Synthesis and acetylcholinesterase inhibitory activity of Mannich base derivatives flavokawain B

Article abstract of DOI:10.1016/j.bmcl.2014.07.087

A novel series of flavokawain B derivatives, chalcone Mannich bases (4-10) were designed, synthesized, characterized, and evaluated for the inhibition activity against acetylcholinesterase (AChE). Biological results revealed that four compounds displayed potent activities against AChE with IC₅₀values below 20 μM. Moreover, the most promising compound 8 was 2-fold more active than rivastigmine, a well-known AChE inhibitor. The log P values of 4-10 were around 2 which indicated that they were sufficiently lipophilic to pass blood brain barriers in vivo. Enzyme kinetic study suggested that the inhibition mechanism of compound 8 was a mixed-type inhibition. Meanwhile, the molecular docking showed that this compound can both bind with the catalytic site and the periphery of AChE.

Full text of DOI:10.1016/j.bmcl.2014.07.087

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4749–4753
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and acetylcholinesterase inhibitory activity of Mannich
base derivatives flavokawain B
⇑
Hao-ran Liu, Xue-qin Huang, Ding-hui Lou, Xian-jun Liu, Wu-kun Liu, Qiu-an Wang
College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of flavokawain B derivatives, chalcone Mannich bases (4–10) were designed, synthesized,
characterized, and evaluated for the inhibition activity against acetylcholinesterase (AChE). Biological
results revealed that four compounds displayed potent activities against AChE with IC50 values below
Received 23 April 2014
Revised 25 July 2014
Accepted 30 July 2014
Available online 27 August 2014
20 lM. Moreover, the most promising compound 8 was 2-fold more active than rivastigmine, a well-
known AChE inhibitor. The logP values of 4–10 were around 2 which indicated that they were sufficiently
lipophilic to pass blood brain barriers in vivo. Enzyme kinetic study suggested that the inhibition mech-
anism of compound 8 was a mixed-type inhibition. Meanwhile, the molecular docking showed that this
compound can both bind with the catalytic site and the periphery of AChE.
Keywords:
Flavokawain B
Chalcone Mannich bases
Synthesis
Ó 2014 Elsevier Ltd. All rights reserved.
AChE inhibitory activity
Alzheimer’s disease (AD), the most common cause of dementia,
is a progressive neurodegenerative disorder characterized by dete-
rioration of memory and cognition in elder patients. The main
cause of the loss of cognitive functions in AD patients is a contin-
uous decline of the cholinergic neurotransmission in cortical and
other regions of the human brain. Cholinergic neurotransmission
is mediated by the neurotransmitter acetylcholine (ACh), which
is released and carried out the effect followed by rapidly hydrolysis
via acetylcholinesterase (AChE).1 So AChE plays an important role
in central and peripheral nervous systems for its main function to
regulate the impulse transmission at cholinergic synapses. Mean-
while, recent studies have identified that AChE could also play a
key role in accelerating the assembly of b-amyloid into amyloid
fibrils which are characteristically found in the brain cells of AD
accumulatively some side effects or demerits such as hepatotoxic-
ity, periphery side effect, short half life, or gastrointestinal tract
excitement cast a shadow in the clinic application.7 Therefore,
the search for novel AChE inhibitors is still of great interest.
Recently, it becomes a trend to discover cholinesterase inhibi-
tors from natural products because of their slight side effects. Chal-
cones, an open-chain flavonoid, exhibit diverse biological
8
0
0
0
activities. Flavokawain B (3), 2 -hydroxy-4 ,6 -dimethoxy chal-
cone, is isolated from the roots of kava and has been shown to
exhibit an interesting spectrum of pharmacological effects.9
Besides its remarkable anti-proliferative activity against different
,2
,10
1
1,12
cancer cell lines,
flavokawain B also exhibited anti-inflamma-
tory, anti-hepatitis, hepatoprotective and antinociceptive
1
3,14
effects.
However, the natural resource of flavokawain B (3) is
3
,4
patients.
limited due to the low content in the plants, which negatively
influenced its further bioactivity evaluation. Therefore, in our
investigation flavokawain B (3) was first total synthesized from
phloroglucinol by the Houben–Hoesch acetylization, regioselective
O-methylation and Claisen–Schmidt condensation with benzalde-
hyde. According to the structure of acetylcholine AChE substrate
and some listed AChE inhibitors illustrated in Figure 1, a novel ser-
ies of flavokawain B (3) derivatives, chalcone Mannich bases
(4–10), were designed and synthesized. Mannich base is possible
to improve the properties of original compounds, such as
One of the major therapeutic strategies adopted for primarily
5
symptomatic AD is based on the cholinergic hypothesis. The
widely used treatment is to inhibiting AChE to increase the ACh
level in brain, which remains the most effective therapeutic
approach against AD for several decades.6 Currently, four AChE
inhibitors have been approved by the European and US regulatory
authorities: tacrine, donepezil, galanthamine and rivastigmine
(Fig. 1). They are important agents for the treatment of AD, but
1
5,16
bioactivity.
Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterases; ACh,
acetylcholine; PAS, peripheral anionic sites; LogP, octanol/water partition; CNS,
central nervous system; BBB, blood brain barrier.
The title compounds were synthesized according to the route
shown in Scheme 1. 2,4,6-Trihydroxyacetophenone (1) was acety-
lated by using the Houben–Hoesch reaction of phloroglucinol
⇑
Corresponding author. Tel.: +86 0731 83860540.
E-mail address: wangqa@hnu.edu.cn (Q.-a. Wang).
http://dx.doi.org/10.1016/j.bmcl.2014.07.087
960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
0

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H.-r. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4749–4753
Table 1
Half-inhibitory concentration against AChE and logP values for compounds 4–10
Compound
–NR1R2
IC50a
(
lM) ± SD
LogPb
4
5
N
N
18.8 ± 1.6
1.61
1.69
15.1 ± 0.89
6
N
54.8 ± 2.21
1.77
7
8
9
N
N
N
16.1 ± 1.13
4.15 ± 0.28
265 ± 18.6
1.74
1.75
1.63
O
N
10
N
12.4 ± 0.83
1.58
—
*
Rivastigmine
—
10.54 ± 0.86
a
IC50 values of compounds represent the concentration that caused 50% enzyme
activity loss.
Figure 1. The chemistry structure of acetylcholine (ACh) and the listed AChE
inhibitors.
b
The partition coefficients of in the octanol/buffer solution at pH 7.4 were
determined by the classical shake-flask method.
*
Used for positive control for IC50
.
according to the reported method.17 Taking advantage of hydrogen
bonding between the carbonyl and an ortho phenolic-OH group,
regioselective O-methylation of compound 1 with dimethyl sulfate
and anhydrous potassium carbonate in dry acetone gained com-
pound 2 in 68% yield. Chalcone flavokawain B (3) was prepared
by the Claisen–Schmidt condensation of compound 2 with 1 equiv
of benzaldehyde in the presence of KOH in a mixture of water and
EtOH (1:4). Mannich reaction of compound 3 with HCHO and dif-
ferent amines in 2-propanol gave novel chalcone Mannich base
derivatives 4–10 in 70–80% yield. The classical conditions of Man-
nich reaction for the hydroxyl compounds are based on the sub-
strate, amine and formaldehyde ratio in alcohol with prolonged
heating. In our experiment, flavokawain B, formaldehyde and
amines in 1:1:1 ratio, respectively, were refluxed and stirred in iso-
propanol for 2–3 h to afford the C-aminomethylated derivatives.
as an important physical chemistry parameter to valuate or predict
a compound’s ability to cross BBB, and widely used in medicinal
chemistry investigation. The logP of 4–10 were determined by
1
8
the classical shake-flask method using reverse phase high perfor-
1
9
mance liquid chromatography (RP-HPLC). Hansch presumed that
the logP with optimum CNS penetration was around 2 ± 0.7. As
showed in Table 1, the logP values of 4–10 were 1.61, 1.69, 1.77,
1.74, 1.75, 1.63 and 1.58, respectively, which indicated 4–10 were
sufficiently lipophilic to pass blood brain barriers in vivo.
All the synthetic compounds were tested for the acetylcholines-
terases (AChE) inhibitory activities in vitro according to the modi-
2
0
fied Ellman’s method.
Rivastigmine, a well-known AChE
inhibitor, was used as the positive control. The inhibit ratio against
AChE was calculated according to the absorbance of the product in
the AChE-catalyzed reaction and the results were shown in Table 1.
Preliminary investigation demonstrated that flavokawain B (3)
0
The regioselectivity of the reaction occurred preferentially at C-3
position of B ring of chalcone backbone. The compounds 4–10 were
1
1
ꢀ4
characterized by MS, H NMR and IR spectra. In the H NMR spectra
showed poor inhibiting effect against AChE (IC50 >10 ), but most
0
of 4–10, the signal for H-3 was disappeared, and a signal at d
of its derivatives revealed potent AChE inhibitory activities. Fur-
thermore, the inhibition was found to be influenced markedly by
4
.1–4.5 indicated the presence of an aminomethyl group at C-8
0
of flavokawain B.
aminomethylene group at C-3 position. Among them, compound
For a compound with potential effect to treat AD, the ability to
penetrate the blood brain barrier (BBB) is vital. Although the fac-
tors that affect the penetration of a drug from the systemic circu-
lation into the central nervous system (CNS) were complicated,
logarithm 1-octanol/water partition coefficient (logP) was thought
8, a flavokawain B derivative contained piperidine group, was
found to have the most potent inhibitory activity with IC50 value
of 4.15 ± 0.28
with rivastigmine (IC50 = 10.54 ± 0.86
dimethylamine, diethylamine, dipropylamine, pyrrolidine and
lM, which was 2-fold of inhibitory effect compared
2
1
l
M, lit. : 9.12 lM). The
HO
OH
HO
OH
O
O
OH
O
O
OH
a
b
c
OH
phloroglucinol
OH
O
O
O
1
2
flavokawain B (3)
CH3
CH3
1
2
NR R
OH
1
2
N
1
2
4
NR R =
5 NR R =
N
N
O
d
1
2
7 NR1R2=
6
8
NR R =
N
N
O
O
1
2
1 2
NR R =
9 NR R =
N
O
1
2
1
0 NR R =
N
N
Scheme 1. The synthetic route of flavokawain B (3) and its Mannich base derivatives 4–10. Reagents and conditions: (a) (1) CH
3
CN, ZnCl
2
, Et
2
O, HCl, 0 °C; (2) concentrated
, isopropanol, reflux 3 h.
HCl, H O, reflux 2 h; (b) (CH SO , K CO , acetone, reflux 2 h; (c) (1) KOH, CH OH, benzaldehyde; (2) HCl (aq); (d) concentrated HCl, HCHO, HNR R
2
3
)
2
4
2
3
3
1 2

H.-r. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4749–4753
4751
Table 2
Kinetic parameters of AChE inhibited by compound 8
Amin 1
ꢀ
K
l
M
K
i
0
/l
M
Compound 8/
lM
Michaelis–Menten equation
K
m
/mM
v
max
/
D
i
/
0
2
4
1/v = 87.80/[S]+23.44
1/v = 145.26/[S]+34.74
1/v = 218.32/[S]+47.13
3.749
4.183
4.632
0.0427
0.0288
0.0212
.2
.4
2.75
4.58
N-methyl piperazine-contained flavokawain B also showed potent
AChE inhibitory activities. However, compound 9, a hydrophilic
morpholine ring-contained flavokawain B, showed significant
reduced AChE inhibitory activity, which might suggest that the
hydrophobic interactions in this binding region of the enzymes is
required for intensive inhibition.22
Compound 8 was selected for further kinetic measurement for
its most potent inhibitory against AChE among these compounds.
The linear Lineweaver–Burk equation of the Michaelis–Menten
was applied to evaluate the inhibition profile.23 The graphical anal-
ysis of the steady-state inhibition data of compound 8 was shown
in Figure 2. These results showed that increasing the concentration
of compound 8 resulted in different slopes and intercepts. Accord-
To elucidate further the binding mechanism of compound 8
with AChE which X-ray crystal structure (PDB code: 1EVE) was
obtained from protein data bank, we performed a docking study
on compound 8 by utilizing the Molecular Operating Environment
(MOE). It is well known that two distinct binding sites exist in the
active pocket of AChE peripheral binding site (PAS) and catalytic
active site (CAS), located at the entrance and the bottom of the
active-site gorge, respectively. These sites are characterized by
two tryptophan residues, Trp 84 at the active site and Trp 279 at
the mouth of the gorge (PAS). Compound 8 manifested binding
mode with AChE in Figure 3. The chalcone moiety adopted an
appropriate orientation for its binding to PAS via the p–p stacking
interactions with Trp 279 and Tyr 334, and their ring-to-ring dis-
tance was between 4.48 and 4.44 Å. Moreover, the conformation
of the side chain conformed to the shape of the mid-gorge, and
in the bottom of the gorge, the charged nitrogen of piperidine ring
25
ing to Table 2, K
m
but not vmax increased with increasing concen-
tration of compound 8, which presented a mixed-type inhibition.
It seemed that compound 8 can bind both with the free enzyme
and the enzyme–substrate complex with different bind equilib-
was observed to bind to the CAS via a cation–
p interaction
rium constants. The inhibition constants for the free enzyme (E),
between Trp 84 and Phe 330. These results suggested that it can
both bind with the catalytic site and the periphery of AChE.
In conclusion, flavokawain B was totally synthesized for the first
time from phloroglucinol by the Houben–Hoesch acetylization,
regioselective O-methylation and Claisen–Schmidt condensation
with benzaldehyde. The Mannich base derivatives of flavokawain
0
K
i
, and that one for enzyme-substrate (ES) complex, K
i
, are
obtained from the replots of the slope and intercept versus concen-
tration of compound 8, respectively, which are both linear in
0
Figure 2. The
K
i
and
K
i
value are 2.75 lM and 4.58 lM,
respectively, which indicates that compound 8 revealed a mixed
inhibiting effect against AChE includes competitive and uncompet-
itive profile. Moreover, competitive inhibition is easier than
uncompetitive inhibition for compound 8.24
B
were designed, synthesized, and subjected to biological
evaluated as the AChE inhibitors. All derivatives exhibited AChE
inhibitory activity compared with flavokawain B in the preliminary
Figure 2. (A) Lineweaver–Burk plot for the inhibition of AChE by compound 8; (B) the replot of the intercept versus concentration of compound 8; (C) the replot of the slope
versus concentration of compound 8.

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H.-r. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4749–4753
Figure 3. Molecular modeling of compound 8 with AChE generated by MOE.
bioassay, which indicates that the base group seemed necessary to
References and notes
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