The first synthesis, carbonic anhydrase inhibition and anticholinergic activities of some bromophenol derivatives with S including natural products

Article abstract of DOI:10.1016/j.bioorg.2018.12.012

Starting from vanillin, known four benzyl bromides with Br were synthesized. The first synthesis of natural product 3,4-dibromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (2) and 3,4,6-tribromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (3) and derivative

Full text of DOI:10.1016/j.bioorg.2018.12.012

Accepted Manuscript
The first synthesis, carbonic anhydrase inhibition and anticholinergic activities
of some bromophenol derivatives with S including natural products
Çetin Bayrak, Parham Taslimi, Halide Sedef Karaman, İlhami Gülçin, Abdullah
Menzek
PII:
S0045-2068(18)31015-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.12.012
YBIOO 2676
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 September 2018
4 December 2018
10 December 2018
Please cite this article as: C. Bayrak, P. Taslimi, H. Sedef Karaman, I. Gülçin, A. Menzek, The first synthesis,
carbonic anhydrase inhibition and anticholinergic activities of some bromophenol derivatives with S including
natural products, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.12.012
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The first synthesis, carbonic anhydrase inhibition and anticholinergic
activities of some bromophenol derivatives with S including natural products
Çetin Bayraka,b, Parham Taslimia, Halide Sedef Karamana, İlhami Gülçina, Abdullah Menzeka,*
aDepartment of Chemistry, Faculty of Science, Ataturk University, 25240-Erzurum, Turkey
bDogubayazit Ahmed-i Hani Vocational School, Agri Ibrahim Cecen University, 04400-Agri, Turkey
ABSTRACT: Starting from vanillin, known four benzyl bromides with Br were synthesized. The first
synthesis of natural product 3,4-dibromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (2) and 3,4,6-
tribromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (3) and derivatives were carried out by
demethylation, acetylatilation, oxidation and hydrolysis reactions of the benzyl bromides. Also, these
compounds were tested against some important enzymes like acetylcholinesterase, butyrylcholinesterase
enzymes, carbonic anhydrase I, and II isoenzyme. The novel bromophenols showed Ki values of in range
of 53.75±12.54-234.68±46.76 nM against hCA I, 42.84±9.36 and 200.54±57.25 nM against hCA II,
0.84±0.12-14.63±3.06 nM against AChE and 0.93±0.20-18.53±5.06 nM against BChE. Induced fit
docking process performed on the compounds inhibiting hCA I, hCA II, AChE, and BChE receptors.
Hydroxyl group should exist at the aromatic ring of the compounds for inhibition of the enzymes. The
moieties reported in this study will be useful for design of more potent and selective inhibitors against the
enzymes.
Keywords: Bromination; bromophenol; carbonic anhydrase; acetylcholinesterase; enzyme inhibition;
molecular docking

1. INTRODUCTION
It is known that various bromophenols are often isolated from red algae of the family Rhodomelaceae in
marine life [1]. Important biological activities such as antioxidant [1,2] and feeding deterrent effects [3] of
the bromophenols were reported. We investigated the carbonic anhydrase (CA), [4] acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) [5] and paraxonase [6] inhibitory properties of bromophenol
derivatives.
Figure 1. Natural phenol compounds having methylsulfonyl group.
As seen Figure1, 5-(methoxymethyl)-4-((methylsulfonyl)methyl)benzene-1,2,3-triol (1), 3,4-dibromo-5-
((methylsulfonyl)methyl)benzene-1,2-diol (2) and 3,4,6-tribromo-5-((methylsulfonyl)methyl)benzene-1,2-
diol (3) are compounds having methylsulfonyl group. These phenol derivatives 1-3, natural product, were
isolated from the marine alga Grateloupia filicina (Wulfen) J. Agardh, [7] marine red alga Rhodomela
confervoides [2c] and marine red alga Symphyocladia latiuscula [2a], respectively. Natural bromophenols
2 and 3 exhibit antioxidant activities [2a,c].
-
The interconversion amongst HCO3 and CO2 is an essential mechanism for the significant flow of
biochemical mechanisms in all-living body cells. The metabolic transformation is performed by the
hydration of carbonic anhydride molecule, that gives rise to the corresponding bicarbonate, soluble ion,
and the emancipation of a proton [8]. CA isoenzymes are metalloenzymes that catalyse the reversible act
of CO2 into protons and bicarbonate and hence are necessary for maintaining extracellular and
intracellular pH [9]. CA isoforms are discovered in all three domains of life and these enzymes belong to
diverse families: α-, β-, γ-, δ-, ζ-, η- and θ-CAs. α-CA isozymes exist in many organisms, such as fungi
cells (together with β-CAs) [10]. β-CA isozymes are also found in archaea, plants, and both prokaryotes
and eukaryotes [11]. γ-CA isozymes are found in bacteria cells. ζ-CAs and δ-CA isoforms exist in some
2

marine planktonic kinds [12]. η-CAs and θ-CAs are found in malaria causative agent, Plasmodium
falciparum, and in marine diatom Phaeodactylum tricornutum, respectively [13]. CA isoforms have Zn2+
ions in their active centre, although certain can have cobalt, cadmium, and iron ions. Human cells have
sixteen CA isozymes but only 12 of them possess Zn2+ ion and are catalytically active. Human CAs
belongs to the α-CA family and are Zn2+ containing metalloenzymes [14].
Alzheimer's disease (AD) is an advanced neurodegenerative pathology disorder with social impact
and intense economic [15]. Nowadays, drug development and research are based on the cholinergic
hypothesis which proposes the elective harm of cholinergic neuron cells outcomes in a deficiency of
acetylcholine (ACh) in special areas of the brain that mediate memory functions and learning [16].
Presently accessible therapy for patients suffering from AD involve approved AChE inhibitor compounds
such as donepezil, rivastigmine, and galantamine, which avoid the hydrolysis of ACh therewith raising its
concentrations [17]. Levels of AChE and BChE manifestly show that these enzymes play a key role in
disease manifestation and progression. Some other studies have pointed to the significant role of AChE
enzyme in the promotion of Aβ oligomerization. Accordingly, the administration of BChE and AChE
inhibitor compounds is most clinically related palliative strategy for decreasing AD symptoms, while
another therapy strategy are failing [18].
To the best of our knowledge, bromophenols 2 and 3, biologically active natural products have not
yet been synthesized. In addition to these, no biological activity was investigated except for the
antioxidant activity of the bromophenols 2 and 3. In the present study, we report the first synthesis of
these natural products 2, 3 and their derivatives and their hCA I, hCA II, AChE and BChE inhibitory
properties.
2. RESULTS AND DISCUSSION
Chemistry. Biologically active natural bromophenols 2 and 3 include methylsufonyl and benzyl whose
aromatic ring are highly brominated groups (Figure 1). Synthesis of benzyl(methyl)sulfane from reaction
of benzyl bromide with NaSMe at mild condition is known [19]. Oxidation of sulfanes with meta-
choloroperbenzoic acide (m-CPBA) to sulfanes is also knonw [20]. For the synthesis of these
bromophenols and their derivatives, favorite method is as follows: (i) preparing benzyl bromides such as 4
and 5 starting with vanillin; (ii) substitutions of the benzyl bromides with NaSMe; (iii) oxidation and
demethylation of the corresponding benzyl(methyl)sulfanes.
3

Scheme 1. Synthesis of benzyl(methyl)sulfanes 8-11 and attempt to obtain natural bromophenol 3
from sulfone 12
After benzyl bromides 4-7 were synthesized by known method, [5a,21] and reactions of them with
NaSMe at room temperature (RT) for 24 h gave corresponding benzyl(methyl)sulfanes 8-11 in high yields
(Scheme 1). By oxidation of the compound 8 with m-CPBA, sulfone 12 was obtained and then its reaction
with BBr3 did not give natural bromophenol 3 (Scheme 1).
As another way for the synthesis of the natural bromophenols and their derivatives,
demethylations, [4,5,22] acetylations [23] and hydrolysis of the benzyl(methyl)sulfanes 8-11 were
thought, respectively. After demethylation of the benzyl(methyl)sulfanes 8-11 with BBr3 in CH2Cl2 were
performed, and then diols which were formed in situ were reacted with Ac2O (acetic acid anhydride)
(Scheme 2). Sulfanes with diacetates 13-16 were synthesized from these reactions. There are characteristic
peaks (as singlet) belonging OMe and Me (in acetates) of products in their NMR spectra. From these
peaks, peaks of OMe in 8-11 and Me in 13-16 were observed in their NMR spectra. Also, all data of them
consist with the proposed structures.
Scheme 2. Bromophenols including (methylsulfonyl)methyl group 2, 3, 21 and 22 from starting
material bromophenol derivatives with (methylthio)methyl group 8-11
4

Oxidation of the diacetates 13-16 with m-CPBA at RT gave their sulfones 17-20 (Scheme 2). In
the compounds, SO2 are more electron-withdrawing group than S. As expected, the chemical shifts of the
hydrogens (CH2 and CH3) in the adjacent carbon atoms of sulfur atom in 17-20 are lower field than those
of sulfur atom in 13-16. These chemical shifts for CH2 and CH3 are 3.61-4.23 ppm and 1.98-2.21 ppm for
13-16 and 4.18-4.96 ppm and 2.82-2.96 ppm for 17-20, respectively.
To synthesize the natural bromophenols 2 and 3, diacetates groups in 17 and 18 should be
hydrolyzed because the OAc also is a protecting group of OH group. Hydrolysis of these groups with
H2SO4/MeOH at reflux temperature gave natural bromophenol 2 and 3 in high yields. Also, bromophenols
21 and 22 were obtained from 19 and 20 by similar reactions. Synthesis of bromophenols 2, 3, 21 and 22
in this work are the first synthesis of them (Scheme 2).
3. Biological Activities
Since unusual amounts or activities of most sixteen hCA isoenzymes have been frequently associated with
various human diseases, these isoforms have raised an intense interest for the synthesis and design of
inhibitor compounds which selectively purpose special isoenzymes, in order to decrease the overall side
efficacy elicited by most non-isoform elective CAIs clinically utilized up to now [24]. These isoforms are
extensively distributed in diverse organs and tissues, where these isoforms are involved in many crucial
physiological mechanisms like CO2 and pH homeostasis, proliferation and cell differentiation, bone
calcification/resorption, electrolyte secretion, neurotransmission in mammals, gluconeogenesis, and tissue
and virulence colonization (in diverse pathogens, like protozoa, bacteria, and fungi [25]. Thus, many of
the CA isoforms are noteworthy aims for many pharmacological applications like diuretics, antiglaucoma
drugs, antiobesity, antitumor and anticonvulsant factors/diagnostic tools. For instance, inhibitor
compounds targeting hCA I are involved in cerebral edema and retinal, inhibitor compounds targeting
hCA II, IV, XII and XIV are utilized in the management edema, antiepileptic drugs, as diuretics and
antiglaucoma agents, and also for the therapy of altitude sickness [26] Recently, the inhibitor compounds
of the hCA IX and XII isoforms have recorded applications as diagnostic factors for imaging hypoxic
tumors and as antimetastatic/antitumor factors [27].
For hCA I isoform, the Ki values were determined in range of 53.75±12.54-234.68±46.76 nM. In
comparison, the Ki for the standard CA inhibitor AZA, a definitive hCA I inhibitor, was 273.61±76.86
nmol/L against hCA I (Table 1). All novel benzyl(methyl)sulfane derivative bromophenols (2, 3, 8-11, 13-
22) had effective inhibition effects than that of AZA. Also, between these compounds, novel natural
product 3-bromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (21), which had two hydroxyl groups (-OH)
and two bromine group (-Br), was the best hCA I inhibitor (Ki 53.75±12.54 nM). It is well known that
5

molecules containing halogen and carbonyl (-CO) groups are effective CA inhibitor compounds. The hCA
I isoform is discovered in many mammalian tissues and happens in high concentrations in the
gastrointestinal tract and blood cells [28]. It is involved in cerebral edema and retinal, and also the
inhibition of this enzyme can be an important factor for fighting these situations or diseases. As shown in
Table 1, IC50 values are in the range of 58.16-204.72 nM towards cytosolic hCA I isoenzyme, and in the
range of 45.04-184.02 nM for hCA II isoenzyme.
Novel benzyl(methyl)sulfane derivative bromophenols (2, 3, 8-11 and 13-22) synthesized in this
paper significantly inhibited hCA II with Ki in the low nanomolar range. Ki values were calculated
between 42.84±9.36 and 200.54±57.25 nM (Table 1 and Figure 2). On the other hand, novel natural
product 3-bromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (21), which had two hydroxyl groups (-OH)
and two bromine group (-Br), is in fact the best inhibitor in this molecules, being 5.34 times a better hCA
II inhibitor compared to the clinical candidate drug (Ki of AZA: 229.08±55.14 nM). The hCA II has key
−
role in diseases such as glaucoma disease. Indeed, HCO3 generation serves as a strategy to transport
sodium ions (Na+) into the eye along with the influx of water leading to an increase in intraocular pressure
−
[29]. Thus, hCA II inhibition reduces HCO3 production and finally aqueous humor secretion, which leads
to reduced pressure in the eye. These bromophenols are also an effective hCA II inhibitor, being almost 3
times more effective than AZA in inhibiting hCA II isoform.
35
28
21
14
7
20
16
12
8
hCA I
hCA II
4
0
0
-1.5
0
1.5
3
4.5
6
7.5
-1
0
1
2
3
4
5
6
7
1/[S] (nM)-1
1/[S] (nM)-1
6

75
60
45
30
15
0
250
200
150
100
50
AChE
BChE
0
-2.5
0
2.5
5
7.5
10
12.5
-150 -50
50
150
1/[S] (nM)-1
250
350
450
1/[S] (nM)-1
Figure 2. Determination of Lineweaver-Burk graphs for excellent inhibitors of hCA I and II (21), AChE
(3), and BChE (2) compounds
AChE is a fundamental enzyme that catalyzes the breakdown of ACh and some other choline ester
molecules that used as neurotransmitter factors, which are recorded as drug aims for AD [30]. The
cholinergic hypothesis was first proposed to describe AD and was based on the finding that the synaptic
depression is hindered due to the prevention of the ACh hydrolysis in the cholinergic neuron cells [31].
The inhibition of AChE results in the obstruction of ACh hydrolysis. Thus, the design of inhibitor
compounds and/or modulator for AChE enzyme is of Intense interests since it is presently one of the most
significant aims to prevent AD, resulting in three commercial drugs recorded by FDA like galantamine,
donepezil, rivastigmine and tacrine [32]. Some of compounds are under clinical evaluation like huperzine
A and ganstigmine [33]. The Ki values of novel benzyl(methyl)sulfane derivative bromophenols (2, 3, 8-
11, 13-22) for BChE and AChE were obtained from Lineweaver-Burk plots. Also, tacrine, which was the
first cholinesterase inhibitor to be discovered for the management of AD symptoms in 1993, had Ki value
of 100.40±15.62 nM. Furthermore, it was considered that donepezil hydrochloride compound, which is
used for the therapy of mild-to-moderate AD and diverse other memory impairments, had been shown to
lower AChE inhibition activity (IC50: 55.0 nM). As seen in Table 1, IC50 values are in the range of 1.63-
18.04 nM towards AChE, for BChE are in the range of 1.84-25.17 nM. In this work, AChE was also
extremely inhibited by novel benzyl(methyl)sulfane derivative bromophenols (2, 3, 8-11 and 13-22) at the
low nanomolar inhibition with Ki values in range of 0.84±0.12-14.63±3.06 nM (Table 1). These results
nicely determined that new synthesized bromophenols had effective AChE inhibition properties. However,
the most powerful AChE inhibition was recorded by novel natural product 3,4,6-tribromo-5-
((methylsulfonyl)methyl)benzene-1,2-diol (3), which had two hydroxyl groups (-OH) and three bromine
group (-Br) with a Ki value of 0.84±0.12 nM. All the remaining newly these compounds reported here
7

were highly efficient inhibition constants against AChE. Recently, evaluation of the crystallographic
structures of AChE enzyme and its complex with inhibitor compounds has provided information regarding
the pharmacophoric features necessary to key interactions and to elucidate the underlying catalytic
mechanism for the discovery of new and powerful AChEIs [34]. Additionally, tacrine (1,2,3,4-
tetrahydroacridin-9-amine), which first centrally acting cholinesterase inhibitor recorded for the therapy of
AD obtained with Ki value of 16.37±5.10 nM against cholinergic AChE. Finally, novel
benzyl(methyl)sulfane derivative bromophenols (2, 3, 8-11, 13-22) inhibited BChE with Ki values in
range of 0.93±0.20-18.53±5.06 nM (Table 1, Figure 2). However, the most powerful BChE inhibition was
recorded by novel natural product 3,4-dibromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (2), which
had two hydroxyl groups (-OH) and one bromine group (-Br) with a Ki value of 0.93±0.20 nM.
In Silico Studies
The ADME analyses of the most active compounds were performed by comprising to properties of a
particular molecule with those of 95% of known drugs using QikProp module. Results of ADME analyses
were used to classify the compounds according to physically significant descriptors and pharmaceutically
relevant properties of the compounds. In order to screening potential drug candidates, the properties
referred to drug-likeness were used. The ADME properties of the compounds are demonstrated in Table 2.
The properties of the compounds were examined whether they are eligible with those of commercial
drugs, in the light of Lipinski's rule of five. All most active compounds have less molecular weight than
500D. All most active compounds have 2 hydrogen bond acceptors (AHB) and 5.5 hydrogen bond donors
(DHB). Octanol/water partition coefficients of the compounds are acceptable ranges in light Lipinski's
rule. The compounds exhibited noteworthy range IC50 value for HERG K+ channel blockage (logHERG)
at values above -5 and range brain/blood partition coefficient (logBB). Human oral absorption percentage
of most active compounds is in appropriate ranges. The MDCK epithelial cell lines are commonly used as
a possible tool for assessing the membrane permeability properties. PMDCK value of drugs should be
high than 500 for great membrane permeability. We think that compounds 2 and 3 from most active
compounds possess great membrane permeability with value higher than 500, however membrane
permeability of c 21 doesn't look good enough. ADME analyses result clearly demonstrated that
physicochemical and pharmacokinetic properties of the most active compounds are compatible with
Lipinski's rule [35].
Catalytic active site
We have used SiteMap to explore the catalytic site on the receptors. The most important property
calculated by SiteMap is an overall SiteScore, which has proven to be effective at identifying known
catalytic sites in co-crystallized complexes. SiteScores of binding sites were calculated as 1.063, 0.971,
8

1.090, and 1.150 for hCA I, hCA II, AChE, and BChE, respectively. The other important property
calculated by SiteMap is Dscore which has indicated whether the catalytic site can be druggable or not.
Dscores of the binding sites were calculated as 1.061, 0.945, 1.113, and 1.217 for hCA I, hCA II, AChE,
and BChE, respectively. SiteMap results indicated that identified binding sites have catalytic sites
properties due to their SiteScores and are druggable sites due to their Dscore. Moreover, we have
presented ligand bonded catalytic sites surface including hydrophilic, hydrophobic, and metal binding
sites in Figure 3.
a
b
c
d
Figure 3. Results of SiteMap analyses. (a) 21-hCA I, (b) 21-hCA II, (c) 3-AChE, and (d) 2-BChE.
Binding site is represented as grey mesh, hydrophilic site is represented as green surface, hydrophobic site
is represented as yellow surface, and metal binding site is represented as blue surface.
Molecular docking
The most active compounds were docked into catalytic active site of hCA I, hCA II, AChE, and BChE
receptors by IFD protocol which provide flexibility both ligand and receptor. We firstly tested reliability
of the IFD protocol by re-docking inhibitor complexed into the crystal structure of the receptors. IFD
results were analyzed in the perspective of ligand-binding pose. The results have shown that co-
crystallized and docked ligands were located in very similar region of the receptors as seen in Figure 4.
After IFD protocol validation, we analyzed IFD results of most active compounds with regard to binding
9

energies of ligand-receptor complex, interactions between ligand and receptor, efficiency of ligand-
binding site, and ligand-binding pose.
a
b
c
d
Figure 4. Docking validation. (a) 21-hCA I, (b) 21-hCA II, (c) 3-AChE, and (d) 2-BChE. The poses of
co-crystallized ligands are represented in black color ball and stick modeling while that of docked ligands
a
b
d
c
f
h
e
g
is represented in grey color ball and stick mode.
10

a
b
c
d
Figure 5. 2D receptor-ligand interaction profile; (a) 21-hCA I, (b) 21-hCA II, (c) 3-AChE, (d) 2-BChE,
(e) AZA-hCA I, (f) AZA-hCA II, (g) TAC-AChE, and (h) TAC-BChE
Figure 6. Superimposed pose of the most active compounds into hCA I, hCA II, AChE, and
BChE receptors. (a) Best-pose of 21 into catalytic active site of hCA I, (b) Best-pose of 21 into catalytic
active site of hCA II, (c) Best-pose of 3 into catalytic active site of AChE, and (d) Best-pose of 2 into
catalytic active site of BChE. Receptors are depicted in the ribbon model which hCA I with grey color,
hCA II with yellow color, AChE with cyan color, and BChE with faded blue color. Catalytic active sites
are represented as grey solid surface. Compounds are represented in ball and stick modeling which
magenta color carbon and residues are represented in thick tube modeling same color with receptors
ribbon model.
Superimposed pose and detailed binding mode of the most active compounds were shown in
Figure 6. The compounds fit into the catalytic active site of the receptors. 3-Bromobenzene-1,2-diol
moiety of the compound 21 was closely surrounded by metal binding site residue including Zn301 and
hydrophilic residues including Gly92, His94, and His96 and methylsulfonyl oxygen moiety was also
closely surrounded by hydrophilic Gly92 residue in catalytic active site of hCA I as seen in Figures 3a and
6a. The moieties of compound 21 were surrounded by almost similar residues in catalytic active site of
hCA II as seen in Figures 3b and 6b. Almost all of 3,4,6-tribromo benzene-1,2-diol and methylsulfonyl
moieties of the compound 3 were surrounded by hydrophobic residues including Tyr72, Tyr124, Trp286,
11

Leu289, Val294, Phe295, Phe297, Tyr337, Phe338, and Tyr341 residues in catalytic active site of AChE
as seen in Figures 3c and 6c. While 3,4-dibromo benzene-1,2-diol moiety of the compound 2 was closely
surrounded by both hydrophilic residues including Glu197 and Ser198 and hydrophobic Trp82 residue,
methylsulfonyl moiety of the compound was closely surrounded by hydrophobic residues including
Met437 and Tyr440 as seen in Figures 3d and 6d.
In conclusion, Starting from vanillin, the known [5a,10] benzyl bromides 4-7 were obtained and
reactions of them with NaSMe gave the corresponding substitution products 8-11. The sulfone 12 was
synthesized from the oxidation of substitution product 8 with m-CPBA, and its reaction with BBr3 did not
give natural bromophenol 3. Demethylation with BBr3, acetylation with Ac2O, oxidation with m-CPBA
and hydrolization with acid of the substitution products 8-11 were performed, respectively. The first
synthesis of the new bromophenols 2, 3, 21 and 22 were realized from these reactions. Two of the
bromophenols are natural products 2 and 3. Additionally, the novel molecules were investigated for
BChE, AChE, hCA I, and hCA II enzymes inhibition effects. As we explained above, novel
benzyl(methyl)sulfane derivative bromophenols (2, 3, 8-11 and 13-22) 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. The binding affinities and interactions of the most active compounds have provided more
insight into the mode of binding of the compounds into the receptors. Hydroxyl group on bromophenol
moiety of the compounds is crucial for inhibition of hCA I, hCA II, AChE, and BChE receptors.
Moreover, oxygen on methylsulfonyl moiety promotes inhibition of particularly hCA II and AChE
receptors.
4. EXPERIMENTAL SECTION
4.1. General Experimental Procedures.
Solvents were purified and dried by known methods. For all compounds, values as well as Mp, IR Spectra,
13
1H and C NMR spectra, chemical shift, elemental analyses, and CA inhibitory properties of samples
were performed as explained previously [5a,36]. PLC (preparative thick-layer chromatography) was used
as 1 mm of silica gel 60 PF (Merck, Darmstadt, Germany) on glass plates. HRMS data were obtained by
LC-MS-TOF electrospray ionization technique (1200/6210, Agilent). The benzyl bromides 4-7 used in the
reactions were synthesized by known ways [5a,21].
4.2. Synthesis
12

4.2.1 Synthesis of methyl(2,3,6-tribromo-4,5-dimethoxybenzyl)sulfane (8), Standard procedure for
substitution reaction with NaSMe: To solution of the bromide 4 (0.4 g, 855.11 µmol) in EtOH (50 mL)
was added NaSMe (62.93 mg, 897.86 µmol), and reaction mixture was stirred at RT for 24 h. After the
solvent was removed under vacuum and water (2 mL) was added, the mixture was neutralized by cold
solution of HCl (%1) and extracted with CH2CI2 (2 x 20 mL). Combined extracts were dried over Na2SO4
and the solvent was evaporated removed under vacuum. The sulfane 8 (1.044 g, %90) as a white solid.
o
1
M.p = 30-32 C; H NMR (400 MHz, CDCl3): 4.22 (s, CH2S, 2H), 3.90 (s, 2x OMe, 6H), 2.22 (s, CH3S
13
3H); C NMR (100 MHz, CDCl3): 150.96 (C), 150.75 (C), 135.90 (C), 122.49 (C), 121.91 (C), 120.40
(C), 60.88 (OMe), 60.82 (OMe), 40.94 (CH2S), 16.16 (CH3S); IR (CH2Cl2, Vmax cm-1): 2935, 1452,
1393, 1366, 1278, 1051, 1010; Rf = 0.59 EtOAc/hexane (5:95); HRMS (APCI – TOF) (m/z) calcd for
C10H1179Br3O2S: 431.8030; found: 431.8037.
4.2.2 Synthesis of (3-bromo-4,5-dimethoxybenzyl)(methyl)sulfane (9): This reaction was performed
according to the standard procedure described in 4.2.1. In the reaction, bromide 5 (0.5 g, 1.61 mmol),
EtOH (40 mL) and NaSMe (124.35 mg, 1.77 mmol) were used. The sulfane 9 (0.4 g, %90) was obtained
1
as a yellow liquid; H NMR (400 MHz, CDCl3): 7.05 (bs, aromatic 1H), 6.84 (d, aromatic, 1H), 3.87 (s,
OCH3, 3H), 3.84 (s, OCH3, 3H), 3.59 (s, CH2, 2H), 2.01 (s, CH3, 3H); 13C NMR (100 MHz, CDCl3):
153.69 (C), 145.46 (C), 135.44 (C), 124.83 (CH), 117.26 (C), 112.15 (CH), 60.54 (OMe), 56.09 (OMe),
37.90 (CH2S), 15.06 (CH3); IR (CH2Cl2, vmax cm-1): 2915, 1595, 1567, 1489, 1411, 1274, 1139, 1047; Rf =
0.40 EtOAc/hexane (5:95); HRMS (APCI-TOF) (m/z) calcd for C10H1379BrO2S: 275.9820; found:
275.9806.
4.2.3 Synthesis of (2,3-dibromo-4,5-dimethoxybenzyl)(methyl)sulfane (10): This reaction was performed
according to the standard procedure described in 4.2.1. In the reaction, bromide 6 (0.30 g, 771.44 µmol),
EtOH (40 mL) and NaSMe (59.47 mg, 848.59 µmol) were used. The sulfane 10 (0.40 g, %90) was
1
obtained as a brown liquid; H NMR (400 MHz, CDCl3): 6.88 (s, aromatic, 1H), 3.80 (s, OMe, 3H), 3.76
(s, CH2, 2H), 3.75 (s, OMe, 3H), 2.00 (s, CH3, 3H); 13C NMR (100 MHz, CDCl3): 152.40 (C), 146.61 (C),
135.05 (C), 122.08 (C), 117.66 (C), 113.27 (CH), 60.51 (OMe), 56.25 (OMe), 40.23 (CH2S), 15.43
(CH3S); IR (CH2Cl2, vmax cm-1): 2917, 2848, 1582, 1548, 1469, 1422, 1372, 1372, 1308, 1263, 1059; Rf =
0.37 EtOAc/hexane (5:95); HRMS (APCI-TOF) (m/z) calcd for C10H1279Br2O2S: 353.8925; found:
353.8918.
4.2.4 Synthesis of 2-bromo-4,5-dimethoxybenzyl)(methyl)sulfane (11): This reaction was performed
according to the standard procedure described in 4.2.1. In the reaction, bromide 7 (1.0 g, 3.23 mmol),
EtOH (40 mL) and NaSMe (248.70 mg, 3.55 mmol) were used. The sulfane 11 (0.79 g, %88) was
13

1
obtained as a yellow liquid; H NMR (400 MHz, CDCl3): 7.00 (s, aromatic, 1H), 6.90 (s, aromatic, 1H),
3.86 (s, OMe, 3H), 3.84 (s, OMe, 3H), 3.75 (s, CH2, 2H), 2.05 (s, CH3, 3H); 13C NMR (100 MHz, CDCl3):
148.59 (C), 148.45 (C), 129.52 (C), 115.50 (CH), 114.40 (C), 113.26 (CH), 56.13 (OMe), 56.05 (OMe).
37.90 (CH2), 15.02 (CH3); IR (CH2Cl2, vmax cm-1): 2914, 1602, 1507, 1438, 1380, 1260, 1217, 1164, 1031;
Rf = 0.31 EtOAc/hexane (5:95); HRMS (APCI-TOF) (m/z) calcd for C10H1379BrO2S: 275.9820; found:
275.9841
4.2.5 Synthesis of 1,2,4-tribromo-5,6-dimethoxy-3-((methylsulfonyl)methyl)benzene (12): Standard
procedure for the oxidation reaction with m-CPBA: To a solution of the sulfane 8 (0.3 g, 689.71 µmol) in
CH2Cl2 (20 mL) was added m-CPBA (0.417 g, 2,41 mmol) and NaHCO3 (100 mg) at RT. The reaction
mixture was stirred for 24 h at room temperature, and then the reaction mixture was stopped. After a
solution of cold NaHCO3 (%5, 0oC, 100 mL) was slowly added, the mixture was extracted with CH2CI2 (2
x 30 mL). Combined organic phases were dried over Na2SO4, and the solvent was removed under vacuum.
The residue was submitted to silica gel (20 g) column chromatography with EtOAc/hexane (5/95) elution.
The sulfone 12 (0.26 g, % 81) was obtained as a white solid. Mp: 98-100 oC; 1H NMR (400 MHz, CDCl3):
5.02 (s, CH2, 2H), 3.94 (s, OMe, 3H), 3.91 (s, OMe, 3H), 3.04 (s, CH3, 3H); 13C NMR (100 MHz, CDCl3):
152.67 (C), 151.12 (C), 126.78 (C), 124.21 (C), 122.64 (C), 122.25 (C), 63.25 (CH2), 60.97 (OMe), 60.93
(OMe), 43.19 (CH3); IR (CH2Cl2, vmax cm-1): 2939, 1454, 1393, 1368, 1312, 1285, 1121, 1052, 1007;
HRMS (APCI-TOF) calcd for [C10H1179Br3O4S + H]+: m/z = 464.8006; found: 464.8020.
4.2.6 Synthesis of 3,4,6-tribromo-5-((methylthio)methyl)-1,2-phenylene diacetate (13): Standard
procedure for the demethylation and acetatilation reactions of sulfanes, respectively: To a stirred solution
of the sulfane 8 (0.30 g, 689.71 µmol) in CH2Cl2 (15 mL) under nitrogen atmosphere added BBr3 (380.13
mg, 1.52 mmol) with the aid of a syringe, and the reaction mixture was allowed to stir at RT for 24 h.
o
After the reaction mixture was cooled at 0 C by ice-water bath, and then MeOH (5 mL) was added
dropwise, the solvent was removed under vacuum, and water (5 mL) was added. The reaction mixture was
extracted with EtOAc (2 × 15 mL), the combined extracts were dried over Na2SO4. The solvent was
removed under vacuum. The crude product of the corresponding diol was reacted with Et3N (2.0 mL) and
Ac2O (2.0 mL) at RT for 24 h, and then CH2Cl2 (40 mL) was added. After the reaction mixture was cooled
to 0 °C and neutralized by cold solution (%1.0) of HCl. Organic phase was separated and washed by cold
solution (%1.0 20 mL) of NaOH and water (20 mL), respectively. After the organic phase was dried over
Na2SO4, the solvent was removed under vacuum, and diacetate 13 (280 mg %92) was obtained as a white
solid. M.p = 85-87 oC; 1H NMR (400 MHz, CDCl3): 4.23 (s, CH2S, 2H), 2.35 (s, 2 x COCH3, 6H), 2.21 (s,
CH3S 3H). 13C NMR (100 MHz, CDCl3): 166.78 (CO), 166.76 (CO), 141.61 (C), 141.58 (C), 138.57 (C),
14

125.46 (C), 121.76 (C), 119.73 (C), 40.86 (CH2S), 20.39 (CH3), 20.35 (CH3), 16.08 (CH3S); IR (CH2Cl2,
vmax cm-1): 2917, 2918, 1783, 1412, 1370, 1275, 1189, 1148, 1033, 1011; Rf = 0.43 EtOAc/Hexane
81
(15:85); HRMS (APCI-TOF) (m/z + H – 2 CH3CO - CH3S) calcd for C7H479Br2 BrO2: 358.7741; found:
358.7729.
4.2.7 Synthesis of 3-bromo-5-((methylthio)methyl)-1,2-phenylene diacetate (14): This reaction was
performed according to the standard procedure described in 4.2.6. In the reaction, sulfane 9 (0.50 g, 1.80
mmol), BBr3 (903.84 mg, 3.61 mmol), Et3N (2.0 mL), Ac2O (2.0 ml)) and CH2Cl2 (15 mL) were used.
Diacetate 14 (0.59 g, % 89) was obtained as a white solid. M.p = 60-62 oC; 1H NMR (400 MHz, CDCl3):
7.44 (s, aromatic, 1H), 7.14 (s, aromatic, 1H), 3.61 (s, CH2, 2H), 2.33 (s, OAc 3H), 2.27 (s, OAc, 3H),
2.00 (s, CH3, 3H); 13C NMR (100 MHz, CDCl3): 167.91 (CO), 167.27 (CO), 143.39 (C), 139.47 (C),
138.24 (C), 130.40 (CH), 123.12 (CH), 117.25 (C), 37.31 (CH2), 20.60 (CH3), 20.31 (CH3), 15.04 (SCH3);
IR (CH2Cl2, vmax cm-1): 2917, 1779, 1574, 1475, 1371, 1278, 1202, 1015; Rf = 0.37 EtOAc/Hexane
(15:85); HRMS (APCI-TOF) (m/z + H) calcd for C12H1379BrO4S: 332.9796; found: 332.9625.
4.2.8 Synthesis of 3,4-dibromo-5-((methylthio)methyl)-1,2-phenylene diacetate (15): This reaction was
performed according to the standard procedure described in 4.2.6. In the reaction, sulfane 10 (1.0 g, 2.81
mmol), BBr3 (1.41 g, 5.62 mmol), Et3N (2.0 mL), Ac2O (2.0 mL) and CH2Cl2 (15 mL) were used.
o
Diacetate 15 (0.93 g, %90) was obtained as a white solid. M.p: 82-84 C; 1H NMR (400 MHz, CDCl3): δ
13
7.20 (s, aromatic, 1H), 3.75 (s, CH2, 2H), 2.26 (s, CH3, 3H), 2.19 (s, CH3, 3H), 1.98 (s, SCH3, 3H); C
NMR (100 MHz, CDCl3): δ 167.69 (CO), 167.02 (CO), 142.21 (C), 140.47 (C), 137.80 (C), 124.34 (C),
123.79 (CH), 122.19 (C), 40.05 (CH2), 20.60 (CH3), 20.36 (CH3), 15.35 (SCH3); IR (CH2Cl2, vmax cm-1):
2917, 1780, 1446, 1370, 1283, 1196, 1135, 1009; Rf = 0.34 EtOAc/Hexane(15:85); HRMS (APCI-TOF)
(m/z + H – 2 CH3CO - CH3S) calcd for C7H579Br81BrO2: 280.8636; found: 280.8625.
4.2.9 Synthesis of 4-bromo-5-((methylthio)methyl)-1,2-phenylene diacetate (16): This reaction was
performed according to the standard procedure described in 4.2.6. In the reaction, sulfane 11 (0.8 g, 2.89
mmol), BBr3 (1.45 g, 5.77 mmol), Et3N (2.0 mL), Ac2O (2.0 mL) and CH2Cl2 (15 mL) were used.
o
1
Diacetate 16 (0.82 g, %83) was obtained as a white solid. M.p = 72-74 C; H NMR (400 MHz, CDCl3):
7.42 (s, aromatic, 1H), 7.23 (s, aromatic, 1H), 3.73 (s, CH2, 2H), 2.26 (s, CH3, 3H), 2.25 (s, CH3, 3H),
13
2.04 (s, SCH3, 3H); C NMR (100 MHz, CDCl3): 168.11 (CO), 168.05 (CO), 141.48 (2XC), 136.40 (C),
127.91 (CH), 125.16 (CH), 120.73 (C), 38.12 (CH2), 20.81 (CH3), 20.75 (CH3), 15.41 (SCH3); IR
15

(CH2Cl2, vmax cm-1): 2917, 1776, 1485, 1370, 1272, 1203, 1133, 1013; Rf = 0.37 EtOAc/Hexane (15:85);
HRMS (APCI-TOF) (m/z - H) calcd for C12H1379BrO4S: 330.9640; found: 330.9635.
4.2.10 Synthesis of 3,4,6-tribromo-5-((methylsulfonyl)methyl)-1,2-phenylene diacetate (17): This reaction
was performed according to the standard procedure described in 4.2.5. In the reaction, the diacetate 13
(0.5 g, 1.02 mmol), m-CPBA (615.06 mg, 3.56 mmol) and NaHCO3 (100 mg) were used. The sulfone 17
o
1
(450 mg, %84) was obtained as a white solid. M.p = 155-157 C; H NMR (400 MHz, CDCl3): 4.96 (s,
13
CH2S, 2H), 2.96 (s, CH3S, 3H), 2.29 (s, 2xCH3, 6H); C NMR (100 MHz, CDCl3): 166.55 (CO), 166.39
(CO), 143.51 (C), 142.21 (C), 129.60 (C), 127.18 (C), 122.67 (C), 121.56 (C), 63.16 (CH2S), 42.94
(CH3S), 20.37 (CH3), 20.31 (CH3); IR (CH2Cl2, vmax cm-1): 2931, 1784, 1417, 1372, 1315, 1186, 1148,
1011; HRMS (APCI-TOF) calcd for [C12H1179Br81Br2O6S + H]+: m/z = 524.7864; found: 524.7851.
4.2.11 Synthesis of 3-bromo-5-((methylsulfonyl)methyl)-1,2-phenylene diacetate (18): This reaction was
performed according to the standard procedure described in 4.2.5. In the reaction, diacetate 14 (1.2 g, 3.60
mmol), m-CPBA (2.35 g, 12.61 mmol) and NaHCO3 (100 mg) were used. The sulfone 18 (1.0 g, %76)
o
1
was obtained as a white solid. M.p = 128-130 C; H NMR (400 MHz, CDCl3): 7.55 (s, aromatic, 1H),
13
7.28 (s, aromatic, 1H), 4.18 (s, CH2, 2H), 2.82 (s, SCH3, 3H), 2.36 (s, OAc, 3H), 2.29 (s, OAc, 3H); C
NMR (100 MHz, CDCl3): 167.71 (CO), 166.93 (CO), 143.82 (C), 141.50 (C), 132.13 (CH), 127.84 (C),
124.97 (CH), 118.21 (C), 59.80 (CH2), 39.44 (SO2CH3), 20.59 (CH3), 20.28 (CH3); IR (CH2Cl2, vmax cm-
1): 2932, 1776, 1576, 1476, 1425, 1371, 1310, 1200, 1121, 1017; Rf = 0.42 EtOAc/Hexane (6:4); HRMS
(APCI-TOF) (m/z) calcd for C12H1379BrO6S: 363.9616; found: 363.9601.
4.2.12 Synthesis of 3,4-dibromo-5-((methylsulfonyl)methyl)-1,2-phenylene diacetate (19): This reaction
was performed according to the standard procedure described in 4.2.5. In the reaction, diacetate 15 (0.5 g,
1.21 mmol), m-CPBA (792.38 mg, 4.25 mmol) and NaHCO3 (100 mg) were used. The sulfone 19 (0.43 g,
o
1
%80) was obtained as a light yellow solid. M.p = 148-150 C; H NMR (400 MHz, CDCl3): 7.52 (s,
13
aromatic, 1H), 4.59 (s, CH2, 2H), 2.87 (s, SO2CH3, 3H), 2.37 (s, CH3, 3H), 2.29 (s, CH3, 3H); C NMR
(100 MHz, CDCl3): 167.46 (CO), 166.68 (CO), 142.79 (C), 142.54 (C), 128.47 (C), 126.21 (CH), 125.17
(C), 122.79 (C), 61.37 (CH2), 40.27 (SO2CH3), 20.55 (CH3), 20.34 (CH3); IR (CH2Cl2, vmax cm-1): 2917,
2849, 1781, 1450, 1372, 1312, 1195, 1141, 1010; Rf = 0.50 EtOAc/Hexane (6:4); HRMS (APCI-TOF)
(m/z + H) calcd for C12H1279Br81BrO6S: 442.8779; found: 442.8770.
4.2.13 Synthesis of 4-bromo-5-((methylsulfonyl)methyl)-1,2-phenylene diacetate (20): This reaction was
performed according to the standard procedure described in 4.2.5. In the reaction, diacetate 16 (0.7 g, 2.10
mmol), m-CPBA (1.37 g, 7.35 mmol) and NaHCO3 (100 mg) were used. The sulfone 20 (0.6 g, %78) was
16

obtained as a white solid. M.p = 145-147 oC; 1H NMR (400 MHz, CDCl3): 7.53 (s, aromatic, 1H), 7.47 (s,
13
aromatic, 1H), 4.46 (s, CH2, 2H), 2.85 (s, SO2CH3, 3H), 2.29 (bs, 2XCH3, 6H); C NMR (100 MHz,
CDCl3): δ 167.70 (CO), 167.55 (CO), 143.18 (C), 141.80 (C), 128.11 (CH), 127.35 (CH), 126.90 (C),
121.11 (C), 59.55 (CH2), 39.99 (SO2CH3), 20.56 (CH3), 20.53 (CH3); IR (CH2Cl2, vmax cm-1): 2924, 1773,
1490, 1371, 1311, 1204, 1138, 1012; Rf = 0.50 EtOAc/Hexane(6:4); HRMS (APCI-TOF) (m/z) calcd for
C12H13Br79O6S: 363.9616; found: 363.9603.
4.2.14 Synthesis of natural product 3,4,6-tribromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (3):
Standard procedure for the synthesis of 2, 21 and 22: A solution of the sulfone 17 (0.2 g, 382.42 µmol) in
MeOH (10 mL) was added H2SO4 (catalytic), and the mixture was stirred for 6 h at 60 °C. The solvent was
removed under vacuum. After H2O (10 mL) was added to this reaction mixture was added, the mixture
was extracted with EtOAc (2 x 10 mL). The combined extracts were dried over Na2SO4, the solvent was
removed under vacuum and natural product 3 (140 mg, 83%) was obtained as a brown solid. M.p: 195-
o
o
1
197 C [2a] 134-136 C; H NMR (400 MHz, Acetone-d6): 9.30-8.70 (m, 2xOH), 4.99 (s, CH2SO2, 2H),
3.07 (s, CH3SO2, 3H); 13C NMR (100 MHz, Acetone-d6): 145.24 (C), 143.65 (C), 122.62 (C), 118.87 (C),
114.12 (C), 113.51 (C), 62.82 (CH2SO2), 42.70 (CH3SO2).
4.2.15 Synthesis of natural product 3-bromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (21):This
reaction was performed according to the standard procedure described in 4.2.14. In the reaction, the
sulfone 18 (0.3 g, 821.48 µmol), CH3OH (20 mL)) and H2SO4 (catalytic) were used. The bromophenol 21
(0.2 g, %87) was obtained as a brown solid. M.p = 152-154 oC; 1H NMR (400 MHz, Acetone-d6): 8.89 (s,
OH, 1H), 8.24 (s, OH, 1H), 7.13 (s, aromatic, 1H), 6.97 (s, aromatic, 1H), 4.24 (s, CH2SO2, 2H), 2.83 (s,
CH3SO2, 3H); 13C NMR (100 MHz, Acetone-d6): 145.95 (C), 143.95 (C), 126.06 (CH), 121.98 (C), 117.16
(CH), 109.26 (C), 59.20 (CH2SO2), 38.82 (CH3SO2); IR (CH2Cl2, vmax cm-1): 3412, 2921, 1706, 1588,
1493, 1435, 1293, 1115; Rf = 0.45 MeOH/CH2Cl2 (5:95); HRMS (APCI-TOF) (m/z) calcd for
C8H979BrO4S: 279.9405; found: 279.9454.
4.2.16 Synthesis of natural product 3,4-dibromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (2):This
reaction was performed according to the standard procedure described in 4.2.14. In the reaction, the
sulfone 19 (0.4 g, 900.72 µmol), CH3OH (20 mL) and H2SO4 (catalytic) were used. The natural
o
1
bromophenol 2 (0.3 g, %92) was obtained as a cream colored solid. M.p = 188-190 C; H NMR (400
MHz, Acetone-d6): 9.18 (s, OH, 1H), 8.65 (s, OH, 1H), 7.16 (s, aromatic, 1H), 4.57 (s, CH2SO2, 2H), 2.91
13
(s, CH3SO2, 3H); C NMR (100 MHz, Acetone-d6): 146.16 (C), 145.56 (C), 122.36 (C), 118.84 (CH),
118.27 (C), 114.06 (C), 61.66 (CH2SO2), 40.59 (CH3SO2); Rf = 0.48 MeOH/CH2Cl2 (5:95); HRMS
(APCI-TOF) (m/z) calcd for C8H879Br2O4S: 357.8510; found: 357.8550.
17

4.2.17 Synthesis of 4-bromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (22): This reaction was
performed according to the standard procedure described in 4.2.14. In the reaction, the sulfone 20 (0.45 g,
1.23 mmol), CH3OH (20 mL) and H2SO4 (catalytic) were used. The bromophenol 22 (0.29 g, %84) was
obtained as a brown solid. M.p = 148-150 oC; 1H NMR (400 MHz, Acetone-d6): 8.47 (s, OH, 1H), 8.31 (s,
OH, 1H), 6.97 (s, aromatic, 1H), 6.95 (s, aromatic, 1H), 4.27 (s, CH2SO2, 2H), 2.72 (s, CH3SO2, 3H); 13C
NMR (100 MHz, Acetone-d6): 147.80 (C), 145.85 (C), 120.84 (C), 120.09 (2xCH), 114.69 (C), 60.07
(CH2SO2), 40.24 (CH3SO2); IR (CH2Cl2, vmax cm-1): 3259, 2918, 1700, 1512, 1425, 1295, 1138, 1115; Rf
= 0.51 MeOH/CH2Cl2 (5:95); HRMS (APC-TOF) (m/z) calcd for C8H979BrO4S: 279.9405; found:
279.9455.
4.3.Biochemical Studies
4.3.1. AChE/BChE activity determination and inhibition studies
The inhibitory efficacy of novel benzyl(methyl)sulfane derivative bromophenols (2, 3, 8-11 and 13-22) on
BChE/AChE activities was obtained conforming to the spectrophotometric procedure of Ellman et al [37].
Acetylthiocholine iodide (AChI) and butrylcholine iodide (BChI) were used as substrate molecules of the
both reactions. In this study, 5,5′-dithio-bis(2-nitro-benzoic)acid (DTNB) was used for the estimation of
the both BChE/AChE enzymes activities [38]. Briefly, 100 µL of buffer solution (pH 8.0, 1.0 M,
Tris/HCl) and diverse concentration of sample solutions (30-300 µL) dissolved in deionized water were
added to 50 µL of both BChE/AChE enzymes solutions (5.32 × 10−3 EU) [39]. Then the mixture was
incubated for 10 min at 20 °C. Finally, 50 µL of DTNB (0.5 mM and 25 mL) of both substrate BChI/AChI
were added to incubated mixture. Also, the reaction was initiated by the addition of 50 µL of BChI/AChI.
Activities of these enzymes were evaluated spectrophotometrically at a wavelength of 412 nm [40].
hCA isoenzyme purification and inhibition studies
For investigating of inhibitory effects of novel benzyl(methyl)sulfane derivative bromophenols (2, 3, 8-11
and 13-22) on hCA isoformes, both hCA isoforms from human erythrocytes were purified via a simple
single-step by method Sepharose-4B-L-Tyrosine-sulphanilamide affinity gel chromatography [41]. For
this purpose, the human erythrocyte samples were centrifuged at 13000 rpm for 25 min. Then, the solution
was filtered to remove precipitate. Both hCA isoenzymes were isolated from the serum, which its pH was
adjusted to 8.7 by adding solid Tris [42]. Affinity column was equilibrated by buffer solution (25 mM
Tris-HCl / 0.1 M Na2SO4) at the pH 8.7. The serum was loaded to affinity gel and washed with buffer
solution (25 mM Tris-HCl/22 mM Na2SO4 at the pH 8.7. The hCA I isoenzyme was eluted by buffer
solution (1.0 M NaCl / 0.25 M sodium phosphate at the pH 6.3. On the other hand, hCA II isoenzyme was
18

eluted by another buffer solution (0.1 M sodium acetate / 0.5 M NaClO4 at the pH 5.6. Both isoenzymes
were taken from the column in fractions of 2 mL [43]. All works was realized at 4 °C. The hCA
isoenzymes activity were measured by following the change at absorbance a specific (348 nm) of p-
o
nitrophenylacetate (PNA) to p-nitrophenolate ion over a period of 3 min at room temperature (25 C)
using a spectrophotometer (Thermo Scientific, UV-Vis Spectrophotometer) according to the method of
Verpoorte et al [44]. There was 0.4 mL of 0.05 M Tris-SO4 buffer (pH 7.4), 03 mL of 3 mM PNA, 0.2 mL
of H2O, and 0.1 mL of enzyme solution in a test tube content of this reaction [45]. Esterase activity assays
were identified from a series of experiments at three different novel benzyl(methyl)sulfane derivative
bromophenols (2, 3, 8-11 and 13-22).
4.4. In Silico Studies
4.4.1. Protein and ligand preparation
The in silico study was conducted using the Small Drug Discovery Suites package (Schrödinger 2017-2,
LLC, USA). X-Ray crystal structures of hCA I, hCA II, AChE, and BChE, (PDB code: 4WR7, 5AML,
4M0E, and 5NN0, respectively) were obtained from RCSB Protein Data Bank (PDB;
http://www.rcsb.org/). The crystal structures of receptors were selected because they possess ligand in
catalytic active site and smallest resolution of 1.5Å, 1.36Å, 2Å, and 2.1Å, respectively. Protein
preparation wizard panel was used for repairing and preparing of the crystal structures of receptors.
Protein preparation workflow was described in summary. Briefly, bond order and charges were assigned
and then missing hydrogen atoms were added to crystal structures. Missing side chains were filled using
Prime module of the program. Amino acids were ionized by setting physiological pH with the help of
Propka software. Water molecules that were formed less than 3 contacts with the protein or ligand were
removed. Finally, energy minimization and geometry optimization have also been performed using OPLC
force field. 3D structures of synthesized compounds were produced with Maestro 11.4 by sketching 2D
structures. 3D structure of ligands was created using LigPrep module of Schrodinger. In order to obtain
correct molecular geometries and protonation state at pH 7.0±2.0, Epik module and OPLS-2005 force
field were used [46,47].
4.4.2. ADME Analysis
To predict pharmacokinetic properties of most active compounds, ADME analysis was performed with
QikProp module in Maestro 11.4. The analysis provides information about absorption, distribution,
metabolism, and excretion of the compounds in comparison to a particular molecule’s properties with
those of 95% of known drugs. Pharmacokinetic properties of prepared most active compounds were
predicted by using QikProp calculation. The drug-likeness of the compounds was determined with
Lipinski’s rule of five (mol_MW, logPo/w, donorHB, and accptHB) [48].
19

4.4.3. Catalytic site prediction
Prediction of catalytic site of the receptors was carried out SiteMap module in Maestro 11.4. The
predicted catalytic site provides to select the target for ligand docking and to evaluate docking hits, by
showing how well the poses display proper complementarity to the receptor. Prepared receptor was
imported into Maestro in PDB file format. SiteMap was run using the default parameter of top-ranked
potential protein binding sites setting. This process was performed for all receptors. After SiteMap
process, catalytic site for each receptor was generated and Sitescore and Dscore were calculated. The site
with the highest Sitescore was selected as catalytic site and induced fit docking (IFD) process was
performed on ligand or residue in the catalytic site [49].
4.4.4. Induced fit docking (IFD)
Molecular docking was carried out to characterize binding affinity and interactions between the most
active compounds and receptors. Induced Fit Docking (IFD) module in Maestro 11.4 was used for docking
process. Centroid of the residues was generated around the selected ligand or residues in the catalytic site
of receptor. After that, side chains were automatically trimmed based on B-factor; closest residues to the
ligand were refined within 3.4 Å of ligand pose in prime refinement. In order to identification accuracy of
the docking process, docking validation was performed with re-docking procedure by evicting inhibitor
complexed in the crystal structure of the receptor, before docking of synthesized compounds. Then, most
active compounds and were docked on the receptors by IFD methodology. Following the docking process,
the types of interactions and interacted residues for best-scored compound results were analyzed [50].
ASSOCIATED CONTENT
*Supporting Information
………………………………………. ……………………………… ………………………………
■ AUTHOR INFORMATION
Corresponding Authors
Tel.: +90 4422314423; fax: +90 4422360948; e-mail address: amenzek@atauni.edu.tr (A. Menzek).
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
20

This study was financed by TÜBİTAK - The Scientific and Technological Research Council of Turkey
(Project No: 113Z702) and realized in Atatürk University, Faculty of Science, Department of Chemistry.
We are grateful to both institutes for their supports.
5. REFERENCES
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25

Table 1. Human carbonic anhydrase I, and II (hCA I, and II) isoenzymes, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes
inhibition effects of novel benzyl(methyl)sulfane derivative bromophenols (2, 3, 8-11 and 13-22)
IC50 (nM)
r2
Ki (nM)
Compounds
r2
hCA II
AChE
r2
BChE
r2
hCA I
hCA II
AChE
BChE
AChE/BChE
hCA I
83.04
69.35
0.9734
0.9804
58.34
50.44
83.18
118.34
104.83
102.07
134.88
91.84
167.23
184.02
121.33
100.62
128.38
108.64
45.04
50.02
248.54
-
0.9812
0.9526
0.9495
0.9975
0.9729
0.9803
0.9645
0.9893
0.9574
0.9830
0.9599
0.9934
0.9655
0.9937
0.9749
0.9727
0.9714
-
2.04
1.63
8.24
9.31
5.12
2.04
13.84
6.53
13.10
10.62
7.14
5.03
12.22
18.04
2.74
3.08
-
0.9714
0.9893
0.9345
0.9811
0.9730
0.9872
0.9937
0.9457
0.9915
0.9693
0.9485
0.9384
0.9476
0.9845
0.9663
0.9305
-
1.84
8.14
14.05
14.95
6.04
9.63
20.64
8.02
11.53
10.77
8.63
9.39
11.03
25.17
3.73
4.18
-
0.9683
0.9937
0.9487
0.9940
0.9374
0.9798
0.9307
0.9748
0.9902
0.9711
0.9837
0.9928
0.9475
0.9742
0.9205
0.9848
-
89.37±11.45
64.12±15.18
116.07±35.88
157.13±29.76
146.11±30.55
103.67±25.13
208.26±43.76
111.43±17.37
186.36±76.23
234.68±46.76
168.34±28.09
103.64±20.64
147.04±35.85
148.34±54.76
53.75±12.54
57.43±10.16
273.61±76.86
-
67.05±14.73
54.76±8.04
1.53±0.23
0.84±0.12
5.64±1.04
6.33±2.01
2.74±0.90
1.99±0.18
9.02±1.05
4.52±1.01
10.64±2.64
7.43±1.54
5.24±0.95
3.58±0.86
8.54±1.76
14.63±3.06
1.05±0.32
1.32±0.43
-
0.93±0.20
3.73±1.03
11.54±1.88
10.40±2.43
4.26±0.93
5.95±1.04
13.65±2.64
5.94±0.99
7.91±1.76
8.64±1.55
6.53±0.93
6.63±1.88
8.35±1.40
18.53±5.06
2.04±0.63
2.75±0.84
-
1.64
0.22
0.48
0.60
0.64
0.33
1.51
0.76
1.34
0.86
0.80
0.54
1.02
0.78
0.51
0.48
-
2
3
105.83 0.9632
137.18 0.9738
123.63 0.9881
109.62 0.9748
174.43 0.9682
94.03±26.47
105.63±24.74
117.04±21.62
100.52±17.38
167.46±43.67
98.64±14.86
154.83±30.57
200.54±57.25
115.85±16.04
94.83±11.65
137.45±25.76
119.46±24.78
42.84±9.36
8
9
10
11
13
98.63
0.9744
14
173.84 0.9814
204.72 0.9602
148.93 0.9034
111.53 0.9738
150.28 0.9836
137.05 0.9508
15
16
17
18
19
20
58.16
61.73
0.9712
0.9814
21
53.75±12.58
229.08±55.14
-
22
AZA*
TACΨ
265.41 0.9954
-
-
32.20
0.9796
40.16
0.9711
16.37±5.10
23.40±3.62
0.70

*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 acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes
Table 2. Physically and pharmaceutically properties of the most active compounds
i
Compound aMW
bDHB cAHB dlogPo/w elogHERG flogBB gMDCK hlogKhsa % Hum. Oral Abs.
360.017
2
5.5
0.988
-3.682
-0.769 571.517
-0.503 1585.065 -0.414
-0.919 287.941 -0.575
-0.499
76.903
2
438.913
281.121
2
2
5.5
5.5
1.490
0.598
-3.551
-3.729
82.390
73.612
3
21
a Molecular weight.
b Number of hydrogen bond donors.
c Number of hydrogen bond acceptors.
d Octanol/water partition coefficient.
e IC50 value for blockage of HERG K+ channels.
f B/blood partition coefficient.
g Cell permeability in nm/sec.
h Binding to human serum albumin.
i Qualitative human oral absorption
Table 3. IFD Glide scores (kcal/mol) of the most active compounds in the catalytic sites of hCA I, and hCA II, AChE, and BChE
IFD Glide Score
Compounds
hCA I
hCA II
AChE
BChE
-
-
-
-
-
-6.625
2
3
-7.500
-
-8.528
-9.016
-
-8.830
-9.560
-
-
-
-
-
21
AZA*
TAC**
-9.579
-8.830
*Acetazolamide (AZA) was used as a standard inhibitor for human carbonic anhydrase isoenzymes I, and II (hCA I, and II).
**Tacrine (TAC) was used as a standard inhibitor for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes.
27

Highlight





A novel serie of some bromophenol derivatives was synthesised.
These bromophenols were firstly shynthesised.
They have been characterized by 1H and 13C NMR, HRMS and FTIR spectroscopies.
Their inhibition effects on hCA I, hCA II, AChE and BChE were determined.
The most active compounds were docked for these enzymes.
28

GRAPHICAL ABSTRACT
29