Design, synthesis and preliminary structure-activity relationship investigation of nitrogen-containing chalcone derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors: A further study based on Flavokawain B Mannich base derivatives

Article abstract of DOI:10.3109/14756366.2015.1050009

In order to study the structure-activity relationship of Flavokawain B Mannich-based derivatives as acetylcholinesterase (AChE) inhibitors in our recent investigation, 20 new nitrogen-containing chalcone derivatives (4 a-8d) were designed, synthesized, and evaluated for AChE inhibitory activity in vitro. The results suggested that amino alkyl side chain of chalcone dramatically influenced the inhibitory activity against AChE. Among them, compound 6c revealed the strongest AChE inhibitory activity (IC₅₀ value: 0.85 μmol/L) and the highest selectivity against AChE over BuChE (ratio: 35.79). Enzyme kinetic study showed that the inhibition mechanism of compound 6c against AChE was a mixed-type inhibition. The molecular docking assay showed that this compound can both bind with the catalytic site and the peripheral site of AChE.

Full text of DOI:10.3109/14756366.2015.1050009

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1050009
!
RESEARCH ARTICLE
Design, synthesis and preliminary structure–activity relationship
investigation of nitrogen-containing chalcone derivatives as
acetylcholinesterase and butyrylcholinesterase inhibitors:
a further study based on Flavokawain B Mannich base derivatives
Haoran Liu1, Haoqun Fan1, Xiaohui Gao2, Xueqing Huang1, Xianjun Liu1, Linbo Liu1, Chao Zhou1, Jingjing Tang1,
Qiuan Wang1, and Wukun Liu1
2
1College of Chemistry and Chemical Engineering, Hu’nan University, Changsha, China and College of Pharmacy, Hu’nan University of Chinese
Medicine, Changsha, China
Abstract
Keywords
In order to study the structure–activity relationship of Flavokawain B Mannich-based derivatives
as acetylcholinesterase (AChE) inhibitors in our recent investigation, 20 new nitrogen-
containing chalcone derivatives (4 a–8d) were designed, synthesized, and evaluated for AChE
inhibitory activity in vitro. The results suggested that amino alkyl side chain of chalcone
dramatically influenced the inhibitory activity against AChE. Among them, compound 6c
revealed the strongest AChE inhibitory activity (IC50 value: 0.85 mmol/L) and the highest
selectivity against AChE over BuChE (ratio: 35.79). Enzyme kinetic study showed that the
inhibition mechanism of compound 6c against AChE was a mixed-type inhibition. The
molecular docking assay showed that this compound can both bind with the catalytic site
and the peripheral site of AChE.
Acetylcholinesterase inhibitors, Alzheimer’s
disease, chalcones, structure–activity
relationship
History
Received 21 February 2015
Revised 7 May 2015
Accepted 7 May 2015
Published online 17 July 2015
Introduction
application or in the development, we supposed that tertiary
amine groups were the possible key pharmacophores for AChE
Alzheimer’s disease (AD), one of the most common diseases in
the elderly population, is a chronic and progressive neurodegen-
erative disorder characterized by memory loss, language impair-
ment, and intellectual ability degression1–3. Although the precise
aetiology of AD is not elucidated enough, the decrease of
acetylcholine level in brain was thought as the widely approved
reason and AChE inhibitors were primary drugs for the therapy of
inhibitory activity13–15
.
Based on our previous investigations, a new series of chalcone
derivatives with different tertiary amine groups were synthesized
and evaluated for their biological activity. Additionally, we
measured their Logarithm 1-octanol/water partition coefficients
(log P values), which can be used to evaluate the ability to
penetrate the blood brain barrier (BBB). Then kinetic experiments
were performed to clarify their binding mode to AChE and
molecular docking studies were carried out to explore their
binding mode with AChE and BuChE.
AD which can improve central cholinergic function4,5
.
In recent years, natural products were considered to be new
promising sources for the treatment of AD. Many natural products
or their derivatives were discovered or synthesized which revealed
AChE inhibitory activity6–11. Flavokawain B, a natural product
with chalcone scaffold, was selected to be as material to
synthesize a series of Flavokawain B Mannich-base derivatives
which revealed a moderate AChE inhibitory activity in our
previous study12. Among them, the derivatives containing
dimethylamine, diethylamine, piperidine, or pyrrolidine groups
showed better inhibitory activity than others. In order to study the
structure–activity relationship (SAR) of Flavokawain B deriva-
tives as AChE inhibitors, we decided to modify the structures. By
studying the characteristics of AChE inhibitors in clinical
Materials and methods
Chemistry
All chemicals and reagents were of analytical reagent grade and
used without further purification. The melting points were
1
measured on a WRS-lA melting point detector. H NMR spectra
were recorded on a Bruker 400 MHz instrument (Bruker Nano,
Inc., Billerica, MA) in CDCl3 with TMS as the internal reference.
Mass spectra were obtained from Finnigan LCQ Advantage MAX
by electrospray ionization (ESI-MS) (ThermoFinnigan, San Jose,
CA). Infrared spectrum was obtained from Shimadzu Infinity-1
infrared spectrometer (Shimadzu Corporation, Kanagawa, Japan).
The purity of compounds was checked by Shimadzu LC-20 A
high-performance liquid chromatography (Shimadzu Corporation,
Kanagawa, Japan).
Address for correspondence: Associate Professor, Haoran Liu, College of
Chemistry and Chemical Engineering, Hu’nan University, Changsha
410082, China. Tel: +86 731 83860540. E-mail: pharmaliu@126.com

2
H. Liu et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Synthesis of 30-hydroxychalcone (2)
J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3033, 2941, 2863, 2756,
1655, 1603, 1588, 1339, 1231, 1175, 835, 769. MS m/z (ESI): 359
[M + H]+.
Benzaldehyde (2.6 mL, 26 mmol), EtOH (20 mL), 20% NaOH
(10 mL) were added into a solution containing 3-hydroxyaceto-
phenone (2.72 g, 20 mmol). After stirring at room temperature for
24 h, 15% HCl was added to adjust the pH of solution to 3,
followed by the appearance of the precipitate. The reaction
mixture was filtrated and a light yellow solid product was gained
with a yield of 87.8%. Mp: 175–177 ꢀC.
(E)-1-(3-(5-bromopentyloxy)phenyl)-3-phenylprop-2-en-1-one (3d)
Compound 3d was synthesized from compound 2 (2.24 g,
10 mmol) with K2CO3 (4.13 g, 30 mmol) in DMF (20 mL) and
1,5-dibromopentane (8.2 mL, 60 mmol), followed by purification
using silica-gel column chromatography with ethyl acetate/
petroleum ether (1:18, v/v) as an eluent. Yield: 70.6%, white
solid. m.p. 91–93 ꢀC. 1H NMR (400 MHz, CDCl3) ꢀ (ppm): 1.63–
1.71 (2H, m, CH2CH2CH2), 1.80–1.87 (2H, m, CH2CH2Br), 1.91–
1.98 (2H, m, OCH2CH2), 3.47 (2H, t, J ¼ 6.0 Hz, CH2CH2Br),
4.08 (2H, t, J ¼ 6.0, OCH2CH2), 7.11–7.16 (1H, m, 40-H), 7.39–
7.45 (5H, m, 2-H and 3-H and 4-H and 5-H and 6-H), 7.52 (1H, d,
J ¼ 16.0 Hz, a-H), 7.58–7.66 (3H, m, 20-H and 50-H and 60-H),
7.82 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3033, 2941,
2863, 2758, 1655, 1603, 1588, 1339, 1231, 1175, 835, 768. MS
m/z (ESI): 373 [M + H]+.
General procedure for the synthesis of 3a–3e
A mixture of compound 2 (2.24 g, 10 mmol), K2CO3 (4.13 g,
30 mmol), and a,!-dibromoalkane (60 mmol) were dissolved in
DMF (20 mL) and stirred at 80 ꢀC until compound 2 disappeared
by monitoring with TLC. Then the mixture was poured into ice
water (120 mL), extracted with ethyl acetate (2 ꢁ 100 mL), and
washed with saturated NaCl solution (2 ꢁ 100 mL). The organic
phase was dried with anhydrous Na2SO4 and concentrated in
vacuum. Then the residue was purified by a silica-gel column
chromatography to afford the final product.
(E)-1-(3-(6-bromohexyloxy)phenyl)-3-phenylprop-2-en-1-one (3e)
1 (E)-1 -(3-(2-bromoethoxy)phenyl)-3-phenylprop-2-en-1-one (3a)
Compound 3e was synthesized from compound 2 (2.24 g,
10 mmol) with K2CO3 (4.13 g, 30 mmol) in DMF (20 mL) and
1,6-dibromohexane (9.2 mL, 60 mmol), followed by purification
using silica-gel column chromatography with ethyl acetate/
petroleum ether (1:20, v/v) as an eluent. Yield: 78.7%, white
solid. m.p. 88–90 ꢀC. 1H NMR (400 MHz, CDCl3) ꢀ (ppm): 1.57–
1.56 (4H, m, CH2CH2CH2CH2 and CH2CH2CH2CH2), 1.78–1.92
(4H, m, CH2CH2Br and OCH2CH2), 3.43 (2H, t, J ¼ 6.0 Hz,
CH2CH2Br), 4.06 (2H, t, J ¼ 6.0, OCH2CH2), 7.11–7.15 (1H, m,
40-H), 7.40–7.45 (5H, m, 2-H and 3-H and 4-H and 5-H and 6-H),
7.51 (1H, d, J ¼ 16.0 Hz, a-H), 7.56–7.66 (3H, m, 20-H and 50-H
and 60-H), 7.82 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3030,
2955, 2877, 2759, 1657, 1609, 1587, 1506, 1341, 1230, 1174,
834, 771. MS m/z (ESI): 387 [M + H]+.
Compound 3a was synthesized from compound 2 (2.24 g,
10 mmol) with K2CO3 (4.13 g, 30 mmol) in DMF (20 mL) and
1,2-dibromoethane (5.2 mL, 60 mmol), followed by purification
using silica-gel column chromatography with ethyl acetate/
petroleum ether (1:12, v/v) as an eluent. Yield: 53.8%, light
yellow solid; m.p. 122–124 ꢀC. 1H NMR (400 MHz, CDCl3) ꢀ
(ppm): 3.69 (2H, t, J ¼ 6.0 Hz, BrCH2), 4.37 (2H, t, J ¼ 6.0 Hz,
OCH2), 7.12–7.16 (1H, m, 40-H), 7.40–7.46 (5H, m, 2-H and 3-H
and 4-H and 5-H and 6-H), 7.51 (1H, d, J ¼ 16.0 Hz, a-H), 7.55–
7.67 (3H, m, 20-H and 50-H and 60-H), 7.82 (1H, d, J ¼ 16.0 Hz,
b-H). IR (KBr) ꢁ/cmꢂ1: IR (KBr) ꢁ/cmꢂ1: 3028, 2952, 2866,
2763, 1659, 1601, 1576, 1341, 1221, 1182, 833, 769. MS m/z
(ESI): 331 [M + H]+.
(E)-1-(3-(3-bromopropoxy)phenyl)-3-phenylprop-2-en-1-one (3b)
General procedure for the synthesis of 4a–8d
Compound 3b was synthesized from compound 2 (2.24 g,
10 mmol) with K2CO3 (4.13 g, 30 mmol) in DMF (20 mL) and
1,3-dibromopropane (6.2 mL, 60 mmol), followed by purification
using silica-gel column chromatography with ethyl acetate/
petroleum ether (1:14, v/v) as an eluent. Yield: 61.3%, light
yellow solid. m.p. 114–116 ꢀC. 1H NMR (400 MHz, CDCl3) ꢀ
(ppm): 2.34–2.53 (2H, m, CH2CH2Br), 3.66 (2H, t, J ¼ 6.0 Hz,
CH2CH2Br), 4.21 (2H, t, J ¼ 6.0, OCH2CH2), 7.12–7.15 (1H, m,
40-H), 7.40–7.44 (5H, m, 2-H and 3-H and 4-H and 5-H and 6-H),
7.52 (1H, d, J ¼ 16.0 Hz, a-H), 7.58–7.67 (3H, m, 20-H and 50-H
and 60-H), 7.83 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3030,
2955, 2877, 2761, 1658, 1609, 1587, 1506, 1338, 1229, 1175,
834, 771. MS m/z (ESI): 345 [M + H]+.
A mixture of compound 3a–3e (1 mmol), secondary amines
(dimethylamine, diethylamine, piperidine, and pyrrolidine),
K2CO3 (0.412 g, 3 mmol), NaI (0.008 g, 0.05 mmol), acetone
(15 mL) were refluxed. The solvent was removed, then the residue
was dissolved with ethyl acetate (30 mL), followed by washing
with saturated NaCl solution (2 ꢁ 20 mL). The organic phase was
dried with anhydrous Na2SO4 and evaporated in vacuum, giving a
crude product which was purified on silica gel with different ratio
of methanol/dichloromethane as an eluent to afford the com-
pounds 4a–8d.
(E)-1-(3-(2-(dimethylamino)ethoxy)phenyl)-3-phenylprop-2-en-1-
one (4a)
(E)-1-(3-(4-bromobutoxy)phenyl)-3-phenylprop-2-en-1-one (3c)
The crude product was gained by the reaction of compound 3a
(0.331 g, 1 mmol) with dimethylamine (0.34 mL, 3 mmol). Then it
was purified using silica-gel column chromatography with
methanol/dichloromethane (1:40, v/v) as an eluent to give light
Compound 3c was synthesized from compound 2 (2.24 g,
10 mmol) with K2CO3 (4.13 g, 30 mmol) in DMF (20 mL) and
1,4-dibromobutane (7.2 mL, 60 mmol), followed by purification
using silica-gel column chromatography with ethyl acetate/
petroleum ether (1:16, v/v) as an eluent. Yield: 65.4%, white
solid. m.p. 108–110 ꢀC. 1H NMR (400 MHz, CDCl3) ꢀ (ppm):
1.92–2.03 (2H, m, OCH2CH2), 2.09–2.15 (2H, m, CH2CH2Br),
3.51 (2H, t, J ¼ 6.0 Hz, CH2CH2Br), 4.09 (2H, t, J ¼ 6.0,
OCH2CH2), 7.11–7.15 (1H, m, 40-H), 7.40–7.45 (5H, m, 2-H
and 3-H and 4-H and 5-H and 6-H), 7.51 (1H, d, J ¼ 16.0 Hz,
a-H), 7.57–7.66 (3H, m, 20-H and 50-H and 60-H), 7.81 (1H, d,
1
yellow solid with a yield of 62.9%. m.p. 123–125 ꢀC. H NMR
(400 MHz, CDCl3) ꢀ (ppm): 2.87 (6H, s, 2 ꢁ NCH3), 3.40 (2H, t,
J ¼ 6.0 Hz, OCH2CH2), 4.55 (2H, t, J ¼ 6.0 Hz, OCH2CH2), 7.16–
7.19 (1H, m, 40-H), 7.42–7.47 (5H, m, 2-H and 3-H and 4-H and
5-H and 6-H), 7.52 (1H, d, J ¼ 16.0 Hz, a-H), 7.57–7.69 (3H, m,
20-H and 50-H and 60-H), 7.83 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr)
ꢁ/cmꢂ1: 3030, 2949, 2875, 2782, 1665, 1606, 1593, 1510, 1339,
1221, 1168, 836, 767. MS m/z (ESI): 296 [M + H]+.

DOI: 10.3109/14756366.2015.1050009
A study based on Flavokawain B Mannich base derivatives
3
(E)-1-(3-(2-(diethylamino)ethoxy)phenyl)-3-phenylprop-2-en-1-one 2877, 2771, 1658, 1609, 1587, 1506, 1348, 1229, 1175, 834, 772.
MS m/z (ESI): 310 [M + H]+.
(4b)
The crude product was gained by the reaction of compound 3a
(0.331 g, 1 mmol) with diethylamine (0.31 mL, 3 mmol). Then it
was purified using silica-gel column chromatography with
(E)-1-(3-(3-(diethylamino)propoxy)phenyl)-3-phenylprop-2-en-1-
one (5b)
methanol/dichloromethane (1:45, v/v) as an eluent to give light The crude product was gained by the reaction of compound 3b
1
yellow solid with a yield of 72.8%. m.p. 105–107 ꢀC. H NMR (0.344 g, 1 mmol) with diethylamine (0.31 mL, 3 mmol). Then it
(400 MHz, CDCl3)
ꢀ
(ppm): 1.49 (6H, t, J ¼ 8.0 Hz, was purified using silica-gel column chromatography with
2 ꢁ NCH2CH3), 3.29 (4H, s, J ¼ 6.0 Hz, 2 ꢁ NCH2CH3), 3.50 methanol/dichloromethane (1:50, v/v) as an eluent to give light
1
(2H, t, J ¼ 6.0 Hz, OCH2CH2), 4.63 (2H, t, J ¼ 6.0 Hz, yellow oil with a yield of 72.7%. H NMR (400 MHz, CDCl3) ꢀ
OCH2CH2), 7.14–7.18 (1H, m, 40-H), 7.42–7.46 (5H, m, 2-H (ppm): 1.12 (6H, t, J ¼ 6.0 Hz, 2 ꢁ NCH2CH3), 1.99–2.03 (2H, m,
and 3-H and 4-H and 5-H and 6-H), 7.49 (1H, d, J ¼ 16.0 Hz, OCH2CH2), 2.56–2.64 (6H, m, 3 ꢁ NCH2), 4.18 (2H, t,
a-H), 7.55–7.68 (3H, m, 20-H and 50-H and 60-H), 7.83 (1H, d, J ¼ 6.0 Hz, OCH2CH2), 7.10–7.15 (1H, m, 40-H), 7.40–7.45
J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3031, 2965, 2869, 2785, (5H, m, 2-H and 3-H and 4-H and 5-H and 6-H), 7.52 (1H, d,
1663. 1606, 1589, 1509, 1337, 1220, 1172, 834, 767. MS m/z J ¼ 16.0 Hz, a-H), 7.61–7.68 (3H, m, 20-H and 50-H and 60-H),
(ESI): 324 [M + H]+.
7.82 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3031, 2955,
2869, 2771, 1653, 1605, 1577, 1509, 1337, 1221, 1173, 831, 769.
MS m/z (ESI): 338 [M + H]+.
(E)-3-phenyl-1-(3-(2-(piperidin-1-yl)ethoxy)phenyl)prop-2-en-1-
one (4c)
(E)-3-phenyl-1-(3-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-
1-one (5c)
The crude product was gained by the reaction of compound 3a
(0.331 g, 1 mmol) with piperidine (0.3 mL, 3 mmol). Then it was
purified using silica-gel column chromatography with methanol/
dichloromethane (1:45, v/v) as an eluent to give light yellow solid
with a yield of 77.4%. m.p. 98–100 ꢀC. 1H NMR (400 MHz,
CDCl3) ꢀ (ppm): 1.48–1.56 (2H, m, CH2CH2CH2), 1.72–1.78
(4H, m, piperidine-H), 2.73 (4H, t, J ¼ 6.0 Hz, 2 ꢁ NCH2CH2),
2.98 (2H, t, J ¼ 6.0 Hz, NCH2CH2), 4.32 (2H, t, J ¼ 6.0 Hz,
OCH2CH2), 7.13–7.16 (1H, m, 40-H), 7.40–7.46 (5H, m, 2-H and
3-H and 4-H and 5-H and 6-H), 7.52 (1H, d, J ¼ 16.0 Hz, a-H),
7.61–7.69 (3H, m, 20-H and 50-H and 60-H), 7.82 (1H, d,
J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3029, 2961, 2875, 2771,
1659, 1608, 1591, 1511, 1339, 1221, 835, 771. MS m/z (ESI): 336
[M + H]+.
The crude product was gained by the reaction of compound 3b
(0.344 g, 1 mmol) with piperidine (0.3 mL, 3 mmol). Then it was
purified using silica-gel column chromatography with methanol/
dichloromethane (1:50, v/v) as an eluent to give light yellow oil
with a yield of 82.3%. 1H NMR (400 MHz, CDCl3) ꢀ (ppm): 1.51–
1.56 (2H, m, CH2CH2CH2), 1.79–1.85 (4H, m, piperidine-H),
2.16–2.21 (2H, m, OCH2CH2), 2.57–2.61 (6H, m, 3 ꢁ NCH2),
4.10 (2H, t, J ¼ 6.0 Hz, OCH2CH2), 7.11–7.14 (1H, m, 40-H),
7.39–7.44 (5H, m, 2-H and 3-H and 4-H and 5-H and 6-H), 7.52
(1H, d, J ¼ 16.0 Hz, a-H), 7.59–7.67 (3H, m, 20-H and 50-H and
60-H), 7.83 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cm ꢂ 1: 3032,
2944, 2861, 2776, 1659, 1610, 1592, 1503, 1338, 1222, 1172,
832, 768. MS m/z (ESI): 350 [M + H]+.
(E)-3-phenyl-1-(3-(2-(pyrrolidin-1-yl)ethoxy)phenyl)prop-2-en-1-
one (4d)
(E)-3-phenyl-1-(3-(3-(pyrrolidin-1-yl)propoxy)phenyl)prop-2-en-
1-one (5d)
The crude product was gained by the reaction of compound 3a
(0.331 g, 1 mmol) with pyrrolidine (0.25 mL, 3 mmol). Then it
was purified using silica-gel column chromatography with
methanol/dichloromethane (1:40, v/v) as an eluent to give light
The crude product was gained by the reaction of compound 3b
(0.344 g, 1 mmol) with pyrrolidine (0.25 mL, 3 mmol). Then it
was purified using silica-gel column chromatography with
methanol/dichloromethane (1:45, v/v) as an eluent to give light
1
1
yellow oil with a yield of 66.7%. H NMR (400 MHz, CDCl3) ꢀ
yellow oil with a yield of 70.4%. H NMR (400 MHz, CDCl3) ꢀ
(ppm): 1.78–1.88 (2H, m, pyrrolidine-H), 2.66–2.74 (4H, m,
2 ꢁ NCH2CH2), 2.97 (2H, t, J ¼ 6.0 Hz, 2 ꢁ NCH2CH2), 4.21 (2H,
t, J ¼ 6.0 Hz, OCH2CH2), 7.14–7.17 (1H, m, 40-H), 7.39–7.44
(5H, m, 2-H and 3-H and 4-H and 5-H and 6-H), 7.51 (1H, d,
J ¼ 16.0 Hz, a-H), 7.59–7.68 (3H, m, 20-H and 50-H and 60-H),
7.81 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3033, 2948,
2859, 2785, 1657, 1605, 1589, 1511, 1340, 1220, 1175, 833, 768.
MS m/z (ESI): 322 [M + H]+.
(ppm): 1.51–1.56 (2H, m, CH2CH2CH2), 1.82–1.88 (4H, m,
pyrrolidine-H), 2.02–2.07 (2H, m, OCH2CH2), 2.61–2.72 (6H, m,
3 ꢁ NCH2), 4.13 (2H, t, J ¼ 6.0 Hz, OCH2CH2), 7.10–7.14 (1H,
m, 40-H), 7.38–7.45 (5H, m, 2-H and 3-H and 4-H and 5-H and 6-
H), 7.51 (1H, d, J ¼ 16.0 Hz, a-H), 7.61–7.68 (3H, m, 20-H andꢂ510-
H and 60-H), 7.82 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cm
:
3031, 2955, 2868, 2767, 1657, 1609, 1589, 1508, 1345, 1226,
1174, 830, 769. MS m/z (ESI): 336 [M + H]+.
(E)-1-(3-(3-(dimethylamino)propoxy)phenyl)-3-phenylprop-2-en-
1-one (5a)
(E)-1-(3-(4-(dimethylamino)butoxy)phenyl)-3-phenylprop-2-en-1-
one (6a)
The crude product was gained by the reaction of compound 3b The crude product was gained by the reaction of compound 3c
(0.344 g, 1 mmol) with dimethylamine (0.34 mL, 3 mmol). Then it (0.358 g, 1 mmol) with dimethylamine (0.34 mL, 3 mmol). Then it
was purified using silica-gel column chromatography with was purified using silica-gel column chromatography with
methanol/dichloromethane (1:45, v/v) as an eluent to give a methanol/dichloromethane (1:50, v/v) as an eluent to give light
light yellow oil with a yield of 75.3%. 1H NMR (400 MHz, yellow solid with a yield of 78.5%. m.p. 85–87 ꢀC. 1H NMR
CDCl3) ꢀ (ppm): 1.99–2.09 (2H, m, OCH2CH2), 2.31 (6H, s, (400 MHz, CDCl3) ꢀ (ppm): 1.75–1.88 (4H, m, OCH2CH2 and
2 ꢁ NCH3), 2.52 (2H, t, J ¼ 6.0 Hz, NCH2CH2), 4.11 (2H, t, NCH2CH2), 2.36 (6H, s, 2 ꢁ NCH3), 2.50 (2H, t, J ¼ 6.0 Hz,
J ¼ 6.0 Hz, OCH2CH2), 7.12–7.15 (1H, m, 40-H), 7.39–7.44 (5H, NCH2CH2), 4.07 (2H, t, J ¼ 6.0 Hz, OCH2CH2), 7.11–7.15 (1H,
m, 2-H and 3-H and 4-H and 5-H and 6-H), 7.52 (1H, d, m, 40-H), 7.39–7.45 (5H, m, 2-H and 3-H and 4-H and 5-H
J ¼ 16.0 Hz, a-H), 7.58–7.67 (3H, m, 20-H and 50-H and 60-H), and 6-H), 7.52 (1H, d, J ¼ 16.0 Hz, a-H), 7.58–7.67 (3H, m,
7.82 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3030, 2955, 20-H and 50-H and 60-H), 7.81 (1H, d, J ¼ 16.0 Hz, b-H).

4
H. Liu et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
IR (KBr) ꢁ/cmꢂ1: 3033, 2941, 2863, 2756, 1655, 1603, 1588, J ¼ 16.0 Hz, a-H), 7.58–7.67 (3H, m, 20-H and 50-H and 60-H),
1339, 1231, 1175, 835, 768. MS m/z (ESI): 324 [M + H]+.
7.82 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ 1: 3031, 2954,
2867, 2779, 1656, 1605, 1589, 1505, 1340, 1225, 1175, 835, 767.
MS m/z (ESI): 338 [M + H]+.
(E)-1-(3-(4-(diethylamino)butoxy)phenyl)-3-phenylprop-2-en-1-
one (6b)
(E)-1-(3-(5-(diethylamino)pentyloxy)phenyl)-3-phenylprop-2-en-
1-one (7b)
The crude product was gained by the reaction of compound 3c
(0.358 g, 1 mmol) with diethylamine (0.31 mL, 3 mmol). Then it
was purified using silica-gel column chromatography with The crude product was gained by the reaction of compound 3d
methanol/dichloromethane (1:55, v/v) as an eluent to give light (0.372 g, 1 mmol) with diethylamine (0.31 mL, 3 mmol). Then it
1
yellow oil with a yield of 68.4%. H NMR (400 MHz, CDCl3) ꢀ was purified using silica-gel column chromatography with
(ppm): 1.21 (6H, t, J ¼ 6.0 Hz, 2 ꢁ NCH2CH3), 1.78–1.90 (4H, m, methanol/dichloromethane (1:60, v/v) as an eluent to give light
1
OCH2CH2 and NCH2CH2), 2.36 (6H, s, 2 ꢁ NCH3), 2.72–2.84 yellow oil with a yield of 62.7%. H NMR (400 MHz, CDCl3) ꢀ
(6H, m, 3 ꢁ NCH2CH2), 4.07 (2H, t, J ¼ 6.0 Hz, OCH2CH2), (ppm): 1.44 (6H, t, J ¼ 6.8 Hz, 2 ꢁ NCH2CH3), 1.58–1.66 (2H, m,
7.11–7.15 (1H, m, 40-H), 7.39–7.45 (5H, m, 2-H and 3-H and 4-H CH2CH2CH2), 1.86–1.99 (4H, m, NCH2CH2 and OCH2CH2),
and 5-H and 6-H), 7.52 (1H, d, J ¼ 16.0 Hz, a-H), 7.59–7.68 (3H, 3.07 (2H, t, J ¼ 6.0 Hz, NCH2CH2), 3.20 (4H, m, 2 ꢁ NCH2CH3),
m, 20-H and 50-H and 60-H), 7.82 (1H, d, J ¼ 16.0 Hz, b-H). IR 4.08 (2H, t, J ¼ 6.0 Hz, OCH2CH2), 7.10–7.14 (1H, m, 40-H),
(KBr) ꢁ/cmꢂ1: 3033, 2948, 2858, 2751, 1658, 1606, 1586, 1341, 7.40–7.47 (5H, m, 2-H and 3-H and 4-H and 5-H and 6-H), 7.52
1230, 1175, 836, 769. MS m/z (ESI): 352 [M+H]+.
(1H, d, J ¼ 16.0 Hz, a-H), 7.58–7.66 (3H, m, 20-H and 50-H and
60-H), 7.82 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3032,
2958, 2875, 2776, 1656, 1605, 1587, 1508, 1342, 1223, 1175,
835, 768. MS m/z (ESI): 366 [M + H]+.
(E)-3-phenyl-1-(3-(4-(piperidin-1-yl)butoxy)phenyl)prop-2-en-1-
one (6c)
The crude product was gained by the reaction of compound 3c
(0.358 g, 1 mmol) with piperidine (0.3 mL, 3 mmol). Then it was
purified using silica-gel column chromatography with methanol/
(E)-3-phenyl-1-(3-(5-(piperidin-1-yl)pentyloxy)phenyl)prop-2-en-
1-one (7c)
dichloromethane (1:55, v/v) as an eluent to give light yellow solid The crude product was gained by the reaction of compound 3d
with a yield of 76.5%. m.p. 73–75 ꢀC. 1H NMR (400 MHz, CDCl3) (0.372 g, 1 mmol) with piperidine (0.30 mL, 3 mmol). Then it was
ꢀ (ppm): 1.46–1.51 (2H, m, CH2CH2CH2), 1.65–1.71 (4H, m, purified using silica-gel column chromatography with methanol/
piperidine-H), 1.74–1.88 (4H, m, OCH2CH2 and NCH2CH2), dichloromethane (1:60, v/v) as an eluent to give light yellow oil
2.47–2.56 (6H, m, 3 ꢁ NCH2CH2), 4.06 (2H, t, J ¼ 6.0 Hz, with a yield of 75.3%. 1H NMR (400 MHz, CDCl3) ꢀ (ppm): 1.43–
OCH2CH2), 7.10–7.13 (1H, m, 40-H), 7.38–7.45 (5H, m, 2-H and 1.54 (4H, m, piperidine-H and CH2CH2CH2), 1.58–1.61 (6H, m,
3-H and 4-H and 5-H and 6-H), 7.51 (1H, d, J ¼ 16.0 Hz, a-H), NCH2CH2 and piperidine-H), 1.79–1.88 (2H, m, OCH2CH2),
7.58–7.67 (3H, m, 20-H and 50-H and 60-H), 7.81 (1H, d, 2.38–2.51 (6H, m, 3 ꢁ NCH2CH2), 4.05 (2H, t, J ¼ 6.0 Hz,
J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3031, 2952, 2861, 2755, OCH2CH2), 7.10–7.13 (1H, m, 40-H), 7.40–7.46 (5H, m, 2-H
1657, 1608, 1589, 1339, 1232, 1174, 838, 771. MS m/z (ESI): 364 and 3-H and 4-H and 5-H and 6-H), 7.51 (1H, d, J ¼ 16.0 Hz,
[M + H]+.
a-H), 7.58–7.67 (3H, m, 20-H and 50-H and 60-H), 7.82 (1H, d,
J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3029, 2956, 2873, 2771,
(ESI): 378 [M + H]+.
(E)-3-phenyl-1-(3-(4-(pyrrolidin-1-yl)butoxy)phenyl)prop-2-en-1- 1657, 1608, 1585, 1508, 1343, 1222, 1175, 834, 767. MS m/z
one (6d)
The crude product was gained by the reaction of compound 3c
(0.358 g, 1 mmol) with pyrrolidine (0.25 mL, 3 mmol). Then it
was purified using silica-gel column chromatography with
(E)-3-phenyl-1-(3-(5-(pyrrolidin-1-yl)pentyloxy)phenyl)prop-2-
en-1-one (7d)
methanol/dichloromethane (1:50, v/v) as an eluent to give light The crude product was gained by the reaction of compound 3d
1
yellow oil with a yield of 64.1%. H NMR (400 MHz, CDCl3) ꢀ (0.372 g, 1 mmol) with pyrrolidine (0.25 mL, 3 mmol). Then it
(ppm): 1.85–1.98 (8H, m, pyrrolidine-H and OCH2CH2 and was purified using silica-gel column chromatography with
NCH2CH2), 2.75–2.92 (6H, m, 3 ꢁ NCH2CH2), 4.07 (2H, t, methanol/dichloromethane (1:55, v/v) as an eluent to give light
1
J ¼ 6.0 Hz, OCH2CH2), 7.10–7.13 (1H, m, 40-H), 7.39–7.46 (5H, yellow oil with a yield of 68.1%. H NMR (400 MHz, CDCl3) ꢀ
m, 2-H and 3-H and 4-H and 5-H and 6-H), 7.52 (1H, d, (ppm): 1.51–1.59 (2H, m, CH2CH2CH2), 1.69–1.82 (6H, m,
J ¼ 16.0 Hz, ꢂ-H), 7.59–7.67 (3H, m, 20-H and 50-H and 60-H), NCH2CH2 and pyrrolidine-H), 1.91–1.96 (2H, m, OCH2CH2),
7.82 (1H, d, J ¼ 16.0 Hz, ꢃ-H). IR (KBr) ꢁ/cmꢂ1: 3032, 2952, 2.68–2.79 (6H, m, 3 ꢁ NCH2CH2), 4.04 (2H, t, J ¼ 6.0 Hz,
2863, 2755, 1656, 1608, 1583, 1341, 1233, 1175, 837, 768. MS OCH2CH2), 7.10–7.13 (1H, m, 40-H), 7.40–7.46 (5H, m, 2-H
m/z (ESI): 350 [M + H]+.
and 3-H and 4-H and 5-H and 6-H), 7.51 (1H, d, J ¼ 16.0 Hz,
a-H), 7.58–7.67 (3H, m, 20-H and 50-H and 60-H), 7.82 (1H, d,
J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3031, 2959, 2878, 2777,
1656, 1607, 1589, 1507, 1341, 1223, 1174, 833, 768. MS m/z
(ESI): 364 [M + H]+.
(E)-1-(3-(5-(dimethylamino)pentyloxy)phenyl)-3-phenylprop-2-
en-1-one (7a)
The crude product was gained by the reaction of compound 3d
(0.372 g, 1 mmol) with dimethylamine (0.34 mL, 3 mmol). Then it
was purified using silica-gel column chromatography with
(E)-1-(3-(6-(dimethylamino)hexyloxy)phenyl)-3-phenylprop-2-en-
1-one (8a)
methanol/dichloromethane (1:55, v/v) as an eluent to give light
1
yellow oil with a yield of 74.2%. H NMR (400 MHz, CDCl3) ꢀ The crude product was gained by the reaction of compound 3e
(ppm): 1.60–1.66 (2H, m, CH2CH2CH2), 1.87–1.92 (2H, m, (0.386 g, 1 mmol) with diethylamine (0.31 mL, 3 mmol). Then it
NCH2CH2), 1.95–2.01 (2H, m, OCH2CH2), 2.86 (6H, s, was purified using silica-gel column chromatography with
2 ꢁ NCH3), 3.11 (2H, t, J ¼ 6.0 Hz, NCH2CH2), 4.08 (2H, t, methanol/dichloromethane (1:55, v/v) as an eluent to give
J ¼ 6.0 Hz, OCH2CH2), 7.09–7.13 (1H, m, 40-H), 7.38–7.46 light yellow oil with a yield of 65.6%. 1H NMR (400 MHz,
(5H, m, 2-H and 3-H and 4-H and 5-H and 6-H), 7.52 (1H, d, CDCl3) ꢀ (ppm): 1.48–1.61 (4H, m, CH2CH2CH2CH2 and

DOI: 10.3109/14756366.2015.1050009
A study based on Flavokawain B Mannich base derivatives
5
CH2CH2CH2CH2), 1.80–1.96 (4H, m, NCH2CH2 and OCH2CH2), PBS (pH ¼ 7.4). Both the octanol and the aqueous phase were
2.86 (6H, s, 3 ꢁ NCH3), 3.09 (2H, t, J ¼ 6.0 Hz, NCH2CH2), 4.05 saturated with each other before use. Different from the described
(2H, t, J ¼ 6.0, OCH2CH2), 7.09–7.13 (1H, m, 40-H), 7.38–7.47 method above, in the present experiment, ultrasonic method was
(5H, m, 2-H and 3-H and 4-H and 5-H and 6-H), 7.51 (1H, d, applied to accelerate this progression. The assay mixture
J ¼ 16.0 Hz, a-H), 7.58–7.67 (3H, m, 20-H and 50-H and 60-H), containing test compounds was shaken at 37 ꢀC over night and
7.82 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) ꢁ/cmꢂ1: 3029, 2961, then centrifuged at 2000 rpm for 20 min, followed by the analysis
2877, 2773, 1657, 1606, 1585, 1507, 1343, 1223, 1174, 833, 771. with HPLC. A C18 column (250 nm ꢁ 4.6 mm, 5 mm) was used
MS m/z (ESI): 352 [M + H]+.
with the mobile phase of methanol–0.1% triethylamine (TEA)
(85:15, v/v) at a flow rate of 1.0 mL minꢂ1 and the detection
(E)-1-(3-(6-(diethylamino)hexyloxy)phenyl)-3-phenylprop-2-en-1- wavelength of 319 nm at 32 ꢀC. Experiments were conducted in
one (8b)
triplicate and log P values were calculated.
The crude product was gained by the reaction of compound 3e
(0.386 g, 1 mmol) with dimethylamine (0.34 mL, 3 mmol). Then it
AChE and butyrylcholinesterase (BuChE) inhibition assay
was purified using silica-gel column chromatography with AChE/BuChE activity assays were conducted by the Ellman
methanol/dichloromethane (1:60, v/v) as an eluent to give light method with slight modification17. Each compound was dissolved
1
yellow oil with a yield of 68.3%. H NMR (400 MHz, CDCl3) ꢀ in Tween 80 and diluted with water to various concentrations
(ppm): 1.44 (6H, t, J ¼ 6.8 Hz, 2 ꢁ NCH2CH3), 1.49–1.60 (4H, m, immediately before use. The reaction mixture containing 40 mL
CH2CH2CH2CH2 and CH2CH2CH2CH2), 1.81–1.95 (4H, m, AChE/BuChE, 100 mL acetylthiocholine iodide/S-butyrylthiocho-
NCH2CH2 and OCH2CH2), 3.05 (2H, t, J ¼ 6.0 Hz, NCH2CH2), line iodide, 2.76 mL Na2HPO4/NaH2PO4 buffer (pH 8.0,
3.19 (4H, m, 2 ꢁ NCH2CH3), 4.04 (2H, t, J ¼ 6.0, OCH2CH2), 0.1 mol/L), and 100 mL different concentrations of tested com-
7.09–7.14 (1H, m, 40-H), 7.38–7.46 (5H, m, 2-H and 3-H and 4-H pounds were incubated at 30 ꢀC for 25 min. Then the reaction was
and 5-H and 6-H), 7.52 (1H, d, J ¼ 16.0 Hz, a-H), 7.58–7.66 (3H, terminated via adding 100 mL 20% sodium dodecylsulfate (SDS)
m, 20-H and 50-H and 60-H), 7.81 (1H, d, J ¼ 16.0 Hz, b-H). IR and 100 mL 10 mmol/L 5,5’-dithiobis-(2-nitrobenzoic acid)
(KBr) ꢁ/cmꢂ1: 3031, 2960, 2875, 2778, 1657, 1605, 1589, 1508, (DTNB) was added to generate the yellow anion 5-thio-2-nitro-
1342, 1225, 1172, 834, 768. MS m/z (ESI): 380 [M + H]+.
benzoic acid. The absorbance of each assay mixture was measured
at 412 nm by UV spectroscopy. The IC50 values were calculated
by the Bliss method and expressed as mean SD of the replicates.
(E)-3-phenyl-1-(3-(6-(piperidin-1-yl)hexyloxy)phenyl)prop-2-en-
1-one (8c)
Kinetic studies
The crude product was gained by the reaction of compound 3e
(0.386 g, 1 mmol) with piperidine (0.3 mL, 3 mmol). Then it was Kinetic characterization of AChE was conducted by a reported
purified using silica-gel column chromatography with methanol/ method with some modifications18. Compound 6c was added into
dichloromethane (1:60, v/v) as an eluent to give light yellow oil the assay solution and preincubated with the enzyme at 30 ꢀC,
with a yield of 70.9%. 1H NMR (400 MHz, CDCl3) ꢀ (ppm): 1.37– followed by the addition of 100 mL acetylthiocholine iodide
1.45 (2H, m, piperidine-H), 1.49–1.57 (4H, m, 4H, m, including five concentrations. The assay solution contained
CH2CH2CH2CH2 and CH2CH2CH2CH2), 1.71–1.88 (8H, piperi- 100 mL compound 6c, 100 mL DTNB, 2.76 mL 0.1 mol/L
dine-H and NCH2CH2 and OCH2CH2), 2.57–2.73 (m, 6H, Na2HPO4/NaH2PO4 buffer (pH 8.0). Kinetic characterization of
3 ꢁ NCH2CH2), 4.05 (2H, t, J ¼ 6.0 Hz, OCH2CH2), 7.09–7.13 the hydrolysis of acetylthiocholine iodide catalyzed by AChE was
(1H, m, 40-H), 7.39–7.47 (5H, m, 2-H and 3-H and 4-H and 5-H conducted spectrometrically at 412 nm. Additionally, the parallel
and 6-H), 7.51 (1H, d, J ¼ 16.0 Hz, a-H), 7.60–7.67 (3H, m, 20-H control experiment was made without compound 6c in the
and 50-H and 60-H), 7.81 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr) mixture.
ꢁ/cmꢂ1: 3033, 2954, 2877, 2773, 1657, 1607, 1589, 1508, 1343,
1223, 1173, 833, 767. MS m/z (ESI): 392 [M + H]+.
Molecular docking
Molecular modelling was performed by Molecular Operating
Environment (MOE) software package (Chemical Computing
Group, Montreal, Canada). The X-ray crystallographic structures
(E)-3-phenyl-1-(3-(6-(pyrrolidin-1-yl)hexyloxy)phenyl)prop-2-en-
1-one (8d)
The crude product was gained by the reaction of compound 3e of AChE (PDB code: 1EVE) and BuChE (PDB code: 1P0I) were
(0.386 g, 1 mmol) with pyrrolidine (0.25 mL, 3 mmol). Then it gained from protein data bank. The 3D structure of the strongest
was purified using silica-gel column chromatography with AChE inhibitor 6c was established by virtue of the builder
methanol/dichloromethane (1:55, v/v) as an eluent to give light interface of MOE program (Chemical Computing Group, London,
1
yellow oil with a yield of 58.5%. H NMR (400 MHz, CDCl3) UK), and docked into the active site of the protein after energy
ꢀ
(ppm): 1.38–1.53 (4H, m, pyrrolidine-H), 1.58–1.65 being minimized. The Dock scoring in MOE software (Chemical
(2H, m, NCH2CH2), 1.79–1.88 (6H, CH2CH2CH2CH2 and Computing Group, Montreal, Canada) was done by ASE scoring
CH2CH2CH2CH2 and OCH2CH2), 2.50–2.61 (6H, function.
3 ꢁ NCH2CH2), 4.04 (2H, t, J ¼ 6.0 Hz, OCH2CH2), 7.10–7.14
(1H, m, 40-H), 7.39–7.47 (5H, m, 2-H and 3-H and 4-H and 5-H Results and discussion
and 6-H), 7.52 (1H, d, J ¼ 16.0 Hz, a-H), 7.60–7.66 (3H, m, 20-H
The synthetic routes to get the target compounds 4a–8d are
and 50-H and 60-H), 7.82 (1H, d, J ¼ 16.0 Hz, b-H). IR (KBr)
ꢁ/cmꢂ1: 3029, 29638, 2875, 2778, 1655, 1606, 1587, 1506, 1341,
1222, 1175, 835, 768. MS m/z (ESI): 378 [M + H]+.
outlined in Scheme 1. Compound 2 was gained from the reaction
of 3-hydroxyacetophenone with benzaldehyde in the presence of
NaOH/EtOH. Then, compound 2 was treated with dibromoalk-
anes and K2CO3 in N,N-dimethylformamide (DMF) at 80 ꢀC to
generate compounds 3a–3e. Finally, compounds 4a–8d were
Log P measurement
Octanol–water partition coefficients of compounds 4a–8d were achieved by refluxing the mixture including compounds 3a–3e
measured by the shake flask method described previously and secondary amines (dimethylamine, diethylamine, piperidine
with slight modification16. The aqueous phase was replaced by and pyrrolidine) in acetone in the presence of K2CO3 and NaI.

6
H. Liu et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Scheme 1. Reagents and conditions: (a) benzaldehyde, NaOH, EtOH, rt; (b) Br(CH2)nBr, K2CO3, DMF, 80 ꢀC; (c) secondary amine, K2CO3, NaI,
acetone, reflux.
Table 1. Inhibition of AChE and BuChE (IC50 values and selectivity) and log P values of nitrogen-containing chalcone derivatives.
a
IC50 (mmol/L)
Compound
R
n
AChE
BuChE
Selectivity for AChEb
Log Pc
2
4a
5a
6a
7a
8a
4b
5b
6b
7b
8b
4c
5c
6c
7c
8c
4500
4500
4500
132.18 10.03
–
415.05
1.04
422.47
4.86
6.34
0.55
4.55
2.62
5.63
6.25
1.70
0.48
35.79
0.65
0.60
0.16
2.34
22.00
1.81
2.38
0.02
–
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
33.23 2.61
127.12 13.56
22.25 1.02
3.82 0.23
5.54 0.29
62.45 3.72
97.47 5.84
25.24 1.33
1.50 0.07
4.41 0.36
25.61 2.12
33.34 0.89
0.85 0.04
2.98 0.21
3.64 0.22
17.36 1.28
33.32 1.64
1.42 0.09
2.48 0.11
3.46 0.24
10.54 0.86
1.54
1.70
1.73
1.65
1.63
1.76
1.79
1.73
1.69
1.81
1.84
1.79
1.77
1.89
1.92
1.88
1.86
1.95
1.98
1.94
1.68
N
4500
18.56 1.94
35.12 7.13
34.26 1.01
443.54 11.20
66.16 13.29
8.44 0.85
27.57 1.86
43.47 2.33
16.01 0.18
30.42 1.71
1.95 0.08
2.19 0.75
2.77 0.03
77.88 7.81
31.24 0.60
4.49 0.17
8.24 1.02
0.26 0.08
N
N
4d
5d
6d
7d
N
8d
Rivastigmine
aIC50 value: 50% inhibitory concentration (means SD of three experiments).
bSelectivity for AChE is defined as IC50 (BuChE)/IC50 (AChE).
cThe partition coefficients between octanol and buffer solution at pH 7.4 were determined by the classical shake-flask method.
The structures of the compounds were characterized by proton in Table 1. The results indicated that all compounds exhibited
nuclear magnetic resonance spectroscopy (1H NMR), infrared higher inhibitory activities against AChE than the precursor
spectrum (IR) and mass spectrometry (MS). In addition, the compound 2 (IC504500 mmol/L). Among them, compounds
purities of all synthesized compounds were confirmed to be 7a, 7b, 6c and 6d with IC50 values of 3.82, 1.50, 0.85, and
higher than 97% by HPLC.
1.42 mmol/L, respectively, showed more potent than Rivastigmine
Inhibiting effects of new synthesized compounds in the present (IC50 ¼ 10.54 mmol/L). Interestingly, compound 6c displayed
study against AChE and BuChE were evaluated by the modified the strongest AChE inhibitory activity (IC50 ¼ 0.85 mmol/L) and
Ellman method, using Rivastigmine as the positive control. The highest selectivity (ratio: 35.79).
half maximal inhibitory concentration (IC50 values) for AChE and Based on the enzyme inhibition data (Figure 1), it seemed that
BuChE as well as the selectivity for AChE are summarized the alteration of the spacer length between amino-alkyl side chain

DOI: 10.3109/14756366.2015.1050009
A study based on Flavokawain B Mannich base derivatives
7
Figure 1. (A) Effects of alkyl chain lengths on anti-AChE activities; (B) effects of alkyl chain lengths on selectivity for AChE.
Figure 2. The comparison of the inhibitory activity between the corresponding Flavokawain B derivatives and chalcone derivatives against AChE.
and chalcone scaffold could dramatically affect the AChE method in the drug discovery and development19–21. This method
inhibitory potency. Generally, the inhibitory potency is benefit for the screening for pharmacophore or important
(¼1/IC50*100) of chalcone derivatives presented a parabola structure domain in natural products, enhancing bioactivity or
profile with the growth of carbon spacer length. For dimethylmine attenuating side effects. For instance, Procaine, simplified from
or diethylamine containing chalcone derivatives, the compounds natural compound Cocaine, had stronger local anaesthesia effect
with a five-carbons spacer (compounds 7a and 7b) revealed the than Cocaine, but had no addiction. Rivastigmine, a simplified
highest inhibitory potency among the homologues. However, for compound from natural alkaloid Eserine, had more potent AChE
piperidine or pyrrolidine containing chalcone derivatives, the inhibition than Eserine as well as satisfactory stability. Thereby,
compounds with a four-carbons spacer (compounds 6c and 6d) simplified nitrogen chalcone derivatives from Flavokawain B
showed the highest inhibition activity among the homologues were more convenient to investigate the structure–activity rela-
against AChE yet. In addition, the compounds with a three- tionship (Appendix Figure A1) According to the results of the
carbons spacer showed the poorest inhibition activity among the present study, it indicated that not only the variations of amino-
homologues. alkyl groups but also the spacer length between amino-alkyl side
As shown in Figure 2, it appeared that some of the new chain and chalcone scaffold were important for AChE inhibitory
synthesized chalcone derivatives (compounds 6c, 6d, 7a and 7b) activity of chalcone derivatives.
exhibited 5–10 folds inhibitory activities against AChE than
Compound 6c, possessing the highest activity against AChE
relative Flavokawain B Mannich base derivatives which were among the new synthesized chalcone derivatives in the present
reported in our previous investigation. For the modification or investigation, was selected for kinetic studies. The linear
optimization of flavokawain B Mannich base derivatives, struc- Lineweaver–Burk equation of the Michaelis–Menten was applied
ture simplification was employed, which was a widely used to evaluate the inhibition profile. The graphical analysis of the

8
H. Liu et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
2. Pivtoraiko VN, Abrahamson EE, Leurgans SE, et al. Cortical
pyroglutamate amyloid-b levels and cognitive decline in
Alzheimer’s disease. Neurobiol Aging 2015;36:12–19.
´
3. Garcıa-Alberca JM. Cognitive intervention therapy as treatment for
behavior disorders in Alzheimer disease: evidence on efficacy and
steady-state inhibition data of compound 6c is shown in Appendix
Figure A2. According to the analysis, Km but not Vmax increased
with the increasing concentration of compound 6c, which
presented a mixed-type inhibition. The competitive inhibition
constant (Ki) and the non-competitive constant (Ki’) are
0.69 mmol/L and 1.19 mmol/L, respectively (Appendix: Table A1).
To explore the possible interacting mode of the chalcone
derivatives with AChE, molecular docking was performed for
compound 6c with software MOE2008 (Chemical Computing
Group, London, UK). As shown in Appendix Figure A3, this
compound exhibited multiple points binding modes with AChE
(Appendix Figure A3). In the top of the gorge, the aromatic
moiety adopted an appropriate orientation for its binding to PAS,
via the ꢄ–ꢄ stacking interaction with Trp279. The conformation
of the side chain conformed to the shape of the mid-gorge and
interacted with Tyr334. In the bottom of the gorge, the charged
nitrogen of piperidine ring was observed to bind to CAS via a
cation–ꢄ interaction between Trp84 and Phe330. That is, com-
pound 6c binds simultaneously to catalytic active sites and
peripheral anionic site of AChE, while it binds with BuChE only
with a cation–ꢄ interaction between Tyr312 and the nitrogen of
piperidine (Appendix Figure A3). These results may partial
explain its potent inhibition for AChE and its high selective
inhibition for AChE over BuChE.
´
neurobiological correlations. Neurologıa (English Edition) 2015;30:
8–15.
4. Molinuevo JL, Gauthier S. Benefits of combined cholinesterase
inhibitor and memantine treatment in moderate–severe Alzheimer’s
disease. Alzheimers Dement 2013;9:326–31.
5. Small G, Bullock R. Defining optimal treatment with cholinesterase
inhibitors in Alzheimer’s disease. Alzheimers Dement 2011;7:
177–84.
6. Silva T, Reis J, Teixeira J, et al. Alzheimer’s disease, enzyme targets
and drug discovery struggles: from natural products to drug
prototypes. Ageing Res Rev 2014;15:116–45.
7. Mukherjee PK, Kumar V, Mal M, et al. Acetylcholinesterase
inhibitors from plants. Phytomedicine 2007;14:289–300.
8. Li RS, Wang XB, Hu XJ, et al. Design, synthesis and evaluation of
flavonoid derivatives as potential multifunctional acetylcholinester-
ase inhibitors against Alzheimer’s disease. Bioorg Med Chem Lett
2013;23:2636–41.
9. Asadipour A, Alipour M, Jafari M, et al. Novel coumarin-3-
carboxamides bearing N-benzylpiperidine moiety as potent acetyl-
cholinesterase inhibitors. Eur J Med Chem 2013;70:623–30.
10. Liew SY, Khaw KY, Murugaiyah V, et al. Natural indole
butyrylcholinesterase inhibitors from Nauclea officinalis.
Phytomedicine 2015;22:45–8.
As a potential compound for treatment of AD, log P was
thought as an important physical chemistry parameter to valuate
or predict the ability to cross blood–brain barrier (BBB). It was
reported that the log P with the optimum central nervous system
(CNS) penetration was around 2 0.722. As shown in Table 1, log
P values of new synthesized compounds ranged from 1.54 to 1.98,
which indicated that all the compounds are possible sufficiently
lipophilic to pass the BBB in vivo.
11. Liu HR, Liu XJ, Fan HQ, et al. Design, synthesis and pharmaco-
logical evaluation of chalcone derivatives as acetylcholinesterase
inhibitors. Bioorg Med Chem 2014;22:6124–33.
12. Liu HR, Huang XQ, Lou DH, et al. Synthesis and acetylcholin-
esterase inhibitory activity of Mannich base derivatives flavokawain
B. Bioorg Med Chem Lett 2014;24:4749–53.
´
13. Konrath EL, Passos CS, Klein-Junior LC, et al. Alkaloids as a source
of potential anticholinesterase inhibitors for the treatment of
Alzheimer’s disease. J Pharm Pharmacol 2013;65:1701–25.
14. Darras FH, Wehle S, Huang G, et al. Amine substitution of
quinazolinones leads to selective nanomolar AChE inhibitors with
‘inverted’ binding mode. Bioorg Med Chem 2014;22:4867–81.
15. Darvesh S, Macdonald LR, Martin E. Selectivity of phenothiazine
cholinesterase inhibitors for neurotransmitter systems. Bioorg Med
Chem Lett 2013;23:3822–5.
16. Sharma J, Singla AK, Dhawan S. Zinc–naproxen complex: synthe-
sis, physicochemical and biological evaluation. Int J Pharm 2003;
260:217–27.
17. Ellman GL, Courtney KD, Andres VJ, et al. A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochem
Pharmacol 1961;7:88–95.
18. Skrzypek A, Matysiak J, Niewiadomy A, et al. Synthesis and
biological evaluation of 1,3,4-thiadiazole analogues as novel AChE
and BuChE inhibitors. Eur J Med Chem 2013;62:311–19.
19. Magedov IV, Kireev AS, Jenkins AR, et al. Structural simplification
of bioactive natural products with multicomponent synthesis. 4: 4H-
pyrano-[2,3-b]naphthoquinones with anticancer activity. Bioorg
Med Chem Lett 2012;22:5195–8.
20. Magedov IV, Manpadi M, Rozhkova E, et al. Structural simplifi-
cation of bioactive natural products with multicomponent synthesis:
dihydropyridopyrazole analogues of podophyllotoxin. Bioorg Med
Chem Lett 2007;17:1381–5.
Conclusions
The present studies indicated that the length of carbon chain-
linked chalcone scaffold and the amino alkyl group had markedly
influence in AChE inhibitory activity. Among them, compound 6c
revealed the strongest AChE inhibitory activity (IC50 value:
0.85 mmol/L) and highest selectivity (ratio: 35.79). Enzyme
kinetic study suggested that the inhibition mechanism of
compound 6c was a mixed-type inhibition. Besides, molecular
docking further explained its potent inhibition of AChE and high
selectivity for AChE over BuChE. Over all, compound 6c might
serve as a potential agent for the treatment of AD.
Declaration of interest
The authors report that that they have no conflicts of interest. The present
investigation was supported by the grant of ‘‘The Natural Science
Foundation of China (No. 21342015)’’, ‘‘The Project of Science and
Technology of Hu’nan Province (No. 2012SK3183)’’and ‘‘The Research
Funds for younger investigators of Hu’nan University (No.
HNU2013066)’’.
21. Yang R, Zhao R, Chen D, et al. Discovering selective agonists of
endothelial target for acetylcholine (ETA) via diversity-guided
pharmacophore simplification and simulation. Bioorg Med Chem
Lett 2004;14:3017–25.
References
1. Anand R, Gill KD, Mahdi AA. Therapeutics of Alzheimer’s disease: 22. Glave WR, Hansch CJ. Relationship between lipophilic character
past, present and future. Neuropharmacology 2014;76:27–50. and anesthetic activity. J Pharm SCI-US 1972;61:589–91.

DOI: 10.3109/14756366.2015.1050009
A study based on Flavokawain B Mannich base derivatives
9
Appendix
Figures A1–A3 and Table A1.
H N
Figure A1. Some examples of the derivatives
from natural products with structural
simplification.
2
H CO C
3
2
CH
O
O
3
N
H
N
O
O
Cocaine
Procaine
CH
H C
H
N
3
3
H
CH
N
3
O
N
O
H C
CH
3
3
H C
N
3
CH
O
3
O
CH
N
3
CH
3
Rivastigmine
Eserine
R R N
2
1
(
)
R R N
O
2
1
n
O
OH
O
O
O
Flavokawain B Mannich base derivatives
Nitrogen-containing chalcone derivatives
Figure A2. (A) Lineaweaver–Burk plot for
the inhibition of AChE by compound 6c; (B)
the replots of the intercept versus concentra-
tion of compound 6c; (C) the replots of the
slope versus concentration of compound 6c.

10 H. Liu et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure A3. Molecular modelling of compound 6c with AChE (A) and BuChE (B) generated with MOE2008.
Table A1. Kinetic parameters of AChE inhibition by compound 6c.
6c/mmol/L
Michaelis–Menten equation
Km/mmol/L
Vmax/DA/min
Ki/mmol/L
Ki’/mmol/L
0
0.58
1.16
1/v ¼ 191.67/[S] + 37.94
1/v ¼ 353.16/[S] + 54.60
1/v ¼ 514.20/[S] + 74.43
5.051
6.467
6.908
0.0264
0.0183
0.0134
0.69
1.19