4- Substituted sampangine derivatives: Novel acetylcholinesterase and β-myloid aggregation inhibitors

Article abstract of DOI:10.1016/j.ijbiomac.2017.10.157

A series of 4- substituted sampangine derivatives (4-aminoalkylaminosampangine Ar–NH(CH₂)_nNR₁R₂) has been designed, synthesized, and tested for their ability to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and β-myloid (Aβ) aggregation. The synthetic compounds exhibited high AChE inhibitory activity and a significant in vitro inhibitory potency toward the self-induced Aβ aggregation. While, treatment of SH-SY5Y cells overexpressing the Swedish mutant form of human β-amyloid precursor protein (APPsw) with derivatives was associated with significant reduction of Aβ42 secretion levels. Moreover, most of the synthetic compounds were predicted to be able to cross the blood-brain barrier (BBB) to reach their targets in the central nervous system (CNS) according to a parallel artificial membrane permeation assay for BBB. The result encourages us to study this class of compounds thoroughly and systematically.

Full text of DOI:10.1016/j.ijbiomac.2017.10.157

Accepted Manuscript
Title: 4- Substituted sampangine derivatives: Novel
acetylcholinesterase and ␤-myloid aggregation inhibitors
Authors: Ke-Lin Chen, Ling Gan, Zhen-Hua Wu, Jin-Fang
Qin, Wen-Xia Liao, Huang Tang
PII:
S0141-8130(17)32978-1
DOI:
Reference:
https://doi.org/10.1016/j.ijbiomac.2017.10.157
BIOMAC 8452
To appear in:
International Journal of Biological Macromolecules
Received date:
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9-8-2017
24-10-2017
25-10-2017
Please cite this article as: Ke-Lin Chen, Ling Gan, Zhen-Hua Wu, Jin-Fang
Qin, Wen-Xia Liao, Huang Tang, 4- Substituted sampangine derivatives: Novel
acetylcholinesterase and ␤-myloid aggregation inhibitors, International Journal of
Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.10.157
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4- Substituted Sampangine Derivatives: Novel Acetylcholinesterase and -myloid
Aggregation Inhibitors
Ke-Lin Chen, Ling Gan, Zhen-Hua Wu, Jin-Fang Qin, Wen-Xia Liao, Huang Tang*
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi
Normal University, Guilin 541004, China
 Author to whom correspondence should be addressed: hyhth@163.com
Research highlights

The new sampangine derivatives exhibited high AChE inhibitory activity
in intro.

Most of the derivatives exhibited a significant in vitro inhibitory activity
toward the self-induced Aβ aggregation and Aβ secretion levels of SH-
SY5Y cells overexpressing the Swedish mutant form of human β-amyloid
precursor protein (APPsw).
Most of the synthetic sampangine derivatives were predicted to be able to cross the
blood-brain barrier to reach their targets in the central nervous system.

Abstract:
A series of 4- substituted sampangine derivatives (4-aminoalkylaminosampangine
Ar–NH(CH2)nNR1R2) has been designed, synthesized, and tested for their ability to
inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and -myloid (A)

aggregation. The synthetic compounds exhibited high AChE inhibitory activity and a
significant in vitro inhibitory potency toward the self-induced A aggregation. While,
treatment of SH-SY5Y cells overexpressing the Swedish mutant form of human -
amyloid precursor protein (APPsw) with derivatives was associated with significant
reduction of A secretion levels. Moreover, most of the synthetic compounds were
predicted to be able to cross the blood-brain barrier (BBB) to reach their targets in the
central nervous system (CNS) according to a parallel artificial membrane permeation
assay for BBB. The result encourages us to study this class of compounds thoroughly
and systematically.
Keywords: Sampangine derivatives; Acetylcholinesterase inhibitors; -Amyloid
aggregation
1. Introduction
Alzheimer’s disease (AD) is a progressive neurological disease of the brain that leads
to the irreversible loss of neurons and dementia. Over the last few decades, a plethora
of targets has been suggested in the attempt to identify the causative factors of
neurodegeneration. Cholinesterases (ChEs) were the earliest research target.
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), regulate cholinergic
neurotransmission, the deterioration of which is responsible for the decline in memory
and cognition in patients suffering from AD[1]. Amyloid-(A) was another vital
target. According to the “amyloid hypothesis,” one of the major neuropathological
hallmarks of AD is the altered production, aggregation, and deposition of A, which
results in amyloid plaque formation[2-5]. Therefore, much effort was directed at

developing drugs to inhibit the production, aggregation, or neurotoxicity of A. Indeed,
AChE and A aggregation is retained as one of the major pathogenic mechanisms in
AD, and since it occurs early in the pathogenesis, it represents an ideal target for
intervention[6].
The copyrine alkaloid sampangine (Fig.1) belongs to the aporphine family of
alkaloids, which was widely distributed in Annonaceae plants, such as
Canangaodorata[7], Cleistophathis patens[8], Duguetia hadrantha[9] and Duguetia
hadrantha[10]. Plant-derived sampangine alkaloid shows broad and potent
antibacterial[11-14], antifungal[8, 15-19], antimalaria[9], anti-inflammatory[20],
antiparasitic[21] and antitumor[10, 22-24] activities. Several A, B-ring substituted and
hetero analogues of sampangine were reported in order to enhance their biological
activity[14, 19, 25]. In the paper, alkylamino were introduced to sampangine on 4-
substituents. A series of 4-aminoalkylaminosampangine derivatives was synthesized.
The inhibitory potency toward cholinesterase and Aβ aggregation of these sampangine
derivatives were first reported.
2. Experimental methods
2.1 Chemistry
Target compounds 5–12 were synthesized as shown in Scheme1. Preparation of
sampangine 3 was carried out by a reported method[11]. Cleistopholine 2 was obtained
through the hetero Diels-Alder reaction of quinone 1 with hydrazine. The condensation
of 2 with dimethylformamide dimethyl acetal provided sampangine 3. The electrophilic

halogenation of 3 with pyridinium bromide perbromide in chloroform produced 4-
bromosampangine 4 in 51 % yield. Subjection of 4 to a refluxing methanolic solution
of sodium methoxide afforded the known 4-methorysampangine 13 in high yield.
Finally, target compounds 5-12 were prepared by amination of 13 with corresponding
diamine in 1-pentanol with moderate yields. Reaction conditions and compound char-
acterisations are given in Supplementary Material.
2.2 In vitro inhibition studies on AChE and BChE
All the assays were under 0.1 M KH2PO4/K2HPO4 buffer, pH 8.0, using a
PerkinElmer LAMBDA 45 Spectrophotometer. AChE from Electrophorus electricus
(Sigma) were prepared to give 2.0 units/ml in 2 ml aliquots. The assay medium
contained phosphate buffer, pH 8.0 (1 mL), 50 L of 0.01 M DTNB, 10 L of enzyme,
and 50 L of 0.01 M substrate (acetylthiocholine chloride). The substrate was added to
the assay medium containing enzyme, buffer, and DTNB with inhibitor after 15 min of
incubation time. The activity was determined by measuring the increase in absorbance
o
at 412 nm for 1 min interval at 37 C. Calculations were performed according to the
method of the equation in Ellman et al[26]. In vitro BChE (from equine serum, Sigma)
assay uses the similar method described above.
2.3 MTT assay
Cell viability was measured in 96-well plates by MTT assay. Briefly, after cells were
treated using vehicle or compounds for indicated times, 500 μg/mL MTT (final
concentration) was added into the medium and the mixture was then incubated at 37 °C

for 3h. The MTT solution was then removed and the colored formazan crystal was
dissolved in dimethyl sulfoxide. The absorbance at 480 nm was measured using an
iMark Microplate Absorbance Reader (Bio-Rad). The cell viability was expressed as
the ratio of the signal obtained from the treatment group to that of the control group.
2.4 ELISA assay
Aβ1-42 secretion was measured in 96-well plates by ELISA assay. Briefly, after cells
were treated using vehicle or compounds for indicated times, the medium was removed,
and the human Aβ1-42 ELISA assay kit was used according to the procedures given in
the manufacturer’s instruction.
2.5 Determination of the inhibitory effect on the self-mediated A(1-42) aggregation
The thioflavin-T fluorescence method was used[27-30], and A(1-42) peptide
(Anaspec Inc) was dissolved in phosphate buffer (pH 7.4, 0.01 M) to give a 40 M
solution. Compounds were firstly prepared in DMSO at a concentration of 10 mM. The
final concentration of A (1-42) and inhibitors were 20 M and 10 M, respectively.
After incubating at 37 oC for 48 h, thioflavin-T (5 M in 50 mM glycine-NaOH buffer,
pH 8.0) was added. Fluorescence was measured at 450 nm (ex) and 485 nm (em). Each
inhibitor was examined in triplicate. The fluorescence intensities were recorded, and
the percentage of inhibition on aggregation was calculated with the following equation:
(1IFi/IFc) × 100%. IFi and IFc were the fluorescence intensities obtained in the
presence and absence of inhibitors, respectively, after subtracting the fluorescence of
corresponding blanks.

2.6 PAMPA-BBB procedure
Brain penetration of new compounds was evaluated using a parallel artificial
membrane permeation assay (PAMPA), in a similar manner as described by Di et
al[31]. Commercial drugs were purchased from Sigma and Aladdin (china). The
porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate
was a 96-well filter plate (PVDF membrane, pore size 0.45 μm) and the acceptor
microplate was an indented 96-well plate, both from Millipore. The 96-well UV plate
(COSTAR®) was from Corning Incorporated. The acceptor 96-well microplate was
filled with 300 L of Phosphate Buffered Saline (PBS) : ethanol (9:1) and The filter
membrane was coated with 4 μL of porcine brain lipid (PBL) in dodecane (20 mg mL-
1). Test compounds were dissolved in DMSO at 5 mg mL-1. Then the compound
solution was diluted 200-fold in PBS : ethanol (9:1) (final concentration 25 g mL-1) to
make secondary stock solution. 300 L of the secondary stock solution were added to
the donor wells. The acceptor filter plate was carefully put on the donor plate to form a
‘sandwich’, which was left undisturbed for 10 hrs at 25 ºC. The concentration of drug
in the acceptor, the donor, and the reference wells was determined using the UV plate
reader (Tecan Infinite® M1000). Pe can be calculated from the following equation as
reported by Faller et al9, 10 and Sugano et al11.




V
d
 V
a
[drug ]acceptor
[drug ]equilibriu
 

  ln 1 




(V
d
 V
a
) A  t
m




Pe=
Vd = volume of donor well, Va = volume in acceptor well, A = filter area, t =
permeation time, [drug]acceptor = the absorbance of compound found in the acceptor

well, [drug]equilibrium = the theoretical equilibrium absorbance. All compounds were
tested in triplicate at pH 7.4 and the results were given as the mean ± standard deviation.
3. Results and discussion
3.1 Inhibition of AChE and BuChE
To evaluate the biological profiles of the synthesized compounds for AD, AChE
(Electrophorus electricus) and BChE (equine serum) inhibition was assayed in
comparison with tacrine as reference compound. The inhibitory potency against AChE
and BChE was evaluated by the method of Ellman et al[26]. The IC50 values for AChE
and BChE inhibition are summarized in Table 1. All the synthesized compounds
demonstrated much higher inhibitory potency against AChE than their precursor
compound 3, with inhibitory activity IC50 values in the submicromolar range. This
result indicated that introduction of the amino group side chains could increase the
inhibitory capacity and selectivity. The structure of terminal groups of side chain has
effects on their inhibitory activities. Highest inhibitory potency was found to be
associated with diethylamine at the end of side chain (compound 8) with IC50 values in
0.23M. Dimethylamine derivatives showed less potency. Variation of chain length (n
= 2 or 3) had less influence on activity than the functional group structure.
3.2 Inhibiton of self-induced A Aggregation
Besides assessing the ability to inhibit AChE and BChE, which is likely to be relevant
on the brain of AD patients, we tested all the compounds (inhibitor:A ratio 1:2) to
assess the structural elements responsible for the in vitro inhibition of the self-assembly

of A(1-42), which is the most amyloidogenic Afragment found in the AD
plaques[32-34]. To determine the amyloid-(1-42) aggregation inhibition of the new
sampangine derivatives (3-12), thioflavin-T (ThT) assay was performed compared with
Curcumin as reference compound[27-29].
Interestingly, all the synthesized compounds presented better inhibitory potency than
curcumin on self-induced A(1-42) aggregation. Data in Table 1 showed that
compounds 3-12 at 10 M inhibited A(1-42) self-aggregation in a range from 46.5%
to 72.3%. The most effective compound is 7, followed by 8 and 9, their inhibitory
potency was 72.3%, 70.5% and 68.8%, respectively. The result revealed a slight trend
of increased efficacy with the reduction of the chain length. As a matter of fact,
increasing the methylene chain length reduced the inhibition of A(1-42) self-
aggregation.
3.3 Derivatives reduced Aβ42 secretion level in APPsw cells
To test the effects of synthesized derivatives on APPsw SH-SY5Y cell viability and
to examine the safety of these compounds, the in vitro cytotoxicity on APPsw SH-
SY5Y cell of compounds 5-12 was evaluated by MTT assay. As shown in Table 1,
introducing the side chain in sampangine alkaloid greatly reduced its toxicity. All the
derivatives (5-12) showed at least 3 times less toxic than sampangine 3. Particularly,
compound 8 showed lowest neurotoxicity with stronger AChE and A(1-42) self-
aggregation inhibitory potency suggesting the wide therapeutic safety ranges.

It is known that AChE inhibitors enhance the release of non-amyloidogenic soluble
derivatives of amyloid precursor protein (APPs) in vitro and in vivo and possibly slow
down the formation of amyloidogenic compounds in brain[35, 36]. Moreover, AChE
directly promotes in vitro the assembly of A peptide into amyloid fibrils forming
stable AChE-A complexes[2]. Thus, the effects of the synthesized compounds on
Aβ42 secretion level in APPsw SH-SY5Y cells were tested by human Aβ42 Elisa assay
kit. The viability of APPsw cells did not change significantly when concentration of
derivatives was less than 10 μM. Thus, two different concentrations (5M and 10M)
of compounds 5-12 were selected for Aβ42 secretion level testing. Since sampangine 3
showed stronger toxicity than its derivatives and had a great influence on cell viability
in 10M, only 5M sampangine was selected. As shown in Fig. 2, after treatment with
derivatives, APPsw cells showed various degrees of reducing the Aβ42 secretion level.
Moreover, the decrease was concentration dependent. The Aβ42 secretion level always
decreased with the increase of derivatives concentration. The most effective compound
is 8 which treatment significantly reduced the A42 production level by 54% (DMSO,
265.1 pg/ml vs compound 8, 146.0 pg/ml) in 10M. The Aβ42 secretion level was even
lower than wild type SH-SY5Y cells (control).
3.4 Prediction of blood-brain barrier (BBB) permeability
While protective in nature, the inability of molecules to permeate the BBB is a
significant source of attrition in central nervous system (CNS) drug discovery[37, 38].
For this reason, BBB permeability properties of CNS drug candidates should be
determined as early as possible in the drug discovery process. To explore whether the

selected compounds would be able to penetrate into the brain, we used a parallel
artificial membrane permeation assay for BBB (PAMPA-BBB). The PAMPA-BBB
model applied in this study was based on the BBB model described by Di et al[31].
This simple and rapid model was capable of identifying compounds as either BBB
permeable (BBB+) or non-permeable (BBB−) with reasonable accuracy by modifying
the lipid composition of the artificial membranes[39]. In this paper, a lipid extract of
porcine brain was used. Assay validation was made comparing experimental
permeabilities of 7 commercial drugs with reported values (Table 2).
A plot of experimental data versus bibliographic values gave a good linear
correlation, Pe(exp.) = 0.79Pe(bibl.) + 0.57 (R2=0.96). From this equation and taking
into account the limit established by Di et al[31] for blood-brain barrier permeation, we
classified compounds as follows:
a）‘CNS +’ (high BBB permeation predicted); Pe (10-6 cm s-1) > 3.7
b) ‘CNS -’ (low BBB permeation predicted); Pe (10-6 cm s-1) < 2.1
c) ‘CNS +/-’ (BBB permeation uncertain); Pe (10-6 cm s-1) from 3.7 to 2.1
Finally, new synthetical derivatives were tested in the PAMPA-BBB assay, and the
results were presented in Table 3. for BBB permeation, we found that molecules with
permeability above 3.7×10-6 cm s-1 would be able to cross the BBB by passive
permeation. Compounds 6-9 and 11 showed greater permeability values than that limit,
pointing out that these molecules would cross the BBB by passive diffusion.
4. Conclusions

Sampangine is a class of alkaloids with a wide range of biological activities.
However, their toxicity and poor water solubility and the scarcity from the natural
source have limited its development and application. In this article, the alkylamino side
chains were first introduced to sampangine on 4-substitution. It not only increased the
water solubility of the derivatives, but also reduced their toxicity. Especially for
compound 8, it had the strongest anti-cholinesterase, anti-A aggregating activity and
high BBB permeability values but its toxicity was weakest. It makes them promising
anti-Alzheimer drug candidates. The results suggested that the novel 4-substitutional
sampangine derivatives herein reported were worthwhile to further research.
Acknowledgments
This work was supported by State Key Laboratory for Chemistry and Molecular
Engineering of Medicinal Resources, Guangxi Normal University (grant No.
CMEMR2017-A09).
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Fig. 1. Structure of sampangine
Fig. 2. APPsw cells were incubated for 24 h in a medium containing DMSO or
compounds 3-12 (5 μM and 10μM). The Aβ42 levels in the cell medium were
determined by ELISA. Control was wild type SH-SY5Y cells.

Scheme 1. Synthesis of 4- Substituted sampangine derivatives.
Table 1. In vitro inhibition of AChE, BChE and self-induced A(1-42) aggregation
activities of compounds 3, 5-12 and their toxicity in APPsw SH-SY5Y cells
IC50
cytotoxicity
IC50(uM) for IC50(uM) for Selectivity for
Inhibition of self-induced
Compound
R
n
(M) against APPsw
AChEa
BChEa
AChE/BChEb
Aβ aggregation (%)d
SH-SY5Y cellsc
12.4
41.2
38.8
42.1
45.8
39.7
40.7
30.7
35.7
n.d.e
n.d.e
46.5 ± 2.3
62.1 ± 2.1
54.2 ± 1.8
72.3 ± 2.3
70.5 ± 1.9
68.8 ± 1.5
66.7 ± 2.2
59.4 ± 1.9
60.9 ± 2.1
n.d.e
﹥100
﹥100
3
2
3
2
3
2
3
2
3
1
12.11 ± 0.66
13.60 ± 0.71
1.82 ± 0.12
0.23 ± 0.04
4.81 ± 0.32
8.86 ± 0.41
1.15 ± 0.09
10.9 ± 0.37
﹥100
5
-N(CH3)2
>7
21
>435
1
6
38.1 ± 0.64
﹥100
7
-N(CH3CH2)2
8
3.39 ± 0.08
12.39 ± 0.36
13.14 ± 0.28
9
1
10
11
11
2
1.65 ± 0.15
0.22 ± 0.02
3.58 ± 0.15
12
Tacrine
0.026 ± 0.001
0.1
Curcumin
n.d.e
n.d.e
44.7 ± 1.9
a IC50: 50% inhibitory concentration (means ± SEM of three experiments) of AChE and BChE.
b Apparent selectivity for AChE is calculated as IC50(BChE)/IC50(AChE).
c Data derived from the mean of three independent assays.
d Inhibition of self-induced A(1-42) aggregation (20 M) produced by the tested compound at 10 M concentration. Values are
expressed as means ± SEM of three experiments.
e Not determined.

Table 2. Permeability (Pe × 10-6 cm s-1) in the PAMPA-BBB assay for 7 commercial
drugs, used in the experiment validation
Compoud
bibliographya experiment b
Imipramine
13
10.2
6.9
5.2
1.3
1.2
4.6
0.6
Chlorpromazine 6.5
Clonidine
5.3
1.9
0.9
5.1
0.2
Hydrocortisone
Enoxacin
Corticosterone
Dopamine
a Taken from ref 29.
b Data are the mean of three independent experiments.
Table 3. Permeability (Pe × 10-6 cm s-1) in the PAMPA-BBB assay for 4- substituted
sampangine derivatives and their predictive penetration in the CNS
Compoud Pe × 10-6 cm s-1 a prediction
CNS -
3
0.2 ± 0.04
0.8 ± 0.1
4.9 ± 0.3
6.4 ± 0.2
5.6 ± 0.1
7.8 ± 0.2
3.1 ± 0.1
CNS -
5
CNS +
CNS +
CNS +
CNS +
CNS +/-
CNS +
CNS +/-
6
7
8
9
10
11
12
7.0 ± 0.3
3.5 ± 0.1
a Data are the mean (n=3) ± SD.