Design and development of a novel chalcone derivative as an anticholinesterase inhibitor for possible treatment of dementia

Article abstract of DOI:10.12659/MSM.901842

Background: Cognitive decline (e.g., memory loss), which mainly occurs in the elderly, is termed dementia. In the present study, we intended to explore the cholinesterase inhibitory activity of some novel synthesized chalcones, together with their effect on β-amyloid anti-aggregation. Material/Methods: A novel class of chalcone derivatives have been synthesized and characterized by FT-IR,¹H-NMR,¹³C-NMR, and mass and elemental analysis. These derivatives were later used for the determination of acetylcholinesterase (AChE) inhibitory and β-amyloid anti-aggregation activity. Results: The results of the study showed that among the developed compounds, 8g inhibits AChE more prominently than BuChE, as suggested by a selectivity index (SI) of 2.88. Furthermore, the most potent compound, 8g, showed considerable action in inhibition of β-secretase and Aβ aggregation, but not as prominent as that of curcumin as a standard. Conclusions: In conclusion, our study revealed a novel class of chalcone derivatives as a selective inhibitor of AChE with considerably action against β-secretase and Aβ aggregation. Our results may be useful in developing AD drug therapy and warrant further investigation to generate more advanced analogues.

Full text of DOI:10.12659/MSM.901842

LAB/IN VITRO RESEARCH
e-ISSN 1643-3750
© Med Sci Monit, 2017; 23: 3311-3317
DOI: 10.12659/MSM.901842
Received: 2016.10.06
Accepted: 2016.11.10
Published: 2017.07.08
Design and Development of a Novel Chalcone
Derivative as an Anticholinesterase Inhibitor for
Possible Treatment of Dementia
Authors’ Contribution:
Study Design A
B 1 Fu-Chun Zhao
DE 2 Yan Wu
A 1 Xiao-Jie Song
1 Department of Neurology, Linyi People’s Hospital, Linyi, Shandong, P.R. China
2 Department of Reproductive Medicine, Linyi, People’s Hospital, Linyi, Shandong,
P.R. China
Data Collection B
Statistical Analysis C
Data Interpretation D
Manuscript Preparation E
Literature Search F
Funds Collection G
Corresponding Author:
Source of support:
Xiao-Jie Song, e-mail: songxiaojie246@hotmail.com
Departmental sources
Background:
Material/Methods:
Results:
Cognitive decline (e.g., memory loss), which mainly occurs in the elderly, is termed dementia. In the present
study, we intended to explore the cholinesterase inhibitory activity of some novel synthesized chalcones, to-
gether with their effect on b-amyloid anti-aggregation.
A novel class of chalcone derivatives have been synthesized and characterized by FT-IR, 1H-NMR, 13C-NMR, and
mass and elemental analysis. These derivatives were later used for the determination of acetylcholinesterase
(AChE) inhibitory and b-amyloid anti-aggregation activity.
The results of the study showed that among the developed compounds, 8g inhibits AChE more prominent-
ly than BuChE, as suggested by a selectivity index (SI) of 2.88. Furthermore, the most potent compound, 8g,
showed considerable action in inhibition of b-secretase and Ab aggregation, but not as prominent as that of
curcumin as a standard.
Conclusions:
In conclusion, our study revealed a novel class of chalcone derivatives as a selective inhibitor of AChE with
considerably action against b-secretase and Ab aggregation. Our results may be useful in developing AD drug
therapy and warrant further investigation to generate more advanced analogues.
MeSH Keywords:
Alzheimer Disease • Cholinesterase Inhibitors • Chemistry Techniques, Synthetic
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Anticholinesterase inhibitor activity of chalcone derivative
© Med Sci Monit, 2017; 23: 3311-3317
LAB/IN VITRO RESEARCH
Background
some novel synthesized chalcones together with their effect
on b-amyloid anti-aggregation.
Cognitive decline (e.g., memory loss), mainly in the elderly, is
termed dementia [1]. It is not as a disease, but rather is a phe-
nomenon considered to be a collection of symptoms related to
problem in thinking and memory, affecting the daily life of the
individual [2,3]. According to the World Health Organisation
(WHO), China has witnessed marked improvement in life ex-
pectancy as result of increased access to better healthcare fa-
cilities [4]. This has increased the number of dementia patients;
in 2010, around 10 million people were suffering from demen-
tia in China, as compared to just 3.68 million in 1990 [5]. The
treatment and care for this huge number of patients is a huge
economic burden, with a cost of 392.6 billion RMB (US$ 63.21
billion) per year for dementia patients in China alone [6]. Among
the diagnosed cases of dementia, nearly 60–80% of cases have
Alzheimer disease (AD) [7]. The etiology of AD is still a mat-
ter of investigation and not yet been fully elucidated [8]. The
available literature suggests that AD may progress because of
decrease in the level of acetyl choline, increase in b-amyloid
plaques [9,10], and increase in oxidative stress, together with
inflammation [11] and Tau-protein aggregation [12].
Material and Methods
Chemistry
The chemicals used in the present study were procured from
Sigma Aldrich (USA). The spectra of 1H NMR and 13C NMR were
recorded on a Bruker Avance 400 and a Bruker Avance 100
Spectrophotometer, respectively. The chemical shifts are ex-
pressed in parts per million (ppm), and coupling constants are
expressed in Hertz (Hz). The low-resolution mass spectrum
(MS) was recorded on a Waters ZQ LC/MS single quadrupole
system equipped with an electrospray ionization (ESI) source.
The elemental analysis of the final derivatives was performed
using a Vario Elemental analyzer. The Thin-layer chromatogra-
phy was performed on 0.25 mm Merck silica gel plates (60F-
254) and visualized under UV light.
General procedure for synthesis of (E)-1-(4-
hydroxyphenyl)-3-phenylprop-2-en-1-one. (3)
Although there is no definitive cure for dementia of the AD
type, various pharmacological approaches have been utilized
to offer symptomatic relief, for instance, acetylcholine replace-
ment therapy [13], acetylcholine precursors [14], cholinergic ag-
onists [15], and enhancement of acetylcholinesterase release
and acetylcholinesterase inhibition [16]. Except for the acetyl-
choline esterase inhibitor (AchIs), all of the above interventions
have been associated with certain disadvantages that render
them ineffective in treating AD [17]. AchIs prevents degrada-
tion of Ach during its transport from one cell to another and
increases the concentration of Ach, causing increase in the
cholinergic neurotransmission in AD patients [18]. However,
these inhibitors have been also associated with certain prob-
lems, such as toxicity, adverse effects, short half-life, and non-
selective inhibition of Ach esterase (AChE) [19]. Thus, much in-
terest has been focussed on the design and development of
novel inhibitors of AChE that are devoid of these shortcomings.
We dissolved 1-(4-hydroxyphenyl)ethanone, 1 (0.01 mol) and
benzaldehyde, 2 (0.01 mol) in 50 mL ethanolic solution and stirred
it for 30 min, followed by dropwise addition of aqueous sodi-
um hydroxide (0.05 mol), followed by further stirring for 24 h.
After completion of the reaction as monitored by TLC using the
mobile phase as n-butanol: acetic acid: water (4: 3: 1), a crude
product was obtained as (E)-1-(4-hydroxyphenyl)-3-phenylprop-
2-en-1-one, 3. The resultant solid was then filtered off, washed
with water, dried, and re-crystallized from ethanol [27].
General procedure for synthesis of (E)-1-(4-(2-
bromoethoxy)phenyl)-3-phenylprop-2-en-1-one (4)
A mixture of 3 (4.95 mmol) and K2CO3 (9.99 mmol) in DMF
(10 mL) was stirred at 80°C for 1 h, then 1,2-dibromoethane
(14.84 mmol) was added dropwise. The mixture was stirred
until the TLC showed completion of the reaction. The reac-
tion mixture was poured into water (20 mL) and was extract-
ed with EtOAc (3×10 mL). The combined organic layers were
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude product was purified by column chroma-
tography over silica gel by using petroleum ether and ethyl
acetate (n/n=2: 1) as eluent to give 4 [28].
Due to ease of synthesis and possibility of numerous structur-
al diversity, chalcones have attracted the attention of medical
chemists [20]. It is extensively reported to exhibit a wide array
of bioactivities, such as antibacterial [21], anticancer [22], an-
timalarial [23], and antifungal [24] effects, particularly AchIs.
Various researchers have successfully developed numerous
novel AchIs belonging to the category of chalcones [25]. These
analogues were also revealed to inhibit the amyloid beta (Ab)
self-assembly and the disassembly of preformed Ab oligo-
mers, suggesting its multifunctional role in treating symptoms
of AD [26]. Encouraged by the above, in the present study we
intended to enumerate the cholinesterase inhibitory effects of
General procedure for synthesis of tert-butyl piperidin-4-
ylcarbamate. (6)
A solution of the appropriate 2- (bromo alkyl)isoin-
dole-1,3-dione or 1-(bromo alkyl)-1H-indole (1 equiv),
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Anticholinesterase inhibitor activity of chalcone derivative
© Med Sci Monit, 2017; 23: 3311-3317
LAB/IN VITRO RESEARCH
4-amino-1-benzylpiperidine (5) (1 equiv), and TEA (1.2 equiv)
in MeCN was refluxed for 4 h. After cooling, the product pre-
cipitated as the hydrobromide salt. This was collected by fil-
tration, washed with MeCN, and dried in vacuo. The product
was dissolved in 30 mL water, and then adjusted to basic pH
by adding 25% NH4OH solution. Then the product was extract-
ed with DCM (3×30 mL) and dried over anhydrous Na2SO4, to
afford the product.
(m, 4H, Ar-H), 8.06-7.57 (m, 2H,Aliphatic-H), 7.59-7.29 (m, 9H,
Ar-H), 4.12-2.96 (m, 4H, CH2x2), 3.69 (s, 2H,Aliphatic-H), 2.63-
1.46 (m, 9H, piperidine-H), 1.97 (s, 1H,NH); 13C-NMR (100 MHz,
CDCl3) d, ppm: 189.9, 165.3, 145.2, 136.8, 135.3, 132.9, 131.2,
130.7, 129.6, 128.7, 128.4, 127.8, 121.3, 114.8, 69.4, 64.3, 59.3,
51.8, 47.5, 30.9; Mass: 476.12 (M+1); Elemental analysis for
C29H31ClN2O2: Calculated: C, 73.33; H, 6.58; N, 5.90; Found: C,
73.38; H, 6.49; N, 5.93.
General procedure for synthesis of substituted
1-benzylpiperidin-4-amine. (7)
(E)-1-(4-(2-((1-(4-bromobenzyl)piperidin-4-yl)amino)
ethoxy)phenyl)-3-phenylprop-2-en-1-one. (8c)
A solution of the appropriate tert-butyl piperidin-4-ylcarba-
mate (6) and substituted benzyl bromide or chloride in THF
was refluxed for 24 h. After completion of the reaction, the
desired substituted 1-benzylpiperidin-4-amine was obtained
by tert-butyloxycarbonyl (BOC)-deprotection in 10% HCl solu-
tion in methanol.
Yield: 76%; MP: 212–213°C; MW: 519.47; Rf: 0.62; FT-IR (nmax;
cm–1 KBr): 3189 (secondary N-H str), 3087 (Ar C-H str), 2971
(Ali C-H str), 1484 (C=C of benzene), 1713 (C=O str), 704 (C-
1
Br), 736 cm–1; H-NMR (400 MHz, DMSO, TMS) d ppm: 8.11-
7.19 (m, 4H, Ar-H), 8.06–7.57 (m, 2H,Aliphatic-H), 7.59-7.29 (m,
9H, Ar-H), 4.12-2.96 (m, 4H, CH2x2), 3.69 (s, 2H,Aliphatic-H),
2.63-1.46 (m, 9H, piperidine-H), 1.97 (s, 1H,NH); 13C-NMR (100
MHz, CDCl3) d, ppm: 189.5, 165.3, 145.4, 137.7, 135.3, 131.3,
131.2, 130.5, 129.7, 128.6, 128.4, 127.8, 121.7, 121.3, 114.8,
69.3, 64.8, 59.3, 51.9, 47.4, 30.8; Mass: 520.54 (M+1); Elemental
analysis for C29H31BrN2O2: Calculated: C, 67.05; H, 6.01; N, 5.39;
Found: C, 67.08; H, 5.96; N, 5.43.
General procedure for synthesis of title compounds. 8(a–h)
A mixture of substituted 1-benzylpiperidin-4-amine (7) and
(E)-1-(4-(2-bromoethoxy)phenyl)-3-phenylprop-2-en-1-one (4)
was dissolved in 1,4-dioxane. The mixture was refluxed for
48 h and monitored by TLC. The reaction mixture was poured
into water (20 mL) and was extracted with EtOAc (3×20 mL),
and the combined organic layers were dried over anhydrous
Na2SO4 and concentrated under reduced pressure to give the
title compounds 8 (a-h).
(E)-1-(4-(2-((1-(4-fluorobenzyl)piperidin-4-yl)amino)
ethoxy)phenyl)-3-phenylprop-2-en-1-one. (8d)
Yield: 71%; MP: 202–203°C; MW: 458.57; Rf: 0.68; FT-IR (nmax
;
cm–1 KBr): 3193 (secondary N-H str), 3089 (Ar C-H str), 2973 (Ali
C-H str), 1489 (C=C of benzene), 1707 (C=O str), 1165 (C-F str),
782, 579 cm–1; 1H-NMR (400MHz, DMSO, TMS) d ppm: 8.14-7.16
(m, 4H, Ar-H), 8.04-7.61 (m, 2H,Aliphatic-H), 7.62-7.12 (m, 9H,
Ar-H), 4.13-2.98 (m, 4H, CH2x2), 3.65 (s, 2H,Aliphatic-H), 2.65-
1.48 (m, 9H, piperidine-H), 1.99 (s, 1H,NH); 13C-NMR (100MHz,
CDCl3) d, ppm: 189.9, 165.3, 161.4, 145.2, 135.4, 134.2, 130.7,
130.4, 129.5, 128.7, 128.4, 127.9, 121.4, 115.3, 114.8, 69.4,
64.8, 59.4, 51.9, 47.3, 30.9; Mass: 459.62 (M+1); Elemental
analysis for C29H31FN2O2: Calculated: C, 75.96; H, 6.81; N, 6.11;
Found: C, 75.99; H, 6.78; N, 6.15.
(E)-1-(4-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)phenyl)-
3-phenylprop-2-en-1-one. (8a)
Yield: 78%; MP: 182–183°C; MW: 440.58; Rf: 0.69; FT-IR
(nmax; cm–1 KBr): 3195 (secondary N-H str), 3082 (Ar C-H str),
2973 (Ali C-H str), 1494 (C=C of benzene), 1704 (C=O str), 882,
1
782 cm–1; H-NMR (400 MHz, DMSO, TMS) d ppm: 8.12-7.16
(m, 4H, Ar-H), 8.05-7.63 (m, 2H,Aliphatic-H), 7.64-7.25 (m, 10H,
Ar-H), 4.16–2.98 (m, 4H, CH2x2), 3.72(s, 2H,Aliphatic-H), 2.65-
1.48(m, 9H, piperidine-H), 1.94 (s, 1H,NH); 13C-NMR (100MHz,
CDCl3) d, ppm: 190.2, 165.3, 145.2, 138.7, 135.3, 130.5, 129.8,
128.9, 128.6, 128.4, 128.3, 127.8, 127.1, 121.4, 114.8, 69.4,
59.3, 51.8, 47.5, 30.7; Mass: 441.63 (M+1); Elemental analysis
for C29H32N2O2: Calculated: C, 79.06; H, 7.32; N, 6.36; Found: C,
79.09; H, 7.28; N, 6.39.
(E)-1-(4-(2-((1-(4-nitrobenzyl)piperidin-4-yl)amino)ethoxy)
phenyl)-3-phenylprop-2-en-1-one. (8e)
Yield: 82%; MP: 224–225°C; MW: 485.57; Rf: 0.78; FT-IR (nmax
;
cm–1 KBr): 3197 (secondary N-H str), 3092 (Ar C-H str), 2975
(Ali C-H str), 1529 (NO2 str), 1484 (C=C of benzene), 1709 (C=O
(E)-1-(4-(2-((1-(4-chlorobenzyl)piperidin-4-yl)amino)
ethoxy)phenyl)-3-phenylprop-2-en-1-one. (8b)
1
str), 789 cm–1; H-NMR (400MHz, DMSO, TMS) d ppm: 8.15-
7.96 (m, 4H, NO2-Ar-H), 8.11-7.18 (m, 4H, Ar-H), 8.06-7.58 (m,
2H,Aliphatic-H), 7.61-7.32 (m, 5H, Ar-H), 4.15-2.95 (m, 4H, CH2x2),
3.67 (s, 2H,Aliphatic-H), 2.64-1.51 (m, 9H, piperidine-H), 1.96 (s,
1H,NH); 13C-NMR (100MHz, CDCl3) d, ppm: 189.9, 165.3, 146.5,
145.2, 144.8, 135.3, 130.5, 129.7, 129.4, 128.7, 128.5, 127.9,
Yield: 82%; MP: 197–198°C; MW: 475.02; Rf: 0.73; FT-IR (nmax
;
cm–1 KBr): 3192 (secondary N-H str), 3084 (Ar C-H str), 2977
(Ali C-H str), 1482 (C=C of benzene), 1709 (C=O str), 815 (C-
Cl), 762 cm–1; 1H-NMR (400 MHz, DMSO, TMS) d ppm: 8.11-7.19
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Anticholinesterase inhibitor activity of chalcone derivative
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LAB/IN VITRO RESEARCH
123.7, 121.4, 114.8, 69.4, 64.8, 59.4, 51.8, 47.5, 30.8; Mass:
486.59 (M+1); Elemental analysis for C29H31N3O4: Calculated: C,
71.73; H, 6.43; N, 8.65; Found: C, 71.79; H, 6.38; N, 8.61.
Biological activity
AChE and BChE inhibition assay
(E)-1-(4-(2-((1-(4-methoxybenzyl)piperidin-4-yl)amino)
ethoxy)phenyl)-3-phenylprop-2-en-1-one. (8f)
Acetylcholinesterase and butylcholinesterase were purchased
from Sigma Aldrich and the other chemicals were obtained
from Fluka. The compound solution was prepared in a mix-
ture of DMSO (5 ml) and methanol (5 ml) and diluted in 0.1 M
KH2PO4/K2HPO4 buffer (pH 8.0) to obtain final assay concentra-
tions. All experiments were performed at controlled tempera-
ture and humidity in triplicate in a 96-well plate reader. Each
well contained 50 µl of potassium phosphate buffer (KH2PO4/
K2HPO4, 0.1 M, pH 8), 25 µl sample dissolved in 50% metha-
nol and 50% DMSO and 25 µl enzyme. They were preincubat-
ed for 15 min at room temperature, and then 125 µl DTNB (3
mM in buffer) was added and determined spectrometrically
at 405 nm. The IC50 values were determined graphically using
Graph Pad Prism [29].
Yield: 75%; MP: 212–213°C; MW: 470.60; Rf: 0.71; FT-IR (nmax
;
cm–1 KBr): 3193 (secondary N-H str), 3098 (Ar C-H str), 2974
(Ali C-H str), 2826 (Ar-OCH3), 1487 (C=C of benzene), 1712
(C=O str), 1232 (C-O str of OCH3), 764 cm–1; 1H-NMR (400MHz,
DMSO, TMS) d ppm: 8.14-7.23 (m, 4H, NO2-Ar-H), 8.08-7.62
(m, 2H,Aliphatic-H), 7.58-6.87 (m, 9H, Ar-H), 4.11-2.93 (m, 4H,
CH2x2), 3.93 (s, 3H, Ar-OCH3), 3.69 (s, 2H,Aliphatic-H), 2.61-1.46
(m, 9H, piperidine-H), 1.95 (s, 1H,NH); 13C-NMR (100MHz, CDCl3)
d, ppm: 189.8, 165.3, 159.2, 145.2, 135.4, 132.5, 130.9, 130.5,
129.6, 128.7, 128.4, 127.9, 121.4, 114.8, 114.1, 69.4, 64.8, 59.3,
55.9, 51.7, 47.4, 30.8; Mass: 471.63 (M+1); Elemental analysis
for C30H34N2O3: Calculated: C, 76.57; H, 7.28; N, 5.95; Found: C,
76.63; H, 7.25; N, 5.98.
Self-mediated Ab (1–42) aggregation assay
(E)-1-(4-(2-((1-(4-methylbenzyl)piperidin-4-yl)amino)
ethoxy)phenyl)-3-phenylprop-2-en-1-one. (8g)
For measuring the effect on self-mediated Ab (1–42) aggrega-
tion, thioflavin T-based fluorometric assay was utilized. Initially,
the stock solutions of 5 mM Ab (1–42) samples (GL Biochem,
Shanghai) were prepared by dissolving in DMSO and freez-
ing at –20°C. Experiments were conducted in the presence/
absence of various concentration of title compounds; peptide
was shaken in 50 mM phosphate buffer (pH 7.4) containing
100 mM NaCl at 37°C for 10 h (final Ab concentration 100 µM)
and incubated. The experiments were performed in triplicate
and the IC50 value for each inhibitor was calculated.
Yield: 71%; MP: 228–229°C; MW: 454.60; Rf: 0.61; FT-IR (nmax
;
cm–1 KBr): 3198 (secondary N-H str), 3092 (Ar C-H str), 2982
(Ali C-H str), 2967 (C-H str of methyl), 1482 (C=C of benzene),
1
1718 (C=O str), 775 cm–1; H-NMR (400MHz, DMSO, TMS) d
ppm: 8.12-7.21 (m, 4H, NO2-Ar-H), 8.06-7.64 (m, 2H,Aliphatic-H),
7.59-7.14 (m, 9H, Ar-H), 4.12-2.95 (m, 4H, CH2x2), 3.68 (s,
2H,Aliphatic-H), 2.63-1.48 (m, 9H, piperidine-H), 2.43 (s, 3H,
Ar-CH3), 1.97 (s, 1H,NH); 13C-NMR (100MHz, CDCl3) d, ppm:
189.8, 165.2, 145.2, 136.8, 135.9, 135.1, 130.6, 130.1, 129.7,
128.7, 128.5, 128.4, 127.8, 121.3, 114.9, 69.4, 64.8, 59.5, 51.8,
47.5, 21.4, 30.7; Mass: 455.57 (M+1); Elemental analysis for
C30H34N2O2: Calculated: C, 79.26; H, 7.54; N, 6.16; Found: C,
79.29; H, 7.59; N, 6.12.
b-secretase inhibition assay
For this assay, a b-secretase fluorescence resonance energy
transfer assay kit (P2985, PanVera) was utilized according to
the manufacturer’s protocol. Due to cleavage catalyzed by b-
Secretase, the weakly fluorescent substrate becomes highly
fluorescent and the rate of proteolysis at the early stage ac-
curately indicates the concentration of the active enzyme. The
fluorescence signal was recorded at lem=585 nm (lexc=535
nm) by use of a microplate reader. The inhibition percentag-
es corresponding to the presence of different concentrations
of test compound were calculated by the following equation:
100×[(Fi/Fo)/100], where Fi and Fo are the fluorescence inten-
sities obtained for b-secretase in the presence and absence of
the desired inhibitor, respectively. The IC50 values compound
6d was estimated after 3 measurements.
(E)-1-(4-(2-((1-(3,4-dichlorobenzyl)piperidin-4-yl)amino)
ethoxy)phenyl)-3-phenylprop-2-en-1-one. (8h)
Yield: 69%; MP: 235-236oC; MW: 509.47; Rf: 0.68; FT-IR (nmax
;
cm–1 KBr): 3194 (secondary N-H str), 3097 (Ar C-H str), 2981
(Ali C-H str), 1487 (C=C of benzene), 1714 (C=O str), 817 (C-Cl),
768 cm–1; 1H-NMR (400MHz, DMSO, TMS) d ppm: 8.13-7.18 (m,
4H, NO2-Ar-H), 8.08-7.58 (m, 2H,Aliphatic-H), 7.64-7.21 (m, 8H,
Ar-H), 4.14-2.98 (m, 4H, CH2x2), 3.69 (s, 2H,Aliphatic-H), 2.65-
1.45 (m, 9H, piperidine-H), 1.95 (s, 1H,NH); 13C-NMR (100MHz,
CDCl3) d,ppm: 189.7, 165.3, 145.2, 135.3, 135.1, 131.8, 130.7,
130.1, 129.8, 129.4, 129.1, 128.8, 128.5, 128.4, 127.9, 121.4,
114.8, 69.4, 64.2, 59.4, 51.8, 47.5, 30.8; Mass: 510.52 (M+1);
Elemental analysis for C29H30ClN2O2: Calculated: C, 68.37; H,
5.94; N, 5.50; Found: C, 68.42; H, 5.91; N, 5.47.
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Anticholinesterase inhibitor activity of chalcone derivative
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LAB/IN VITRO RESEARCH
Table 1. In vitro inhibition of AChE and BuChE of compound 8(a–h).
AChE inhibition
(IC50, µM)*
BuChE inhibition
Selectivity Index
(SI)**
Code
(IC50, µM)*
8a
H
32.34 0.26
10.03 0.54
18.11 0.33
14.23 0.15
28.62 0.22
4.22 0.34
1.12 0.13
24.54 0.21
0.02 0.006
21.72 0.11
0.67
1.93
1.56
1.86
0.87
2.23
2.88
1.06
105.5
8b
4-Cl
19.45 0.15
28.42 0.42
26.50 0.28
25.01 0.31
9.45 0.25
3.23 0.12
26.11 0.18
2.11 0.33
8c
4-Br
8d
4-F
8e
4-NO2
4-OCH3
4-CH3
3,4-Cl
8f
8g
8h
Donepezil
* Data are expressed as means standard deviation (SD) of at least three independent experiments; ** The selectivity index of
inhibitor has been determined by BuChE/AChE.
Results and Discussion
were characterized by the appearance of strong bands at
3198–3189 cm–1, which is attributed to the N-H group. The band
corresponds to the C-H benzene ring that appears at 3098–
3082 cm–1. Furthermore, another band at 2826 cm–1 is attrib-
uted to the stretching vibrations of the aromatic OCH3group.
The CH3 group of aromatic rings appears at 2982 cm–1. Many
strong absorption bands appear at 1718–1704 cm–1, which
confirm the existence of a C=O group. Another band at 1529
cm–1 is attributed to the stretching vibrations of the aromatic
NO2 group. Furthermore, another band at 817–815 cm–1 is at-
tributed to the stretching vibrations of the aromatic Cl group.
The 1H NMR spectra of title compounds 8(a–h) reveal a mul-
tiple corresponding to the phenyl ring at 8.11–7.18 ppm. The
aliphatic CH2 proton appear at 4.13–2.97 ppm. A resonance
due to piperidine proton was found in the range of 2.65–1.45
ppm. The substituted benzene ring proton appeared as a sin-
glet multiple at 7.63–7.24 ppm. Finally, all the structures of ti-
tle derivatives 8 (a–h) were confirmed by elemental analysis
and mass spectra.
Chemistry
Scheme 1: Synthesis of designed analogues of chalcones, 8
(a–h). Reagents and condition: EtOH, NaOH, stirring 24 h; (b)
1,2-di bromo ethane, K2CO3, DMF, 80°C; (c): BOC2O, TEA, THF,
rt, 12 h; (d) 10% Pd/C, H2, methanol, 24 h; (e) substituted ben-
zyl bromide or chloride, THF, reflux, 24 h; (f) 10% HCl in meth-
anol, 24 h; (g) 1,4-dioxane, reflux, 48 h.
To obtain the designed chalcones, a cost-effective synthesis
was devised, as presented in scheme 1. The synthesis was
initially started with condensation of benzaldehyde (1) with
4-hydroxy acetophenone (2) under reflux in the presence of
ethanol to yield compound 3. It was later allowed to undergo
nucleophilic substitution reaction with 1,2-dibromoethane in
DMF for 8 h at 80°C to furnish compound 4. In the next step,
the synthesis has been aimed at development of substitut-
ed 4-amino-1-benzylpiperidines. The primary amine of ben-
zyl-4-aminopiperidine was BOC-protected with the help of
tert-butyloxy carbonyl in the presence of tetrahydrofuran to
furnish a compound which then was debenzylated in the pres-
ence of methanol and alkylated with the corresponding sub-
stituted benzyl bromide in tetrahydrofuran. Compound 7 was
obtained by means of BOC-deprotection in 10% alc HCl solu-
tion. The last step corresponds to etherification of compound
4 with 7 in the presence of KOH in DMF at 40°C, as indicated
by TLC, for the appropriate time.
Pharmacological activity
Due to involvement of choline esterase enzyme in the degra-
dation of Ach in AD patients, the designed target compound
was rigorously analyzed for the determination of inhibitory
activity against AChE and BuChE. As presented in Table 1, the
entire set of target compounds showed considerable inhibi-
tion of AChE and BuChE, with varying degrees of inhibition
pattern. Among the series, the most potent inhibition was in
the compound 8g, which has the p-methyl as substituent on
the piperidine against both of the tested enzymes (AChE and
BuChE). The inhibitory activity showed that 8g inhibits AChE
more prominently than BuChE, as interpreted with the help of
The structure of the title compound has been ascertained
with the help of various spectroscopic analyses. It has been
found that the FT-IR spectra of all title compounds 8(a–h)
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Anticholinesterase inhibitor activity of chalcone derivative
© Med Sci Monit, 2017; 23: 3311-3317
LAB/IN VITRO RESEARCH
Table 2. Compound 8g showed considerably affinity in inhib-
iting the b-secretase and Ab aggregation.
O
H
N
Ab aggregation
(IC50, µM)a
b-secretase
(IC50, µM)**
O
Compound
N
8 (a–h)
8g
18.21 2.3
11.3 0.3
23.14 1.8
6.1 0.8
Order of activity
4-CH3>4-OCH3>4-CL>4-F>4-Br>3,4-di chloro>4-NO2>H
Curcumin
* Data are expressed as means standard deviation (SD) of at
least three independent experiments.
Figure 1. Structure-activity relationship of target compound for
choline esterase inhibitory activity.
a selectivity index (SI) of 2.88. The conversion of methyl to me-
thoxy, as in the case of 8f, renders the compound approximate-
ly 3-fold less active against AChE and BuChE than the parent
compound. The least activity was observed in compound 8a,
which contains no substituent with IC50 of 32.34 µM against
AChE, with improved inhibition against BuChE. With the in-
troduction of an electron-withdrawing group, p-chloro led to
marked improvement in the activity against AChE, with mar-
ginal improvement against BuChE. The activity against AChE
was markedly reduced in compound 8c, with SI index of 1.56.
The activity against ChE was further enhanced, together with
mild improvement against BuChE, in the case of compound
8d. Compound 8e showed significant reduction in the inhibi-
tory activity against AChE, with mild improvement in the case
of BuChE, with SI of 0.87. The di-chloro-containing compound
8h showed drastic reduction in the activity as compared to its
mono-chloro substituted derivative 8b, together with notice-
able change in SI value. In comparison to the inhibitory activ-
ity of Donepezil, none of the synthesized compounds showed
equal or better activity.
The most potent compound, 8g, has been further evaluated to
assess inhibitory activity against b-secretase and Ab aggrega-
tion. This assay is important because current therapeutic ap-
proaches to treat AD have focussed on targeting the various
pathways that leads to AD progression. Several studies con-
firmed that b-secretase plays a significant role in the cleavage
of amyloid precursor, which leads to the production of Ab pep-
tide. Particularly, in the case of AD, this pathway is involved
in cognitive decline and offers beneficial and symptomatic ef-
fects when targeted specifically. As presented in Table 2, com-
pound 8g showed considerable action in inhibiting b-secre-
tase and Ab aggregation, but not as prominent as compared
to curcumin as a standard.
Conclusions
A novel class of chalcone derivatives has been synthesized and
characterized with the aid of various spectroscopic methods.
Our results show that this novel class of chalcone derivative is
a selective inhibitor of AChE, with considerable action against
b-secretase and Ab aggregation. This study demonstrates po-
tential value in developing drugs against AD and warrants
further investigation to generate more advanced analogues.
Results of the structure-activity relationship study suggest that
the activity is greatly dependent on the type and nature of the
substituents (Figure 1). On close inspection, the inhibitory ac-
tivity of the compounds containing an electron-donating group
showed much stronger activity than in the electron-withdraw-
ing group. It was surprising to find that the di-chloro-substitut-
ed compound is less potent than the mono-chloro derivative.
Conflict of interest
The authors have declared no conflict of interest.
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