:+90 4422314109 (Ilhami Gulcin).

ABSTRACT
In this study, a series of novel bis-thiomethylcyclohexanone compounds (3a-3j) were
synthesized by the addition of thio-Michael to the bis-chalcones under mild reaction
conditions. The bis-thiomethylcyclohexanone derivatives (bis-sulfides) were characterized by
1H-NMR, 13C-NMR, FTIR and elemental analysis techniques. Furthermore, the molecular and
crystal structures of 3h, 3i and 3j compounds were determined by single crystal X-ray
diffraction studies. In this study, X-ray crystallography provided an alternative and often-
complementary means for elucidating functional groups at the enzyme inhibitory site.
Acetylcholinesterase (AChE) is a member of the hydrolase protein super family and has a
significant role in acetylcholine-mediated neurotransmission. Here, we report the synthesis
and determining of novel bis-thiomethylcyclohexanone compounds based hybrid scaffold of
AChE inhibitors. The newly synthesized bis-thiomethylcyclohexanone compounds showed Ki
values of in range of 39.14-183.23 nM against human carbonic anhydrase I isoenzyme (hCA
I), 46.03-194.02 nM against human carbonic anhydrase II isoenzyme (hCA II), 4.55-32.64
nM against AChE and 12.77-37.38 nM against butyrylcholinesterase (BChE). As a result,
novel bis-thiomethylcyclohexanone compounds can have promising anti Alzheimer drug
potential and record novel hCA I, and hCA II enzymes inhibitor.
Keywords: bis-thiomethylcyclohexanone; carbonic anhydrase; acetylcholinesterase; crystal
structure
2

1. INTRODUCTION
Carbon-sulfur bond formation is an important methodology used in the synthesis of sulfur-
containing natural and pharmaceutical products [1-5]. The thio-Michael addition reaction is
one of the most important processes for C-S bond formation [6]. Generally, C-S bond
formation is performed with the addition of a thiol, 1,4-conjugate on the acceptor α,β-
unsaturated carbonyl compounds by Lewis acid or deprotonation of thiol [7,8]. It is known
that this method plays an important role in the synthesis of bioactive compounds [8-11].
Sulfide and bis-sulfide compounds have been reported to possess important biological
activities in organic, pharmaceutical and biological applications (Scheme 1) [12-15].
Scheme 1
It is well known that the studies related to enzyme inhibition and activation is one of
the most common biological activities. Carbonic anhydrases (CAs) are extensively distributed
zinc-comprising metalloenzymes available in all life phyla, which hold pH homeostasis factor
in the human body by catalyzing the carbon dioxide (CO2) hydration action to bicarbonate
-
(HCO3 ) and proton (H+) plus other hydrolytic reactions [16-19]. Also, depending on their
localization in diverse organisms, capacity, and catalytic activity to various classes of
inhibitors, CAs are divided in seven genetically separate classes, α-, β-, δ-, γ-, ζ-, η- and θ-
CAs [20,21]. Indeed, only the α-class is recorded to be present in human bodies, in which
sisteen α-CA isoenzymes were explained which differ in their distribution in tissues,
subcellular localization, and kinetic and molecular properties [22-24]. The CA isoenzymes are
involved in multiple physiological and biochemical processes like calcification, acid-base
regulation, ureagenesis, bone resorption, tumorigenicity, and gluconeogenesis, hence
representing interesting biochemical aims for the design of CA inhibitors (CAIs) with many
biological applications [25,26]. The ubiquitous hCA I isoenzyme is involved in cerebral
edema, and retinal, and its inhibition action can be a valuable factor in fighting these
situations. The cytosolic hCA II isozyme is involved in epilepsy, edema, glaucoma, and
among others [27,28]. The clinical usage of CAIs has been established as anti-glaucoma
agents, antiepileptics and diuretics. Also, they frequently used in the treatment of mountain
sickness, duodenal and gastric ulcers, neurological disorders, idiopathic intracranial
hypertension and osteoporosis [29,30].
On the other hand, Alzheimer’s disease (AD) causes an advanced harm on the central
neural mechanism, such as the decline in language, behavioral disorders, and memory loss
[31,32]. Various anomalies in the brain cells including inflammation, oxidative stress,
neuronal cell death and protein aggregates are relevant to the effects of the AD [33]. The
3

heterogeneous etiology and complexity of AD play a key role in novel drug development to
avoid the progress of AD [34-36]. One significant therapeutic palliative-process to minimize
the AD effects has been utilized of AChE inhibitors (AChEIs) such as rivastigmine,
donepezil, tacrine, and galantamine. Recently, it was reported that some identified AChEIs as
promising molecules to treat dementia, withdrawned from the clinical trials due to their
toxicity and poor central nervous system stability [37,38]. Therefore, the design, discovery
and development of novel AChEIs are still a big challenge for medicinal chemist. Thus, the
selectivity of the inhibition has a great importance and presents a significant consideration for
developing novel kinds of AChEIs [39-42].
In this study, we synthesized new bis-sulfide derivatives using bis-
benzylidenecyclohexanone (bis-chalcone) and evaluated their biological activites. Also, we
aimed to investigate the inhibitory effect of these novel bis-thiomethylcyclohexanone
derivatives (3a-j) using bis-benzylidenecyclohexanone (2a-j) derivatives on the hCA I, hCA
II, AChE and BChE enzymes for alternative and compared to standard and clinically used
inhibitors.
2.RESULTS AND DISCUSSION
2.1. Chemistry
Firstly, the bis-chalcone compounds were synthesized according to previous studies [43,44].
For this purpose, the substituted aromatic aldehydes and cyclohexanones were subjected to
the Claisen-Schmidt condensation reaction in ethanol with potassium hydroxide (Scheme 2).
Then, suitable reaction conditions for the synthesis of bis-sulfide compounds were
determined. For this, the reaction of addition of thiophenol to bis-chalcone using different
Lewis acids and bases was studied and the most suitable reaction condition was found to be
metallic Na in DCM (Table 1).
Scheme 2 and Table 1
TLC detected the product mixture in experiments with iodine. However, any products were
not isolated from this mixture. Also, in the experiments with other catalysts (Table 1, entry 3-
7), product formation was not observed. A single product was formed in the experiments with
sodium at room temperature in DCM. This compounds was detected by TLC and purified by
crystallization (Table 1). The bis-chalcone based sulfides were synthesized by reacting bis-
chalcones with thiophenol in the presence of sodium-metal in dichloromethane at room
temperature via thio-Michael addition reaction (Scheme 3). Spectrometric data (1H-NMR,
4

13C-NMR, FTIR and elemental analysis) and physical properties (color and melting point) of
these products have been confirmed by comparison with similar studies reported in the
literature [45,46].
Scheme 3
2.2. Enzyme inhibition studies
Evaluation of the effects of novel bis-sulfide derivatives (3a-j) on both cholinergic enzymes
of AChE and BChE, and both hCA isoenzymes was the main objective of this study. The
inhibition results are summarised in Table 2 and related Figures 1 and 2. The cytosolic hCA I,
and II isoenzymes are present in the human body and are involved in the secretion of
-
electrolytes in a plenty of tissue cells, like the HCO3 rich aqueous humor in the anterior
chamber of the cerebrospinal fluid or the eyes, as well as hold CO2 homeostasis and pH all the
body [47,48]. Indeed, dysregulation of hCA I, and II isoenzymes in tissue cells lead to some
pathologic conditions including edema, glaucoma and epilepcy [49,50]. For hCA I isozyme,
the Ki values were found in range of 39.14±9.94-183.23±28.18 nM. In this study, the Ki for
the positive control CA inhibitor acetazolamide (AZA), a recorded hCA I inhibitor, was
273.61±76.86 nM against hCA I isoenzyme (Table 2). All novel bis-sulfide compounds (3a-j)
had effective inhibition effects against hCA I isoenzyme. In addition, between these novel
molecules, 2,6-bis((4-chlorophenyl)(phenylthio)methyl)cyclohexanone (3h) and 2,6-bis((3-
bromophenyl)(phenylthio)methyl)cyclohexanone (3i), which possessed chlorophenyl and
bromophenyl groups demonstrated the best hCA I inhibitor (Kis of 39.14±9.94 and
47.92±7.20 nM, respectively) (Figure 1). It is well known that the molecules comprising
chlorophenyl and bromophenyl groups are efficient CA inhibitors [51]. As seen in Table 2,
IC50 values were found in the range of 38.63-180.34 nM towards hCA I, and 42.84-201.58
nM for hCA II. Novel bis-sulfide derivatives (3a-j) synthesized in this study significantly
inhibited the slow cytosolic hCA II isozyme with Ki in the low nanomolar levels. Ki values
were obtained between 46.03±10.47 and 194.02±28.55 nM (Table 2). Also, the compounds of
3h and 3i are, in fact, the best inhibitor among these molecules (Ki 46.03±10.47 and
48.18±19.10 nM, respectively) compared to the AZA, which is known as specific inhibitor of
CA isoenzymes as a standard (Ki: 229.08±55.14 nM). For hCA I isoform, its excellent
inhibitors were (3h) and (3i), which were the best hCA I inhibitor with IC50: 38.63 nM (r2:
0.9812)
and
IC50:
44.28
nM
(r2:
0.9590),
respectively.
2,6-Bis((3-
nitrophenyl)(phenylthio)methyl) cyclohexanone (3c) compound had relativelly weak
5

inhibition effects when compared to other compounds for this isoenzyme IC50: 180.34 nM (r2:
0.9598) for hCA I, IC50: 201.58 nM (r2: 0.9902) for hCA II isoenzyme.
Table 2, Figure 1 and 2
In this work, AChE and BChE were also extremely inhibited by novel bis-sulfide compounds
(3a-j) at the nanomolar range inhibition. Ki values of this novel compounds were found in the
range of 4.55±0.94-32.64±6.86 nM for AChE and 12.77±2.20-37.38±11.53 nM for AChE
(Table 2). These inhibition results clearly designated that novel synthesized compounds had
efficient both cholinergic enzymes inhibition properties. Indeed, the most potent AChE and
BChE
inhibition
were
obtained
by
novel
compound
2,6-bis((3-
fluorophenyl)(phenylthio)methyl)cyclohexanone (3e) with Ki values of 4.55±0.94 and
12.77±2.20 nM, respectively (Figure 1). Also, tacrine (1,2,3,4-tetrahydroacridin-9-amine),
which the first centrally acting cholinesterase inhibitor approved for the treatment of AD,
possesed Ki values of 56.37±15.10 and 63.40±13.62 nM against both cholinergic AChE and
BChE enzymes, respectively. Eventually, the Ki values of these derivatives for AChE and
BChE were calculated from Lineweaver-Burk plots. Additionally, as seen in Table 2, IC50
values are in the range of 7.18-41.77 nM towards AChE and in the range of 17.90-61.38 nM
for BChE. 2,6-Bis((4-nitrophenyl)(phenylthio)methyl)cyclohexanone (3d) is weak inhibitor
compared to other compounds for these enzymes. IC50 were found as 41.77 nM (r2:0.9732)
for AChE and 61.38 nM for BChE (r2:0.9682).
CA inhibitors targeting hCAs are clinically used in recent years for the management of
diverse diseases among which obesity, glaucoma, intracranial hypertension epilepsy, and as
diuretics [52]. Recently, they started to be utilized for the therapy of hypoxic tumours, were
also evaluated as possible drugs for cerebral ischemia, neuropathic pain, and arthritis.
Additionally, plenty of work has been done on the synthesis and characterization of CAIs
belonging to diverse groups, such as coumarins, carboxylic acids, sulfonamides, phenols,
dithiocarbamates, heterocyclic derivatives, etc [53]. Indeed, sulfonamides and their
bioisosteres such as the sulfamides and the sulfamates are powerful active site coordinating
CAIs, which in deprotonated form, bind to the Zn2+ ion present within the active site of
enzyme [54]. Many sulfonamide-based drugs, such as methazolamide, acetazolamide,
dorzolamide, ethoxzolamide, brinzolamide and celecoxib are recorded. Some of them are in
clinical use as antiepileptics (targeting hCAs VII and XIV), diuretics (targeting hCAs II, IV,
XII and XIV), antiglaucoma (targeting hCAs II, IV and XII), or in clinical trials as antitumor
or antimetastatic agents (targeting hCAs IX and XII) [55,56].
6

Many researches have recorded that by raising ACh level through inhibiting AChE
and BChE enzymes in the body offer a potential treatment for therapy AD and hence improve
both memory and mental symptoms in that disease [57]. Control AChEIs such as
galantamine, donepezil, tacrine, and rivastigmine are involved in improving symptoms for
most patients by enhancing cholinergic neurotransmission range in the human body [58].
Thus, these inhibitors are expensive as well as they are long-term utilize give rise to diverse
harmful side effects and adverse of symptoms. Recently, naturally occurring BChE and AChE
inhibitors have been isolated from the animals and plants and utilized as the natural treatment
for the AD [59-61]. In this study, novel bis-sulfide compounds (3a-j) have been reported as
potent both cholinesterase enzymes inhibitors.
2.3 Crystallographic studies
Thermal ellipsoid views of the 3h, 3i and 3j are shown in Figure 3. Selected bond lengths and
angles are illustrated in Table 3, mostly consistent with the similar molecules in the literature
[62-65].
Figure 3 and Table 3
As depicted in Figure 3, the asymmetric unit of the molecule 3h has one-half-molecule and it
is completed with a twofold symmetry axis [symmetry code: x, 1−y, z]. Compound 3h
crystallizes in a monoclinic system with space group lm (Table 4). The dihedral angle
between the phenylthio ring and cyclohexanone ring is 60.0 (3) while the phenylthio rings
°
are inclined to chlorophenyl ring by 34.8 (3) . The cyclohexanone ring is nearly planar to the
°
molecule plane making dihedral angle is 10.4 (2) . In the crystal structure, there is only weak
°
intramolecular interaction (Table 5). The packing diagram of the molecule is formed by
stacking interactions along the a axis (Figure 4).
Tables 4 and 5
Compound 3i crystallizes in a triclinic system with space group P−1 (Table 4). The
dihedral angle between the phenylthio rings and bromophenyl rings are 25.2(4) (left groups),
°
52.8(3) (right groups). Phenylthio ring (C1/C6) is nearly perpendicular to molecule plane
°
with 87.1(3) . The dihedral angle between bromophenyl ring (C21/C26) and cyclohexanone
°
ring is 87.3(3) while the another ring (C8/C13) are inclined to cyclohexanone ring by 57.8
°
(4) . In addition, phenylthio rings are nearly perpendicular cyclohexanone ring with 82.8(4)
°
°
and 77.2(4) . In the crystal structure, molecules are linked by strong intermolecular C–H⋯S
°
hydrogen bond to form an infinite chain along the b axis. This hydrogen bond also generates
7

푅2
2(10) ring motif with dimeric structure (Figure 4) [65]. Moreover, there is a strong C–H⋯pi
interaction between the C25 atom of the bromophenyl ring and another bromophenyl ring
[Cg: C8/C9/C10/C11/C12/C13; C25–Cg 3.374(9) Å, H25⋯Cg 2.803(9) Å, C25–H25⋯Cg
120.7(8)°, symmetry code: 1−x, 1−y, 1−z].
Figure 4
Compound 3j crystallizes in a monoclinic system with lm space group (Table 4). The
molecule has crystallographic mirror symmetry with x, 1-y, z symmetry operator. The
dihedral angle between the phenylthio ring and cyclohexanone ring is 57.6 (2) while the
°
phenylthio rings are inclined to bromophenyl ring by 33.3 (2) . The cyclohexanone ring is
°
nearly planar to the molecule plane making dihedral angle is 9.36 (14) . The packing
°
structure of the 3j is stabilised by stacking interactions along the b axis (Figure 4).
The expected absolute configuration for molecule 3h and 3j were confirmed by
refinement of the Flack parameters (its value 0.01(11) A° for 3h, -0.027(9) A° for 3j). Details
of the molecular geometry having two stereogenic centers which is C5 atoms in molecule 3h
and 3j, C7 and C20 atoms in molecule 3i reveal that there are two enantiomeric forms (R and
S) named with respect to the majority of chiral centers in the crystal structure of the
compounds.
To examine the chemical details of enzymes function, the first step is to determine
what functional groups are required for enzyme activity and to ascertain these functional
groups [66]. In this study, X-ray crystallography provides an alternative and often-
complementary means for elucidating functional groups at the enzyme active site. According
to enzyme inhibition studies, comprising chlorophenyl and bromophenyl groups in 3h and 3i
respectively are efficient both CA isoenzymes inhibitors. The X-ray crystallography reveals
that the electron density of these groups in compounds less than similar groups in molecule 3j.
Therefore, the enzymes inhibition effects can be directly related to electron density of binding
site of molecules [67].
3. CONCLUSION
The novel bis-thiomethylcyclohexanone compounds were investigated for AChE, BChE, hCA
I, and hCA II enzymes inhibition effects. As we explained above, novel bis-sulfide
compounds (3a-j) can be good candidate drugs, the same as AChE, BChE, and carbonic
anhydrase inhibitor compounds, for therapy of some diseases like AD, epilepsy, glaucoma,
gastric and duodenal ulcers, mountain sickness, osteoporosis or neurological disorders.
8

4. EXPERIMENTALS
4.1. General
All chemicals were obtained from Sigma-Aldrich, Fluka, Merck or Alfa Aesar and were used
without further purification. Melting points were determined on a Gallenkamp melting point
apparatus. FTIR spectra were recorded on Bruker Tensor27 FTIR spectrometer using the KBr
disc in the range of 4000-400 cm-1. 1H-NMR and 13C-NMR spectra were recorded on a Varian
200 MHz or Bruker Avance DPX 400 MHz spectrometer using CDCl3 as a solvent. Splitting
patterns are designated as follows; s, singlet; d, doublet; m, multiplet. Chemical shift (δ)
values are given in ppm. Elemental analysis was done on a LECO CHNS-932 instrument.
4.2. Chemistry
Bis-chalcones (2a-j) were synthesized following literature procedures by the condensation of
cyclohexanone with aromatic aldehydes (1a-j) [43,44].
4.2.1. General procedure for synthesis of bis-thiomethylcyclohexanone derivatives (3a-j)
Bis-chalcone (1 eq.) was dissolved in 20 mL of DCM and Na (catalitic amount) added. After
five minutes shaking in DCM, to reaction mixture was slowly added thiophenol (2.4 eq.) and
the reaction was stirred at room temperature overnight. After completion of reaction, the
mixture evaporated in vacuo. Pure bis-sulfıde derivatives were obtained by recrystallization
from methanol.
2,6-Bis(phenyl(phenylthio)methyl)cyclohexanone (3a). Yield 62%; White solid; mp: 168-
169oC; FT-IR (KBr, cm-1) ν: 3040-3025 (arom. C-H stretch.), 2958-2861 (alif. C-H stretch.),
1724 (C=O stretch.), 1622 (C=C stretch.), 780-700 (C-S stretch.); 1H-NMR(400 MHz, CDCl3,
δ, ppm): 7.19-7.06 (m, 20H), 4.67 (d, J=7.6 Hz, 2H, CH-S), 2.89 (m, 2H, CH-C=O), 2.65 (m,
13
2H), 2.10 (m, 2H), 1.70-1.68 (m, 2H); C-NMR(100 MHz, CDCl3, δ, ppm): 208.6, 142.1,
134.9, 132.6, 128.8, 128.2, 128.1, 127.2, 126.8, 57.5, 52.9, 33.1, 25.6; Elemental Analysis
Calculated: C, 77.73; H, 6.07; S, 12.96. Found: C, 77.64; H, 6.12; S, 12.83.
2,6-Bis(naphthalen-2-yl(phenylthio)methyl)cyclohexanone (3b). Yield 43%; White solid; mp
166-167oC; FT-IR (KBr, cm-1) ν: 3051(arom. C-H stretch.), 2923-2854 (alif. C-H stretch.),
1
1707 (C=O stretch.), 1630 (C=C stretch.), 742-692 (C-S stretch.); H-NMR (400 MHz,
CDCl3, δ, ppm): 7.63-7.00 (m, 24H), 4.79 (d, J= 7.6 Hz, 2H, CH-S), 3.00-2.97 (m, 2H, CH-
C=O), 2.73-2.60 (m, 2H), 2.07-2.05 (m, 2H), 1.76-1.72 (m, 2H); 13C-NMR (100 MHz,
9

CDCl3, δ, ppm): 208.3, 139.5, 134.9, 133.1, 132.5, 128.8, 128.0, 127.9, 127.6, 127.2, 126.8,
126.4, 126.0, 125.6, 57.6, 53.0, 33.2, 25.6; Elemental Analysis Calculated: C, 80.80; H, 5.72;
S, 10.77. Found: C, 80.83; H, 5.80; S, 10.68.
2,6-Bis((3-nitrophenyl)(phenylthio)methyl)cyclohexanone (3c). Yield 36%; white solid; mp
138-139oC; FT-IR (KBr, cm-1) ν: 3081 (arom. C-H), 2943-2895 (alif. C-H), 1710 (C=O),
1
1534 (C=C), 1450 (NO2 asym.), 1350 (NO2 sym.), 732-687 (C-S); H-NMR (400 MHz,
CDCl3, δ, ppm): 7.78 (d, J=8.0 Hz, 2H), 7.69 (s, 2H), 7.23 (d, J=8.0 Hz, 2H), 7.20-7.00 (m,
12H,), 4.44 (d, J= 9.6 Hz, 2H, CH-S), 3.00-2.97 (m, 2H), 2.90-2.87 (m, 2H), 2.15-2.00 (m,
2H), 1.91-1.88 (m, 1H), 1.70-1.60 (m, 1H); 13C-NMR (100 MHz, CDCl3, δ, ppm): 209.3,
147.8, 144.6, 134.3, 133.8, 132.6, 129.2, 128.7, 128.4, 122.5, 121.7, 57.3, 52.7, 35.1, 25.5;
Elemental Analysis Calculated: C, 65.75; H, 4.79; S, 10.96. Found: C, 65.82; H, 4.90; S,
10.95.
2,6-Bis((4-nitrophenyl)(phenylthio)methyl)cyclohexanone (3d). Yield 44%; White solid; mp
162-164oC; FT-IR (KBr, cm-1) ν: 3083-3075 (arom. C-H), 2924-2857- (alif. C-H), 1711
1
(C=O), 1599 (C=C), 1519 (NO2 asym.), 1347 (NO2 sym.), 749-694 (C-S); H-NMR(400
MHz, CDCl3, δ, ppm): 7.86 (d, J=8.4 Hz, 4H), 7.31-7.05 (m, 14H), 4.47 (d, J=8.4 Hz, 2H,
CH-S), 2.98-2.92 (m, 2H, CH-C=O), 2.81-2.79 (m, 2H), 2.15-2.12 (m, 2H), 1.86-1.71 (m,
13
2H); C-NMR(100 MHz, CDCl3, δ, ppm): 209.2, 149.8, 146.0, 133.7, 132.6, 129.2, 128.8,
128.4, 123.3, 57.0, 53.0, 34.1, 25.5; Elemental Analysis Calculated: C, 65.75; H, 4.79; S,
10.96. Found: C, 65.82; H, 4.92; S, 11.00.
2,6-Bis((3-fluorophenyl)(phenylthio)methyl)cyclohexanone (3e). Yield 40%; White solid; mp
168oC; FT-IR (KBr, cm-1) ν: 3056-3035 (arom. C-H), 2924-2855 (alif. C-H), 1697 (C=O),
1
1590 (C=C), 1146 (Ar-F), 763-690 (C-S); H-NMR (400 MHz, CDCl3, δ, ppm): 7.16 (m,
10H), 6.99 (m, 2H), 6.77 (m, 2H), 6.75 (m, 2H), 6.73 (m, 2H), 4.50 (d, J= 8.0 Hz, 2H, CH-S),
13
2.93-2.86 (m, 2H, CH-C=O), 2.73-2.70 (m, 2H), 2.08-2.04 (m, 1H), 1.75-1.63 (m, 3H); C-
NMR (100 MHz, CDCl3, δ, ppm): 208.6, 163.9, 145.0, 144.7, 133.1, 129.4, 128.9, 127.7,
123.8, 114.9, 113.7, 57.4, 52.7, 33.9, 25.5; Elemental Analysis Calculated: C, 72,45; H, 5.28;
S, 12.08. Found: C, 72.60; H, 5.43; S, 12.20.
2,6-Bis((4-fluorophenyl)(phenylthio)methyl)cyclohexanone (3f). Yield 38%; White solid; mp
144-145oC; FT-IR (KBr, cm-1) ν: 3068-3054 (arom. C-H), 2956-2857 (alif. C-H), 1706
10

(C=O), 1603 (C=C), 1226 (Ar-F), 750-690 (C-S); 1H-NMR (400 MHz, CDCl3, δ, ppm): 7.19-
7.09 (m, 10H), 6.97 (m, 4H), 6.72 (t, J= 8.4 Hz, 4H), 4.46 (d, J= 8.8 Hz, 2H, CH-S), 2.90-
13
2.83 (m, 2H, CH-C=O), 2.73-2.70 (m, 2H), 2.07-2.03 (m, 1H), 1.75-1.60 (m, 3H); C-NMR
(100 MHz, CDCl3, δ, ppm): 208.4, 160.4, 137.9, 133.1, 129.6, 128.9, 127.6, 115.0, 114.8,
57.8, 52.3, 34.2, 25.6; Elemental Analysis Calculated: C, 72,45; H, 5.28; S, 12.08. Found: C,
72.53; H, 5.42; S, 11.95.
2,6-Bis((3-chlorophenyl)(phenylthio)methyl)cyclohexanone (3g). Yield 35%; White solid; mp
115-116oC; FT-IR (KBr, cm-1) ν: 3064-3058 (arom. C-H), 2948-2857 (alif. C-H), 1709
1
(C=O), 1570 (C=C), 1072 (Ar-Cl), 745-688 (C-S); H-NMR (400 MHz, CDCl3, δ, ppm):
7.20-7.10 (m, 10H), 7.06 (m, 2H), 7.02 (m, 2H), 6.98 (m, 2H), 6.88 (m, 2H), 4.48 (d, J= 8.0
Hz, 2H, CH-S), 2.91-2.85 (m, 2H, CH-C=O), 2.71-2.68 (m, 2H), 2.08-2.04 (m, 1H), 1.77-1.62
(m, 3H); 13C-NMR (100 MHz, CDCl3, δ, ppm): 208.5, 144.2, 134.0, 133.1, 129.3, 129.0,
128.1, 127.7, 127.1, 126.3, 57.3, 52.7, 33.7, 25.5; Elemental Analysis Calculated: C, 68.21;
H, 4.97; S, 11.37. Found: C, 68.25; H, 5.12; S, 11.29.
2,6-Bis((4-chlorophenyl)(phenylthio)methyl)cyclohexanone (3h). Yield 52%; White solid;
decomposition after 240oC; FT-IR (KBr, cm-1) ν: 3068-3054 (arom. C-H), 2926-2857 (alif. C-
1
H), 1704 (C=O), 1600 (C=C), 1091 (Ar-Cl), 746-688 (C-S); H-NMR (400 MHz, CDCl3, δ,
ppm): 7.15 (m, 10H), 7.02 (d, J=8.8 Hz, 4H), 6.93 (d, J=8.4 Hz, 4H), 4.46 (d, J= 8.4 Hz, 2H,
CH-S), 2.90-2.83 (m, 2H, CH-C=O), 2.74-2.70 (m, 2H), 2.08-2.03 (m, 1H), 1.74-1.61 (m,
13
3H); C-NMR (100 MHz, CDCl3, δ, ppm): 209.1, 140.7, 134.0, 133.1, 132.5, 129.3, 129.0,
128.2, 127.7, 57.7, 52.4, 34.2, 25.6; Elemental Analysis Calculated: C, 68.21; H, 4.97; S,
11.37. Found: C, 68.12; H, 5.13; S, 11.22.
2,6-Bis((3-bromophenyl)(phenylthio)methyl)cyclohexanone (3i). Yield 36%; White solid; mp
120-121oC; FT-IR (KBr, cm-1) ν: 3062-3058 (arom. C-H), 2917-2856 (alif. C-H), 1709
1
(C=O), 1566 (C=C), 1069 (Ar-Br), 747-688 (C-S); H-NMR (400 MHz, CDCl3, δ, ppm):
7.26-6.91 (m, 18H), 4.49 (d, J= 8.4 Hz, 2H, CH-S), 2.91-2.85 (m, 2H, CH-C=O), 2.71-2.68
(m, 2H), 2.07-2.00 (m, 1H), 1.74-1.62 (m, 3H); 13C-NMR (100 MHz, CDCl3, δ, ppm): 208.5,
144.5, 134.0, 133.1, 131.0, 130.0, 129.7, 129.0, 127.8, 126.8, 122.3, 57.3, 52.7, 33.7, 25.5;
Elemental Analysis Calculated: C, 58.90; H, 4.29; S, 9.82. Found: C, 58.84; H, 4.33; S, 9.96.
11

2,6-Bis((4-bromophenyl)(phenylthio)methyl)cyclohexanone (3j). Yield 48%; White solid; mp
166-167oC; FT-IR (KBr, cm-1) ν: 3066-3052 (arom. C-H), 2937-2853 (alif. C-H), 1706
1
(C=O), 1577 (C=C), 1068 (Ar-Br), 751-688 (C-S); H-NMR (400 MHz, CDCl3, δ, ppm):
7.18-6.89 (m, 14H), 6.87 (d, J= 8.4 Hz, 4H), 4.44 (d, J= 8.8 Hz, 2H, CH-S), 2.89-2.82 (m,
2H, CH-C=O), 2.74-2.70 (m, 2H), 2.07-2.00 (m, 1H), 1.77-1.60 (m, 3H); 13C-NMR (100
MHz, CDCl3, δ, ppm): 209.1, 141.2, 133.9, 133.1, 131.2, 129.7, 129.0, 127.7, 120.6, 57.6,
52.4, 34.2, 25.6; Elemental Analysis Calculated: C, 58.90; H, 4.29 S, 9.82. Found: C, 58.86;
H, 4.42; S, 9.76.
4.3. X-ray Crystallography
Single crystal X-ray diffraction data of 3h, 3i and 3j were collected at room temperature on an Rigaku-
Oxford Xcalibur diffractometer with an Eos-CCD detector, operated at 50 kV and 40 mA using
graphite monochromated Mo-Kα (λ = 0.71073 Å) radiation. Utilising CrysAlisPro software package,
data collections and reductions along with absorption corrections were performed [68]. Structure
solutions were performed using SHELXT [69] embedded in the Olex2 [70]. Refinement of coordinates
and anisotropic thermal parameters of non-hydrogen atoms were carried out by the full-matrix least-
squares method in SHELXL [71]. For the compounds, all hydrogen atoms were placed in
geometrically idealized positions (C-H=0.97 A° for methylene groups and C-H=0.93 A° for aromatic
groups). Detailed crystallographic data and refinement results are summarized in Table 4.
4.4. Biochemical Studies
Both hCA isoenzymes inhibition effects of novel bis-sulfide compounds (3a-j) was measured
according to Verpoorte et al. [72] conforming to previous studies [73-77] and recorded at 348
nm spectrophotometrically using p-nitrophenylacetate substrate (PNA) [78]. On the other
hand, AChE and BChE inhibitory effects of novel bis-sulfide compounds (3a-j) were
determined according to procedure of Ellman et al. [79] conforming to previous studies [80-
85] and recorded at 412 nm spectrophotometrically using acetylthiocholine iodide and
butyrylthiocholine iodide as substrates for the enzymatic reaction. 5,5’-Dithio-bis(2-nitro-
benzoic) acid compound was used for the measurement of the AChE and BChE activities,
respectivelly [86].
Supplementary material
Crystallographic data as .cif files for the structures reported in this paper have been deposited
at the Cambridge Crystallographic Data Center with CCDC 1865288, 1865295 and 1865296 for the
molecule 3h, 3i and 3j respectively. Copies of the data can be obtained free of charge at
12

www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Center, 12,
Union Road, Cambridge CB2 1EZ, UK). Email: deposit@ccdc.cam.ac.uk.
Acknowledgement: The authors acknowledge Scientific Research Projects Coordination Unit of
Akdeniz University (Project Number FDK-2016-1541) for financial support and Faculty of Science,
Ataturk University.
REFERENCES
[1] Z. Qiao, X. Jiang, Recent developments in sulfur–carbon bond formation reaction involving
thiosulfates, Org. Biomol. Chem. 15 (2017) 1942-1946.
[2] K. L. Dunbar, D. H. Scharf, A. Litomska, C. Hertweck, Enzymatic carbon-sulfur bond formation
in natural product biosynthesis, Chem. Rev. 117 (2017) 5521-5577.
[3] E. H. Krenske, R. C. Petter, K. N. Houk, Kinetics and thermodynamics of reversible thiol additions
to mono- and diactivated michael acceptors: Implications for the design of drugs that bind
covalently to cysteines, J. Org. Chem. 81 (2016) 11726-11733.
[4] M. Feng, B. Tang, S.H. Liang, X. Jiang, Sulfur containing scaffolds in drugs: Synthesis and
application in medicinal chemistry, Curr. Top. Med. Chem. 16 (2016) 1200-1216.
[5] D. Enders, K. Luettgen, A.A. Narine, Asymmetric sulfa-Michael additions, Synthesis 7 (2007)
959-980.
[6] P. Espeel, F.E. Du Prez, One-pot multi-step reactions based on thiolactone chemistry: A powerful
synthetic tool in polymer science, Eur. Polym. J. 62 (2015) 247-272.
[7] J.P. Phelan, J.A. Ellman, Conjugate addition-enantioselective protonation reactions, Beilstein J.
Org. Chem. 12 (2016) 1203-1228.
[8] R. Dalpozzo, G. Bartoli, G. Bencivenni, Asymmetric organocatalytic reactions of α,β-unsaturated
cyclic ketones, Symmetry 3 (2011) 84-125.
[9] N. Azizi, A. Khajeh-Amiri, H. Ghafuri, M. Bolourtchian, A highly efficient, operationally simple
and selective thia-Michael addition under solvent-free condition, Green Chem. Lett. Rev. 2 (2009)
43-46.
[10] G. Yerli, H. Gezegen, M. Ceylan, Iodine-catalyzed addition of 2-mercaptoethanol to chalcone
derivatives: Synthesis of the novel β-mercapto carbonyl compounds, Org. Commun. 5 (2012) 70-
76.
[11] S.S. Pandit, S.S. Pardhe, Y.B. Pandit, Guanidine hydrochloride: An efficient catalyst for the
synthesisof 2-hydrazolyl-4-thiazolidinone derivatives under solvent free conditions, Iran. Chem.
Commun. 5 (2017) 227-236.
[12] B.L. Zhao, D.M. Du, Enantioselective squaramide-catalyzed trifluoromethylthiolation-sulfur-
Michael/Aldol cascade reaction: One-pot synthesis of CF3S containing spiro cyclopentanone-
thiochromane, Org. Lett. 19 (2017) 1036-1039.
13

[13] X. Gu, Y.Z. Zhu, Therapeutic applications of organosulfur compounds as novel hydrogen sulfide
donors and/or mediators, Expert Rev. Clin. Pharmacol. 4 (2011) 123-133.
[14] M. Feng, B. Tang, S.H. Liang, X. Jiang, Sulfur containing scaffolds in drugs: synthesis and
application in medicinal chemistry, Curr. Top. Med. Chem. 16 (2016) 1200-1216.
[15] S. Parcell, Sulfur in human nutrition and applications in medicine, Altern. Med. Rev. 7 (2002) 22-
44.
[16] A. Akıncıoğlu, Y. Akbaba, H. Göçer, S. Göksu, İ. Gülçin, C.T. Supuran, Novel sulfamides as
potential carbonic anhydrase isoenzymes inhibitors, Bioorg. Med. Chem. 21 (2013) 1379-1385.
[17] K. Aksu, M. Nar, M. Tanç, D. Vullo, İ. Gülçin, S. Göksu, F. Tümer, C.T. Supuran, Synthesis and
carbonic anhydrase inhibitory properties of sulfamides structurally related to dopamine, Bioorg.
Med. Chem. 21 (2013) 2925-2931.
[18] M. Güney, A. Coşkun, F. Topal, A. Daştan, İ. Gülçin, C.T. Supuran, Oxidation of
cyanobenzocycloheptatrienes: Synthesis, photooxygenation reaction and carbonic anhydrase
isoenzymes inhibition properties of some new benzotropone derivatives, Bioorg. Med. Chem. 22
(2014) 3537-3543.
[19] S. Göksu, A. Naderi, Y. Akbaba, P. Kalın, A. Akıncıoğlu, İ. Gulcin, S. Durdaği, R.E. Salmas,
Carbonic anhydrase inhibitory properties of novel benzylsulfamides using molecular modeling and
experimental studies, Bioorg. Chem. 56 (2014) 75-82.
[20] 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.
[21] 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.
[22] A. Akıncıoğlu, H. Akıncıoğlu, I. Gülçin, S. Durdağı, C.T. Supuran, S. Göksu, Discovery of potent
carbonic anhydrase and acetylcholine esterase inhibitors: Novel sulfamoylcarbamates and
sulfamides derived from acetophenones, Bioorg. Med. Chem. 23 (2015) 3592-3602.
[23] P. Taslimi, İ. Gulcin, B. Ozgeris, S. Goksu, F. Tumer, S.H. Alwasel, C.T. Supuran, The human
carbonic anhydrase isoenzymes I and II (hCA I and II) inhibition effects of trimethoxyindane
derivatives, J. Enzyme Inhib. Med. Chem. 31 (2016) 152-157.
[24] M. Küçük, İ. Gulcin, Purification and characterization of carbonic anhydrase enzyme from black
sea trout (Salmo trutta Labrax Coruhensis) kidney and inhibition effects of some metal ions on the
enzyme activity, Environ. Toxicol. Pharmacol. 44 (2016) 134-139.
[25] P. Taslimi, İ. Gülçin, N. Öztaşkın, Y. Çetinkaya, S. Göksu, S.H. Alwasel, C.T. Supuran, The
effects of some bromophenol derivatives on human carbonic anhydrase isoenzymes, J. Enzyme
Inhib. Med. Chem. 31 (2016) 603-607.
14

[26] D. Ozmen Ozgun, C. Yamali, H.İ. Gül, P. Taslimi, İ. Gülçin, T. Yanik, C.T. Supuran, Inhibitory
effects of isatin mannich bases on carbonic anhydrases, acetylcholinesterase and
butyrylcholinesterase, J. Enzyme Inhib. Med. Chem. 31 (2016) 1498-1501.
[27] A. Sujayev, E. Garibov, P. Taslimi, İ. Gülçin, S. Gojayeva, V. Farzaliyev, S.H. Alwasel, C.T.
Supuran, Synthesis of some tetrahydropyrimidine-5-carboxylates, determination of their metal
chelating effects and inhibition profiles against acetylcholinesterase, butyrylcholinesterase and
carbonic anhydrase, J. Enzyme Inhib. Med. Chem. 31 (2016) 1531-1539.
[28] K. Aksu, B. Özgeriş, P. Taslimi, A. Naderi, İ. Gülçin, S. Göksu, Antioxidant activity,
acetylcholinesterase and carbonic anhydrase inhibitory properties of novel ureas derived from
phenethylamines, Arch. Pharm. 349 (2016) 944-954.
[29] 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.
[30] 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.
[31] F. Topal, İ. Gulcin, A. Dastan, M. Guney, Novel eugenol derivatives: potent acetylcholinesterase
and carbonic anhydrase inhibitors, Int. J. Biol. Macromol. 94 (2017) 845-851.
[32] M. Işık, S. Beydemir, A. Yılmaz, M.E. Naldan, H.E. Aslan, İ. Gülçin, Oxidative stress and
mRNA expression of acetylcholinesterase in the leukocytes of ischemic patients, Biomed.
Pharmacother. 87 (2017) 561-567.
[33] U.M. Kocyigit, Y. Budak, M.B. Gürdere, Ş. Tekin, T. Kul Köprülü, F. Ertürk, K. Özcan, İ.
Gülçin, M. Ceylan, Synthesis, characterization, anticancer, antimicrobial and carbonic anhydrase
inhibition profiles of novel (3aR,4S,7R,7aS)-2-(4-((E)-3-(3-aryl)acryloyl) phenyl)-3a,4,7,7a-
tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione derivatives, Bioorg. Chem. 70 (2017) 118-125.
[34] H.I. Gül, A. Demirtas, G. Ucar, P. Taslimi, İ. Gülçin, Synthesis of Mannich bases by two
different methods and evaluation of their acetylcholine esterase and carbonic anhydrase inhibitory
activities, Lett. Drug Des. Discov. 14 (2017) 573-580.
[35] 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.
[36] A. Aktaş, P. Taslimi, I. Gülçin, Y. Gök, Novel NHC Precursors: Synthesis, characterization and
carbonic anhydrase and acetylcholinesterase inhibitory properties, Arch. Pharm. 350 (2017)
e1700045.
15

[37] 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.
[38] 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.
[39] P. Taslimi, A. Sujayev, E. Garibov, N. Nazarov, Z. Huyut, S.H. Alwasel, İ. Gülçin, The synthesis
of new cyclic thioureas and evaluation of their metal-chelating activity, acetylcholinesterase,
butyrylcholinesterase and carbonic anhydrase inhibition profiles, J. Biochem. Mol. Toxicol. 31
(2017) e21897.
[40] 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.
[41] 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.
[42] P. Taslimi, S. Osmanova, İ. Gulçin, S. Sardarova, V. Farzaliyev, A. Sujayev, R. Kaya, F. Koc, S.
Beydemir, S.H. Alwasel, O.I. Kufrevioglu, Discovery of potent carbonic anhydrase,
acetylcholinesterase and butyrylcholinesterase enzymes inhibitors: the new amides and
thiazolidineones synthesized on an acetophenone base, J. Biochem. Mol. Toxicol. 31 (2017)
e21931.
[43] N. Nohut Maslakcı, A. Biçer, G. Turgut Cin, A. Uygun Oksüz, Electrochromic properties of some
bis-chalcone derivatives-based nanofibers, J. Appl. Polym. Sci. 135 (2018) 46010.
[44] G. Yakalı, A. Biçer, C. Eke, G. Turgut Cin, Solid state structural investigations of the
bis(chalcone) compound with single crystal X-ray crystallography, DFT, gamma-ray spectroscopy
and chemical spectroscopy methods, Radiat. Phys. Chem. 145 (2018) 89-96.
[45] N.K. Konduru, S. Dey, M. Sajid, M. Owais, N. Ahmed, Synthesis and antibacterial and antifungal
evaluation of some chalcone based sulfones and bisulfones, Eur. J. Med. Chem. 59 (2013) 23-30.
[46] C. Guha, N. Sepay, T. Halder, A.K. Mallik, Remarkable Diastereoselectivity of the Thia-Michael
Reaction on α,α′-di[(E)-benzylidene]alkanones: Exclusive formation of a meso product, Synlett 29
(2018) 1161-1166.
[47] 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 (2011)
4259-4262.
16

[48] M. Şentürk, İ. Gülçin, Ş. Beydemir, Ö.İ. Küfrevioğlu, C.T. Supuran, In vitro inhibition of human
carbonic anhydrase I and II isozymes with natural phenolic compounds, Chem. Biol. Drug Des. 77
(2011) 494-499.
[49] 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.
Lett. 20 (2010) 5050-5053.
[50] 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 (2010) 2159-2164.
[51] P. Taslimi, H.E. Aslan, Y. Demir, N. Öztaşkın, A. Maraş, İ. Gulçin, Ş. Beydemir, S. 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. Biol. Macromol. 119 (2018) 857-863.
[52] T.A. Coban, S. Beydemir, İ. Gülcin, D. Ekinci, A. Innocenti, D. Vullo, C.T. Supuran, Sildenafil is
a strong activator of mammalian carbonic anhydrase isoforms I-XIV, Bioorg. Med. Chem. 17
(2009) 5791-5795.
[53] M. Şentürk, İ. Gülçin, A. Daştan, Ö.İ. Küfrevioğlu, C.T. Supuran, Carbonic anhydrase inhibitors.
Inhibition of human erythrocyte isozymes I and II with a series of antioxidant phenols, Bioorg.
Med. Chem. 17 (2009) 3207-3211.
[54] A. Akıncıoğlu, M. Topal, İ. Gülçin, S. Göksu, Novel sulfamides and sulfonamides incorporating
tetralin scaffold as carbonic anhydrase and acetylcholine esterase inhibitors, Arch. Pharm. 347
(2014) 68-76.
[55] H.İ. Gül, K. Kucukoglu, C. Yamali, S. Bilginer, H. Yuca, İ. Ozturk, P. Taslimi, İ. Gülçin, C.T.
Supuran, Synthesis of 4-(2-substitutedhydrazinyl)benzenesulfonamides and their carbonic
anhydrase inhibitory effects, J. Enzyme Inhib. Med. Chem. 31 (2016) 568-573.
[56] H.İ. Gül, M. Tuğrak, H. Sakagami, P. Taslimi, İ. Gülçin, C.T. Supuran, Synthesis and bioactivity
studies
on
new
4-(3-(4-substitutedphenyl)-3a,4-dihydro-3h-indeno[1,2-c]pyrazol-2-yl)
benzenesulfonamides, J. Enzyme Inhib. Med. Chem. 31 (2016) 1619-1624.
[57] F. Özbey, P. Taslimi, İ. Gulcin, A. Maraş, S. Goksu, C.T. Supuran, Synthesis,
acetylcholinesterase, butyrilcholinesterase, carbonic anhydrase inhibitory and metal chelating
properties of some novel diaryl ether, J. Enzyme Inhib. Med. Chem. 31 (2016) 79-85.
[58] 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.
[59] 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 aminomethyl and
alkoxymethyl derivatives as carbonic anhydrase, acetylcholinesterase and butyrylcholinesterase
inhibitors, J. Enzyme Inhib. Med. Chem. 32 (2017) 1174-1182.
17

[60] 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
(2018) 854-866.
[61] 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 (2018) 808-825.
[62] S. Naveen, B. Dinesh, K. Abiraj, D. Channe Gowda, M. A. Sridhar, J. Shashidhara Prasad,
Synthesis and crystal structure of tert-butyl 2-((phenylthio)-carbonyl) pyrrolidine-1-carboxylate, J.
Chem Crystallogr. 37 (2007) 721-725.
[63] A.N. Chekhlov, Crystal structure of 1-(2-pyridiniomethyl)-2,4-bis(phenylsulfonyl)benzene
chloride ethanol solvate, Russ. J. Gen. Chem. 74 (2004) 944-948.
[64] R.N. Osorio-Yanez, C. Crisostomo-Lucas, E. Santacruz-Juarez, R. Reyes-Martıneza, D. Morales-
Morales, Acta Cryst. Sect. E 70 (2014) o529.
[65] J. Bernstein, R.E Davis, L. Shimoni, N.L. Chang, Hydrogen-bond Pattern Functionality and
Graph Sets, Angew. Chem. Int. Ed. Engl. 34 (1995) 1555-1573.
[66] J.F. Darby, M. Atobe, J.D. Firth, P. Bond, G.J. Davies, P. O'Brien, R.E. Hubbard, Increase of
enzyme activity through specific covalent modification with fragments, Chem. Sci. 8 (2017) 7772-
7779.
[67] X.Y. Meng, H. X. Zhang, M. Mezei, M. Cui, Molecular docking: a powerful approach for
structure-based drug discovery, Curr. Comput. Aided Drug Des. 7 (2011) 146-157.
[68] Agilent Technologies, CrysAlis PRO and CrysAlis RED, Yarnton, Oxfordshire England, 2002.
[69] G.M. Sheldrick, SHELXT-Integrated space-group and crystal-structure determination, Acta
Crystallogr. Sect. A 71 (2015) 3-8.
[70] O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A.K. Howard and H. Puschmann, OLEX2: A
complete structure solution, refinement and analysis program, J. Appl. Cryst. 42 (2009) 339-341.
[71] G.M. Sheldrick, Crystal structure refinement with SHELXL, Acta Crystallogr. Sect. C 71 (2015)
3-8.
[72] J.A. Verpoorte, S. Mehta, J.T. Edsall, Esterase activities of human carbonic anhydrases B and C,
J. Biol. Chem. 242 (1967) 4221-4229.
[73] P. Taslimi, İ. Gulçin, Antioxidant and anticholinergic properties of olivetol, J. Food Biochem. 42
(2018) e12516.
[74] F. Türker, D. Barut Celepci, A. Aktaş, P. Taslimi, Y. Gök, M. Aygün, İ. Gulçin, Meta-
cyanobenzyl substituted benzimidazole: Synthesis, characterization, crystal structure and carbonic
anhydrase, α-glycosidase, butyrylcholinesterase, acetylcholinesterase inhibitory properties, Arch.
Pharm. 351 (2018) e201800029.
18

[75] İ. Gulçin, P. Taslimi, Sulfonamide inhibitors: A patent review 2013-present, Exp. Opin. Ther. Pat.
28 (2018) 541-549.
[76] B. Yiğit, M. Yiğit, D. Barut Celepci, Y. Gök, A. Aktaş, M. Aygün, P. Taslimi, İ. Gulçin, Novel
benzylic substituted imidazolinium, tetrahydropyrimidinium and tetrahydrodiazepinium salts-
potent carbonic anhydrase and acetylcholinesterase inhibitors, ChemistrySelect 3 (2018) 7976-
7982.
[77] 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.
[78] F. Turkan, Z. Huyut, P. Taslimi, İ. Gulçin, The in vivo effects of cefazolin, cefuroxime, and
cefoperazon on the carbonic anhydrase in different rat tissues, J. Biochem. Mol. Toxicol. 32 (2018)
e22041.
[79] 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.
[80] P. Taslimi, C. Çağlayan, F. Farzaliyev, O. Nabiyev, A. Sujayev, F. Türkan, R. Kaya, İ. Gulçin,
Synthesis and discovery of potent carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase
and α-glycosidase enzymes inhibitors: the novel N,N′-bis-cyanomethylamine and
alkoxymethylamine derivatives, J. Biochem. Mol. Toxicol. 32 (2018) e22042.
[81] M.U. Koçyigit, P. Taslimi, F. Gurses, S. Soylu, S. Durna Dastan, İ. Gulçin, The effects of
wireless electromagnetic fields on the activities of carbonic anhydrase and acetylcholinesterase
enzymes in various tissues of rats, J. Biochem. Mol. Toxicol. 32 (2018) e22031.
[82] P. Taslimi, E. Sujayev, F. Turkan, E. Garibov, Z. Huyut, F. Farzaliyev, S. Mamedova, İ. Gulçin,
Synthesis and investigation of the conversion reactions of pyrimidine-thiones with nucleophilic
reagent and evaluation of their acetylcholinesterase, carbonic anhydrase inhibition and antioxidant
activities, J. Biochem. Mol. Toxicol. 32 (2018) e22019.
[83] S. Burmaoglu, A.O. Yilmaz, P. Taslimi, O. Algul, D. Kılıç, İ., Gulçin, Synthesis and biological
evaluation of phloroglucinol derivatives possessing α-glycosidase, acetylcholinesterase,
butyrylcholinesterase, carbonic anhydrase inhibitory activity, Arch. Pharm. 351 (2018) e1700314.
[84] 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 (2018) e22009.
[85] 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.
19

[86] 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. 3 (2017) e21973.
20

Table 1. Optimization of reaction conditions for addition of thiophenol to bis-chalcones
Entry Catalyzer
Temperature
Room temperature
Reflux
Room temperature
Room temperature
Reflux
Room temperature
Reflux
Room temperature
Solvent
DCM
DCM
DCM
DCM
EtOH
BuOH
DCM
DCM
Time
1 day
1 day
1 day
1 day
1 day
1 day
1 day
1 day
Yield
1
2
3
4
5
6
7
8
I2 (%10 mol)
I2 (%10 mol)
FeCl3 (%2 mol)
AlCl3 (%2 mol)
Al2O3 (5 mol)
t-BuOK (2 mol)
NEt3 (5 mol)
Na (1 mol)
Isomer mixture
Isomer mixture
No reaction
No reaction
No reaction
No reaction
No reaction
Product observed
21

Table 2. Inhibition patrameters including half maximal inhibitory concentration (IC50) and inhibition constants (Ki) of a dousen of novel bis-
sulfide compounds (3a-j) against human carbonic anhydrase I, and II isoenzymes (hCA I, and II), acetylcholinesterase (AChE), and
butyrylcholinesterase (BChE) enzymes
IC50 (nM)
Ki (nM)
Compounds
hCA I
109.43
147.05
180.34
117.03
71.66
55.08
61.38
38.63
44.28
69.43
265.41
-
r2
hCA II
138.55
170.82
201.58
143.84
80.03
74.22
68.52
42.84
64.03
72.71
248.54
-
r2
AChE
12.73
10.03
19.39
41.77
7.18
r2
BChE
28.93
31.53
42.04
61.38
17.90
24.83
20.28
28.11
29.43
31.47
-
r2
hCA I
87.53±14.07
183.23±28.18
167.34±22.72
137.03±43.94
80.48±17.11
50.66±9.17
64.83±19.33
47.92±7.20
39.14±9.94
54.37±11.10
273.61±76.86
-
hCA II
127.44±31.84
157.99±50.72
194.02±28.55
154.93±36.04
88.32±18.13
80.12±21.98
61.34±13.81
48.18±19.10
46.03±10.47
60.52±18.52
229.08±55.14
-
AChE
8.63±1.83
7.14±1.05
12.74±2.95
32.64±6.86
4.55±0.94
11.42±1.98
13.56±2.64
17.81±4.66
6.06±2.01
14.37±3.33
-
BChE
19.73±3.64
22.84±4.93
20.51±3.02
37.38±11.53
12.77±2.20
18.53±5.94
14.82±4.08
19.73±3.98
17.43±2.25
20.28±4.90
-
AChE/BChE
0.43
0.9736
0.9927
0.9598
0.9819
0.9816
0.9743
0.9891
0.9812
0.9590
0.9711
0.9954
-
0.9308
0.9673
0.9902
0.9819
0.9772
0.9609
0.9923
0.9904
0.9891
0.9690
0.9714
-
0.9563
0.9604
0.9912
0.9732
0.9208
0.9714
0.9843
0.9672
0.9840
0.9531
-
0.9892
0.9722
0.9831
0.9682
0.9801
0.9492
0.9618
0.9910
0.9733
0.9562
-
3a
3b
0.31
0.62
3c
0.87
3d
0.35
3e
15.71
15.02
23.83
9.83
0.61
3f
0.91
3g
0.90
3h
0.34
3i
17.47
-
0.70
3j
-
AZA*
TACΨ
92.20
0.9796
105.16
0.9711
56.37±15.10
63.40±13.62
0.88
*Acetazolamide (AZA) was used as a standard inhibitor for both carbonic anhydrases I, and II (hCA I and II) isoenzymes
ΨTacrine (TAC) was used as a standard inhibitor for both acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes
22

Table 3. Selected experimental parameters of the novel bis-sulfide compounds (3h, 3i and 3j)
Å)
Bond Lengths (
3i
3h
3j
O1-C4
Cl1-C9
S1-C5
1.214(7)
1.735(5)
1.852(4)
1.778(5)
O1-C19
S2-C20
S2-C27
Br2-C23
Br1-C12
S1-C1
1.206(8)
1.861(8)
1.782(9)
1.864(8)
1.912(9)
1.770(9)
1.834(8)
Br1-C9
O1-C1
S1-C5
1.899(5)
1.221(8)
1.857(5)
1.771(5)
S1-C12
S1-C12
S1-C7
°)
Bond Angles (
3i
3h
3j
C5-S1-C12
O1-C4-C3
Cl1-C9-C8
101.4(2)
123.6(2)
120.0(5)
C1-S1-C7
C20-S2-C27
O1-C19-C18
Br1-C12-C13
Br2-C23-C24
101.1(4)
102.3(4)
123.2(8)
117.3(7)
119.8(6)
C12-S1-C5
S1-C5-C2
O1-C1-C2
Br1-C9-C8
101.6(2)
106.1(3)
122.7(3)
120.4(4)
23

Table 4. Crystal data and structure refinement parameters for the novel bis-sulfide compounds (3h, 3i and 3j)
Crystal data
Empirical formula
Formula weight
Temperature (K)
Crystal system
Space group
Unit cell dimensions
a (Å)
3h
C32H28Cl2OS2
563.56
293(2)
monoclinic
lm
3i
C32H28Br2OS2
652.48
3j
C32H28Br2OS2
652.48
294(2)
monoclinic
lm
292(2)
triclinic
P-1
b (Å)
c (Å)
5.1312(5)
25.692(2)
10.9554(11)
90
97.941(9)
90
10.399(2)
10.431(3)
14.373(4)
108.01(2)
101.662(19)
97.05(2)
5.0984(4)
25.8212(19)
11.2384(6)
90
98.313(6)
90
α (⁰)
β (⁰)
γ (⁰)
Volume (Å3)
Z
Dcalc (g/cm-3)
1430.4(2)
2
1.308
0.397
588
1423.2(6)
2
1.523
3.019
660.0
1463.94(18)
2
1.480
2.935
660.0
Absorption coefficient (mm-1)
F (000)
Crystal size (mm)
0.659 × 0.395 × 0.053
‒ 6 ≤ ℎ ≤ 3,
‒ 31 ≤ 푘 ≤ 27,
‒ 13 ≤ 푙 ≤ 13
2733/1791
1791/2/172
1.029
0.335 × 0.242 × 0.166
‒ 12 ≤ ℎ ≤ 12,
‒ 12 ≤ 푘 ≤ 12,
‒ 17 ≤ 푙 ≤ 17
7467/7467
7467/12/335
0.848
0.241 × 0.211 × 0.092
‒ 6 ≤ ℎ ≤ 6,
‒ 31 ≤ 푘 ≤ 31,
‒ 8 ≤ 푙 ≤ 13
4554/2187
2187/2/172
1.023
Index ranges
Reflections collected/unique
Data / restrains / parameters
Goodness of fit on F2
푅1 = 0.0465,
푤푅2 = 0.1151
푅1 = 0.0523
푤푅2 = 0.1226
푅1 = 0.0340,
푤푅2 = 0.0603
푅1 = 0.0616
푤푅2 = 0.0685
푅1 = 0.0543,
푤푅2 = 0.1462
푅1 = 0.1367
푤푅2 = 0.1704
Final R indices [I > 2σ(I)]
R indices (all data)
Largest difference peak and hole (e Å-3)
0.23/-0.19
0.39/-0.39
0.24/-0.28
24

Table 5. Interactions geometry ( ) for the novel bis-sulfide compounds (3h, 3i and 3j)
Å,°
Compounds
3h
D–H∙∙∙A
D–H
H∙∙∙A
D∙∙∙A
D–H∙∙∙A
C2-H2B…S1
C15-H15B…S1
C17-H17A…O1i
C17-H17B…S2
C25-H25…Cg2
C3-H3B…S1
0.97
2.74
3.1821(5)
3.178(8)
3.544(10)
3.103(8)
3.374(9)
3.192(4)
108
0.970(11)
0.969(12)
0.971(12)
0.932(10)
0.970(6)
2.719(8)
2.592(10)
2.683(8)
2.803(9)
2.757(5)
109.5(7)
167.4(8)
106.6(7)
120.7(8)
108.0(4)
3i
3j
25

FIGURE LEGENDS
Figure 1. Determination of inhibition constant values (Ki) of novel bis-sulfide compounds
(3a-j) obtained from Lineweaver-Burk graphs against human carbonic anhydrases
isoenzyme I and II (hCA I and II) (3i), and acetylcholinesterase (AChE) and
butrylcholinesterase (BChE) enzymes (3e)
Figure 2. Inhibition constant values (Ki) of novel bis-sulfide compounds (3a-j) for human
carbonic anhydrases isoenzyme I, and II (hCA I, and II) (A) and acetylcholinesterase
(AChE) and butrylcholinesterase (BChE) enzymes (B)
Figure 3. Thermal ellipsoid view of the molecules with the atom numbering scheme and 75%
probability
Figure 4. Packing structure of the novel bis-sulfide compound of 3h via stacking interactions
along the a axis, (3i) via strong C-H…O interctions along the b axis, (3j) via stacking
interactions along the b axis
26

40
30
20
10
0
24
18
12
6
hCA II
hCA I
0
-1
0
1
2
3
4
5
6
7
-1
0
1
2
3
4
5
6
7
1/[S] (nM)-1
1/[S] (nM)-1
64
48
32
16
0
440
330
220
110
0
AChE
BChE
-3
0
3
6
9
12
-100
0
100
200
300
400
1/[S] (mM)-1
1/[S] (mM)-1
Figure 1
27

300
225
150
75
3a
3b
3d
3f
3h
3j
3c
3e
3g
3i
AZA
0
hCA I
h CA II
A
64
48
32
16
0
3a
3b
3c
3d
3e
3f
AChE
BChE
B
Figure 2
28

3h
3i
29