Design, synthesis and biological evaluation of selected 3-[3-(amino) propoxy] benzenamines as acetylcholinesterase inhibitors

Article abstract of DOI:10.1080/07391102.2016.1220330

The present paper describes design, synthesis, and biological evaluation of a series of some 3-[3-(amino)propoxy]benzenamines as acetylcholinesterase inhibitors using mice as a model and piracetam as a reference drug. The structures of these compounds were confirmed by spectral analysis and compounds were tested for memory enhancing activity using elevated plus maze test and acetylcholinesterase inhibitory assay. The inhibitory range of synthesized compounds was from 8.99 to 28.31?μM. The synthesized compounds possessed higher or equivalent percent retention as compared to piracetam at 1?mg/kg with no other CNS-related activities (locomotor and muscle relaxant, analgesic and anticonvulsant activities). Compound 3-[3-(imidazolo)propoxy]benzenamine has shown significant dose-dependent (1 and 3?mg/kg) memory enhancing activity, while 3-[3-(pyrrolidino)propoxy]benzenamine also showed activity equivalent to reference drug piracetam at 1?mg/kg. Both compounds 3-[3-(pyrrolidino)propoxy]benzenamine and 3-[3-(imidazolo)propoxy]benzenamine were also found to show AChE inhibition with IC₅₀ value of 8.99 and 17.87?μM. The molecular docking, MM-GBSA and molecular dynamics simulation studies were performed in order to establish a relationship between the biological results. RMSD, root-mean-square fluctuations, and interaction patterns of 10a–AChE and Sck–AChE complexes proved that the binding affinity of 10a toward AChE was highly stable with the proposed binding orientations.

Full text of DOI:10.1080/07391102.2016.1220330

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Design, synthesis and biological evaluation of
selected 3-[3-(amino) propoxy] benzenamines as
acetylcholinesterase inhibitors
Ruchi Malik, Richa Gupta, Shubham Srivastava, Bhanwar Singh Choudhary &
Manish Sharma
To cite this article: Ruchi Malik, Richa Gupta, Shubham Srivastava, Bhanwar Singh Choudhary
& Manish Sharma (2016): Design, synthesis and biological evaluation of selected 3-[3-(amino)
propoxy] benzenamines as acetylcholinesterase inhibitors, Journal of Biomolecular Structure
and Dynamics, DOI: 10.1080/07391102.2016.1220330
To link to this article: http://dx.doi.org/10.1080/07391102.2016.1220330
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Date: 20 August 2016, At: 12:41

Journal of Biomolecular Structure and Dynamics, 2016
http://dx.doi.org/10.1080/07391102.2016.1220330
Design, synthesis and biological evaluation of selected 3-[3-(amino) propoxy] benzenamines as
acetylcholinesterase inhibitors
Ruchi Malika*, Richa Guptab, Shubham Srivastavaa, Bhanwar Singh Choudharya and Manish Sharmac
aDepartment of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Bandarsindri, Kishangarh,
b
Ajmer 305817, India; Department of Medicinal and Pharmaceutical Chemistry, B.R. Nahata College of Pharmacy, Mandsaur,
c
Madhya Pradesh 458001, India; School of Pharmacy, MM University, Sadopur, Ambala, Haryana 134007, India
Communicated by Ramaswamy H. Sarma
(Received 1 March 2016; accepted 30 July 2016)
The present paper describes design, synthesis, and biological evaluation of a series of some 3-[3-(amino)propoxy]ben-
zenamines as acetylcholinesterase inhibitors using mice as a model and piracetam as a reference drug. The structures of
these compounds were confirmed by spectral analysis and compounds were tested for memory enhancing activity using
elevated plus maze test and acetylcholinesterase inhibitory assay. The inhibitory range of synthesized compounds was
from 8.99 to 28.31 μM. The synthesized compounds possessed higher or equivalent percent retention as compared to
piracetam at 1 mg/kg with no other CNS-related activities (locomotor and muscle relaxant, analgesic and anticonvulsant
activities). Compound 3-[3-(imidazolo)propoxy]benzenamine has shown significant dose-dependent (1 and 3 mg/kg)
memory enhancing activity, while 3-[3-(pyrrolidino)propoxy]benzenamine also showed activity equivalent to reference
drug piracetam at 1 mg/kg. Both compounds 3-[3-(pyrrolidino)propoxy]benzenamine and 3-[3-(imidazolo)propoxy]ben-
zenamine were also found to show AChE inhibition with IC50 value of 8.99 and 17.87 μM. The molecular docking,
MM-GBSA and molecular dynamics simulation studies were performed in order to establish a relationship between the
biological results. RMSD, root-mean-square fluctuations, and interaction patterns of 10a–AChE and Sck–AChE com-
plexes proved that the binding affinity of 10a toward AChE was highly stable with the proposed binding orientations.
Keywords: acetylcholinesterase inhibitors; molecular dynamics; docking; Alzheimer’s disease; memory enhancers
Introduction
but have different mechanism of action for producing the
same biological effect. Additionally, these agents encom-
pass different pharmacological effects beyond acetyl-
cholinesterase (AChE).
With the advancement in research in therapies relating to
neurological disorders, acetylcholinestease inhibitors
(AChEIs) have held upper hand in treatment of cognitive
disorders and several other disorders at different stages
of severity. An emergent curiosity in drug-facilitated
memory performance (Malik, Sangwan, Saihgal, Jindal,
O
O
O
N
N
O
&
Piplani, 2007; Reneerkens, Rutten, Steinbusch,
Blokland, & Prickaerts, 2009; Wallace, Ballard, Pouzet,
Riedel, & Wettstein, 2011) has emerged from basic
research on learning and memory as well as therapeutic
hunt for neurological impairments due to Alzheimer’s
disease (AD), aging, and brain injury (Cumin, Bandle,
Gamzu, & Haefely, 1982). Yet, specificity of these drugs
on memory processes has proven tricky (Heise, 1981).
Cognitive neuropharmacology, which provides theoretical
support and matrix of reliable assays for the discovery of
cognitive enhancers, is still in its formative years (Merrer
& Noguès, 2000). Four AChEIs approved by US-FDA
for treatment of AD are donepezil (Aricept) 1, rivastig-
mine (Exelon) 2, galantamine (Reminyl) 3, and tacrine
(Cognex) 4. These four agents possess AChEI activity
N
O
1
2
OH
H
NH2
O
O
N
N
3
4
The anticipatory action underlying AChE inhibition
to act as cognitive enhancer is increased level of acetyl-
choline (ACh) in central cholinergic synapses involved
in memory circuit. Despite, being familiar in mechanism
*Corresponding author. Email: ruchimalik1976@curaj.ac.in
© 2016 Informa UK Limited, trading as Taylor & Francis Group

2
R. Malik et al.
of action, these agents represent entirely heterogeneous
groups based on their marked difference in chemical
structures (Piplani, Singh, & Sharma, 2013). Based on
SAR studies of these pharmacological divergent classes,
some structural features can be deduced: a basic nitrogen
which may be a part of a heteroaromatic ring or cyclic/
acyclic system for interacting electrostatically with the
appropriate target, a hydrogen bond acceptor, an appro-
priate linker group giving optimal spacing to the hydro-
gen bond acceptor function from the basic nitrogen.
Some marketed cognition enhancing drugs such as pirac-
etam 5, indeloxazine 6, bifemelane 7 have oxygen and
nitrogen linked by 2–4 carbon chain and attached to cyc-
lic aromatic/nonaromatic ring (Martini et al., 2009;
Romanelli et al., 2006). Several reported acetyl-
cholinesterase inhibitors possess basic side chains and a
free amino group as an integral part of the structure
(Refolo & Fillit, 2004).
indicated by symbols, s (singlet), d (doublet), dd (double
doublet), t (triplet), m (multiplet), q (quartet), and quin
(quintet). The purity of compounds was established by
thin-layer chromatography (TLC). Plates for TLC were
prepared with silica gel G according to Stahl’s method
(E. Merck) using ethyl acetate as the solvent and acti-
vated at 110°C for 30 min. Iodine was used to develop
the TLC plates. All solvents were distilled prior to use
according to standard procedures. Anhydrous sodium
sulfate was used as a drying agent.
General procedure for preparation of 3-[3-(amino)
propoxy]benzenamine derivatives (10a–10e)
3-Aminophenol 8 (1.00 g, 9.16 mmol) was dissolved in
ethyl methyl ketone (100 ml) and anhydrous potassium
carbonate (2.00 g) was added to the solution. Reaction
mixture was magnetically stirred and refluxed for half an
hour.
Then,
1-bromo-3-chloropropane
(2.50 ml,
H
N
16.40 mmol) was added, continued refluxing for 15 h,
reaction being continuously monitored with the help of
TLC. The slurry was filtered and solvent was recovered
under reduced pressure to obtain 3-(3-chloropropoxy)
benzenamine 9 which was further stirred with or without
heating for 4–8 h with corresponding cyclic/acyclic
amine (1.00 ml)/imidazole (100 mg). Crushed ice was
added to the reaction mixture and the solid obtained was
crystallized from acetone to afford 3-[3-amino)propoxy]
benzenamines 10a–10e.
O
O
CH3
O
O
N
NH
NH2
O
5
6
7
Previously, compounds possessing basic nitrogen
(acyclic/cyclic amine), carboxamide group, and amino
ether function separated by two to three carbon alkyl
chain using dialkylaminoethoxy/propoxy moiety which
have been designed and synthesized possessing good
pharmacological activity favorable to acetylcholinesterase
enzyme inhibition (Piplani, Malik, Kaur, & Kaplish,
2012; Piplani et al., 2004, 2013). With this knowledge
and encouragement, we designed a series of compounds
having selected groups and compared the effect of differ-
ent structural modifications on activity. 3-aminophenol
was used as initial material, the free hydroxyl group
allowed introduction of dialkylaminopropoxy side chain
at the phenolic end, heterocyclic and acyclic moieties at
the other end of propoxy chain, and free amino group of
3-amino phenol facilitated in maintaining basicity.
3-(3-Chloropropoxy)benzenamine (9)
Yield .84 g, 69.41%. FTIRνmax (KBr): 3504.42, 3340.48,
3055.00, 1323.00, 1264.60, 1070.40, 979.70 and
696.25 cm−1; H NMR (CDCl3 + DMSO): δ 7.89 (t, 1H,
1
Ar proton 5-CH, J = 2.3 Hz), 6.88 (d,1H, Ar proton 4-
CH, J = 2.3 Hz), 6.24 (d, 1H, J = 1.9 Hz, Ar proton 6-
CH), 5.65 (s, 1H, Ar proton 2-CH), 4.16 (s, 2H, protons
of amino group, disappeared on deuterium exchange),
3.91 (t, J = 2 Hz, 2H, –OCH2−), 3.38 (t, J = 2 Hz, 2H,
ClCH2−) and 1.98 ppm (quintet, 2H, J = 4 Hz,
–
CH2CH2CH2−); 13C NMR (CDCl3 + DMSO) : δ 154.70
(Ar carbon 3-CH), 148.96 (Ar carbon 1-CH), 131.01 (Ar
carbon 5-CH), 110.07 (Ar carbon 6-CH), 104.12 (Ar
carbon 4-CH), 98.99 (Ar carbon 2-CH), 66.05
(–OCH2CH2−), 48.75 (–CH2CH2N<), and 30.95 ppm
(–CH2CH2CH2−). Mass (m/z): 185/186 (M+/M++1);
Anal. Calc. For C9H12ClNO (mol. wt. 185.65): C: 58.23,
H: 6.52, N: 7.54. Found: C: 58.17, H: 6.48, N: 7.52.
Methods
Chemistry
Melting points were determined on a Veego melting
point apparatus and were uncorrected. Infrared (IR) spec-
tra were recorded on a Perkin-Elmer 882 and Perkin-
Elmer spectrum RX 1 FT-IR spectrophotometer models
using potassium bromide (νmax in cm–1). Proton NMR
spectra were recorded on Brucker multinuclear FT NMR
spectrometer model AV-400, 400 MHz using deuterated
chloroform or deuterated dimethylsulfoxide (DMSO)
containing tetramethylsilane (Me4Si) as internal standard
(chemical shifts in δ, ppm). The spin multiplicities are
3-[3-(Pyrrolidino)propoxy]benzenamine (10a)
Yield .88 g, 67.05%, mp 176–178°C. FTIRνmax
(KBr): 3301.91, 3031.91, 1332.72, 1255.57, 1114.37,
1
1071.70 and 908.41 cm−1; H NMR (CDCl3 + DMSO): δ

Design, synthesis and biological evaluation of some acetylcholinesterase inhibitors
3
7.84 (t, 1H, Ar proton 5-CH, J = 2.4 Hz), 6.82 (d,1H, Ar
proton 4-CH, J = 2.4 Hz), 6.12 (d, 1H, J = 1.6 Hz, Ar
proton 6-CH), 5.55 (s, 1H, Ar proton 2-CH), 4.56 (s, 2H,
protons of amino group, disappeared on deuterium
exchange), 3.84 (t, J = 2 Hz, 2H, –OCH2−), 2.82 (t, 4H,
J = 1.6 Hz, (–NCH2CH2−)2 pyrrolidino protons), 2.23 (t,
2H, J = 2 Hz, –CH2N<)], 1.88 (quintet, 2H, J = 4 Hz,
–CH2CH2CH2−), and 1.54 ppm (quintet, 4H, J = 4 Hz,
(–N(CH2CH2−)2 pyrrolidino protons); 13C NMR
(CDCl3 + DMSO) : δ 154.80 (Ar carbon 3-C), 149.02
(Ar carbon 1-C), 130.02 (Ar carbon 5-CH), 107.90 (Ar
carbon 6-CH), 104.10 (Ar carbon 4-CH), 99.70 (Ar car-
bon 2-CH), 66.62 (–OCH2CH2−), 58.31 (–NCH2CH2−)2
2H, protons of amino group and disappeared on deu-
terium exchange), 3.82 (t, 4H, J = 2.4 Hz, –OCH2−, and
–CH2N<) and 2.13 ppm (quintet, 2H, J = 4.0 Hz,
–CH2CH2CH2−); 13C NMR (CDCl3 + DMSO): δ 155.20
(Ar carbon 3-C), 149.20 (Ar carbon 1-C), 135.50 (imida-
zolyl carbon 2′-CH), 130.00 (Ar carbon 5-CH), 126.10
(imidazolyl carbon 4′-CH), 120.00 (imidazolyl carbon
5′- CH), 109.70 (Ar carbon 6-CH), 103.80 (Ar carbon
4-CH), 99.90 (Ar carbon 2-CH), 66.80 (–OCH2CH2−),
49.50 (–CH2CH2N<), and 31.50 ppm (–CH2CH2CH2−);
Mass (m/z): 217/218 (M+/M++1); Anal. Calc. For
C12H15N3O (mol. wt. 217.27): C: 66.34, H: 6.96, N:
19.34. Found: C: 66.17, H: 7.01, N: 19.01.
pyrrolidino
carbons),
49.43(–CH2CH2N<),
28.32
(–NCH2CH2−)2 pyrrolidino carbons), and 26.14 ppm
(–CH2CH2CH2−). Mass (m/z): 220/221 (M+/M++1);
Anal. Calc. For C13H20N2O (mol. wt. 220.16): C: 70.87,
H: 9.15, N: 12.72. Found: C: 70.84, H: 9.12, N: 12.69.
3-[3-(Dimethylamine)propoxy]benzenamine (10d)
Yield .58 g, 75.24%, mp 128–130°C. FTIRνmax (KBr):
3310.00, 3074.92, 1317.29, 1260.00, 1153.35, 1089.71,
1
968.56 cm−1; H NMR (CDCl3 + DMSO): δ 7.83 (t, 1H,
Ar proton 5-CH, J = 2.8 Hz), 6.82 (d,1H, Ar proton
4-CH, J = 2.4 Hz), 6.56 (d, 1H, J = 4.0 Hz, Ar proton
6-CH), 5.42 (s, 1H, Ar proton 2-CH), 4.63 (s, 2H, pro-
tons of amino group and disappeared on deteurium
exchange), 3.52 (t, J = 1.6 Hz, 2H, –OCH2−), 2.93 (s,
6H, –N(CH3)2), 2.21 (t, 2H, J = 4.8 Hz, –CH2N<), and
1.62 ppm (quintet, 2H, J = 4 Hz, –CH2CH2CH2−). 13C
NMR (CDCl3 + DMSO): δ 154.70 (Ar carbon 3-C),
149.32(Ar carbon 1-C), 130.08 (Ar carbon 5-CH),
109.10 (Ar carbon 6-CH), 104.10 (Ar carbon 4-CH),
99.91 (Ar carbon 2-CH), 66.69 (–OCH2CH2−), 48.98 (–
CH2CH2N<), 46.10 (–N(CH3)2 carbons), and 27.24 ppm
(–CH2CH2CH2−). Mass (m/z): 194/195(M+/M++1); Anal.
Calc. For C11H18N2O (mol. wt. 194.27): C: 68.01, H:
9.34, N: 14.42. Found: C: 67.93, H: 9.51, N: 14.36.
3-[3-(Morpholino)propoxy]benzenamine (10b)
Yield .68 g, 73.54%, mp 182–184°C. FTIRνmax
(KBr):3479.30, 3413.27, 3058.89, 1320.79, 1261.36,
1164.92, 1085.85, 1031.85, and 911.23 cm−1; H NMR
1
(CDCl3 + DMSO): δ 7.82 (t, 1H, Ar proton 5-CH,
J = 3.2 Hz), 6.84 (t, 1H, Ar proton 4-CH, J = 1.6 Hz),
6.42 (d, 1H, J = 1.6 Hz, Ar proton 6-CH), 5.54 (s,
1H, Ar proton 2-CH), 4.55 (s, 2H, protons of amino
group, disappeared on deuterium exchange), 3.83
(t, J = 2 Hz, 2H, –OCH2−), 3.12 (t, 4H, J = 1.4 Hz,
(–CH2CH2)2O- morpholino protons), 2.16 (t, 4H,
J = 4 Hz –N(CH2CH2)2- morpholino protons), 2.05
(t, 2H, J = 4 Hz, –CH2N<), and 1.45 ppm (quintet, 2H,
–CH2CH2CH2−). 13C NMR (CDCl3 + DMSO)
: δ
154.90 (Ar carbon 3-C), 149.01 (Ar carbon 1-C), 130.02
(Ar carbon 5-CH), 108.90 (Ar carbon 6-CH), 103.90 (Ar
carbon 4-CH), 99.80 (Ar carbon 2-CH), 66.84
(–OCH2CH2−), 66.63 (–CH2CH2)2O- morpholino car-
bons), 53.71 (–N(CH2CH2)2- morpholino carbons), 49.13
(–CH2CH2N<), and 27.84 ppm (–CH2CH2CH2−). Mass
(m/z): 236/237 (M+/M++1); Anal. Calc. For C13H20N2O2
(mol. wt. 236.31): C: 66.07, H: 8.53, N: 11.85. Found:
C: 66.04, H: 8.54, N: 11.81.
3-[3-(Diethylamine)propoxy]benzenamine (10e)
Yield 1.20 g, 71.67%, mp 162–164°C. FTIRνmax (KBr):
3548.78, 3267.40, 3068.53, 1342.36, 1269.07, 1124.42,
1074.13, and 945.05 cm−1;1H NMR (CDCl3 + DMSO):
δ 7.82 (t, 1H, Ar proton 5-CH, J = 1.6 Hz), 6.88 (d,2H,
Ar proton 4-CH and 6-CH, J = 4.0 Hz), 5.42 (s, 1H, Ar
proton 2-CH), 4.82 (s, 2H, protons of amino group and
disappeared on deteurium exchange), 3.58 (t, 2H,
J = 4 Hz, –OCH2−), 2.86 (q, 4H, J = 4 Hz, –N(CH2−)2),
2.16 (t, 2H, J = 2.4 Hz, –CH2N<), 1.57 (quin, 2H,
J = 4 Hz, –CH2CH2CH2−), and .72 ppm (t, 6H, J = 4 Hz
(–NCH2CH3)2). 13C NMR (CDCl3+DMSO): δ 154.92
(Ar carbon 3-C), 148.96 (Ar carbon 1-C), 130.06 (Ar
carbon 5-CH), 108.91 (Ar carbon 6-CH), 104.10 (Ar
carbon 4-CH), 99.74 (Ar carbon 2-CH), 66.62
(–OCH2CH2−), 49.13 (–CH2CH2N<), 48.10 (–N(CH2)2
carbons), 27.24 (–CH2CH2CH2−), and 13.41 ppm
(–NCH2CH3)2). Mass (m/z): 222/223(M+/M++1); Anal.
3-[3-(Imidazolo)propoxy]benzenamine (10c)
Yield .66 g, 66.41%, mp 174–176°C. FTIRνmax (KBr):
3370.40, 3294.19, 3087.82, 1494.90, 1450.30, 1261.36,
1097.42, 1022.20, and 902.62 cm−1; 1H NMR
(CDCl3 + DMSO): δ 8.43 (s, 1H, imidazolyl proton 2′-
CH), 7.82(t, 1H, J = 1.6 Hz, Ar proton 5-CH), 7.42(d,
1H, J = 2.4 Hz, imidazolyl proton 5′-CH), 6.82 (d, 1H,
J = 2.4 Hz, imidazolyl proton 4′-CH), 6.76 (d, 1H,
J = 1.6 Hz, Ar proton 4-CH), 6.22(d, 1H, J = 4.0 Hz, Ar
proton 6-CH), 5.05 (s, 1H, Ar proton 2-CH), 4.69 (s,

4
R. Malik et al.
Calc. For C13H22N2O (mol. wt. 222.33): C: 70.23, H:
9.97, N: 12.60. Found: C: 70.11, H: 10.03, N: 12.36.
training, the animals were administered the test com-
pounds intraperitoneally (i.p.). On the second day, i.e.
24 h after the first exposure, TL was again noted. The
results are expressed as % retention (mean (S.E.M)
calculated as:
Pharmacological methods
All the animal experiments were carried out at Pharma-
cology Department of B.R. Nahata College of Pharmacy,
Mandsaur (M.P.) and permission for conducting these
experiments was obtained from Institutional Animal Ethi-
cal Committee. All the compounds except for their inter-
mediates were tested. Control group did not receive any
drug treatment. Albino mice (LAKA strain) weighing
20–25 g of either sex, taken from animal House of B. R.
Nahata College of Pharmacy were housed in groups of
six and kept under 12-h light and dark cycle. They were
fed with standard food and water available ad libitum.
The experiments were performed between 9:00 and
12:00 h. Piracetam (Ranbaxy, Ponta Sahib, Himachal
Pradesh) was used as the reference drug against which
all the test compounds were compared.
TLon 1st day ꢀ TLon 2nd day
¼
TL on1st day
The mean S.E.M. value for control was 23.47 2.16
(s). All the behavioral data were analyzed using one-way
analysis of variance (ANOVA) followed by Dunnet’s test
using SigmaStat program (Figure 1).
Rota rod test
The apparatus consisted of a base platform and a rotating
rod of 3-cm diameter with a non-slippery surface. The
rod was placed at a height of 15 cm from the base. The
rod, 30 cm in length, was divided into five equal sections
by six disks. Upto five mice were tested simultaneously
on the apparatus with a rod-rotating speed of 16 rpm.
The integrity of motor coordination was assessed on the
basis of the number of falls from the rod in 30 s accord-
ing to Vaught (Vaught, Pelley, Costa, Setler, & Enna,
1985) before and after treatment with test compound.
The experimental results are given as the mean S.E.M.
An ANOVA, followed by Fisher’s Protected Least
Significant Difference procedure for post hoc comparison
was used to verify significance between two means.
Data were analyzed by the StatView software for the
Macintosh (1992). p values of less than .01 were
considered significant.
Elevated plus maze test
Transfer latency (TL) (the time in which mouse moves
from the open arms to the enclosed arm) was utilized
as an index of learning and memory processes (Itoh,
Nabeshima, & Kameyama, 1991). Briefly, the apparatus
consisted of two open arms (50 cm × 10 cm) and two
enclosed arms (50 cm × 10 cm × 40 cm) elevated to a
height of 25 cm from the floor. The animals were
placed individually at the end of either of the open
arms and the TL was noted on the first day up to 90 s.
The animals were allowed to explore the apparatus for
20 s to acquaint with the maze. Immediately after the
Figure 1. Effect of various compounds (10a-10e) and reference drug (piracetam) (1, 3 and 5 mg/kg, i.p.) on % retention measured on
elevated plus maze in mice. p° ≤ 0.001 as compared to control group, p* ≤ 0.001 as compared to reference drug (1 mg/kg). p** ≤ 0.001 as
compared to reference drug (3 mg/kg). p*** ≤ 0.001 as compared to reference drug (5 mg/kg). (ANOVA followed by Dunnett’s test).
n = 6, S.E.M., Standard Error of Mean

Design, synthesis and biological evaluation of some acetylcholinesterase inhibitors
5
Writhing test
allowed to stand for 30 min at room temperature. The
absorbance was measured at 750 nm. The protein con-
centration was calculated from the standard curve made
by taking different concentrations of the BSA standard.
It was observed that incubation of drugs at a concentra-
tion of 10 μM resulted in inhibiting the AChE activity.
The experimental results are expressed as mean S.E.M.
value of nmole product/min/mg protein. All the data
were analyzed using t-test using SigmaStat program. p
values ≤.05 as compared to control group were consid-
ered significant.
Mice were injected intraperitoneally with a .6% solution
of acetic acid (10 ml/kg). Compounds were administered
perorally 30 min before test. The number of stretching
movements was counted for 10 min, starting 5 min after
acetic acid injection. The experimental results are given
as the mean S.E.M. An ANOVA, followed by Fisher’s
Protected Least Significant Difference procedure for post
hoc comparison was used to verify significance between
two means. Data were analyzed with the StatView soft-
ware for the p values of less than .01 were considered
significant.
Computational studies
Anticonvulsant activity
Following steps were undertaken to carry out molecular
docking simulations on acetylcholinesterase enzyme
antagonists using Schrodinger (Release 2015-1).
All the compounds were screened for anticonvulsant
activity using pentylenetetrazole as control. Drugs were
dissolved in normal saline and administered intraperito-
nially. After 30 min, noted the severity of convulsions.
The experimental results are given as the mean S.E.M.
An ANOVA, followed by Fisher’s Protected Least Sig-
nificant Difference procedure for post hoc comparison
was used to verify significance between two means. Data
were analyzed with the StatView software for the Macin-
tosh (1992). p values of less than .01 were considered
significant.
Protein preparation
The X-ray crystal structure (PDB Id: 2HA2) of acetyl-
cholinesterase enzyme bound with succinyldicholine
(Sck) was retrieved from Brookhaven Protein Data Bank
(http://www.rcsb.org/pdb). Bond orders were assigned,
hydrogens were added and missing side chains and loops
were added using Prime v3.9. The protein was then sub-
jected to optimization in which h-bond assignment is
performed at neutral pH. Minimization was performed at
last in order to achieve local minima using OPLS-2005
force field. Chain A was selected for our computational
studies.
Biochemical method
Assay of acetylcholinestrase enzyme
The activity of acetylcholinesterase was determined in
the mouse brain homogenate according to the Ellman
method (Ellman, Courtney, Andres, & Featherstone,
1961). The animals were fasted overnight and sacrificed
by decapitation. The brains were removed, rinsed in ice-
cold isotonic saline (.9% w/v NaCl), blotted dry,
weighed separately. A 10% (w/v) tissue homogenate was
prepared in 50 mM phosphate buffer saline (pH 7.4)
using Potter-Elvehjem glass homogenizer. The reaction
mixture consisted of 2.8 ml of .1 M phosphate buffer
(pH 8.0), .1 ml of Ellman’s reagent, .3 ml of drug, and
10 μl of brain homogenate. The reaction was initiated by
the addition of .1 ml of acetylthiocholine iodide and the
rate of change in absorbance was measured at 412 nm
for 2 min. The protein content was estimated according
to the method given by Lowry (Lowry, Rosebrough,
Farr, & Randall, 1951). .1 ml of suitably diluted sample
of brain homogenate, BSA standard, and water (for
blank) were taken separately in different test tubes and
the final volume was made 1.0 ml with double distilled
water. 3.0 ml of Lowry’s reagent was added to all the
tubes, vortexed, and allowed to stand for 10 min at room
temperature. Further, .5 ml of Folin–Phenol reagent was
added to each of the tubes, vortexed for 30 s, and
Ligand preparation
The ligands were prepared using LigPrep v3.3. All possi-
ble ionization states were generated using Epik v3.1 at
pH 7.0 2.0. Stereoisomers were generated and the
result was obtained in maestro (.mae) format.
Grid box formation
The binding pocket in the receptor was defined at the
binding site of co-crystallized molecule. Van der Waals
scaling factor was kept 1.0 and partial charge cut-off as
.25. The size of the grid box was kept in similar with
the co-crystallized ligand size.
Docking method validation
To ensure that the ligand orientations and positions
obtained from the docking studies represent valid and
reasonable potential binding modes of the inhibitors, the
docking methods and parameters used were validated by

6
R. Malik et al.
followed by the Broyden–Fletcher–Goldfarb–Shanno
algorithm with convergence threshold of 2.0 kcal/mol
and overall 1000 iterations. MD simulations were carried
out at normal temperature and pressure (300 K and
1.01325 bar, respectively). Nose–Hoover thermostat and
Martyna–Tobias–Klein barostat method was opted with
ensemble pathway comprising NVT (constant number of
particles, volume, and temperature) and isotropic cou-
pling method. Overall model system was relaxed before
10 ns, simulation and columbic interactions were defined
by short-range cut-off radius of 9.0 Å and by long-range
smooth particle mesh Ewald tolerance to 1e−09. The sim-
ulated system was analyzed for stability of the docked
complex. Energy fluctuations and RMSD of the complex
in each trajectory were analyzed with respect to simula-
tion time. The root-mean-square fluctuations (RMSF) of
side chains of AChE–ligand complex were analyzed for
each residue. The docked complex was analyzed and
monitored for consistency in various interactions.
Figure 2. Overlay of SCK molecules for validating docking
protocol
redocking the crystallized SCK and overlaying the
docked and co-crystallized Sck chemical structures
(Figure 2) and calculating the RMSD value which was
found to be 1.32 Å.
Docking parameters
Results and discussion
Glide was utilized for carrying out molecular docking. The
parameters of the docking were kept default as provided
by the software vendor, i.e. Schrodinger. The complexes
obtained as output was subjected to MM-GBSA analysis
keeping 5 Å of residues nearby ligand as flexible.
Chemistry
The general synthetic method shown in Scheme 1 was
employed to prepare 3-[3-(amino)propoxy]benzenamines
10a–10e. The key intermediate 3-(3-chloropropoxy)ben-
zenamine 9 involved in the synthesis of all the com-
pounds in the series was prepared by condensing
m-aminophenol 8 with 1-bromo-3-choropropane in ethyl
methyl ketone in the presence of anhydrous potassium
carbonate. In the infrared spectrum, asymmetrical and
symmetrical N–H stretch at 3504.42 and 3340.80 cm−1
and absence of broad –OH stretch confirmed the pres-
ence of intact free primary amino group in the product.
The chloro compound 9 on treatment with requisite cyc-
lic (pyrrolidine, morpholine, imidazole) and acyclic ami-
nes (dimethylamine, diethylamine) gave 10a–10e,
respectively, as detailed in the “Methods” section. The
Molecular dynamics simulation
The protein–ligand docked complex obtained from MM-
GBSA was subjected to system builder, in which SPC
was used as a water model for molecular dynamics simu-
lations. The cubic box of 10 Å radius was used to define
core and overall complex was neutralized by adding four
Cl- counter ion for simulation. The model system to be
used for molecular dynamics contained 55,368 molecules
which included 15,637 water molecules. Further, this
complex was minimized by steepest decent method
Scheme 1. Synthetic procedure of 3-[3-(amino)propoxy]benzenamines 10a–10e. Reagents and conditions (i) ClCH2CH2CH2Br,
K2CO3, condense; (ii) amines, stirring with or without heating for 4–8 h.

Design, synthesis and biological evaluation of some acetylcholinesterase inhibitors
7
Table 1. Effect of compounds 10a–10e and reference drug (piracetam) (1, 3 and 5 mg/kg, i.p.) on % retention measured on elevated
plus maze in mice.
% Retention (Mean S.E.M)
Compound
(1 mg/kg)
(3 mg/kg)
(5 mg/kg)
Piracetam
10a
10b
10c
10d
37.24 .023°
37.81 3.791°
36.66 3.370°
47.72 3.890°
39.91 5.100°
42.71 2.670°
57.60 .015°
67.83 3.950°
27.56 1.860°,**
40.11 5.200°,**
54.02 2.016°
11.91 2.570***
06.29 1.800***
10.92 2.960***
02.32 1.600***
11.64 2.560***
29.08 2.360°,**
33.96 2.040°,**
10e
Note: All drugs were administered i.p. 30 min after training session. Each value represents the mean of six mice.
*p ≤ .001 as compared to the reference drug (1 mg/kg).
**p ≤ .001 as compared to reference drug (3 mg/kg).
***p ≤ .001 as compared to reference drug (5 mg/kg). po≤ .001 as compared to control.
Table 2. Effect of compounds in the Rota Rod apparatus.
Compound
No. of falls before treatment (Mean S.E.M)
No. of falls after treatment (Mean S.E.M)
CMC
10a
10b
10c
10d
10e
46.20 7.08
46.20 3.70
53.40 6.34
51.60 4.39
57.00 1.87
55.22 2.16
32.20 5.54
31.60 6.35
40.00 5.78
38.80 8.70
37.40 5.68
25.20 1.30
Notes: All drugs were administered i.p. 30 min before test, n = 6, as compared to vehicle treated mice, vehicle: normal saline.
completion of reaction was monitored with the help of
TLC. The structures of the compounds were character-
ized using various spectral analyses. FT-IR spectra of all
compounds showed a characteristic C–O–C stretching
vibrations at ~1261 (asymmetric stretch) and
~1083 cm−1(symmetric stretch) and the absence of ali-
phatic C–Cl stretch at 696.25 cm−1. Proton resonance
signals in the NMR spectrum showed quintet at δ 1.88
(–NCH2CH2)2 and a triplet at 2.82 ppm [–N(CH2)2] for
pyrrolidino methylenes, morpholino protons appeared as
triplets at δ 2.16 [4H, –N(CH2CH2)2−], and 3.12 ppm
[4H, (–CH2CH2)2O−], dimethylamino as a six protons
singlet at δ 2.93 ppm, diethyl amino protons showed a
quartet at δ 2.86 (4H, J = 4 Hz) and a triplet at δ
.72 ppm (6H, J = 4 Hz), concordant J values showed that
they are coupling with each other. In all the compounds
10a–10e, aromatic protons resonated near δ 7.83 (t,
5-CH), 6.82 (d, 4-CH), 6.44 (d, 6-CH), and 5.40 ppm
(s, 2-CH). Protons of amino group appeared as singlet
near 4.65 ppm which disappeared on deteurium
exchange. Methylene protons of the propylene chain res-
onated at δ ~3.69 (–OCH2−), δ ~2.16 (–CH2N<), and δ
~1.54 ppm (–CH2CH2CH2−) for all these derivatives
except in imidazolyl derivative where downfield values
appeared at δ 3.82 (–OCH2−, –CH2N<) and 2.13 ppm
(–CH2CH2CH2−), respectively, probably because of the
vicinity of two nitrogens in conjugation. Imidazole ring
protons occured at δ 8.43 (s, 2′-CH), 7.82 (d, 5′-CH),
and 6.82 ppm (d, 4′-CH), both these doublets have J
value ~2 Hz indicating protons must be coupling with
each other. 13C NMR signals for imidazolyl carbons
appeared at δ135.50 (2′-CH), 126.10 (5′-CH) and
120.00 ppm (4′-CH). HRMS (FAB) found 218.20
[M + 1] calcd for C12H15N3O 217.20.
Pharmacology
The compounds were screened for memory enhancing
potential using elevated plus maze model (Table 1,
Figure 1). All the synthesized compounds showed higher
or equivalent percent retention as compared to piracetam
at 1 mg/kg, compounds 10a, 10b, 10c showed significant
activity in comparison to control and reference drug
piracetam and compounds 10a and 10b showed dose
dependency (1–3 mg/kg) as well. Of all the compounds,
compound 10c has shown significant dose-dependent
(1–3 mg/kg) memory enhancing activity, while 10a also
showed activity equivalent to reference drug piracetam at
1 mg/kg. The drugs when compared at 5 mg/kg showed
significant activity but with a tremendous decrease in
percent retention, the decrease in activity at a higher
dose suggested these drugs might have muscle relaxant

8
R. Malik et al.
Table 3. Effect of compounds in the mice abdominal constric-
tion test (Writhing test).
Table 6. Docking score and binding energies of synthesized
compounds.
Compound
No. of mice
No. of writhes (Mean S.E.M)
Docking
score
Binding
energy
Compound
CMC
10a
10b
10c
10d
10e
06
06
06
06
06
06
6.0 1.23
7.4 1.14
8.4 .89
7.8 1.30
7.6 1.14
7.8 1.30
10a
10b
10c
10d
10e
Sck
−10.118
−9.672
−7.244
−8.851
−7.992
−13.998
−50.160
−64.289
−64.430
−27.938
−53.442
−97.222
Notes: All drugs were administered i.p. 30 min before test, n = 6,
as compared to vehicle treated mice, vehicle: normal saline.
Molecular modeling study results
Table 4. Effect of compounds against pentylenetetrazol-
induced clonic convulsions in mice.
Docking experiments were employed to answer various
queries and predict the position and orientation of
inhibitor, i.e. whether the molecules had affinity to
protein. Docking in XP mode in Glide gave us an
insight to the relative binding affinity expressed in kcal/-
mol in terms of docking score. The description obtained
from the docking studies was further utilized for identi-
fying underlying mechanism for inhibition. Docking
scores of benzenamines 10a–10e have been mentioned
in Table 6.
Compound Onset action (Mean S.E.M) Death/recovered
PTZ
10a
10b
10c
10d
10e
126.00 3.85
125.50 1.64
134.20 5.04
131.70 3.83
139.00 3.26
145.00 2.19
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Notes: PTZ: Pentylenetetrazole. All drugs were administered i.p.
30 min before test, n = 6, as compared to vehicle treated mice, vehicle:
normal saline.
MM-GBSA calculations were performed which gave
us an insight for binding energy expressed in kcal/mol.
The relative binding energies of benzenamines 10a–10e
are mentioned in Table 6. The complex obtained from
MM-GBSA calculations for molecule 10a was subjected
to molecular dynamics simulations. A 10 ns simulation
yielded us with results which could enhance our under-
standing relating acetylcholinesterase inhibition. RMSD
and RMSF for complex 10a revealed a stable conforma-
tion of the complex. MD simulations are carried in near
physiological environment condition. Therefore, the
binding orientations of lead molecules show better corre-
lation to their biologically active states (Karplus &
McCammon, 2002).
Table 5. Acetylcholinesterase inhibitory concentrations for
compound 10a–10e (μM).
Compound
IC50 (μM)
Piracetam
10a
58.69
8.99
10b
10c
10d
10e
28.31
17.87
10.57
14.69
During MD simulations, the interaction patterns of
AChE–10a and AChE–Sck complex were produced giv-
ing us an insight of mechanism underlying the inhibition
(Figure 3). The analysis of the RMSD plot for Cα atoms
(Figure 3(a)) showed that after a small rearrangement
from the initial conformation, the complex was relatively
stable during entire MD simulation period. The RMSD
for atoms after 300 ps remained within the limit of 2 Å
(Figure 3(a)). The average RMSD of Cα atoms, for the
AChE–10a docked complex during 5000 ps simulation
time was 1.6 Å. The RMSD range for Cα atoms varied
from 1.4 to 1.8 Å. The lower RMSD during MD simula-
tions for AChE–10a docked complex showed consistent
nature of docking conformations. The RMSF of all resi-
dues in the MD trajectories were calculated by averaging
over all the atoms of the given residues (Figure 3(b)).
activity at this dose. So the compounds were tested for
muscle relaxant activity using rota rod test (Table 2), but
all these compounds did not show any significant activ-
ity. The compounds were further screened for other
CNS-related activities like analgesic property (Table 3)
and anticonvulsant activity (Table 4). Compounds did
not show any effect in the mice abdominal constriction
test and pentylenetetrazole-induced convulsions suggest-
ing that these drugs did not possess analgesic properties
and anticonvulsant activity. Synthesized 3-[3-amino)pro-
poxy]benzenamines 10a–10e were acetylcholine esterase
inhibitors with IC50 values ranging from 8.99 to
28.31 μM (Table 5). Compounds 10a and 10c were
found to show AChE inhibition with IC50 value of 8.99
and 17.87 μM.

Design, synthesis and biological evaluation of some acetylcholinesterase inhibitors
9
RSMF of most of the residues were within the limit of
2.5 Å. Fluctuations for a few residues were exceeded
3 Å. The lower atomic fluctuations of the active site and
the backbone atoms indicated small conformational
changes. The energy plot, RMSD plot, and RSMF plot
analysis revealed that the AChE–10a docked complex
was stable during 5000 ps MD simulation time. In
AChE–10a complex; Trp86, Glu202, His447, Trp286,
Asp74, Tyr341 were found to be the key interacting
residues for AChE–10a complex. Trp86 was found to
interact hydrophobically through Π–Π stacking. Trp286
and Tyr 341 showed hydrophobic contact through
Π–cation interaction. His447 showed polar contact
through Π–cation interaction. Glu202 was found impor-
tant residue interacting via forming a hydrogen bond
required for stability of the complex. Asp74 was found
interacting through hydrogen bonding, ionic as well as
through water bridges.
Figure 3. Molecular dynamics simulation results of 10a and Sck molecule complexed with AChE. (a) RMSD of protein ligand com-
plex showing RMSD pattern for 10a-AChE; (b) RMSF of protein ligand complex showing RMSF pattern for 10a-AChE; (c) RMSD
of protein ligand complex showing RMSD pattern for Sck-AChE; (d) RMSF of protein ligand complex showing RMSD pattern for
Sck-AChE.

10
R. Malik et al.
Figure 3. (Continued)
In AChE–Sck complex RMSD was found to be
1.75 Å although upon whole trajectory analysis one
could observe the rising nature in RMSD plot
which gives our synthesized molecule 10a an advantage
over already published enzyme–inhibitor complex
(Figure 3(c)). RSMF of most of the residues were within
the limit of 2.5 Å. Fluctuations for a few residues were
exceeded 3 Å (Figure 3(d)). Important residues in inter-
action of Sck are mentioned in Figure 4(c) and (d).
Trp86, His447, Phe295, Trp286, Asp74 were found to
be key interacting residues (Figure 4(a)). Trp86 was
found to interact hydrophobically through Π–cation inter-
action. His447 showed polar contact through Π–cation
interaction as observed with 10a molecule. Trp286
showed hydrophobic contact through Π–cation interac-
tion. Asp74 was found interacting through a salt bridge
(Figure 4(b)).
The molecular docking (Figures 4, 5, and 6) and MD
simulation (Figure 3) results such as RMSD, RMSF, and
interaction patterns of both the complexes proved that

Design, synthesis and biological evaluation of some acetylcholinesterase inhibitors
11
Figure 4. Ligand Protein interactions for 10a and Sck molecule with amino acid residues of AChE.

12
R. Malik et al.
Figure 5. 3D molecular interaction diagram of 10a with AChE.
Figure 6. 3D Superimposed molecular interactions of 10a-10e with AChE

Design, synthesis and biological evaluation of some acetylcholinesterase inhibitors
the binding affinity of 10a toward AChE was highly
13
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Acknowledgements
The authors are grateful to University Grants Commission,
New Delhi, India for providing financial assistance (Grant num-
ber-No.F.30-18/2014). The authors are also grateful to Schro-
dinger LLc, USA and Central University of Rajasthan for
providing assistance for software.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the University Grants Commis-
sion, New Delhi, India for providing financial assistance [grant
number-No.F.30-18/2014].