Synthesis, biological evaluation, and molecular docking of N' -(Aryl/alkylsulfonyl) -1-(Phenylsulfonyl) piperidine-4-Carbohydrazide derivatives

Article abstract of DOI:10.3906/kim-1303-89

A series of new N'-[(alkyl/aryl)sulfonyl^-1-(phenylsulfonyl) piperidine-4-carbohydrazide derivatives were synthesized. Starting from ethyl piperidine-4-carboxylate (a), first ethyl 1-(phenylsulfonyl)piperidine-4- carboxylate (1), second 1-(pheny

Full text of DOI:10.3906/kim-1303-89

Turk J Chem
Turkish Journal of Chemistry
(2014) 38: 189 – 201
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1303-89
Research Article
Synthesis, biological evaluation, and molecular docking of
N′ -(Aryl/alkylsulfonyl)-1-(phenylsulfonyl) piperidine-4-carbohydrazide derivatives
Hira KHALID1, Aziz UR REHMAN1,∗, Muhammad Athar ABBASI1,
Rashad HUSSAIN2, Khalid MOHAMMAD KHAN3, Muhammed ASHRAF4,
Syeda Abida EJAZ5, Muhammad Qaiser FATMI2
1Department of Chemistry, Government College University, Lahore, Pakistan
2Department of Biosciences, COMSATS Institute of Information Technology, Chak Shahzad, Islamabad, Pakistan
3HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi, Pakistan
4Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
5Department of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
Received: 27.03.2013
•
Accepted: 03.07.2013
•
Published Online: 14.03.2014
•
Printed: 11.04.2014
Abstract: A series of new N′ -[(alkyl/aryl)sulfonyl]-1-(phenylsulfonyl)piperidine-4-carbohydrazide derivatives were syn-
thesized. Starting from ethyl piperidine-4-carboxylate (a), first ethyl 1-(phenylsulfonyl)piperidine-4-carboxylate (1), sec-
ond 1-(phenylsulfonyl)piperidine-4-carbohydrazide (2), and finally N′ -[(alkyl/aryl)sulfonyl]-1-(phenylsulfonyl)piperidine-
4-carbohydrazides (4a–n) were synthesized by reacting 2 with alkyl/aryl sulfonyl chlorides (3a–n). The structures of
the synthesized compounds were characterized by IR, 1 H-NMR, and EI-MS spectra and all were screened in vitro for
their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme inhibition activities. Molecular docking was
accomplished for these compounds to examine their binding interactions with AChE and BChE human proteins. The
strategy we applied for this purpose was a direct receptor-based approach. The binding modes of the inhibitors under
study were determined using an automated docking program (AutoDock) and were compared with antienzymatic IC50
values. Both studies confirmed the potential of compounds as excellent inhibitors for AChE and BChE.
Key words: Ethyl piperidine-4-carboxylate, benzenesulfonyl chloride, piperidine-4-carbohydrazide, enzyme inhibition,
molecular docking, spectral analysis, AChE/BChE inhibitors
1. Introduction
Animal-source cholinesterases are prevalent enzymes present in body fluids and cholinergic as well as noncholin-
ergic tissues.1−3 Cholinesterase inhibitors are important for treatment of various diseases like organophosphate
poisoning,4 myasthenia gravis, glaucoma, and promisingly Alzheimer’s disease (AD).5,6
AD is a neurodegenerative disorder that is attributed to a deficit in acetylcholine (ACho)7 among other
neurotransmitters, which affects the elderly population. The most prevalent strategy to treat AD is based on the
cholinergic hypothesis and cholinesterase inhibitors, which enhance the level of ACho at cholinergic synapses.8,9
Acetylcholinesterase (AChE; C. 3.1.1.7) and butyrylcholinesterase (BChE; E.C. 3.1.1.8) represent 2 main classes
of cholinesterases on the basis of their specificity, behavior to substrate, and vulnerability to inhibitors. AChE
and BChE are specific in their tissue distribution. AChE is profuse in the brain, erythrocyte membrane, and
∗Correspondence: azizryk@yahoo.com
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KHALID et al./Turk J Chem
muscle, whereas BChE is distributed in the liver, intestine, heart, kidney, and lungs.10,11 AChE catalytic activity
involves acylation or deacylation of serine moiety present in the active center, which consists of 2 subsites, i.e.
esteratic and anionic.12 It has been demonstrated that AChE activity involves allosteric regulation of the ligand
binding to an anionic site.13 BChE favorably acts on butyrylcholine (BCho) as a substrate but also hydrolyzes
ACho.14,15
At present, tacrine, donepezil, galantamine, and rivastigmine are the most frequently prescribed anti-
cholinesterase drugs.16 However, they have adverse effect profiles and some toxicity. Therefore, there is a great
need for cholinesterase inhibitor drug candidates with high potency and reduced toxicity. Recently, a number
of active compounds have been identified with potential therapeutic activity against cholinergic disorders.17
The present work is a continuation of our successful efforts for the synthesis of potentially bioactive
compounds bearing the piperidine nucleus.18 The reported derivatives were found to be potent against AChE
and BChE. Molecular docking studies were carried out to rationalize these activities at molecular level and
to identify the binding patterns and affinities of ligands in the binding pockets of AChE and BChE. Binding
models of compounds 4g, 4m, 4i, and 4n are shown in Figure 1 (a–d), which were the most potent compounds
of this series.
2. Experimental
2.1. General
Melting points of the synthesized compounds were recorded on a Griffin and George melting point apparatus by
open capillary tube and were uncorrected. Purity was checked by thin layer chromatography (TLC) on precoated
silica gel G-25-UV254 plates with different polarity solvent systems using ethyl acetate and n-hexane giving a
single spot. Identification of spots was carried out at 254 nm with a UV lamp, and by ceric sulfate reagent. The
IR spectra were recorded by KBr pellet method on a Jasco-320-A spectrophotometer (wave number in cm−1).
1 H-NMR spectra were recorded in CDCl3 on a Bruker spectrometer operating at 300 MHz. Chemical shifts are
given in ppm taking TMS as reference. Mass spectra (EIMS) were recorded on a JMS-HX-110 spectrometer,
with a data system (Scheme).
2.2. Preparation of ethyl 1-(phenylsulfonyl)piperidine-4-carboxylate in aqueous media (1)
Ethyl piperidine-4-carboxylate (a; 20.0 mL, 10.0 mmol) was suspended in 50 mL of water and the pH was
maintained at 9.0 during the whole reaction by adding basic aqueous solution of Na2 CO3 (5%) at 0–5 ◦ C.
Then benzenesulfonyl chloride (b; 29.0 mL, 10.0 mmol) was slowly added over 10–15 min. After complete
addition of b, the temperature was allowed to rise slowly to 30 ◦ C. The reaction mixture was stirred and
monitored with TLC. After the reaction was finished, conc. HCl (2.0 mL, 11 M) was added slowly to adjust
the pH to 2.0. The precipitate was filtered, washed with distilled water, and dried to afford the off-white solid
compound 1.
Ethyl 1-(phenylsulfonyl)piperidine-4-carboxylate (1): IR (KBr, cm−1):vmax : 3015 (Ar-CH), 2925
(C-H), 1760 (C=O), 1531 (C=C), 1329 (S=O); 1 H-NMR (300 MHz, CDCl3 , δ/ppm): 7.78 (dd, J = 7.8, 1.8
Hz, 2H, H-2’, H-6’), 7.68–7.65 (m, 1H, H-4’), 7.62 (dd, J = 7.5, 1.8 Hz, 2H, H-3’, H-5’), 4.11 (q, J = 7.2 Hz,
2H, O-CH2), 3.64 (t, J = 3.6 Hz, 2H, Heq -2, Heq -6), 3.59 (t, J = 3.6 Hz, 2H, Hax -2, Hax -6), 2.13–2.10 (m,
1H, H-4), 1.75–1.72 (m, 4H, H-3, H-5), 1.20 (t, J = 6.9 Hz, CH3); EIMS (m/z): 297 (7%) [M]+ , 224 (10%),
156 (90%), 141 (14%), 77 (32%), 82 (100%).
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KHALID et al./Turk J Chem
Figure 1. Binding models of compounds 4g (a), 4m (b), 4i (c), and 4n (d). (a): Compound 4g is nicely bound to
AChE through Tyr124 and Tyr337, Gly122, Phe295, His447 and 3 π − π interactions with Trp86, Trp286, and Tyr341.
(b): Compound 4m is well bound to AChE through Gly122, Tyr341, His447, Tyr124, and Tyr337, Trp86, Trp286, and
Phe297 and 3 π − π interactions. (c): Compound 4i is nicely bound to BChE with Tyr332, Gly117, and Ser207 and
4 π − π interactions with Trp82, Trp231, Phe329, and Phe398 (d): Compound 4n is adequately bound to BChE with
Tyr332, Gly117, and Leu286. There are 4 π − π interactions with Trp82, Trp231, Phe329, and His438.
2.3. Preparation of 1-(phenylsulfonyl)piperidine-4-carbohydrazide (2)
Ethyl 1-(phenylsulfonyl)piperidine-4-carboxylate (1; 0.03 mol, 10 g) was dissolved in 50 mL of methanol as
solvent in a 250-mL RB flask and the mixture was cooled to 0–5 ◦ C. Then 30.0 mL of hydrazine hydrate (80%)
was added dropwise to the reaction mixture and the solution was stirred for 1 h at 0–5 ◦ C. Reaction completion
was monitored by TLC until a single spot was obtained. After the reaction was finished, excess solvent was
removed from the reaction mixture by distillation and white crystalline product 2 was formed, filtered off, and
washed with n-hexane.19,20
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KHALID et al./Turk J Chem
O
O
S
O
O
O
O
S
5% NaCO soln.
3
HN
N
Cl
pH 9 to 10
Stirr at RT
OC2H5
OC2H5
Reflux 1hr
a
1
b
NH .NH /MeOH
2 2
5% NaCO soln.
3
O O
S
O O
S
O
O
pH 9 to 10
3
O
2
N
NH
S
R
N
Stirr at RT
O
3'
1'
NH2
NH
5'
N
5
O
6
H
Cl
S
R
2
4a-n
O
3a-n
Compd.
R
Compd.
4e
R
Compd.
4i
R
Compd.
4m
R
4a
4b
4c
4f
4j
4n
6''
O
2''
4''
C
H
3C
CH3
8''
4g
4k
H3C
9''
6''
3''
H2C
10''
O
4d
4h
4l
Scheme. Outline for the synthesis of N′ -(1-(phenylsulfonyl)piperidine-4-carbonyl)sulfonohydrazide derivatives.
(2): IR (KBr, cm−1):vmax : 3310 (N-H), 3018 (Ar-CH), 2926 (C-H), 1630 (C=O), 1529 (C=C), 1325
(S=O); 1 H-NMR (300 MHz, CDCl3 , δ/ppm): 7.85 (s, 1H, NH-CO), 7.78 (dd, J = 7.8, 1.8 Hz, 2H, H-2’, H-6’),
7.66–7.63 (m, 3H, H-3’ to H-5’), 3.79 (t, J = 3.6 Hz, 2H, Heq -2, Heq -6), 3.75 (t, J = 3.6 Hz, 2H, Hax -2,
Hax -6), 2.13-2.11 (m, 1H, H-4), 1.9 (s, 2H, NH2) 1.79–1.75 (m, 4H, H-3, H-5); EIMS (m/z): 283 (13%) [M]+ ,
252 (10%), 224 (12%), 156 (12%), 84 (95%), 77 (100%).
2.4. General procedure for the synthesis of N′ -[(alkyl/aryl)sulfonyl]-1-(phenylsulfonyl)piperidine-
4-carbohydrazides derivatives in aqueous media (4a–n)
1-(Phenylsulfonyl) piperidine-4-carbohydrazide (2; 0.2 g, 0.007 mol) was suspended in 10.0 mL of water
and the pH was maintained at 9.0 by adding basic aqueous solution of a Na2 CO3 at 0–5 ◦ C. Then the
alkyl/alkylaryl/aryl sulfonyl chlorides (3a–n; 0.007 mol) were slowly added to the reaction mixture. After the
addition was finished, the temperature was allowed to rise slowly to room temperature. The reaction mixture
was kept on stirring for 2–3 h and was monitored via TLC until a single spot was obtained. Then conc. HCl
(around 0.5 mL) was slowly added to adjust the pH to 2.0. The formed precipitate was filtered and washed with
distilled water to afford the title compounds 4a–n on drying. Recrystallization was performed using methanol.
N′ , 1-bis (phenylsulfonyl)piperidine-4-carbohydrazide (4a): IR (KBr, cm−1):vmax : 3449 (N-H),
3011 (Ar-CH), 2924 (C-H), 1639 (C=O), 1530 (C=C), 1328 (S=O), 828 (N-S); 1 H-NMR (300 MHz, CDCl3 ,
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KHALID et al./Turk J Chem
δ/ppm): 8.02 (s, 1H, NH-CO), 7.85 (d, J = 7.2 Hz, 2H, H-2” & H-6”), 7.73 (dd, J = 6.8, 1.6 Hz, 2H, H-2’ &
H-6’), 7.66–7.61 (m, 2H, H-4’ & H-4”), 7.61–7.59 (m, 2H, H-3’ & H-5’), 7.51 (d, J = 8.8 Hz, 2H, H-3” & H-5”),
3.61–3.58 (m, 2H, Heq -2 & Heq -6), 2.34 (dt, J = 11.6, 2.8 Hz, 2H, Hax -2 & Hax -6), 2.04–2.00 (m, 1H, H-4),
1.65–1.45 (m, 4H, H-3 & H-5); EIMS (m/z): 423 (1.2%) [M]+ , 281 (28%), 252 (46%), 224 (55%), 170 (14%),
156 (71%), 77 (100%), 82 (56%).
N′ -[(4-methylphenylsulfonyl)]-1-(phenylsulfonyl)piperidine-4-carbohydrazide (4b): IR (KBr,
cm−1):vmax : 3443 (N-H), 3015 (Ar-CH), 2910 (C-H), 1640 (C=O), 1531 (C=C), 1329 (S=O), 838 (N-S); 1 H-
NMR (300 MHz, CDCl3 , δ/ppm): 8.05 (s, 1H, NH-CO), 7.75 (d, J = 7.6 Hz, 2H, H-2” & H-6”), 7.73–7.67 (m,
2H, H-2’ & H-6’), 7.66–7.64 (m, 1H, H-4’), 7.61–7.59 (m, 2H, H-3’ & H-5’), 7.30 (d, J = 8.0 Hz, 2H, H-3” &
H-5”), 3.66–3.63 (m, 2H, Heq -2 & Heq -6), 2.41 (s, 3H, -CH3), 2.32 (dt, J = 11.6, 2.4 Hz, 2H, Hax -2 & Hax -6),
2.02–1.62 (m, 1H, H-4), 1.49–1.47 (m, 4H, H-3 & H-5); EIMS (m/z): 437 (0.7%) [M]+ , 280 (27%), 252 (47%),
224 (50%), 170 (11%), 156 (100%), 91 (72%), 77 (50%), 84 (44%).
N′ -(benzylsulfonyl)-1-(phenylsulfonyl)piperidine-4-carbohydrazide (4c): IR (KBr, cm−1):vmax
3440 (N-H), 3013 (Ar-CH), 2924 (C-H), 1638 (C=O), 1530 (C=C), 1327 (S=O), 841 (N-S); 1 H-NMR (300 MHz,
CDCl3 , δ/ppm): 8.07 (s, 1H, NH-CO), 7.79 (d, J = 6.8 Hz, 2H, H-2’ & H-6’), 7.64 (d, J = 7.2 Hz, 1H, H-4’),
7.60 (d, J = 7.6 Hz, 2H, H-3’ & H-5’), 7.43–7.33 (m, 5H, H-2” to H-6”), 4.30 (s, 2H, H-7”), 3.77–3.74 (m, 2H,
Heq -2 & Heq -6), 2.43–2.38 (m, 2H, Hax -2 & Hax -6), 2.21–2.18 (m, 1H, H-4), 1.85–1.77 (m, 4H, H-3 & H-5);
EIMS (m/z): 437 (0.8%) [M]+ , 280 (25%), 252 (49%), 224 (51%), 141 (65%), 91 (100), 84 (40%), 42 (43%).
:
N′ -{[4-(bromomethyl)phenyl]sulfonyl} -1-(phenylsulfonyl)piperidin-4-carbohydrazide (4d):
IR (KBr, cm−1):vmax : 3439 (N-H), 3017 (Ar-CH), 2921 (C-H), 1635 (C=O), 1533 (C=C), 1322 (S=O), 842
(N-S); 1 H-NMR (300 MHz, CDCl3 , δ/ppm): 8.05 (s, 1H, NH-CO), 7.77 (d, J = 8.4 Hz, 2H, H-2” & H-6”),
7.76 (d, J = 7.2 Hz, 2H, H-2’ & H-6’), 7.64–7.61 (m, 2H, H-3’ & H-5’), 7.59 (m, 1H, H-4’), 7.45 (d, J = 8.0
Hz, 2H, H-3” & H-5”), 4.57 (s, 2H, C4”-CH2), 3.61–3.58 (m, 2H, Heq -2 & Heq -6), 2.47–2.41 (m, 2H, Hax -2 &
Hax -6), 2.29–2.24 (m, 1H, H-4), 1.97–1.69 (m, 4H, H-3 & H-5); EIMS (m/z): 516 (0.7%) [M]+ , 280 (2%), 279
(8%), 167 (33%), 149 (100%), 71 (46%), 57 (65%), 43 (50%).
N′ -{[4-(acetamido)phenyl]sulfonyl} -1-(phenylsulfonyl)piperidin-4-carbohydrazide (4e): IR
(KBr, cm−1):vmax : 3431 (N-H), 3019 (Ar-CH), 2920 (C-H), 1631 (C=O), 1529 (C=C), 1319 (S=O), 835 (N-S);
1 H-NMR (300 MHz, CDCl3 , δ/ppm): 8.09 (s, 1H, NH-CO), 7.86 (d, J = 7.6 Hz, 2H, H-2” & H-6”), 7.77–7.75
(m, 2H, H-2’ & H-6’), 7.70–7.69 (m, 1H, H-4’), 7.69–7.64 (m, 2H, H-3’ & H-5’), 7.58 (d, J = 8.0 Hz, 2H, H-3”
& H-5”), 7.33 (s, 1H, NH-COCH3), 3.65–3.62 (m, 2H, Heq -2 & Heq -6), 2.32 (dt, J = 11.6, 2.4 Hz, 2H, Hax -2
& Hax -6), 2.15 (s, 3H, -CH3), 2.03–1.61 (m, 1H, H-4), 1.53–1.47 (m, 4H, H-3 & H-5); EIMS (m/z): 480 (0.5%)
[M]+ , 282 (17%), 252 (44%), 224 (62%), 134 (78%), 77(65%), 55 (33%), 42 (47%).
N′ -[(4-(acetylphenyl)sulfonyl]-1-(phenylsulfonyl)piperidin-4-carbohydrazide (4f): IR (KBr,
cm−1):vmax : 3432 (N-H), 3011 (Ar-CH), 2897 (C-H), 1705 (R-C=O-R) 1624 (C=O), 1522 (C=C), 1317 (S=O),
833 (N-S); 1 H-NMR (300 MHz, CDCl3 , δ/ppm): 8.11 (s, 1H, NH-CO), 7.77 (d, J = 7.2 Hz, 2H, H-2” & H-6”),
7.75 (d, J = 7.2 Hz, 2H, H-2’ & H-6’), 7.64 (m, 1H, H-4’), 7.64–7.61 (m, 2H, H-3’ & H-5’), 7.59 (d, J = 7.6
Hz, 2H, H-3” & H-5”), 3.62–3.57 (m, 2H, Heq -2 & Heq -6), 2.54–2.47 (m, 2H, Hax -2 & Hax -6), 1.96–1.69 (m,
1H, H-4), 1.57–1.28 (m, 4H, H-3 & H-5); EIMS (m/z): 465 (1.5%) [M]+ , 282 (15%), 252 (49%), 224 (99%),
112 (22%), 84 (31%), 77 (68%), 42 (55%).
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N′ -[(2,4,6-trimethylphenyl)sulfonyl]-1-(phenylsulfonyl)piperidin-4-carbohydrazide (4g): IR
(KBr, cm−1):vmax : 3437 (N-H), 3013 (Ar-CH), 2923 (C-H), 1620 (C=O), 1521 (C=C), 1319 (S=O), 855 (N-
S); 1 H-NMR (300 MHz, CDCl3 , δ/ppm): 8.09 (s, 1H, NH-CO), 7.76–7.72 (m, 2H, H-2’ & H-6’), 7.65–7.61 (m,
1H, H-4’), 7.60–7.58 (m, 2H, H-3’ & H-5’), 6.96 (s, 2H, H-3” & H-5”), 3.69–3.60 (m, 2H, Heq -2 & Heq -6), 2.62
(s, 6H, CH3 -2”, CH3 -6”), 2.28 (s, 3H, CH3 -4”), 2.32-2.22 (m, 2H, Hax -2 & Hax -6), 1.99–1.94 (m, 1H, H-4),
1.56–1.46 (m, 4H, H-3 & H-5); EIMS (m/z): 466 (2%) [M]+ , 281 (28%), 252 (77%), 224 (100%), 119 (14%),
84 (25%), 55 (70%), 42 (63%).
N′ -[(4-chlorophenyl)sulfonyl]-1-(phenylsulfonyl)piperidin-4-carbohydrazide (4h): IR (KBr,
cm−1):vmax : 3429 (N-H), 3025 (Ar-CH), 2921 (C-H), 1637 (C=O), 1524 (C=C), 1318 (S=O), 847 (N-S);
1 H-NMR (300 MHz, CDCl3 , δ/ppm): 8.07 (s, 1H, NH-CO), 7.82 (d, J = 8.4 Hz, 2H, H-2” & H-6”), 7.73 (dd,
J = 8.4, 1.6 Hz, 2H, H-2’ & H-6’), 7.66–7.64 (m, 1H, H-4’), 7.61–7.59 (m, 2H, H-3’ & H-5’), 7.51 (d, J = 8.8
Hz, 2H, H-3” & H-5”), 3.65–3.62 (m, 2H, Heq -2 & Heq -6), 2.34 (dt, J = 11.6, 2.8 Hz, 2H, Hax -2 & Hax -6),
2.02–1.60 (m, 1H, H-4), 1.52–1.45 (m, 4H, H-3 & H-5); EIMS (m/z): 458 (0.8%) [M]+ , 282 (68%), 252 (86%),
224 (100%), 111 (15%), 77 (67%), 55 (58%), 43 (73%).
N′ -[(4-bromophenyl)sulfonyl]-1-(phenylsulfonyl)piperidin-4-carbohydrazide (4i): IR (KBr,
cm−1):vmax : 3427 (N-H), 3023 (Ar-CH), 2925 (C-H), 1640 (C=O), 1529 (C=C), 1316 (S=O), 849 (N-S);
1 H-NMR (300 MHz, CDCl3 , δ/ppm): 8.11 (s, 1H, NH-CO), 7.85 (d, J = 8.4 Hz, 2H, H-2” & H-6”), 7.77 (d,
J = 6.8 Hz, 2H, H-2’ & H-6’), 7.65–7.64 (m, 1H, H-4’), 7.59 (d, J = 6.8 Hz, 2H, H-3’ & H-5’), 7.50 (d, J = 8.8
Hz, 2H, H-3” & H-5”), 3.61–3.58 (m, 2H, Heq -2 & Heq -6), 2.52–2.47 (m, 2H, Hax -2 & Hax -6), 2.38–2.35 (m,
1H, H-4), 2.34–1.96 (m, 4H, H-3 & H-5); EIMS (m/z): 502 (1.7%) [M]+ , 282 (67%), 252 (83%), 224 (78%),
156 (100%), 77 (54%), 54 (41%), 43 (57%).
N′ -[(3,5-dichloro-2-hydroxyphenyl)sulfonyl)-1-(phenylsulfonyl) piperidin-4-carbohydrazide
(4j): IR (KBr, cm−1):vmax : 3440 (N-H), 3320 (O-H), 3017 (Ar-CH), 2921 (C-H), 1632 (C=O), 1526 (C=C),
1320 (S=O), 855 (N-S); 1 H-NMR (300 MHz, CDCl3 , δ/ppm): 8.11 (s, 1H, NH-CO), 7.73 (d, J = 7.2 Hz, 2H,
H-2’ & H-6’), 7.64 (d, J = 2.4 Hz, 1H, H-6”), 7.63 (d, J = 2.4 Hz, 1H, H-4”), 7.64–7.61 (m, 1H, H-4’), 7.59
(d, J = 6.8 Hz, 2H, H-3’ & H-5’), 3.60–3.57 (m, 2H, Heq -2 & Heq -6), 2.40–2.35 (m, 2H, Hax -2 & Hax -6),
2.10–1.65 (m, 1H, H-4), 1.52–1.50 (m, 4H, H-3 & H-5); EIMS (m/z): 508 (1.2%) [M]+ , 282 (23%), 252 (47%),
224 (82%), 161 (52%), 150 (100%), 77 (63%), 55 (48%).
N′ -{[(1R,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]hept-1-yl)methyl] sulfonyl} -1-(phenylsulfonyl)
piperidine-4-carbohydrazide (4k): IR (KBr, cm−1):vmax : 3448 (N-H), 3014 (Ar-CH), 2924 (C-H), 1715 (R-
C=O-R), 1637 (C=O), 1530 (C=C), 1328 (S=O), 828 (N-S), 860 (N-S); 1 H-NMR (300 MHz, CDCl3 , δ/ppm):
8.12 (s, 1H, NH-CO), 7.77–7.76 (m, 2H, H-2’ & H-6’), 7.66–7.64 (m, 1H, H-4’), 7.61–7.60 (m, 2H, H-3’ & H-5’),
3.71 (brs, 2H, H-10”), 3.61–3.58 (m, 2H, Heq -2 & Heq -6), 2.52–2.47 (m, 2H, Hax -2 & Hax -6), 2.37–2.31 (m,
2H, H-3”), 2.30–2.28 (m, 1H, H-4), 2.19–2.16 (m, 4H, H-3 & H-5), 1.97–1.94 (m, 2H, H-6”), 1.86-1.81 (m, 2H,
H-5”), 1.83–1.69 (m, 1H, H-4”), 1.06 (s, 3H, H-9”), 0.74 (s, 3H, H-8”); EIMS (m/z): 497 (0.8%) [M]+ , 252
(49%), 224 (68%), 215 (42%), 175 (51%), 151 (100%), 84 (35%), 77 (27%).
N′ -(butylsulfonyl)-1-(phenylsulfonyl)piperidine-4-carbohydrazide (4l): IR (KBr, cm−1):vmax
:
3445 (N-H), 3011 (Ar-CH), 2920 (C-H), 1638 (C=O), 1525 (C=C), 1317 (S=O), 878 (N-S); 1 H- NMR (300
MHz, CDCl3 , δ/ppm): 8.10 (s, 1H, NH-CO), 7.77 (d, J = 6.8 Hz, 2H, H-2’ & H-6’), 7.66–7.64 (m, 1H, H-4’),
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KHALID et al./Turk J Chem
7.59 (d, J = 6.8 Hz, 2H, H-3’ & H-5’), 3.74 (brs, 2H, H-1”), 3.61–3.58 (m, 2H, Heq -2 & Heq -6), 2.52–2.47 (m,
2H, Hax -2 & Hax -6), 2.38–2.35 (m, 1H, H-4), 2.34–1.96 (m, 4H, H-3 & H-5), 1.89–1.77 (m, 2H, H-3”), 1.76–1.69
(m, 2H, H-2”), 1.28 (brs, 3H, H-4”); EIMS (m/z): 403 (2.2%) [M]+ , 282 (66%), 252 (49%), 224 (80%), 121
(32%), 84 (21%), 77 (39%), 54 (29%).
N′ -(naphthalene-1-ylsulfonyl)-1-(phenylsulfonyl)piperidine-4-carbohydrazide (4m): IR (KBr,
cm−1):vmax : 3447 (N-H), 3016 (Ar-CH), 2923 (C-H), 1637 (C=O), 1529 (C=C), 1324 (S=O), 831 (N-S); 1 H-
NMR (300 MHz, CDCl3δ/ppm): 8.71 (s, 1H, NH-CO), 8.53–8.02 (m, 7H, H-2” to H-8”), 7.98 (d, J = 6.8 Hz,
2H, H-2’ & H-6’), 7.85–7.80 (m, 1H, H-4’), 7.75 (d, J = 6.8 Hz, 2H, H-3’ & H-5’), 3.61–3.58 (m, 2H, Heq -2
& Heq -6), 2.52–2.47 (m, 2H, Hax -2 & Hax -6), 2.38-2.35 (m, 1H, H-4), 2.34–1.96 (m, 4H, H-3 & H-5); EIMS
(m/z): 473 (2.5%) [M]+ , 252 (52%), 224 (44%), 127 (89%), 119 (39%), 101 (100%), 84 (21%), 77 (15%).
N′ -(naphthalene-2-ylsulfonyl)-1-(phenylsulfonyl)piperidine-4-carboohydrazide (4n): IR (KBr,
cm−1):vmax : 3443 (N-H), 3018 (Ar-CH), 2921 (C-H), 1635 (C=O), 1525 (C=C), 1327 (S=O), 834 (N-S); 1 H-
NMR (300 MHz, CDCl3δ/ppm): 8.70 (s, 1H, NH-CO), 8.68 (s, 1H, H-1”), 8.51–8.00 (m, 6H, H-3” to H-8”),
7.96 (d, J = 6.8 Hz, 2H, H-2’ & H-6’), 7.82–7.78 (m, 1H, H-4’), 7.73 (d, J = 6.8 Hz, 2H, H-3’ & H-5’), 3.61–3.58
(m, 2H, Heq -2 & Heq -6), 2.52–2.47 (m, 2H, Hax -2 & Hax -6), 2.38–2.35 (m, 1H, H-4), 2.34–1.96 (m, 4H, H-3
& H-5); EIMS (m/z): 473 (2%) [M]+ , 252 (51%), 224 (42%), 127 (88%), 119 (38%), 101 (100%), 84 (21%), 77
(17%).
2.5. Cholinesterase assays
The AChE and BChE inhibition activities were determined according to the spectrophotometric method
of Ellman21 with small modifications. A volume of 100 µL comprising 60 µL of Na2 HPO4 buffer with
concentration of 50 mM (pH 7.7), 10 µL of test compound (0.5 mM well−1), and 10 µL (0.005 unit well−1 for
AChE and 0.5 unit well−1 for BChE) of enzyme was developed. This homogeneous mixture was pre-read at
405 nm followed by pre-incubation for 10 min at 37 ◦ C. The reaction started by the addition of 10 µL of 0.5
mM well−1 substrate (acetylthiocholine iodide for AChE and butyrylthiocholine chloride for BChE) and the
addition of 10 µL of DTNB (0.5 mM well−1). After 15 min of incubation at 37 ◦ C, absorbance was measured
at 405 nm using a 96-well plate reader (Synergy HT, Biotek, USA). All experiments were carried out with their
respective controls in triplicate. Eserine (0.5 mM well−1) was used as a positive control. The percent inhibition
was calculated by the following equation: Inhibition (%) = (Abs of Control – Abs of Test Comp)/Abs of Control
× 100,
where control is the activity without inhibitor and test is the activity in the presence of test compound.
IC50 values were calculated using EZ–Fit Enzyme kinetics software (Perrella Scientific Inc., Amherst, NH,
USA). The IC50 values were the average of 3 independent experiments.
All the measurements were executed in triplicate and statistical analysis was performed using Microsoft
Excel 2010. Results are presented as mean ꢀ SEM.
2.6. Molecular docking
The AChE crystal structure (PDB accession code, 1B41)22 was retrieved from the Protein Databank (PDB).
The missing residues in the crystal structure were constructed by using the program UCSF Chimera 1.6.16.23
The pdb file (PDB accession code, 2WID) was retrieved from the protein databank and its missing residues
195

KHALID et al./Turk J Chem
were constructed by aligning it to the other pdb file (PDB accession code, 1P0P). All water molecules were
removed from the retrieved crystal structures using the program Visual Molecular Dynamics, VMD 1.9.24 Both
AChE and BChE were allowed to dock to experimentally synthesize 16 active compounds including parent
compounds. The 3D structures of all compounds were constructed in pdb format and subsequently optimized
at semiempirical RM1 level of theory using by the programs Gabedit25 and MOPAC 2012,26 respectively.
The docking study of all compounds was accomplished by the software AutoDock Vina,27 using the built-
in Lamarckian genetic algorithm method. A total of 20 runs were performed for each docking and rests of
parameters were set to default values.
The search space was restricted to a grid box size of 46 × 46 × 46 in the x, y, and z dimensions,
respectively, centered on the binding site of protein with x, y, and z coordinates of 120.491, 106.059, and –
˚
136.443 A, respectively. All the docking runs were performed on Intel Core i5-2410M CPU @ 2.30 GHz of Sony
origin, with 6.0 GB DDR RAM. AutoDock Vina was compiled and run under the Windows 7 Professional 64-bit
operating system.
3. Result and discussion
3.1. Chemistry
In the present work, N′ -(aryl/alkylsulfonyl)(1-(phenylsulfonyl)piperidine-4-carbohydrazide derivatives were
prepared in a 3-step synthesis. Then they were screened against AChE and BChE enzymes. The parent com-
pound, ethyl-1-(phenylsulfonyl)piperidine-4-carboxylate (1) was prepared by the reaction of ethyl piperidine-4-
carboxylate (a) and benzenesulfonyl chloride (b) at dynamic pH control in aqueous media.28−32 The reaction
was processed in basic media at pH 9 to neutralize the hydrochloric acid developed by sulfonyl chloride during
the reaction. The produced acid suppresses the nucleophilic character of the amine by capturing the lone pair of
nitrogen, thus rendering the corresponding salt and influencing also the rate of reaction. At the end, the reaction
medium was acidified to remove the unreacted amine in the form of salt and also to convert the salt form of
sulfonamide into acidic form. The excess of acid should be restrained because it lessens yield due to another
salt formation of sulfonamide. Further, the compound (1) was converted to 1-(phenylsulfonyl)piperidine-4-
carbohydrazide (2) by refluxing with hydrated hydrazine (80%) in methanol as solvent for 3–4 h. The product
was collected by filtration after the evaporation of half of the solvent. The third and last step comprises the
synthesis of all derivatives 4a–n by coupling 2 with the different alkyl/aryl sulfonyl chlorides (3a–n) in the
aqueous media under low basic pH. The final products were filtered off after acidifying the reaction mixture.
The structures of the synthesized compounds 1, 2, and 4a–n were elucidated by spectral data as described in
the experimental section. The physical data of the synthesized compounds are provided in Table 1.
Compound 2 was synthesized as a white crystalline solid with melting point of 119 ◦ C and 80% yield. The
molecular formula C12 H19 N3 O3 S was established by molecular ion peak at m/z 283 in EI-MS and by counting
the number of protons in its 1 H-NMR spectrum. The infrared spectrum showed absorption bands at 3310 cm−1
,
3018 cm−1 , 2926 cm−1 , 1630 cm−1 , 1529 cm−1 , and 1325 cm−1 , which were assigned to N-H (stretching),
C-H (aromatic stretching), C-H (aliphatic stretching), C=O (stretching), C=C (aromatic stretching), and S=O
(stretching of sulfonyl group), respectively. The EI-MS gave characteristic peaks at m/z224 and 156, which
were attributed to the loss of (phenylsulfonyl)piperidine and benzene sulfonamide groups, respectively. In the
aromatic region of the 1 H-NMR spectrum, signals appearing at δ 7.78 (dd, J = 7.8, 1.8 Hz, 2H, H-2’, H-6’)
and 7.66–7.63 (m, 3H, H-3’ to H-5’) were assigned to the benzenesulfonyl ring. In the aliphatic region of the
196

KHALID et al./Turk J Chem
1 H-NMR spectrum, signals appearing at δ 3.79 (t, J = 3.6 Hz, 2H, Heq -2’, Heq -6’), 3.75 (t, J = 3.6 Hz, 2H,
Hax -2’, Hax -6’), 2.12–1.99 (m, 1H, H-4’), and 1.79–1.75 (m, 4H, H-3’ & H-5’) for δ 3.79 (t, J = 3.6 Hz, 2H,
Heq -2, Heq -6’), 3.75 (t, J = 3.6 Hz, 2H, Hax -2’ Hax -6’), 2.12–1.99 (m, 1H, H-4), and 1.79–1.75 (m, 4H, H-3 &
H-5) indicated the presence of a piperidine nucleus in the molecule. One signal emerging at δ 7.85 as a singlet
was specified for the proton of nitrogen directly attached to the carbonyl group. On the basis of these data,
the structure of 2 was assigned as 1-(phenylsulfonyl)piperidine-4-carbohydrazide. Similarly, the structures of
other compounds were characterized by 1 H-NMR, IR, and mass spectral data as described in the experimental
section.
Table 1. Physical data of synthesized compounds.
Compound Appearance
Melting point (◦C) Molecular formula %Yield
1
2
Creamy white powder 60–62
White crystals 119
Creamy white powder 150–152
C14H19NO4S
90
80
88
75
78
77
78
73
72
78
80
C12H19N3O3S
C18H21N3O5S2
C19H23N3O5S2
C19H23N3O5S2
C19H22BrN3O5S2
C20H24N4O6S2
C20H23N3O6S2
C21H27N3O5S2
C18H20ClN3O5S2
C18H20BrN3O5S2
4a
4b
4c
4d
4e
4f
4g
4h
4i
White powder
White fibrous solid
White powder
White powder
Off white powder
176–178
168–169
140–142
115–117
110–112
Creamy white powder 72–74
White powder
165–166
Creamy white powder 149–150
4j
4k
4l
4m
4n
White powder
210–212
–
–
59–60
126–127
C18H19Cl2N3O5S2 83
Lemon yellow liquid
Mustard sticky liquid
Light yellow powder
Off white powder
C22H31N3O6S2
C16H25N3O5S2
C22H23N3O5S2
C22H23N3O5S2
76
79
82
76
*Melting points of the synthesized compounds were recorded on a Griffin and George melting point apparatus by open
capillary tube; Molecular formulas were confirmed by EI-MS calculations
3.2. Biological assay and docking analysis
In order to elucidate the probable mechanism by which the title compounds could induce anticholinesterase
activity and to rationalize the ligand–protein interaction at molecular level for establishing structure activity
relationships, molecular docking of the potent inhibitors from a series of compounds was accomplished into the
receptor site of the crystal structures of AChE and BChE. The highly potent compounds from the series were
selected on the basis of IC50 , which was determined experimentally. The enzyme inhibition data are provided
for all the synthesized compounds in Table 2. Compounds 4g and 4m were found to be the most active
against AChE and compounds 4i and 4n against BChE among the series of the compounds. Their candidacy
of being highly potent was also supported by docking score as these compounds had good binding energy with
the respective proteins. Compounds 4g and 4m had docking scores of –10.00 kcal/mol and –11.60 kcal/mol,
respectively, against AChE as compared to the reference inhibitor, eserine, i.e. –7.10 kcal/mol. Compounds
4i and 4n had docking scores of –9.80 kcal/mol and –11.50 kcal/mol, respectively, against BChE as compared
to the reference inhibitor, eserine, i.e. –7.50 kcal/mol. The binding energies of the highlighted compounds are
given in Table 3.
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KHALID et al./Turk J Chem
Table 2. Enzymatic activity profile of the synthesized compounds.
AChE
BChE
Conc./well
(mM)
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
IC50
µM
-
-
-
Conc./well
(mM)
0.5
0.5
0.5
IC50
µM
-
Sample code
Inhibition (%)
Inhibition (%)
1
2
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
41.00 ꢀ 0.15
43.33 ꢀ 0.15
48.96 ꢀ 0.61
78.86 ꢀ 0.94
62.31 ꢀ 0.69
57.61 ꢀ 0.44
60.46 ꢀ 0.31
73.71 ꢀ 0.11
91.57 ꢀ 0.78
68.02 ꢀ 0.14
87.51 ꢀ 0.22
38.12 ꢀ 0.18
76.01 ꢀ 0.14
11.72 ꢀ 0.47
90.80 ꢀ 0.55
87.73 ꢀ 0.87
Eserine
22.48 ꢀ 0.16
26.68 ꢀ 0.16
41.21 ꢀ 0.54
51.54 ꢀ 0.11
32.46 ꢀ 0.15
81.85 ꢀ 0.22
47.54 ꢀ 0.55
28.41 ꢀ 0.17
86.92 ꢀ 0.62
52.46 ꢀ 0.77
92.31 ꢀ 0.21
43.96 ꢀ 0.69
66.62 ꢀ 0.89
27.64 ꢀ 0.18
72.46 ꢀ 0.47
93.85 ꢀ 0.64
Eserine
-
-
> 400
-
104.21 ꢀ 0.11 0.5
212.31 ꢀ 0.11 0.5
256.81 ꢀ 0.14 0.5
230.14 ꢀ 0.91 0.5
121.41 ꢀ 0.04 0.5
68.71 ꢀ 0.51
-
-
58.91 ꢀ 0.14
0.5
63.21 ꢀ 0.91
141.51 ꢀ 0.17 0.5
> 400
48.91 ꢀ 0.11
-
72.41 ꢀ 0.71
0.5
0.5
-
116.31 ꢀ 0.12 0.5
202.31 ꢀ 0.14
-
0.5
0.5
0.5
-
4m
4n
Positive
control
64.11 ꢀ 0.04
103.21 ꢀ 0.11
44.52 ꢀ 0.12
0.85 ꢀ 0.0001
0.5
78.51 ꢀ 0.91
0.25
0.04 ꢀ 0.0001 0.25
91.29 ꢀ 1.17
82.82 ꢀ 1.09
Note: IC50 values (concentration at which there is 50% enzyme inhibition) of compounds were calculated using EZ–Fit
Enzyme kinetics software (Perella Scientific Inc., Amherst, NH, USA).
AChE = Acetylcholinesterase.
BChE = Butyrylcholinesterase.
Table 3. Results obtained from the molecular docking of most active compounds from series.
–H
–
Cation–
Electrostatic
H-Bonding
VDW
interaction interaction interaction interactions
B.E .
(kcal/mol)
Enzyme Compd.
Interacting
residues
Distance
(Å)
Tyr124
Tyr337
Phe295
Gly122
His447
Tyr124
Tyr337
*Tyr341
Gly122
*His447
2.839
2.360
3.704
3.148
3.017
3.209
3.305
5.760
3.148
4.551
Trp286
Trp86
4g
–10.00
–11.60
Tyr341
Phe297
-
-
-
-
Tyr449
AChE
Trp286
Trp86
Tyr449
Phe295
4m
Tyr332
Gly117
Ser287
2.893
3.724
4.077
Trp231
Trp82
Phe329
4i
BChE
–9.80
Phe398
-
-
-
-
His438
-
Tyr332
Gly117
Leu286
3.037
3.692
4.220
Trp231
Trp82
Phe329
His438
4n
–11.50
˚
* The binding energies >4.5 A show weak H-bonding AChE = Acetyl cholinesterase BChE = Butyrylcholinesterase
B.E = Binding energy VDW = Van der Waal interaction
198

KHALID et al./Turk J Chem
Compounds 4g and 4m were the most active against AChE with IC50 values of 58.91 ꢀ 0.14 µM and
64.11 ꢀ 0.04 µM, respectively, with respect to the reference standard eserine, having an IC50 value of 0.04 ꢀ
0.0001 µM. The binding models of compounds 4g and 4m in the active site of AChE are depicted in Figure
1 (a and b, respectively). Compound 4g was nicely bound to AChE through H-bonds involving amino and
˚
amido groups projecting toward the hydroxyl oxygen of Tyr124 (2.59 A) and Tyr337 (3.84 A). The oxygen of
˚
˚
both sulfonyl groups formed H-bonds with the backbone nitrogen atom of Gly122 (3.08 A) and Phe295 (3.70
˚
A). Protons of methyl group of mesitylene were also involved in H-bonding with carboxylic oxygen of His447
˚
(3.02 A). There are 2 π − π interactions among Trp86, Trp286, and the inhibitor (Figure 1a). Compound 4m
was well bound to the AChE with its oxygen atoms of the sulfonyl group projecting toward the amino group
˚
of Gly122 (3.15 A) and His447 (4.55 A), the protons of amino and amido groups bound to oxygen atoms of
˚
˚
˚
hydroxyl groups of Tyr124 (3.21 A) and Tyr337 (3.31 A). Trp86 and Trp286 formed 2 π − π interactions with
the inhibitor (Figure 1b). The binding of ligands with common residues Tyr124, Tyr337, Gly122, and His447
is vital for their activities as AChE inhibitors. All these residues were found in the binding model of both 4g
and 4m compounds, making them highly potent for AChE (Table 3).
Compounds 4i and 4n were the most potent inhibitors of BChE with IC50 values of 48.91 ꢀ 0.11 µM
and 44.52 ꢀ 0.12 µM, respectively, with eserine, the reference standard having IC50 value of 0.85 ꢀ 0.0001
µM. The binding models of compounds 4i and 4n in the active site of BChE are depicted in Figure 1(c and d,
respectively). Compound 4i is bound to the BChE in an adequate manner through its amino group projecting
˚
toward the hydroxyl oxygen of Tyr 332 (2.89 A) and oxygen atoms of the sulfonyl group projected toward the
˚
˚
amino group of Gly 117 (3.72 A) and Ser 287 (4.08 A). The bromine at the para position of the benzene ring
is bound to the amino group of His438 through weak electrostatic interactions. Three π − π interactions were
formed among Trp82, Trp231, Phe329, and Phe398 and the inhibitor (Figure 1c). Compound 4n is nicely bound
˚
to BChE through its amino group with hydroxyl oxygen of Tyr332 (3.04 A) and the oxygen atoms of the sulfonyl
˚
˚
group are bound to the amino group of Gly117 (3.69 A) and Leu286 (4.22 A). There are 3 π − π interactions
between Trp82, Trp231, Phe329, and the inhibitor (Figure 1d). The binding of ligands with common residues
Tyr332, Gly117, Trp82, Trp231, and Phe329 is vital for their activities as BChE inhibitors. All these residues
were found in the binding model of both 4i and 4n compounds, making them rather potent for AChE (Table 3).
4. Conclusion
The synthesis of this series of compounds bearing a heterocyclic moiety of piperidine and 2 different function-
alities involving sulfonamide and amide linkages was proved to be highly active due to remarkable inhibitory
action for AChE and BChE enzymes. In this study, insights into the interaction of cholinesterase with syn-
thesized inhibitory compounds were elucidated through the use of molecular docking. AutoDock was used to
model the interaction between cholinesterase (AChE and BChE) and its inhibitors. The results revealed that
Tyr124, Tyr337, Gly122, and His447 and Tyr332, Gly117, Trp82, Trp231, and Phe329 residues were important
for the interaction of inhibitors with both AChE and BChE. The knowledge accomplished with this study
has important implications for the design of new AChE and BuChE inhibitors. To further enhance the anti-
cholinesterase efficiency of piperidine bearing sulfonamides, further structural modifications of existing scaffolds
or development of new structures is a promising venue to explore.
199

KHALID et al./Turk J Chem
Acknowledgment
The authors are thankful to the Higher Education Commission of Pakistan for the financial support regarding
this research work and the spectroscopic analysis.
References
1. Massoulie, J.; Pezzementi, L.; Bon, S.; Krejci, E.; Valette, F. M. Prog. Neurobiol. 1993, 41, 31–91.
2. Chatonnet, A.; Lockridge, O. Biochem. J. 1989, 260, 625–634.
3. C¸oku˘gra¸s, N. A. Turk. J. Biochem. 2003, 28, 54–61.
4. Sussman, L. J.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Protein Sci. 1991, 253,
872–878.
5. Gilman, A. G.; Rall, T. W.; Nies, A.; Taylor, P. In The Pharmacological Basis of Therapeutics; Eds. Pergamon,
New York 1990, pp. 131–149.
6. Hallak, M.; Giacobini, E. Neuropharmacology 1989, 28, 199–206.
7. Scarpini, E.; Scheltens, P.; Feldman, H. Lancet Neurol. 2003, 2, 539–547.
8. Perry, E. K.; Tomlinson, B. E.; Blessed, G.; Bergman, K.; Gibson, P. H.; Perry, R. H. Brit. Med. J. 1978, 25,
1457–1459.
9. Holzgrabe, U.; Kapkova, P.; Alptuzun, V.; Scheiber, J.; Kugelmann, E. Expert. Opin. Ther. Targets 2007, 11,
161–179.
10. Butini, S.; Campiani, G.; Borriello, M.; Gemma, S.; Panico, A.; Persico, M.; Catalanotti, B.; Ros, S.; Brindisi, M.;
Agnusdei, M.; et al. J. Med. Chem. 2008, 51, 3154–3170.
11. Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. Biochem. Pharmacol. 2003, 65, 407–416.
12. Shafferman, A.; Velan, B.; Ordentlich, A.; Kronman, C.; Grosfeld, H.; Leitner, M.; Flashner, Y.; Cohen, S.; Barak,
D.; Ariel, N. EMBO J. 1992, 11, 3561–3568.
13. Changeux, J. P. Mol. Pharmacol. 1966, 2, 369–392.
14. Cochard, P.; Coltey, P. Dev. Biol. 1983, 98, 221–238.
15. Chatonnet, A.; Lockridge, O. Biochem. J. 1989, 260, 625–634.
16. Sikorav, J. L.; Krejci, E.; Massoulie, J. EMBO J. 1987, 6, 1865–1873.
17. Solis, A.; Nava, A.; Perez, H. I.; Manjarrez, N.; Luna, H.; Cassani, J. J. Mex. Chem. Soc. 2008, 52, 181–184.
18. Khalid, H. Aziz-ur-Rehman; Abbasi, M. A.; Khan, K. M. Int. J. Pharm. Pharm. Sci. 2012, 4, 443–448.
19. Aziz-ur-Rehman; Nafeesa, K.; Abbasi, M. A.; Khalid, H.; Khan, K. M.; Ashraf, M.; Ahmad, I.; Ejaz, S. A. Asian.
J. Pharm. Hea. Sci. 2012, 2, 370–376.
20. Aziz-ur-Rehman; Siddiqui, S. Z.; Abbas, N.; Abbasi, M. A.; Khan, K. M.; Shahid, M.; Mahmood, Y.; Akhtar, N.
M.; Lajis, N. H. Int. J. Pharm. Pharm. Sci. 2012, 4, 676–680.
21. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88–95.
22. Kryger, G.; Harel, M.; Giles, K.; Toker, L.; Velan, B.; Lazar, A.; Kronman, C.; Barak, D.; Ariel, N.; Shafferman,
A.; et al. Acta. Crystallogr. D. 2000, 56, 1385–1394.
23. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. J.
Comput. Chem. 2004, 25, 1605–1612.
24. Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33–38.
25. Allouche, A. R. J. Comput. Chem. 2010, 32, 174–182.
26. Stewart, J. P. J. Mol. Model. 2007, 13, 1173–1213.
27. Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455–461.
200

KHALID et al./Turk J Chem
28. Deng, X; Mani, N. S. Green Chem. 2006, 8, 835–838.
29. Jafarpour, M; Rezaeifard, A; Golshani, T. J. Phosphorous, Sulfur and Silicon. 2011, 186, 140–148.
30. Aziz-ur-Rehman; Afroz, S; Abbasi, M. A; Tanveer, W; Khan, K. M; Asraf, M; Afzal, I; Ambreen. Pak. J. Pharm.
Sci. 2012, 25, 809–814.
31. Aziz-ur-Rehman; Rasool, S; Abbasi, M. A; Khan, K. M; Asraf, M.; Afzal, I. Asian J. Phram. Bio. Res. 2012, 2,
100–105.
32. Aziz-ur-Rehman; Awais-ur-Rehman; Abbasi, M. A; Khalid, H; Khan, K. M; Dar, P. Asian J. Pharm. Hea. Sci.
2012, 2, 384–389.
201

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