Tryptamine–Triazole Hybrid Compounds for Selective Butyrylcholinesterase Inhibition

Article abstract of DOI:10.1002/bkcs.11729

Tryptamine–triazole hybrid compounds (11–18) were synthesized via click reaction between tryptamine azide and propargylated natural compounds. Their cholinesterase inhibitory activity was evaluated. Among the eight compounds, compound 11 showed the most p

Full text of DOI:10.1002/bkcs.11729

Article
DOI: 10.1002/bkcs.11729
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M. Son et al.
KOREAN CHEMICAL SOCIETY
Tryptamine–Triazole Hybrid Compounds for Selective
Butyrylcholinesterase Inhibition
Minky Son,†,† Haneul Lee,‡,† Cheolmin Jeon,‡ Yujung Kang,‡ Chanin Park,‡ Keun Woo Lee,†, and
*
Jeong Ho Park‡,
*
†Division of Life Science, Division of Applied Life Science (BK21 Plus), Plant Molecular Biology and
Biotechnology Research Center (PMBBRC), Systems and Synthetic Agrobiotech Center (SSAC), Research
Institute of Natural Science (RINS), Gyeongsang National University (GNU), Jinju 52828, Republic of
Korea. *E-mail: kwlee@gnu.ac.kr
‡Department of Chemical & Biological Engineering, Hanbat National University, Daejeon 34158, South
Korea. *E-mail: jhpark@hanbat.ac.kr
†These authors contributed equally to this work.
Received February 23, 2019, Accepted March 29, 2019
Tryptamine–triazole hybrid compounds (11–18) were synthesized via click reaction between tryptamine
azide and propargylated natural compounds. Their cholinesterase inhibitory activity was evaluated.
Among the eight compounds, compound 11 showed the most potent inhibitory activity
[IC50 = 0.42 ꢀ 0.29 μM for equine butyrylcholinesterase (BuChE) and 1.96 ꢀ 0.15 μM for human
BuChE]. From the molecular modeling study, compound 11 was bound to the catalytic anionic site,
anionic subsite, peripheral anionic subsite, acyl-binding pocket, and oxyanion hole of human or horse
BuChE by forming a hairpin or U-shaped structure. The Lineweaver-Burk plot of compound 11 against
BuChE suggests a mixed type of inhibition which corresponds well with the molecular modeling study.
Keywords: Tryptamine–triazole hybrid compounds, Cholinesterase inhibitory activity, Alzheimer’s dis-
ease, Molecular docking calculation, Molecular dynamics simulation
Introduction
developing AD therapeutic compounds. In this study, we
have synthesized derivatives of natural compounds such as
lipoic acid, polyphenols, or paeonol with various physio-
logical activities and studied whether they can act as selec-
tive BuChE inhibitors.
α-Lipoic acid (ALA) and its reduced form, dihydrolipoic
acid, show various physiological activities. ALA is often
referred to as an “antioxidant of antioxidants” because of
its ability to act as a multifunctional antioxidant against a
variety of reactive oxygen species (ROS)5,6 and to promote
new synthesis of endogenous antioxidants such as glutathi-
one, α-tocopherol, and vitamin C.5 ALA conjugated com-
pounds such as ALA-tacrin conjugate (Lipocrine;
IC50 = 6.96 ꢀ 0.45 nM for AChE)7 and ALA-triazole-
coumarin conjugate (IC50 = 7.8 ꢀ 3.6 μM for BuChE)8
showed cholinesterase inhibitory activity.
Natural polyphenols (PPs) are known as natural antioxi-
dants by reducing ROS levels in vivo.9 PP also reduces the
inflammation effect on coronary artery disease10 and slows
down the process of wrinkling the skin.11
Paeonol is a natural compound isolated from Paeonia
suffruicosa.12 Paeonol and its derivatives have many bene-
ficial anticancer, antimicrobial, antimutagenic, anti-inflam-
matory, and analgesic properties.13
For the last 10 years, new drugs have not been introduced
to the market for Alzheimer’s disease (AD) treatment.
Therefore, strategies are needed to develop drug candidates
for AD. There are many targets for AD drugs such as beta-
amyloid, tau-protein, cholinesterase, γ- and β-secretase,
neurotransmitter systems (serotoninergic 5-HT6 and hista-
minergic H3), etc.1 Among them, cholinesterase inhibitors
based on the cholinergic hypothesis are still the most valu-
able targets for novel AD drugs.2 Currently commercially
available AD drugs such as donepezil, rivastigmine, and
galantamine are targeted primarily to acetylcholinesterase
(AChE). However, they are not a complete remedy for AD
and have many side effects that require a new type of target
for cholinergic hypothesis. The activity of AChE in AD
patients is decreases but that of BuChE is increased to
40–90%.3,4 When a selective inhibitor of BuChE, such as a
cymserine analogue, is applied to rodents, the concentration
of acetylcholine (ACh) in the brain increases.4 Thus, selec-
tive inhibition of BuChE may increase ACh levels in AD
patients with superior drug candidates.
Synthesis of derivatives of natural or secondary metabo-
lites with physiological activities such as antioxidant, anti-
inflammatory, or cytoprotective and verifying that they
meet cholinergic hypothesis can be a good strategy for
Previously, we have reported that a benzoisoxazole-
tryptamine hybrid compound (1) showed good BuChE
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inhibitory value (IC50 = 0.72 ꢀ 0.11 μM) (Figure 1).14 In
this study, compound 1 derivatives were synthesized by
converting the benzoisoxazole moiety to biologically active
natural compounds. The click reaction between trytamine
azide and propargylated lipoic acid, polyphenols, and
paeonol was carried out. We also evaluated the effect of
compound 1-derivatives on BuChE inhibition in vitro.
Molecular modeling studies using compound 11 in human
or horse BuChE have been performed to understand the
binding mode.
added at room temperature to a dichloromethane (DCM)
solution of the carboxylic acids. The reaction mixture is
stirred at room temperature for 4 h and then water is added.
The solution was extracted three times with DCM and dried
over anh. MgSO4. The solvent was removed in vacuo and
the residue was purified by silica gel column chromatogra-
phy (DCM: EA = 4: 1) to give the propargylated compound
(3, 7–10).
General Procedure (B). The following is a general reac-
tion procedure for the synthesis of propargylated deriva-
tives (5 and 6). Propargyl bromide (1.5 equiv) was added
to the mixture of 2,4-dihydroxyacetophenone or paeonol
(1.1 equiv), and Cs2CO3 (1.1 equiv) in acetone at room
temperature. The reaction mixture was refluxed for 24 h
and water was added. The solution was extracted three
times with DCM and dried over anh. MgSO4. The solvent
was removed in vacuo, and the residues were purified by
silica gel column chromatography (EA: Hex = 1: 6) to give
propargylated compounds (5 and 6), respectively.
Experimental
General Methods. 1H NMR and 13C NMR spectra were
recorded on a Varian Mercury 400 (400 MHz) spectrometer
(Varian, Palo Alto, California, USA) and mass spectra were
taken using an Agilent G1956B mass spectrometer (Agilent,
Santa Clara, California, USA). Flash column chromatography
was performed using E. Merck silica gel (60, particle size
0.040–0.063 mm) and analytical thin layer chromatography
(TLC) was performed using pre-coated TLC plates with silica
gel 60 F254 (Merck, Merck, Darmstadt, Germany). The
microwave synthesis reaction was carried out using a Biotage
microwave reactor (Model: Initiator Exp Eu, Sweden).
Unless otherwise noted, the reagents used were reagent grade
and all synthetic reactions were carried out under an argon
atmosphere using a dry solvent. (α)-Lipoic acid was pur-
chased from Xi’an Union Pharmpro Co., Ltd., (Xi’an,
China). Caffeic acid, 3,4-dimethoxy cinnamic acid, ferulic
acid, syringic acid, honokiol, 2,4-dihydroxyacetophenone,
acetic anhydride, N,N-dimethylpyridin-4-amine (DMAP), N-
(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochlo-
ride (EDC), triethylamine (TEA), Cs2CO3, PPh3, H3PO4, KI,
NaOH, acetylcholinesterase [electric eel, cat. (C2888)], and
butyrylcholinesterase [equine serum, cat. (C-7512) and
human serum, cat. (B4168)] were purchased from Sigma-
Aldrich Korea (Yongin, Korea), Merck, or ThermoFisher
Scientific (Seoul, Korea) and used without purification.
General Procedure (C). The following is a general reac-
tion procedure for the synthesis of compound (1)-
derivatives by the click reaction using a microwave reactor.
Tryptamine azide (2, 1.1 equiv) was prepared by treating
tryptamine with imidazole sulfonyl azide,15 which was
added to a solution of propargylated compounds (3–10,
1.0 equiv) in acetone in the presence of Cu(PPh3)3Br
(0.1 equiv).16 The reaction mixture was reacted in a micro-
wave reactor at 65 ꢁC for 30 min and then water was
added. The solution was extracted three times with DCM
and dried over anh. MgSO4. The solvent was removed in
vacuo and the tryptamine derivatives (11–18) were purified
using silica gel column chromatography (DCM: MeOH = 9:
1), respectively.
(R)-N-((1-(2-(1H-indol-3-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methyl)-5-(1,2-dithiolan-3-yl)pentanamide (11). Click reac-
tion between compounds 3 and 2 resulted in compound 11
with 89% yield via general procedure (C).
Compound 3: White solid; yield: 85%; 1H NMR
(400 MHz, CDCl3): δ 1.43 (m, 2H), 1.64 (m, 4H), 1.87 (m,
1H), 2.16 (m, 3H), 2.41 (m, 1H), 3.27 (m, 2H), 3.76 (m,
1H), 4.01 (m, 2H), 5.56 (s, 1H).
General Synthetic Procedure
General Procedure (A). The following is a general reac-
tion procedure for the synthesis of propargylated deriva-
tives (3, 7–10). EDC (1.5 equiv), hydroxybenzotriazole
(HOBT, 0.1 equiv), and propargylamine (1.1 equiv) were
Compound 11: Yellow oily product; yield: 89%; 1H
NMR (400 MHz, CDCl3): δ 1.38 (m, 2H), 1.62 (m, 4H),
1.85 (m, 1H), 2.12 (t, J = 7.2 Hz, 2H), 2.43 (m, 1H), 3.09
(m, 2H), 3.33 (t, J = 6.8 Hz, 2H), 3.51 (m, 1H), 4.38 (d,
J = 5.6 Hz, 2H), 4.60 (t, J = 6.8 Hz, 2H), 6.15 (s, 1H), 6.87
(s, 1H), 7.08 (t, J = 8.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H),
7.19 (s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz,
1H), 8.24 (s, 1H); 13C NMR (100 MHz, DMSO‑d6): δ
25.3, 26.5, 28.8, 34.5, 34.8, 36.0, 38.4, 40.2, 50.9, 56.4,
110.6, 111.6, 118.1, 119.3, 122.0, 122.8, 122.9, 126.9,
136.3, 144.5, 173.2; ESI-MS: m/z [M]+ 460.63
(calcd 460.1).
(R)-1-(4-(1,2-dithiolan-3-yl)butyl)-3-((1-(2-(1H-indol-3-yl)
ethyl)-1H-1,2,3-triazol-4-yl)methyl)urea (12). Compound
Figure 1. Structures of benzoisoxazole moiety-tryptamine hybrid
compound (1).
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4 was synthesized by Curtius rearrangement of lipoic acid
using diphenyl phosphorazidate (1.0 equiv) and propargyl
amine. Click reaction between compound 4 and 2 resulted
in compound 12 with 89% yield via general procedure (C).
Compound 4: White solid; yield: 87%; 1H NMR
(400 MHz, CDCl3): δ 1.45 (m, 4H), 1.65 (m, 3H), 1.89 (m,
1H), 2.18 (s, 1H), 2.43 (m, 1H), 3.07 (m, 2H), 3.19 (m,
2H), 3.50 (m, 1H), 3.94 (m, 2H), 4.65 (bs, 1H), 4.73
(bs, 1H).
4-((E)-2-((1-(2-(1H-indol-3-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methylcarbamoyl)vinyl)-2-methoxyphenyl acetate (17). Click
reaction between compounds 9 and 2 resulted in compound
17 with 95% yield via general procedure (C).
Compound 9: Yellow solid; yield: 66%; 1H NMR
(400 MHz, CDCl3): δ 1.99 (d, J = 2.8 Hz, 1H), 2.25 (s,
3H), 3.82 (s, 3H), 4.16 (q, J = 2.8 Hz, 2H), 5.93 (bs, 1H),
6.30 (d, J = 15.2 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 7.03
(d, J = 4.4 Hz, 1H), 7.06 (dd, J = 8.8, 4.4 Hz, 1H), 7.57 (d,
J = 15.2 Hz, 1H).
Compound 12: Yellow oily product; yield: 89%; 1H
NMR (400 MHz, DMSO‑d6): δ 1.30 (m, 3H), 1.48 (m,
1H), 1.60 (m, 1H), 1.81 (m, 1H), 2.34 (m, 1H), 2.93 (m,
2H), 3.06 (m, 1H), 3.11 (m, 1H), 3.18 (t, J = 7.6 Hz, 2H),
3.54 (m, 1H), 4.14 (d, J = 5.6 Hz, 2H), 4.54 (t, J = 7.6 Hz,
2H), 5.88 (t, J = 5.6 Hz, 1H), 6.17 (t, J = 5.6 Hz, 1H), 6.93
(t, J = 7.4 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 7.05 (d,
J = 2.4 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.49 (d,
J = 8.0 Hz, 1H), 7.65 (m, 1H), 7.85 (s, 1H), 10.82 (s, 1H);
13C NMR (100 MHz, CDCl3): δ 21.61, 25.16, 25.20,
29.73, 29.73, 30.64, 33.62, 35.28, 35.42, 45.99, 51.75,
105.72, 106.82, 113.26, 114.61, 117.24, 118.14, 121.97,
125.08, 128.53, 131.48, 153.97; ESI-MS: m/z [M]+ 445.1
(calcd 445.1).
Compound 17: White solid; yield: 95%; 1H NMR
(400 MHz, DMSO‑d6): δ 2.24 (s, 3H), 3.22 (t, J = 7.5 Hz,
2H), 3.78 (s, 3H), 4.39 (d, J = 5.2 Hz, 2H), 4.58 (t, J = 7.5 Hz,
2H), 6.63 (d, J = 15.6 Hz, 1H), 6.95 (t, J = 7.2 Hz, 1H), 7.04
(t, J = 7.2 Hz, 1H), 7.08 (d, J = 6.8 Hz, 1H), 7.10 (s, 1H),
7.13 (d, J = 6.8 Hz, 1H), 7.28 (s, 1H), 7.30 (d, J = 8.0 Hz,
1H), 7.42 (d, J = 15.6 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 8.10
(s, 1H), 8.54 (t, J = 5.2 Hz, 1H), 10.84 (s, 1H); 13C NMR
(100 MHz, DMSO‑d6): δ 20.8, 26.49, 34.83, 50.37, 56.20,
110.45, 111.85, 112.04, 118.63, 118.85, 120.41, 121.50,
122.68, 123.57, 123.72, 127.32, 134.32, 136.56, 138.81,
140.58, 151.48, 165.22, 168.88; ESI-MS: m/z [M]+ 460.5
(calcd 460.1); mp: 175 ꢁC.
1-(4-((1-(2-(1H-indol-3-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methoxy)-2-hydroxyphenyl)ethenone (13). Click reaction
between compounds 5 and 2 resulted in compound 13
with 79% yield via general procedure (C). Analytical
data can be found in Ref. 17.
1-(2-((1-(2-(1H-indol-3-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methoxy)-4-methoxyphenyl)ethenone (14). Click reaction
between compounds 6 and 2 resulted in compound 14 with
92% yield via general procedure (C). Analytical data can
be found in Ref. 17.
4-((1-(2-(1H-indol-3-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methylcarbamoyl)-2,6-dimethoxyphenyl acetate (15). Click
reaction between compound 7 and 2 resulted in compound
15 with 70% yield via general procedure (C). Analytical
data can be found in Ref. 17.
(E)-N-((1-(2-(1H-indol-3-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methyl)-3-(3,4-dimethoxyphenyl)acrylamide (16). Click
reaction between compounds 8 and 2 resulted in com-
pound 16 with 85% yield via general procedure (C).
Compound 8: Analytical data can be found in Ref. 14.
Compound 16: White solid; yield: 85%; 1H NMR
(400 MHz, DMSO‑d6): δ 3.19 (t, J = 7.6 Hz, 2H), 3.73 (s,
6H), 4.35 (d, J = 2.4 Hz, 2H), 4.55 (t, J = 7.6 Hz, 2H),
6.49 (d, J = 15.6 Hz, 1H), 6.93 (s, J = 4.0 Hz, 1H), 6.94 (s,
1H), 7.02 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 12.0 Hz, 1H),
7.06 (s, 1H), 7.08 (d, J = 12.0 Hz, 1H), 7.28 (d, J = 7.6 Hz,
1H), 7.39 (d, J = 15.6 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H),
7.95 (s, 1H), 8.42 (t, J = 2.4 Hz, 1H), 10.83 (s, 1H); 13C
NMR (100 MHz, DMSO‑d6): δ 26.5, 34.8, 50.3, 55.8,
56.0, 110.5(2C), 111.9. 112.2, 118.6, 118.9, 120.1, 121.5,
121.7, 123.3, 123.6, 127.3, 128.1, 136.6, 139.5, 145.1,
149.3, 150.6, 165.6; ESI-MS: m/z [M]+ 432.49 (calcd
432.2); mp: 160 ꢁC.
4-((E)-2-((1-(2-(1H-indol-3-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methylcarbamoyl)vinyl)phenyl-1,2-diacetate (18). Click reac-
tion between compounds 10 and 2 resulted in compound 18
with 70% yield via general procedure (C).
Compound 10: White solid; yield: 20%; 1H NMR
(400 MHz, CDCl3): δ 2.20 (d, J = 2.8 Hz, 1H), 2.26 (s,
6H), 4.13 (q, J = 2.8 Hz, 2H), 5.86 (bs, 1H), 6.25 (d,
J = 15.6 Hz, 1H), 7.14 (d, J = 8 Hz, 1H), 7.28 (d, J = 2 Hz,
1H), 7.31 (dd, J = 8, 2 Hz, 1H), 7.53 (d, J = 15.6 Hz, 1H).
Compound 18: White solid; yield: 70%; 1H NMR
(400 MHz, DMSO‑d6): δ 2.26 (s, 6H), 3.22 (t, J = 7.6 Hz,
2H), 4.38 (d, J = 5.7 Hz, 2H), 4.58 (t, J = 7.6 Hz, 2H),
6.61 (d, J = 15.6 Hz, 1H), 6.95 (t, J = 7.5 Hz, 1H), 7.03 (t,
J = 7.5 Hz, 1H), 7.08 (s, 1H), 7.28 (d, J = 7.5 Hz, 1H),
7.30 (d, J = 7.5 Hz, 1H), 7.41 (d, J = 15.6 Hz, 1H), 7.44 (s,
1H), 7.47 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H),
7.97 (s, 1H), 8.58 (t, J = 5.7 Hz, 1H), 10.83 (s, 1H); 13C
NMR (100 MHz, DMSO‑d6): δ 20.57, 26.46, 50.43, 56.56
(2C), 104.69, 110.48, 110.51, 111.83, 111.90, 118.63,
118.84, 118.93, 121.50, 123.56, 123.67, 127.34, 130.67,
132.63, 132.65, 151.93, 152.02, 165.71, 168.31(2C); ESI-
MS: m/z [M]+ 488.51 (calcd 488.2); mp: 79 ꢁC.
Cholinesterase Assay. Measurements of inhibitory activ-
ity against AChE and BuChE were performed using the
Ellman method.18
Homology Modeling. Amino acid sequence for Equus
caballus BuChE (horse BuChE, accession no: P81908) was
obtained from UniProtKB (http://www.uniprot.org/). Tem-
plate structure was searched on PSI-BLAST and sequence
alignment with the selected template was performed using
Discovery Studio (DS) 2016 (BIOVIA, San Diego, CA,
USA). The homology model structure was generated by
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NH
using the Build Homology Models protocol available in
DS. The energy minimization for the model structure was
performed using GROMACS 5.0.6 package with
CHARMM 27 force field. The steepest descent algorithm
was used and the maximum number of minimization steps
was set to 10 000. The stereochemical quality of the
predicted structure was checked by PROCHECK19 ProSA
(Protein Structure Analysis),20 and ERRAT.21
N3
2
O
H
H
N
N
N
H
S
S
O
S
S
3
4
6
O
O
O
Molecular Docking Calculation. Molecular docking cal-
culation of 11 to BuChE was performed using Genetic
Optimization for Ligand Docking (GOLD) version 5.2.22
The X-ray crystal structure of human BuChE (PDB ID:
4AQD)23 from the RCSB Protein Data Bank (www.rcsb.
org) and the homological model structure of horse BuChE
were used. All co-crystal water and ligand molecules were
removed from the structure. The protonation states of all
titratable residues in the protein were set to pH 7 using
Clean Protein tool in DS 2016. The 3D structure of 11 was
prepared by DS. The geometry of the compound was opti-
mized by energy minimization through Minimize Ligands
tool in DS. All atoms within 15 Å from the center of the
binding site were defined as docking sites. The number of
docking runs was set to 100. The most populated confor-
mations were selected for further investigation.
O
OH
MeO
5
O
O
MeO
AcO
N
MeO
MeO
N
H
H
7
8
OMe
O
O
MeO
AcO
AcO
AcO
N
N
H
H
9
10
Figure 2. The structures of tryptamine azide and propargylated
compounds prepared for click reaction.
Molecular Dynamics Simulation. Molecular dynamics
(MD) simulation of the protein-ligand complex was per-
formed with AMBER03 force field24 using GROMACS
5.0.6 package. The complex structure obtained from the
molecular docking calculation was used as the initial struc-
ture for MD simulation. The ligand topology was generated
by ACPYPE (AnteChamber Python Parser interface).25 The
complex structure was immersed in a water box of TIP3P
water molecules26 to make an aqueous environment.
Counter-ions were added to neutralize the system and energy
minimization with steepest descent algorithm was carried out
until the maximum force converged to less than 1000 kJ/
mol. The system was then subjected to 100 ps NVT equili-
bration followed by 100 ps NPT equilibration. Finally, a
10 ns production run was carried out under NPT ensemble.
A constant temperature of 300 K and a pressure of 1 bar was
controlled by Nose–Hoover thermostat27 and Parrinello–
Rahman barostat,28 respectively. All bond lengths and the
geometry of water molecules were restrained by LINCS29
and SETTLE algorithms,30 respectively. Cut-off values for
short-range electrostatic and van der Waals interactions were
1.2 nm while long-range electrostatic interactions were cal-
culated using particle mesh Ewald method.31 The simulation
was performed under periodic boundary conditions to avoid
edge effects. The time step of the simulation was 2 fs and
the coordinates were saved to trajectory files every 1 ps.
polyphenols, and paeonol used in the click reactions are
shown in Figure 2.
The representative triazole-linked tryptamine derivatives
11–18 were, respectively, synthesized by the click reactions
between tryptamine-azide (2) and the propargyl compounds
3–10 using a microwave reactor (Scheme 1). All tryptamine
hybrid compounds synthesized in this work are listed in
Figure 3.
Inhibitory Results (IC50 Value). The inhibitory effects
(IC50 value) of tryptamine derivatives on AChE and BuChE
are shown in Table 1 and galantamine was used as a
Results
Chemistry. The structures of tryptamine azide (2) and
propargylated derivatives (3−10) of lipoic acid,
Scheme 1. Synthesis of triazole-linked tryptamine hybrid com-
pounds by click reaction.
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Figure 3. The structures of the triazol-linked tryptamine hybrid compounds synthesized in this work.
positive control compound. The starting compounds (trypt-
amine, lipoic acid, paeonol, syringic acid, ferulic acid, and
caffeic acid) did not show any inhibitory effect for cholin-
esterases (data not shown). All tryptamine hybrid com-
pounds had the same pattern as compound 1. They
selectively inhibited BuChE compared to AChE.
The Binding Mode of Compound 11 in Horse BuChE.
Homology Modeling of Horse (Equus caballus) BuChE. A
homology model of horse BuChE was constructed using
the 3D structure of human BuChE as a template (PDB ID:
4AQD). Sequence alignment displayed that horse BuChE
was highly homologous to human BuChE with a sequence
identity of 90.2% and the similarity of 94.3% (Figure 4(a)).
The structural alignment of horse BuChE to the template
showed that the overall tertiary structures are very similar
with a root-mean-square deviation (RMSD) of 0.16 Å for
the backbone atoms in the proteins (Figure 4(b)). They
shared the same active site residues, except that Ala277 of
human BuChE is replaced by Val277 in horse BuChE. The
homology model was subjected to energy minimization to
refine intramolecular contacts followed by the assessment
of its stereochemical quality. Ramachandran plot from
PROCHECK revealed that 91.2% of the residues were in
the most favored regions, 8.4% were in additional allowed
regions, and only two residues were found in disallowed
regions (Figure 4(c)). The ProSA Z-Score value of −10.6
was within the range of scores typically found for native
proteins of similar size. Moreover, a quality factor score
obtained from ERRAT was 95.93, which represents a high
quality model (Figure 4(d)). As a result, the quality of our
homology model for horse BuChE was evaluated using
three different criteria. These results indicated that the
model structure is reliable for further modeling studies.
Binding Conformation of 11 at the Active Site of Horse
BuChE. Molecular docking of 11 to horse BuChE was also
performed. The GOLD fitness score for all the generated
100 poses ranged from 59.08 to 82.1. The result showed
that 62 conformations were similar to that of human
BuChE. The top-scored conformation was selected as a
starting structure for MD simulation. To check the stability
of the system, RMSD and potential energies were measured
during the 10 ns simulation time. The Cα RMSD value for
the protein and the potential energies were constant and
remained less than 0.15 nm and −1 030 000 kJ/mol,
respectively (Figure 5(a) and (b)). Structural analysis rev-
ealed that the binding mode of 11 in horse BuChE was
analogous to that of human BuChE (Figure 5(c) and (d)).
Table 1. ChE inhibition IC50 values for tryptamine hybrid
compounds.a
LogP
AChE inh.
IC50(μM)
BuChE inh.
IC50(μM)
Sample
valuec
Gal.
1
1.41
3.88
3.32
0.64 ꢀ 0.1
91.30 ꢀ 0.97
>480
>480
>480
229.8 ꢀ 1.26
>450
8.4 ꢀ 0.1
0.72 ꢀ 0.11
0.42 ꢀ 0.29
1.96 ꢀ 0.15
7.69 ꢀ 0.82
>450
5.49 ꢀ 0.41
9.04 ꢀ 0.62
>450
11
11b
ꢀ12
13
2.98
2.48
2.75
2.16
2.92
2.63
2.34
14
15
16
17
>450
>450
>450
>450
41.79 ꢀ 1.30
18
>450
a AChE (from electric eel) and BuChE (from horse serum) were used.
b BuChE (from human serum). IC50 values represent the inhibitor
concentration necessary to decrease enzyme activity by 50% and are
calculated using the mean of the triplicate measurements.
c LogP values were calculated by using ChemDraw Program.
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Figure 4. Homology modeling for horse BuChE. (a) Sequence alignment between horse and human BuChEs. (b) Superimposed structure
of homology model (green) and template (gray) of BuChE. The catalytic triad of horse BuChE was displayed as gray stick models.
(c) Ramachandran plot of the backbone dihedral angles (φ and ψ) for the model structure of horse BuChE. (d) ProSA plot of model struc-
ture. The Z-score of −10.6 was represented as a black dot.
Binding Conformation of 11 at the Active Site of Human
BuChE. Molecular docking calculation was performed to
explore the binding mode of 11 in the active site of human
BuChE. From the top cluster including 48 out of 100 dock-
ing poses, a pose with a GOLD fitness score of 80.45 was
selected as a representative pose of the compound based on
their molecular interactions (Figure 6). The binding confor-
mation of 11 showed that the indole group of the com-
pound was headed for Trp82 and the end of lipoamide
group was toward to Ser198. The complex structure was
subjected to MD simulation in order to alleviate unfavor-
able contacts and check the binding stability. Prior to trajec-
tory analysis, the RMSD for the Cα atoms in the protein
and the potential energy of the system were calculated. The
RMSD was converged within 0.15 nm during 10 ns MD
simulation (Figure 7(a)). The potential energy was also
maintained at about −1 075 000 kJ/mol (Figure 7(b)). Both
plots showed that there were no abnormal behaviors in the
structure during the entire simulation time. A snapshot with
the lowest potential energy was taken as a representative
structure for further analysis. Compound 11 bound to
human BuChE by forming a hairpin or U-shaped structure
in the active site (Figure 7(c) and (d)).
Discussion
Compound 11 exhibited slightly higher inhibitory activity
against BuChE (from horse serum, IC50 = 0.42 ꢀ 0.29 μM)
than compound 1. The IC50 value (1.96 ꢀ 0.15 μM) was a
little higher for the BuChe originating from human serum
compared to horse serum. Molecular modeling study for
BuChE originating from both human and horse were also
done on compound 11.
Lipoic acid transferred to compound 4, which has one
less carbon, through Curtius rearrangement reaction
quenching with propargyl amine. The click reaction
between
4
and
2
resulted in compound 12
(IC50 = 7.69 ꢀ 0.82 μM). Compound 11, which has an
amide functional group, had an IC50 value 10 times less
than compound 12, which has an urea functional group.
Compounds 13 and 14 are derivatives of paeonol. When
anti-inflammatory activity was measured using compounds
13, 14, and 15, compound 13 and 14 independently
inhibited NO (nitric oxide) production in BV2 cells. How-
ever, compound 15 suppressed NO generation to a mini-
mum. This means that the paeonol moiety plays an
important role in the anti-inflammatory effect.17 Starting
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Figure 5. The results of 10 ns MD simulation for horse BuChE in complex with compound 11. (a) The RMSD of Cα atoms and (b) potential
energy were calculated during the simulation time. (c) Detailed binding mode of compound 11 at the active site of horse BuChE. The protein
and compound 11 were shown as green line ribbons and blue stick models, respectively. The active site residues were displayed as different
stick colors based on their subsites, PAS (magenta), ABP (orange), OH (yellow), CAS (purple), and AS (green), respectively. The residues
not belonging to any subsite were shown as gray stick models. Hydrogen bonds were represented as black dashed lines. (d) The surface repre-
sentation for 11 binding in horse BuChE. The protein surface was colored by its electrostatic potential (blue, positive; red, negative).
Figure 6. The most populated conformations of compound 11 in human BuChE. (a) Binding pattern of 11 in the active site of human
BuChE. The protein and the compound were drawn as sky blue line ribbons and blue stick models. The active site residues were displayed
as different stick colors based on their subsites, PAS (magenta), ABP (orange), OH (yellow), CAS (purple), and AS (green), respectively.
(b) The docking poses for compound 11 were shown with the electrostatic potential surface of human BuChE (blue, positive; red,
negative).
compound
5
was synthesized from the selective
4-methoxy group of paeonol with a propargyl group.
Starting compound 6 was obtained from the propargylation
of paeonol. Compounds 13 and 14 were synthesized by the
propargylation between 2,4-dihydroxyacetophenone and
propargyl chloride, which replaced the methyl group of
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Figure 7. The results of 10 ns MD simulation for human BuChE in complex with compound 11. (a) The RMSD of Cα atoms and
(b) potential energy were calculated during the simulation time. (c) Detailed binding mode of compound 11 at the active site of human
BuChE. The protein and compound 11 were shown as sky blue line ribbons and blue stick models, respectively. The active site residues
displayed as different stick colors based on their subsites, PAS (magenta), ABP (orange), OH (yellow), CAS (purple), and AS (green),
respectively. The residues not belonging to any subsite were shown as gray stick models. Hydrogen bonds were represented as black
dashed lines. (d) The surface representation for 11 binding in human BuChE. The protein surface was colored by its electrostatic potential
(blue, positive; red, negative).
click reactions between compounds 5 or 6 and tryptamine
azide, respectively. Compound 13 exhibited weak inhibi-
tory activity against AChE, but there was no inhibitory
activity against BuChE. Compound 14 showed moderate
inhibitory activity against BuChE only (IC50 = 5.49
ꢀ 0.41 μM). Compounds 15–18 coupled between trypt-
amine azide and propargylated polyphenols such as acetyl
syringic acid, 3,4-dimethoxy cinnamic acid, acetyl ferulic
acid, and diacetyl caffeic acid showed moderate or no
inhibitory activity against BuChE. This may be due to the
size effect of polyphenol. Compound 15 has the benzoic
acid moiety, but compounds 16–18 have the cinnamic acid
moiety. The size of cinnamic acid is bigger than that of
benzoic acid. When anti-inflammatory activity was mea-
sured using compounds 13, 14, and 15, compound 13 and
14 showed similar inhibitory effects on LPS-induced NO
production, while compound 15 had little anti-inflammatory
activity. However, compound 14 was less cytotoxic than
13.17 In the measurement of cholinesterase inhibitory activ-
ity using these compounds, compound 13 weakly inhibited
AChE, while compounds 14 and 15 selectively inhibited
BuChE.
(BuChE)] inhibitory activity.32 In ALA-polyphenol com-
pounds, ALA-acetylated caffeic acid compounds showed
the highest BuChE inhibitory activity, while in the trypt-
amine derivatives acetylated syringic acid derivative
showed the highest activity. In ALA-polyphenol com-
pounds, activity was analyzed using various QSAR param-
eters [hydrophobicity parameter (π), Hammett electronic
substituent constant (σm & σp), and molar refractiv-
ity (MR)].32
In the tryptamine derivative, the size of the propargyl
moiety and the position of its functional group appear to be
important for anti-inflammatory activity and cholinesterase
inhibitory activity.
The binding mode of compound 11 was analyzed. It was
observed that the indole group interacted with Trp82 and
Tyr332 though π–π stacking interactions with horse
BuChE. The middle triazole ring also formed π–π stacking
with Phe329. The lipoamide group was stabilized by hydro-
phobic interactions with the active site residues including
Glu197, Ser198, Trp231, Leu285, Leu286, and Val288.
The middle NH moiety and carbonyl group in lipoamide
group made hydrogen bonds with Leu285 and Gln119,
respectively. Furthermore, the sulfur atom in a terminal
dithiolane ring of lipoamide group formed hydrogen bond
interactions with Gly116 and Gly117. The middle amide
group of compound 11 was toward the outside, and indole
Previous we synthesized hybrid molecules between (R)-
lipoic acid (ALA) and acetylated or methylated polyphenol
compounds and evaluated their in vitro cholinesterases
[acetylcholinesterase (AChE) and butyrylcholinesterase
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25
20
15
10
5
and lipoamide groups at both ends were buried inside of
human BuChE. The indole group of compound 11 was
placed near a catalytic anionic site (CAS) and anionic sub-
site (AS). It showed hydrophobic interactions with Trp82
and Tyr128 on the AS and His438 on the CAS. The NH
group of the indole ring formed a hydrogen bond with the
carboxyl group of Glu197. On the other hand, the
lipoamide group was positioned within the pocket sur-
rounded by residues that belong to the peripheral anionic
subsite (PAS), acyl binding pocket (ABP), and oxyanion
hole (OH). It formed hydrophobic interactions with Gln119
and Tyr332 on PAS, Leu286, Val288, and Trp231 on ABP,
as well as Gly116 and Gly117 on OH subsites. Addition-
ally, a terminal dithiolane ring in the lipoamide group was
involved in sulfur-π interactions with Phe329 and Phe398.
The middle NH moiety and sulfur atom in the dithiolane
ring were hydrogen bonded to the backbone carbonyl group
of Pro285 and the hydroxyl group of Ser198, one of the
catalytic triad, respectively.
5
0
0
0.2
[I] µM
0
-3
-2
-1
0
1
2
3
4
5
6
1/S
Figure 8. Lineweaver–Burk plot of the kinetic study of the inhibi-
tory effect of compound 11 against BuChE = 001 μM,
= 0.08 μM, = 0.10 μM). The inset is a plot of [I] vs.
KM/Vmax
(
.
Rural Development Administration (RDA) of Republic of
Korea and by Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by
the Ministry of Education (2017R1D1A1B04028757).
A kinetic study of BuChE was performed at different
concentrations of compound 11 to explore inhibition mech-
anisms (Figure 8), and the Lineweaver–Burk plot suggested
a mixed type of inhibition. The mixed type of inhibition
corresponds well with the molecular modeling study, in
which compound 11 was bound to the CAS, AS, PAS,
ABP, and OH.
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