Active site directed docking studies: Synthesis and pharmacological evaluation of cis-2,6-dimethyl piperidine sulfonamides as inhibitors of acetylcholinesterase

Article abstract of DOI:10.1016/j.ejmech.2009.04.042

Hypocholinergic function associated with Alzheimer's disease (AD) is well-accepted hypothesis, in this regard, many research attempts have been made to elevate the reduced cholinergic neurotransmission, among them two main treatment strategies were widely

Full text of DOI:10.1016/j.ejmech.2009.04.042

European Journal of Medicinal Chemistry 44 (2009) 4057–4062
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Active site directed docking studies: Synthesis and pharmacological evaluation of
cis-2,6-dimethyl piperidine sulfonamides as inhibitors of acetylcholinesterase
H.R. Girisha, J.N. Narendra Sharath Chandra, Sriramamurthy Boppana, Manish Malviya, C.T. Sadashiva,
*
Kanchugarakoppal S. Rangappa
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore - 570006, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Hypocholinergic function associated with Alzheimer’s disease (AD) is well-accepted hypothesis, in this
regard, many research attempts have been made to elevate the reduced cholinergic neurotransmission,
among them two main treatment strategies were widely explored, namely stimulation of muscarinic
receptor 1 and/or reversible inhibition of acetylcholinesterase (AChE) enzyme. In an attempt to improve
the efficacy and to minimize general side effects of these AChE inhibitors, many lead molecules are
developed in research; one among them is piperidine derivative. Donazepil is a widely prescribed AChE
inhibitor which displays a piperidine ring in its structure. In the present study, we have docked cis-2,6-
dimethyl piperidine sulfonamides (3a–i) on AChE enzyme and synthesized by nucleophilic substitution
reaction between cis-2,6-dimethyl piperidine and alkyl/aryl sulfonyl chlorides in the presence of trie-
thylamine. These piperidine sulfonamides were subjected to in vitro AChE enzyme inhibition studies and
in vivo antiamnesic study to reverse scopolamine induced memory loss in rats. Two derivatives (3a and f)
in this class of piperidines (3a–i) showed considerable inhibition against different sources of AChE in
vitro and reduced average number of mistakes done by wistar rats as compared to scopolamine treated
group in vivo (rodent memory evaluation).
Received 1 May 2007
Received in revised form
10 April 2009
Accepted 17 April 2009
Available online 8 May 2009
Keywords:
Alzheimer’s diseases
AChE
Docking
Ó 2009 Published by Elsevier Masson SAS.
1. Introduction
The cholinergic hypothesis of AD has provided the rationale for
the current major therapeutic approach to the disease, which is
Modern methods for computer-assisted drug design fall into
two major categories generally known as ligand-based and target-
based methods. The former, which include conventional quantita-
tive structure–activity relationships (QSARs) [1], active analog
approach [2], and recently comparative molecular field analysis
(CoMFA) [3], are based entirely on experimental structure–activity
relationship for receptor ligands or enzyme inhibitors, and their
application from past 30 years has led to several drugs now in
clinical practice [4]. The latter methods which include docking
and advanced molecular simulations require the structural infor-
mation about target as provided by X-ray crystallography, NMR or
protein homology model building. This strategy has become
available only recently, with rapid advances in structure elucidation
methods yielding several promising drug candidates in clinical
practice [5].
aimed to correct the cognitive decline by enhancing cholinergic
neurotransmission [6,7]. In this regard, multiple possible strategies
(muscarinic 1 receptor activation, muscarinic 2 receptor blocking,
nicotinic receptor activation and AChE inhibition) were used.
Among them, cholinesterase inhibition has been so for the most
extensively used approach for the treatment of Alzheimer’s disease.
In our present study, we have investigated the interaction of alkyl
and aryl cis-2,6-dimethyl piperidine sulfonamides (3a–i) with
mouse AChE by docking study, along with their in vitro inhibition of
AChE and in vivo evaluation of memory and learning in male wistar
rats (rodent memory evaluation study).
2. Materials and methods
2.1. Experimental section
2.1.1. Protein–ligand docking
The ligands including all hydrogen atoms were built and opti-
mized with Chemsketch software suite (Advanced Chemistry
Development, Inc). Extremely Fast Rigid Exhaustive Docking (FRED)
version 2.1 was used for docking studies (Open Eye Scientific
* Corresponding author. Tel.: þ91 821 2419661; fax: þ91 821 2412191.
E-mail addresses: rangappaks@gmail.com, rangappaks@chemistry.uni-mysore.
ac.in (K.S. Rangappa).
0223-5234/$ – see front matter Ó 2009 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2009.04.042

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H.R. Girisha et al. / European Journal of Medicinal Chemistry 44 (2009) 4057–4062
Software, Santa Fe, NM). This program generates an ensemble of
different rigid body orientations (poses) for each compound
conformer within the binding pocket and then passes each mole-
cule against a negative image of the binding site. Poses clashing
with this ‘bump map’ are eliminated. Poses surviving the bump test
are then scored and ranked with a Gaussian shape function. We
defined the binding pocket using the ligand-free protein structure
and a box enclosing the binding site of 4 Å. One unique pose for
each of the best-scored compounds was saved for the subsequent
steps. The compounds used to dock were converted in 3-dimension
with Omega (Open Eye Scientific Software, Santa Fe, NM). To this
set, the substrate (generation of multiconformer with Omega)
corresponding to the modeled protein was added. It is an
implementation of multiconformer docking, meaning that
a conformational search of the ligand is first carried out and all
relevant low-energy conformations are then rigidly placed in the
binding site. This two-step process allows only the remaining six
rotational and translational degrees of freedom for the rigid
conformer to be considered. The FRED (Fast Rigid Exhaustive
Docking) process uses a series of shape-based filters and the default
scoring function is based on Gaussian shape fitting.
2.2.1.3. Synthesis of 1-(4-methyl benzene)-sulfonyl-cis-2,6-dimethyl
piperidine (3c). The general synthetic method described above
affords 3c as a colorless crystalline solid from piperidine 1 (1 g,
8.85 mmol) and toluene-4-sulfonyl chloride 2c (2.023 g,
10.62 mmol). IR nmax 1370, 1465, 1350, 1150, 1600, 1480 cmꢂ1 1H
.
NMR (CDCl3, 400 MHz): 1.3–1.8 (m, 6H, –CH2), 2.3–2.8 (m, 2H,
–CH), 1.0–1.1 (d, 6H, –CH3), 7.7–8.0 (d, 2H, –ArH), 7.2–7.4 (d, 2H,
–ArH), 7.2–7.4 (d, 2H, –ArH), 2.3 (s, 3H, –ArCH3). mp 162–164 ꢀC
(Yield ¼ 85%). Anal. (C14H21N1O2S1) C, H, N.
2.2.1.4. Synthesis of 1-(4-tert-butyl benzene)-sulfonyl-cis-2,6-dime
thyl piperidine (3d). The general synthetic method described above
affords 3d as a colorless crystalline solid from piperidine 1 (1 g,
8.85 mmol) and 4-tert-butylbenzene sulfonyl chloride 2d (2.46 g,
10.62 mmol). IR nmax 1370, 1465, 1350, 1150, 1600, 1480 cmꢂ1 1H
.
NMR (CDCl3, 400 MHz): 1.3–1.8 (m, 6H, –CH2), 2.3–2.8 (m, 2H,
–CH), 1.0–1.1 (d, 6H, –CH3), 7.7–8.0 (d, 2H, –ArH), 7.3–7.4 (d, 2H,
–ArH), 1.4 (s, 9H, –(CH3)3). mp 138–141 ꢀC (Yield ¼ 78%). Anal.
(C17H27N1O2S1) C, H, N.
2.2.1.5. Synthesis of 1-(4-chloro benzene)-sulfonyl-cis-2,6-dimethyl
piperidine (3e). The general synthetic method described above
affords 3e as a colorless crystalline solid from piperidine 1 (1 g,
8.85 mmol) and 4-chloro benzene sulfonyl chloride 2e (2.240,
2.2. Chemistry
10.62 mmol). IR nmax 1370, 1465, 1350, 1150, 1600, 1450, 1050 cmꢂ1
.
2.2.1. General procedure for the synthesis of cis-2,6-dimethyl
piperidine sulfonamides derivatives (3a–i)
1H NMR (CDCl3, 400 MHz): 1.3–1.8 (m, 6H, –CH2), 2.3–2.8 (m, 2H,
–CH), 1.0–1.1 (d, 6H, –CH3), 7.75–7.8 (d, 2H, –ArH), 7.5–7.6 (d, 2H,
–ArH). mp 173–175 ꢀC (Yield ¼ 80%). Anal. (C13H18Cl1N1O2S1)
C, H, N.
To a solution of cis-2,6-dimethyl piperidine 1 (1 eq) and trie-
thylamine (3 eq) in dry dichloromethane at 0 ꢀC awas dded alkyl/
aryl sulfonyl chlorides (1.2 eq). The reaction mixture was stirred at
0 ꢀC for about 2 h and stirring was continued at room temperature
for about 4–5 h (completion of the reaction was monitored by TLC).
After the completion of the reaction, the reaction mass was
quenched with distilled water and extracted with dichloromethane
(3 ꢁ15 mL). Finally, the combined organic layer was washed with
distilled water again and dried over anhydrous Na2SO4. After
removal of the solvent in vacuum, the residue was purified by
recrystallisation.
The melting point was recorded on a SELACO-650 hot stage
apparatus and is uncorrected. IR (KBr) spectra recorded on a Jusco
FTIR-4100 Fourier transform infrared spectrometer, 1H NMR were
recorded on a Shimadzu AMX, spectrometer by using CDCl3 as
solvent and TMS as an internal standard (Chemical shift in ppm).
TLC was conducted on 0.25 mm silica gel plates (60F254, Merck).
Visualization was made in ultraviolet light. All extracted solvents
were dried over anhydrous Na2SO4 and evaporated with a BUCHI
rotary evaporator. Reagents were obtained commercially and used
as received.
2.2.1.6. Synthesis of 1-(2,5-dichloro benzene)-sulfonyl-cis-2,6-
dimethyl piperidine (3f). The general synthetic method described
above affords 3f as a colorless crystalline solid from piperidine 1
(1 g, 8.85 mmol) and 2,5-dichloro benzene sulfonyl chloride 2f
(2.607 g, 10.62 mmol). IR nmax 1370, 1465, 1350, 1150, 1600, 1480,
1050 cmꢂ1. 1H NMR (CDCl3, 400 MHz): 1.3–1.8 (m, 6H, –CH2), 2.3–
2.8 (m, 2H, –CH), 1.0–1.1 (d, 6H, –CH3), 8.1–8.4 (t, 1H, –ArH), 7.2–7.5
(t, 1H, –ArH), 8.5–8.7 (d, 1H, –ArH). mp 178–181 ꢀC (Yield ¼ 82%).
Anal. (C13H17Cl2N1O2S1) C, H, N.
2.2.1.7. Synthesis of 1-(2-nitro benzene)-sulfonyl-cis-2,6-dimethyl
piperidine (3g). The general synthetic method described above
affords 3g as a yellow crystalline solid from piperidine 1 (1 g,
8.85 mmol) and 2-nitro benzene sulfonyl chloride 2g (2.352 g,
10.62 mmol). IR nmax 1370, 1465, 1350, 1150, 1600, 1480, 1530,
1330 cmꢂ1 1H NMR (CDCl3, 400 MHz): 1.3–1.8 (m, 6H, –CH2),
.
2.3–2.8 (m, 2H, –CH), 1.0–1.1 (d, 6H, –CH3), 7.8–8.0 (d, 1H, –ArH),
8.1–8.4 (t,1H, –ArH), 7.2–7.5 (d,1H, –ArH), 8.5–8.7 (d,1H, –ArH). mp
180–183 ꢀC (Yield ¼ 75%). Anal. (C13H18N2O4S1) C, H, N.
2.2.1.1. Synthesis of cis-2,6-dimethyl-1-methyl sulfonyl piperidine
(3a). The general synthetic method described above affords 3a as
a colorless crystalline solid from piperidine 1 (1 g, 8.85 mmol) and
methane sulfonyl chloride 2a (1.215 g, 10.62 mmol). IR nmax 1370,
1465, 1150 cmꢂ1. 1H NMR (CDCl3, 400 MHz): 1.3–1.8 (m, 6H, –CH2),
2.3–2.8 (m, 2H, –CH), 1.0–1.1 (d, 6H, –CH3), 2.9 (s, 3H, –SO2, –CH3).
mp 155–157 ꢀC (Yield ¼ 82%). Anal. (C8H17N1O2S1) C, H, N.
2.2.1.8. Synthesis of 1-(3-nitro benzene)-sulfonyl-cis-2,6-dimethyl
piperidine (3h). The general synthetic method described above
affords 3h as a yellow crystalline solid from piperidine 1 (1 g,
8.85 mmol) and 3-nitro benzene sulfonyl chloride 2h (2.352 g,
10.62 mmol). IR nmax 1370, 1465, 1350, 1150, 1600, 1480, 1525,
1335 cmꢂ1 1H NMR (CDCl3, 400 MHz): 1.3–1.8 (m, 6H, –CH2),
.
2.3–2.8 (m, 2H, –CH), 1.0–1.1 (d, 6H, –CH3), 7.5–8.3 (m, 4H, –ArH).
mp 181–183 ꢀC (Yield ¼ 77%). Anal. (C13H18N2O4S1) C, H, N.
2.2.1.2. Synthesis of 1-benzene sulfonyl-cis-2,6-dimethyl piperidine
(3b). The general synthetic method described above affords 3b as
a colorless crystalline solid from piperidine 1 (1 g, 8.85 mmol) and
benzene sulfonyl chloride 2b (1.874 g, 10.62 mmol). IR nmax 1370,
1465, 1350, 1150, 1600, 1480 cmꢂ1. 1H NMR (CDCl3, 400 MHz): 1.3–
1.8 (m, 6H, –CH2), 2.3–2.8 (m, 2H, –CH), 1.0–1.1 (d, 6H, –CH3), 7.8–
7.9 (d, 2H, –ArH), 7.5–7.6 (t, 2H, –ArH), 7.2–7.3 (t, 1H, –ArH). mp
163–165 ꢀC (Yield ¼ 83%). Anal. (C13H19N1O2S1) C, H, N.
2.2.1.9. Synthesis of 1-(4-nitro benzene)-sulfonyl-cis-2,6-dimethyl
piperidine (3i). The general synthetic method described above
affords 3i as a yellow crystalline solid from piperidine 1 (1 g,
8.85 mmol) and 4-nitro benzene sulfonyl chloride 2i (2.352 g,
10.62 mmol). IR nmax 1370, 1465, 1350, 1150, 1600, 1480, 1530,
1330 cmꢂ1 1H NMR (CDCl3, 400 MHz): 1.3–1.8 (m, 6H, –CH2),
.

H.R. Girisha et al. / European Journal of Medicinal Chemistry 44 (2009) 4057–4062
4059
Table 1
The total energies of chemguass score, chem score, PLP score and shapeguass score of the best-docked conformations.
Compd
Chemguass score
Chem score
PLP score
Screen score
Shapeguass score
Total score
IC50 (nM) (in vitro assay)
3a
3b
3c
3d
3e
3f
3g
3h
3i
ꢂ34.89
ꢂ47.56
ꢂ50.41
ꢂ56.66
ꢂ50.65
ꢂ52.32
NF
ꢂ15.45
ꢂ15.90
ꢂ13.86
ꢂ14.04
ꢂ13.22
ꢂ18.65
NF
ꢂ36.54
ꢂ44.17
ꢂ43.46
ꢂ43.04
ꢂ42.23
ꢂ47.58
NF
ꢂ77.95
ꢂ118.89
ꢂ107.73
ꢂ86.89
ꢂ107.00
ꢂ125.17
NF
ꢂ225.21
ꢂ324.96
ꢂ331.54
ꢂ385.61
ꢂ336.48
ꢂ348.53
NF
ꢂ390.04
ꢂ551.48
ꢂ547.00
ꢂ586.24
ꢂ549.58
ꢂ592.25
NF
85, 75, 90
362, 392, 388
362, 368, 365
463, 458, 450
325, 318, 312
75, 90, 81
195, 185, 180
186, 192, 200
1200, 1150, 1210
ꢂ51.36
ꢂ13.15
ꢂ43.56
ꢂ112.88
ꢂ379.42
ꢂ600.37
ꢂ54.75
ꢂ9.57
ꢂ38.78
ꢂ101.13
ꢂ360.43
ꢂ564.66
NF ¼ no fit.
2.3–2.8 (m, 2H, –CH), 1.0–1.1 (d, 6H, –CH3), 8.1–8.7 (m, 4H, –ArH).
mp 182–184 ꢀC (Yield ¼ 79%). Anal. (C13H18 N2O4S1) C, H, N.
2.2.3. Experimental conditions and kinetics
Enzyme activity was measured using a Shimadzu Spectropho-
tometer. The assay medium contained phosphate buffer, pH 8.0
2.2.2. In vitro acetylcholinesterase (AChE) assay
(2.6 mL), DTNB (0.1 mL), 5 mL of enzyme, 20 mL of 0.075 M substrate.
The acetylcholinesterase assay method of Ellman et al. [8] was
used to determine the in vitro acetylcholinesterase (AChE) activity.
The activity was measured by the increase in absorbance at 412 nm
due to the yellow color produced from the reaction of acetylth-
iocholine iodide with the dithiobisnitrobenzoate (DTNB) ion. AChE
was obtained from the brain of wistar rats by homogenizing with
a Teflon blender for 5 min in 0.1 M KH2PO4 buffer pH 8. A stock
solution of the enzyme in 0.1 M KH2PO4 buffer (pH 8) was kept
frozen. The protein concentration was estimated by the method
described by Lowry et al. [9] using bovine serum albumin as stan-
The activity was determined by measuring the increase in absor-
bance at 412 nm at 1 min intervals for 10 min at 37 ꢀC. In dose
dependent inhibition studies, the substrate was added to the assay
medium containing enzyme, buffer and DTNB with inhibitor
after 10 min of incubation time. Calculations were performed as
described by Ellman et al. [8]. All the experiments were carried out
in duplicate and the mean values are reported here. The relative
activity is expressed as percentage ratio of enzyme activity in the
absence of inhibitor.
dard. For each assay 300
mg of enzyme was used. Acetylthiocholine
2.2.4. IC50 determination
iodide was prepared freshly using 0.1 M KH2PO4 buffer (pH 7). A
0.01 M solution of DTNB was prepared in 0.1 M KH2PO4 buffer (pH
7). Crude human AChE was prepared by mixing 9 mL of fresh blood
(collected from healthy volunteers by vein puncture) with 1 ml of
3.8% (w/v) trisodium citrate and centrifuging at 3000 rpm at 4 ꢀC
for 20 min. The supernatant was used as a source of AChE. Electric
eel AChE was obtained from Sigma Laboratory and similar proce-
dure was employed for the assay as that of rat brain AChE.
AChE inhibitor neostigmine (a reversible cholinesterase inhib-
itor) was used in the concentration range 25–500 nM to inhibit
AChE in electric eel, human serum, and rat brain homogenate.
Inhibition by piperidine 3a–i derivatives was studied in the pres-
ence of different concentrations of compounds and the percentage
inhibition of enzyme activity was calculated. The inhibition of AChE
by piperidine derivatives was analyzed with values obtained in
comparison to that of neostigmine.
Fig. 1. Docking of cis-2,6-dimethyl-1-methyl sulfonyl piperidine (3a) on active site of mouse AChE.

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H.R. Girisha et al. / European Journal of Medicinal Chemistry 44 (2009) 4057–4062
Fig. 2. Docking of 1-(2,5-chloro benzene)-sulfonyl-cis-2,6-dimethyl piperidine (3f) on active site of mouse AChE.
2.2.5. Antiamnesic effect
evident from the figures that inhibitors are stabilized by hydrogen
bonding interactions (Figs. 1 and 2). Table 1 shows the interaction
energies’ score including chemguass score [12], chem score [13],
PLP score [14], screen score [15], shapeguass score, and consensus
score for all the residues in the active site of enzyme–inhibitor
complex. The hydrogen bonds present in enzyme–inhibitor
complex along with their distances and angles are listed Table 2.
This identification, compared with a definition based on the
distance from the inhibitors can clearly show the relative signifi-
cance for every residue. Hydrogen bonding interactions show that
3a molecule is binding with nitrogen atom of Gly120 and Gly121, 3f
molecule is binding with OH group of Tyr124 as shown in Table 2
along with their distance and angles. Comparison of interaction
energies of these inhibitors with other molecules shows that 3f
molecule has better binding affinity next to 3h with a chemguass
score of ꢂ52.32, chem score of ꢂ18.65, PLP score of ꢂ47.58, screen
score of ꢂ125.17 and, shapeguass score of ꢂ348.53 as shown in
Table 1 compared to other molecules. These results show that 3f
molecule is a very good inhibitor against the mouse AChE. These
results in general also indicate that steric interactions and ligand
acceptor–enzyme donor interactions, contribution from ligand
Antiamnesic behavioural studies were carried out for piperidine
derivatives (3a–i) to check their ability to reverse scopolamine
induced memory loss according to the method described by
Sharma and Kulkarni [10,11].
2.2.6. Statistical analysis
The data was analyzed by ANOVA followed by Tukey’s multiple
comparisons test for significant differences and the correlations
between rat brain, human serum and electric eel anticholinesterase
activities were calculated by Pearson correlation using SPSS 16.0
computer software. The IC50 values were calculated by Boltzmann’s
dose–response analysis using Origin 6.1 computer software. The
values were considered significant at p ꢃ 0.01.
3. Results and discussion
3.1. Docking studies
Docking studies of piperidine sulfonamides (3a–i) on the active
site of mouse AChE inhibited by tabun (PDB: 2C0P) revealed their
mode of interaction, structural and positional requirements for
potential AChE inhibition. These studies were further extended to
in vitro AChE enzyme inhibition and in vivo pharmacological
studies (rodent memory evaluation). To understand the interaction
between mouse AChE and inhibitors, the AChE–inhibitor complex
was generated using the OPENEYE software suite (Open Eye
Scientific Software, Santa Fe, NM). Docking studies with these
inhibitors show that 3a–i are binding with a total score of ꢂ390.04,
ꢂ551.48, ꢂ547.00, ꢂ586.24, ꢂ549.58, ꢂ592.25, ꢂ600.37 and
ꢂ564.66 (more negative better the fit). These studies show that 3h
molecule is binding with high affinity with a chemguass score of
ꢂ51.36, chem score of ꢂ13.15, PLP score of ꢂ43.56, screen score of
ꢂ112.58 and, shapeguass score of ꢂ372.42 as shown in Table 1. It is
CH3
NH
CH3
N
O
S
2(a-i) RSO2Cl
R
Triethylamine
Dichloromethane
O
CH3
CH3
3(a-i)
1
O2N
R=
3a
3b
3c
CH3
3d
3g
3h
3i
NO2
Table 2
Cl
3e
Type of interaction between mouse AChE amino acids with 3a and f. Significant
binding key residues in the active site of the model were determined.
Cl
Molecule
Ligand atom
Atom protein
Distance (Å)
Angle (ꢀ)
3f
NO2
3a
3a
3f
Lig O(10)
Lig O(10)
Lig O(15)
N GLY120
N GLY121
OH TYR124
2.71
3.01
3.27
134.18
101.73
106.09
Cl
Scheme 1.

H.R. Girisha et al. / European Journal of Medicinal Chemistry 44 (2009) 4057–4062
4061
Table 3
Comparative inhibitory activities of cis-2,6-dimethyl piperidine sulfonamides (3a–i) against AChE from different sources.
Compound
Rat brain homogenate AChE IC50 (nM)
Human serum AChE IC50 (nM)
Electric eel AChE IC50 (nM)
3a
3b
3c
3d
3e
3f
3g
3h
85 ꢄ 13.09
362 ꢄ 23.71
362 ꢄ 21.51
463 ꢄ 27.73
325 ꢄ 28.90
75 ꢄ 7.35
75 ꢄ 10.14
392 ꢄ 25.30
368 ꢄ 23.78
458 ꢄ 25.51
318 ꢄ 25.87
90 ꢄ 7.35
90 ꢄ 8.85
388 ꢄ 21.76
365 ꢄ 38.50
450 ꢄ 28.75
312 ꢄ 27.33
81 ꢄ 7.71
195 ꢄ 15.50
186 ꢄ 12.33
1200 ꢄ 55.73
40 ꢄ 5.32
185 ꢄ 11.10
192 ꢄ 10.55
1150 ꢄ 53.71
41 ꢄ 4.17
180 ꢄ 12.23
200 ꢄ 13.13
1210 ꢄ 49.56
53 ꢄ 6.73
3i
Neostigmine
Results are expressed as mean (ꢄSEM).
donors’ interaction with enzyme acceptors, aromatic–aromatic
interactions, frozen rotatable bond penalty, lipophilic–lipophilic
interactions, hydrogen bond interactions, penalty for ligand clashes
with the enzyme, ligand hydrogen bond donors and acceptors,
ligand non-polar atoms, interactions of ligand sulphur atoms, lip-
ophlic–polar and polar–polar interactions, clash penalty, Piecewise
Linear Potential, phenyl interactions with amides, methyl and aryl
CH groups and Shape complementarity between ligand and active
site of AChE are important for the protein interaction and complex
interaction. Through the interaction analysis, it was found that
Gly120, Gly121 and TYR124 were important anchoring residues for
the inhibitor and are main contributors to the inhibitor interaction.
Though the interaction energy does not include the contribution
from the water or the extended enzyme structure, this preliminary
data along with the list of hydrogen bond interactions between the
enzyme and the active site residues clearly supports Gly120, Gly121
and TYR124 are more preferred residues in the inhibitors’ binding.
From the literature it is inferred that piperidine class of molecules
form noncovalent bond with active site and also in vitro kinetic
assay performed for these class compounds (3a–i) revealed that
inhibition is of reversible type.
of S]O at 1320 cmꢂ1, bending absorption at 1370 cmꢂ1 for terminal
methyl group (3a, c and d), absorption at 1050 cmꢂ1 for chloro aryl
group (3e and f) and aromatic nitro group absorbs at 1525 and
1350 cmꢂ1 (3g–i). We obtained all the products (3a–i) in good yield
as mentioned in Scheme 1.
3.3. Experimental studies
Structure–activity relationship (SAR) of the synthesized piperi-
dine derivatives (3a–i) can be drawn from in vitro findings (Scheme
1 and Table 3) that introduction of methyl group on sulfonyl-cis-
2,6-dimethyl piperidine (3a) shows moderate AChE inhibition
(IC50 ¼ 85, 75 and 90 nm), whereas introducing electronegative
chloro atom at positions 2 and 5 of phenyl ring (3f) on sulfonyl-cis-
2,6-dimethyl piperidine demonstrated significant inhibitory
activity (IC50 ¼ 75, 90 and 81 nM). In a similar way electronegative
nitro group at meta position (3h) of phenyl ring and nitro group at
ortho position (3g) of the phenyl ring exerted prominent AChE
inhibition (IC50 ¼ 186, 192, 200 and 195, 185, 180 nM respectively).
Although nitro group at para position (3i) of phenyl ring is found to
be least active (IC50 ¼ 1200, 1150 and 1210 nM), chloro atom at para
position of phenyl ring (3e) weakens the inhibitory activity
(IC50 ¼ 325, 318 and 312 nM) as compared to ortho, meta substitu-
tion of chlorine atom (3f). Alkyl substitution at para position
(methyl -3c and tert-butyl–3d) detrimental to AChE inhibitory
activity (IC50 ¼ 362, 368, 365, 463, 458 and 450 nM respectively).
Docking studies also show that 3f molecule was a better inhibitor
compared to other molecules as shown in Table 1. A significant
correlation (r ¼ 0.971, p ꢃ 0.01) was observed between brain,
human serum and electric eel anticholinesterase activities of all the
compounds indicating effective inhibition of AChE from all the
three biological sources.
3.2. Synthetic chemistry
The reaction of cis-2,6-dimethyl piperidine 1 with different
alkyl/aryl sulfonyl chlorides was carried out in the presence of
triethylamine and dichloromethane as a solvent. The chemical
structures of all the synthesized compounds (3a–i) are given in
Scheme 1. The presence of N–H proton at 3.38 value in starting
material cis-2,6-dimethyl piperidine 1 and the absence of this
proton peak in proton NMR spectra confirm our products (3a–i) and
also by the appearance of aromatic peaks at 7.3 to 8.7
d value
(except 3i). Apart from this, IR data showed asymmetric stretching
Table 4
Study of antiamnesic effect cis-2,6-dimethyl piperidine sulfonamides (3a–i) against scopolamine induced memory loss.
Sl. No.
Experimental groups
Treatment (dose) mg/kg i.p.
Basal latency (s) of rat to reach
shock free zone (SFZ)
Memory parameters
Latency (s)
I
II
III
0.7
08
0.7
Mistakes
1.
Control group*
Scopolamine treated group
3a þ Scop.
Saline (0.9%)
0.4
16
35
20
26
27
28
25
16
17
22
34
03
09
04
07
08
09
08
04
05
06
09
01
05
02
04
04
04
03
02
02
04
03
07 ꢄ 2.11
38 ꢄ 2.43
10 ꢄ 2.22
22 ꢄ 3.25
18 ꢄ 2.14
25 ꢄ 1.85
17 ꢄ 2.81
8 ꢄ 2.43
2.
3.
4
5.
6.
7.
8.
9.
10.
11.
0.1 þ 0.4
0.1 þ 0.4
0.1 þ 0.4
0.1 þ 0.4
0.1 þ 0.4
0.1 þ 0.4
0.1 þ 0.4
0.1 þ 0.4
0.1 þ 0.4
3b þ Scop.
04
05
06
05
3c þ Scop.
3d þ Scop.
3e þ Scop.
3f þ Scop.
0.8
0.9
04
07
3g þ Scop.
15 ꢄ 2.84
14 ꢄ 2.74
36 ꢄ 2.73
3h þ Scop.
3i þ Scop.
Results are expressed as mean (ꢄSEM), n ¼ 8 (Scop. ¼ Scopolamine).

4062
H.R. Girisha et al. / European Journal of Medicinal Chemistry 44 (2009) 4057–4062
In vitro AChE inhibition studies were done to support docking
be summarized that substitution of electronegative group at ortho
or meta position of phenyl ring as R need to be explored for better
AChE inhibitory activity.
studies and extended these studies to in vivo pharmacological task
involving the reversing amnesic effect of scopolamine induced
memory loss, in passive avoidance step down task paradigm using
rat as animal model, although slight structural differences are
found in the active site of mouse and rat brain AChE, but showed
comparable results. Compound 3a with methyl substituent and 3f
with 2,5-dichloro substituent effectively reversed the average
number of mistakes done by rats from 38 (scopolamine) to 10 and 8
respectively considerable extent (Table 4). Whereas ortho and meta
nitro groups in 3g and h respectively reversed the average number
of mistakes (38 to 15 and 14 respectively) done by rats to moderate
level. The in vivo and in vitro experimental data were correlated
with the results obtained computationally.
Acknowledgments
The authors are grateful to the Department of Science and
Technology (DST), New Delhi for financial support under the
project SR/SO/HS-58/2003. The CHNS, IR and other data obtained
from the instruments granted under DST-FIST and UGC-SAP (phase
I) programmes are greatly acknowledged. Manish Malviya is
grateful to the Indian Council of Medical Research (Ref. No. 45/51/
2007/Pha/BMS) for providing the Senior Research Fellowship (IRIS
Cell No. 2007-04110).
4. Conclusion
References
As a part of our continued efforts in the design, synthesis and
development of bioactive heterocyclic sulfonamides, herein, we
have described the docking study and synthesis of alkyl and aryl
cis-2,6-dimethyl piperidine sulfonamides along with their in vitro
AChE inhibition and in vivo antidementia activity against scopol-
amine induced memory loss. cis-2,6-Dimethyl piperidine sulfon-
amides (3a–i) have been shown to be potentially useful,
bioavailable and reversible inhibitors of AChE. These derivatives
(3a–h, except 3g) were superimposed on mouse AChE and was
clearly shown that hydrogen bonding interactions of two deriva-
tives (3a and 3f) with active site of mouse AChE, whereas six
derivatives (3b–e, h and i) just fit into active site of the enzyme and
one derivative (3g) unable to occupy active site of the mouse AChE.
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tives of cis-2,6-dimethyl piperidine exerted considerable AChE
inhibition against different sources of AChE in vitro. Therefore it can
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