Synthesis of calix[4]azacrown substituted sulphonamides with antioxidant, acetylcholinesterase, butyrylcholinesterase, tyrosinase and carbonic anhydrase inhibitory action

Article abstract of DOI:10.1080/14756366.2020.1765166

A series of novel calix[4]azacrown substituted sulphonamide Schiff bases was synthesised by the reaction of calix[4]azacrown aldehydes with different substituted primary and secondary sulphonamides. The obtained novel compounds were investigated as inhibitors of six human (h) isoforms of carbonic anhydrases (CA, EC 4.2.1.1). Their antioxidant profile was assayed by various bioanalytical methods. The calix[4]azacrown substituted sulphonamide Schiff bases were also investigated as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase enzymes, associated with several diseases such as Alzheimer, Parkinson, and pigmentation disorders. The new sulphonamides showed low to moderate inhibition against hCAs, AChE, BChE, and tyrosinase enzymes. However, some of them possessed relevant antioxidant activity, comparable with standard antioxidants used in the study.

Full text of DOI:10.1080/14756366.2020.1765166

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis of calix[4]azacrown substituted
sulphonamides with antioxidant,
acetylcholinesterase, butyrylcholinesterase,
tyrosinase and carbonic anhydrase inhibitory
action
Mehmet Oguz, Erbay Kalay, Suleyman Akocak, Alessio Nocentini, Nebih
Lolak, Mehmet Boga, Mustafa Yilmaz & Claudiu T. Supuran
To cite this article: Mehmet Oguz, Erbay Kalay, Suleyman Akocak, Alessio Nocentini, Nebih
Lolak, Mehmet Boga, Mustafa Yilmaz & Claudiu T. Supuran (2020) Synthesis of calix[4]azacrown
substituted sulphonamides with antioxidant, acetylcholinesterase, butyrylcholinesterase, tyrosinase
and carbonic anhydrase inhibitory action, Journal of Enzyme Inhibition and Medicinal Chemistry,
35:1, 1215-1223, DOI: 10.1080/14756366.2020.1765166
To link to this article: https://doi.org/10.1080/14756366.2020.1765166
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UK Limited, trading as Taylor & Francis
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1215–1223
https://doi.org/10.1080/14756366.2020.1765166
RESEARCH PAPER
Synthesis of calix[4]azacrown substituted sulphonamides with antioxidant,
acetylcholinesterase, butyrylcholinesterase, tyrosinase and carbonic anhydrase
inhibitory action
Mehmet Oguza,b , Erbay Kalayc, Suleyman Akocakd, Alessio Nocentinie , Nebih Lolakd, Mehmet Bogaf,
Mustafa Yilmaza and Claudiu T. Supurane
b
aDepartment of Chemistry, University of Selcuk, Konya, Turkey; Department of Advanced Material and Nanotechnology, Selcuk University,
c
d
Konya, Turkey; Kars Vocational School, Kafkas University, Kars, Turkey; Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
e
ꢀ
Adiyaman University, Adiyaman, Turkey; NEUROFARBA Department, Sezione di Scienze Farmaceutiche, Universita degli Studi di Firenze,
f
Florence, Italy; Department of Analytical Chemistry, Faculty of Pharmacy, Dicle University, Diyarbakir, Turkey
ABSTRACT
ARTICLE HISTORY
Received 2 April 2020
Revised 29 April 2020
Accepted 30 April 2020
A series of novel calix[4]azacrown substituted sulphonamide Schiff bases was synthesised by the reaction
of calix[4]azacrown aldehydes with different substituted primary and secondary sulphonamides. The
obtained novel compounds were investigated as inhibitors of six human (h) isoforms of carbonic anhy-
drases (CA, EC 4.2.1.1). Their antioxidant profile was assayed by various bioanalytical methods. The calix[4]-
azacrown substituted sulphonamide Schiff bases were also investigated as inhibitors of
acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase enzymes, associated with several
diseases such as Alzheimer, Parkinson, and pigmentation disorders. The new sulphonamides showed low
to moderate inhibition against hCAs, AChE, BChE, and tyrosinase enzymes. However, some of them pos-
sessed relevant antioxidant activity, comparable with standard antioxidants used in the study.
KEYWORDS
Calix[4]azacrown; enzyme
inhibition; antioxidant;
carbonic anhydrase
1. Introduction
Several major classes of macrocyclic derivatives such as the
crown ethers, cyclodextrins, and calixarenes are important third-
generation derivatives in supramolecular chemistry13,14. Among
them, calixarenes may have biomedical applications thanks to
their exceptional structural properties, which may be exploited for
designing antitumor, antiviral, antimicrobial, anti-thrombotic, and
antifungal derivatives15–17. From this perspective, synthesising
new derivatised calixarenes as antioxidant and enzyme inhibitors,
and exploring the molecular mechanisms underlying their effect
are ongoing new fields.
In continuation of our recent interest in developing new metal-
loenzymes inhibitors18–21, in this study, we report the first calix[4]-
azacrown substituted sulphonamide Schiff bases (to the best of
our knowledge) acting as an antioxidant and metalloenzyme
inhibitors (such as carbonic anhydrase, acetylcholinesterase, butyr-
ylcholinesterase, and tyrosinase inhibitors).
Carbonic anhydrases are metalloenzymes present in Archaea, pro-
karyotes, and eukaryotes, and catalyse a physiologically very sim-
ple but relevant reaction, i.e. the interconversion of CO2 to HCO3
ꢀ
and protons via a ping-pong mechanism1–6. In humans, 16 differ-
ent isoforms have been described from eight genetically distinct
CA families (a-, b-, c-, d-, f-, g-, h-, and i-CAs)1–6. The isozymes
have different subcellular localisation, catalytic activity, and inhibi-
tory properties in body fluid and tissues. Since these isoforms play
an important role in acid–base regulation, gluconeogenesis and
other biosynthetic reactions, electrolyte secretion, bone resorp-
tion/calcification, and tumorigenicity, their inhibition/activation
may be exploited in several diseases, including glaucoma, obesity,
neuropathic pain, arthritis, Alzheimersꢀ disease and more
recently cancer1–6
.
Acetylcholinesterase (AChE; EC 3.1.1.7), which hydrolyses the
neurotransmitter acetylcholine, is found in high concentrations
over the peripheral and central nervous systems but also in other
tissues7,8. The imbalance in AChE activity can cause various types
of neurodegenerative pathologies such as Alzheimer’s disease
(AD) and Parkinson’s disease (PD). Butyrylcholinesterase (BChE) is a
non-specific cholinesterase enzyme that hydrolyses many different
choline-based esters9,10. Thus, AChE and BChE inhibition have
been documented as an interesting approach in the palliative
2. Materials and methods
2.1. General
All materials were purchased from commercial suppliers and used
without further purification. All reactions were conducted under
an atmosphere of nitrogen unless noted otherwise. Anhydrous sol-
vents were distilled over appropriate drying agents before use. H
NMR spectra were recorded on a Varian 400 MHz spectrometer in
DMSO-d6 solution with the internal solvent signal peak at
1
treatment of AD11,12
.
CONTACT Mustafa Yilmaz
claudiu.supuran@unifi.it
Florence, 50019, Italy
myilmaz42@gmail.com
Department of Chemistry, University of Selcuk, Campus, Konya, 42031, Turkey; Claudiu T. Supuran
ꢀ
NEUROFARBA Department, Sezione di Scienze Farmaceutiche, Universita degli Studi di Firenze, Via Ugo Schiff 6, Sesto Fiorentino,
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1216
M. OGUZ ET AL.
2.50 ppm. 13C NMR was recorded at 100 MHz spectrometer in and the reaction mixture was refluksed for 2 h before being
cooled down to room temperature. Then the reaction solution
was quenched with 2 M HCl to approximately pH 6, resulting in a
large number of white solids. To obtain the desired product, the
crude product was filtered, washed with water, and then dried in
the desiccator (white solid, yield 68%).
DMSO-d6 solution and referenced to the internal solvent signal at
39.5 ppm. Proton NMR data are reported as follows: chemical shift
(ppm),
multiplicity
(s ¼ singlet,
d ¼ doublet,
t ¼ triplet,
q ¼ quartette, dd ¼ doublet of doublets, dt ¼ triplet of doublets,
m ¼ multiplet, brs. ¼ broad singlet), and coupling constants (Hz).
Infra-red spectra were measured using a Bruker Spectrometer
transform-infra-red (FT-IR) spectrometer. The progress of the reac-
tions was monitored by thin layer chromatography. For the detec-
tion of the spots, the plates were exposed to UV light (254
and 365 nm).
2.2.3. Synthesis of SA-3
2.2. Synthesis procedure for preparation of sulphonamides
2.2.1. Synthesis of SA-1
The SA-3 was synthesised as described in literature24. Briefly, 3-
amino-5-methylisoxazole (1.18 g, 12 mmol) was added to a 50 ml
round-bottom flask and dissolved in a solution of THF (20 ml) and
pyridine (1 ml). p-Acetamidobenzensulfonyl chloride (6 g,
2.81 mmol) was then added to the reaction solution. The reaction
mixture was stirred at room temperature for 12 h. The reaction
was followed by TLC. After completion of the reaction, the THF
was removed in the evaporator. Subsequently, 30 ml 0.5 M HCl
was added to the crude mixture and a large amount of precipitate
was formed. The precipitate was filtrated, washed with water, and
directly used in the next step. The Intermediate was dissolved in a
20% NaOH solution and the reaction mixture was refluxed for 2 h.
After the reaction was completed, the mixture was quenched to
approximately pH 7.4 with 2 M HCl, resulting in a large number of
white solids. The precipitate was obtained and washed with water,
then dried overnight in a desiccator (white solid, yield 60%).
The SA-1 was synthesised as described in literature22. Briefly, pyri-
midin-2-amine (1.14 g, 12 mmol) was added to a 50 ml round-bot-
tom flask and dissolved in a solution of THF (20 ml) and pyridine
(1 ml). p-Acetamidobenzenesulfonyl chloride (2.81 g, 12 mmol) (6 g,
2.81 mmol) was then added to the reaction solution. After comple-
tion of the reaction followed by TLC, the THF was removed in the
evaporator. Subsequently, 30 ml 0.5 M HCl was added and a large
amount of precipitate was formed. The precipitate was filtrated
and then washed with water and used directly in the next step.
The Intermediate was dissolved in a 20% NaOH solution and the
reaction mixture was refluxed for 2 h. After the reaction was com-
pleted, the mixture was quenched to approximately pH 7.4 with
2 M HCl, resulting in a large number of white solids. The precipi-
tate was obtained and washed with water, then dried overnight in
a desiccator (white solid, yield 65%).
2.2.4. Synthesis of SA-4 and SA-5
2.2.2. Synthesis of SA-2
General Procedure: The SA-5 and SA-6 were synthesised as
described in literature25. Briefly, a solution of an arylsulfonyl chlor-
ide (10 mmol) in tetrahydrofuran (40 ml) was added hydrazine
monohydrate (1.25 ml, 2.5 equiv.) dropwise at 0 ꢁC. The reaction
mixture was stirred vigorously for 30 min at 0 ꢁC. Upon comple-
tion, the reaction mixture was added ethyl acetate (50 ml) and
extracted with saturated brine (3 ꢂ 50 ml). The organic layer was
separated, dried over anhydrous Na2SO4, filtrated, concentrated in
an evaporator and added to hexane (10 ml) over 5 min. The pre-
cipitate was filtered, collected, and dried in vacuum.
The SA-2 was synthesised as described in literature23. Briefly, in a
50 ml round-bottom flask, to a stirred solution of benzo[d]thiazol-
2-amine (1.0 g, 6.66 mmol) in dry pyridine (6 ml) was added 4-acet-
amidobenzenesulfonyl chloride (1.55 g, 6.66 mmol). The reaction
mixture was refluksed for 2 h. The reaction mixture was poured
onto acidic crushed ice and filtered. The crude solid was washed
with water and directly used in the next step. The intermediate
2.3. General procedure for the synthesis of compounds CX (1–6)
In a 25-ml round-bottomed flask equipped with a magnetic stirrer,
sulphonamide derivative (0.4 mmol) was added to the solution of
calix[4]arene-aldehyde (0.2 mmol, 146.6 mg) in a mixture of 10 ml
was dissolved in 1 M NaOH solution (3.5 ekv. of the intermediate) CHCl3/MeOH (1:1). The resulting mixture heated to reflux

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1217
overnight. After the reaction was complete, the solvent was J ¼ 9.0 Hz, 4H, ArH), 7.73 (d, J ¼ 9.0 Hz, 4H, ArH), 7.42 (s, 4H, calix-
removed. The crude mixture was dissolved with 2 ml of methylene ArH), 7.11 (s, 4H, calix-ArH), 2.30 (s, 6H, Me), 4.46 (s, 4H, OCH2),
4.10 (d, J ¼ 13.1 Hz, 4H, ArCH2Ar), 3.57 (d, J ¼ 13.1 Hz, 4H, ArCH2Ar),
chloride. Upon the addition of hexane to the solution, the target
t
3.47 (m, 4H, CH2CH2), 2.03 (s, 6H, Me), 1.06 (s, 18H, Bu); 13C NMR
product was precipitated. Then, the product was filtered off and
dried under vacuum at 40 ꢁC. The obtained final pure compounds
CX(1–6) were fully characterised by 1H-NMR and 13C-
NMR techniques.
(100 MHz, DMSO-d6) d 168.2, 167.6, 156.9, 152.4, 147.9, 143.0,
141.2, 132.2, 131.0, 129.7, 129.2, 128.4, 127.5, 126.4, 124.9, 75.2,
44.1, 38.0, 34.0, 30.1, 23.8; Anal. Calcd for C60H66N8O12S2 (1155.34):
C, 62.37; H, 5.76; N, 9.70; S, 5.55. Found: C, 62.59; H, 5.76; N, 9.73;
S, 5.54.
CX-1: A white solid, yield 717%. 1H NMR (400 MHz, DMSO-d6)
d; 9.19 (s, 2H, CONH), 8.34 (s, 2H, CH ¼ N), 7.88 (d, J ¼ 8.5 Hz, 4H,
ArH) , 7.57 (d, J ¼ 8.5 Hz, 4H, ArH) , 7.55 (s, 4H, calix-ArH), 7.11 (s,
4H, calix-ArH), 4.51 (s, 4H, OCH2), 4.20 (d, J ¼ 13.5 Hz, 4H, ArCH2Ar),
3.69 (d, J ¼ 13.5 Hz, 4H, ArCH2Ar), 3.49 (m, 4H, CH2CH2), 1.10 (s,
18H, tBu); 13C NMR (100 MHz, DMSO-d6) d 166.9, 161.4, 153.2,
150.0, 141.9, 140.3, 131.7, 130.3, 129.6, 129.0, 128.3, 127.6, 126.8,
125.8, 74.1, 43.6, 38.6, 34.4, 32.5; Anal. Calcd for C64H66N8O12S2
(1041.24): C, 64.60; H, 5.81; N, 8.07; S, 6.16. Found: C, 64.83; H,
5.80; N, 8.07; S, 6.14.
2.4. CA inhibition assay
An SX.18 MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the inhibition of various CA
isozymes26. Phenol Red (at a concentration of 0.2 mM) has been
used as an indicator, working at the absorbance maximum of
557 nm, with 10 mM Hepes (pH 7.4) as a buffer, 0.1 M Na2SO4 or
NaClO4 (for maintaining constant the ionic strength; these anions
are not inhibitory in the used concentration), following the CA-cat-
alyzed CO2 hydration reaction for a period of 5–10 s. Saturated
CO2 solutions in water at 25 ꢁC were used as substrate. Stock solu-
tions of inhibitors were prepared at a concentration of 10 mM (in
DMSO-water 1:1, v/v) and dilutions up to 0.01 nM done with the
assay buffer mentioned above. At least 7 different inhibitor con-
centrations have been used for measuring the inhibition constant.
Inhibitor and enzyme solutions were pre-incubated together for
10 min at room temperature before assay, to allow for the forma-
tion of the E-I complex. Triplicate experiments were done for each
inhibitor concentration, and the value reported throughout the
paper is the mean of such results. The inhibition constants were
obtained by nonlinear least-squares methods using the Cheng-
Prusoff equation, as reported earlier, and represent the mean from
at least three different determinations127–32. All CA isozymes used
here were recombinant proteins obtained as reported earlier by
our group.
1
CX-2: A yellow solid, yield 70%. H NMR (400 MHz, DMSO-d6) d
9.31 (s, 2H, CONH), 8.30 (s, 2H, CH ¼ N), 7.92 (d, J ¼ 8.5 Hz, 4H,
ArH), 7.60 (d, J ¼ 8.5 Hz, 4H, ArH), 7.56 (s, 4H, calix-ArH), 7.15 (s,
4H, calix-ArH), 6.07 (s, 2H, Het-ArH), 4.50 (s, 4H, OCH2), 4.21 (d,
J ¼ 13.5 Hz, 4H, ArCH2Ar), 3.68 (d, J ¼ 13.5 Hz, 4H, ArCH2Ar), 3.50
(m, 4H, CH2CH2), 2.25 (s, 6H, Me), 1.12 (s, 18H, tBu); 13C NMR
(100 MHz, DMSO-d6) d 168.8, 167.7, 160.5, 152.2, 151.1, 150.4,
143.3, 141.9, 132.4, 130.1,129.7, 129.2, 128.7, 127.1, 126.3, 124.6,
95.6, 74.7, 42.0, 38.7, 34.4, 32.0, 12.9; Anal. Calcd for
C64H66N8O12S2 (1197.38): C, 63.88; H, 5.53; N, 9.31; S, 5.33. Found:
C, 63.97; H, 5.50; N, 9.29; S, 5.35.
1
CX-3: A yellow solid, yield 72%. H NMR (400 MHz, DMSO-d6) d
9.42 (s, 2H, CONH), 8.45 (d, J ¼ 8.8 Hz, 4H, Het-ArH), 8.33 (s, 2H,
CH ¼ N), 7.96 (d, J ¼ 8.8 Hz, 4H, ArH), 7.60 (d, J ¼ 8.8 Hz, 4H, ArH),
7.58 (s, 4H, calix-ArH), 7.15 (s, 4H, calix-ArH), 6.97 (t, J ¼ 4.8 Hz, 2H,
ArH), 4.50 (s, 4H, OCH2), 4.21 (d, J ¼ 13.4 Hz, 4H, ArCH2Ar), 3.70 (d,
t
J ¼ 13.4 Hz, 4H, ArCH2Ar), 3.50 (m, 4H, CH2CH2), 1.12 (s, 18H, Bu);
13C NMR (100 MHz, DMSO-d6) d 167.4, 162.6, 159.7, 158.0, 157.4,
157.1, 155.9, 144.5, 142.2, 138.8,130.1, 129.9, 129.2, 128.7, 128.3,
127.7, 126.0, 75.6, 44.0, 38.2, 34.2, 31.1; Anal. Calcd for
C64H64N10O10S2 (1197.38): C, 64.20; H, 5.39; N, 11.70; S, 5.36.
Found: C, 64.42; H, 5.41; N, 11.74; S, 5.37.
2.5. Determination of antioxidant, anticholinesterase and
tyrosinase activity of calix[4]arene sulphonamides CX(1–6)
1
CX-4: A yellow solid, yield 70%. H NMR (400 MHz, DMSO-d6) d
2.5.1. Dpph radical scavenging assay
9.41 (bs, 2H, CONH), 8.31 (s, 2H, CH ¼ N), 7.92 (d, J ¼ 8.5 Hz, 4H,
ArH), 7.85–7.70 (m, 4H, Het-ArH), 7.57 (s, 4H, calix-ArH), 7.55–7.48
(m, 8H, ArH), 7.14 (s, 4H, calix-ArH), 4.50 (s, 4H, OCH2) 4.21 (d,
J ¼ 13.5 Hz, 4H, ArCH2Ar), 3.69 (d, J ¼ 13.5 Hz, 4H, ArCH2Ar), 3.49
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activ-
ity of the synthesised compounds was determined by a spectro-
photometric method based on the reduction of an ethanol
solution of DPPH33,34. 2, 5, 10, 20 mL of 1 mM stock solution of
each compound was completed to 40 mL with the DMSO and
mixed with 160 mL of 0.1 mM of DPPH free radical solution. The
mixture was led to stand for 30 min in the dark and the absorb-
ance was then measured at 517 nm against a blank. Inhibition of
free radical, DPPH, in percent (I %), was calculated according to
the formula:
t
(m, 4H, CH2CH2), 1.12 (s, 18H, Bu); 13C NMR (100 MHz, DMSO-d6) d
169.6, 166.1, 161.9, 152.6, 152.0, 151.4, 147.3, 144.2, 132.5, 130.9,
130.0, 129.4, 128.7, 128.2, 127.9, 127,4, 127.4, 127.0, 126.8, 126.2,
125.4, 75.9, 44.0, 38.5, 34.6, 31.0; Anal. Calcd for C70H66N8O10S4
(1307.58): C, 64.30; H, 5.09; N, 8.57; S, 9.81. Found: C, 64.49; H,
5.09; N, 8.59; S, 9.79.
CX-5: As a yellow solid, yield 90%. 1H NMR (400 MHz, DMSO-
d6) d 8.92 (s, 2H, CONH), 8.31 (s, 2H, CH ¼ N), 7.72 (d, J ¼ 8.5 Hz,
4H, ArH), 7.45 (s, 4H, calix-ArH), 7.34 (d, J ¼ 8.5 Hz, 4H, ArH), 7.15
(s, 4H, calix-ArH), 2.30 (s, 6H, Me), 4.46 (s, 4H, OCH2), 4.10 (d,
J ¼ 13.2 Hz, 4H, ArCH2Ar), 3.58 (d, J ¼ 13.2 Hz, 4H, ArCH2Ar), 3.47
(m, 4H, CH2CH2), 2.30 (s, 6H, Me), 1.09 (s, 18H, tBu); 13C NMR
(100 MHz, DMSO-d6) d 167.6, 153.1, 151.9, 148.2, 143.7, 141.4,
133.6, 131.8, 130.0, 129.5, 129.1, 128.2, 127.3, 125.6, 75.5, 44.3,
I % ¼ Acontrol ꢀ Asample =Acontrol ꢂ 100
ð
Þ
where Acontrol is the absorbance of the control reaction (contain-
ing all reagents except for the tested compounds), and Asample is
the absorbance of the test compounds. Tests were carried out in
triplicate. BHA and BHT were used as positive control.
38.2, 34.5, 30.9, 21.8; Anal. Calcd for C58H64N6O10S2 (1069.29): C, 2.5.2. Abts cation radical decolorisation assay
65.15; H, 6.03; N, 7.86; S, 6.00. Found: C, 65.39; H, 6.02; N, 7.86; The percent inhibition of decolorisation of ABTS [2,20-azino-bis(3-
S, 5.59.
ethylbenzothiazoline-6-sulfonic acid)] cation radical is obtained as
1
CX-6: As a yellow solid. (203 mg, yield 88%); H NMR (400 MHz, a function of time and concentration and evaluated by compari-
DMSO-d6) d 8.89 (s, 2H, CONH), 8.30 (s, 2H, CH ¼ N), 7.78 (d, son with the BHT and BHA compounds used as standard35,36. The

1218
M. OGUZ ET AL.
tested compounds at different concentrations are added to each 2.6. Statistical analysis
well and 160 mL of 7 mM ABTS solution is added. After 6 min at
The results of the antioxidant, anticholinesterase, and tyrosinase
activity assays are expressed as the mean SD of three parallel
measurements. The statistical significance was estimated using a
room temperature, the absorbances were measured at 734 nm.
ABTS cation radical decolorisation activities were determined by
using the equation below:
Student’s t-test, where
p values < 0.05 were considered
significant.
%Inhibition ¼
A
control ꢀ Asample =A
ꢂ 100
ð
Þ
control
where A is the absorbance. Tests were carried out in triplicate.
BHA and BHT were used as positive control.
3. Results and discussion
3.1. Chemistry
2.5.3. Metal chelating activity
To develop novel and effective enzyme inhibitors and antioxidant
agents based on calixarenes, we used the calix[4](aza)crown dia-
ldehyde as a scaffold to design a series of new derivatives bearing
different sulphonamide moieties. The sulphonamide-substituted
calix[4]zacrown derivatives CX(1–6) were obtained in four steps
(Scheme 1). The required starting compound p-tert-butylcalix[4]ar-
ene was synthesised according to literature procedure16 and then
The chelating ability of synthesised compounds was examined
according to the method of Dinis et al.37. The tested compounds
at different concentrations were added to each well and 4 mL of
2 mM ferrous (II) chloride was added. Then 8 mL of 5 mM ferrozine
was added and the reaction was started. After 10 min at room
temperature, the absorbance was measured at 562 nm against a
blank. The results were expressed as a percentage of inhibition of the calix[4]arene diester was obtained in satisfactory yield by
the ferrozine-Fe2þ complex formation. The percentage inhibition
reflux with bromomethyl acetate in the presence of K2CO3 in
CH3CN16. The diester derivative of calixarene was reacted with
ethylenediamine in CH3OH/CHCl3 (1:1) at room temperature, being
converted to p-tert-butycalix[4](aza-)crown derivative which was
isolated by recrystallisation from the methanol16. The desired p-
tert-butycalix[4](aza)crown dialdehyde derivative was prepared by
treating p-tert-butycalix[4](aza)crown with hexamethylenetetra-
amine with refluxing trifluoroacetic acid16. In the last step, the sul-
phonamide derivatives (SA-1–5) were reacted with calix(aza)crown
dialdehyde in absolute ethanol to synthesise the novel calix(aza)-
crown substituted sulphonamide derivatives CX(1–6). All the syn-
of the ferrozine -Fe2þ complex formation was calculated using the
formula given below:
Chelating ability ð%Þ ¼ Acontrol ꢀ Asample =Acontrol ꢂ 100
ð
Þ
where A is the absorbance. Tests were carried out in triplicate.
EDTA was used as a positive control.
2.5.4. Anticholinesterase assay
The inhibitory effect of novel calix[4]azacrown substituted sul-
phonamide Schiff bases CX(1–6) on AChE and BChE activities was thesised compounds were fully characterised by using
spectroscopic techniques (see the experimental part for details).
determined according to the slightly modified spectrophotometric
method of Ellman et al.38. All compounds were dissolved in DMSO
to prepare stock solutions at 4 mM concentration. Aliquots of
150 mL of 100 mM sodium phosphate buffer (pH 8.0), 10 mL of sam-
ple solution and 20 mL AChE (or BChE) solution were mixed and
incubated for 15 min at 25 ꢁC, and DTNB [5,50-Dithio-bis(2-nitro-
benzoic)acid] (10 mL) is added. The reaction was then initiated by
3.2. CA inhibition studies
The newly synthesised calix[4]azocrown substituted sulphonamide
Schiff bases CX(1–6) were assessed as inhibitors of six physiologic-
ally relevant CA isoforms, the cytosolic hCA I, hCA II, and hCA VII,
the addition of acetylthiocholine iodide (or butyrylthiocholine iod- membrane-bound hCA IV, and the transmembrane tumor-associ-
ated hCA IX and hCA XII, by a stopped flow CO2 hydrase assay26.
ide) (10 mL). The final concentration of the tested compounds’
The clinically used sulphonamide acetazolamide (AAZ) was used
as a positive control. The compounds, in general, showed low
inhibition potency against the tested CA isoforms. Specifically, all
the secondary sulphonamides exhibited inhibition constants
>100 mM activity against the tested CA isoforms, except the iso-
form hCA IX, for which some of the compounds (CX-2, CX-3 and
CX-6) showed moderate activity with KIs of 67.6, 46.0, and
64.6 mM, respectively. On the other hand, the primary sulphona-
mide substituted derivative (CX-1) showed medium potency activ-
ity against the tumor-associated isoforms hCA IX and hCA XII with
solution was 200 mM.
%Inhibition ¼ Acontrol ꢀ Asample =Acontrol ꢂ 100
ð
Þ
where A is the absorbance. Tests were carried out in triplicate.
Galantamine was used as positive control.
2.5.5. Anti-tyrosinase activity
Anti-tyrosinase activity of the compounds was performed accord-
ing to the method designed by Hearing and Jimenez39. Firstly, the
inhibition of diphenolase function of the compounds was eval- KIs in the range of 0.15 to 0.27 mM, respectively, also inhibiting in
the micromolar ramge the other isoforms (Table 1).
uated and L-DOPA was used as substrate. Tyrosinase from mush-
room (E.C. 1.14.18.1) (30 U, 28 nM) was dissolved in Na-phosphate
buffer (pH ¼ 6.8, 50 nM) and the compounds were added to the
solution for pre-incubation at room temperature for ten minutes.
After incubation, 0.5 mM L-DOPA was added to the mixture and
the change in absorbance was measured at 475 nm at 37 ꢁC. For
positive control, kojic acid was used. The following formula was
used to calculate the percentage of all enzyme inhibitions:
3.3. Antioxidant activity
The antioxidant capacities of the newly synthesised compounds
CX(1–6) were demonstrated by using three different methods,
namely, DPPH free radical scavenging, ABTS cation radical scav-
enging, and metal chelating methods. All of the compounds
showed antioxidant activities in a dose-dependent manner and
shown in Table 2, and the IC50 values were compared with the
standards BHA, BHT, and EDTA. The three compounds (CX-1, CX-
Inhibition ð%Þ ¼
A
control–Asample =A
ꢂ 100
ð
Þ
control
A: Absorbance.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1219
Scheme 1. General synthetic route for the synthesis of the calix[4]arene substituted sulphonamide Schiff base derivatives CX(1–6).
Table 1. In vitro hCA I, hCA II, hCA IV, hCA VII, hCA IX, and hCA XII inhibition
data with calix[4]azacrown substituted sulphonamide Schiff base derivatives
CX(1–6) investigated here, and standard sulphonamide inhibitor Acetazolamide
Table 2. The antioxidant activity of calix[4]azacrown substituted sulphonamide
Schiff base derivatives CX(1–6) and controls BHA, BHT, and EDTA.
IC50 values (lM)a
(AAZ) by a stopped flow CO2 hydrase assay26
.
KIa (mM)
Samples
DPPH Free Radical
ABTS Cation Radical
Metal Chelate
CX-1
CX-2
CX-3
CX-4
CX-5
CX-6
BHAb
BHTb
EDTAb
>1000
>1000
>1000
520.33 0.89
16.79 0.85
9.02 0.05
7.88 0.20
58.86 0.50
–
769.97 0.22
>1000
121.03 0.95
>1000
9.79 0.09
7.74 0.04
17.59 0.10
13.25 0.27
–
>1000
>1000
>1000
>1000
>1000
>1000
–
Compound
hCA I
hCA II
hCA IV
hCA VII
hCA IX
hCA XII
CX-1
CX-2
CX-3
CX-4
CX-5
CX-6
AAZ
5.55
>100
>100
>100
>100
>100
0.25
0.82
>100
>100
>100
>100
>100
0.01
4.36
>100
>100
>100
>100
>100
0.07
1.21
0.15
67.6
46.0
>100
>100
64.6
0.27
>100
10.2
>100
>100
>100
0.006
>100
>100
>100
>100
>100
0.002
–
0.02
26.82 0.10
aMean from 3 different assays, by a stopped flow technique (errors were in the
aIC50 values represent the means (standard deviation of three parallel measure-
range of 5–10% of the reported values).
ments (p < 0.05).
bReference compounds.
2, and CX-3) showed no activity against DPPH free radical assay
with IC50 values of >1000 lM, but CX-5 and CX-6 had an activity
comparable with standards, having IC50 values of 16.79 0.85 and
9.02 0.05 lM, respectively. Interestingly, these two compounds
(CX-5 and CX-6) were also sensitive to ABTS radical scavenging
activity with IC50 values of 9.79 0.09 and 7.74 0.04 lM, respect-
ively. On the other hand, none of the tested compounds showed
any metal chelating activity.
3.4. Acetylcholinesterase, butyrylcholinesterase, and
tyrosinase activity
The calix[4]azacrown substituted sulphonamide Schiff bases
CX(1–6) were also evaluated for their anti-cholinesterase (AChE
and BChE) and anti-tyrosinase activities. None of the compounds
from the series showed any inhibition potency against AChE and
BChE enzymes, except for compounds CX-6, which showed

1220
M. OGUZ ET AL.
Table 3. Anti-cholinesterase and anti-tyrosinase activity of calix[4]azacrown sub-
stituted sulphonamide Schiff base derivatives CX(1–6) and controls galantamine
and kojik acid.
the alpha-beta- and gamma-carbonic anhydrases from bac-
teria: can bacterial carbonic anhydrases shed new light on
evolution of bacteria? J Enzyme Inhib Med Chem 2015;30:
325–32. (c) Supuran CT. Carbon-versus sulphur-based zinc
binding groups for carbonic anhydrase inhibitors? J Enzyme
Inhib Med Chem 2018;33:485–95.
Samples
AChE assaya
BChE assaya
Tyrosinase activitya
CX-1
CX-2
CX-3
CX-4
NA
NA
NA
NA
NA
NA
NA
NA
NA
24.46 0.53
19.55 0.43
35.52 0.82
16.48 0.21
NA
2. (a) Supuran CT, Capasso C. The eta-class carbonic anhy-
drases as drug targets for antimalarial agents. Expert Opin
Ther Targets 2015;19:551–63. (b) Del Prete S, De Luca V, De
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malaria pathogen Plasmodium falciparum. Bioorg Med
Chem 2015;23:526–31.
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mechanisms exist? J Enzyme Inhib Med Chem 2016;31:
345–60. (b) Supuran CT. Structure and function of carbonic
anhydrases. Biochem J 2016;473:2023–32. (c) Supuran CT.
Carbonic anhydrases: novel therapeutic applications for
inhibitors and activators. Nat Rev Drug Discov 2008;7:
168–81. (d) Neri D, Supuran CT. Interfering with pH regula-
tion in tumours as a therapeutic strategy. Nat Rev Drug
Discov 2011;10:767–77. (e) Supuran CT, Vullo D, Manole G,
et al. Designing of novel carbonic anhydrase inhibitors and
activators. Curr Med Chem Cardiovasc Hematol Agents 2004;
2:49–68.
4. (a) Akocak S, Ilies MA. Next-generation primary sulfonamide
carbonic anhydrase inhibitors. In: Supuran CT, Cappasso C,
eds. Targeting carbonic anhydrases. London: Future Science;
2014:35–51. (b) Akocak S, Alam MR, Shabana AM, et al.
PEGylated bis-sulfonamide carbonic anhydrase inhibitors can
efficiently control the growth of several carbonic anhydrase
IX-expressing carcinomas. J Med Chem 2016;59:5077–88. (c)
Shabana AM, Mondal UK, Alam R, et al. pH-Senstive multili-
gand gold nanoplatform targeting carbonic anhydrase IX
enhances the delivery of Doxorubicin to hypoxic tumor
spheroids and overcomes the hypoxia-induced chemoresist-
ance. ACS Appl Mater Interfaces 2018;10:17792–808. (d)
Akocak S, Supuran CT. Activation of a-, b-, c-, d-, f-, and g-
class of carbonic anhydrases with amines and amino acids: a
review. J Enzyme Inhib Med Chem 2019;34:1652–9.
5. (a) Supuran CT. Exploring the multiple binding modes of
inhibitors to carbonic anhydrases for novel drug discovery.
Expert Opin Drug Discov 2020. (b) Nocentini A, Supuran CT.
Advances in the structural annotation of human carbonic
anhydrases and impact on future drug discovery. Expert
Opin Drug Discov 2019; 14:1175–97. (c) Berrino E, Supuran
CT. Novel approaches for designing drugs that interfere
with pH regulation. Expert Opin Drug Discov 2019;14:
231–48.
CX-5
NA
CX-6
NA
80.69 0.59
–
35.41 0.90
76.50 1.28
–
28.15 0.74
–
95.26 0.23
Galantamineb
Kojik acidb
a% inhibition values at 200 mM.
bStandard drugs. NA: not active.
moderate activity against BChE with
%
inhibition value of
35.41 0.90. The tyrosinase activity of the compounds was also
moderate and close the each other, with % inhibition values in
the range of 16.48 0.21 to 35.52 0.82, except compound CX-5,
which showed no activity against tyrosinase (Table 3).
4. Conclusion
In the current work, we report a novel series of six calix[4]aza-
crown substituted sulphonamide Schiff bases which were synthes-
ised by the reaction of calix[4]arene dialdehydes with different
substituted primary and secondary sulphonamide derivatives. The
newly synthesised novel compounds were investigated as antioxi-
dant and metabolic enzyme inhibitors namely, carbonic anhy-
drase, acetylcholinesterase, butyrylcholinesterase, and tyrosinase
enzymes. The results revealed that these calix[4]azacrown based
sulphonamides CX(1–6) show, in general, low to moderate meta-
bolic enzyme inhibition against hCAs, AChE, BChE, and tyrosinase
enzymes. More specifically, only primary sulphonamide substituted
compound (CX-1) showed moderate activity against six different
isoforms of carbonic anhydrases with Ki values ranging from 0.15
to 5.55 mM. On the other hand, some of the synthesised com-
pounds showed great antioxidant activity comparable with stand-
ards used in the study, such as CX-6 (IC50 of 9.02 0.05 lM) for
DPPH radical scavenging assay and CX-5 and CX-6 (IC50
9.79 0.09 and 7.74 0.04 lM, respectively) for ABTS radical deco-
larisation assay.
Acknowledgements
The authors thank the Research Foundation of Selcuk University
for financial support of this work.
Disclosure statement
No potential conflict of interest was reported by the author(s).
6. (a) Gul HI, Yamali C, Yesilyurt F, et al. Microwave-assisted
synthesis and bioevaluation of new sulfonamides. J Enzyme
Inhib Med Chem 2017;32:369–74. (b) Supuran CT. Carbonic
anhydrases and metabolism. Metabolites 2018;8:25. (c)
Guzel-Akdemir O, Akdemir A, Karali N, et al. Discovery of
novel isatin-based sulfonamides with potent and selective
inhibition of the tumor-associated carbonic anhydrase iso-
forms IX and XII. Org Biomol Chem 2015;13:6493–9. (d)
Alterio V, Di Fiore A, D’Ambrosio K, et al. Multiple binding
modes of inhibitors to carbonic anhydrases: how to design
specific drugs targeting 15 different isoforms? Chem Rev
ORCID
Mehmet Oguz
Alessio Nocentini
Claudiu T. Supuran
http://orcid.org/0000-0002-3999-620X
http://orcid.org/0000-0003-3342-702X
http://orcid.org/0000-0003-4262-0323
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