Sulphonamides incorporating 1,3,5-triazine structural motifs show antioxidant, acetylcholinesterase, butyrylcholinesterase, and tyrosinase inhibitory profile

Article abstract of DOI:10.1080/14756366.2019.1707196

A series of 16 novel benzenesulfonamides incorporating 1,3,5-triazine moieties substituted with aromatic amines, dimethylamine, morpholine and piperidine were investigated. These compounds were assayed for antioxidant properties by using 1,1-diphenyl-2-pi

Full text of DOI:10.1080/14756366.2019.1707196

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Sulphonamides incorporating 1,3,5-
triazine structural motifs show antioxidant,
acetylcholinesterase, butyrylcholinesterase, and
tyrosinase inhibitory profile
Nabih Lolak, Mehmet Boga, Muhammed Tuneg, Gulcin Karakoc, Suleyman
Akocak & Claudiu T. Supuran
To cite this article: Nabih Lolak, Mehmet Boga, Muhammed Tuneg, Gulcin Karakoc,
Suleyman Akocak & Claudiu T. Supuran (2020) Sulphonamides incorporating 1,3,5-triazine
structural motifs show antioxidant, acetylcholinesterase, butyrylcholinesterase, and tyrosinase
inhibitory profile, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 424-431, DOI:
10.1080/14756366.2019.1707196
To link to this article: https://doi.org/10.1080/14756366.2019.1707196
©
2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 03 Jan 2020.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
020, VOL. 35, NO. 1, 424–431
2
https://doi.org/10.1080/14756366.2019.1707196
RESEARCH PAPER
Sulphonamides incorporating 1,3,5-triazine structural motifs show antioxidant,
acetylcholinesterase, butyrylcholinesterase, and tyrosinase inhibitory profile
a
b
b
a
a
c
Nabih Lolak , Mehmet Boga , Muhammed Tuneg , Gulcin Karakoc , Suleyman Akocak and Claudiu T. Supuran
a
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Adiyaman University, Adiyaman, Turkey; Department of Analytical Chemistry,
c
Faculty of Pharmacy, Dicle University, Diyarbakir, Turkey; NEUROFARBA Department, Sezione di Scienze Farmaceutiche, Universit ꢀa degli Studi di
Firenze, Florence, Italy
ABSTRACT
ARTICLE HISTORY
A series of 16 novel benzenesulfonamides incorporating 1,3,5-triazine moieties substituted with aromatic
amines, dimethylamine, morpholine and piperidine were investigated. These compounds were assayed for
antioxidant properties by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, 2, 2ꢀ -azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical decolarisation assay and metal chelating methods.
They were also investigated as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and
tyrosinase, which are associated with several diseases such as Alzheimer, Parkinson and pigmentation
disorders. These benzenesulfonamides showed moderate DPPH radical scavenging and metal chelating
activity, and low ABTS cation radical scavenging activity. Compounds 2 b, 3d and 3 h showed inhibitory
potency against AChE with % inhibition values of >90. BChE was also effectively inhibited by most of
the synthesised compounds with >90% inhibition potency. Tyrosinase was less inhibited by these compounds.
Received 23 November 2019
Revised 14 December 2019
Accepted 16 December 2019
KEYWORDS
Benzenesulfonamides; 1,3,5-
triazine; enzyme inhibition;
Alzheimer’s dis-
ease; tyrosinase
1
. Introduction
Thus, AChE and BChE inhibition are being considered as a one of
2
1,22
the most possible approaches for the treatment of AD
. There
The 1,3,5-triazine scaffold, also known as s-triazine, and its deriva-
tives have a wide range of applications due to broad biological
activities including antiviral, antibacterial, anti-inflammatory, anti-
HIV and more recently anti-cancer activity . Specifically, sulpho-
namides incorporating 1,3,5-triazine moieties were extensively
studied as a potent and selective carbonic anhydrase inhibi-
are several cholinesterase inhibitors used clinically for the treat-
ment of AD such as, donepezil, tacrine, rivastigmine and galanth-
amine. However, these drugs have a limited efficacy, are toxic and
1
–4
have unfavourable side effects such as dizziness, hepatotoxicity,
21,22
diarrhoea, vomiting and nausea
. For these reasons, there is an
urgent need for more potent and highly efficient cholinesterase
inhibitors for the management of AD.
5–10
tors
. Some of these compounds showed the best selectivity
ratio for tumour over-expressed membrane-bound carbonic anhy-
drase isoform IX (hCA IX) over the off-target isoform II (hCA II),
between 166 to 706 fold . More recently, ureido benzenesulfo-
namides incorporating 1,3,5-triazine motifs were investigated as a
potent class of inhibitors of the cancer related isoform human car-
It has recently been documented that 1,3,5-triazine derivatives,
specifically sulphonamide substituted ones, may show interesting
5–7
5–12
biological properties as enzyme inhibitors
. To the best of our
knowledge, there are no literature reports on the antioxidant, anti-
cholinesterase and tyrosinase activities of 1,3,5-triazines containing
sulphonamides. Therefore, in this study, we aimed to evaluate the
antioxidant, anticholinesterase and tyrosinase activities of novel
sulphonamides incorporating 1,3,5-triazine moieties.
1
1,12
bonic anhydrase IX (hCA IX) by our group
.
Alzheimer’s disease (AD), a progressive neurodegenerative dis-
order of the brain, is characterised by cognitive dysfunction, mem-
ory decrease, speech impairment and dementia, which is the
1
3–15
leading cause of disability in elderly people in the world
. It
affects about 50 million people worldwide and this number might 2. Materials and methods
1
6
triplicate to 152 million by 2050 (World Alzheimer report) , as life
expectancy is increasing worldwide. Acetylcholine (ACh), a neuro-
2.1. Chemistry
transmitter responsible for the conduction of electrical impulses All chemicals and anhydrous solvents were purchased from
from one nerve to another, is one of the main biochemical alter- Sigma-Aldrich, Merck, Alfa Aesar and TCI and used without further
16–18
ation in the brain of patients with Alzheimer
. The level of purification. FT-IR spectra were obtained by using Perkin Elmer
ACh decreases due to its hydrolysis by acetylcholinesterase Spectrum 100 FT-IR spectrometer. Nuclear Magnetic Resonance
1
13
(
AChE), which is a terminator enzyme of nerve impulse transmis- ( H-NMR and
C-NMR) spectra of compounds were recorded
sion. Butyrylcholinesterase (BChE) is a non-specific cholinesterase using a Bruker Advance III 300 MHz spectrometer in DMSO-d and
6
1
9,20
1
enzyme that hydrolyses many different choline-based esters
.
TMS as an internal standard operating at 300 MHz for H-NMR and
CONTACT Mehmet Boga
Suleyman Akocak akocaksuleyman@gmail.com; sakocak@adiyaman.edu.tr;
University, Adiyaman, Turkey; Claudiu T. Supuran
di Firenze, Florence, Italy
mehmetboga1980@gmail.com
Department of Analytical Chemistry, Faculty of Pharmacy, Dicle University, Diyarbakir, Turkey;
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Adiyaman
NEUROFARBA Department, Sezione di Scienze Farmaceutiche, Universit ꢀa degli Studi
claudiu.supuran@unifi.it
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.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
425
1
3
7
5 MHz for C-NMR. Thin-layer chromatography (TLC) was carried 4-((4-(dimethylamino)-6-((4-fluorophenyl)amino)-1,3,5-triazin-2-
out on Merck silica gel 60 F254 plates.
yl)amino) benzenesulfonamide (3a). Yield: 77%; Colour: white
ꢀ
ꢁ1
solid; m.p.: 222–225 C; FT-IR (cm ): 3366, 3325, 3262, 1625, 1485
1
(
asymmetric), 1337, 1159 (symmetric) (S ¼ O); H-NMR (DMSO-d
6
,
2
.1.1. General procedure for the synthesis of compounds 2(a–d)
300 MHz, d ppm): 8.05 (d, 2H, J ¼ 6.3 Hz, Ar-H), 7.91 (d, 2H,
ꢀ
At 0–5 C, a 10 mmol solution of R1 (aromatic amine derivatives, J ¼ 6.3 Hz, Ar-H), 7.72 (d, 2H, J ¼ 6.6 Hz, Ar-H), 7. 58 (d, 2H,
1
3
-
4 F, -4MeO, -3,4diCl, -3NO
2
) was added to 5 mmol of compound 1 J ¼ 6.6 Hz, Ar-H), 7.49 (s, 2H, -SO
, 75 MHz, d ppm): 167.2, 165.6, 164.5, 157.2, 143.5, 136.4,
30.8, 129.3, 120.9, 117.2, 36.8;
2 2 3
NH ), 3.05 (s, 6H, -CH ): C-NMR
in DMF under stirring. After complete addition, the mixture was (DMSO-d
6
1
allowed to warm to room temperature for 1 h, after that the reac-
ꢀ
tion mixture was heated to 30–40 C for 6–8 h. Then, the product
was filtered off, washed with water and dried under vacuum at 4-((4-((4-fluorophenyl)amino)-6-morpholino-1,3,5-triazin-2-yl)ami-
ꢀ
4
0 C. The obtained final pure products were fully characterised no)benzenesulfonamide (3 b). Yield: 83%; Colour: white solid; m.p.:
1
13
ꢀ
ꢁ1
by FT-IR, H-NMR, C-NMR, and melting points.
249–252 C; FT-IR (cm ): 3334, 3263, 3202, 1605, 1484 (asymmet-
1
ric), 1356, 1152 (symmetric) (S ¼ O); H-NMR (DMSO-d , 300 MHz, d
6
ppm): 8.01 (d, 2H, J ¼ 6.6 Hz, Ar-H), 7.89 (d, 2H, J ¼ 6.3 Hz, Ar-H),
4
-((4-chloro-6-((4-fluorophenyl)amino)-1,3,5-triazin-2-yl)amino)ben-
7
.70 (d, 2H, J ¼ 6.6 Hz, Ar-H), 7. 55 (d, 2H, J ¼ 6.6 Hz, Ar-H), 7.46 (s,
13
zenesulfonamide (2a). Yield: 75%; Colour: white solid; m.p.:
62–265 C; FT-IR (cm ): 3418, 3309, 3248, 1617, 1496 (asymmet-
ric), 1322, 1157 (symmetric) (S ¼ O); H-NMR (DMSO-d
ppm): 7.92 (d, 2H, J ¼ 6.3, Ar-H), 7.85 (d, 2H, J ¼ 6.3, Ar-H), 7.76 (d,
H, J ¼ 6.6, Ar-H), 7. 55 (d, 2H, J ¼ 6.3, Ar-H), 7.38 (s, 2H, -SO NH ):
C-NMR (DMSO-d , 75 MHz, d ppm): 169.8 (C-Cl), 167.2, 165.5
ꢀ
ꢁ1
2 2 6
2H, -SO NH ), 3.80–3.42 (m, 8H, morpholine): C-NMR (DMSO-d ,
2
1
75 MHz, d ppm): 166.9, 165.3, 164.2, 157.5, 143.1, 136.6, 130.3,
6
, 300 MHz, d
1
29.5, 120.7, 117.6, 65.4, 42.5;
2
2
2
1
3
4-((4-((4-fluorophenyl)amino)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)a-
6
mino) benzenesulfonamide (3c). Yield: 85%; Colour: white solid;
(
Ctriaz-N), 157.4 (C-F), 143.7, 135.4, 130.9, 130.1, 120.7, 116.4, 113.7;
ꢀ
ꢁ1
m.p.: 228–230 C; FT-IR (cm ): 3319, 3260, 1603, 1494 (asymmet-
1
ric), 1336, 1151 (symmetric) (S ¼ O); H-NMR (DMSO-d
6
, 300 MHz, d
4
-((4-chloro-6-((4-methoxyphenyl)amino)-1,3,5-triazin-2-yl)amino)-
ppm): 8.04 (d, 2H, J ¼ 6.6 Hz, Ar-H), 7.88 (d, 2H, J ¼ 6.3 Hz, Ar-H),
benzenesulfonamide (2 b). Yield: 68%; Colour: white solid; FT-IR
7
.72 (d, 2H, J ¼ 6.6 Hz, Ar-H), 7. 56 (d, 2H, J ¼ 6.6 Hz, Ar-H), 7.47 (s,
H, -SO NH ), 3.43–3.21 (m, 4H, piperidine), 1.75–1.45 (m, 6H,
ꢁ
1
(
(
2
cm ): 3447, 3316, 3255, 1623, 1505 (asymmetric), 1329, 1162
2
1
2
2
13
symmetric) (S ¼ O); H-NMR (DMSO-d
H, J ¼ 6.3, Ar-H), 7.81 (d, 2H, J ¼ 6.3, Ar-H), 7.74 (d, 2H, J ¼ 6.6, Ar-
H), 7. 45 (d, 2H, J ¼ 6.3, Ar-H), 7.33 (s, 2H, -SO NH ), 3.85 (s, 3H,
, 75 MHz, d ppm): 169.5 (C-Cl), 167.1,
65.2 (Ctriaz-N), 156.3, 143.2, 135.8, 131.4, 130.3, 120.4, 116.2,
13.3, 56.5;
6
, 300 MHz, d ppm): 7.89 (d,
piperidine):
6
C-NMR (DMSO-d , 75 MHz, d ppm): 166.7, 165.4,
1
2
64.1, 157.6, 143.0, 136.8, 130.5, 129.4, 120.8, 117.3, 42.7,
5.4, 24.2;
2
2
1
3
-
1
1
3 6
OCH ): C-NMR (DMSO-d
4-((4-(dimethylamino)-6-((4-methoxyphenyl)amino)-1,3,5-triazin-2-
yl)amino) benzenesulfonamide (3d). Yield: 77%; Colour: cream
ꢁ
1
solid; FT-IR (cm ): 3447, 3310, 3265, 1613, 1495 (asymmetric),
4
-((4-chloro-6-((3,4-dichlorophenyl)amino)-1,3,5-triazin-2-yl)ami-
1
1
325, 1160 (symmetric) (S ¼ O); H-NMR (DMSO-d , 300 MHz, d
6
no)benzenesulfonamide (2c). Yield: 55%; Colour: white solid; m.p.:
1
ppm): 7.87 (d, 2H, J ¼ 6.6, Ar-H), 7.79 (d, 2H, J ¼ 6.3, Ar-H), 7.70 (d,
ꢀ
ꢁ1
93–196 C; FT-IR (cm ): 3430, 3306, 3262, 1620, 1508 (asymmet-
2
3
H, J ¼ 6.6, Ar-H), 7. 46 (d, 2H, J ¼ 6.3, Ar-H), 7.38 (s, 2H, -SO
2
NH
2
),
1
ric), 1336, 1160 (symmetric) (S ¼ O); H-NMR (DMSO-d
6
, 300 MHz, d
13
.87 (s, 3H, -OCH ), 3.03 (s, 6H, -CH ): C-NMR (DMSO-d , 75 MHz,
3 3 6
ppm): 8.06 (d, 2H, J ¼ 6.3, Ar-H), 7.92 (d, 2H, J ¼ 6.3, Ar-H), 7.60 (s,
d ppm): 169.3, 167.0, 165.4, 156.9, 143.8, 136.2, 131.2, 129.9, 120.6,
1
3
H, Ar-H), 7. 58 (d, 2H, J ¼ 6.3, Ar-H), 7.45 (s, 2H, -SO
DMSO-d , 75 MHz, d ppm): 170.2, 167.5, 165.7, 164.1, 157.2, 150.4,
42.9, 136.3, 130.6, 129.9, 126.4, 120.6, 118.1;
2 2
NH ): C-NMR
1
16.9, 56.8, 36.9;
(
1
6
4-((4-((4-methoxyphenyl)amino)-6-morpholino-1,3,5-triazin-2-yl)a-
mino) benzenesulfonamide (3e). Yield: 80%; Colour: cream solid,
m.p.: 209–212 C; FT-IR (cm ): 3437, 3305, 3255, 1610, 1502
4
-((4-chloro-6-((3-nitrophenyl)amino)-1,3,5-triazin-2-yl)amino)ben-
ꢀ
ꢁ1
1
zenesulfonamide (2d). Yield: 68%; Colour: white solid; FT-IR
(
(
(
asymmetric), 1328, 1159 (symmetric) (S ¼ O); H-NMR (DMSO-d
6
,
ꢁ
1
cm ): 3337, 3272, 3132, 1614, 1490 (asymmetric), 1350, 1156 300 MHz, d ppm): 7.90 (d, 2H, J ¼ 6.3, Ar-H), 7.82 (d, 2H, J ¼ 6.0, Ar-
1
symmetric) (S ¼ O); H-NMR (DMSO-d , 300 MHz, d ppm): 7.95 (d, H), 7.73 (d, 2H, J ¼ 6.6, Ar-H), 7. 49 (d, 2H, J ¼ 6.3, Ar-H), 7.39 (s,
6
2
H, J ¼ 6.0, Ar-H), 7.89 (d, 2H, J ¼ 6.0, Ar-H), 7.58 (s, H, Ar-H), 7. 56 2H, -SO NH ), 3.86 (s, 3H, -OCH ), 3.79–3.43 (m, 8H, morpholine):
2
2
3
13
13
(m, 3H, Ar-H), 7.42 (s, 2H, -SO
2
NH
2
): C-NMR (DMSO-d
6
, 75 MHz, d
6
C-NMR (DMSO-d , 75 MHz, d ppm): 169.7, 167.3, 165.2, 157.3,
ppm): 169.8, 167.2, 165.1, 163.8, 157.0, 150.8, 142.3, 135.9, 130.2, 143.2, 136.4, 131.0, 129.8, 120.2, 116.6, 65.3, 56.7, 42.2;
1
29.5, 125.8, 120.1, 117.5;
4-((4-((4-methoxyphenyl)amino)-6-(piperidin-1-yl)-1,3,5-triazin-2-
yl)amino) benzenesulfonamide (3f). Yield: 82%; Colour: cream
solid, m.p.: 252–254 C; FT-IR (cm ): 3425, 3312, 3248, 1605, 1501
ꢀ
ꢁ
1
2
.1.2. General procedure for the synthesis of compounds 3(a-l)
1
2
Under stirring, a 2 mmol solution of R -H (dimethyl amine, mor-
(
asymmetric), 1332, 1160 (symmetric) (S ¼ O); H-NMR (DMSO-d6,
pholine and piperidine) was added to 1 mmol of 2(a-d) in DMF at 300 MHz, d ppm): 7.88 (d, 2H, J ¼ 6.3, Ar-H), 7.80 (d, 2H, J ¼ 6.0, Ar-
room temperature for 1 h. Then, the reaction temperature was H), 7.71 (d, 2H, J ¼ 6.6, Ar-H), 7. 47 (d, 2H, J ¼ 6.3, Ar-H), 7.40 (s,
ꢀ
raised to 90 C for 5 h. After cooling to room temperature, the 2H, -SO NH ), 3.88 (s, 3H, -OCH ), 3.45–3.28 (m, 4H, piperidine),
2
2
3
1
3
mixture was filtered and the precipitate was washed with water 1.77–1.46 (m, 6H, piperidine):):
C-NMR (DMSO-d , 75 MHz, d
6
ꢀ
and dried at 50 C. The obtained final pure products 3(a-o) were ppm): 169.1, 167.7, 165.1, 157.8, 143.6, 136.2, 130.6, 129.7, 120.1,
fully characterised by FT-IR, H-NMR, C-NMR, and melting points. 116.2, 56.8, 42.4, 25.1, 24.4;
1
13

4
26
N. LOLAK ET AL.
4
2
-((4-((3,4-dichlorophenyl)amino)-6-(dimethylamino)-1,3,5-triazin-
2.2. Determination of the antioxidant, anticholinesterase and
-yl)amino) benzenesulfonamide (3 g). Yield: 75%; Colour: white tyrosinase activity of benzenesulfonamides 2(a-d) and 3(a-l)
ꢀ
ꢁ1
solid; m.p.: 254–257 C; FT-IR (cm ): 3353, 3270, 3262, 1600, 1502
2.2.1. DPPH radical scavenging assay
1
(
3
asymmetric), 1327, 1150 (symmetric) (S ¼ O); H-NMR (DMSO-d
6
,
The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activ-
ity of the synthesised compounds was determined by spectro-
00 MHz, d ppm): 8.04 (d, 2H, J ¼ 6.6, Ar-H), 7.94 (d, 2H, J ¼ 6.3, Ar-
H), 7.58 (s, H, Ar-H), 7. 53 (d, 2H, J ¼ 6.3, Ar-H), 7.42 (s, 2H,
photometric method based on the reduction of an ethanol
13
-
SO NH ), 3.08 (s, 6H, -CH ): C-NMR (DMSO-d , 75 MHz, d ppm):
23,24
2
2
3
6
solution of DPPH
. 2, 5, 10, 20 mL of 1 mM stock solution of
1
1
69.8, 167.3, 165.6, 164.4, 157.5, 150.2, 141.7, 136.2, 130.3, 129.5,
26.6, 120.8, 118.3, 36.6;
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 as follows:
4
-((4-((3,4-dichlorophenyl)amino)-6-morpholino-1,3,5-triazin-2-yl)a-
mino) benzenesulfonamide (3 h). Yield: 78%; Colour: white solid;
m.p.: 199–201 C; FT-IR (cm ): 3329, 3211, 1625, 1490 (asymmet-
ꢀ
ꢁ1
1
I% ¼ ðAcontrol–AsampleÞ=Acontrol ꢂ 100,
ric), 1337, 1153 (symmetric) (S ¼ O); H-NMR (DMSO-d , 300 MHz, d
6
ppm): 8.06 (d, 2H, J ¼ 6.6, Ar-H), 7.92 (d, 2H, J ¼ 6.3, Ar-H), 7.57 (s, where Acontrol is the absorbance of the control reaction (contain-
H, Ar-H), 7. 52 (d, 2H, J ¼ 6.3, Ar-H), 7.44 (s, 2H, -SO
2
NH
, 75 MHz, d the absorbance of the test compounds. Tests were carried out in
ppm): 169.4, 167.1, 165.8, 164.3, 157.2, 150.0, 141.9, 136.6, 130.8, triplicate. BHA, BHT and a-Tocopherol were used as positive
2
), ing all reagents except for the tested compounds), and Asample is
13
3
.81–3.46 (m, 8H, morpholine):
C-NMR (DMSO-d
6
2
5–28
1
29.2, 126.5, 120.2, 118.6, 65.6, 42.2;
control
.
4
-((4-((3,4-dichlorophenyl)amino)-6-(piperidin-1-yl)-1,3,5-triazin-2-
2
.2.2. ABTS cation radical decolorisation assay
yl)amino) benzenesulfonamide (3i). Yield: 81%; Colour: white solid;
m.p.: 245–248 C; FT-IR (cm ): 3340, 3211, 2935, 2854, 1603, 1496
ꢁ1
The percent inhibition of decolorisation of ABTS (2,2ꢀ-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid)) cation radical is obtained as
a function of time and concentration, and evaluated by compari-
son with the BHT, BHA and a-Tocopherol compounds used as
ꢀ
1
(
3
asymmetric), 1361, 1155 (symmetric) (S ¼ O); H-NMR (DMSO-d
00 MHz, d ppm): 8.02 (d, 2H, J ¼ 6.3, Ar-H), 7.89 (d, 2H, J ¼ 6.0, Ar-
H), 7.54 (s, H, Ar-H), 7. 50 (d, 2H, J ¼ 6.0, Ar-H), 7.41 (s, 2H,
SO NH ), 3.44–3.27 (m, 4H, piperidine), 1.72–1.41 (m, 6H, piperi-
6
,
2
9,30
standard
. The tested compounds at different concentrations
-
2
2
1
3
are added to each well and 160 mL of 7 mM ABTS solution is
added. After 6 min at room temperature, the absorbances were
measured at 734 nm. ABTS cation radical decolorisation activities
were determined by
dine): C-NMR (DMSO-d
6
, 75 MHz, d ppm): 168.2, 166.9, 165.2,
1
4
64.1, 157.8, 150.3, 141.5, 136.4, 130.3, 129.6, 126.2, 120.4, 118.7,
2.5, 25.1, 24.4;
%Inhibition ¼ ðAcontrol ꢁ AsampleÞ=Acontrol ꢂ 100
4
-((4-(dimethylamino)-6-((3-nitrophenyl)amino)-1,3,5-triazin-2-yl)a-
mino) benzenesulfonamide (3j). Yield: 78%; Colour: light yellow
solid; m.p.: 233–235 C; FT-IR (cm ): 3345, 3282, 3135, 1612, 1498
where A is the absorbance. Tests were carried out in triplicate.
BHA, BHT and a-Tocopherol were used as positive control.
ꢀ
ꢁ1
1
(
3
asymmetric), 1345, 1158 (symmetric) (S ¼ O); H-NMR (DMSO-d6,
00 MHz, d ppm): 7.97 (d, 2H, J ¼ 6.3, Ar-H), 7.87 (d, 2H, J ¼ 6.3, Ar-
H), 7.55 (s, H, Ar-H), 7. 51 (m, 3H, Ar-H), 7.42 (s, 2H, -SO NH
), 3.05 2.2.3. Metal chelating activity
, 75 MHz, d ppm): 169.6, 167.5, The chelating ability of synthesised compounds was examined
2
2
1
3
(
1
1
s, 6H, -CH
3
): C-NMR (DMSO-d
64.9, 163.5, 157.2, 150.5, 142.1, 135.4, 130.7, 129.8, 125.4, 120.8, according to the method of Dinis et al. The tested compounds
17.2, 36.8;
at different concentrations were added to each well and 4 mL of
mM ferrous (II) chloride was added. Then 8 mL of 5 mM ferrozine
6
3
1
2
was added and the reaction was started. After 10 min at room
temperature, the absorbance was measured at 562 nm against
blank. The results were expressed as percentage of inhibition of
4
-((4-morpholino-6-((3-nitrophenyl)amino)-1,3,5-triazin-2-yl)ami-
no)benzenesulfonamide (3k). Yield: 86%; Colour: light yellow solid;
m.p.: 215–218 C; FT-IR (cm ): 3338, 3280, 3145, 1615, 1502
ꢀ
ꢁ1
2þ
1
the ferrozine-Fe complex formation. The percentage inhibition
(
3
asymmetric), 1348, 1159 (symmetric) (S ¼ O); H-NMR (DMSO-d
00 MHz, d ppm): 7.99 (d, 2H, J ¼ 6.3, Ar-H), 7.85 (d, 2H, J ¼ 6.3, Ar-
H), 7.59 (s, H, Ar-H), 7. 52 (m, 3H, Ar-H), 7.44 (s, 2H, -SO NH ),
6
,
2þ
of the ferrozine -Fe complex formation was calculated as fol-
lows:
2
2
13
3
.83–3.48 (m, 8H, morpholine):
C-NMR (DMSO-d
6
, 75 MHz, d
Chelating abilityð%Þ ¼ ðAcontrol ꢁ AsampleÞ=Acontrol ꢂ 100,
ppm): 169.0, 167.8, 164.5, 163.2, 157.8, 150.3, 142.4, 135.8, 130.1,
where A is the absorbance. Tests were carried out in triplicate.
1
29.3, 125.6, 120.5, 117.8, 65.5, 42.4;
3
2–34
EDTA was used as a positive control
.
4
-((4-((3-nitrophenyl)amino)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)a-
mino) benzenesulfonamide (3 l). Yield: 84%; Colour: light yellow 2.2.4. Anti-cholinesterase assay
ꢀ
ꢁ1
solid; m.p.: 181–184 C; FT-IR (cm ): 3341, 3215, 3125, 1602, 1502 The inhibitory effect of novel benzenesulfoanmides incorporating
(
3
1
asymmetric), 1350, 1154 (symmetric) (S ¼ O); H-NMR (DMSO-d6, 1,3,5-triazine moieties 2(a-d) and 3(a-l) on AChE and BChE activ-
00 MHz, d ppm): 7.96 (d, 2H, J ¼ 6.0, Ar-H), 7.82 (d, 2H, J ¼ 6.3, Ar- ities was determined according to the slightly modified spectro-
35
2 2
H), 7.58 (s, H, Ar-H), 7. 50 (m, 3H, Ar-H), 7.41 (s, 2H, -SO NH ), photometric method of Ellman et al. . All compounds were
1
3
3
.46–3.29 (m, 4H, piperidine), 1.74–1.45 (m, 6H, piperidine): C- dissolved in DMSO to prepare stock solutions at 4 mM concentra-
NMR (DMSO-d , 75 MHz, d ppm): 168.8, 167.3, 164.6, 162.7, 157.4, tion. Aliquots of 150 mL of 100 mM sodium phosphate buffer (pH
6
1
2
50.6, 142.1, 135.4, 130.7, 129.2, 125.1, 120.0, 117.2, 42.3, 8.0), 10 mL of sample solution and 20 mL AChE (or BChE) solution
ꢀ
5.0, 24.2;
were mixed and incubated for 15 min at 25 C, and DTNB (5,5’-

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
427
based on our previous work which showed efficient carbonic
anhydrase IX (tumour over-expressed isozyme) inhibition potency
5–12
associated with such derivatives
. A number of structurally
diverse benzenesulfonamides incorporating 1,3,5-triazine moieties
were synthesised according to the general synthetic route
depicted in Scheme 1. In order to generate chemical diversity, dif-
ferent substituted aromatic amines (-4 F, -4MeO, -3,4diCl and
-
3NO substituted anilines) were chosen and reacted at one side
2
of the triazine moiety, whereas on the other side the derivatisa-
tion was achieved by using dimethlyamine, morpholine and
piperidine functionalities.
The synthesis of benzenesulfonamides incorporating 1,3,5-tria-
zine moieties 2(a-d) and 3(a-l) was carried out according to the
1
1,12
procedure described in our previous papers
ing key intermediate compound 1 was coupled with substituted
aromatic anilines (-4 F, -4MeO, -3,4diCl and -3NO ), leading to for-
. Briefly, the start-
2
mation of compounds 2(a-d). After that, the third chloride atom
of the starting material 1,3,5-triazine (cyanuric chloride) was deri-
vatised with dimethylamine, morpholine and piperidine to pro-
duce compounds 3(a-l). The structures of benzenesulfonamides
incorporating 1,3,5-triazine moieties 2(a-d) and 3(a-l) were con-
Scheme 1. General synthetic route for the synthesis of benzenesulfonamides
incorporating 1,3,5-triazine moieties. Reagents and conditions: (i) R (–4 F, –4MeO,
1
ꢀ
ꢀ
–
3,4diCl, and –3NO
2
), DMF, 0 to 5 C, 1 h, then 30–40 C, 8 h, (ii) R
2
H (dimethyl-
1
ꢀ
firmed by using several analytical and spectral data (FT-IR, H-
amine, morpholine and piperidine), DMF, room temperature, 1 h, then 90 C, 5 h.
1
3
NMR, C-NMR, and melting points) as described in the experi-
mental part.
Dithio-bis(2-nitro-benzoic)acid) (10 mL) is added. The reaction was
then initiated by the addition of acetylthiocholine iodide (or
butyrylthiocholine iodide) (10 mL). The final concentration of the
tested compounds’ solution was 200 mM.
3.2. Antioxidant activity
The benzenesulfonamides incorporating 1,3,5-triazine moieties
were screened for their antioxidant activity by three methods,
namely DPPH free radical scavenging, ABTS cation radical scaveng-
ing, and metal chelating activity. All of the compounds showed
antioxidant activities in a dose-dependent manner and the results
were summarised in Table 1, which demonstrates the IC50 values
of the synthesised derivatives and standard compounds (BHA,
%
Inhibition ¼ ðAcontrol ꢁ AsampleÞ=Acontrol ꢂ 100,
where A is the absorbance. Tests were carried out in triplicate.
Galantamine was used as positive control.
2
.2.5. Anti-tyrosinase activity
Anti-tyrosinase activity of the compounds was performed accord- BHT, a-tocopherol, and EDTA).
36
ing to the method designed by Hearing and Jimenez . The inhib-
The results revealed that benzenesulfonamides incorporating
ition of the diaphanous function of the compounds was evaluated 1,3,5-triazine moieties 2(a-d) and 3(a-l) shows, in general, moder-
with L-DOPA as substrate. Tyrosinase from mushroom (E.C. ate DPPH radical scavenging and metal chelating activity, and low
1
¼
.14.18.1) (30 U, 28 nM) was dissolved in Na-phosphate buffer (pH ABTS cation radical scavenging activity. Specifically, three com-
6.8, 50 nM) and the compounds were added to the solution for pounds from the synthesised derivatives (3 b, 3 h and 3j) indicates
pre-incubation at room temperature for ten minutes. After incuba- high DPPH radical scavenging activity with IC50 values of 73.25,
tion, 0.5 mM L-DOPA was added to the mixture and the change in 60.18, and 58.59 mM, respectively. These compounds have better
ꢀ
absorbance was measured at 475 nm at 37 C. For the positive antioxidant activity than standards BHA (IC50: 61.72 mM) and BHT
control, kojic acid was used as an inhibitor. The following formula (IC50: 232.11 mM). On the other hand, compounds 3a, 3c, 3e, and
was used to calculate the percentage of all enzyme inhibitions:
3f showed any activity with IC50 values of >1000 mM.
Furthermore, the ABTS cation radical scavenging activity of the
compounds was also assayed and compared with standards BHT,
BHA, and a-Tocopherol. All compounds showed weak activity
with IC50 values of <1000 mM, except the compounds 3d, 3f and
Inhibitionð%Þ ¼ ðAcontrol–AsampleÞ=Acontrol ꢂ 100,
where A is absorbance.
3
k exhibited moderate activity with IC50 values of 294.12, 408.44
2
.3. Statistical analysis
and 481.21 mM, respectively (Table 1). The metal chelating activity
of the synthesised compounds was also screened and compared
with standard EDTA. None of the compounds showed better
activity than standard. Moreover, several derivatives (3a, 3e and
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
Student’s t-test, where
significant.
3
k) displayed close metal chelating activity to EDTA with IC50 val-
p values < 0.05 were considered
ues of 84.98, 84.32 and 88.42 mM, respectively. The remaining
compounds were shown to have moderate metal chelating activ-
ity with IC50 values ranging from 98.84 to 488.29 mM.
3
. Result and discussion
In the present study, sulphonamides incorporating 1,3,5-triazine
moieties 2(a-d) and 3(a-l) were also evaluated for their anticholi-
nesterase (AChE and BChE) activities (Table 2). In general, all com-
3.1. Chemistry
The rationale for designing this novel benzenesulfonamides incor- pounds showed better BChE inhibitory activity than AChE, except
porating 1,3,5-triazine structural motifs presented in this work are the compound 2 b, which displayed more AChE inhibitory activity

4
28
N. LOLAK ET AL.
Table 1. Antioxidant activity of sulphonamides 2 and 3.
a
IC50 (mM)
DPPH free radical
Scavenging activity
ABTS cation radical
Scavenging activity
Metal chelating
Activity
Comp.
R1
–4F
–4MeO
–3,4diCl
–3NO
R2
2
2
2
2
3
a
b
c
d
a
Cl
Cl
Cl
Cl
469.75 ± 1.17
500.97 ± 1.17
304.52 ± 1.38
443.26 ± 1.38
>1000
>1000
>1000
>1000
>1000
>1000
488.29 ± 0.84
164.26 ± 0.68
109.63 ± 0.80
296.78 ± 0.52
84.98 ± 1.14
2
–4F
–N(Me)
–N(Me)
–N(Me)
–N(Me)
2
3
b
–4F
–4F
73.25 ± 0.52
>1000
148.03 ± 0.61
3c
>1000
>1000
338.90 ± 0.59
3
d
e
–4MeO
–4MeO
2
102.65 ± 1.17
294.12 ± 1.20
337.51 ± 0.55
84.32 ± 0.39
3
>1000
>1000
3f
–4MeO
>1000
408.44 ± 1.67
98.84 ± 0.90
3
g
h
–3,4diCl
–3,4diCl
2
609.35 ± 0.98
60.18 ± 0.59
>1000
>1000
139.15 ± 1.15
147.60 ± 0.82
3
3i
–3,4diCl
351.97 ± 1.33
>1000
99.10 ± 0.52
3j
–3NO
–3NO
2
2
2
58.59 ± 0.12
>1000
98.84 ± 0.90
88.42 ± 0.75
3k
336.28 ± 1.43
481.21 ± 0.97
3l
–3NO
2
114.38 ± 0.60
>1000
115.46 ± 0.87
b
BHA
BHT
–
–
–
–
–
–
–
–
61.72 ± 0.85
232.11 ± 3.01
56.86 ± 0.77
–
45.40 ± 1.08
26.54 ± 0.18
34.12 ± 0.41
–
–
–
–
b
b
a-Tocopherol
EDTA
b
52.35 ± 1.15
a
IC values represent the means (standard deviation of three parallel measurements (p < 0.05).
5
0
b
Reference compounds.
(% inhibition 96.37) than BChE (91.99). Compounds 2 b, 3d and and 3 h) displayed higher activity against both enzyme (AChE and
3
h found to have most potent AChE inhibitors, having better % BChE) together than standard drug galantamine, which is a one of
inhibition than standard drug galantamine, with % inhibition val- the most important findings of the current study. These three
ues of 96.37, 91.10 and 93.19, respectively. Some compounds compounds gave promising anticholinesterase activity and might
from the series (2a, 2c, 3a, 3e, 3f, 3 g, 3i, 3j, and 3 l) showed any be improved and used as powerful cholinesterase inhibitors.
activity (NA) against AChE enzyme. The remaining compounds
Tyrosinase is a bi-functional copper containing enzyme which
(
2d, 3 b, 3c, and 3k) showed low activity to AChE enzyme with % is widely distributed in different organisms such as animals, plants,
3
7
inhibition values ranging from 14.18 to 36.55. On the other hand, and microorganisms . This enzyme catalyses hydroxylation of
BChE enzyme was effectively inhibited by most of the synthesised monophenols to o-diphenols which generates melanin
3
7,38
.
compounds. Specifically, compounds 2 b, 2d, 3d, 3f, 3 h, 3j, and Tyrosinase inhibitors capable of inhibiting the biosynthesis of mel-
3
9
40
3
l showed >90% inhibition, which is higher than standard drug anin are used for various applications in the food , cosmetics
3
7,38
galantamine with a % inhibition value of 87.86. Among the and medicinal industries
. In the present study, tyrosinase
remaining compounds, some of them (2c, 3 b, 3c, and 3i) showed enzyme inhibition activity was also evaluated and moderate activ-
close % inhibition scale to standard drug with % inhibition values ity was observed against this enzyme (Table 2). In general, none
of 87.44, 88.76, 89.21, and 88.48, respectively. Only one non active of the compounds showed better inhibition potency than stand-
compound (3e) was observed against BChE enzyme, in which this ard Kojic acid, which has % inhibition value of 95.26. Two com-
compound also inactive against AChE enzyme, too (Table 2). pounds showed high activity, 3f and 3j, with % inhibition values
Interestingly, three compounds from the current series (2 b, 3d of 86.35 and 77.30, respectively. The remaining compounds

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
429
Table 2. Anti-cholinesterase and anti-tyrosinase activity of compounds 2 and 3.
Anticholinesterase activity
a
a
Comp.
R1
–4F
–4MeO
–3,4diCl
–3NO
–4F
R2
AChE assay
NA
96.37 ± 1.71
NA
22.38 ± 1.09
NA
BChE assay
Tyrosinase activity
2
2
2
2
3
a
b
c
d
a
Cl
Cl
Cl
Cl
77.41 ± 1.19
91.99 ± 0.60
87.44 ± 1.46
91.40 ± 1.48
34.67 ± 0.83
38.96 ± 0.12
14.21 ± 0.40
33.44 ± 0.82
NA
2
–N(Me)
–N(Me)
–N(Me)
–N(Me)
2
2
2
2
13.50 ± 0.04
3
b
c
–4F
–4F
14.18 ± 0.41
36.55 ± 1.17
88.76 ± 0.97
15.34 ± 0.87
3
89.21 ± 1.75
6.29 ± 0.39
3
3
d
e
–4MeO
–4MeO
91.10 ± 1.42
NA
96.94 ± 1.61
NA
27.76 ± 0.22
30.37 ± 0.17
3f
–4MeO
NA
95.14 ± 1.17
86.35 ± 1.39
3
3
g
h
–3,4diCl
–3,4diCl
NA
10.23 ± 0.70
98.50 ± 1.63
58.49 ± 0.86
24.13 ± 0.52
93.19 ± 2.33
3
3
i
j
–3,4diCl
NA
88.48 ± 1.29
23.21 ± 0.97
–3NO
3NO
2
2
NA
92.14 ± 1.02
65.44 ± 1.19
77.30 ± 1.42
18.40 ± 0.43
–
28.42 ± 0.76
3l
–3NO
2
NA
94.94 ± 0.24
58.59 ± 0.89
b
Galantamine
–
–
84.20 ± 0.74
–
87.86 ± 0.24
–
b
Kojic Acid
–
95.26 ± 0.23
a
%
inhibition values at 200 mM.
bStandard drugs. NA: not active.
indicated moderate activity depends on the substitution on com- Disclosure statement
pounds with % inhibition values ranging from 6.29 to 58.59. Only
one compound (2d) was none active against this enzyme.
No potential conflict of interest was reported by the authors.
Funding
4
. Conclusions
This work was supported by the Scientific and Technological
Research Council of Turkey (TUBITAK) [grant number 315S103].
In the present study, we report a novel series of benzenesulfona-
mides incorporating 1,3,5-triazine moieties substituted with aro-
matic amine derivatives, dimethylamine, morpholine and
piperidine. The novel compounds were investigated as an antioxi- ORCID
dant and inhibitors of AChE, BChE and tyrosinase enzymes. The
Claudiu T. Supuran
http://orcid.org/0000-0003-4262-0323
results revealed that benzenesulfonamides incorporating 1,3,5-tria-
zine moieties 2(a-d) and 3(a-l) show, in general, moderate DPPH
radical scavenging and metal chelating activity, and low ABTS cat-
ion radical scavenging activity. Compounds 2 b, 3d and 3 h dis-
played great inhibition potency against AChE with % inhibition
values of 96.37, 91.10 and 93.19, respectively. BChE also effectively
inhibited by most of the synthesised compounds having >90%
inhibition potency which is a one of the most important findings
from the current work. Several lead compounds were also
obtained against tyrosinase enzyme and they might be improved
and used as effective inhibitors of these enzymes.
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