Novel biphenyl bis-sulfonamides as acetyl and butyrylcholinesterase inhibitors: Synthesis, biological evaluation and molecular modeling studies

Article abstract of DOI:10.1016/j.bioorg.2015.11.002

A series of new biphenyl bis-sulfonamide derivatives 2a-3p were synthesized in good to excellent yield (76-98%). The inhibitory potential of the synthesized compounds on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was investigated. Most

Full text of DOI:10.1016/j.bioorg.2015.11.002

Bioorganic Chemistry 64 (2016) 13–20
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel biphenyl bis-sulfonamides as acetyl and butyrylcholinesterase
inhibitors: Synthesis, biological evaluation and molecular modeling
studies
a
b
b
a,
a
⇑
´
Sadaf Mutahir , Jakub Jonczyk , Marek Bajda , Islam Ullah Khan , Muhammad Asim Khan ,
Nisar Ullah c, Muhammad Ashraf d, Qurat-ul-Ain d, Sadaf Riaz a, Sajjad Hussain a, Muhammad Yar e,
⇑
a Department of Chemistry, Government College University, Lahore 54000, Pakistan
b Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Cracow, Poland
c Department of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia
d Department of Chemistry, and Department of Biochemistry & Biotechnology, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
e Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new biphenyl bis-sulfonamide derivatives 2a–3p were synthesized in good to excellent yield
(76–98%). The inhibitory potential of the synthesized compounds on acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) was investigated. Most of the screened compounds showed modest
in vitro inhibition for both AChE and BChE. Compared to the reference compound eserine (IC50
Received 6 September 2015
Revised 5 November 2015
Accepted 7 November 2015
Available online 10 November 2015
0.04 0.0001
were ranged from 2.27 0.01 to 123.11 0.04
Among the tested compounds, 3p was found to be the most potent against AChE (IC50 2.27 0.01
whereas 3g exhibited the highest inhibition for BChE (IC50 7.74 0.07 M). Structure–activity relation-
l
M for AChE) and (IC50 0.85 0.0001
l
M for BChE), the IC50 values of these compounds
M for BuChE.
M),
l
M for AChE and 7.74 0.07 to <400
l
Keywords:
l
Alzheimer’s disease
Acetylcholinesterase
Butyrylcholinesterase
Biphenyl sulfonamides
l
ship (SAR) of these compounds was developed and elaborated with the help of molecular docking studies.
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
Therefore the existing remedies that are used to alleviate the
symptoms of AD such as donepezil, [11] rivastigmine, [12] and
Alzheimer’s disease (AD), a progressive neurodegenerative dis-
ease, associated with selective loss of cholinergic neurons and
reduced level of acetylcholine neurotransmitter. The disorder is
characterized by multiple cognitive impairments including gradual
loss of memory, judgment and learning ability [1,2]. Reports have
revealed that an estimated 35.6 million people worldwide suffer
from this ailment [3]. The amyloid b-peptide (Ab) plaques, intracel-
lular neurofibrillary tangles, loss of central cholinergic function are
the typical pathological hallmarks of AD [4,5]. The cholinergic
hypothesis manifests that AChE plays a major role in functioning
of cholinergic system and is associated with AD through involve-
ment in the acetylcholine metabolism [6]. In addition, cholinergic
neurotransmission is co-regulated by BChE, whose activity is
increased in AD. The current therapeutic approach implies inhibi-
tion of AChE and BChE [7–9] to enhance central cholinergic func-
tion [10] which in turn increase acetylcholine level in the brain.
more recently galantamine [13] belong to the class of cholinester-
ase inhibitors (Fig. 1).
Unfortunately, the potential effectiveness of these inhibitors is
often limited as they suffer from central and peripheral side effects.
Clinical studies have indicated that tacrine has hepatotoxic liability
[14,15] and due to adverse events, it was discontinued [16]. Studies
have revealed that inhibition of AChE-induced Ab aggregation [17]
by cholinesterase inhibitors employ additional benefits for AD
treatment [18,19]. In recent years, a significant number of studies
have shown advancement in the development of dual cholinester-
ase inhibitors [20,21].
Sulfonamides, an important class of pharmacophores, consti-
tute nearly 200 drugs currently in the market. Numerous
sulfonamide-based medicines have been developed as diuretics,
anti-migraine agents, cyclooxygenase-II (COX-2)-specific anti-
inflammatory drugs recently as AChE [22,23]. In addition, aro-
matic/heterocyclic sulfonamides possessing free amino function
have shown effective inhibition of three carbonic anhydrase iso-
zymes [24]. Since biphenyl based sulfonamides are considered
great pharmacophores in medicinal chemistry, and recently we
⇑
Corresponding authors.
E-mail addresses: iuklodhi@yahoo.com (I.U. Khan), drmyar@ciitlahore.edu.pk
(M. Yar).
http://dx.doi.org/10.1016/j.bioorg.2015.11.002
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

14
S. Mutahir et al. / Bioorganic Chemistry 64 (2016) 13–20
bis-sulfonamides analogues (2a–3p) (Scheme 1) and their AChE
and BChE inhibition activities along with molecular modeling
investigations.
O
NH2
N
H3CO
H3CO
N
Donepezil
2. Results and discussion
Tacrine
H
OH
H
O
2.1. Chemistry
H3CO
A series of structurally related sulfonamides derivatives (2a–3p)
were synthesized as outlined in Scheme 1. Condensation of appro-
priate benzidines (1a or 1b) with benzenesulfonyl chloride yielded
the corresponding sulfonamides 2a and 2b. N-alkylation or acyla-
tion of the intermediates 2a and 2b with appropriate alkyl or acyl
halides in DMF at room temperature, using pyridine as a base,
afforded the desired biphenyl bis(benzenesulfonamides) 3a–3p in
good to excellent yield (Scheme 1).
CH3
O
H3C
CH3
CH3
N
N
H3C
N
CH3
O
Galantamine
Rivastigmine
Fig. 1. Chemical structures of potent AChE inhibitors.
2.2. AChE and BChE inhibition
reported their LOX inhibition activity [25] and as part of our
extensive efforts in the search of potent cholinesterase inhibitors
[26]. Herein we report synthesis of a series of new biphenyl
All synthesized compound (2a–3p) were screened for their
in vitro inhibition against AChE and BChE using commercially
Scheme 1. Synthesis of biphenyl bis-sulfonamides (2a–3p).

S. Mutahir et al. / Bioorganic Chemistry 64 (2016) 13–20
15
available eserine as control [27]. The results summarized in Table 1
indicated that compounds 3p, 3o, 3k, 3g and 3c were found to be
more potent AChE inhibitors as compared to others with moderate
affinity. Compound 3a possessing electron withdrawing acetyl
moiety on nitrogens of sulfonamide, exhibited weak activity (IC50
appreciable part of the active site, and this caused the shift of the
biphenyl fragment toward the exit from the gorge of enzyme.
Docking results did not show distinct differences between the
compounds having
a methyl group at the R1 position and
not-substituted derivatives.
103.21 0.11
propyl groups, yielding 3b, moderately improved the activity (IC50
73.21 0.11 M). Likewise, compound 3c (IC50 8.41 0.07 M)
bearing n-butyl substituent at nitrogens, significantly enhanced
the activity. This indicated that electronic releasing alkyl moieties
on nitrogen are important for the higher activity of these com-
pounds. This hypothesis was further supported by the observation
of increase activity in the case of 3,30-dimethylbiphenyl analogues.
For instance when acetyl moieties on nitrogens in 3i (IC50
l
M), however replacement of acetyl moiety with iso-
Similar results were observed in case of docking to the active
site of BChE. Most of the compounds revealed common binding
mode, and hydrophobic interactions between the inhibitors and
aromatic amino acids such as Trp82 and Tyr332 seemed to be cru-
cial for binding. The pose adopted by compound 3k which was the
second compound in BChE inhibitory activity order is shown in
Fig. 3. Compounds 3f, 3n, 3h and 3p could additionally bind with
hydrophobic pocket consisting of Phe398, Trp231, Leu286 and
Phe329. As in case of AChE, inhibitors 3g and 3o bound partially
inside and partially beyond the enzyme active site due to the long
alkyl substituents. Binding at the entry to the gorge could block the
access of substrate to the catalytic site.
Summing up, it can be assumed that the binding of the tested
compounds with AChE and BChE was mainly provided due to the
presence of hydrophobic interactions. We did not observe any
strong hydrogen bonds as well as salt bridges. However, the
obtained compounds are interesting starting point for their further
development and synthesis of potent cholinesterase inhibitors.
Structural modifications leading to increase of number of hydrogen
bond donors and acceptors should augment the strength and speci-
ficity of binding to enzymes.
l
l
96.31 0.14
lM) were replaced with n-butyl group, yielding 3k
(IC50 3.72 0.01
lM), significantly increased the activity. Interest-
ingly, the activity started diminishing, both in the biphenyl and
3,30-dimethylbiphenyl analogues (3d–3f and 3l–3n) alike, when
substituents on nitrogens of sulfonamide were changed from n-
butyl group to the next higher homologues i.e. n-pentyl, n-hexyl
and n-heptyl groups. This revealed that the bulkiness of the
attached function on nitrogens cause diminishing activity. Surpris-
ingly, the activity of compounds 3g (IC50 4.63 0.05
(IC50 2.45 0.01 M) increased enormously when substituents on
nitrogens were replaced to n-hexadecanyl group. Whereas
compound 3h (IC50 61.57 0.05 M), possessing benzyl group on
nitrogens showed moderate activity, the 3,30-dimethylbiphenyl
analogue 3p (IC50 2.27 0.01 M), bearing benzyl moiety on nitro-
lM) and 3o
l
l
l
3. Conclusions
gens turned out to be the most potent among the compounds
screened for AChE inhibition. This revealed that both the 3,30-
dimethylbiphenyl functionality as well as benzyl moiety on nitro-
gens are crucial for the higher activity of 3p.
In case of BuChE inhibition studies, compound 3g (IC50
2.27 0.01 lM) was turned out to be the most active. The higher
activity of 3g, bearing n-hexadecanyl moiety on nitrogens, is a sim-
ilar trend as was the case in AChE inhibition and could be attribu-
ted to the hydrophobic bulkiness of the n-hexadecanyl group.
In summary, 18 new biphenyl bis-sulfonamides analogues (2a–
3p) have been successfully synthesized and were evaluated for
their potentially in vitro AChE and BuChE inhibition. Among the
series, compounds 3p and 3o have shown the highest activity for
AChE. In case of BChE inhibition assay, compound 3g turned out
to be the most potent. Compounds 3p and 3o are quite promising
in the sense that they have shown higher activity both for AChE
and BChE as well as good selectivity for AChE. Molecular modeling
studies further demonstrated the binding modes of this series into
both cholinesterases. Based on these results we believe the present
research would lead to the development of more potent AChE and
BChE inhibitors to treat AD.
Compounds 3k (IC50 20.27 0.32
lM), 3o (IC50 24.74 0.01
lM),
3p (IC50 31.31 0.05 M) and 3h (IC50 35.82 0.05
l
lM) have
shown good to moderate activity whereas the rest of the com-
pounds have shown either low activity or were inactive against
BChE.
Compound 3c showed moderate activity for both AChE and
BChE but possessed the highest selectivity toward AChE
(14.8525). The most potent AChE inhibitor Compound 3p, being
in the series, has also shown good selectivity (13.792). Likewise
compound 3o also exhibited good and moderate inhibition for
BChE and AChE, respectively, have also shown good selectivity
(10.097).
4. Experimental
4.1. Material and methods
All chemicals and solvents used are of analytical grade and were
purchased from Sigma, Aldrich and Merck Chemical Co. and were
used without further purification. Melting points were taken in
open capillary tubes and are uncorrected. TLC was performed on
silica coated aluminum sheets (6F254, 0.2 mm) and visualized in
low UV light. IR spectra in KBr pellets were recorded on FT-IR Per-
kin Elmer spectrum BX spectrophotometer. 1H NMR and 13C NMR
spectra were recorded in DMSO-d6 using Bruker NMR 500 MHz.
Splitting patterns were as follows s (singlet), d (doublet), dd (dou-
ble doublets), t (triplet), and m (multiplet). Chemical shifts are
reported in d (ppm) and coupling constants are given in Hz.
2.3. Docking studies
All synthesized compounds were docked to the active site of
both cholinesterases. The docking results showed that the whole
group of the analyzed biphenyl derivatives except 3g and 3o dis-
played similar binding mode with AChE. Ligand binding within
the active site of AChE was limited to hydrophobic interactions
with Trp84, Phe330, and Phe331 from anionic sub-site, Phe290
from acyl pocket as well as Tyr121, Trp279 and Tyr334 from
peripheral anionic site (PAS). Biphenyl fragment was engaged in
4.2. General procedure for the precursor sulfonamides (2a, 2b)
more specific
p–p and CH–p interactions with Trp279, Phe331
and Tyr334. Oxygen atoms in sulfonamide groups might create
weak H-bonds with hydroxyl group of Tyr70 or unionized form
of Asp72. The arrangement of the most active compound 3p in
the active gorge of AChE is shown in Fig. 2. In case of compounds
3g and 3o long aliphatic chain in R2 position occupied the
To a
stirred solution of benzidine 1a or o,o0-tolidine 1b
(0.01 mol) in dichloromethane (50 mL) pyridine (around 1 mL, to
adjust pH 8) was added. Then benzenesulfonyl chloride
(0.02 mol, 0.443 g) was added to the reaction and the mixture
was stirred at room temperature while maintaining the pH of the

16
S. Mutahir et al. / Bioorganic Chemistry 64 (2016) 13–20
Table 1
In vitro AChE & BChE inhibitory activity of compounds 2a–3p (inhibition percentage and IC50 values are means given with SEM).
R1
R1
O
S
O
O
S
O
R2
N
O
S
O
O
S
O
H
N
N
H
N
R2
R1
R1
3a-3p
2a,2b
Entry
Sample code
Conc. (mM)
AChE inhibition
BuChE inhibition
Selectivity
AChE/BuChE
4.8005
Inhibition (% SEM)
IC50
(
l
M
SEM)
Inhibition (% SEM)
IC50
(
l
M
SEM)
1
2
3
4
5
6
7
8
2a
2b
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
96.54 0.12
76.08 0.21
78.51 0.34
89.61 0.25
98.74 0.15
88.47 0.52
89.05 0.33
95.61 0.36
98.55 0.13
92.91 0.19
85.61 0.24
83.29 0.51
97.82 0.21
92.51 0.25
93.37 0.32
95.97 0.38
97.37 0.13
98.94 0.12
91.29 1.17
42.31 0.11
123.11 0.04
103.21 0.11
73.21 0.11
8.41 0.07
79.61 0.21
78.61 0.17
58.61 0.12
4.63 0.05
61.57 0.05
96.31 0.14
101.21 0.11
3.72 0.01
51.21 0.08
49.61 0.11
43.21 0.14
2.45 0.02
60.26 0.16
28.15 0.61
72.19 0.45
35.98 0.64
92.72 0.21
40.95 0.47
44.81 0.61
33.61 0.25
98.67 0.13
97.72 0.11
34.11 0.31
22.74 0.61
93.21 0.18
40.62 0.18
50.66 0.62
94.71 0.14
94.35 0.19
95.23 0.14
82.82 1.09
203.11 0.31
–
156.21 0.31
–
124.91 0.08
–
–
–
1.5135
14.8525
9
7.74 0.07
35.82 0.05
–
1.617
0.5817
10
11
12
16
13
14
15
17
18
Control
–
3k
3l
20.32 0.08
–
<400
48.71 0.11
24.74 0.09
31.31 0.05
0.85 0.0001
5.4623
3m
3n
3o
3p
Eserineb
1.127
0.5
0.5
0.25 mM
10.097
13.792
21.25
2.27 0.01
0.04 0.0001
a All samples were tested in triplicates and averaged. SEM is standard mean error of the three experiments.
b
Standard used for AChE and BChE inhibition.
Fig. 2. Binding mode of compound 3p within the active site of AChE.
mixture to ꢀ8.0 with occasional addition of pyridine. After comple-
tion of reaction in 4 h (monitored by TLC), pH of the reaction mix-
ture was adjusted to 2.0 by adding 1 M HCl where the desired
product 2a or 2b was solidified which was filtered and washed
with distilled water (50 mL). Recrystallization from methanol
afforded the required product in essential pure form.
(2H, m, ArH), 7.62–7.60 (4H, m, ArH), 7.54 (4H, d, J = 8.5 Hz),6.69
(4H, d, J = 9 Hz); 13C NMR (500 MHz; DMSO-d6): 140.6 (2C),
133.4 (2C), 131.3 (2C), 129.7 (4C), 128.8 (2C), 127.3 (4C), 127.20
(4C), 120.7 (4C); Anal. Calcd for C24H20N2O4S2: C, 62.05; H, 4.34;
N, 6.03; O, 13.78; S, 13.80. Found: C, 62.13; H, 4.38; N, 6.14 S,
13.34.
4.2.1. N,N0-(biphenyl-4,40-diyl)dibenzenesulfonamide (2a)
4.2.2. N,N0-(3,30-dimethylbiphenyl-4,40-diyl) dibenzenesulfonamide
Violet solid; Yield: 98%; m.p.: 249 °C; IR (
t
max, KBr, cmꢁ1): 3350
(2b)
(NAH str), 3029 (CAH str Ar), 1330 (SAO str); 1H NMR (500 MHz,
DMSO-d6): 10.36 (2H,s,NH), 7.82 (4H,d, J = 10 Hz, ArH), 7.77–7.75
White solid; Yield: 98%; m.p.:251 °C; IR (
(NAH str), 3027 (CAH str Ar), 1335 (SAO str); 1H NMR (500 MHz,
t
max, KBr, cmꢁ1): 3376

S. Mutahir et al. / Bioorganic Chemistry 64 (2016) 13–20
17
Fig. 3. Binding mode of compound 3k within the active site of BuChE.
DMSO-d6): 9.62 (2H, s, NH), 7.88 (4H, d, J = 10 Hz, ArH), 7.72–7.70
(2H, m, ArH), 7.62–7.60 (4H, m, ArH), 7.54 (2H, s, ArH), 7.38 (2H, d,
J = 8.5 Hz, ArH), 6.33 (2H, d, J = 8.2 Hz, ArH), 1.9 (6H, s, CH3); 13C
NMR (500 MHz; DMSO-d6): 140.6 (2C), 137.1 (2C), 134.5 (2C),
132.9 (4C), 129.3 (2C), 128.8 (4C), 126.7 (4C), 126.6 (2C), 124.4
(2C), 17.7 (2C); Anal. Calcd for: C26H24N2O4S2: C, 63.39; H, 4.91;
N, 5.69; O, 12.99; S, 13.02. Found: C, 63.45; H, 5.52; N, 5.56; S,
13.06.
(2C), 20.2 (4C). Anal. Calcd for C30H32N2O4S2: C, 65.67; H, 5.88; N,
5.11; S, 11.69. Found: C, 65.99; H, 5.55; N, 5.13; S, 12.57.
4.3.3. N,N0-(biphenyl-4,40-diyl)bis(N-butylbenzenesulfonamide) (3c)
White solid; Yield: 84%; m.p.:224 °C; IR (t
max, KBr, cmꢁ1): 3021
(CH str Ar), 2940 (CH2 str), 1334 (SAO str); 1H NMR (500 MHz,
DMSO-d6): 7.82 (4H, d, J = 10 Hz, ArH), 7.77–7.75 (2H, m, ArH),
7.62–7.60 (4H, m, ArH), 7.54 (4H, d, J = 8.5 Hz), 6.69 (4H, d,
J = 9 Hz), 3.50 (4H, t, J = 7.1 Hz, CH2), 1.57 (4H, m, CH2), 1.23 (4H,
m, CH2), 0.91 (6H, t, J = 7.1 Hz, CH3); 13C NMR (500 MHz; DMSOd6):
140.6 (2C), 133.4 (2C), 131.3 (2C), 129.7 (4C), 128.8 (2C), 127.3
(4C), 127.2 (4C), 120.7 (4C), 48.7 (2C), 27.3 (2C), 24.1 (2C), 14.1
(2C). Anal. Calcd for C31H34N2O4S2: C, 66.16; H, 6.09; N, 4.98; S,
11.40. Found: C, 66.97; H, 5.96; N, 5.9; S, 11.97.
4.3. General procedure A for the Synthesis of 3a–3p
To a solution of compound 2a or 2b (10 mmol) in DMF (10 mL)
n-hexane washed sodium hydride (2.5 mmol, 0.6 g) was added and
after being stirred for 40 min at room temperature, the corre-
sponding alkylating/acylating reagent (20 mmol) was added to
the mixture. The mixture was stirred until the completion of reac-
tion which was monitored by TLC. When the reaction was com-
pleted the reaction mixture was poured on the crushed ice. The
pH of the mixture was adjusted to 4.0 with 1 N HCl and the precip-
itated product was filtered and washed twice with distilled water.
4.3.4. N,N0-(biphenyl-4,40-diyl)bis(N-pentylbenzenesulfonamide) (3d)
Light pink solid; Yield: 89%; m.p.: 227 °C; IR (t
max, KBr, cmꢁ1):
3021 (CH str Ar), 2938 (CH2 str), 1334 (SAO str); 1H NMR
(500 MHz, DMSO-d6): 7.82 (4H, d, J = 10 Hz, ArH), 7.77–7.75 (2H,
m, ArH), 7.62–7.60 (4H, m, ArH), 7.54 (4H, d, J = 8.5 Hz), 6.69 (4H,
d, J = 9 Hz), 3.53 (4H, t, J = 7.1 Hz, CH2), 1.50 (4H, m, CH2), 1.30–
1.29 (8H, m, CH2), 0.85 (6H, t, J = 7.1 Hz, CH3); 13C NMR
(500 MHz; DMSO-d6): 140.6 (2C), 133.4 (2C), 131.3 (2C), 129.7
(4C), 128.8 (2C), 127.3 (4C), 127.2 (4C), 120.7 (4C), 49.0 (2C),
28.5 (2C), 27.7 (2C), 22.0 (2C), 13.6 (2C). Anal. Calcd for C34H40N2-
O4S2: C, 67.52; H, 6.67; N, 4.63; S, 10.60. Found: C, 67.61; H, 6.42;
N, 4.92; S, 11.06.
4.3.1. N,N0-(biphenyl-4,40-diyl)bis(N-(phenylsulfonyl)acetamide) (3a)
Pink solid; Yield: 90%; m.p.: 259 °C; IR (t
max, KBr, cmꢁ1): 3029
(CAH str Ar), 1330 (SAO str), 1707 (C@O); 1H NMR (500 MHz,
DMSO-d6): 7.82–7.79 (12H, m, ArH), 7.75–7.73 (2H, m, ArH),
7.62–7.60 (4H, m, ArH); 2.04 (6H, s, CH3); 13C NMR (500 MHz;
DMSO-d6): 171.1 (2C), 140.6 (2C), 137.9 (2C), 133.8 (2C), 133.0
(2C), 129.0 (4C), 128.7 (4C), 127.3 (4C), 120.0 (4C), 28.3 (2C). Anal.
Calcd for C28H24N2O6S2: C, 61.30; H, 4.41; N, 5.11; S, 11.69. Found:
C, 61.34; H, 4.35; N, 5.08; S, 11.71.
4.3.5. N,N0-(biphenyl-4,40-diyl)bis(N-hexylbenzenesulfonamide) (3e)
Pink solid; Yield: 79%; m.p.: 251 °C. IR (t
max, KBr, cmꢁ1): 3021
4.3.2. N,N0-(biphenyl-4,40-diyl)bis(N-isopropylbenzenesulfonamide)
(CH str Ar), 2932 (CH2 str), 1334 (SAO str); 1H NMR (500 MHz,
CDCl3): 7.82 (4H, d, J = 10 Hz, ArH), 7.77–7.75 (2H, m, ArH), 7.62–
7.60 (4H, m, ArH), 7.54 (4H, d, J = 8.5 Hz), 6.69 (4H, d, J = 9 Hz),
3.5 (4H, t, J = 7.1 Hz, CH2), 1.5 (4H, m, CH2), 1.27–1.25 (12H, m,
CH2), 0.85 (6H, t, J = 7.1 Hz, CH3); 13C NMR (500 MHz; DMSOd6):
140.6 (2C), 133.4 (2C), 131.3 (2C), 129.7 (4C), 128.8 (2C), 127.3
(4C), 127.2 (4C), 120.7 (4C), 40.0 (2C), 31.4 (2C), 29.5 (2C), 27.3
(2C), 24.1 (2C), 14.1 (2C). Anal. Calcd for C36H44N2O4S2: C, 68.32;
H, 7.01; N, 4.43; S, 10.13. Found: C, 68.44; H, 7.77; N, 4.38; S, 10.94.
(3b)
Brown solid; Yield: 76%; m.p.: 235 °C; IR (t
max, KBr, cmꢁ1): 3029
(CH str Ar), 2927 (CH str), 1332 (SAO str); 1H NMR (500 MHz,
DMSO-d6): 7.82 (4H, d, J = 10 Hz, ArH), 7.77–7.75 (2H, m, ArH),
7.62–7.60 (4H, m, ArH), 7.54 (4H, d, J = 8.5 Hz), 6.69 (4H, d,
J = 9 Hz), 2.75–2.74 (2H, m, CH), 1.25 (12H, d, J = 6.2 Hz, CH3); 13C
NMR (500 MHz; DMSOd6): 140.6 (2C), 133.4 (2C), 131.3 (2C),
129.7 (4C), 128.8 (2C), 127.3 (4C), 127.20 (4C), 120.7 (4C), 43.0

18
S. Mutahir et al. / Bioorganic Chemistry 64 (2016) 13–20
4.3.6. N,N0-(biphenyl-4,40-diyl)bis(N-heptylbenzenesulfonamide) (3f)
Light yellow solid; Yield: 82%; m.p.: 252 °C; IR ( max, KBr,
4.3.11. N,N0-(3,30-dimethylbiphenyl-4,40-diyl)bis(N-
butylbenzenesulfonamide) (3k)
t
cmꢁ1): 3021 (CH str Ar), 1333 (SAO str); 1H NMR (500 MHz,
DMSO-d6): 7.82 (4H, d, J = 10 Hz, ArH), 7.77–7.75 (2H, m, ArH),
7.62–7.60 (4H, m, ArH), 7.54 (4H, d, J = 8.5 Hz), 6.69 (4H, d,
J = 9 Hz), 3.51 (4H, t, J = 7.1 Hz, CH2), 1.59–1.57 (4H, m, CH2),
1.29–1.27 (16H, m, CH2), 0.88 (6H, t, J = 7.1 Hz, CH3); 13C NMR;
(500 MHz; DMSO-d6): 140.6 (2C), 133.3 (4C), 132.2 (2C), 130.9
(2C), 129.7 (4C), 127.2 (2C), 126.9 (4C), 120.7 (4C), 40.0 (2C),
39.8 (2C), 39.7 (2C), 39.5 (2C), 39.3 (2C), 39.1 (2C), 14.1 (2C). Anal.
Calcd for C38H48N2O4S2: C, 69.06; H, 7.32; N, 4.24; S, 9.70. Found: C,
69.76; H, 4.38; N, 4.20; S, 9.97.
Light Brown solid; Yield: 85%; m.p.: 215 °C; IR (v
max, KBr, cmꢁ1):
3021 (CH str Ar), 2940 (CH2 str), 1333 (SAO str); 1H NMR (500 MHz,
DMSO-d6): 7.88 (4H, d, J = 10 Hz, ArH), 7.72–7.70 (2H, m, ArH),
7.62–7.60 (4H, m, ArH), 7.54 (2H, s, ArH), 7.38 (2H, d, J = 8.5 Hz,
ArH), 6.33 (2H, d, J = 8.2 Hz, ArH), 3.16 (4H, t, J = 7.1 Hz, CH2),
2.01 (6H, s, ArACH3), 1.49 (4H, m, CH2), 1.32 (4H, m, CH2), 0.90
(6H, t, J = 7.1 Hz, CH3); 13C NMR (500 MHz; DMSO-d6): 140.6
(2C), 137.1 (2C), 134.5 (2C), 132.9 (4C), 129.3 (2C), 128.8 (4C),
126.7 (4C), 126.6 (2C), 124.4 (2C), 44.0 (2C), 25.7 (2C), 18.9 (2C),
17.7 (2C), 13.8 (2C). Anal. Calcd for C33H38N2O4S2: C, 67.09; H,
6.48; N, 4.74; S, 10.85. Found: C, 67.05; H, 6.57; N, 4.98; S, 10.73.
4.3.7. N,N’-(biphenyl-4,4’-diyl)bis(N-hexadecylbenzenesulfonamide)
4.3.12. N,N0-(3,30-dimethylbiphenyl-4,40-diyl)bis(N-
pentylbenzenesulfonamide) (3l)
(3g)
Light Brown solid; Yield: 80%; m.p.: 257 °C; IR (t
max, KBr, cmꢁ1):
3021 (CH str Ar), 1350 (SAO str); 1H NMR (500 MHz, DMSO-d6):
7.82 (4H, d, J = 10 Hz, ArH), 7.77–7.75 (2H, m, ArH), 7.62–7.60
(4H, m, ArH), 7.54 (4H, d, J = 8.5 Hz, ArH), 6.69 (4H, d, J = 9 Hz,
ArH), 3.51 (4H, t, J = 7.1 Hz, CH2), 1.59–1.57 (4H, m, CH2), 1.29–
1.27 (52H, br s, CH2), 0.88 (6H, t, J = 7.1 Hz, CH3); 13C NMR
(500 MHz; DMSO-d6): 140.6 (2C), 133.4 (2C), 131.3 (2C), 129.7
(4C), 128.8 (2C), 127.3 (4C), 127.2 (4C), 120.7 (4C), 47.8 (2C),
31.8 (2C), 29.6 (16C), 29.3 (4C), 27.7 (2C), 26.5 (2C), 23.7 (2C),
17.71 (2C), 14.0 (2C). Anal. Calcd for C56H84N2O4S2: C, 73.64; H,
9.27; N, 3.07; S, 7.02. Found: C, 73.66; H, 9.77; N, 3.18; S, 7.28.
Light yellow solid; Yield: 84%; m.p.: 224 °C; IR (tmax, KBr,
cmꢁ1): 3021 (CH str Ar), 2940 (CH2 str), 1335 (SAO str); 1H NMR
(500 MHz, DMSO-d6): 7.88 (4H, d, J = 10 Hz, ArH), 7.72–7.70 (2H,
m, ArH), 7.62–7.60 (4H, m, ArH), 7.54 (2H, s, ArH), 7.38 (2H, d,
J = 8.5 Hz, ArH), 6.33 (2H, d, J = 8.2 Hz, ArH), 3.16 (4H, t,
J = 7.1 Hz, CH2), 2.31 (6H, s, ArACH3), 1.39–1.38 (4H, m, CH2),
1.2–1.18 (8H, m, CH2), 0.77 (6H, t, J = 7.1 Hz, CH3); 13C NMR
(500 MHz; DMSO-d6): 140.6 (2C), 137.1 (2C), 134.5 (2C), 132.9
(4C), 129.3 (2C), 128.8 (4C), 126.7 (4C), 126.6 (2C), 124.4 (2C)
51.2 (2C), 28.2 (2C), 27.5 (2C), 21.7 (2C), 17.7 (2C), 14.0 (2C). Anal.
Calcd for C36H44N2O4S2: C, 68.32; H, 7.01; N, 4.43; S, 10.13. Found:
C, 68.60; H, 7.67; N, 4.41; S, 9.91.
4.3.8. N,N0-(biphenyl-4,40-diyl)bis(N-benzylbenzenesulfonamide) (3h)
Brown solid; Yield: 90%; m.p.: 262 °C; IR (t
max, KBr, cmꢁ1): 3021
4.3.13. N,N0-(3,30-dimethylbiphenyl-4,40-diyl)bis(N-
(CH str Ar), 2940 (CH2 str), 1345 (SAO str); 1H NMR (500 MHz,
DMSO-d6): 7.82 (4H, d, J = 10 Hz, ArH), 7.77–7.75 (2H, m, ArH),
7.62–7.60 (4H, m, ArH), 7.54 (4H, d, J = 8.5 Hz), 6.69 (4H, d,
J = 9 Hz) 7.33–7.23 (10H, m, ArH), 4.41 (4H, s, CH2); 13C NMR
(500 MHz; DMSO d6): 145.1 (2C), 140.5 (2C), 142.0 (2C), 139.6
(2C), 132.74 (2C), 129.3 (4C), 129.3 (4C), 128.7 (4C), 127.4 (4C),
hexylbenzenesulfonamide) (3m)
Light Brown solid; Yield: 81%; m.p.: 205 °C; IR (t
max, KBr, cmꢁ1):
3021 (CH str Ar), 2940 (CH2 str), 1335 (SAO str); 1H NMR (500 MHz,
DMSO-d6): 7.88 (4H, d, J = 10 Hz, ArH), 7.72–7.70 (2H, m, ArH),
7.62–7.60 (4H, m, ArH), 7.54 (2H, s, ArH), 7.38 (2H, d, J = 8.5 Hz,
ArH), 6.33 (2H, d, J = 8.2 Hz, ArH), 3.16 (4H, t, J = 7.1 Hz, CH2),
2.01 (6H, s, ArACH3), 1.48–1.46 (4H, m, CH2), 1.23 (12H, br s,
CH2), 0.79 (6H, t, J = 7.1 Hz CH3); 13C NMR (500 MHz, DMSO-d6):
140.6 (2C), 137.1 (2C), 134.5 (2C), 132.9 (4C), 129.3 (2C), 128.8
(4C), 126.7 (4C), 126.6 (2C), 124.4 (2C) 48.0 (2C), 32.3 (2C), 27.2
(2C), 22.0 (2C), 17.7 (2C), 14.0 (2C). Anal. Calcd for C38H48N2O4S2:
C, 69.06; H, 7.32; N, 4.24; S, 9.70. Found: C, 69.41; H, 7.79; N,
4.56; S, 9.78.
126.3 (4C), 126.0 (2C), 124.7 (4C), 47.2 (2C). Anal. Calcd for C38H32
N2O4S2: C, 70.78; H, 5.00; N, 4.34; S, 9.95. Found: C, 70.54; H, 4.68;
N, 4.43; S, 9.96.
-
4.3.9. N,N0-(3,30-dimethylbiphenyl-4,40-diyl)bis(N-(phenylsulfonyl)
acetamide) (3i)
White solid; Yield: 82%; m.p.: 257 °C. IR (t
max, KBr, cmꢁ1): 3028
(CH str Ar), 1330 (SAO str), 1713 (C@O); 1H NMR (500 MHz, DMSO-
d6): 7.88 (4H, d, J = 10 Hz, ArH), 7.77 (2H, d J = 5 Hz, ArH), 7.73 (2H,
d, J = 8.2 Hz, ArH) 7.72–7.70 (2H, m, ArH), 7.64–7.62 (4H, m, ArH),
7.53 (2H, d, J = 10 Hz, ArH), 2.49 (6H, s, ArACH3), 1.9 (6H, s, CH3);
13C NMR (500 MHz; DMSO-d6): 171.1 (2C), 140.6 (2C), 137.1
(2C), 134.5 (2C), 132.9 (4C), 129.3 (2C), 128.8 (4C), 126.7 (4C),
4.3.14. N,N0-(3,30-dimethylbiphenyl-4,40-diyl)bis(N-heptyl-
benzenesulfonamide) (3n)
Yellow solid; Yield: 89%; m.p.: 240 °C; IR (t
max, KBr, cmꢁ1):
3021 (CH str Ar), 2940 (CH2 str), 1335 (SAO str); 1H NMR
(500 MHz, DMSO-d6): 7.88 (4H, d, J = 10 Hz, ArH), 7.72–7.70 (2H,
m, ArH), 7.62–7.60 (4H, m, ArH), 7.54 (2H, s, ArH), 7.38 (2H, d,
J = 8.5 Hz, ArH), 6.33 (2H,d, J = 8.2 Hz, ArH), 3.16 (4H, t, J = 7.1 Hz,
CH2), 2.03 (6H, s, ArACH3), 1.49–1.47 (4H, m, CH2), 1.31–1.29
(16H, br s, CH2), 0.88 (6H, t, J = 7.1 Hz, CH3); 13C NMR (500 MHz;
DMSO-d6): 140.6 (2C), 137.1 (2C), 134.5 (2C), 132.9 (4C), 129.3
(2C), 128.8 (4C), 126.7 (4C), 126.6 (2C), 124.4 (2C), 50.1 (2C),
32.8 (2C), 30.1 (2C), 28.0 (2C), 26.5 (2C), 23.7 (2C), 17.7 (2C), 14.0
(2C). Anal. Calcd for C40H52N2O4S2: C, 69.73; H, 7.61; N, 4.07; S,
9.31. Found: C, 69.53; H, 7.82; N, 4.27; S, 9.39.
126.6 (2C), 124.4 (2C), 18.9 (2C), 17.7 (2C). Anal. Calcd for C30H28
N2O6S2: C, 62.48; H, 4.89; N, 4.86; S, 11.12. Found: C, 62.56; H,
4.75; N, 5.07; S, 11.41.
-
4.3.10. N,N’-(3,3’-dimethylbiphenyl-4,4’-diyl)bis(N-
isoprpylbenzenesulfonamide) (3j)
White solid; Yield: 76%; m.p.: 234 °C; IR (t
max, KBr, cmꢁ1): 3021
(CH str Ar), 2940 (CH2 str), 1332 (SAO str); 1H NMR (500 MHz,
DMSO-d6): 7.88 (4H, d, J = 10 Hz, ArH), 7.72–7.70 (2H, m, ArH),
7.62 7.60 (4H, m, ArH), 7.54 (2H, s, ArH), 7.38 (2H, d, J = 8.5 Hz,
ArH), 6.33 (2H, d, J = 8.2 Hz, ArH), 2.97–2.95 (2H, m, CH), 1.57
(6H, s, ArACH3), 1.25 (12H, d, J = 6.2 Hz, CH3); 13C NMR
(500 MHz; DMSO-d6): 140.6 (2C), 137.1 (2C), 134.5 (2C), 132.9
(4C), 129.3 (2C), 128.8 (4C), 126.7 (4C), 126.6 (2C), 124.4 (2C),
44.0 (2C), 20.9 (4C), 17.9 (2C). Anal. Calcd for: C32H36N2O4S2 C,
66.64; H, 6.29; N, 4.86; S, 11.12. Found: C, 66.59; H, 6.17; N,
4.72; S, 12.0.
4.3.15. N,N0-(3,30-dimethylbiphenyl-4,40-diyl)bis(N-
hexadecylbenzenesulfonamide) (3o)
White solid; Yield: 83%; m.p.: 250 °C; IR (v
max, KBr, cmꢁ1): 3021
(CH str Ar), 2940 (CH2 str), 1350 (SAO str); 1H NMR (500 MHz,
DMSO-d6): 7.88 (4H, d, J = 10 Hz, ArH), 7.72–7.70 (2H, m, ArH),
7.62–7.60 (4H, m, ArH), 7.54 (2H, s, ArH), 7.38 (2H, d, J = 8.5 Hz,
ArH), 6.33 (2H, d, J = 8.2 Hz, ArH), 3.16 (4H, t, J = 7.1 Hz, CH2),

S. Mutahir et al. / Bioorganic Chemistry 64 (2016) 13–20
19
2.01 (6H,s, Ar CH3), 1.50–1.49 (4H, m, CH2), 1.31–1.29 (48H, br s,
4.6. Molecular modeling
CH2), 0.84 (6H, t, J = 7.1 Hz, CH3); 13C NMR (500 MHz; DMSO-d6):
140.6 (2C), 137.1 (2C), 134.5 (2C), 132.9 (4C), 129.3 (2C), 128.8
(4C), 126.7 (4C), 126.6 (2C), 124.4 (2C), 50.0 (2C), 31.8 (2C), 29.6
(16C), 29.3 (4C), 27.72 (2C), 26.5 (2C), 23.7 (2C), 17.7 (2C), 14.0
(2C). Anal. Calcd for C58H88N2O4S2: C, 73.99; H, 9.42; N, 2.98; S,
6.81. Found: C, 73.69; H, 9.74; N, 3.10; S, 7.54.
The docking studies were performed according to the previ-
ously described method [29]. The three-dimensional structures of
compounds were built with Corina on-line tool (Molecular Net-
works). Protonation states were predicted at neutral pH by Marvin
(Chemaxon). The atom and bond types were checked and Gastei-
ger–Marsili charges were assigned by Sybyl-X 1.1 (Tripos). Ligand
structures were saved in the mol2 format. The structures of AChE
from Torpedo californica (resolution 2.5 Å, PDB code: 1EVE) and
butyrylcholinesterase from Homo sapiens (resolution 2.0 Å, PDB
code: 1P0I) were downloaded from Protein Data Bank. All histidine
4.3.16. N,N0-(3,30-dimethylbiphenyl-4,40-diyl)bis(N-
benzylbenzenesulfonamide) (3p)
Brown solid; Yield: 91%; m.p.: 232 °C; IR (v
max, KBr, cmꢁ1): 3021
(CH str Ar), 2940 (CH2 str), 1332 (SAO str); 1H NMR (500 MHz,
DMSO-d6): 7.88 (4H, d, J = 10 Hz, ArH), 7.72–7.70 (2H, m, ArH),
7.62–7.60 (4H, m, ArH), 7.54 (2H, s, ArH), 7.49–7.24 (12H, m,
ArH), 6.33 (2H, d, J = 8.2 Hz, ArH), 4.33 (4H, s, CH2), 2.14 (6H, s,
CH3); 13C NMR (500 MHz; DMSO-d6): 145.1 (2C), 142.0 (2C),
140.5 (2C), 139.6 (2C), 132.74 (2C), 131.2 (C), 129.3 (4C), 128.7
(4C), 127.4 (4C), 126.3 (4C), 126.0 (4C), 122.7 (2C), 47.8 (2C),
17.7 (2C). Anal. Calcd for C40H36N2O4S2: C, 71.40; H, 5.39; N,
4.16; S, 9.53. Found: C, 71.50; H, 4.98; N, 4.31; S, 9.85.
residues were protonated at Ne atom (HSE tautomer); ligand and
needless water molecules were removed. Missing hydrogens were
added using Hermes 1.5 (CCDC). The binding site was defined as all
amino acid residues within 10 Å from donepezil molecule in AChE
complex and 20 Å from glycerol in BChE complex. Dockings were
performed with Gold 5.1 software (CCDC) using genetic algorithm
with default settings. Chem Score function and analysis of binding
mode were applied to find final ligand poses. Results were visual-
ized by PyMOL 0.99rc2 (DeLano Scientific LLC).
4.4. Acetylcholinesterase assay
Acknowledgments
The AChE inhibition activity was determined according to the
Ellman’s method [28] with slight modifications. Total volume of
We acknowledge Higher Education Commission and Ministry of
Science and Technology Pakistan for financial support. Docking
studies were financially supported by the Jagiellonian University
Medical College – Poland grant no. K/DSC/002860.
the reaction mixture was 100
l
L. It contained 60
lL Na2HPO4 buf-
fer with concentration of 50 mM and pH 7.7. Ten
l
L test compound
(0.5 mM wellꢁ1) was added, followed by the addition of 10
lL
(0.005 unit wellꢁ1) enzyme. The contents were mixed and pre-
read at 405 nm. Then contents were pre-incubated for 10 min at
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