Synthesis, biological evaluation and molecular docking study of novel piperidine and piperazine derivatives as multi-targeted agents to treat Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2014.12.057

Development of Multi-Target Directed Ligands (MTDLs) has emerged as a promising approach for targeting complex etiology of Alzheimer's disease (AD). Following this approach, a new series of N′-(4-benzylpiperidin-/piperazin-/benzhydrylpiperazin-1-yl)alkylamine derivatives were designed, synthesized and biologically evaluated as inhibitors of cholinesterases (ChEs), amyloid-beta (Aβ) self aggregation and also for their radical scavenging activity. The in vitro studies showed that the majority of synthesized derivatives strongly inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC₅₀ values in the low-nanomolar range, and were clearly more potent than the reference compound donepezil in this regard. Among them, inhibitors 5h and 5k, strongly inhibited AChE, with IC₅₀ value of 6.83 nM and 2.13 nM, respectively, and particularly, compound 5k was found to be highly selective for AChE (～38-fold). Moreover, both kinetic analysis of AChE inhibition and the docking study suggested that 5k binds simultaneously to catalytic active site and peripheral anionic site of AChE. Besides, these compounds also exhibited greater ability to inhibit self-induced Aβ_1-42 aggregation at 25 μM with percentage inhibition from ～54% to 89% and specially compound 5k provided highest inhibition (88.81%). Also, the derivatives containing methoxy and hydroxy groups showed potent oxygen radical absorbance capacity (ORAC) ranging from 2.2- to 4.4-fold of the Trolox value. Furthermore, results of ADMET studies suggested that all compounds exhibited appropriate drug like properties. Taken together, these results suggest that 5k might be a promising lead compound for further AD drug development.

Full text of DOI:10.1016/j.bmc.2014.12.057

Accepted Manuscript
Synthesis, biological evaluation and molecular docking study of novel piperi-
dine and piperazine derivatives as multi-targeted agents to treat Alzheimer’s
disease
Poonam Meena, Vishal Nemaysh, Manisha Khatri, Apra Manral, Pratibha
Mehta Luthra, Manisha Tiwari
PII:
S0968-0896(14)00915-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.12.057
BMC 11989
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
13 November 2014
22 December 2014
23 December 2014
Please cite this article as: Meena, P., Nemaysh, V., Khatri, M., Manral, A., Luthra, P.M., Tiwari, M., Synthesis,
biological evaluation and molecular docking study of novel piperidine and piperazine derivatives as multi-targeted
agents to treat Alzheimer’s disease, Bioorganic & Medicinal Chemistry (2014), doi:http://dx.doi.org/10.1016/j.bmc.
2014.12.057
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Synthesis, biological evaluation and molecular docking study of novel piperidine and
piperazine derivatives as multi-targeted agents to treat Alzheimer’s disease
Poonam Meenaa, Vishal Nemayshb, Manisha Khatric, Apra Manrala, Pratibha Mehta Luthrab, Manisha
Tiwaria *
aBio-Organic Chemistry Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, University
of Delhi, Delhi-110007, India.
bNeuropharmaceutical Chemistry Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research,
University of Delhi, Delhi-110007, India.
cShaheed Rajguru College of Applied Sciences for Women, University of Delhi, Delhi-110096, India.
Correspondence to:
Dr. Manisha Tiwari
Bio-Organic Chemistry laboratory
Add: Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi-110007, India
Tel: 91-11-27666272, Fax: 91-11-27666248
*E-mail: mtiwari07@gmail.com
1

Abstract
Development of Multi-Target Directed Ligands (MTDLs) has emerged as a promising
approach for targeting complex etiology of Alzheimer's disease (AD). Following this
approach, new series of N'-(4-benzylpiperidin-/piperazin-/benzhydrylpiperazin-1-
a
yl)alkylamine derivatives were designed, synthesized and biologically evaluated as inhibitors
of cholinesterases (ChEs), amyloid-beta (Aβ) self aggregation and also for their radical
scavenging activity. The in vitro studies showed that the majority of synthesized derivatives
strongly inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC50
values in the low-nanomolar range, and were clearly more potent than the reference
compound donepezil in this regard. Among them, inhibitors 5h and 5k, strongly inhibited
AChE, with IC50 value of 6.83 nM and 2.13 nM respectively, and particularly, compound 5k
was found to be highly selective for AChE (~38-fold). Moreover, both kinetic analysis of
AChE inhibition and the docking study suggested that 5k binds simultaneously to catalytic
active site and peripheral anionic site of AChE. Besides, these compounds also exhibited
greater ability to inhibit self-induced Aβ1-42 aggregation at 25 µM with percentage inhibition
from ~ 54% to 89% and specially compound 5k provided highest inhibition (88.81%). Also,
the derivatives containing methoxy and hydroxy groups showed potent oxygen radical
absorbance capacity (ORAC) ranging from 2.2- to 4.4-fold of the Trolox value. Furthermore,
results of ADMET studies suggested that all compounds exhibited appropriate drug like
properties. Taken together, these results suggest that 5k might be a promising lead compound
for further AD drug development.
Keywords:
Alzheimer's disease
Multi-target directed ligands
Cholinesterases
Aβ aggregation
Antioxidants
Molecular docking
2

1. Introduction
Alzheimer’s disease (AD) is the most common cause of progressive dementia in the elderly
population and the fourth leading cause of death in the western countries. The disorder is
reaching epidemic proportions, with a large human, social, and economic burden in
developed countries.1 According to World Alzheimer Report 2010,2 more than 30 million
people worldwide are afflicted with the disease and with the considerable increase in the
aging population this figure is expected to rise beyond 100 million in 2050. Over the past few
decades enormous efforts have been put forth to understand the complex pathophysiology of
AD, but its etiology remains elusive. Several hypotheses have been proposed which
contribute towards the development of AD and these mainly include deposits of abnormal
proteins (amyloid-beta (Aβ) and τ-protein), oxidative stress, dyshomeostasis of biometals,
loss of synapses and degeneration of cholinergic neurons in many areas of the CNS.
Cholinergic hypothesis represents one of the classical hypothesis associated with AD which
suggests that the substantial decrease in the levels of neurotransmitter acetylcholine (ACh)
leads to cognitive and memory deficits associated in AD patients.3 Acetylcholinesterase
(AChE) is the main enzyme responsible for the hydrolysis of the ACh at the cholinergic
synapses, while butyrylcholinesterase (BuChE) acts as a co-regulator of the activity of AChE.
Under normal physiological condition maximum cholinesterase activity is due to AChE
however as the disease progresses AChE activity decreases in specific brain regions, whereas
BuChE activity increases which compensate some of the functions of AChE in cholinergic
neurons.4 Consequently, therapeutic agents that serve as inhibitors of both these enzymes
could provide additional benefits in AD. Although several strategies have been adopted in the
last few years but current therapies for AD mainly focus on the use of three FDA approved
acetylcholinesterase inhibitors (AChEIs),5 namely donepezil, rivastigmine, galantamine and
one N-methyl-D-aspartate (NMDA) receptor antagonist, memantine.6 These medications are
regarded as merely symptomatic and therefore there is need to find more efficient strategies
to stop the disease progression.
Among the multiple factors that contribute towards AD progression, Aβ plays a
primary role, as its production and accumulation in the brain is a central event in the
pathogenesis of AD.7 The development of therapeutics which target inhibition of Aβ fibril
3

aggregation is currently viewed as a promising approach for the treatment of AD. The other
associated hypothesis, namely oxidative stress, occurs early in the progression of AD.8 Aging
of the brain is the main risk factor for disease development, resulting from an imbalance
between ROS production and antioxidant defences. Multiple lines of evidence suggest that
oxidative stress enhances the manifestation of major disease hallmarks namely, the
development of amyloid plaques and neurofibrillary tangles.9 Therefore, drugs that aimed at
clearing or preventing the formation of the free radicals in the brain might be beneficial for
AD.
In recent years, research on the ‘non-classical’ roles of AChE, has strengthened its
importance in the phenomenon of neurodegeneration, a key player in AD. This action of
AChE is primarily mediated by the peripheral anionic site (PAS) through which it colocalizes
with the Aβ peptide and promote Aβ fibrillogenesis by forming stable AChE-Aβ
complexes.10-12 These findings have led to development of novel class of AChEIs inhibitors
with dual specificity, being directed to both the active and peripheral sites of AChE.
Insights into AD complexity and the multiple etiologies contributing to the AD has
gradually shifted the traditional approach of drug discovery based on a 'one molecule, one
target' paradigm to the “multi-target-directed agents" which are aimed at targeting multiple
pathological processes simultaneously.13,14 The designing of multifunctional agents usually
involves the use of synthetic hybrid compounds, rationally designed to incorporate into a
single framework at least two different pharmacophoric moieties.15-17 Therefore, development
of hybrids that simultaneously inhibit cholinesterases (ChEs) along with decreased Aβ fibril
self-aggregation and antioxidant capacity might be considered an important advancement in
the treatment of AD. In recent years, a significant number of studies have shown
advancement in the development of AD multifunctional agents with two or more
complementary biological activities.18-20
Therefore, in the view of the above mentioned reasons, we aimed to exploit multi-
target-directed drug design strategy, to combine benzylpiperidine and piperazine derivatives
with appropriate electron rich aromatic rings using alkyl spacer of different lengths. The
strategy for designing the compounds was based on the selection of different pharmacophores
present in the structures of various anti-Alzheimer agents. Literature described that presence
of benzyl piperidine/piperazine and benzhydrylpiperazine moiety in AChE inhibitors (Fig. 1)
mainly contribute to inhibitory activity by interacting with the active site of AChE and has
also been efficiently used for design of dual AChE inhibitors.21-26 In addition, many
4

researchers have reported that the presence of electron rich substituents such as hydroxy, di-
and tri-methoxy groups on aromatic ring and nitrogen containing heterocyclic system may
play an important role in inhibiting Aβ aggregation and scavenging a variety of reactive
oxygen species.27,28
Taking these factors into consideration, we rationally designed and synthesized a
novel series of N'-(4-benzylpiperidin-/piperazin-/benzhydrylpiperazin-1-yl)alkylamine
derivatives as potential multifunctional agents for the treatment of AD. All synthesized
compounds were investigated for their biological activities against inhibition of AChE and
BuChE, self-induced Aβ aggregation and antioxidant properties. Further, mechanism of
inhibition for the most active derivatives were explored through molecular docking and
kinetic studies.
2. Results and discussion
2.1. Chemistry
The synthetic methodology employed to develop target compounds [5(a-l), 6(a-l), 7(a-l)] is
outlined in Scheme 1. The key intermediates were prepared according to our previously
reported procedure29 with slight modifications. The commercially available secondary amines
were N-alkylated with bromoalkylnitriles under reflux in acetone in the presence of K2CO3
to afford the corresponding nitrile derivatives 3(a-i) which were further reduced to
corresponding amines 4(a-i) in tetrahydrofuran using LiAlH4 as reducing agent. In the next
step, the target compounds were obtained by the reaction of different substituted aromatic
aldehydes in methanol with intermediate 4(a-i) followed with reductive amination using
NaBH4 in methanol.30
2.2. In vitro inhibition studies of AChE and BuChE
To determine the potential application of target compounds for the treatment of AD, their
inhibition towards AChE (from electric eel) and BuChE (from equine serum) were
determined using the the method of Ellman et al.31 with donepezil as reference compound.
The IC50 values of all tested compounds for ChE inhibition and their selectivity index are
summarized in Table 1. With the aim of optimizing the interaction of the synthesized
compounds with catalytic and peripheral binding sites of AChE, both the benzyl substituted
piperazine / piperidine and the position of the electron rich substituents on benzyl group were
5

varied. From Table 1, it could been seen that most of the target compounds inhibited both
ChEs exhibiting IC50 values in the nanomolar range.
The inhibitory activity of 4-benzylpiperidine series 5(a-l) against AChE demonstrated
that compounds with 3,4,5-trimethoxy and vanillyl group on benzyl ring in their side chains
were stronger inhibitors of AChE than those with the 3,4-dimethoxy group. For instance,
compound 5b and 5h showed 23- and 29-folds stronger inhibition for AChE (IC50 = 8.56 nM
& IC50 = 6.83 nM) than compound 5f, with an IC50 value of 201.8 nM. Moreover, the
introduction of pyridine ring at the end showed improved anti-AChE activity as observed in
case of compound 5k (IC50 = 2.13 nM) and 5l (IC50 = 7.72 nM) which were found to be 22-
and 6-fold more active than reference drug donepezil (IC50 = 48 nM). In addition, compound
5k also demonstrated a high selectivity for AChE over BuChE (~38-fold). With regard to
BuChE inhibition in 4-benzylpiperidine series, compounds 5g, 5h & 5i containing vanillyl
group showed strongest inhibition with IC50 values of 4.20 nM, 7.61 nM & 3.87 nM
respectively. Replacement of piperidine group in the benzyl piperidine series with a
piperazine group 6(a-l) led to a slight decrease in AChE inhibition for the same length of
alkyl chain linker. The IC50 values observed for this series range between 13.52 nM to 682.3
nM. The compounds with vanillyl group 6h (IC50 = 13.52 nM) and pyridine ring 6k (IC50
=
23.10 nM) in their side chains were among the best inhibitors, just like the derivatives of 4-
benzylpiperidine series. Interestingly, some of the compounds mainly 6c , 6g, & 6i displayed
4-9 times higher selectivity towards BuChE than for AChE. The target compounds 7(a-l)
showed better inhibition for AChE as compared to the corresponding benzylpiperazine series,
indicating that the introduction of a (C6H5)2CH- group might contribute to the inhibitory
activity observed for this series. Compounds which contain terminal vanillyl group 7h (IC50 =
9.79 nM), was the most effective in inhibiting AChE within this series followed by
compounds 7c, 7i and 7k whose IC50 values were very close to those of compound 7h. The
results showed that AChE inhibition was influenced by the type and position of substituent on
the benzyl group. For AChE inhibition, the order of activity for four terminal substituted
groups on benzyl ring is as follows: pyridine > vanillyl > 3,4,5-trimethoxy > 3,4-dimethoxy.
On the other hand, derivatives of all the three series were uniformly more potent than
donepezil against BuChE. The general trend for BuChE inhibition in terms of substitution at
benzyl ring was vanillyl > 3,4,5-trimethoxy > pyridine > 3,4-dimethoxy. These results
suggested that the electron density of aromatic ring might be associated with the extent of
inhibition of enzyme activity. From the IC50 values of all the three series, it appears that both
6

two and three methylene groups are of an appropriate length for a linker between the two
terminal pharmacophore groups in comparison to one methylene group. Taking into
consideration, the structure activity relationship (SAR), we conclude that 4-benzyl piperidine
derivatives bearing a terminal pyridine ring 5k and vanillyl group 5h on benzyl ring were
most effective in inhibiting the activity of both ChEs.
2.3. Kinetic chracterization of AChE inhibition
Based on the in vitro results of AChE inhibition, we further selected the most potent inhibitor
5k for kinetic analysis to investigate the type of enzyme inhibition. Graphical analysis of the
reciprocal Lineweaver-Burk plot (Fig. 2) showed both increased slopes (decreased Vmax) and
intercepts (higher Km) at higher concentration of the inhibitor 5k. This pattern indicated a
mixed-type inhibition and therefore supported the dual site (CAS and PAS) binding of this
compound.
2.4. Molecular docking studies
In order to further explore the mechanism of inhibition, a molecular docking study was
performed based on the available crystal structure of Torpedo californica AChE (TcAChE).
The 3D structure of TcAChE enzyme revealed that the active site is located at the bottom of a
deep and narrow gorge containing Trp84 and Phe330 residues in the catalytic active site
(CAS). In addition, a peripheral anionic site (PAS) is present near the active site gorge
mainly comprising of aromatic residues Tyr70, Tyr121, Tyr334, Trp279 and Asp72.32 On the
basis of the in vitro analysis of AChE inhibition, the most active derivatives were selected for
further docking studies. Their calculated free energies of binding (∆Gb) is summarized in
Table 2. For each compound, the best pose with the lowest docked energy, belonging to the
top-ranked conformations was selected for further post-docking analysis with
AutoDockTools and PyMOL. It was observed that the most potent AChE inhibitor in our
studies 5k (Table 2) displayed significant interactions in the CAS and PAS binding sites of
the enzyme to provide maximal inhibitory profile. The position of compound 5k with respect
to the key residues in the binding site is shown in Figure 3. The interaction of compound 5k
with CAS in the binding model suggests that the pyridine ring is firmly bound to the catalytic
site of AChE in the bottom of the gorge, through a favorable π-π interaction with the active
site residue Trp84 with the distance of 3.5 Å. Furthermore, the binding is assisted by the
formation of strong hydrogen bond between the pyridine nitrogen atom and the hydroxyl
7

group of Ser122 with an average distance of 3.3 Å. The NH group of substituted benzylamine
interacts at the PAS of AChE through hydrogen bond formation with the carboxylate group
of Asp72, and the distance from the amino nitrogen atom to the carboxylate oxygen of Asp72
is 1.6 Å. At the top of the gorge, the benzyl ring is stacked against the indole ring of Trp279
through π–π stacking interactions, with a ring to ring distance of 4.6 Å. The piperidine ring
may have van der Waals contacts with the phenyl rings of Phe331 and Tyr334 (distance
between the closest carbon atoms of 3.9 Å and 3.8 Å). The result of docking study revealed
that the dual binding profile for selected compound was found due to hydrogen bond, π–π
(aromatic) and hydrophobic interactions with both CAS and PAS of AChE.
A similar docking study was performed on reported crystal structure of Human
BuChE (HuBuChE).33 In the absence of X-ray structure of equine serum BuChE, a
HuBuChE (PDB code 1POI) with a resolution of 2.0 Å was used for docking because of
its high sequence homology with that of equine serum BuChE. The active site of
HuBuChE is located at the bottom of a 20 Å gorge consisting of aromatic amino acid residues
almost half of that found in TcAChE. In addition, the CAS residues of human BuChE
includes Trp82, Ala328, with His438, which is an important residue of catalytic triad, while
the PAS located at the mouth of the gorge primarily consists of Asn68, Asp70, Glu197,
Ala277, Tyr332 residues. As shown in Figure 4, it was found that the compound 5k could
reside in the large catalytic cavity of HuBuChE, through π-π stacking interaction between
pyridine ring and Trp82 with an average distance of 3.2 Å . In addition, a hydrogen bond was
also observed between the carbonyl group of Glu197 and the NH of substituted benzylamine.
Moreover, the calculated binding free energy of compound 5k with TcAChE was lower (-
12.9 Kcal/mol) than the HuBuChE (-10.12 Kcal/mol) (Table 2) which was found to correlate
well with the in vitro inhibition study. On the basis of in vitro result, compound 5i was
further selected for docking with HuBuChE because it exhibited better activity against
BuChE as compared to donepezil. The compound 5i (-11.97 Kcal/mole) fits into the active
site of HuBuChE through π-π and hydrogen bonds interactions which are found to be
essential for binding. As shown in Figure 5, it was found that the terminal benzyl group
(vanillyl) occupied the catalytic site of HuBuChE through π-π stacking interactions with
Trp82 of the active site and the approximate distance was 3.7 Å. Furthermore, the binding is
also supported by the formation of hydrogen bonds between the hydroxyl group of vanillyl
moiety and His438 with an average distance of 2.0 Å. In addition, at the beginning of gorge
the NH group of substituted benzylamine establishes hydrogen bond interaction with Asp70
8

(key residue of PAS) with an average distance 1.8 Å. All these interactions contribute to a
strong affinity of 5i toward HuBuChE.
2.5. Antioxidant activity in vitro
Oxidative stress is an important player of neuronal degeneration in AD. Its reduction is
another crucial aspect in designing multifunctional agents for AD treatment. The antioxidant
activities of all synthesized compounds were determined by measuring the oxygen radical
absorbance capacity of fluorescein (ORAC-FL), and the results are presented in Table 1.
Their ability to scavenge radicals is expressed as Trolox (a vitamin E analog) equivalent
(µmol of Trolox equivalents / µmol of tested compound), in a relative scale where ORAC
(trolox) = 1. Our results showed that introduction of methoxy and hydroxy substituents at 3,4
position of benzyl ring seems to be important for radical absorbance capacity. This could be
clearly seen in all the three series where compounds bearing these groups had antioxidant
activity ranging from 1.28- to 4.45-fold the value of Trolox. Among tested compounds,
compound 5g and 5i, gave the best results with 4.04 and 4.45 Trolox equivalents.
Furthermore, compounds 5a-5c, 6a-6c and 7a-7c which features three methoxy groups on
benzyl ring also showed higher antioxidant activities than their corresponding dimethoxy
derivatives. However, compounds containing pyridine ring mainly 7j-7l and 6j-6l showed
relatively lower antioxidant activity. Overall our results indicated that the presence of vanillyl
ring at the terminal end is crucial for providing strong radical scavenging ability.
2.6. Inhibition of self-mediated Aβ1−42 aggregation
Aβ generated from amyloid precursor protein is a critical player in the pathology of AD. Aβ1-
and Aβ1-40 are the major forms of Aβ peptide found in senile plaques. The Aβ1-42 is
42
generally considered to be more pathogenic because it has greater propensity towards fibrillar
aggregates which are neurotoxic in nature. Therefore, its inhibition could possibly be another
therapeutic strategy for AD treatment. With this aim, synthesized compounds were selected
to assess their ability to inhibit Aβ1-42 peptide aggregation employing the thioflavin-T
fluorescence method using curcumin (a known anti-amyloidogenic agent) as reference
compound. Table 3 summarizes the data for their effects on Aβ1-42 peptide aggregation at
concentration of 25 µM. The results indicated that most of target compounds showed higher
potencies ranging from ~ 54 % to 89 % relative to that of the curcumin (51.8 %) indicating an
inhibition of the Aβ1-42 self-aggregation process. Based on the results shown in Table 3,
9

compounds of series 5(a-l) were found to be more effective in blocking the Aβ1-42 self-
aggregation at 25 µM, as most of them presented aggregation inhibition values above 70%.
Among this series, compound 5k, which exhibited excellent inhibitory activity and high
selectivity to AChE, also showed good inhibitiory potency against Aβ1-42 self-aggregation
(~1.71 fold increase w.r.t curcumin). In the screening results of second series 6(a-l), the most
effective compound was found out to be 6d, followed by 6c, 6b, 6e and 6a, their inhibitory
potency are ~ 80%, 80%, 79%, 79% and 75%, respectively. On the other hand, compounds of
series 7(a-l) gave slightly weaker inhibitory activity than compounds of series I and II but
their inhibition was comparable to that of positive control curcumin. These results implied
that the presence of electron rich groups on benzyl ring and the terminal benzylpiperidine
seems to be beneficial for the inhibition of Aβ1-42 self-aggregation.
2.7. ADMET prediction
To predict the drug-likeness of the designed compounds, a few indicators of their
pharmacokinetic profiles were predicted using QikProp, v. 3.5.34 About 45 physically
significant descriptors and pharmacologically relevant properties of these compounds were
predicted. In the present study some of these important properties were considered and are
reported in Table 4 (supplementary file). Fundamental physiochemical features of CNS drugs
are mainly related to their ability to penetrate the blood-brain barrier (BBB) and exhibit CNS
activity. Our results indicated that compounds from all the three series (5a-5l), (6a-6l) and
(7a-7l) showed drug like characteristics based on Lipinski’s rule of five (mol_MW < 500,
QPlogPo/w < 5, donorHB ≤ 5, accptHB ≤ 10). Based on the predicted values for QPlogBB
and CNS activity, all compounds might be able to penetrate into the CNS. PSA is another
important predictor for BBB. Generally, drugs aimed at the CNS tends to have lower polar
surface area (PSA) usually between 60-70 Å2.35 The calculated theoretical PSA values for all
compounds may support their ability to penetrate the blood–brain barrier. Among calculated
parameters, QPPCaco, QPlogBB, QPLogKhsa, and QPPMDCK mainly account for the
ability of the compound’s distribution inside the body. Most of the compounds showed
moderate permeability for both in vitro Caco-2 cells and in vitro MDCK cells. Only
compounds of series (5a-5f) exhibited significant results in Caco-2 permeability. Moreover,
all compounds displayed good oral absorption. The amount of protein binding of CNS drugs
is an important consideration which tends to be rather high. The predicted values of
QPLogKhsa for all compounds indicate their strong binding with plasma protein. Target
10

compounds were further analysed for toxicity prediction using OSIRIS program. The
predictions were based on predetermined set of structural fragments that give rise to toxicity
alerts in case they are encountered in the structure. Interestingly, all compounds presented
low in silico possible toxicity risks in most cases as shown in Table 5 (supplementary file).
The predicted ADME properties revealed that all compounds fulfil drug-like criteria and
could be considered as good candidate for drug development.
2.8. PASS prediction
The PASS prediction results of test compounds for nootropic activity, relevant to
neurological disorders are listed in Table 6. The probability of the test compounds for being
active with Pa values greater than 0.50, considered as potent nootropic. Among tested
compounds, 5k, 5l and 7k have shown significant nootropic activity (Pa > 0.60) in
comparison to donepezil. This predicted nootropic activity for synthesized compounds
directly correlates with the memory and learning aspect of AD and can be considered for
further biological evaluation.
3. Conclusion
In the present study, a new series of N'-(4-benzylpiperidin-/piperazin-/benzhydrylpiperazin-1-
yl)alkylamine derivatives have been developed for targeting the multifaceted nature of AD.
These compounds were evaluated in vitro and in silico against important biological targets
including, inhibition of AChE, BuChE, amyloid β-aggregation, as well as their radical
scavenging activity. Most of the synthesized compounds effectively inhibited both AChE and
BuChE in the nanomolar range in vitro. The structure activity relationship studies revealed
that the extent of AChE inhibition was mainly influenced by the presence of both the electron
rich aromatic group at the end of the compound, as well as the benzyl substituted piperazine /
piperidine. The compounds of benzylpiperidine series, mainly 5h and 5k ( IC50 = 6.83 nM &
2.13 nM respectively) displayed about 7- and 22-fold higher potency for AChE than
reference compound donepezil. Similarly, compounds 5g, 5h, 5i and 7i showed good
inhibitory potency towards BuChE. Further investigation of kinetic characterization and
molecular modeling study indicated that compound 5k exhibited mixed-type inhibition mode,
suggesting that it simultaneously binds with both CAS and PAS of active site gorge. These
results are in agreement with in vitro AChE inhibition. On the other hand, Aβ1-42 self-
aggregation results showed that most of the compounds exhibited greater activity towards Aβ
11

aggregation than curcumin, especially compounds 5b, 5c, 5e-5h, 5j-5l and 6b-6e ( between
1.51- to 1.71-fold of curcumin). With regard to antioxidant activity among the three series,
the best results were obtained with compounds containing vanillyl group, which showed
about 1.28-4.45 fold radical capture capacity than Trolox. The promising results of
compound 5k in all assays demonstrated that the strategy behind the designing of compounds
was rational and favourable. In summary, these preliminary findings suggested that these new
compounds could be further investigated for pharmacological development in AD therapy.
4. Experimental section
4.1. Chemistry
All organic solvents and common reagents were procured from Merck India Ltd. All the
piperazines and piperidines were procured from Aldrich Chemical Company, Inc. USA.
Solvents were dried according to standard methods.36 The chemical reactions were monitored
by TLC using commercially available alumina plates coated with silica gel 60 F254 (Merck).
1
The purity of all organic compounds was confirmed by TLC, H-NMR, 13C-NMR and Mass
1
spectroscopy. H-NMR and 13C-NMR spectras were recorded in Jeol instrument operating at
400 MHz (1H) in CDCl3 and at 100 MHz (13C) using TMS as an internal standard. The
chemical shifts are reported in parts per million (δ) downfield from TMS and coupling
constants are reported in hertz (Hz). Proton coupling patterns are abbreviated as single (s),
double (d), triplet (t) and multiple (m). Mass spectra were recorded on a Qstar (Applied
biosystem) ESI-MS mass spectrometer. Elemental analyses were carried out by a EuroVector
Euro-E 3000 elemental analyzer instrument.
4.1.1. General procedures for the synthesis of intermediate nitriles 3(a-i)
Potassium carbonate (15 mM) was added to a stirred solution of 4-benzylpiperidine/1-
benzylpiperazine/1- benzhydrylpiperazine (10 mM) in acetone (20 ml). Then bromo alkyl
nitrile (10 mM) was added dropwise and the mixture was refluxed for 2 h. After cooling to
room temperature and filtration, the solution was evaporated to dryness and then residue was
extracted with diethylether and water. The organic layer was dried over anhydrous sodium
sulfate and concentrated on a rotary evaporator to afford the desired product as pale yellow
12

oil in a 80–90% yield that was identified through ESI-MS and NMR and used without further
purification.
4.1.2. General procedures for the synthesis of intermediate amines 4(a-i)
A solution of (3a-3i, 5 mM) in tetrahydrofuran (15 ml) was added dropwise to a suspension
of lithium aluminium hydride (380 mg, 10 mM) in dry tetrahydrofuran (20 ml) at 0º under
inert atmosphere followed by vigorous stirring. The temperature of reaction mixture was then
allowed to rise to room temperature and stirred for 2-3 h. The reaction was then quenched by
addition of water (0.47 ml), 15% aqueous NaOH (0.47 ml) and water (1.41 ml) sequentially.
The resulting granular precipitate was vaccum filtered and filtrate was washed with ethyl
acetate. The resulting organic layer was dried over anhydrous sodium sulfate and
concentrated on a rotary evaporator to afford the corresponding amines as pale yellow oil.
The residue obtained was identified through ESI-MS and NMR and used without further
purification.
4.1.3. General procedures for the synthesis of final compounds 5(a-l), 6(a-l) and 7(a-l)
Aromatic aldehydes (5 mmol) and intermediate amines 4(a-i) (5 mmol) were stirred in
MeOH (25 ml) at room temperature under nitrogen atmosphere. The mixture was stirred at
room temperature for 3 h, until the aldimine formation was completed. The aldimine in
MeOH was carefully treated with solid NaBH4 (20 mmol) at 0ºC. The reaction mixture was
stirred for 15 min and then quenched with water followed by extraction with EtOAc (3 x 10
ml). The organic layer was washed with saturated aqueous NaCl and then dried over
anhydrous sodium sulfate. The solvent was further evaporated to afford the crude product as
yellow oil, which was further purified through silica gel column chromatography using a
mixture of CH2Cl2 and MeOH (9.8:0.2) as eluent to afford the desired product (Scheme 1).
4.1.3.1. 2-(4-benzylpiperidin-1-yl)-N-(3,4,5-trimethoxybenzyl)ethanamine (5a)
Intermediate 4a was treated with 3,4,5-trimethoxybenzaldehyde according to general
1
procedure to afford the desired product 5a as light yellow oil (66% yield). H NMR (400
MHz, CDCl3): δ 7.26 (t, J = 7.36, 2H), 7.19-7.15 (m, 1H), 7.13 (d, J = 6.6, 2H), 6.60 (s, 1H),
6.55 (s, 1H), 4.60 (s, 1H), 3.85 (d, J = 4, 9H), 3.72 (s, 2H), 2.83 (br, d, J = 11.7, 2H), 2.70 (t, J
= 6.6, 2H), 2.53 (d, J = 7.32, 4H), 2.46 (t, J = 6.6, 2H), 1.89 (td, J = 11.7,2.2, 2H), 1.61 (br, d,
J = 12.4, 2H), 1.54-1.47 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 153.27, 140.62, 136.25,
13

129.10, 128.16, 125.80, 104.97, 103.59, 60.77, 58.07, 56.0, 54.28, 45.95, 43.24, 37.99, 32.25.
LC-MS m/z: 399.8 [M+H]+. Anal. Calcd for C24H34N2O3: C, 72.33; H, 8.60; N,7.03; O,
12.04. Found: C, 72.78; H, 8.23; N,7.83; O, 12.64.
4.1.3.2. 3-(4-benzylpiperidin-1-yl)-N-(3,4,5-trimethoxybenzyl)propan-1-amine (5b)
Intermediate 4b was treated with 3,4,5-trimethoxybenzaldehyde according to general
1
procedure to afford the desired product 5b as light yellow oil (70% yield). H NMR (400
MHz, CDCl3): δ 7.28 (t, J = 8.0, 2H), 7.19-7.11 (m, 3H), 6.60 (s, 2H), 4.61 (s, 1H), 3.85 (s,
9H), 3.83 (s, 2H), 3.05 (br, d, J = 12.4, 2H), 2.86-2.78 (m, 2H), 2.67 (t, J = 7.32, 2H), 2.52
(d, J = 5.12, 4H), 1.88-1.82 (m, 2H), 1.63 (br, d, J = 13.2, 4H), 1.55-1.45 (m, 1H). 13C NMR
(100 MHz, CDCl3): δ 153.28, 140.73, 137.24, 129.11, 129.96, 125.80, 105.85, 103.64, 64.99,
60.84, 56.06, 55.86, 53.85, 46.37, 43.67, 38.17, 37.92, 32.97, 32.12. LC-MS m/z: 413.1
[M+H]+. Anal. Calcd for C25H36N2O3: C, 72.78; H, 8.80; N, 6.79; O, 11.63. Found: C, 71.78;
H, 8.21; N, 6.09; O, 11.93.
4.1.3.3. 4-(4-benzylpiperidin-1-yl)-N-(3,4,5-trimethoxybenzyl)butan-1-amine (5c)
Intermediate 4c was treated with 3,4,5-trimethoxybenzaldehyde according to general
1
procedure to afford the desired product 5c as light yellow oil (72% yield). H NMR (400
MHz, CDCl3): δ 7.20 (t, J = 7.32, 2H), 7.11-7.04 (m, 3H), 6.53 (s, 1H), 6.49 (s, 1H), 4.51 (s,
1H), 3.78 (d, J = 4, 9H), 3.72 (s, 2H), 2.82 (br, d, J = 11, 2H), 2.58 (t, J = 6.6, 2H), 2.44 (d, J
= 6.6, 4H), 2.23 (t, J = 7.32, 2H), 1.78 (t, J = 7.32, 2H), 1.55 (br, d, J = 12.48, 2H), 1.45-1.42
(m, 5H). 13C NMR (100 MHz, CDCl3): δ 152.30, 139.64, 135.98, 135.39, 128.14, 127.24,
124.86, 104.11, 102.60, 63.25, 59.28, 57.96, 55.0, 53.30, 53.03, 48.43, 42.31, 37.09, 31.24,
27.20, 23.97. LC-MS m/z: 427.6 [M+H]+. Anal. Calcd for C26H38N2O3: C, 73.20; H, 8.98; N,
6.57; O, 11.25. Found: C, 73.59; H, 8.08; N, 5.87; O, 11.65.
4.1.3.4. 2-(4-benzylpiperidin-1-yl)-N-(3,4-dimethoxybenzyl)ethanamine (5d)
Intermediate 4a was treated with 3,4-dimethoxybenzaldehyde according to general procedure
to afford the desired product 5d as light yellow oil (68% yield). 1H NMR (400 MHz, CDCl3):
δ 7.24 (t, J = 8.04, 2H), 7.15-7.07 (m, 3H), 6.84 (s, 1H), 6.81-6.75 (m, 2H), 4.53 (s, 1H), 3.83
(d, J = 4.76, 6H), 3.68 (s, 2H), 2.79 (br, d, J = 11, 2H), 2.65 (t, J = 6.24, 2H), 2.48 (d, J = 6.6,
4H), 2.41 (t, J = 6.6, 2H), 1.84 (t, J = 10.64, 2H), 1.57 (br, d, J = 12.48, 2H), 1.49-1.42 (m,
1H). 13C NMR (100 MHz, CDCl3): δ 148.68, 147.67, 140.40, 132.84, 128.83, 127.87, 125.50,
119.97, 111.07, 110.70, 57.83, 55.63, 55.55, 53.75, 53.52, 45.64, 42.96, 37.70, 31.97. LC-MS
14

m/z: 369.6 [M+H]+. Anal. Calcd for C23H32N2O2: C,74.96; H, 8.75; N, 7.60; O, 8.68. Found:
C,74.28; H, 8.07; N, 7.11; O, 8.10.
4.1.3.5. 3-(4-benzylpiperidin-1-yl)-N-(3,4-dimethoxybenzyl)propan-1-amine (5e)
Intermediate 4b was treated with 3,4-dimethoxybenzaldehyde according to general procedure
to afford the desired product 5d as light yellow oil (74% yield). 1H NMR (400 MHz, CDCl3):
δ 7.05-6.92 (m, 5H), 6.76-6.58 (m, 3H), 4.38 (s, 1H), 3.84 (d, J = 4.76, 6H), 3.70 (s, 2H),
2.78 (br, d, J = 12.48, 1H), 2.68 ( d, J = 8.8, 1H), 2.44 (t, J = 6.6, 2H), 2.30-2.23 (m, 4H),
2.14 (t, J = 5.88, 2H), 1.70-1.58 (m, 1H), 1.50 (t, J = 7.32, 2H), 1.39 (d, J = 7.32, 2H), 1.32-
1.29 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 148.82, 147.84, 140.55, 132.86, 128.97,
128.01, 125.63, 120.12, 111.22, 110.83, 57.92, 55.79, 53.88, 45.74, 43.08, 37.82, 32.09,
26.87. LC-MS m/z: 383.5 [M+H]+. Anal. Calcd for C24H34N2O2: C,75.35; H, 8.96; N, 7.32;
O, 8.36. Found: C,75.86; H, 8.16; N, 7.01; O, 8.96.
4.1.3.6. 4-(4-benzylpiperidin-1-yl)-N-(3,4-dimethoxybenzyl)butan-1-amine (5f)
Intermediate 4c was treated with 3,4-dimethoxybenzaldehyde according to general procedure
1
to afford the desired product 5f as light yellow oil (70% yield). H NMR (400 MHz, CDCl3):
δ 7.26 (t, J = 7.32, 2H), 7.17-7.10 (m, 3H), 6.88 (s, 1H), 6.82-6.79 (m, 2H), 4.57 (s, 1H), 3.86
(d, J = 3.68, 6H), 3.70 (s, 2H), 2.88 (br, d, J = 11.7, 2H), 2.63 (t, J = 6.9, 2H), 2.51 (d, J = 6.6,
4H), 2.28 (t, J = 7.32, 2H), 1.84 (td, J = 9.52, 1.48, 2H), 1.61 (br, d, J = 12.4, 2H), 1.51-1.49
(m, 5H). 13C NMR (100 MHz, CDCl3): δ 148.92, 148.93, 147.93, 140.66, 134.54, 133.09,
129.06, 128.10, 125.71, 120.16, 111.35, 110.97, 60.31, 58.91, 55.86, 55.79, 53.93, 53.77,
49.27, 43.20, 37.96, 32.14, 28.13, 24.90. LC-MS m/z: 397.6 [M+H]+. Anal. Calcd for
C25H36N2O2: C,75.72; H, 9.15; N, 7.06; O, 8.07. Found: C,74.85; H, 9.90; N, 7.42; O, 8.71.
4.1.3.7. 4-(((2-(4-benzylpiperidin-1-yl)ethyl)amino)methyl)-2-methoxyphenol (5g)
Intermediate 4a was treated with 4-hydroxy-3-methoxybenzaldehyde according to general
1
procedure to afford the desired product 5g as light yellow oil (66% yield). H NMR (400
MHz, CDCl3): δ 7.25-7.20 (m, 5H), 6.87-6.65 (m, 3H), 5.32 (br, 1H), 4.52 (s, 1H), 3.76 (s,
3H), 3.69 (s, 2H), 2.84 (br, d, J = 10.9, 2H), 2.67 (t, J = 6.6, 2H), 2.47-2.37 (m, 6H), 1.85-
1.78 (m, 2H), 1.59-1.45 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 147.23, 145.41, 145.17,
140.28, 130.57, 128.73, 127.83, 125.50, 125.43, 120.63, 119.49, 114.87, 110.03, 64.22,
60.04, 57.30, 56.80, 55.29, 55.21, 53.61, 53.34, 45.57, 45.06, 42.80, 42.72, 37.55, 37.43,
15

35.86, 32.16, 31.70. LC-MS m/z: 355.6 [M+H]+. Anal. Calcd for C22H30N2O2: C, 74.54; H,
8.53; N, 7.90; O, 9.03. Found: C, 74.19; H, 8.03; N, 7.61; O, 9.93.
4.1.3.8. 4-(((3-(4-benzylpiperidin-1-yl)propyl)amino)methyl)-2-methoxyphenol (5h)
Intermediate 4b was treated with 4-hydroxy-3-methoxybenzaldehyde according to general
1
procedure to afford the desired product 5h as light yellow oil (75% yield). H NMR (400
MHz, CDCl3): δ 7.15-7.0 (m, 6H), 6.80-6.62 (m, 3H), 4.9 (br, 1H), 4.45 (s, 1H), 3.71 (s, 3H),
2.97 (d, J = 11, 2H), 2.79 (s, 2H), 2.55 (t, J = 6.6, 1H), 2.45-2.39 (m, 4H), 2.25 (t, J = 6.6,
2H), 1.76-1.71 (m, 1H), 1.57-1.38 (m, 6H). 13C NMR (100 MHz, CDCl3): δ 146.97, 145.44,
140.45, 132.96, 129.03, 128.19, 125.92, 120.01, 114.57, 110.13, 65.17, 63.16, 55.76, 53.78,
48.06, 47.11, 45.31, 43.19, 37.85, 37.49, 31.89. MS m/z: 369.5 [M+H]+. Anal. Calcd for
C23H32N2O2:C, 74.96; H, 8.75; N, 7.60; O, 8.68. Found: C, 74.25; H, 8.65; N, 7.68; O, 8.98.
4.1.3.9. 4-(((4-(4-benzylpiperidin-1-yl)butyl)amino)methyl)-2-methoxyphenol (5i)
Intermediate 4c was treated with 4-hydroxy-3-methoxybenzaldehyde according to general
1
procedure to afford the desired product 5i as light yellow oil (62% yield). H NMR (400
MHz, CDCl3): δ 7.27-7.10 (m, 5H), 6.81 (s, 1H), 6.75 (d, J = 8.0, 1H), 6.71 (d, J = 8.0, 1H),
4.87 (br, 1H), 4.53 (s, 1H), 3.80 (s, 3H), 3.66 (s, 2H), 2.90 (d, J = 10.9, 2H), 2.63 (t, J = 6.56,
2H), 2.52 (d, J = 6.6, 4H), 2.28-2.27 (m, 2H), 1.88-1.80 (m, 2H), 1.61 (br, d, J = 12.4, 2H),
1.50-1.37 (m, 5H). 13C NMR (100 MHz, CDCl3): δ 146.71, 144.76, 140.06, 130.52, 128.46,
127.50, 125.18, 120.33, 114.29, 110.68, 58.19, 55.05, 53.26, 48.43, 42.52, 37.31, 31.33,
27.21, 24.06. MS m/z: 383.1 [M+H]+. Anal. Calcd for C24H34N2O2: C, 75.35; H, 8.96; N,
7.32; O, 8.36. Found: C, 75.72; H, 8.16; N, 7.05; O, 8.59.
4.1.3.10. 2-(4-benzylpiperidin-1-yl)-N-(pyridin-3-ylmethyl)ethanamine (5j)
Intermediate 4a was treated with 3-pyridinecarboxaldehyde according to general procedure to
1
afford the desired product 5j as light yellow oil (76% yield). H NMR (400 MHz, CDCl3): δ
8.56 (s, 1H), 8.48 (d, J = 5.12, 1H), 7.71-7.65 (m, 1H),7.28-7.11 (m, 6H), 4.68 (s, 1H), 3.78
(s, 2H), 2.81 (br, d, J = 11.7, 2H), 2.68 (t, J = 6.6, 2H), 2.52 (d, J = 6.6, 4H), 2.45 (t, J = 6.6,
2H), 1.89 (t, J = 9.5, 2H), 1.61 (br, d, J = 12.4, 2H), 1.53-1.42 (m, 1H). 13C NMR (100 MHz,
CDCl3): δ 149.23, 148.02, 140.32, 135.63, 134.43, 128.80, 127.84, 125.47, 123.14, 57.64,
53.72, 50.92, 45.65, 42.87, 37.62, 31.87. MS m/z: 310.4 [M+H]+. Anal. Calcd for C20H27N3:
C, 77.63; H, 8.79; N, 13.58. Found: C, 77.80; H, 8.21; N, 13.03.
16

4.1.3.11. 3-(4-benzylpiperidin-1-yl)-N-(pyridin-3-ylmethyl)propan-1-amine (5k)
Intermediate 4b was treated with 3-pyridinecarboxaldehyde according to general procedure to
afford the desired product 5k as light yellow oil (68% yield). 1H NMR (400 MHz, CDCl3): δ
8.45 (s, 1H), 8.34 (t, J = 5.88, 1H), 7.61 (dd, J = 8.08, 2.2, 1H), 7.16-7.0 (m, 6H), 4.57 (s,
1H), 3.33 (s, 2H), 2.77 (br, d, J = 11.7, 2H), 2.53 (t, J = 7.32, 2H), 2.41 (t, J = 6.6, 4H), 2.24
(t, J = 7.32, 2H), 1.84-1.68 (m, 2H), 1.60-1.38 (m, 5H). 13C NMR (100 MHz, CDCl3): δ
149.91, 147.89, 140.18, 136.51, 134.36, 128.70, 127.77, 125.40, 123.02, 60.07, 56.90, 53.59,
53.36, 50.82, 47.86, 42.76, 37.37, 31.68, 26.51. MS m/z: 324.5 [M+H]+. Anal. Calcd for
C21H29N3:C, 77.97; H, 9.04; N, 12.99. Found: C, 77.87; H, 9.21; N, 12.90.
4.1.3.12. 4-(4-benzylpiperidin-1-yl)-N-(pyridin-3-ylmethyl)butan-1-amine (5l)
Intermediate 4c was treated with 3-pyridinecarboxaldehyde according to general procedure to
afford the desired product 5l as light yellow oil (76% yield). 1H NMR (400 MHz, CDCl3): δ
8.44 (s, 1H), 8.40 (s, 1H), 7.59 (d, J = 8.0, 1H), 7.14-6.98 (m, 6H), 4.53 (s, 1H), 3.62 (s, 2H),
2.75 (br, d, J = 11.7, 2H), 2.49 (t, J = 6.6, 2H), 2.39 (d, J = 5.12, 4H), 2.16 (t, J = 7.32, 2H),
1.76-1.61 (m, 3H), 1.49 (br, d, J = 12.4, 2H), 1.38-1.36 (m, 4H). 13C NMR (100 MHz,
CDCl3): δ 149.11, 147.91, 140.19, 135.46, 134.24, 128.66, 127.71, 125.33, 122.98, 122.93,
61.20, 58.39, 56.28, 53.48, 53.41, 50.76, 48.80, 42.75, 37.49, 31.71, 31.65, 27.63, 24.33. MS
m/z: 338.7 [M+H]+. Anal. Calcd for C22H31N3:C, 78.29; H, 9.26; N, 12.45. Found: C, 78.94;
H, 9.76; N, 12.21.
4.1.3.13. 2-(4-benzylpiperazin-1-yl)-N-(3,4,5-trimethoxybenzyl)ethanamine (6a)
Intermediate 4d was treated with 3,4,5-trimethoxybenzaldehyde according to general
1
procedure to afford the desired product 6a as light yellow oil (76% yield). H NMR (400
MHz, CDCl3): δ 7.25-7.19 (m, 5H), 6.54 (s, 1H), 6.49 (s, 1H), 4.54 (s, 1H), 3.79 (d, J = 4.36,
6H), 3.76 (s, 3H), 3.66 (s, 2H), 3.43 (s, 2H), 2.65 (t, J = 6.6, 2H), 2.45 (t, J = 5.88, 2H), 2.39
(br, s, 8H). 13C NMR (100 MHz, CDCl3): δ 152.61, 137.43, 136.27, 128.53, 127.57, 126.41,
104.41, 102.97, 63.83, 62.42, 59.63, 57.02, 55.37, 53.55, 52.57, 44.97. MS m/z: 400.5
[M+H]+. Anal. Calcd for C23H33N3O3:C, 69.14; H, 8.33; N,10.52; O, 12.01. Found: C, 69.49;
H, 8.11; N,9.52; O, 12.41.
4.1.3.14. 3-(4-benzylpiperazin-1-yl)-N-(3,4,5-trimethoxybenzyl)propan-1-amine (6b)
17

Intermediate 4e was treated with 3,4,5-trimethoxybenzaldehyde according to general
1
procedure to afford the desired product 6b as light yellow oil (69% yield). H NMR (400
MHz, CDCl3): δ 7.30-7.23 (m, 5H), 6.59 (s, 2H), 4.59 (s, 1H), 3.84 (s, 9H), 3.82 (s, 2H), 3.49
(t, J = 6.6, 2H), 2.85 (t, J = 6.8, 2H), 2.45-2.39 (m, 12H), 2.23 (2, 2H). 13C NMR (100 MHz,
CDCl3): δ 152.57, 137.14, 136.21, 128.58, 127.53, 126.39, 102.88, 63.80, 62.94, 59.97,
59.61, 55.29, 53.54, 53.0, 52.36, 45.14, 26.05. MS m/z: 414.5 [M+H]+. Anal. Calcd for
C24H35N3O3: C, 69.70; H, 8.53; N, 10.16; O, 11.61. Found: C, 69.32; H, 8.05; N, 10.22; O,
11.52.
4.1.3.15. 4-(4-benzylpiperazin-1-yl)-N-(3,4,5-trimethoxybenzyl)butan-1-amine (6c)
Intermediate 4f was treated with 3,4,5-trimethoxybenzaldehyde according to general
1
procedure to afford the desired product 6c as light yellow oil (69% yield). H NMR (400
MHz, CDCl3): δ 7.25-7.18 (m, 5H), 6.53 (s, 1H), 6.50 (s, 1H), 4.53 (s, 1H), 3.79 (d, J = 5.12,
9H), 3.75 (s, 2H), 3.43 (s, 2H), 2.58-2.27 (m, 12H), 1.46-1.30 (m, 4H). 13C NMR (100 MHz,
CDCl3): δ 153.02, 152.97, 137.73, 137.15, 135.90, 129.02, 127.98, 126.82, 104.70, 103.41,
64.55, 62.84, 58.29, 55.85, 55.81, 54.08, 52.93, 49.14, 27.81, 24.47. MS m/z: 428.7 [M+H]+.
Anal. Calcd for C25H37N3O3: C, 70.22; H, 8.72; N, 9.83; O, 11.23. Found: C, 70.39; H, 8.02;
N, 9.62; O, 11.46.
4.1.3.16. 2-(4-benzylpiperazin-1-yl)-N-(3,4-dimethoxybenzyl)ethanamine (6d)
Intermediate 4d was treated with 3,4-dimethoxybenzaldehyde according to general procedure
to afford the desired product 6d as light yellow oil (76% yield). 1H NMR (400 MHz, CDCl3):
δ 7.09-7.02 (m, 5H), 6.73-6.55 (m, 3H), 4.37 (s, 1H), 3.66 (d, J = 5.12 , 6H), 3.49 (d, J =
7.32, 2H), 3.26 (d, J = 8.08, 2H), 2.45 (t, J = 6.6, 2H), 2.26-2.21 (m, 10H), 1.79 (d, J = 8.80,
1H). 13C NMR (100 MHz, CDCl3): δ 148.18, 147.26, 137.11, 131.93, 128.40, 127.39, 126.25,
119.55, 110.73, 109.62, 63.27, 62.17, 59.46, 56.67, 55.03, 52.72, 52.16, 44.49. MS m/z:
370.7 [M+H]+. Anal. Calcd for C22H31N3O2: C, 71.51; H, 8.46; N, 11.37; O, 8.66. Found: C,
71.16; H, 8.98; N, 11.07; O, 8.61.
4.1.3.17. 3-(4-benzylpiperazin-1-yl)-N-(3,4-dimethoxybenzyl)propan-1-amine (6e)
Intermediate 4e was treated with 3,4-dimethoxybenzaldehyde according to general procedure
1
to afford the desired product 6e as light yellow oil (69% yield). H NMR (400 MHz, CDCl3):
δ 7.28-7.21 (m, 5H), 6.91 (s, 1H), 6.85 (d, J = 8.04, 1H), 6.79 (d, J = 8.04, 1H), 4.56 (s, 1H),
3.84 (s, 6H), 3.82 (s, 2H), 3.46-3.43 (m, 2H), 2.80 (t, J = 5.12, 2H), 2.62 (t, J = 6.6, 2H),
18

2.43-2.32 (m, 8H), 1.67-1.64 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 148.57, 147.80,
137.45, 134.07, 128.83, 127.78, 126.65, 118.68, 110.66, 109.99, 63.93, 62.59, 59.94, 55.48,
53.66, 52.68, 52.50, 45.24, 26.14. MS m/z: 384.6 [M+H]+. Anal. Calcd for C23H33N3O2: C,
72.03; H, 8.67; N, 10.96; O, 8.34. Found: C, 72.13; H, 8.77; N, 10.53; O, 8.14.
4.1.3.18. 4-(4-benzylpiperazin-1-yl)-N-(3,4-dimethoxybenzyl)butan-1-amine (6f)
Intermediate 4f was treated with 3,4-dimethoxybenzaldehyde according to general procedure
1
to afford the desired product 6f as light yellow oil (76% yield). H NMR (400 MHz, CDCl3):
δ 7.34-7.20 (m, 5H), 6.92-6.76 (m, 3H), 4.55 (s, 1H), 3.84 (s, 3H), 3.81(s, 3H), 3.69 (s, 2H),
3.46 (s, 2H), 2.82-2.31 (m, 12H), 1.67-1.50 (m, 2H), 1.49-1.20 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ 148.79, 147.82, 137.95, 132.94, 129.10, 128.06, 126.88, 120.04, 111.21, 110.82,
64.58, 62.96, 58.42, 55.78, 53.70, 53.08, 49.17, 45.91, 27.94, 24.63. MS m/z: 398.6 [M+H]+.
Anal. Calcd for C24H35N3O2: C,72.51; H, 8.87; N, 10.57; O, 8.05. Found: C,72.59; H, 8.49;
N, 10.37; O, 8.22.
4.1.3.19. 4-(((2-(4-benzylpiperazin-1-yl)ethyl)amino)methyl)-2-methoxyphenol (6g)
Intermediate 4d was treated with 4-hydroxy-3-methoxybenzaldehyde according to general
1
procedure to afford the desired product 6g as light yellow oil (70% yield). H NMR (400
MHz, CDCl3): δ 7.25-7.18 (m, 5H), 6.76-6.66 (m, 3H), 4.50 (s, 1H), 3.75 (s, 3H), 3.68 (s,
2H), 3.43 (s, 2H), 2.67 (t, J = 6.24, 2H), 2.47 (t, J = 6.56, 2H), 2.39 (br, s, 8H).. 13C NMR
(100 MHz, CDCl3): δ 147.05, 145.08, 137.67, 130.98, 129.15, 128.06, 126.94, 120.84,
114.68, 111.06, 62.88, 57.14, 55.49, 53.51, 52.90, 45.04. MS m/z: 356.7 [M+H]+. Anal.
Calcd for C21H29N3O2: C, 70.95; H, 8.22; N, 11.82; O, 9.0. Found: C, 70.85; H, 8.29; N,
11.68; O, 9.09.
4.1.3.20. 4-(((3-(4-benzylpiperazin-1-yl)propyl)amino)methyl)-2-methoxyphenol (6h)
Intermediate 4e was treated with 4-hydroxy-3-methoxybenzaldehyde according to general
1
procedure to afford the desired product 6h as light yellow oil (73% yield). H NMR (400
MHz, CDCl3): δ 7.12-7.04 (m, 5H), 6.73-6.52 (m, 3H), 5.49 (br, 1H), 4.38 (s, 1H), 3.58 (s,
3H), 3.52 (s, 2H), 3.29 (s, 2H), 2.70 (t, J = 5.12, 2H), 2.47-2.17 (m, 10H), 1.54-1.48 (m, 2H).
13C NMR (100 MHz, CDCl3): δ 147.28, 145.99, 138.20, 135.57, 129.47, 128.69, 127.61,
120.45, 115.38, 110.77, 65.30, 64.01, 63.48, 56.19, 53.93, 49.13, 45.93. MS m/z: 370.6
[M+H]+. Anal. Calcd for C22H31N3O2: C, 71.51; H, 8.46; N, 11.37; O, 8.66. Found: C, 71.72;
H, 8.23; N, 10.77; O, 8.56.
19

4.1.3.21. 4-(((4-(4-benzylpiperazin-1-yl)butyl)amino)methyl)-2-methoxyphenol (6i)
Intermediate 4f was treated with 4-hydroxy-3-methoxybenzaldehyde according to general
1
procedure to afford the desired product 6i as light yellow oil (76% yield). H NMR (400
MHz, CDCl3): δ 7.25-7.19 (m, 5H), 6.74-6.68 (m, 3H), 4.61 (br, 1H), 4.51 (s, 1H), 3.73 (s,
3H), 3.64 (s, 2H), 3.44 (s, 2H), 2.60 (t, J = 6.72, 2H), 2.42 (br, s, 8H), 2.29 (t, J = 6.72, 2H),
1.48-1.39 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 146.92, 145.03, 137.77, 131.08, 129.15,
128.06, 126.93, 120.89, 114.72, 111.13, 64.75, 62.88, 58.29, 55.54, 53.60, 52.93, 48.89,
27.57, 24.41. MS m/z: 384.5 [M+H]+. Anal. Calcd for C23H33N3O2: C, 72.03; H, 8.67; N,
10.96; O, 8.34. Found: C, 72.53; H, 8.70; N, 10.04; O, 8.49.
4.1.3.22. 2-(4-benzylpiperazin-1-yl)-N-(pyridin-3-ylmethyl)ethanamine (6j)
Intermediate 4d was treated with 3-pyridinecarboxaldehyde according to general procedure to
1
afford the desired product 6j as light yellow oil (75% yield). H NMR (400 MHz, CDCl3): δ
8.45-8.35 (m, 2H), 7.60-7.54 (m, 1H), 7.20-7.11 (m, 6H), 4.57 (s, 1H), 3.72 (s, 2H), 3.38 (s,
2H), 2.58 (t, J = 5.84, 2H), 2.39 (t J = 5.88, 2H), 2.34 (br, s). 13C NMR (100 MHz, CDCl3): δ
149.17, 147.97, 137.59, 135.58, 134.37, 128.86, 127.84, 126.70, 123.09, 62.65, 57.17, 52.78,
52.63, 50.80, 45.24. MS m/z: 311.5 [M+H]+. Anal. Calcd for C19H26N4 : C, 73.51; H, 8.44;
N, 18.05. Found: C, 73.67; H, 8.14; N, 18.30.
4.1.3.23. 3-(4-benzylpiperazin-1-yl)-N-(pyridin-3-ylmethyl)propan-1-amine (6k)
Intermediate 4e was treated with 3-pyridinecarboxaldehyde according to general procedure to
afford the desired product 6k as light yellow oil (65% yield). 1H NMR (400 MHz, CDCl3): δ
8.47-8.38 (m, 2H), 7.66-7.59 (m, 1H), 7.24-7.18 (m, 5H), 4.61 (s, 1H), 3.69 (s, 2H), 3.44-
3.35 (m, 2H), 2.79 (t, J = 5.48, 2H), 2.59 (t, J = 6.6, 2H), 2.40-2.30 (m, 8H), 1.63-1.60 (m,
2H). 13C NMR (100 MHz, CDCl3): δ 150.08, 148.16, 137.57, 137.03, 134.69, 129.10, 128.03,
126.92, 123.30, 63.42, 61.69, 53.97, 52.76, 51.04, 45.57, 30.76. MS m/z: 325.5 [M+H]+.
Anal. Calcd for C20H28N4 : C, 74.03; H, 8.70; N, 17.27. Found: C, 74.32; H, 8.31; N, 16.77.
4.1.3.24. 4-(4-benzylpiperazin-1-yl)-N-(pyridin-3-ylmethyl)butan-1-amine (6l)
Intermediate 4f was treated with 3-pyridinecarboxaldehyde according to general procedure to
afford the desired product 6l as light yellow oil (72% yield). 1H NMR (400 MHz, CDCl3): δ
8.43 (s, 1H), 8.38 (d, J = 5.12, 1H), 7.57 (d, J = 5.16, 1H), 7.21-7.12 (m, 5H), 4.57 (s, 1H),
20

3.68 (s, 2H), 3.39 (s, 2H), 2.89-2.16 (m, 12H), 1.59-1.33 (m, 4H). 13C NMR (100 MHz,
CDCl3): δ 149.31, 148.06, 137.72, 135.57, 128.94, 127.90, 126.73, 123.11, 62.76, 58.15,
52.87, 52.70, 50.94, 48.94, 27.69, 24.33. MS m/z: 339.4 [M+H]+. Anal. Calcd for C21H30N4:
C, 74.51; H, 8.93; N, 16.55. Found: C, 74.58; H, 8.43; N, 16.11.
4.1.3.25. 2-(4-benzhydrylpiperazin-1-yl)-N-(3,4,5-trimethoxybenzyl)ethanamine (7a)
Intermediate 4g was treated with 3,4,5-trimethoxybenzaldehyde according to general
1
procedure to afford the desired product 7a as light yellow oil (76% yield). H NMR (400
MHz, CDCl3): δ 7.40 (d, J = 7.32 Hz, 2 x 2H), 7.25 (t, J = 7.32 Hz, 2 x 2H), 7.16 (t, J = 7.32
Hz, 2H), 6.57 (s,1H), 6.52 (s,1H), 4.58 (s, 1H), 4.19 (s, 1H), 3.81 (s, 6H), 3.79 (s, 3H), 3.70
(s, 2H), 3.79 (s, 2H), 2.69 (t, J = 6.4, 2H), 2.50 (t, J = 6.12, 2H), 2.42 (br, s, 8H). 13C NMR
(100 MHz, CDCl3): δ 153.09, 142.60, 136.12, 128.36, 127.83, 126.81, 104.81, 103.60, 76.18,
65.05, 57.62, 55.98, 54.23, 53.40, 51.8, 45.64. MS m/z: 476.7 [M+H]+. Anal. Calcd for
C29H37N3O3: C,73.23; H,7.84; N, 8.83; O,10.09. Found: C,73.61; H,7.40; N, 8.33; O, 10.13.
4.1.3.26. 3-(4-benzhydrylpiperazin-1-yl)-N-(3,4,5-trimethoxybenzyl)propan-1-amine (7b)
Intermediate 4h was treated with 3,4,5-trimethoxybenzaldehyde according to general
1
procedure to afford the desired product 7b as light yellow oil (72% yield). H NMR (400
MHz, CDCl3): δ 7.38 (d, J = 7.36 Hz, 2 x 2H), 7.25 (t, J = 7.32 Hz, 2 x 2H), 7.14 (t, J = 7.32
Hz, 2H), 6.56 (s,1H), 6.51 (s,1H), 4.56 (s, 1H), 4.16 (s, 1H), 3.79 (d, J = 4.36, 9H), 3.66 (s,
2H), 2.82 (t, J = 5.12, 2H), 2.40 (br, s, 4H), 2.38 (t, J = 7.32, 2H), 2.31 (br, s, 4H), 1.68-1.64
(m, 2H). 13C NMR (100 MHz, CDCl3): δ 153.04, 142.56, 142.40, 136.77, 128.26, 127.76,
126.71, 103.37, 76.52, 64.56, 55.84, 53.32, 52.98, 51.71, 45.91, 26.64. MS m/z: 490.8
[M+H]+. Anal. Calcd for C30H39N3O3: C, 73.59; H, 8.03; N, 8.58; O, 9.80. Found: C, 73.95;
H, 8.14; N, 8.68; O, 9.75.
4.1.3.27. 4-(4-benzhydrylpiperazin-1-yl)-N-(3,4,5-trimethoxybenzyl)butan-1-amine (7c)
Intermediate 4i was treated with 3,4,5-trimethoxybenzaldehyde according to general
1
procedure to afford the desired product 7c as light yellow oil (75% yield). H NMR (400
MHz, CDCl3): δ 7.41 (d, J = 7.36 Hz, 2 x 2H), 7.27 (t, J = 7.32 Hz, 2 x 2H), 7.17 (t, J = 7.32
Hz, 2H), 6.59 (s,1H), 6.54 (s,1H), 4.61 (s, 1H), 4.12 (s, 1H), 3.85 (d, J = 5.88, 9H), 3.82 (s,
2H), 2.88 (t, J = 5.88, 2H), 2.68 (t, J = 6.96, 2H), 2.43 (br, s, 4H), 2.42 (t, J = 7.32, 2H), 2.34
13
(br, s, 4H), 1.71 (t, J = 6.6, 2H), 1.42-1.34 (m, 2H). C NMR (100 MHz, CDCl3): δ 153.12,
21

142.72, 142.61, 136.19, 128.38, 127.12, 126.81, 104.76, 103.62, 76.29, 64.74, 56.94, 56.02,
54.25, 53.50, 51.90, 48.18, 46.22, 26.97. MS m/z: 504.6 [M+H]+. Anal. Calcd for
C31H41N3O3: C, 73.92; H, 8.20; N, 8.34; O, 9.53. Found: C, 72.92; H, 8.11; N, 8.82; O, 9.09.
4.1.3.28. 2-(4-benzhydrylpiperazin-1-yl)-N-(3,4-dimethoxybenzyl)ethanamine (7d)
Intermediate 4g was treated with 3,4-dimethoxybenzaldehyde according to general procedure
to afford the desired product 7d as light yellow oil (70% yield). 1H NMR (400 MHz, CDCl3):
δ 7.29 (d, J = 7.32 Hz, 2 x 2H), 7.12 (t, J = 8.04 Hz, 2 x 2H), 7.02 (t, J = 8.08 Hz, 2H),6.80-
6.61 (m, 3H), 4.44 (s, 1H), 4.06 (s, 1H), 3.68 (d, J = 5.12, 3H), 3.64 (d, J = 5.12, 3H ), 3.54
(s, 2H), 2.52 (t, J = 5.88, 2H), 2.34 (t, J = 6.6, 2H), 2.27 (br, s, 8H). 13C NMR (100 MHz,
CDCl3): δ 148.90, 147.82, 142.61, 133.91, 132.80, 128.32, 127.78, 126.77, 120.09, 110.83,
76.15, 64. 71, 57.51, 55.79, 55.69, 53.58, 53.31, 51.79, 45.37. MS m/z: 446.6 [M+H]+. Anal.
Calcd for C28H35N3O2: C, 75.47; H, 7.92; N, 9.43; O,7.18. Found: C, 75.53; H, 7.80; N, 9.48;
O, 7.08.
4.1.3.29. 3-(4-benzhydrylpiperazin-1-yl)-N-(3,4-dimethoxybenzyl)propan-1-amine (7e)
Intermediate 4h was treated with 3,4-dimethoxybenzaldehyde according to general procedure
1
to afford the desired product 7e as light yellow oil (75% yield). H NMR (400 MHz, CDCl3):
δ 7.38 (d, J = 7.36 Hz, 2 x 2H), 7.24 (t, J = 7.36 Hz, 2 x 2H), 7.13 (t, J = 6.60 Hz, 2H),6.90 (s,
1H), 6.84 (d, J = 8.8, 1H), 6.78 (d, J = 8.08, 1H), 4.54 (s, 1H), 4.14 (s, 1H), 3.82 (d, J = 4.4,
8H), 2.79 (t, J = 5.12, 2H), 2.61 (t, J = 6.6, 2H), 2.38-2.31 (m, 8H), 1.63-1.52 (m, 2H). 13C
NMR (100 MHz, CDCl3): δ 149.64, 148.99, 143.40, 134.66, 129.09, 128.49, 127.54, 119.86,
111.61, 111.02, 77.00, 65.45, 57.59, 56.53, 56.42, 54.17, 53.64, 52.46, 46.59, 27.10. MS m/z:
460.6 [M+H]+. Anal. Calcd for C29H37N3O2: C, 75.78; H, 8.11; N, 9.14; O, 6.96. Found: C,
75.28; H, 8.20; N, 9.19; O, 6.76.
4.1.3.30. 4-(4-benzhydrylpiperazin-1-yl)-N-(3,4-dimethoxybenzyl)butan-1-amine (7f)
Intermediate 4i was treated with 3,4-dimethoxybenzaldehyde according to general procedure
1
to afford the desired product 7e as light yellow oil (72% yield). H NMR (400 MHz, CDCl3):
δ 7.24 (d, J = 6.6 Hz, 2 x 2H), 7.06 (t, J = 6.6 Hz, 2 x 2H), 6.96 (t, J = 6.6 Hz, 2H), 6.78-6.56
(m, 3H), 4.41 (s, 1H), 4.0 (s, 1H), 3.65 (d, J = 4.33, 6H), 3.52 (s, 2H), 2.58-2.13 (m, 12H),
1.79-1.67 (m, 2H), 1.45-1.24 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 148.82, 147.90,
142.63, 132.44, 128.31, 127.75, 126.75, 120.13, 111.37, 110.24, 76.15, 64.62, 58.34, 55.75,
22

53.53, 51.66, 49.03, 27.79, 24.53. MS m/z: 474.7 [M+H]+. Anal. Calcd for C30H39N3O2: C,
76.07; H, 8.30; N; 8.87; O, 6.76. Found: C, 76.16; H, 8.39; N; 8.28; O, 6.51.
4.1.3.31. 4-(((2-(4-benzhydrylpiperazin-1-yl)ethyl)amino)methyl)-2-methoxyphenol (7g)
Intermediate 4g was treated with 4-hydroxy-3-methoxybenzaldehydeaccording to general
1
procedure to afford the desired product 7g as light yellow oil (76% yield). H NMR (400
MHz, CDCl3): δ 7.41 (d, J = 7.32 Hz, 2 x 2H), 7.27 (t, J = 7.32 Hz, 2 x 2H), 7.18 (t, J = 7.32,
2H), 6.80-6.67 (m, 3H), 4.55 (s, 1H), 4.21 (s, 1H), 3.80 (s, 3H), 3.71 (s, 2H), 2.70 (t, J =
6.24, 2H), 2.52 (t, J = 5.88, 2H), 2.44 (br, s, 8H). 13C NMR (100 MHz, CDCl3): δ 146.84,
145.02, 142.66, 128.41, 127.84, 126.86, 121.13, 114.32, 111.02, 76.18, 55.79, 53.37, 51.77,
44.92. MS m/z: 432.8 [M+H]+. Anal. Calcd for C27H33N3O2: C, 75.14; H, 7.71; N, 9.74; O,
7.41. Found: C, 75.11; H, 7.49; N, 9.62; O, 7.35.
4.1.3.32. 4-(((3-(4-benzhydrylpiperazin-1-yl)propyl)amino)methyl)-2-methoxyphenol (7h)
Intermediate 4h was treated with 4-hydroxy-3-methoxybenzaldehyde according to general
1
procedure to afford the desired product 7h as light yellow oil (72% yield). H NMR (400
MHz, CDCl3): δ 7.41 (d, J = 7.32 Hz, 2 x 2H), 7.29-7.13 (m, 6H), 6.89-6.70 (m, 3H), 5.26
(br,1H), 4.55 (s, 1H), 4.20 (s, 1H), 3.83 (s, 3H), 3.77 (s, 2H), 2.88 (t, J = 5.12, 2H), 2.65 (t, J
= 7.32, 2H), 2.44-2.26 (m,8H), 1.69-1.66 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 142.75,
142.58, 131.84, 128.39, 127.92, 126.82, 120.83, 114.44, 110.93, 76.27, 55.74, 53.47, 53.23,
51.85, 46.09, 34.86. MS m/z: 446.7 [M+H]+. Anal. Calcd for C28H35N3O2: C, 75.47; H, 7.92;
N, 9.43, O, 7.18. Found: C, 75.22; H, 7.72; N, 9.47, O, 7.04.
4.1.3.33. 4-(((4-(4-benzhydrylpiperazin-1-yl)butyl)amino)methyl)-2-methoxyphenol (7i)
Intermediate 4i was treated with 4-hydroxy-3-methoxybenzaldehyde according to general
1
procedure to afford the desired product 7i as light yellow oil (68% yield). H NMR (400
MHz, CDCl3): δ 7.39 (d, J = 7.32 Hz, 2 x 2H), 7.21 (t, J = 7.36, 2 x 2H), 7.14 (t, J = 7.32,
2H), 6.79-6.68 (m, 3H), 4.55 (s, 1H), 4.16 (s, 1H), 3.71 (s, 3H), 3.65-3.56 (m, 2H), 2.59-2.30
(m, 12H), 1.48-1.22 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 148.13, 146.17, 143.57,
131.83, 129.20, 128.62, 127.61, 121.71, 115.77, 112.12, 77.0, 59.19, 56.3, 54.49, 54.17,
52.46, 49.75, 46.65, 28.45, 25.23. MS m/z: 460.1[M+H]+. Anal. Calcd for C29H37N3O2: C,
75.78; H, 8.11; N, 9.14; O, 6.96. Found: C, 75.65; H, 8.30; N, 9.44; O, 5.96.
4.1.3.34. 2-(4-benzhydrylpiperazin-1-yl)-N-(pyridin-3-ylmethyl)ethanamine (7j)
23

Intermediate 4g was treated with 3-pyridinecarboxaldehyde according to general procedure to
1
afford the desired product 7j as light yellow oil (76% yield). H NMR (400 MHz, CDCl3): δ
8.54 (s, 1H), 8.49-8.43 (m, 1H), 7.69 (dd, J = 8.04, 3.68 1H), 7.40 (d, J = 7.32 Hz, 2 x 2H),
7.28 (t, J = 8.04, 2 x 2H), 7.16 (t, J = 7.32, 2H), 7.21-7.18 (m, 1H), 4.66 (s, 1H), 4.21 (s, 1H),
3.70 (s, 2H), 2.66 (t, J = 5.84, 2H), 2.49 (t, J = 6.6, 2H), 2.43 (br, s, 8H). 13C NMR (100
MHz, CDCl3): δ 149.38, 148.32, 142.58, 135.72, 134.59, 128.29, 127.75, 126.74, 123.27,
76.09, 57.43, 53.22, 51.05, 51.73, 45.50. MS m/z: 387.5 [M+H]+. Anal. Calcd for C25H30N4:
C, 77.68; H, 7.82; N, 14.49. Found: C, 76.88; H, 7.32; N, 14.53.
4.1.3.35. 3-(4-benzhydrylpiperazin-1-yl)-N-(pyridin-3-ylmethyl)propan-1-amine (7k)
Intermediate 4h was treated with 3-pyridinecarboxaldehyde according to general procedure to
afford the desired product 7k as light yellow oil (72% yield). 1H NMR (400 MHz, CDCl3): δ
8.54-8.41 (m, 2H), 8.50 (dd, J = 9.52, 5.16, 1H), 7.66 (d, J = 8.04, 1H), 7.39-7.10 (m, 11H),
4.61 (s, 1H), 4.20 (s, 1H), 3.71 (s, 2H), 2.82 (t, J = 4.4, 2H), 2.62-2.22 (m, 10H), 1.65-1.62
(m, 2H). 13C NMR (100 MHz, CDCl3): δ 149.12, 148.89, 143.50, 136.35, 135.32, 129.41,
128.69, 127.60, 77.43, 57.68, 54.28, 53.99, 52.65, 46.91, 27.59. MS m/z: 401.7 [M+H]+.
Anal. Calcd for C26H32N4: C, 77.96; H, 8.05; N, 13.99. Found: C, 77.66; H, 8.10; N, 13.72.
4.1.3.36. 4-(4-benzhydrylpiperazin-1-yl)-N-(pyridin-3-ylmethyl)butan-1-amine (7l)
Intermediate 4i was treated with 3-pyridinecarboxaldehyde according to general procedure to
afford the desired product 7l as light yellow oil (73% yield). 1H NMR (400 MHz, CDCl3): δ
8.50 (s, 1H), 8.43 (dd, J = 9.52, 4.4, 1H), 7.60 (d, J = 7.36, 1H), 7.41 (d, J = 7.32, 4H), 7.24-
7.11 (m,7H), 4.63 (s, 1H), 4.20 (s, 1H), 3.73 (s, 2H), 2.50-2.22 (m, 12H), 1.63-1.42 (m, 4H).
13C NMR (100 MHz, CDCl3): δ 149.02, 147.71, 142.24, 135.30, 134.09, 127.93, 127.38,
126.36, 122.86, 75.70, 57.91, 52.94, 51.32, 50.64, 48.70, 27.46, 24.05. MS m/z: 415.6
[M+H]+. Anal. Calcd for C27H34N4: C, 78.22; H, 8.27; N, 13.51. Found: C, 78.16; H, 8.20; N,
13.38.
4.2. Biological activity
4.2.1. In vitro inhibition of AChE and BuChE
The inhibitory potency of target compounds on AChE and BuChE was determined using
spectroscopic method of Ellman et al.31 and was expressed as IC50 i.e. inhibitory
concentration that reduces the cholinesterase activity by 50%. Acetylcholinesterase (AChE,
24

E.C. 3.1.1.7, from electric eel), butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine
serum), 5,5'- dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acetylthiocholine
iodide (ATC), butyryllthiocholine iodide (BTC), donepezil was purchased from Sigma
Aldrich. Stock solutions of tested compounds (10 mM) were prepared in ethanol and diluted
using 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0) to afford a final concentration range between
(0.001-100) µM. Enzyme solutions were prepared by dissolving lyophilized powder in
double-distilled water. The assay solution consisted of 1 ml of 0.1 M KH2PO4/K2HPO4
phosphate buffer, 25µl of AChE (0.22 U/ml) or 25 µl of BuChE (0.06 U/ml) and 100 µl of
various concentrations of test compounds which was allowed to stand for 5 min before 100 µl
of 0.01 M DTNB were added. A positive control of donepezil was used in the same range of
concentrations. The reaction was started by addition of 20 µl of the 0.075 M substrate
solution (ATC/BTC) and exactly 2 min after substrate addition the absorption was measured
at 25 °C at 412 nm. Assays were carried out with a blank containing all components except
AChE or BuChE in order to account for non-enzymatic reaction. Each concentration was
analysed in triplicate at 25°C. Data from concentration-inhibition experiments of the
inhibitors were calculated by nonlinear regression analysis, using the Graph Pad Prism 5
program.
4.2.2. Kinetic characterization of AChE inhibition
To obtain kinetic study on the mechanism of AChE inhibition by tested compound, a
reciprocal plot of 1/velocity versus 1/substrate were constructed at relatively low
concentrations of substrate by using Ellman’s method.31 Three concentrations of compound
were selected for this study: 2, 6, and 10 nM. Vmax and Km values of the Michaelis-Menten
kinetics were calculated by nonlinear regression from substrate−velocity curves using Graph
Pad Prism 5. Linear regression was used for calculating the Lineweaver-Burk plots.
4.2.3. Molecular docking studies
To understand the binding interactions and selectivity of our synthesized compounds against
two ChEs, molecular docking simulation was carried out with AUTODOCK 4.2.37 The
crystal structure of AChE complexed with E2020 (code ID: 1EVE; resolution 2.5-Å) and
BuChE complexed with Echothiophate (code ID: 1POI resolution 2.0-Å) used in the docking
study were obtained from the Protein Data Bank (http://www.rcsb.org/pdb). The original
inhibitor, heteroatoms and water molecules in the PDB files were eliminated in the beginning
25

of docking study. The enzyme was set up for docking with standard protocol. ADT; version
1.5.4, was used to add polar hydrogen atoms to amino acids residues and for assigning
Gasteiger charges to all atoms of the enzyme. 3D structures of the AChEI donepezil and
synthesized compounds were generated and energy optimized by Discovery Studio 2.5
package (Accelrys Inc., San Diego, CA) employing CHARMm force field. Full hydrogens
were added to the ligands and Gasteiger-Marsili partial atomic charges were caculated using
the BABEL-2.2.338 program and saved in the PDBQT format for further analysis. All
possible rotatable bonds of the resultant ligand molecules were defined by using standerd
protocol. The prepared PDBQT format of ligands were used as input files for AutoDock 4.2
subsequently. The docking simulation was performed using the Lamarckian Genetic
Algorithm.39 To analyse binding energy in the docking step, three terms were considered, the
van der Waals interaction represented as a Lennard-Jones 12–6 dispersion/repulsion term, the
hydrogen bonding represented as a directional 12–10 term, and the Coulombic electrostatic
potential. The resultant docking orientations within 2.0 Å in root mean square deviation
(rmsd) in tolerence of each other were clustered together to represent the most favourable
free energy of binding. Finally, the top-posed docking conformations were submitted to post-
docking energy minimization on Discovery Studio 2.5. The resultant structure files were
evaluated using PyMOL visualization program.40
4.2.4. Oxygen Radical Absorbance Capacity (ORAC-FL) Assay
The antioxidant activity was determined based on the oxygen radical absorbance capacity-
fluorescein (ORAC-FL) assay.41,42 2,2′-Azobis- (amidinopropane) dihydrochloride (AAPH),
( )-6-hydroxy-2,5,7,8- tetramethylchromane-2-carboxylic acid (trolox), and fluorescein (FL)
were purchased from Sigma-Aldrich. The reaction was carried out in 75 mM phosphate
buffer (pH 7.4), and the final reaction mixture was 200 µl. Antioxidant (20 µl) and FL (120
µl; 70 mM, final concentration) solutions were placed in a black 96-well microplate (96F
untreat, Nunc). The mixture was preincubated for 15 min at 37 °C, and then AAPH solution
(60 µl, 12 mM, final concentration) was added rapidly using a multichannel pipette. The
microplate was immediately placed in the reader and the fluorescence recorded every minute
for 80 min (excitation, 485 nm; emission, 520 nm). Samples were measured at ten different
concentrations (1-10 µM). A blank (FL + AAPH) using phosphate buffer instead of the tested
compound and eight calibration solutions using Trolox (1-8 µM, final concentration) as
antioxidant were also carried out in each assay. All reaction mixtures were prepared in
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duplicate, and at least three independent runs were performed for each sample. The
antioxidant curves (fluorescence versus time) were normalized to the curve of the blank. The
area under the fluorescence decay curve (AUC) was calculated using following equation:
i = 80
AUC = 1 + ∑ (fi/f0)
i = 1
where f 0 is the initial fluorescence reading at 0 min and f i is the fluorescence reading at time
i. The net AUC corresponding to a sample was calculated by subtracting the AUC
corresponding to the blank. Regression equations between net AUC and antioxidant
concentration were calculated for all the samples. ORAC-FL values were expressed as trolox
equivalents by using the standard curve calculated for each assay, where the ORAC-FL value
of trolox was taken as 1.
4.2.5. Self-mediated Aβ1–42 aggregation assay
Commercially available peptides were first treated with hexafluoroisopropanol (HFIP) at
5mg/ml to avoid self-aggregation. The clear solution containing the dissolved peptide was
then aliquoted in microcentrifuge tube. The HFIP was allowed to evaporate under a stream of
nitrogen until a clear film remained in the test tube. The pretreated Aβ1-42 samples were then
dissolved in DMSO in order to have a stable stock solution (Aβ = 5 mM). For the inhibition
of self-mediated Aβ1–42 aggregation experiment, the Aβ stock solution was diluted with 50
mM phosphate buffer (pH 7.4) to 50 µM before use. A mixture of the peptide (10 µl, 50 µM,
final concentration) with or without the tested compound (25 µM) was incubated at 37oC for
48 h. To quantify amyloid fibril formation, the thioflavin-T fluorescence method was
used.43,44 Blanks containing 50 mM phosphate buffer (pH 7.4) instead of Aβ with or without
inhibitors were also carried out. After incubation, samples were diluted to a final volume of
200 µl with 50 mM glycine–NaOH buffer (pH 8.0) containing thioflavin-T (5 µM). Then the
fluorescence intensities were recorded five minutes later (excitation, 450 nm; emission, 485
nm). The percent inhibition of aggregation was calculated by the expression (1-IFi/IFc) ×
100% in which IFi and IFc are the fluorescence intensities obtained for Aβ in the presence
and absence of inhibitors after subtracting the background, respectively. Each measurement
was run in triplicate.
4.2.6. ADME property prediction
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To analyze the potential of the new compounds as anti-AD agents, their ADME (Absorption,
Distribution, Metabolism and excretion) properties were predicted using QikProp program, v.
3.5 of the Schrödinger software.34 This gave an estimate of the physicochemical properties
and the bioavailability of the compounds. Parameters such as polar surface area (PSA),
solvent accessible surface area (SASA), CNS activity (Predicted central nervous system
activity on a -2 (inactive) to +2 (active) scale), QPPCaco (Predicted apparent Caco-2 cell
permeability in nm/s. Caco-2 cells is a model for the gut blood barrier), QPlogBB (Predicted
brain/blood partition coefficient), QPPMDCK (Predicted apparent MDCK cell permeability
in nm/s. MDCK cells are considered to be a good mimic for the blood–brain barrier),
QPLogKhsa (Prediction of binding to human serum albumin), QPlogS (Predicted aqueous
solubility) and percent human oral absorption (Predicted human oral absorption on 0–100%
scale) were calculated. The acceptability of the compounds based on the Lipinski’s rule of
five45 were also estimated from the results. Besides, targeted compounds were analysed for
toxicity prediction such as mutagenic, tumorigenic, irritant and reproductive effects using
OSIRIS property explorer.46
4.2.7. PASS prediction
Prediction of biological activity spectra for selected compounds with potent ChEs inhibitiory
activity was perform by PASS (available at http //pass.ibmh.msk.su/). This web based tool
predicts a spectrum of biological activities based on structure-activity relationships of the
training set containing more than 260,000 compounds with known biological activities and
for each activity, it gives two values namely probable activity (Pa) and probable inactivity
(Pi).47,48
5. Conflict of Interest
The authors declare that there is no conflict of interest.
Acknowledgements
The author Poonam Meena wishes to acknowledge the Rajiv Gandhi National Fellowship
received from the University Grants Commission. The author Manisha Tiwari wishes to
acknowledge the financial assistance received from the Department of Science & Technology
and the facilities provided by University of Delhi. The USIC-CIF, University of Delhi is
acknowledged for providing access to NMR facilitiy. The authors wish to acknowledge the
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facilities provided by ACBR including the Bioinformatics facility. The authors also thank
Mr. R. Raghu, vice president, Schrödinger, India, for providing valuable suggestions
regarding QikProp module. Scientific contributions from Prof. Vani Brahmachari are
gratefully acknowledged.
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