Synthesis of α, β-unsaturated carbonyl based compounds as acetylcholinesterase and butyrylcholinesterase inhibitors: Characterization, molecular modeling, QSAR studies and effect against amyloid β-induced cytotoxicity

Article abstract of DOI:10.1016/j.ejmech.2014.06.034

A series of novel carbonyl compounds was synthesized by a simple, eco-friendly and efficient method. These compounds were screened for anti-oxidant activity, in vitro cytotoxicity and for inhibitory activity for acetylcholinesterase and butyrylcholinesterase. The effect of these compounds against amyloid βinduced cytotoxicity was also investigated. Among them, compound 14 exhibited strong free radical scavenging activity (18.39 μM) while six compounds (1, 3, 4, 13, 14, and 19) were found to be the most protective against Aβ-induced neuronal cell death in PC12 cells. Compounds 4 and 14, containing N-methyl-4-piperidone linker, showed high acetylcholinesterase inhibitory activity as compared to reference drug donepezil. Molecular docking and QSAR (Quantitative StructureeActivity Relationship) studies were also carried out to determine the structural features that are responsible for the acetylcholinesterase and butyrylcholinesterase inhibitory activity.

Full text of DOI:10.1016/j.ejmech.2014.06.034

Accepted Manuscript
Synthesis of α, β-Unsaturated Carbonyl Based Compounds as Acetylcholinesterase
and Butyrylcholinesterase Inhibitors: Characterization, Molecular Modeling, QSAR
Studies and Effect Against Amyloid β-Induced Cytotoxicity
Syed Nasir Abbas Bukhari, Ibrahim Jantan, Vijay H. Masand, Devidas T. Mahajan,
Muhammad Sher, M. Naeem-ul-Hassan, Muhammad Wahab Amjad, Oya Unsal Tan
PII:
S0223-5234(14)00553-4
10.1016/j.ejmech.2014.06.034
EJMECH 7077
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 February 2014
Revised Date: 12 June 2014
Accepted Date: 17 June 2014
Please cite this article as: S.N.A. Bukhari, I. Jantan, V.H. Masand, D.T. Mahajan, M. Sher, M. Naeem-
ul-Hassan, M.W. Amjad, O. Unsal Tan, Synthesis of α, β-Unsaturated Carbonyl Based Compounds as
Acetylcholinesterase and Butyrylcholinesterase Inhibitors: Characterization, Molecular Modeling, QSAR
Studies and Effect Against Amyloid β-Induced Cytotoxicity, European Journal of Medicinal Chemistry
(2014), doi: 10.1016/j.ejmech.2014.06.034.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis of α, β-Unsaturated Carbonyl Based Compounds as Acetylcholinesterase and
Butyrylcholinesterase Inhibitors: Characterization, Molecular Modeling, QSAR Studies
and Effect Against Amyloid β-Induced Cytotoxicity
,
Syed Nasir Abbas Bukhari, Ibrahim Jantan, Vijay H. Masand, Devidas T. Mahajan
Muhammad Sher, M.Naeem-ul-Hassan, Muhammad Wahab Amjad, Oya Unsal Tan

ACCEPTED MANUSCRIPT
Synthesis of α, β-Unsaturated Carbonyl Based Compounds as Acetylcholinesterase and
Butyrylcholinesterase Inhibitors: Characterization, Molecular Modeling, QSAR Studies
and Effect Against Amyloid β-Induced Cytotoxicity
Syed Nasir Abbas Bukhari, Ibrahim Jantan, Vijay H. Masand, Devidas T. Mahajan , Muhammad
Sher, M.Naeem-ul-Hassan, Muhammad Wahab Amjad, Oya Unsal Tan
Highlights:
•
•
•
•
We synthesized and characterized thirty novel molecules.
Compound 14 exhibited strong free radical scavenging activity (18.39µM).
Six compounds showed strong neuroprotective effect.
Compounds 4 and 14, containing N-methyl-4-piperidone linker, showed high
acetylcholinesterase inhibitory activity.

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Synthesis of α, β-Unsaturated Carbonyl Based Compounds as Acetylcholinesterase and
Butyrylcholinesterase Inhibitors: Characterization, Molecular Modeling, QSAR Studies
and Effect Against Amyloid β-Induced Cytotoxicity
Syed Nasir Abbas Bukharia*, Ibrahim Jantana*, Vijay H. Masandb, Devidas T. Mahajanb,
Muhammad Sherc, M.Naeem-ul-Hassanc, Muhammad Wahab Amjada, Oya Unsal Tand
a. Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan
Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia
b. Department of Chemistry, Vidya Bharati Mahavidyalaya, Amravati, Maharashtra, India-
444 602.
c. Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan.
d. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University,
Ankara 06100, Turkey
* Authors to whom correspondence should be addressed;
E mail: (SNAB) snab@pharmacy.ukm.my; snab_hussaini@yahoo.com
(IJ) ibj@pharmacy.ukm.my
Tel: +6-01123695295
Fax: +6-0326983271

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ABSTRACT
A series of novel carbonyl compounds was synthesized by a simple, eco-friendly and efficient
method. These compounds were screened for anti-oxidant activity, in vitro cytotoxicity and for
inhibitory activity for acetylcholinesterase and butyrylcholinesterase. The effect of these
compounds against amyloid β-induced cytotoxicity was also investigated. Among them,
compound 14 exhibited strong free radical scavenging activity (18.39µM) while six compounds
(1, 3, 4, 13, 14, and 19) were found to be the most protective against Aβ-induced neuronal cell
death in PC12 cells. Compounds 4 and 14, containing N-methyl-4-piperidone linker, showed
high acetylcholinesterase inhibitory activity as compared to reference drug donepezil. Molecular
docking and QSAR (Quantitative Structure-Activity Relationship) studies were also carried out
to determine the structural features that are responsible for the acetylcholinesterase and
butyrylcholinesterase inhibitory activity.
KEYWORDS: Alzheimer's disease, neuroprotection, oxidative stress, 4-piperidone, synthesis.

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1. INTRODUCTION
According to the World Alzheimer Report 2013, Alzheimer’s disease and other forms of
dementia are among the biggest global public health issues facing our generation. Currently, over
35 million people around the world live with these conditions and this number is likely to
increase threefold by 2050, to 115 million people [1]. Alzheimer’s disease (AD), the most
common form of dementia amongst the aged, is a deadly neurodegenerative disease categorized
by the loss of mental abilities and a diversity of neuropsychiatric symptoms and behavioral
disorders [2, 3]. Neurofibrillary tangles and amyloid β (Aβ) plaques are found in the brain. The
cholinergic system is predominantly vulnerable to synapse loss, particularly in cortical regions
related with memory and executive function [4]. Most treatment approaches to date have been
based on the cholinergic hypothesis which assumes that memory loss in patients suffering from
this disease result from a loss of cholinergic function in brain for cholinergic neurotransmission
is specifically affected in patients suffering from Alzheimer’s disease.
For the treatment of AD, one of the most common methods is to increase the
acetylcholine levels in brain with acetylcholinesterase inhibitors [5]. The best characterized
therapeutic effect of cholinesterase inhibitors (ChE) in Alzheimer disease patients is to maintain
cognitive function for a 1-year period in about 50% of the patients [6]. Two kinds of
cholinesterase enzymes are found in the central nervous system; acetylcholinesterase (AChE)
and butyrylcholinesterase (BuChE). Both enzymes are capable of hydrolyzing acetylcholine, but
AChE has a 1013-fold higher hydrolytic acetylcholine activity than BuChE, at the similar
temperature and pH [7]. In the healthy brain, BuChE is thought to play a trivial role in regulating
brain acetylcholine levels. In the AD brain, BuChE activity rises while AChE activity remains
unchanged or declines. Hence, both enzymes may contribute to regulating acetylcholine levels
and are appropriate therapeutic targets to enhance the cholinergic deficit. The two enzymes vary

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in location, substrate specificity and kinetics. The most recent studies suggest that BuChE might
also have a part in the etiology and development of AD beyond the regulation of synaptic
acetylcholine levels. Experimental evidence from the use of agents with improved selectivity for
BuChE and ChE, such as rivastigmine (having dual inhibitory action on both AChE and BuChE),
shows that there are possible therapeutic benefits of inhibiting both AChE and BuChE in AD and
associated dementias. The development of specific small molecule drugs BuChE inhibitors with
the capability to inhibit BuChE together with AChE should lead to better clinical outcomes [8].
In our previous studies, we synthesized and screened the series of novel chalcone
derivatives and curcumin analogues with α, β-carbonyl linkers for their activity against
phagocytic chemotaxis and the production of reactive oxygen species (ROS) from zymosan
stimulated human phagocytes. On the basis of these findings and a those of other scientists, it
was determined that chalcone derivatives and curcumin-like compounds bearing α, β-carbonyl
group have a wide diversity of pharmacological activities [9-14]. Our latest studies showed that a
range of compounds with a α, β-unsaturated moiety are capable of inhibiting both AChE and
BuChE and can be possible candidates for the treatment of Alzheimer’s disease [15, 16].
Numerous studies have documented increased protein oxidation [17] and reactive oxygen
species (ROS) formation [18] in the brain tissue of AD patients. Aβ is considered to be a cause
of lipid peroxidation in brain cell membranes that may contribute to neurodegeneration [19, 20].
Encouraged by these results, we continued our work in this area, and in the current study we
synthesized thirty novel compounds bearing the α, β-carbonyl moiety and have screened them
for their effects on the AChE/BuChE activities in addition to amyloid β-Induced cytotoxicity in
PC12 cells. Lastly, molecular modeling calculations and QSAR studies were performed to
understand the structural basis for the biological activity of compounds.

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2. RESULTS AND DISCUSSION
2.1. Chemistry
Thirty novel compounds of ten different types were synthesized in the current study as reported
previously [21, 22]. To synthesize the desired α, β-unsaturated carbonyl based compounds,
Claisen-Schmidt condensation was used [23] between different ketones and suitable aryl
aldehydes at a molar ratio 1:2, (twenty one compounds, 1-7; 11-17; 21-27) in the presence of
NaOH in ethanol. Some compounds were synthesized in acetic acid in the presence of dry HCl
gas. Nine compounds (8-10; 18-20; 28-30) resembling chalcone analogues were synthesized by
same Claisen-Schmidt condensation using molar ratio 1:1 of ketone and aldehyde (Scheme 1).
Eight compounds (7, 8, 10, 17, 18, 20, 27, 28) were synthesized using both types of catalytic
systems comprising NaOH and the dry HCl gas mixture. As compared to NaOH, the catalytic
system with HCl gas mixture produced compounds more efficiently with a higher yield. When
using NaOH, a mixture of numerous unidentified products was obtained requiring extensive
purification by column chromatography.
All synthesized new compounds were characterized by spectrophotometric techniques as
well as elemental analysis of C, H, and N, and melting points. Carbon nuclear magnetic
resonance spectra were also obtained. For compounds 3, 13 and 23, the NH absorption peaks
were not seen in the 1H NMR spectra of the compounds. Absence of NH absorption peaks is also
reported previously by other researchers [24, 25].
2.2. Antioxidant activity of α, β-unsaturated carbonyl based compounds
The DPPH (1,1-diphenyl-1-picrylhydrazyl) assay was performed to assess the compounds for
their antioxidant activity. The data of the antioxidant assay is summarized in Table 1. According
to the data, five compounds (1, 3, 4, 14 and 19) were found to possess potent DPPH radical

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scavenging activity with IC50 in the range of 18.39 to 22.99 µM. Compound 14, having a N-
methyl-4-piperidone linker and diethoxymethyl substitution at position 4 of aromatic rings,
showed the strongest antioxidant activity (18.39 µM), even more than the positive control
(ascorbic acid; 19.36 µM). Among all thirty compounds, only eleven compounds have poor
antioxidant activities, exhibiting IC50 values greater than 50µM.
2.3.Cytotoxicity of synthetic compounds in PC12 cells
To investigate the effect of the new compounds on cell viability, the MTT assay was conducted
on PC12 cells. The cells were incubated with varying concentrations (0.01-100 ꢀM) of the test
compounds for 24 h and under these conditions the α, β-unsaturated carbonyl based compounds
were nontoxic to PC12 cells at any of the concentrations tested.
2.4.Neuroprotective effect of synthetic compounds against Aβ-induced cell cytotoxicity
The MTT assay was performed to evaluate the effect of novel synthetic compounds on Aβ-
induced PC12 cell toxicity. The cells treated with Aβ exhibited significantly reduced cell
viability as compared to control cells (decreased cell viability by 48%). Pretreatment of cells
with the synthetic compounds protected against Aβ-induced cell death up to 87%, at a concentra-
tion of 100 ꢀM. Six compounds (1, 3, 4, 13, 14, 19) were the most protective against Aβ-induced
PC12 cell toxicity (Figure 1). As compared to the positive control selegiline, the protective effect
of the synthetic compounds was significantly higher with compound (14) being the best.
Amongst the tested compounds, all those possessing 2-nitro and 4-dimethylamine groups (11-20)
had extremely high protective activities, while a diethoxymethyl group at position 4 of the rings
(1-10) lead to significantly less protective activity. The presence of pyrolidine at position 4 of
compounds (21-30) decreased the protective activity against Aβ-induced cytotoxicity.
Compounds having a 4-piperidone (3, 13) and those possessing N-methyl-4-piperidone (4, 14)

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linker exhibited somewhat more protection as compared to all other compounds. These results
suggest that a few of the novel α, β-unsaturated carbonyl based compounds may reduce cell
damage caused by Aβ-induced cytotoxicity.
2.5. Acetylcholinesterase and butyrylcholinesterase inhibition activity
Evaluation of new compounds for their inhibitory effects on AChE and BChE was done by
Ellman’s method. The screening of novel α, β-unsaturated carbonyl based compounds against
AChE and BChE demonstrated that nearly all these were moderate to strongly active against
AChE (Table 1). The compounds were found to be less active on BChE but most inhibited AChE
to various extents. The IC50 values of the compounds 8, 9, 15-18, 21, 22, 25, 26, and 28-30 could
not be calculated owing to their weak inhibition potency, which did not surpass 50% at the
highest concentration (100 ꢀM). In contrast, the compounds 3, 4, 12-14, 23 and 24 exhibited IC50
values ranging from 0.042 to 9.52 ꢀM for inhibition of AChE. In addition, the compounds 3, 4,
13 and 14 showed substantial AChE inhibitory activity below 1µM, which is similar to that of
donepezil (0.062µM). The compounds 4 and 14, having N-methyl-4-piperidone linker, had a
better inhibitory potency than the reference drug donepezil. For the inhibition of BuChE,
compounds 3, 7 and 12 displayed IC50 below 20µM, but none was found more active than
donepezil (6.92 µM).
These findings encouraged us to explore the relationship between AChE inhibitory activity and
the chemical structures of the compounds. Ten varying types of ketones were used as linkers, but
only three types of linkers present in compounds (3, 4, 12, 13, 14, 23, and 24) were found to be
the most effective. The presence of tetrahydropyran-4-one linker displayed good inhibition but
only in the presence of 2-nitro and 4-dimethylamine substitution patterns (Compound 12). Table
1 shows that the compounds (4, 14, and 24) having N-methyl-4-piperidone linker were strong

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inhibitors of AChE. Synthetic α, β-unsaturated carbonyl based compounds possessing the 4-
piperidone moiety (3, 13, and 23) were also good inhibitors but relatively weaker than those with
4-piperidone linker. In an earlier study, Yalda et al. reported the synthesis of piperidone-grafted
novel mono- and bisspiro heterocyclic hybrids containing functionalized piperidine, pyrrolizine
and oxindole rings. The cholinesterase inhibitory activity of those cycloadducts revealed that
monospiripyrrolizines were more active with IC50 in the range of 3.36 to 20.07 µM than
dipolarophiles or bisspiropyrrolizines [26]. The current investigation shows that the increased
activity is the result of specific linkers, whereas substitution patterns on the linked aromatic rings
participated to lesser extent. The comparison amongst the three active compounds with same
linker (4, 14, 24) revealed that compound 4 with diethoxymethyl group at position 4 of the
aromatic rings showed more activity than compound 14 possessing a 2-nitro and 4-
dimethylamine substitution pattern. The introduction of pyrrolidine at position 4 of compound 24
reduced the inhibition of AChE. A similar like activity relationship was also observed amongst
compounds (3, 13 and 23), having 4-piperidone groups on the aromatic rings. The activities of
these compounds showed that the linkers derived from ketones during synthesis play key roles in
terms of inhibitory activity. Compounds (1-10) have similar substitution pattern, but only two
compounds (3, 4) with 4-piperidone and N-methyl-4-piperidone moieties are potent inhibitors.
Similar results were seen amongst other two series of synthetic compounds (11-20 and 21-30).
Contreras et al. reported the synthesis of novel compounds by using 1-benzyl-4-piperidone and a
conventional structure-activity relationship examination proposed that the presence of a central
pyridazine ring is vital for high AChE inhibition [27]. The data in our current investigation
support the significance of the piperidone moiety for the inhibition of AChE.

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2.6. Docking and QSAR analyses
Molecular docking analysis was performed to obtain better insight into mechanism of action of
the AChE inhibitors. The active site of AChE is located at the base of ~20Ao deep and narrow
gorge, from the surface of the enzyme and is composed by two subsites [28, 29]: (1) catalytic
esteratic site (CES) and (2) peripheral anionic site (PAS). The active site consists of Gln71-Tyr-
Val-Asp-Thr-Leu76, Gly82-Thr-Glu84, Trp86-Asn-Pro88, Tyr121, Leu130, Tyr133, Glu199,
Ser200, Glu202-Ser-Ala204, Trp279, Trp286, Phe295, Phe297, Glu327, Phe330, Tyr334,
Tyr337-Phe338, Tyr341, Trp439, His447-Gly-Tyr449, and Ile451. The mechanism of
acetylcholine (ACh) hydrolysis involves trapping of ACh to PAS, followed by the transfer of
ACh to the active site.
[Insert figure 2 and 3 here]
From figure 2 and 3, it is clear that the most active AChE inhibitor (Compound 4) interacts with
the receptor mainly due to hydrophobic and mild polar interactions. The close proximity of
compound 4 with Tyr334 and Trp279 is due to mild polar interactions, indicating that it binds
reversibly with the receptor.
QSAR analysis: For QSAR analysis, a myriad number of descriptors were calculated followed
by elimination of redundant descriptors using QSARINS. Then, GA-MLR (Genetic Algorithm-
Multilinear Regression) was performed to develop robust QSAR model. The best two parametric
QSAR model along with its statistical parameters is as following:
pIC50 = 126.3811 (±28.0863) -7.2606 (±2.3866) RDF090m -4.8896 (±2.0119) F01[C-N]

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Ntr = 14, Nex = 3, R2 = 0.8558, R2adj = 0.8296, CCCtr = 0.9223, F = 32.6359, Q2LOO = 0.7924,
CCCcv = 0.8897, R2ex = 0.7718, CCCex = 0.8578
The symbols have the usual meaning [30-33]. The high value of R2, R2adj, CCCtr, F, Q2
,
LOO
CCCcv and R2 indicates that the QSAR model is not only robust but also possesses good
ex
predictive ability [30-35]. Since the model is based on a data set of seventeen molecules only,
further improvements in the external predictive ability are also possible. From the QSAR model,
it is clear that activity has correlation with RDF090m (Radial Distribution Function - 9.0 /
weighted by atomic masses, RDF descriptors) and F01[C-N] (frequency of C - N at topological
distance of 01, a 2D frequency fingerprints descriptor). It is quite clear that the docking and the
QSAR analyses not only support each other but are complementary to each other also.
3. CONCLUSION
In the work described above, we report the synthesis, pharmacological evaluation and molecular
modeling of thirty novel α, β-unsaturated carbonyl based compounds. From docking and QSAR
analyses, it can be stated that lipophilicity steers the AChE activity which is additionally
supported by the hydrophobic interactions between the ligand and the receptor. We propose that
the free radical scavenging activity of these novel compounds is responsible for their
neuroprotective effects as it was seen that all strong antioxidant compounds possess more
protective effect against Aβ-induced PC12 cell death. The most potent AChE inhibitors in this
series correspond to N-methyl-4-piperidone and 4-piperidone moieties. The type of the
substituent at the aromatic rings in compounds does not appear to have a strong influence on the
inhibitory activity. Overall, the substitution of benzene rings by diethoxymethyl group at position
4 and presence of piperidone moiety imparts strong acetylcholinesterase inhibition. The multiple

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activities of these compounds suggest that they may be useful for the treatment of
neurodegenerative diseases such as AD that involve a loss of cholinergic neurons.
4. Experimental
4.1.Materials
All chemicals and reagents were purchased from Sigma-Aldrich, Merck and Acros Organics
(above 98% purity) and were used without additional purification. MTT [3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide] was procured from Sigma-Aldrich (St. Louis, MO,
USA). AChE, (E.C.3.1.1.7 from Electric Eel, 500 units), BChE, (E.C. 3.1.1.8, from horse serum,
1000 units) and donepezil hydrochloride were also purchased from Sigma–Aldrich. Aβ1-42 was
purchased from Bachem (Bubendorf, Switzerland). Potassium dihydrogen phosphate, 5,5'-
dithiobis-(2-nitrobenzoic acid (DTNB), potassium hydroxide, sodium hydrogen carbonate,
gelatine, acetylthiocholine iodide (ATC) and butrylthiocholine iodide (BTC) were supplied by
Fluka (Buchs, Switzerland). Spectrophotometric analyses were performed using a Shimadzu UV-
1700, UV–Vis spectrophotometer. Phosphate buffer (100mM, pH 8.0) at 25°C was used to
measure cholinesterase activity of the compounds, using ATC and BTC (75 mM) as substrates.
In both cases, DTNB (10 mM) was used to observe absorbance changes at 412 nm. Donepezil
hydrochloride was used as a positive control [36].
4.2.General Procedures
1H and 13C NMR spectra were recorded on a JEOL ECP spectrometer operating at 500 MHz,
with Me4Si as internal standard and CDCl3 or DMSO-d6 as the solvent. Electrospray ionization
mass spectrometry (ESI-MS) on MicroTOF-Q mass spectrometer (Bruker) was used to obtain
high resolution mass spectra (HRMS). Microanalyses data was obtained using Fison EA 1108

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elemental analyzer. Infrared spectra using KBr disc were recorded on a Perkin Elmer 400 (FTIR)
spectrometer. Flash column chromatography was carried out with silica gel 60 (230-400mesh)
(Merck) and thin layer chromatography (TLC) was performed on pre-coated silica plates (kiesel
gel 60 F254, BDH). Melting points were determined using an electrothermal instrument and are
uncorrected. The compounds were visualized by illumination under ultraviolet (UV) light (254
nm) or by vanillin stain followed by charring on a hotplate.
4.3.Synthesis of α, β-Unsaturated Carbonyl based Compounds
Twenty one α, β-unsaturated carbonyl based compounds (1-7; 11-17; 21-27) were synthesized by
direct coupling of the appropriate aromatic aldehyde with the ten different types of ketones at a
molar ratio of 1:2, and the compounds (8-10; 18-20; 28-30) were synthesized at a molar ratio of
1:1 under base catalyzed Claisen–Schmidt condensation reaction conditions. Scheme 1
demonstrates the general synthesis of α, β-unsaturated carbonyl based compounds d. Concisely,
the appropriate aromatic aldehyde (20 mmol, 2 equivalant) and the suitable ketone (10 mmol, 1
equivalent) were mixed and dissolved in 15 mL of ethanol in single necked round bottomed
flask, and stirred at 5°C for a couple of minutes. Afterwards, a 40% NaOH solution in ethanol
was then added drop wise for several minutes. The mixture was left stirring at room temperature
(27 °C) for 1-24 h. The precipitate formation and color changes of the reaction mixture served as
an indication of product formation. The reaction was monitored by TLC and upon completion;
the reaction was quenched by the addition of acidified ice to the mixture. The α, β-
unsaturated carbonyl based compounds were isolated by column chromatography or by
recrystallization.

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4.3.1. 2,6-Bis[4-(diethoxymethyl)benzylidene]cyclohexanone (1)
1
Yellow crystals (2.59 g, 54%). mp: 112-114 ˚C; H NMR (500 MHz, CDCl3) δ: 7.89 (s, 2H),
7.29 (d, J=8Hz, 4H), 6.82 (d, J=8Hz, 4H), 5.89 (s, 2H), 3.42 (q, J=7.5, 8H), 2.32 (t, J=12.0 Hz,
4H), 1.82 (m, 2H), 1.24 (t, J=7.5, 12H); 13C NMR (500 MHz, CDCl3) δ: 186.4, 152.9, 144.4,
136.5, 132.5, 127.3, 126.5, 101.1, 55.0, 28.3, 27.8, 16.2 ; HRMS (ESI) m/z: 479.68 [M+H]+,
Microanalysis calculated for C30H38O5 (478.62), C: 75.28%, H: 8.00%. Found C: 75.42%, H:
8.12%.
4.3.2. 3,5-Bis-(4-diethoxymethyl-benzylidene)-tetrahydro-pyran-4-one (2)
Pale Yellow crystals (3.06 g, 64%). mp: 106-108 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.85 (s, 2H),
7.32 (d, J=8Hz, 4H), 6.95 (d, J=8Hz, 4H), 5.52 (s, 2H), 3.62 (q, J=7.5, 8H), 2.69 (s, 4H), 1.18 (t,
J=8, 12H); 13C NMR (500 MHz, CDCl3) δ: 187.2, 152.7, 143.8, 136.7, 131.9, 126.2, 125.2,
101.5, 62.4, 55.2, 16.1; HRMS (ESI) m/z: 481.65 [M+H]+, Microanalysis calculated for
C29H36O6 (480.59), C: 72.48%, H: 7.55%. Found C: 72.62%, H: 7.72%.
4.3.3. 3,5-Bis[4-(diethoxymethyl)benzylidene]piperidin-4-one (3)
1
Yellow solid (2.89 g, 60%). mp: 101-102 ˚C; H NMR (500 MHz, CDCl3) δ: 7.68 (s, 2H), 7.12
(d, J=7.5Hz, 4H), 6.93 (d, J=7.5Hz, 4H), 5.65 (s, 2H), 3.36 (q, J=7.5, 8H), 2.65 (s, 4H), 1.27 (t,
J=7.0, 12H); 13C NMR (500 MHz, CDCl3) δ: 185.2, 145.1, 144.5, 138.5, 136.2, 128.1, 127.8,
102.1, 55.1, 48.7, 16.4 ; HRMS (ESI) m/z: 480.71 [M+H]+, Microanalysis calculated for
C29H37NO5 (479.61), C: 72.62%, H: 7.78%, N: 2.92%. Found C: 72.71%, H: 7.86%, N: 3.10%.
4.3.4. 3,5-Bis[4-(diethoxymethyl)benzylidene]-1-methyl-piperidin-4-one (4)

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Light Yellow solid (2.12 g, 43%). mp: 142-144 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.63 (s, 2H),
7.32 (d, J=8Hz, 4H), 7.15 (d, J=8Hz, 4H), 5.59 (s, 2H), 3.29 (q, J=7.5, 8H), 2.71 (s, 4H), 2.12 (s,
3H), 1.20 (t, J=7.0, 12H); 13C NMR (500 MHz, CDCl3) δ: 189.7, 148.2, 147.5, 139.8, 135.1,
128.4, 127.1, 101.4, 56.6, 51.2, 40.1, 16.7 ; HRMS (ESI) m/z: 494.65 [M+H]+, Microanalysis
calculated for C30H39NO5 (493.63), C: 72.99%, H: 7.96%, N: 2.84%. Found C: 72.95%, H:
7.99%, N: 2.87%.
4.3.5. 1-Benzyl-3,5-bis[4-(diethoxymethyl)benzylidene]piperidin-4-one (5)
Brown semisolid (2.76 g, 48%). mp: 112-114 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.89 (d, J=8Hz,
2H), 7.65 (s, 2H), 7.52 (d, J=8Hz, 2H), 7.22 (d, J=7Hz, 4H), 7.12 (d, J=7Hz, 4H), 6.69 (t,
J=6.5Hz, H), 5.17 (s, 2H), 4.17 (s, 2H), 3.38 (q, J=7.5Hz, 8H), 2.95 (s, 4H), 1.14 (t, J=7.0, 12H);
13C NMR (500 MHz, CDCl3) δ: 191.5, 148.2, 147.9, 139.5, 139.1, 132.5, 128.4, 127.6, 127.0,
125.2, 124.9, 101.8, 64.3, 56.2, 50.92, 17.1 ; HRMS (ESI) m/z: 570.84 [M+H]+, Microanalysis
calculated for C36H43NO5 (569.73), C: 75.89%, H: 7.61%, N: 2.46%. Found C: 75.92%, H:
7.64%, N: 2.44%.
4.3.6. 1,5-Bis-(4-diethoxymethyl-phenyl)-penta-1,4-dien-3-one (6)
1
Pale brownish solid (1.96 g, 45%). mp: 98-100 ˚C; H NMR (500 MHz, CDCl3) δ: 7.82 (d,
J=6.5Hz, 4H), 7.71 (d, J=8Hz, 2H) , 7.49 (d, J=6.5Hz, 2H), 7.16 (d, J=7Hz, 4H), 5.25 (s, 2H),
13
3.52 (q, J=7Hz, 8H), 1.17 (t, J=7.0, 12H); C NMR (500 MHz, CDCl3) δ: 190.5, 152.2, 148.6,
139.7, 136.4, 135.2, 132.8, 102.2, 55.5, 16.9 ; HRMS (ESI) m/z: 439.62 [M+H]+, Microanalysis
calculated for C27H34O5 (438.56), C: 73.94%, H: 7.81%. Found C: 74.12%, H: 7.89%.
4.3.7. 2,5-Bis-(4-diethoxymethyl-benzylidene)-cyclopentanone (7)

ACCEPTED MANUSCRIPT
1
Light yellow crystals (3.12 g, 67%). mp: 88-89 ˚C; H NMR (500 MHz, CDCl3) δ: 7.69 (d,
J=6.5Hz, 4H), 7.42 (s, 2H), 7.21 (d, J=6.5Hz, 4H), 5.45 (s, 2H), 3.62 (q, J=7Hz, 8H), 2.38 (t,
J=7Hz, 4H), 1.22 (t, J=7.0, 12H); 13C NMR (500 MHz, CDCl3) δ: 189.7, 149.4, 145.5, 138.7,
136.4, 133.9, 129.1, 100.9, 56.2, 32.5, 16.1 ; HRMS (ESI) m/z: 465.72 [M+H]+, Microanalysis
calculated for C29H36O5 (464.59), C: 74.97%, H: 7.81%. Found C: 75.18%, H: 7.87%.
4.3.8. 2-(4-Diethoxymethyl-benzylidene)-indan-1-one (8)
1
White powder (2.52 g, 78%). mp: 152-154 ˚C; H NMR (500 MHz, CDCl3) δ: 7.80 (d, J=8Hz,
2H), 7.69 (s, H), 7.48 (d, J=8Hz, 2H), 7.25 (d, J=7.5Hz, H), 7.20 (d, J=7.5Hz, H), 7.05 (t,
J=7.5Hz, H), 6.94 (t, J=7Hz, H), 5.34 (s, H), 3.49 (q, J=7Hz, 4H), 2.75 (s, 2H), 1.29 (t, J=6Hz,
6H); 13C NMR (500 MHz, CDCl3) δ: 193.2, 144.5, 142.2, 140.1, 139.2, 138.8, 136.5, 130.4,
129.6, 128.1, 125.6, 125.1, 124.2, 102.2, 56.3, 29.9, 16.4 ; HRMS (ESI) m/z: 323.67 [M+H]+,
Microanalysis calculated for C21H22O3 (322.40), C: 78.23%, H: 6.88%. Found C: 78.44%, H:
6.92%.
4.3.9. 2-(4-Diethoxymethyl-benzylidene)-3,4-dihydro-2H-naphthalen-1-one (9)
1
Light yellow solid (2.67g, 80%). mp: 94-95 ˚C; H NMR (500 MHz, CDCl3) δ: 7.84 (d, J=8Hz,
2H), 7.59 (s, H), 7.47 (d, J=8.5Hz, 2H), 7.29 (d, J=7.5Hz, H), 7.22 (d, J=7.5Hz, H), 7.12 (t,
J=7.5Hz, H), 7.02 (t, J=7Hz, H), 5.49 (s, H), 3.35 (q, J=7Hz, 4H), 2.29 (t, J=7Hz, 2H), 2.05 (t,
J=7Hz, 2H), 1.17 (t, J=6Hz, 6H); 13C NMR (500 MHz, CDCl3) δ: 188.3, 152.2, 150.1, 148.5,
146.2, 143.9, 137.5, 134.4, 129.2, 127.2, 126.2, 124.8, 117.2, 101.7, 56.2, 29.5, 28.2, 16.0 ;
HRMS (ESI) m/z: 337.51 [M+H]+, Microanalysis calculated for C22H24O3 (336.42), C: 78.54%,
H: 7.19%. Found C: 78.33%, H: 7.12%.
4.3.10. 2-[3-(4-Diethoxymethyl-phenyl)-acryloyl]-3,4-dihydro-2H-naphthalen-1-one (10)

ACCEPTED MANUSCRIPT
Yellow solid (3.16 g, 83%). mp: 121-122 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.75 (d, J=6Hz, H),
7.52 (d, J=6Hz, H), 7.42 (d, J=8Hz, 2H), 7.39 (d, J=8Hz, 2H), 7.32 (d, J=7Hz, H), 7.29 (d, J=7Hz,
H), 7.17 (t, J=7Hz, H), 6.89 (t, J=7Hz, H), 5.29 (s, H), 3.47 (q, J=7Hz, 4H), 3.29 (t, J=8.5Hz, H),
2.12 (t, J=8Hz, 2H), 1.95 (t, J=7Hz, 2H), 1.24 (t, J=6.5Hz, 6H); 13C NMR (500 MHz, CDCl3) δ:
194.6, 151.1, 148.9, 144.6, 142.3, 139.2, 138.4, 136.2, 135.5, 132.4, 130.6, 129.1, 125.2, 124.2,
103.1, 66.1, 55.7, 30.2, 28.6, 16.7 ; HRMS (ESI) m/z: 379.52 [M+H]+, Microanalysis calculated
for C24H26O4 (378.46), C: 76.17%, H: 6.92%. Found C: 76.21%, H: 6.91%.
4.3.11. 2,6-Bis-(4-dimethylamino-2-nitro-benzylidene)-cyclohexanone (11)
White powder (2.78 g, 62%). mp: 139-140 ˚C; δ: 7.94 (s, 2H), 7.55 (d, J=8Hz, 2H), 7.41 (d,
J=8Hz, 2H), 7.13 (s, 2H), 3.15 (s, 12H), 2.35 (t, J=12.0 Hz, 4H), 1.87 (m, 2H) ; 13C NMR (500
MHz, CDCl3) δ: 190.7, 149.5, 145.7, 145.6, 140.1, 128.2, 118.6, 117.6, 106.5, 46.8, 29.1, 27.5;
HRMS (ESI) m/z: 451.64 [M+H]+, Microanalysis calculated for C24H26N4O5 (450.49), C:
63.99%, H: 5.82%, N: 12.44%. Found C: 64.12%, H: 5.72%, N: 12.42%.
4.3.12. 3,5-Bis[4-(dimethylamino)2-nitro-benzylidene]tetrahydro-pyran-4-one (12)
White powder (2.92 g, 65%). mp: 188- 190 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.89 (s, 2H), 7.46
13
(d, J=8Hz, 2H), 7.14 (d, J=8Hz, 2H), 7.05 (s, 2H), 3.13 (s, 12H), 2.93 (s, 4H); C NMR (500
MHz, CDCl3) δ: 190.5, 149.2, 146.9, 145.1, 139.9, 128.4, 118.9, 118.1, 107.8, 65.5, 46.2; HRMS
(ESI) m/z: 475.52 [M+Na]+, Microanalysis calculated for C23H24N4O6 (452.46), C: 61.05%, H:
5.35%, N: 12.38%. Found C: 61.24%, H: 5.59%, N: 12.42%.
4.3.13. 3,5-Bis[4-(dimethylamino)2-nitro-benzylidene]piperidin-4-one (13)

ACCEPTED MANUSCRIPT
Light yellow solid (2.28 g, 51%). mp: 192- 194 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.82 (s, 2H),
7.72 (d, J=8Hz, 2H), 7.21 (d, J=8Hz, 2H), 6.92 (s, 2H), 3.17 (s, 12H), 3.13 (s, 4H); 13C NMR
(500 MHz, CDCl3) δ: 189.4, 149.7, 148.6, 142.5, 140.7, 127.3, 118.8, 118.1, 106.5, 49.4, 46.9;
HRMS (ESI) m/z: 452.52 [M+H]+, Microanalysis calculated for C23H25N5O5 (451.48), C:
61.19%, H: 5.58%, N: 15.51%. Found C: 61.42%, H: 5.62%, N: 15.89%.
4.3.14. 3,5-Bis[4-(dimethylamino)2-nitro-benzylidene]-1-methyl-piperidin-4-one (14)
White powder (2.56 g, 55%). mp: 181- 182 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.76 (s, 2H), 7.45
13
(d, J=8Hz, 2H), 7.23 (d, J=8Hz, 2H), 7.13 (s, 2H), 3.21 (s, 12H), 3.03 (s, 4H), 2.19 (s, 3H); C
NMR (500 MHz, CDCl3) δ: 187.5, 148.9, 148.2, 143.2, 141.8, 126.7, 119.6, 118.9, 106.2, 44.5,
46.1, 38.8; HRMS (ESI) m/z: 466.72 [M+H]+, Microanalysis calculated for C24H27N5O5 (465.50),
C: 61.92%, H: 5.85%, N: 15.04%. Found C: 61.99%, H: 5.91%, N: 15.19%.
4.3.15. 1-Benzyl-3,5-bis-(4-dimethylamino-2-nitro-benzylidene)-piperidin-4-one (15)
1
White crystals (3.22 g, 60%). mp: 165-166 ˚C; H NMR (500 MHz, CDCl3) δ: 7.82 (d, J=8Hz,
2H), 7.72 (s, 2H), 7.59 (d, J=8Hz, 2H), 7.32 (d, J=7Hz, 2H), 7.25 (t, J=7Hz, 2H), 7.02 (s, 2H),
6.92 (t, J=6.5Hz, H), 4.22 (s, 2H), 3.19 (s, 12H), 2.72 (s, 4H), ; 13C NMR (500 MHz, CDCl3) δ:
189.6, 148.9, 147.2, 139.5, 139.0, 131.9, 128.6, 126.5, 126.0, 125.3, 124.5, 119.2, 104.9, 64.8,
53.8, 45.9; HRMS (ESI) m/z: 542.65 [M+H]+, Microanalysis calculated for C30H31N5O5 (541.60),
C: 66.53%, H: 5.77%, N: 12.93%. Found C: 66.48%, H: 5.79%, N: 12.54%.
4.3.16. 1,5-Bis-(4-dimethylamino-2-nitro-phenyl)-penta-1,4-dien-3-one (16)
Pale yellow solid (2.97 g, 68%). mp: 177-179 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.70 (d, J=6Hz,
2H), 7.68 (d, J=8Hz, 2H), 7.44 (d, J=6Hz, 2H), 7.25 (d, J=8Hz, 2H), 7.12 (s, 2H), 3.15 (s, 12H);

ACCEPTED MANUSCRIPT
13C NMR (500 MHz, CDCl3) δ: 187.5, 150.9, 148.6, 144.4, 132.8, 128.1, 117.5, 113.8, 107.9,
46.7; HRMS (ESI) m/z: 409.45 [M-H]+, Microanalysis calculated for C21H22N4O5 (410.42), C:
61.45%, H: 5.40%, N: 13.65%. Found C: 61.48%, H: 5.62%, N: 13.77%.
4.3.17. 2,5-Bis-(4-dimethylamino-2-nitro-benzylidene)-cyclopentanone (17)
White powder (2.41 g, 55%). mp: 156-157 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.92 (d, J=6.5Hz,
2H), 7.88 (d, J=6Hz, 2H), 7.75 (d, J=6.5Hz, 2H), 7.44 (s, 2H), 3.10 (s, 12H), 2.45 (t, J=7Hz,
4H) ; 13C NMR (500 MHz, CDCl3) δ: 188.9, 153.2, 149.9, 144.2, 143.8, 125.8, 118.1, 116.4,
108.3, 46.9, 32.6; HRMS (ESI) m/z: 437.72 [M+H]+, Microanalysis calculated for C23H24N4O5
(436.46), C: 63.29%, H: 5.54%, N: 12.84%. Found C: 63.42%, H: 5.48%, N: 12.72%.
4.3.18. 2-(4-Dimethylamino-2-nitro-benzylidene)-indan-1-one (18)
Yellow semisolid (1.95 g, 63%). mp: 169-171 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.56 (d, J=8Hz,
H), 7.48 (s, H), 7.32 (d, J=8Hz, H), 7.12 (s, H), 6.98 (d, J=7.5Hz, H), 6.91 (d, J=7.5Hz, H), 6.74
13
(t, J=7.5Hz, H), 6.65 (t, J=7Hz, H), 3.22 (s, 6H), 2.81 (s, 2H); C NMR (500 MHz, CDCl3) δ:
192.8, 145.6, 142.6, 141.3, 139.8, 138.2, 137.4, 130.9, 128.7, 128.0, 125.9, 123.8, 119.5, 117.6,
108.6, 46.8, 28.3 ; HRMS (ESI) m/z: 309.55 [M+H]+, Microanalysis calculated for C18H16N2O3
(308.33), C: 70.12%, H: 5.23%, N: 9.09%. Found C: 70.18%, H: 5.59%, N: 9.18%.
4.3.19. 2-(4-Dimethylamino-2-nitro-benzylidene)-3,4-dihydro-2H-naphthalen-1-one (19)
White powder (2.50 g, 78%). mp: 138-139 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.82 (d, J=6.5Hz,
H), 7.57 (s, H), 7.42 (d, J=6.5Hz, H), 7.35 (s, H), 7.28 (d, J=7Hz, H), 7.20 (d, J=7Hz, H), 7.19 (t,
J=7.5Hz, H), 6.92 (t, J=8Hz, H), 3.14 (s, 6H), 2.47 (t, J=7Hz, 2H), 2.14 (t, J=7Hz, 2H) ; 13C
NMR (500 MHz, CDCl3) δ: 188.7, 152.8, 151.3, 148.9, 146.8, 144.2, 136.8, 134.6, 129.9, 127.6,

ACCEPTED MANUSCRIPT
126.1, 125.3, 124.2, 107.8, 46.2, 29.9, 27.9; HRMS (ESI) m/z: 323.45 [M+H]+, Microanalysis
calculated for C19H18N2O3 (322.36), C: 70.79%, H: 5.63%, N: 8.69%. Found C: 71.12%, H:
5.52%, N: 8.72%.
4.3.20. 2-[3-(4-Dimethylamino-2-nitro-phenyl)-acryloyl]-3,4-dihydro-2H-naphthalen-1-one
(20)
1
Pale yellow powder (2.93 g, 80%). mp: 102-103 ˚C; H NMR (500 MHz, CDCl3) δ: 7.79 (d,
J=6.5Hz, H), 7.62 (d, J=6.5Hz, H), 7.45 (d, J=7.5Hz, H), 7.32 (s, H), 7.29 (d, J=7.5Hz, H), 7.21
(d, J=7.5Hz, H), 7.14 (d, J=7.5Hz, H), 7.02 (t, J=7.5Hz, H), 6.94 (t, J=7.5Hz, H), 3.45 (t,
J=8.5Hz, H), 3.10 (s, 6H), 2.22 (t, J=8Hz, 2H), 2.15 (t, J=8Hz, 2H) ; 13C NMR (500 MHz, CDCl3)
δ: 193.8, 152.2, 148.7, 145.2, 142.8, 139.6, 137.1, 136.8, 135.6, 131.9, 130.5, 129.4, 124.8, 123.5,
116.5, 106.2, 65.8, 46.8, 31.3, 28.7 ; HRMS (ESI) m/z: 365.55 [M+H]+, Microanalysis calculated
for C21H20N2O4 (364.39), C: 69.22%, H: 5.53%, N: 7.69%. Found C: 69.34%, H: 5.59%, N:
7.81%.
4.3.21. 2,6-Bis-(4-pyrrolidin-1-yl-benzylidene)-cyclohexanone (21)
1
Brownish powder (3.12 g, 76%). mp: 105-107 ˚C; H NMR (500 MHz, CDCl3) δ: 7.92 (s, 2H),
7.45 (d, J=8Hz, 4H), 6.99 (d, J=8Hz, 4H), 2.37 (t, J=10.0 Hz, 4H), 2.10 (m, J=6.5Hz, 8H), 1.84
(m, J=5.5, 2H), ; 13C NMR (500 MHz, CDCl3) δ: 188.9, 152.2, 145.2, 136.7, 131.9, 127.5, 126.9,
58.1, 28.8, 27.2, 25.6 ; HRMS (ESI) m/z: 413.62 [M+H]+, Microanalysis calculated for
C28H32N2O (412.57), C: 81.51%, H: 7.82%, N: 6.79%. Found C: 81.67%, H: 7.99%, N: 6.95%.
4.3.22. 3,5-Bis-(4-pyrrolidin-1-yl-benzylidene)-tetrahydro-pyran-4-one (22)

ACCEPTED MANUSCRIPT
1
Light Brown solid (2.40 g, 58%). mp: 99-101 ˚C; H NMR (500 MHz, CDCl3) δ: 7.81 (s, 2H),
13
7.37 (d, J=6.5Hz, 4H), 7.17 (d, J=6.5Hz, 4H), 2.94 (s, 4H), 1.92 (m, J=6.5Hz, 8H) ; C NMR
(500 MHz, CDCl3) δ: 189.0, 150.3, 144.7, 136.8, 130.1, 126.8, 125.6, 62.9, 57.1, 25.5; HRMS
(ESI) m/z: 415.49 [M+H]+, Microanalysis calculated for C27H30N2O2 (414.54), C: 78.23%, H:
7.29%, N: 6.76%. Found C: 78.45%, H: 7.52%, N: 6.72%.
4.3.23. 3,5-Bis-(4-pyrrolidin-1-yl-benzylidene)-piperidin-4-one (23)
1
Pale yellow powder (2.83 g, 69%). mp: 92-93 ˚C; H NMR (500 MHz, CDCl3) δ: 7.72 (s, 2H),
7.17 (d, J=7.5Hz, 4H), 6.81 (d, J=7.5Hz, 4H), 2.88 (s, 4H), 1.74 (m, J=6Hz, 8H) ; 13C NMR (500
MHz, CDCl3) δ: 185.7, 145.9, 143.4, 137.1, 136.5, 128.7, 119.9, 56.4, 48.2, 24.9; HRMS (ESI)
m/z: 436.62 [M+Na]+, Microanalysis calculated for C27H31N3O (413.55), C: 78.42%, H: 7.56%,
N: 10.16%. Found C: 78.44%, H: 7.60%, N: 10.25%.
4.3.24. 1-Methyl-3,5-bis[4-(pyrrolidinyl)benzylidene]piperidin-4-one (24)
1
Yellow powder (1.99 g, 47%). mp: 122- 124 ˚C; H NMR (500 MHz, CDCl3) δ: 7.89 (d,
J=8.5Hz, 4H), 7.65 (s, 2H), 7.52 (d, J=8.5Hz, 4H), 2.99 (s, 4H), 2.43 (s, 3H), 1.96 (m, J=6Hz,
8H); 13C NMR (500 MHz, CDCl3) δ: 190.1, 146.4, 144.7, 141.9, 131.8, 128.2, 109.6, 57.4, 56.5,
38.2, 25.5; HRMS (ESI) m/z: 428.71 [M+H]+, Microanalysis calculated for C28H33N3O (427.58),
C: 78.65%, H: 7.78%, N: 9.83%. Found C: 78.82%, H: 7.95%, N: 10.12%.
4.3.25. 1-Benzyl-3,5-bis-(4-pyrrolidin-1-yl-benzylidene)-piperidin-4-one (25)
1
Light yellow crystals (2.16 g, 43%). mp: 88-89 ˚C; H NMR (500 MHz, CDCl3) δ: 7.71 (d,
J=8Hz, 2H), 7.45 (s, 2H), 7.31 (d, J=8Hz, 2H), 7.25 (d, J=7Hz, 4H), 7.19 (d, J=7Hz, 4H), 6.81 (t,
13
J=6.5Hz, H), 4.19 (s, 2H), 2.92 (s, 4H), 1.78 (m, J=6Hz, 8H); C NMR (500 MHz, CDCl3) δ:
192.2, 148.8, 146.3, 138.2, 137.7, 132.7, 128.5, 126.2, 125.4, 124.9, 118.9, 65.1, 58.1, 50.2, 25.7;

ACCEPTED MANUSCRIPT
HRMS (ESI) m/z: 504.69 [M+H]+, Microanalysis calculated for C34H37N3O (503.68), C: 81.08%,
H: 7.40%, N: 8.34%. Found C: 81.25%, H: 7.69%, N: 8.17%.
4.3.26. 1,5-Bis-(4-pyrrolidin-1-yl-phenyl)-penta-1,4-dien-3-one (26)
1
Light brownish solid (2.02 g, 54%). mp: 111-113 ˚C; H NMR (500 MHz, CDCl3) δ: 7.76 (d,
J=6Hz, 4H), 7.52 (d, J=6Hz, 2H) , 7.12 (d, J=8Hz, 2H), 7.05 (d, J=8Hz, 4H), 1.85 (m, J=6Hz,
8H); 13C NMR (500 MHz, CDCl3) δ: 190.0, 151.8, 148.2, 138.2, 136.5, 134.9, 131.2, 56.9, 26.6;
HRMS (ESI) m/z: 373.82 [M+H]+, Microanalysis calculated for C25H28N2O (372.50), C: 80.61%,
H: 7.58%, N: 7.52%. Found C: 80.88%, H: 7.74%, N: 7.85%.
4.3.27. 2,5-Bis-(4-pyrrolidin-1-yl-benzylidene)-cyclopentanone (27)
1
Yellow powder (2.96 g, 74%). mp: 94-95 ˚C; H NMR (500 MHz, CDCl3) δ: 7.87 (d, J=8Hz,
4H), 7.59 (s, 2H), 7.12 (d, J=8Hz, 4H), 2.42 (t, J=7Hz, 4H), 1.91 (m, J=6Hz, 8H); 13C NMR
(500 MHz, CDCl3) δ: 190.1, 149.5, 145.8, 139.1, 136.8, 134.5, 129.8, 58.1, 32.9, 26.2 ; HRMS
(ESI) m/z: 399.51 [M+H]+, Microanalysis calculated for C27H30N2O (398.54), C: 81.37%, H:
7.59%, N: 7.03%. Found C: 81.42%, H: 7.62%, N: 7.12%.
4.3.28. 2-(4-Pyrrolidin-1-yl-benzylidene)-indan-1-one (28)
White powder (1.94g, 67%). mp: 136-137 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.94 (d, J=8Hz,
2H), 7.81 (s, H), 7.43 (d, J=8Hz, 2H), 7.37 (d, J=7.5Hz, H), 7.01 (d, J=7.5Hz, H), 6.92 (t,
J=7.5Hz, H), 6.79 (t, J=7Hz, H), 2.82 (s, 2H), 1.84 (m, J=6Hz, 4H); 13C NMR (500 MHz, CDCl3)
δ: 192.8, 144.7, 142.9, 141.2, 139.5, 137.9, 136.6, 130.8, 129.8, 128.6, 125.2, 124.5, 122.9, 57.7,
28.5, 27.4; HRMS (ESI) m/z: 290.40 [M+H]+, Microanalysis calculated for C20H19NO (289.37),
C: 83.01%, H: 6.62%, N: 4.84%. Found C: 83.25%, H: 6.75%, N: 4.82%.

ACCEPTED MANUSCRIPT
4.3.29. 2-(4-Pyrrolidin-1-yl-benzylidene)-3,4-dihydro-2H-naphthalen-1-one (29)
1
Brown solid (2.41 g, 80%). mp: 123- 125 ˚C; H NMR (500 MHz, CDCl3) δ: 7.76 (d, J=8Hz,
2H), 7.52 (s, H), 7.42 (d, J=8Hz, 2H), 7.21 (d, J=7.5Hz, H), 7.15 (d, J=7.5Hz, H), 7.02 (t,
J=7.5Hz, H), 6.99 (t, J=7Hz, H), 2.24 (t, J=7Hz, 2H), 1.97 (t, J=7Hz, 2H), 1.81 (m, J=6Hz, 4H);
13C NMR (500 MHz, CDCl3) δ: 188.8, 150.5, 149.2, 148.7, 147.4, 142.6, 135.1, 132.4, 129.8,
128.9, 125.5, 124.2, 117.5, 58.9, 29.7, 27.9, 25.9; HRMS (ESI) m/z: 304.56 [M+H]+,
Microanalysis calculated for C21H21NO (303.40), C: 83.13%, H: 6.98%, N: 4.62%. Found C:
83.06%, H: 6.91%, N: 4.89%.
4.3.30. 2-[3-(4-Pyrrolidin-1-yl-phenyl)-acryloyl]-3,4-dihydro-2H-naphthalen-1-one (30)
Pale yellow solid (2.52 g, 73%). mp: 118-120 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.70 (d, J=6Hz,
H), 7.48 (d, J=6Hz, H), 7.33 (d, J=6.5Hz, 2H), 7.21 (d, J=6.5Hz, 2H), 7.17 (d, J=6.5Hz, H), 7.02
(d, J=6.5Hz, H), 6.98 (t, J=6.5Hz, H), 6.81 (t, J=6Hz, H), 3.24 (t, J=6Hz, H), 2.29 (t, J=8Hz, 2H),
13
1.98 (t, J=7Hz, 2H), 1.94 (m, J=6Hz, 4H); C NMR (500 MHz, CDCl3) δ: 192.5, 151.8, 149.1,
145.7, 142.5, 140.4, 138.7, 136.9, 135.6, 131.9, 130.8, 129.5, 126.1, 119.1 , 65.5, 58.2, 32.5, 28.7,
25.9; HRMS (ESI) m/z: 346.57 [M+H]+, Microanalysis calculated for C23H23NO2 (345.43), C:
79.97%, H: 6.71%, N: 4.05%. Found C: 80.19%, H: 6.55%, N: 4.09%.
4.4. DPPH (1,1-diphenyl-2-picrylhydrazyl) assay
The reported DPPH method was applied to assess the scavenging ability of the compounds [37].
The compounds were tested in the range of 0-25 µg/mL in methanol. To 2.5 ml of compound in
5 different concentrations, 1 ml of 0.3 mM DPPH ethanol solution was added. Then 1 ml of
methanol was added to the solution and allowed to react for 30 min in the dark at room
temperature. The change in the absorbance was read at 518 nm. The blank was comprised of 2.5

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ml of test compound and 1 ml methanol, while the mixture of 1 ml DPPH and 2.5 ml of
methanol served as negative control. The percentage antioxidant activity was calculated as
follows:
ꢆꢇ − ꢆꢈ
% ꢀꢁℎꢂꢃꢂꢄꢂꢅꢁ =
× 100
ꢆꢈ
Where: AB: absorption of blank sample, AA: absorption of test samples. The IC50 value was
calculated and compared with that of ascorbic acid as a reference.
4.5. Cell culture
The rat pheochromocytoma (PC12) cell line was purchased from the ATCC. The cells were
maintained in RPMI-1640 medium consisting of 10% heat-inactivated FBS and 1% penicillin-
streptomycin. The cells were incubated at 37˚C in a 5% CO2 atmosphere with 95% humidity.
4.6. Cell viability assay
To investigate the cytotoxicity or neuroprotective effects of synthetic compounds in PC12 cells,
the MTT assay was performed. The cells (1x104cells/well) were seeded in 96-well cell culture
plates and treated with different concentrations of compounds for 24 h. The cell viability was
expressed as a relative percentage against control cultures. To assess the Aβ-induced
neuroprotective activity, different concentrations of all compounds were individually added to
cells for 24 h, before treatment with Aβ. The positive control drug selegiline was also evaluated
in the same manner for comparison. 2.0 mg/ml MTT solution was added to each well and
incubated for 4 h at 37°C. The medium was removed and the formazan crystals were solubilized
in 100 ꢀl dimethyl sulfoxide (DMSO). A microplate reader was used to determine the optical
density (excitation at 570 nm, emission at 630 nm) and data are plotted as percent of control.
4.7. AChE and BuChE inhibition assay

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To evaluate the potency of compounds to inhibit AChE and BuChE, all compounds were
subjected to a slightly modified method of Ellman’s test [38]. The reaction of released
thiocholine to give a coloured product with a chromogenic reagent 5,5-dithio-bis (2-
nitrobenzoic) acid (DTNB) is the basis of the spectrophotometric method. The measurements
were performed on a 1700 Shimadzu UV-1700, UV-Vis spectrophotometer. At a concentration
of 2.5 units/mL, the enzyme solutions were prepared in gelatin solution (1%). AChE or BChE
solution (50 ꢀL) and compound solution (50 ꢀL), prepared in 2% DMSO at a concentration
range of 10-1- 10-6 mM, were added to 3.0 mL phosphate buffer (pH 8 ± 0.1) and were incubated
at 25°C for 5 min. DTNB (50 ꢀL) and ATC (10 ꢀL) were added to the enzyme-inhibitor mixture
to start the reaction. For 10 min, the production of the yellow anion was recorded at 412 nm.
Serving as a control, an identical solution of the enzyme without the inhibitor was processed
using the same protocol. The blank reading consisted of 3.0 mL buffer, 50 ꢀL 2% DMSO, 50 ꢀL
DTNB and 10 ꢀL substrate. All the processes were assayed in triplicate. The percentage
inhibition rate was calculated by the following equation:
ꢆꢉ − ꢆꢊ
% ꢀꢁℎꢂꢃꢂꢄꢂꢅꢁ =
× 100
ꢆꢉ
Where AI is the absorbance in the presence of the inhibitor, AC is the absorbance of the control
and AB is the absorbance of blank. Both values were corrected with blank-reading value. The
data was expressed as Mean ± SD.
4.8. Molecular docking and QSAR analysis
For docking, the molecules were drawn using ACD Chemsketch 12 freeware followed by energy
optimization using MMFF94 force field in TINKER [32]. The protein was first subjected to
energy minimization, protonation followed by detection of site detection. The receptor and the
drug candidates were optimized before actual docking followed by docking in MOE using the

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standard procedure found in the manual of software. The molecules were docked in the active
site using ‘force field’ as a refinement technique with default settings in MOE [29]. All of the
newly synthesized molecules were docked in the active site of AChE (pdb- 1GQR, x-ray
resolution = 2.20 Ao). For the sake of convenience and comparison, as a representative, we report
the docking poses for the most active compound of the series. For QSAR analysis, e-Dragon was
used to calculate myriad number of descriptors, followed by random splitting of dataset into
training set (80%) and test set (20%). QSARINS v1.2 was used to eliminate redundant
descriptors and for building GA-MLR (Genetic Algorithm-Multilinear regression) model [31,
32].
ACKNOWLEDGMENTS
SNAB thanks University Kebangsaan Malaysia for post-doctoral contract and highly obliged to
Professor Dr. David Schubert, Head of cellular neurobiology laboratory, Salk Institute (USA),
for his kind suggestions and reading the manuscript. VHM is thankful to Dr. Paola Gramatica,
Italy for providing evaluation copy of QSARINS and to NCL, Pune for allowing to perform the
computational work. Authors gratefully acknowledge the generous support provided by the
University Kebangsaan Malaysia (UKM-DLP-2013-013).

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