Sulfonamides incorporating ketene N,S-acetal bioisosteres as potent carbonic anhydrase and acetylcholinesterase inhibitors

Article abstract of DOI:10.1002/ardp.201900383

In this study, 15 novel compounds in a series of sulfonamide-based ketenes (7a–o) were synthesized and characterized using Fourier-transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry. All compounds were tested for their ability to inhibit the human carbonic anhydrase (hCA) isoforms I and II, and acetylcholinesterase (AChE). The halogen-appended compounds, 7g, 7o, and 7i, exhibited the highest hCA I/II and AChE inhibition, with the K_I values in the low nanomolar range (K_I = 9.01 ± 0.08, 7.41 ± 0.03, and 7.37 ± 0.31 nM, respectively), as compared with their corresponding parent 2-[2,2-dicyano-1-(phenylamino)vinylthio]-N-(4-sulfamoylphenyl)acetamide analogs 7a–o. Besides, derivatives 7c and 7e selectively inhibited the isoform hCA I, whereas compounds 7m and 7n selectively inhibited isoform hCA II. These findings indicated that all compounds can inhibit metabolic dysfunctions, such as edema, epilepsy, glaucoma, and Alzheimer's disease, by specifically targeting both the hCA isoforms and AChE expression. Herein, also the interactions between ligands and receptors were highlighted through in silico molecular docking studies. The molecular mechanics–generalized Born surface area method was utilized to compute the binding free energy and the energy contribution of the critical residues in the active site was estimated. All these results would help us to perfectly understand the relationship between activity and structural characteristics of derivatives and to further improve newly and highly effective analogs targeting hCA and AChE.

Full text of DOI:10.1002/ardp.201900383

Received: 30 December 2019
DOI: 10.1002/ardp.201900383
Revised: 28 February 2020
Accepted: 17 March 2020
|
|
F U L L P A P E R
Sulfonamides incorporating ketene N,S‐acetal bioisosteres as
potent carbonic anhydrase and acetylcholinesterase
inhibitors
1
2
3
4
Qëndresa Istrefi | Cüneyt Türkeş
| Mustafa Arslan | Yeliz Demir
|
1
5
6
Arleta R. Nixha | Şükrü Beydemir | Ömer İ. Küfrevioğlu
1
Department of Chemistry, Faculty of
Mathematical and Natural Sciences, University
Abstract
of Prishtina, Prishtina, Republic of Kosovo,
In this study, 15 novel compounds in a series of sulfonamide‐based ketenes (7a–o)
were synthesized and characterized using Fourier‐transform infrared spectroscopy,
nuclear magnetic resonance spectroscopy, and mass spectrometry. All compounds
were tested for their ability to inhibit the human carbonic anhydrase (hCA) isoforms
I and II, and acetylcholinesterase (AChE). The halogen‐appended compounds, 7g, 7o,
2
Department of Biochemistry, Faculty of
Pharmacy, Erzincan Binali Yıldırım University,
Erzincan, Turkey
3
Department of Chemistry, Faculty of Arts and
Sciences, Sakarya University, Sakarya, Turkey
4
Department of Pharmacy Services, Nihat
Delibalta Göle Vocational High School,
Ardahan University, Ardahan, Turkey
and 7i, exhibited the highest hCA I/II and AChE inhibition, with the K
I
values in the
5
low nanomolar range (K = 9.01 ± 0.08, 7.41 ± 0.03, and 7.37 ± 0.31 nM, respectively),
I
Department of Biochemistry, Faculty of
Pharmacy, Anadolu University, Eskişehir,
Turkey
as compared with their corresponding parent 2‐[2,2‐dicyano‐1‐(phenylamino)
vinylthio]‐N‐(4‐sulfamoylphenyl)acetamide analogs 7a–o. Besides, derivatives 7c
and 7e selectively inhibited the isoform hCA I, whereas compounds 7m and 7n
selectively inhibited isoform hCA II. These findings indicated that all compounds can
inhibit metabolic dysfunctions, such as edema, epilepsy, glaucoma, and Alzheimer's
disease, by specifically targeting both the hCA isoforms and AChE expression.
Herein, also the interactions between ligands and receptors were highlighted
through in silico molecular docking studies. The molecular mechanics–generalized
Born surface area method was utilized to compute the binding free energy and the
energy contribution of the critical residues in the active site was estimated. All these
results would help us to perfectly understand the relationship between activity and
structural characteristics of derivatives and to further improve newly and highly
effective analogs targeting hCA and AChE.
6
Department of Chemistry, Faculty of
Sciences, Atatürk University, Erzurum, Turkey
Correspondence
Cüneyt Türkeş, Department of Biochemistry,
Faculty of Pharmacy, Erzincan Binali Yıldırım
University, Erzincan 24100, Turkey.
Email: cuneyt.turkes@erzincan.edu.tr
Mustafa Arslan, Department of Chemistry,
Faculty of Arts and Sciences, Sakarya
University, Sakarya, 54187, Turkey.
Email: marslan@sakarya.edu.tr
Funding information
Research Fund of Erzincan Binali Yildirim
University, Grant/Award Number: FBA‐2017‐
501; Research Fund of Anadolu University,
Grant/Award Number: 1610S681; Research
Fund of Sakarya University,
K E Y W O R D S
Grant/Award Number: 2016‐02‐04‐018
acetylcholinesterase, carbonic anhydrase, ketene N,S‐acetal, molecular docking, sulfonamide
1
| INTRODUCTION
ketene N,S‐acetal is the biggest family, and it is utilized in cyclization
and multicomponent reactions for the synthesis of various fused
Electron‐donating and ‐accepting groups containing alkenes, which
are called push–pull alkenes, are very important in organic synthesis
and synthetic intermediates. Among the ketene acetal compounds,
heterocyclic systems and the concerned natural products. Sig-
nificantly, the reactivity and performance of ketene N,S‐acetals are
different and prominent as compared with ketene O,O‐, N,N‐, S,S‐, and
Abbreviations: AAZ, acetazolamide; ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer's disease; ADMET, adsorption, distribution, metabolism, excretion, and toxicity; CA, carbonic
anhydrase; MM–GBSA, molecular mechanics–generalized Born surface area; SAR, structure–activity relationship; TAC, tacrine.
|
Arch Pharm. 2020;e1900383.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201900383

2
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ISTREFI ET AL.
|
[
1–3]
[38]
N,O‐acetals.
Thiazolidinones that possess many biological activ-
hepatotoxicity reactions,
which severely affect therapeutic targets
[4,5]
ities were synthesized from ketene N,S‐acetals,
together with
induced by AChEIs during treatment.
[6]
thienopyrroles that have interesting biological properties. Some
CAs, are the metalloenzymes, have a crucial role in many me-
[
39]
[40]
nucleic acid bases and vitamins that are physiologically important are
tabolic processes like bone resorption,
calcification,
[43]
carbox-
[44]
[
7]
[41]
[42]
pyrimidine derivatives, and anilinopyrazolo[1,5‐a]pyrimidine ana-
logs were synthesized from S,S and N,S‐acetals, and they are used as
ylation,
photosynthesis,
pH regulation,
respiration,
and
[
45]
ureagenesis,
2
etc and catalyzing the CO hydration/dehydration
[8]
[46,47]
c‐Src kinase inhibitors for the treatment of acute ischemic stroke.
biochemical reaction a reversible.
Although human CAs belong
[48]
Furthermore, cyclic ketene N,S‐acetal derivatives, ralitoline and
to the α‐class, they are classified into seven different families : α‐,
etozolin, are used as drugs in the treatment of hypertension and
β‐, γ‐, δ‐, ζ‐, η‐, and θ‐CA. Human carbonic anhydrase I and II (hCA I/II)
[
9]
[49]
neurological diseases. Nithiazine was the first lead structure of
are cytosolic isoforms,
and irregular or overexpression of these
neonicotinoid insecticides and it is widely used as an insecticide in
enzymes is associated with some pathological disorders. hCA I iso-
[50]
[
10,11]
the world.
lized probes for nucleic acids in G‐quadruplex.
Sulfonamides are –SO NH– functional group‐containing com-
Thiazole orange and SYBR safe are the widely uti-
enzyme is correlated with retinal and cerebral edema,
whereas
[12,13]
[51]
[52]
hCA II isoform is associated with edema,
epilepsy,
and glau-
[
53]
2
coma.
Thus, hCAs are an important drug target enzyme, and
pounds, and they play a significant role in medicinal chemistry. The
derivatives of sulfonamide form an important class of sulfur‐
containing compounds having primary, secondary, and tertiary
several CA inhibitors are currently in clinical use in different phar-
maceutical forms. Therefore, the design and synthesis of novel in-
hibitors with high‐order isoform selectivity index is an excellent
approach to contribute to the field of toxicology with drug discovery
and development technology. In this context, CA inhibitors, mainly
sulfonamides, are the most targeted and researched ligands in drug
design because they are effectively zinc‐binders. The sulfonamides
are especially known as dual CA and AChE inhibitors. On the basis of
the above information, we focused on the design and synthesis of
15 new ketene N,S‐acetal sulfonamides (7a–o) displaying selectivity
as CA inhibitors. We appended differently substituted functional
groups, such as fluorine, chlorine, bromine, iodine, methyl, methoxy,
and nitro, as a tail to the heterocyclic scaffolds of compounds to
maximize the interaction of derivatives with the binding sites of hCA
isoforms I, II, and AChE, and researched their in vitro and in silico
biological effects on these enzymes. Furthermore, we extended this
study to understand the structure–activity relationships (SARs) of
this class of analogs.
[
14]
amides.
Sulfonamides resembling 4‐amino benzoic acid were dis-
covered in the early 1930s as the first synthetic antibacterial agent,
which was the first effective agent against most Gram‐positive and
many Gram‐negative organisms; also, it was employed for bacterial
[
15,16]
infections.
therapeutic agents to treat other diseases. Due to the SO
moiety, sulfamide and sulfamic acid are responsible for binding to the
Their applications have been further extended as
2
NH
2
[
17]
Zn(II) ion within carbonic anhydrase (CA; EC 4.2.1.1) binding site.
Sulfonamides have shown several interesting biological activities,
[
18]
including inhibition of CA (acetazolamide, AAZ),
anticancer ac-
[20]
[
19]
tivity (agent E7070),
antibacterial activity (sulfathiazole),
and
[
21]
HIV protease inhibitory activity.
Furthermore, these compounds
are a significant class of drugs with several types of pharmacological
agents having antitumor, CA inhibitory activity, antidiabetic, diuretic,
hypoglycemic, antithyroid, or protease inhibitory activity, as reported
[
22]
in pharmacological and clinical studies.
There are many clinically
used sulfonamide compounds, for instance, AAZ and brinzolamide as
[
23,24]
a CA inhibitor for the treatment of glaucoma and seizures,
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
[
25]
benzothiadiazine and chlorothiazide for diuretic activity,
hydro-
benzthiazide for
diazoxide for potassium
and polythiazide for the treatment of con-
gestive heart failure, hypertension, diabetes insipidus, renal tubular
[26]
flumethiazide for chronic vascular hypertension,
[27]
high blood pressure as well as edema,
[
28]
channel activation,
A new series of ketene N,S‐acetal sulfonamide compounds (7a–o)
was synthesized, and it is presented in Schemes
1 and 2.
[
29]
acidosis, edema, and the prevention of kidney stones.
Chloroacetylchloride‐substituted sulfonamide (3) was prepared from
sulfanilamide and acetylchloride in the presence of triethylamine
(TEA) in dimethylformamide (DMF) at 0°C for 5 hrs. The ketene N,S‐
acetal potassium salt was synthesized from isothiocyanate com-
pounds (4) and malononitrile with potassium hydroxide in DMF. The
targeted compounds were prepared from the ketene N,S‐acetal po-
tassium salt and acetylchloride‐substituted sulfonamide in DMF at
room temperature. The prepared analogs were characterized by
Alzheimer's disease (AD) is an irreversible neurodegenerative
[
30]
disease
that is known to be the most common type of de-
accounting for approximately 60–80% of all cases in the
Acetylcholinesterase (AChE) is an important type of cho-
[
31]
mentia,
[
32]
world.
[33]
[34]
linesterase
that is responsible for the regulation
and degrada-
[
35]
tion
of acetylcholine (ACh) in the central nervous system. An
increment in ACh expression in the metabolism, owing to the in-
1
hibition of the AChE enzyme, leads to the enhancement of the cog-
Fourier‐transform infrared spectroscopy, H NMR (nuclear magnetic
[36]
13
nitive abilities
functions in AD.
such as language skills, attention span, and memory
resonance), C NMR, and elemental analysis.
[
37]
1
Thus, the development of effective AChE in-
From the H NMR spectra, N–H hydrogen atoms' resonance is
hibitors (AChEIs) may be a critical approach for AD treatment and for
preventing the adverse reactions such as digestive tract and
observed at 10.60 and 10.80 ppm, sulfanilamide NH
2
hydrogen
atoms' resonance is observed at around 7.30 ppm, and resonance of

ISTREFI ET AL.
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SCHEME 1 General synthetic procedure for target compounds
SCHEME 2 Synthesis of ketene N,S‐acetal‐substituted sulfonamide derivatives 7a–o from the compounds 6a–o and the acetylchloride‐
substituted sulfonamide 3

4
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ISTREFI ET AL.
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CH
2
hydrogen atoms attached to a sulfur atom is observed at around
II, and AChE. The SARs of compounds were generated from the in-
13
4
.10 ppm. From the C NMR spectra, the resonance of carbonyl
I
hibition data (IC50 values, K values, and inhibition types) listed in the
carbon atoms is observed at around 167 ppm, and for an SCH car-
2
tables.
bon atom, it is observed at around 38 ppm. In the infrared spectra of
All the newly synthesized ketene N,S‐acetal sulfonamides, 7a–o,
inhibited cytosolic isoform hCA I, which is found everywhere and
known to be linked with edema, in a potent way, with IC50 values
compounds 7a–o, it was possible to observe the absorptions between
−
1
3
,200 and 3,350 cm , corresponding to NH and NH
2
peaks. Nitrile
and amide carbonyl peaks are seen around 2,220 and
I
ranging from 11.47 ± 0.27 to 31.20 ± 0.41 nM and K values ranging
−
1
[54]
1
,660–1,700 cm , respectively. As seen in the literature,
there
from 9.01 ± 0.08 to 107.43 ± 7.76 nM. Furthermore, all analogs
showed the best inhibition, compared to AAZ, which is a standard
reference drug, and they were found to be more effective inhibitors
are two peaks assigned to S═O, symmetric and asymmetric stretch-
−
1
ing, that are observed around 1,330 and 1,150 cm . All spectra and
elemental analyses support the structure of the synthesized
compounds.
in the fold between
475.55 ± 10.62 nM). Compound 7g (2‐[1‐(3‐bromophenylamino)‐2,2‐
dicyanovinylthio]‐N‐(4‐sulfamoylphenyl)acetamide) bearing 3‐
bromine showed the best inhibition (K 9.01 ± 0.08 nM). The re-
placement of the 3‐methoxy (7d, K 27.31 ± 0.32 nM), 3‐fluorine (7l,
16.64 ± 0.16 nM), and 3‐trifluoromethyl (7n, K 22.43 ± 0.50 nM)
4 and 52, as compared with AAZ (K
I
a
I
2
.2 | Biological evaluation
I
K
I
I
The newly synthesized 15 sulfonamides, 7a–o, as detailed above,
were screened against physiologically significant cytosolic isoforms
hCA I, II (associated with some complications like edema and glau-
coma, especially), and AChE (associated with AD) for their efficacy as
both CA inhibitors and AChE inhibitors by using the esterase and
Ellman's assay methods. The standard inhibitors, AAZ and tacrine
moieties with a chlorine atom (compound 7g) led to a reduction in the
inhibition. Furthermore, the displacement of the bromine from po-
sition 3 to positions 2 and 4 also decreased the inhibition (7f and 7h,
I
K s 107.43 ± 7.76 and 96.92 ± 7.19 nM, respectively). On the con-
trary, the presence of two chlorine atoms (compounds 7j,k, K
I
s
12.16 ± 0.10 and 21.04 ± 0.20 nM, respectively) produced an increase
in the inhibition when compared to both 7f and 7h derivatives. The
replacement of the 3‐bromine of compound 7g with a methyl, nitro,
chlorine, fluorine, and iodine groups (analogs 7b, 7e, 7i, 7m, and 7o,
(TAC), were used as a reference for hCA isoforms and AChE in the
tests, respectively. According to the data summarized in Tables 1–3,
it is revealed that all derivatives are potent inhibitors against hCA I,
TABLE 1 IC50 values of hCA isoforms
hCA I
hCA II
AChE
(I–II) and AChE with compounds 7a–o, AAZ,
2
2
2
Compound ID
IC50 (nM)
R
IC50 (nM)
R
IC50 (nM)
R
and TAC
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
23.75 ± 0.29
25.45 ± 0.43
17.17 ± 0.10
31.20 ± 0.41
18.95 ± 0.28
16.29 ± 0.07
11.47 ± 0.27
15.79 ± 0.40
11.90 ± 0.05
15.01 ± 0.53
22.76 ± 0.12
25.96 ± 0.18
21.15 ± 0.21
15.22 ± 0.34
12.65 ± 0.20
223.90 ± 3.91
–
0.9983
0.9974
0.9996
0.9991
0.9989
0.9998
0.9942
0.9969
0.9999
0.9947
0.9998
0.9996
0.9992
0.9973
0.9979
0.9988
–
12.81 ± 0.05
14.24 ± 0.14
17.74 ± 0.27
19.88 ± 0.27
15.09 ± 0.16
16.94 ± 0.22
8.44 ± 0.06
15.12 ± 0.11
13.90 ± 0.18
10.86 ± 0.05
18.59 ± 0.19
14.55 ± 0.21
14.60 ± 0.16
8.83 ± 0.14
10.07 ± 0.25
96.67 ± 0.55
–
0.9998
0.9992
0.9987
0.9985
0.9991
0.9971
0.9997
0.9991
0.9978
0.9998
0.9994
0.9973
0.9992
0.9982
0.9968
0.9999
–
24.37 ± 0.38
19.32 ± 0.58
9.77 ± 0.13
15.59 ± 0.34
9.39 ± 0.23
15.76 ± 0.33
13.96 ± 0.27
16.36 ± 0.08
6.81 ± 0.11
9.34 ± 0.12
15.88 ± 0.18
15.16 ± 0.16
12.73 ± 0.11
14.76 ± 0.23
10.54 ± 0.08
–
0.9986
0.9967
0.9983
0.9942
0.9963
0.9959
0.9951
0.9999
0.9988
0.9977
0.9981
0.9986
0.9993
0.9987
0.9995
–
g
h
i
j
k
l
m
n
o
AAZ
TAC
430.10 ± 1.45
0.9998
Abbreviations: AAZ, acetazolamide; AChE, acetylcholinesterase; hCA, human carbonic anhydrase;
TAC, tacrine.

ISTREFI ET AL.
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|
TABLE 2 Inhibition data of hCA isoforms
hCA I
(nM)
hCA II
(nM)
AChE
(nM)
(I–II) and AChE with compounds 7a–o, AAZ,
2
2
2
and TAC
Compound ID
K
I
R
K
I
R
K
I
R
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
22.60 ± 0.22
23.42 ± 0.23
48.28 ± 1.40
27.31 ± 0.32
105.94 ± 7.12
107.43 ± 7.76
9.01 ± 0.08
96.92 ± 7.19
20.12 ± 0.48
12.16 ± 0.10
21.04 ± 0.20
16.64 ± 0.16
21.41 ± 0.46
22.43 ± 0.50
16.30 ± 0.44
475.55 ± 10.62
–
0.9999
0.9999
0.9999
0.9998
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9998
0.9989
–
24.14 ± 0.57
10.53 ± 0.17
13.07 ± 0.19
17.59 ± 0.10
19.46 ± 0.16
33.58 ± 0.62
12.58 ± 0.22
54.65 ± 0.87
32.35 ± 0.67
24.60 ± 0.31
16.09 ± 0.06
17.84 ± 0.12
56.75 ± 0.20
49.92 ± 2.42
7.41 ± 0.03
97.73 ± 1.62
–
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9999
0.9993
–
22.18 ± 0.52
22.62 ± 1.07
12.70 ± 0.66
23.68 ± 1.56
22.14 ± 2.47
10.84 ± 0.16
17.40 ± 0.92
15.07 ± 0.35
7.37 ± 0.31
12.23 ± 0.93
18.94 ± 1.50
29.69 ± 3.10
15.23 ± 0.60
11.16 ± 0.54
32.89 ± 5.14
–
0.9989
0.9995
0.9994
0.9993
0.9985
0.9996
0.9993
0.9989
0.9995
0.9988
0.9984
0.9985
0.9996
0.9993
0.9982
–
g
h
i
j
k
l
m
n
o
AAZ
TAC
159.64 ± 0.87
0.9999
Abbreviations: AAZ, acetazolamide; AChE, acetylcholinesterase; hCA, human carbonic anhydrase;
TAC, tacrine.
TABLE 3 Selectivity index values for K
compounds 7a–o
I
constants of the
I
K s ranging between 16.30 ± 0.44 and 105.94 ± 7.12 nM) also induced
a decrease in the inhibition. Thus, in the ketene N,S‐acetal sulfona-
mides, the inhibitory activity is related to the presence of sub-
stituents on the aryl ring; as a matter of fact, compound 7f displayed
the lowest inhibition in this new series. The best derivative was 7g,
bearing a 3‐bromine group. The activities for the substituted ketene
N,S‐acetal‐based sulfonamides (7a–o) against hCA I were reduced in
the following order: 7g > 7i > 7o > 7j > 7n > 7h > 7f > 7c > 7e > 7m > 7
k > 7a > 7b > 7l > 7d (Tables 1–3).
K
I
K
I
(hCA II/
K
I
(AAZ/
K
I
(AAZ/
(TAC/
AChE)
Compound ID hCA I)
hCA I)
21.04
20.31
9.85
hCA II)
4.05
9.28
7.48
5.56
5.02
2.91
7.77
1.79
3.02
3.97
6.07
5.48
1.72
1.96
13.19
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
1.07
0.45
0.27
0.64
0.18
0.31
1.40
0.56
1.61
2.02
0.76
1.07
2.65
2.23
0.45
7.20
7.06
12.57
6.74
17.41
4.49
The isoform hCA II, being linked with glaucoma, was strongly
inhibited by all the newly synthesized ketene N,S‐acetal sulfona-
7.21
4.43
14.73
9.17
mides, 7a–o, with IC50 and K
range (IC50 ranging from 8.44 ± 0.06 to 19.88 ± 0.27 nM and K
ranging from 7.41 ± 0.03 to 56.75 ± 0.20 nM). These 15 analogs
7a–o) indicated a better inhibition capacity than standard drug
AAZ (K 97.73 ± 1.62 nM). Compound 7o (2‐[2,2‐dicyano‐1‐(4‐
iodophenylamino)vinylthio]‐N‐(4‐sulfamoylphenyl)acetamide) con-
taining 4‐iodine (K 7.41 ± 0.03 nM) was found to have the most
potent inhibition profile, whereas derivative 7m containing
‐fluorine (K 56.75 ± 0.20 nM) was found to have the weakest in-
I
values spanning in the low nanomolar
g
h
i
52.78
4.91
I
10.59
21.66
13.05
8.43
(
23.64
39.11
22.60
28.58
22.21
21.20
29.17
I
j
k
l
I
5.38
4
I
m
n
o
10.48
14.30
4.85
hibition profile against physiological isoform hCA II. The comparison
of inhibitory activity of these sulfonamides (7o and 7m) revealed
that the replacement of the 4‐iodine of compound 7o by a fluorine
group of analog 7m caused an approximately 7.5‐fold reduction in
the inhibition. On the contrary, the displacement of the fluorine
Abbreviations: AAZ, acetazolamide; AChE, acetylcholinesterase; hCA,
human carbonic anhydrase; TAC, tacrine.

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ISTREFI ET AL.
|
from position 4 to position 3 repristinated the inhibition (compound
l, K 17.84 ± 0.12 nM). As a general trend, the replacement of the 4‐
iodine atom of compound 7o by other groups, such as methyl (7b, K
(7a–o), the compounds 7i, 7f, and 7n containing 4‐fluorine,
2‐bromine, and 3‐trifluoromethyl groups were found to be the
most effective AChE inhibitors amongst their respective groups of
derivatives. In this respect, the order of the compound inhibitory
7
I
I
I
1
0.53 ± 0.17 nM), nitro (7e, K
4.65 ± 0.87 nM), chlorine (7i, K
56.75 ± 0.20 nM), led to a decline in the inhibition. However, the
presence of two chlorine atoms (compounds 7j and 7k,
4.60 ± 0.31 and 16.09 ± 0.06 nM, respectively) caused a reduction
in inhibition when compared with 7o analog. The replacement of the
I
19.46 ± 0.16 nM), bromine (7h, K
5
I
32.35 ± 0.67 nM), and fluorine (7m,
I
strength of K constants is as follows: 7i > 7f > 7n > 7j > 7c > 7h > 7
K
I
m > 7g > 7k > 7e > 7a > 7b > 7d > 7l > 7o (Tables 1–3).
I
K s
2
2.3 | In silico studies
2.3.1 | ADMET study
4
1
‐iodine of compound 7o by
3.07 ± 0.19 nM) reduced the inhibition level by 2‐fold; also, the
33.58 ± 0.62 nM) con-
a 2‐methoxy group (7c, K
I
replacement by a 2‐bromine atom (7f, K
I
tributed an approximately 4.5‐fold reduction in inhibition. In gen-
eral, it was found that the ketene N,S‐acetal sulfonamides having
the functional groups of substituents on the aryl ring were found to
be potent inhibitors of isoform hCA II. Concerning the effect of
substitution of the phenylamino moiety (7a–o), the hCA II inhibitory
activities were reduced in the following order: 7o > 7b > 7g > 7c >
The ADMET properties (adsorption, distribution, metabolism, ex-
cretion, and toxicity) and some pharmacokinetic parameters of the
new synthesized ketene N,S‐acetal sulfonamide derivatives (7a–o)
were estimated by using the QikProp module in Maestro. The overall
predicted values are summarized in Table 4. The molecular weights
(MWs 413.47–539.37), total solvent‐accessible surface areas (SASAs
7
k > 7d > 7l > 7e > 7a > 7j > 7i > 7f > 7n > 7h > 7m. Interestingly, the
analogs bearing methoxy and nitro groups (7c and 7e, S s 3.69 and,
.44, respectively) on the aryl ring exhibited the highest selectivity
index for hCA I, whereas the derivatives containing fluorine and
trifluoromethyl moiety (7m and 7n, S s 2.65 and 2.23, respectively)
666.60–752.60),
and
total
solvent‐accessible
volumes
I
(1,196.43–1,208.88) of the compounds (7a–o) have been determined
to be in the permissible values. The logP values (QPlogPw and
QPlogPo/w), which indicate the hydrophilicity/lipophilicity of the
analogs (7a–o), are in the acceptable range. All the compounds (7a–o)
have displayed good skin permeability ranges (QPlogKps ranging
from −4.43 to −6.81) and the van der Waals surface area values
(PSAs ranging between 135.74 and 199.67). QPlogS, which is an
aqueous solubility descriptor, was found in the permissible values,
compared with standard ranges. Brain/blood partition coefficient
(QPlogBB) values range from −2.73 to −3.87, which indicates that
these compounds (7a–o) have a moderate capability. Human serum
albumin‐binding coefficient (QPlogKhsa ranging from −0.32 to −0.68)
5
I
displayed a remarkable selectivity profile for hCA II, compared with
the other substituents in this study (Tables 1–3).
All of the synthesized new series of ketene N,S‐acetal sulfona-
mides (7a–o) displayed a good inhibitory activity against AChE en-
zyme (linked with AD), with IC50 values ranging between 6.81 ± 0.11
and 24.37 ± 0.38 nM and K
2.89 ± 5.14 nM. Here, analog 7i (2‐[1‐(4‐chlorophenylamino)‐2,2‐
dicyanovinylthio]‐N‐(4‐sulfamoylphenyl)acetamide) containing 4‐
chlorine exhibited the highest inhibition with constant of
.37 ± 0.31 nM, almost 21‐fold stronger than the standard drug (TAC,
I
constants ranging from 7.37 ± 0.31 to
3
+
K
I
values and IC50 value for the blockage of HERG K channels (QPlo-
7
gHERG ranging from −5.34 to −6.76) values are in the acceptable
range (−1.5 to 1.5 and less than −5, respectively) for these target
derivatives (7a–o). The estimated values of human oral absorption
(HOA) are between 16.70% and 64.97%, indicating that all com-
pounds (7a–o, except 7e) are in an acceptable range. In summary, the
ADMET results revealed that the synthesized active analogs (7a–o)
possess the drug‐likeness criteria conformed by both Jorgensen's
K
I
159.64 ± 0.87 nM). Herein, the different substituents on the aryl
ring (7a–o) played a crucial role in the AChE inhibition activity. The
replacement of chlorine by bromine, fluorine, and iodine at the same
position of aryl ring decreased enzyme inhibition in such a manner
that K
values: 15.07 ± 0.35, 15.23 ± 0.60, and 32.89 ± 5.14 nM, respectively.
However, the presence of bromine at position (7f,
0.84 ± 0.16 nM) increased antienzyme activity than 4‐substituted
compound 7h. Compound 7n possessing 3‐trifluoromethyl moiety
also exhibited a high inhibition activity (K 11.16 ± 0.54 nM). Also,
I
constants for compounds 7h, 7m, and 7o have the following
[
55]
[56]
2
K
I
rule of three
and Lipinski's rule of five.
These compounds can
1
be regarded as promising lead compounds for designing more potent
hCA I, II, and AChE inhibitors that may be novel drug candidates.
I
compounds 7j and 7k possessing two Cl groups at 2‐, 4‐ and 3‐, 5‐
positions of aryl ring showed a lower inhibition profile than analog 7i
2.3.2 | Molecular docking study
(
7j and 7k, K
thermore, derivatives 7b and 7e possessing methyl and nitro groups
at position 3 displayed a moderate similar activity (K s 22.62 ± 1.07
I
s 12.23 ± 0.93 and 18.94 ± 1.50 nM, respectively). Fur-
To describe the trends determined for the observed relative potency
and selectivity of our designed and synthesized new compounds
(7a–o), docking and molecular mechanics–generalized Born surface
area (MM–GBSA)‐based refinements studies were performed for the
selected analogs as representative of the derivatives. Before mole-
cular docking, key residues in the active sites and the noncovalent
contacts between the receptor–ligand complexes were investigated
I
and 22.14 ± 2.47 nM, respectively). Interestingly, the transposal of
the 2‐methoxy group from the aryl ring of compound 7c to position 3
to give isomeric 7d led to about a twofold decrease in the inhibition
activity (K
I
s 12.70 ± 0.66 and 23.68 ± 1.56 nM, respectively). Out of
the newly synthesized ketene N,S‐acetal sulfonamide derivatives

ISTREFI ET AL.
7 of 16
|

8
of 16
ISTREFI ET AL.
|
by the Protein Contacts Atlas (http://www.mrc‐lmb.cam.ac.uk/pca).
The X‐ray crystal structures of 5E2M (for hCA I) and 4M0E (for
AChE) were present in the form of a homodimer chain; hence, their
chain A was chosen for in silico studies. V14 (PubChem CID
thioacetamide skeleton as H‐bond acceptor formed two hydrogen
bonds with Asn62 and Gln92.
In fact, a first look at the active sites of hCA I and hCA II, we can
obtain some useful information. The main difference among the
isoforms is the size of the active sites. The smaller binding site of hCA
I can explain the lower activity of the compound 7o on this isoform.
Second prominent data, the structural difference between hCA I and
the hCA II is residues, which is His64 in hCA I, whereas Gly92 in hCA
II. As in most of the poses of the docked analogs used in this study,
there is an interaction between the ligand and His64, and the Gly/His
residues probably lead to the ligands' higher activity toward hCA I.
1YL, which is previously reported as the native ligand, and analog
73774785, C16
23 3 2 5 2
H F N O S , 3‐(cyclooctylamino)‐2,5,6‐trifluoro‐4‐
[(2‐hydroxyethyl)sulfonyl]benzenesulfonamide), FK8 (PubChem CID
12136,
C H
8 9
2
NO ,
benzylcarbamate), and 1YL (PubChem CID
11425923, C18
14 3
H O , (1R)‐1,6‐dimethyl‐1,2‐dihydronaphtho[1,2‐g]
[1]benzofuran‐10,11‐dione) were the cocrystallized ligands with
5E2M, 6H29, and 4M0E, respectively. The molecular docking pro-
tocol for these proteins was validated by extracting the bound li-
gands (V14, FK8, and 1YL) from the receptors and again redocking
[
57]
them on the same sites.
and root‐mean‐square deviation values were computed to be 1.14,
.99, and 0.10 Å, respectively. The most active compounds (7a–o),
The docking poses were superimposed,
I
7i (K 7.37 ± 0.31 nM), which is the most active analog of the series,
were analyzed in terms of interactions with AChE. Unsurprisingly,
the docking modes into 4M0E determined for the two ligands were
very similar to that estimated for their other analogs, as both 1YL
and compound 7i displayed the same interactions with Trp286, and
the docking scores were −10.36 and −7.42, respectively. Within
4M0E active site, the chlorine atom on the aryl ring and the amino
moiety of the benzenesulfonamide group of compound 7i showed
potential two H‐bonds with Tyr133 and Ser293, respectively,
whereas 1YL established an H‐bond by the carbonyl group with
Phe295. A hydrophobic interaction was monitored between deriva-
tive 7i and Tyr72, Trp86, Tyr124, Leu289, Val294, Phe295, Phe297,
Tyr337, Phe338, Tyr341, and Ile451. Apart from these, derivative 7i
also exhibited a polar interaction with Asn87 and Ser125. The
docking score of compound 7n was −9.74, which was better than
compound 7i because compound 7n formed more interactions with
residues and also formed an H‐bond with Ser293 (Figure 3).
Furthermore, Prime MM–GBSA (ΔG Bind) ranges were found as
−15.40 and −47.43 kcal/mol (compounds 7h and 7f) for 5E2M,
−13.45 and −35.29 kcal/mol (analogs 7k and 7b) for 6H29, and −7.86
and −65.44 kcal/mol (derivatives 7i and 7g) for 4M0E. Among the
analogs, 7f (with 5E2M), 7b (with 6H29), and 7g (with 4M0E) were
determined to exhibit more binding‐free energy, −47.43, −35.29, and
−65.44 kcal/mol, respectively; hence, they are more effective than
other compounds.
0
against hCA I, hCA II, and AChE, were docked into the binding sites of
the receptors complexed with the V14 (PDB iD 5E2M), FK8 (PDB iD
6
H29), and 1YL (PDB iD 4M0E).
According to the literature, the native ligand (V14) displays three
major interactions, like H‐bond interaction with His67, Thr199, and
Pro201, π–π stacking with His67, and His94 and a salt bridge be-
tween Leu198 and the Zinc ion, and the docking score was −10.12 in
the catalytic domain of 5E2M. For the most active compound (7g, K
I
9
.01 ± 0.08 nM), the docking score (−5.81) was found low for the
cocrystallized ligand. Compound 7g exhibited H‐bond interactions
with Trp5, Val62, His64, His67, and Pro201. It also displayed π–π
stacking with His64 and His 200, and it made both π–cation inter-
action and salt bridge with Zn metal (Figure 1). But according to the
docking scores, derivative 7f displayed the best docking score (−7.90)
among the analogs. Compound 7f made H‐bond with Trp5, His64,
Gln92, and Pro201. It also showed π–π stacking with His64, Phe91,
and His 200.
The 6H29 complexed with FK8 shows two H‐bonds with Asn67.
Apart from this, FK8 showed hydrophobic interactions with Ala65,
Val121, Leu141, Val143, Leu198, Val207, and Trp209. The docking
score was −4.40, which was surprisingly low, as it was expected to
range between −5.00 and −6.00. It may be possible that interaction
with Zinc ion was not monitored in the docked pose, so the docking
score might be lower. Compound 7o, which is the most inhibitory
activity compound (K
I
7.41 ± 0.03), revealed interaction modes be-
3 | CONCLUSION
tween the 6H29 and ligands, which further deepened the under-
standing of SAR. As shown in Figure 2, compound 7o formed H‐bonds
with the active site residues; for example, the carbonyl group of the
acetamide skeleton as H‐bond acceptor formed an H‐bond with
Gln92, the aromatic portion of the benzenesulfonamide core ex-
hibited π–π stacking with His94, and also the oxygen atom of the
sulfonamide formed metal coordination with the zinc metal, as re-
In summary, this study reports the design, synthesis, and char-
acterization of 15 new ketene N,S‐acetal benzenesulfonamides and
their evaluation as inhibitors of hCA isoforms I, II, and AChE. In
general, all the synthesized derivatives inhibited hCA I, II, and AChE,
which were found in the low nanomolar range. Furthermore, all
analogs showed a better inhibition profile than the standard drugs
AAZ and TAC for hCA isoforms and AChE. Compounds 7c and 7e
selectively inhibited edema‐associated isoform hCA I, whereas de-
rivatives 7m and 7n selectively inhibited edema, epilepsy, and
glaucoma‐linked isoform hCA II. It is further found that halogen‐
appended analogs, 7g (for hCA I), 7o (for hCA II), and 7i (for AChE),
are stronger inhibitors of tested enzymes as compared to their
[
58]
ported by Ahmed et al.
pound 7e exhibited the best docking score (−5.82) among the
5 derivatives used for this investigation. Compound 7e formed four
However, based on the dock score, com-
1
H‐bonds with the active site residues, that is, the amino moiety of the
sulfonamide skeleton as H‐bond donor formed two H‐bonds with
Phe70 and Asp72, the nitrogen atoms at position
2 of the

ISTREFI ET AL.
9 of 16
|
FIGURE 1 Interaction of the ligands with the key amino acids within the binding site of hCA I (human carbonic anhydrase; PDB ID 5E2M). (a)
Docking pose of the native ligand V14 (3‐(cyclooctylamino)‐2,5,6‐trifluoro‐4‐[(2‐hydroxyethyl)sulfonyl]benzenesulfonamide). (b) Docking pose
of compound 7g (2‐[1‐(3‐bromophenylamino)‐2,2‐dicyanovinylthio]‐N‐(4‐sulfamoylphenyl)acetamide)
corresponding parent 2‐[2,2‐dicyano‐1‐(phenylamino)vinylthio]‐N‐(4‐
sulfamoylphenyl)acetamide derivatives 7a–o. In this context, mole-
cular docking and MM–GBSA studies of compounds 7g, 7o, and 7i in
the binding sites of hCA I, II, and AChE provide insights into the details
of the binding interactions producing the inhibition profiles. Also, the
SAR study helped us gain an understanding of the relationship be-
tween structure and activity. So, it may be concluded that these shreds
of evidence may prove precious in the design and synthesis of more
selective and more effective CA and AChE inhibitors.
4 | EXPERIMENTAL
4.1 | Chemistry
4.1.1 | General
All starting materials and reagents were commercially available and
used without further purification except where indicated. Melting
points were determined on a Yanagimoto micro‐melting point

10 of 16
ISTREFI ET AL.
|
FIGURE 2 Interaction of the ligands with the key amino acids within the binding site of hCA II (human carbonic anhydrase; PDB ID 6H29).
a) Docking pose of the native ligand FK8 (benzylcarbamate). (b) Docking pose of compound 7o (2‐[2,2‐dicyano‐1‐(4‐iodophenylamino)
(
vinylthio]‐N‐(4‐sulfamoylphenyl)acetamide)
apparatus and were uncorrected. Infrared (IR) spectra were mea-
4.1.2 | Synthesis of acetylchloride‐substituted
sulfonamide 3
1
sured on a Shimadzu Prestige‐21 (200 VCE) spectrometer. H and
1
3
C NMR spectra were recorded on a Varian Infinity Plus 300 spec-
1
13
trometer at 75 MHz. H and C chemical shifts are referenced to the
internal deuterated solvent. Chemical shift values (δ) are given in
ppm. The elemental analysis was carried out with a Leco CHNS‐932
instrument. All chemicals were purchased from Merck and Sigma‐
Aldrich.
A solution of sulfanilamide (1) (2 mmol) in DMF (5 ml) and TEA
(2 mmol) was cooled down to 0°C, and then acetylchloride (2)
(3 mmol) was added dropwise while stirring. The whole reaction was
maintained at 0°C in an ice bath for 5 hr and then poured into a
beaker containing ice/water mixture with a few drops of hydrochloric
acid. The solid product was washed with water and collected by fil-
tration, dried, and crystallized from ethanol.
The InChI keys of the investigated compounds are provided as
Supporting Information.

ISTREFI ET AL.
11 of 16
|
FIGURE 3 Interaction of the ligands with the key amino acids within the binding site of AChE (acetylcholinesterase; PDB ID 4M0E). (a)
Docking pose of the native ligand 1YL ((1R)‐1,6‐dimethyl‐1,2‐dihydronaphtho[1,2‐g][1]benzofuran‐10,11‐dione)). (b) Docking pose of compound
7i (2‐[1‐(4‐chlorophenylamino)‐2,2‐dicyanovinylthio]‐N‐(4‐sulfamoylphenyl)acetamide)
4
.1.3 | General procedure for the synthesis of
compound 6
4.1.4 | General procedure for the synthesis of
ketene N,S‐acetal‐substituted sulfonamide
compounds 7a–o
A solution of isocyanate compound 4 (2 mmol) in DMF (5 ml) was
mixed at room temperature and then malononitrile (2 mmol) and
potassium hydroxide (1.3 mmol) were added while stirring. The whole
reaction mixture was mixed at room temperature overnight.
Compound 3 (2 mmol) was added to compound 6 (2 mmol) and
was stirred overnight at room temperature. Ice‐cold water was
added into the reaction mixture with a few drops of hydrochloric

12 of 16
ISTREFI ET AL.
|
acid. The solid product formed was washed with water and col-
lected by filtration, dried, and crystallized from hexane/acetone.
139.54, 130.7, 128, 127.5, 119.6, 117, 114.3, 113.2, 109.7, 55.9, and
38 (Figure S12). Anal. calcd. for C19
5.79; O, 14.43; S, 14.46. Found: C, 51.52; H, 3.89; N, 15.85; O,
14.49; S, 14.48.
17 5 4 2
H N O S : C, 51.46; H, 3.86; N,
1
2
‐[2,2‐Dicyano‐1‐(phenylamino)vinylthio)]‐N‐(4‐sulfamoylphenyl)-
acetamide (7a)
Yield 86.6%, m.p. 200°C; IR (cm ): 3,349 (–NH
−
1
2
), 3,202 (–NH), 3,133
2‐[2,2‐Dicyano‐1‐(4‐nitrophenylamino)vinylthio]‐N‐(4‐
(
═C–H, aromatic), 2,988 (–CH
2
, aliphatic), 2,207 (CN), 1,660 (C═O),
sulfamoylphenyl)acetamide (7e)
1
−1
1
,621 (C═C), 1,320, and 1,152 (S═O; Figure S1); H NMR (300 MHz,
2
Yield 86.5%, m.p. 215°C; IR (cm ): 3,300 (–NH ), 3,200 (–NH), 3,077
dimethyl sulfoxide [DMSO]‐d
6
, ppm): 10.6–10.8 (2H, s, –NH), 7.76
(═C–H, aromatic), 2,948, (–CH
1,614 (C═C), 1,496 (NO
NMR (300 MHz, DMSO‐d
═CH, J = 8.7 Hz), 7.65 (2H, d, ═CH, J = 8.8 Hz), 7.42 (2H, d, ═CH,
J = 8.4 Hz), 7.40 (2H, d, ═CH, J = 8.3 Hz), 7.20 (2H, s, –NH ), and 4.1
, ppm): 166.4,
152.5, 149.5, 141.8, 139.58, 131.9, 128, 127.5, 125.7, 125.5, 122.98,
19.6, and 39.3 (Figure S15). Anal. calcd. for C18 : C, 47.16;
2
, aliphatic), 2,225 (CN), 1,653 (C═O),
1
(2H, d, ═CH, J = 8.6 Hz), 7.68 (2H, d, ═CH, J = 8.7 Hz), 7.2–7.5 (5H, m,
2
), 1,309, and 1,151 (S═O; Figure S13);
H
1
3
═
CH), 7.30 (2H, s, –NH2), and 4.1 (2H, s, –CH
75 MHz, DMSO‐d , ppm): 169.9, 166.6, 141.9, 139.6, 138.9, 129.9,
27.5, 127.3, 124.2, 119.6, 117, 114.3, and 38 (Figure S3). Anal. calcd.
: C, 52.29; H, 3.66; N, 16.94; O, 11.61; S, 15.51.
Found: C, 52.35; H, 3.76; N, 16.99; O, 11.70; S, 15.56.
2
; Figure S2); C NMR
6
, ppm): 10.6–11.2 (2H, s, –NH), 7.74 (2H, d,
(
6
1
2
1
3
for C18
15
H N
5 3
O S
2
(2H, s, –CH
2
; Figure S14); C NMR (75 MHz, DMSO‐d
6
1
14 6 5 2
H N O S
2
‐[1‐(p‐Toluidino)‐2,2‐dicyanovinylthio]‐N‐(4‐sulfamoylphenyl)-
acetamide (7b)
Yield 66.5%, m.p. 206°C; IR (cm ): 3,305 (–NH
H, 3.08; N, 18.33; O, 17.45; S, 13.99. Found: C, 47.19; H, 3.12; N,
18.39; O, 17.48; S, 14.05.
−
1
2
), 3,203 (–NH), 3,133
(
═C–H, aromatic), 2,988 (–CH
2
, aliphatic), 2,220 (CN), 1,665 (C═O),
2‐[1‐(2‐Bromophenylamino)‐2,2‐dicyanovinylthio]‐N‐(4‐
sulfamoylphenyl)acetamide (7f)
1
1
,626 (C═C), 1,320, and 1,153 (S═O; Figure S4); H NMR (300 MHz,
−1
DMSO‐d
6
,
ppm): 10.6–10.8 (2H, s, –NH), 7.77 (2H, d, ═CH,
2
Yield 76.7%, m.p. 205°C; IR (cm ): 3,405 (–NH ), 3,200 (–NH), 3,127
J = 8.6 Hz), 7.69 (2H, d, ═CH, J = 8.7 Hz), 7.0 (2H, d, ═CH, J = 8.4 Hz),
(═C–H, aromatic), 2,984, (–CH
2
, aliphatic), 2,220 (CN), 1,665 (C═O),
1
7.42 (2H, d, ═CH, J = 8.3 Hz), 7.30 (2H, s, –NH
2
), 4.1 (2H, s, –CH
2
),
1,600 (C═C), 1,334, and 1,162 (S═O; Figure S16); H NMR (300 MHz,
1
3
and 2.3 (3H, s, –CH
3
; Figure S5); C NMR (75 MHz, DMSO‐d , ppm):
6
DMSO‐d
6
, ppm): 10.6–10.8 (2H, s, –NH), 7.81 (2H, d, ═CH,
1
70.1, 166.6, 141.9, 139.54, 136.6, 136.4, 130.3, 127.5, 127.4, 124.1,
23.3, 119.6, 37.9, and 21.3 (Figure S6). Anal. calcd. for
: C, 52.38; H, 4.01; N, 16.38; O, 11.23; S, 15.00. Found:
C, 52.45; H, 4.11; N, 16.44; O, 11.29; S, 15.08.
J = 8.6 Hz), 7.76 (2H, d, ═CH, J = 8.7 Hz), 7.2–7.6 (4H, m, ═CH), 7.20
1
3
1
(2H, s, –NH
DMSO‐d , ppm): 170.8, 167.1, 141.8, 139.7, 137.5, 134.2, 133.8,
130.2, 129.9, 128.8, 128.5, 125.7, 127.5, 121.98, 119.6, and 38.2
Figure S18). Anal. calcd. for C18 14BrN : C, 43.91; H, 2.87; Br,
2
), and 4.1 (2H, s, –CH2; Figure S17); C NMR (75 MHz,
C
19
H N
17 5
O S
3 2
6
(
H
5 3 2
O S
2
‐[2,2‐Dicyano‐1‐(2‐methoxyphenylamino)vinylthio]‐N‐(4‐
16.23; N, 14.22; O, 9.75; S, 13.02. Found: C, 43.98; H, 2.89; Br, 16.27;
N, 14.26; O, 9.79; S, 13.12.
sulfamoylphenyl)acetamide (7c)
−
1
2
Yield 69.2%, m.p. 217°C; IR (cm ): 3,308 (–NH ), 3,205 (–NH), 3,133
(
═C–H, aromatic), 2,981 (–CH
2
, aliphatic), 2,214 (CN), 1,670 (C═O),
2‐[1‐(3‐Bromophenylamino)‐2,2‐dicyanovinylthio]‐N‐(4‐
sulfamoylphenyl)acetamide (7g)
1
1
,622 (C═C), 1,343, and 1,155 (S═O; Figure S7); H NMR (300 MHz,
−1
DMSO‐d
6
,
ppm): 10.5–10.7 (2H, s, –NH), 7.77 (2H, d, ═CH,
2
Yield 73.7%, m.p. 215°C; IR (cm ): 3,361 (–NH ), 3,271 (–NH), 3,118
J = 8.9 Hz), 7.72 (2H, d, ═CH, J = 8.8 Hz), 6.9–7.4 (4H, m, ═CH), 7.30
(═C–H, aromatic), 2,988, (–CH
2
, aliphatic), 2,213 (CN), 1,705 (C═O),
1
3
1
(
2H, s, –NH
NMR (75 MHz, DMSO‐d
30.3, 127.5, 126.15, 119.6, 117, 116.3, 114.3, 113.2, 109.7, 55.3,
and 37.95 (Figure S9). Anal. calcd. for C19 : C, 51.46; H,
.86; N, 15.79; O, 14.43; S, 14.46. Found: C, 51.49; H, 3.89; N, 15.84;
O, 14,49; S, 14.51.
2
), 4.2 (2H, s, –CH
2
), and 3.8 (3H, s, –CH
3
; Figure S8);
C
1,658 (C═C), 1,326, and 1,153 (S═O; Figure S19); H NMR (300 MHz,
6
, ppm): 169.8, 166.6, 160.4, 142.3, 140.7,
DMSO‐d
6
, ppm): 10.6–10.8 (2H, s, –NH), 7.78 (2H, d, ═CH,
1
J = 8.9 Hz), 7.70 (2H, d, ═CH, J = 8.7 Hz), 7.2–7.6 (4H, m, ═CH), 7.20
1
3
H N
17 5
O S
4 2
(2H, s, –NH
DMSO‐d , ppm): 170.2, 166.6, 141.9, 140.6, 139.62, 131.8, 129.8,
127.5, 127.4, 126.4, 125.5, 122.9, 122.4, 119.6, and 38.4 (Figure S21).
Anal. calcd. for C18 14BrN : C, 43.91; H, 2.87; Br, 16.23; N,
2 2
), and 4.1 (2H, s, –CH ; Figure S20); C NMR (75 MHz,
3
6
H
5 3 2
O S
2
‐[2,2‐Dicyano‐1‐(3‐methoxyphenylamino)vinylthio]‐N‐(4‐
14.22; O, 9.75; S, 13.02. Found: C, 43.98; H, 2.89; Br, 16.28; N, 14.27;
O, 9.79; S, 13.09.
sulfamoylphenyl)acetamide (7d)
−
1
2
Yield 73.5%, m.p. 210°C; IR (cm ): 3,265 (–NH ), 3,202 (–NH), 3,077
(
═C–H, aromatic), 2,988 (–CH
2
, aliphatic), 2,217 (CN), 1,660 (C═O),
2‐[1‐(4‐Bromophenylamino)‐2,2‐dicyanovinylthio]‐N‐(4‐
sulfamoylphenyl)acetamide (7h)
1
1
,615 (C═C), 1,319, and 1,149 (S═O; Figure S10); H NMR (300 MHz,
−
1
DMSO‐d
6
,
ppm): 10.6–10.8 (2H, s, –NH), 7.76 (2H, d, ═CH,
), 6.8–7.3
2
Yield 77.7%, m.p. 190°C; IR (cm ): 3,305 (–NH ), 3,211 (–NH), 3,139
J = 8.6 Hz), 7.70 (2H, d, ═CH, J = 8.6 Hz), 7.30 (2H, s, –NH
2
(═C–H, aromatic), 2,988, (–CH
2
, aliphatic), 2,221 (CN), 1,660 (C═O),
1
3
1
2
(4H, m, ═CH), 4.1 (2H, s, –CH ), and 3.7 (3H, s, –CH3; Figure S11);
C
1,628 (C═C), 1,314, and 1,151 (S═O; Figure S22); H NMR (300 MHz,
NMR (75 MHz, DMSO‐d
6
, ppm): 169.8, 166.6, 160.3, 141.9, 140,
6
DMSO‐d , ppm): 10.6–10.8 (2H, s, –NH), 7.76 (2H, d, ═CH,

ISTREFI ET AL.
13 of 16
|
1
J = 8.6 Hz), 7.68 (2H, d, ═CH, J = 8.8 Hz), 7.58 (2H, d, ═CH, J = 7.8 Hz),
1,655 (C═C), 1,322, and 1,152 (S═O; Figure S34); H NMR (300 MHz,
DMSO‐d ppm): 10.6–10.8 (2H, s, –NH), 7.76 (2H, d, ═CH,
J = 8.8 Hz), 7.68 (2H, d, ═CH, J = 8.8 Hz), 7.0–7.6 (4H, m, ═CH), 7.3
7
.31 (2H, d, ═CH, J = 7.9 Hz), 7.20 (2H, s, –NH
2
), and 4.1 (2H, s, –CH
, ppm): 170.2, 166.6, 141.9,
39.62, 138.4, 133.3, 132.8, 132, 127.5, 125.9, 119.6, 119.5, and 38.3
Figure S24). Anal. calcd. for C18 14BrN : C, 43.91; H, 2.87; Br,
6.23; N, 14.22; O, 9.75; S, 13.02. Found: C, 43.95; H, 2.89; Br, 16.28;
2
;
6
,
13
Figure S23); C NMR (75 MHz, DMSO‐d
6
1
3
1
(2H, s, –NH
DMSO‐d , ppm): 170.2, 166.64, 161.2, 141.9, 139.6, 131.7, 131.6,
127.5, 120, 119.6, 113.9, 113.6, 111.2, 110.9, and 35.4 (Figure S36).
Anal. calcd. for C18 14FN : C, 50.11; H, 3.27; F, 4.4; N, 16.23; O,
1.12; S, 14.86. Found: C, 50.18; H, 3.29; F, 4.47; N, 16.28; O, 11.17;
S, 14.89.
2 2
), and 4.1 (2H, s, –CH ; Figure S35); C NMR (75 MHz,
(
H
5
O S
3 2
6
1
N, 14.29; O, 9.79; S, 13.10.
H
5 3 2
O S
1
2
‐[1‐(4‐Chlorophenylamino)‐2,2‐dicyanovinylthio]‐N‐(4‐
sulfamoylphenyl)acetamide (7i)
−
1
Yield 79.9%, m.p. 188°C; IR (cm ): 3,260 (–NH
2
), 3,202 (–NH), 3,143
2‐[2,2‐Dicyano‐1‐(4‐fluorophenylamino)vinylthio]‐N‐(4‐
(═C–H, aromatic), 2,805 (–CH
2
, aliphatic), 2,220 (CN), 1,660 (C═O),
sulfamoylphenyl)acetamide (7m)
1
−1
1
,628 (C═C), 1,319, and 1,152 (S═O; Figure S25); H NMR (300 MHz,
2
Yield 78.5%, m.p. 212°C; IR (cm ): 3,271 (–NH ), 3,208 (–NH), 3,136
DMSO‐d
J = 8.6 Hz), 7.68 (2H, d, ═CH, J = 8.7 Hz), 7.32 (2H, d, ═CH, J = 8.1 Hz),
.28 (2H, d, ═CH, J = 8.1 Hz), 7.20 (2H, s, –NH ), and 4.1 (2H, s, –CH
, ppm): 170.2, 166.5, 141.9,
39.62, 138, 131.2, 129.9, 129.8, 127.5, 127.4, 125.5, 119.6, and 38.3
Figure S27). Anal. calcd. for C18 14ClN : C, 48.27; H, 3.15; Cl,
.92; N, 15.64; O, 10.72; S, 14.32. Found: C, 48.31; H, 3.19; Cl, 7.98;
6
,
ppm): 10.6–10.8 (2H, s, –NH), 7.76 (2H, d, ═CH,
(═C–H, aromatic), 2,988 (–CH
2
, aliphatic), 2,220 (CN), 1,660 (C═O),
1
1,622 (C═C), 1,319, and 1,151 (S═O; Figure S37); H NMR (300 MHz,
7
2
2
;
DMSO‐d
J = 8.7 Hz), 7.69 (2H, d, ═CH, J = 8.7 Hz), 7.20 (2H, d, ═CH, J = 8.4 Hz),
7.13 (2H, d, ═CH, J = 8.3 Hz), 7.3 (2H, s, –NH ), and 4.1 (2H, s, –CH
, ppm): 170.3, 166.6, 141.9,
139.6, 137.4, 127.5, 127.4, 126.8, 126.7, 119.6, 117, 116.6, and 37.98
(Figure S39). Anal. calcd. for C18 14FN : C, 50.11; H, 3.27; F,
.4; N, 16.23; O, 11.12; S, 14.86. Found: C, 50.16; H, 3.29; F, 4.45; N,
16.26; O, 11.19; S, 14.89.
6
, ppm): 10.6–10.8 (2H, s, –NH), 7.76 (2H, d, ═CH,
13
Figure S26); C NMR (75 MHz, DMSO‐d
6
1
2
2
;
1
3
(
H
5
O S
3 2
Figure S38); C NMR (75 MHz, DMSO‐d
6
7
N, 15.69; O, 10.75; S, 14.36.
H
5 3 2
O S
4
2
‐[2,2‐Dicyano‐1‐(2,4‐dichlorophenylamino)vinylthio]‐N‐(4‐
sulfamoylphenyl)acetamide (7j)
−1
Yield 79.9%, m.p. 207°C; IR (cm ): 3,308 (–NH
2
), 3,211 (–NH), 3,071
2‐[2,2‐Dicyano‐1‐(3‐(trifluoromethyl)phenylamino)vinylthio]‐N‐(4‐
(
═C–H, aromatic), 2,957 (–CH , aliphatic), 2,222 (CN), 1,662 (C═O),
2
sulfamoylphenyl)acetamide (7n)
1
−1
1
,621 (C═C), 1,331, and 1,149 (S═O; Figure S28); H NMR (300 MHz,
Yield 68.5%, m.p. 188°C; IR (cm ): 3,264 (–NH
2
), 3,200 (–NH),
6
DMSO‐d , ppm): 10.6–10.8 (2H, s, –NH), 7.77 (2H, d, ═CH, J = 8.8 Hz),
3,068 (═C–H, aromatic), 2,988 (–CH , aliphatic), 2,217 (CN),
2
7
4
1
1
.73 (2H, d, ═CH, J = 8.8 Hz), 7.4 (3H, m, ═CH), 7.20 (2H, s, –NH
2
), and
1,660 (C═O), 1,622 (C═C), 1,330, and 1,150 (S═O; Figure S40);
13
1
.1 (2H, s, –CH
2
; Figure S29); C NMR (75 MHz, DMSO‐d
6
, ppm):
6
H NMR (300 MHz, DMSO‐d , ppm): 10.6–10.8 (2H, s, –NH), 7.74
67.6, 141.8, 139.9, 133.4, 131.4, 130.2, 130.1, 129.6, 129.1 127.5,
27.4, 119.7, 119.6, and 38.2 (Figure S30). Anal. calcd. for
(2H, d, ═CH, J = 8.7 Hz), 7.67 (2H, d, ═CH, J = 8.6 Hz), 7.4 (4H, m,
1
3
═CH), 7.3 (2H, s, –NH
NMR (75 MHz, DMSO‐d
139.5, 131.2, 130.7, 127.8, 127.4, 126.2, 123.5, 122.6, 120.4,
19.5, 116.6, and 38.2 (Figure S42). Anal. calcd. for
: C, 47.4; H, 2.93; F, 11.84; N, 14.55; O, 9.97; S,
2
), and 4.1 (2H, s, –CH
2
; Figure S41);
C
C
18
H
13Cl
2
N
5
O
3
S
2
: C, 44.82; H, 2.72; Cl, 14.70; N, 14.52; O, 9.95; S,
6
, ppm): 170.69, 166.6, 141.9, 139.8,
13.29. Found: C, 44.88; H, 2.77; Cl, 14.74; N, 14.56; O, 9.99; S, 13.34.
1
2
‐[2,2‐Dicyano‐1‐(3,5‐dichlorophenylamino)vinylthio]‐N‐(4‐
19 14 3 5 3 2
C H F N O S
sulfamoylphenyl)acetamide (7k)
13.32. Found: C, 47.44; H, 2.98; F, 11.89; N, 14.58; O, 9.99;
S, 13.37.
−
1
2
Yield 70.9%, m.p. 235°C; IR (cm ): 3,275 (–NH ), 3,214 (–NH), 3,086
(═C–H, aromatic), 2,989 (–CH
2
, aliphatic), 2,216 (CN), 1,662 (C═O),
1
1
,621 (C═C), 1,317, and 1,160 (S═O; Figure S31); H NMR (300 MHz,
2‐[2,2‐Dicyano‐1‐(4‐iodophenylamino)vinylthio]‐N‐(4‐
sulfamoylphenyl)acetamide (7o)
DMSO‐d
6
, ppm): 10.6–10.8 (2H, s, –NH), 7.76 (2H, d, ═CH,
−
1
J = 8.4 Hz), 7.67 (2H, d, ═CH, J = 8.4 Hz), 7.2–7.6 (3H, ═CH), 7.4 (2H,
2
Yield 78.5%, m.p. 213°C; IR (cm ): 3,264 (–NH ), 3,205 (–NH),
1
3
s, –NH
ppm): 170.5, 166.06, 141.8, 139.64, 135.3, 135.1, 131.7, 129.1, 128.1,
27.5, 126.2, 122.3, 119.6, 113.1, and 39.3 (Figure S33). Anal. calcd.
for C18 13Cl : C, 44.82; H, 2.72; Cl, 14.70; N, 14.52; O, 9.95;
2
), 4.1 (2H, s, –CH
2
; Figure S32); C NMR (75 MHz, DMSO‐d
6
,
3,136 (═C–H, aromatic), 2,988 (–CH2, aliphatic), 2,219 (CN),
1,660 (C═O), 1,622 (C═C), 1,330, and 1,150 (S═O; Figure S43);
1
1
6
H NMR (300 MHz, DMSO‐d , ppm): 10.6–10.8 (2H, s, –NH), 7.75
H
N O
2 5 3
S
2
(2H, d, ═CH, J = 8.5 Hz), 7.67 (2H, d, ═CH, J = 8.5 Hz), 7.22 (2H, d,
S, 13.29. Found: C, 44.86; H, 2.77; Cl, 14.75; N, 14.58; O, 9.99;
S, 13.38.
═CH, J = 8.1 Hz), 7.00 (2H, d, ═CH, J = 8.1 Hz), 7.3 (2H, s, –NH ),
2
1
3
6
and 4.1 (2H, s, –CH2; Figure S44); C NMR (75 MHz, DMSO‐d ,
ppm): 170.1, 166.5, 141.9, 139.5, 139.4, 138.7, 138.6, 127.8,
127.5, 125.9, 119.6, 119.5, and 38.0 (Figure S45). Anal. calcd. for
2
‐[2,2‐Dicyano‐1‐(3‐fluorophenylamino)vinylthio]‐N‐(4‐
sulfamoylphenyl)acetamide (7l)
18 5 3 2
C H14IN O S : C, 40.08; H, 2.62; I, 23.53; N, 19.98; O, 8.9; S,
−
1
Yield 80.9%, m.p. 210°C; IR (cm ): 3,270 (–NH
2
), 3,200 (–NH), 3,086
11.89. Found: C, 40.18; H, 2.65; I, 23.55; N, 20.05; O, 8.94;
S, 11.93.
(
═C–H, aromatic), 2,889 (–CH , aliphatic), 2,209 (CN), 1,705 (C═O),
2

14 of 16
ISTREFI ET AL.
|
4
.2 | Biological studies
.2.1 | CA activity assay
partition coefficient (QPlogBB), human serum albumin‐binding
+
(QPlogKhsa), IC50 value for blockage of HERG K channels (QPlo-
4
gHERG), human oral absorption (HOA), the van der Waals surface
area of polar nitrogen and oxygen atoms (PSA), the number of
[
73]
The chromatography media used in the experiments and all chemical
compounds, including 4‐nitrophenyl acetate (N8130), were of ana-
lytical grade and purchased from Sigma‐Aldrich Chemie GmbH
violations of Lipinski's rule of five,
and the number of violations
[
74]
of Jorgensen's rule of three,
which are significant in the novel
drug discovery and development process.
(Taufkirchen, Germany). Prestained protein MW standard and mar-
[
59]
kers
(26617, a mixture of recombinant proteins ranging from
[
60]
10 to 180 kDa)
were obtained from Thermo Fisher Scientific Inc.
4.3.2 | Molecular docking study
(Waltham, MA). AAZ was used as the reference drug. Solutions of all
newly synthesized analogs (7a–o) and AAZ were prepared in DMSO
at an initial concentration of 1 mg/ml. The concentration of DMSO in
the final reaction mixture was approximately 1%. CA isoforms (I and
II) were obtained with purification from human erythrocytes by
Sepharose‐4B‐L‐tyrosine‐sulfanilamide affinity chromatography, as in
The crystal structures of the receptors, including hCA I (5E2M, re-
[75]
solution 1.41 Å, complex with V14),
hCA II (6H29, resolution 1.46 Å,
[
76]
complex with FK8),
and AChE (4M0E, resolution 2.00 Å, complex
[78]
[77]
with 1YL),
which are downloaded from the RCSB PDB (rcsb.org),
[
79]
were prepared using the Protein Preparation Wizard
[80]
in Schrö-
[
61]
our previous work.
The protein concentration of the eluates was
dinger Suite 2019‐3. OPLS3e force field
was used for the optimi-
[
62]
determined by a straightforward analytical procedure at 595 nm,
zation of both the synthesized ketene N,S‐acetal sulfonamide analogs
[
63]
[64]
according to the Bradford method,
spectrophotometrically.
The
(7a–o) and the receptors. The grid box accounting for the binding site
[
81]
purity of the purified enzyme fractions of both isoenzymes was de-
termined by sodium dodecyl sulfate polyacrylamide gel electro-
residues of hCA isoforms I, II, and AChE was generated
by the
[82]
Receptor Grid Generation module.
Three‐dimensional ligand
[
65]
[66]
[83]
phoresis,
which was realized on 8% slab gels,
as explained by
structures were prepared by the LigPrep tool
and implemented for
[84]
[
67]
Laemmli.
Esterase activity of human erythrocyte CA isoforms was
their minimization states at neutral pH (7.0 ± 0.5) with Epik.
Extra
[
85]
[86]
determined by following the change in absorbance at 348 nm over a
period of 3 min at 25°C, according to the method defined by Ver-
precision Glide protocol
in Schrödinger Suite
was performed and
bioactive compound structures were treated as flexible to obtain five
[87]
[68]
poorte et al.
IC50 and K values were computed as in our previous work.
inhibition type for each of the derivatives was found using
All rate measurements were conducted in triplicate.
poses for each ligand. The Prime MM–GBSA module
[88]
(Schrödinger
[
69]
I
The
Release 2019‐3) using the VSGB solvent model
and OPLS3e force
[89]
field
was applied to calculate the binding free energy (ΔG Bind) of
[
70]
Lineweaver–Burk curves.
the synthesized derivatives (7a–o) toward the receptors (5E2M, 6H29,
and 4M0E).
4
.2.2 | AChE activity assay
4
.4 | Statistical studies
Acetylcholinesterase from Electrophorus electricus (C2888, Type V‐S),
which is a tetramer composed of four equal subunits of 70 kDa each,
The analysis of the data and drawing of graphs were realized using
GraphPad Prism version 6 for Mac, GraphPad Software, La Jolla, CA.
5,5′‐dithiobis(2‐nitrobenzoic acid) (D8130, DTNB), and acet-
ylthiocholine iodide (01480, AChI) were acquired from Sigma‐Aldrich
Chemie GmbH (Taufkirchen, Germany). In vitro effects on AChE
activity of the target derivatives (7a–o) were evaluated by the
I
Also, K constants were determined using SigmaPlot version 12, from
Systat Software, San Jose, CA. The results were exhibited as
mean ± standard deviation (95% confidence intervals). Differences
between data sets were considered as statistically significant when
the p value was <0.05.
[71]
method of Ellman et al.
TAC was used as the reference drug. All
the measurements were repeated three times.
ACKNOWLEDGMENTS
4
.3 | In silico studies
.3.1 | ADMET study
This study was supported by the Research Fund of Erzincan Binali
Yıldırım University (Grant number FBA‐2017‐501), the Research
Fund of Sakarya University (Grant number 2016‐02‐04–018), and
the Research Fund of Anadolu University (Grant number 1610S681).
4
[
72]
Qikprop software,
contained in the Schrodinger Suite 2019‐3,
was used to predict pharmaceutically relevant and varied ADMET
and drug‐likeness parameters of the synthesized ketene N,S‐acetal
sulfonamides (7a–o), such as water/gas partition coefficient
CONFLICT OF INTERESTS
The authors declare that there are no conflicts of interests.
(
QPlogPw), octanol/water partition coefficient (QPlogPo/w), aqu-
ORCID
eous solubility (QPlogS), skin permeability (QPlogKp), brain/blood
Cüneyt Türkeş
http://orcid.org/0000-0002-2932-2789

ISTREFI ET AL.
15 of 16
|
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