Galantamine derivatives as acetylcholinesterase inhibitors: Docking, design, synthesis, and inhibitory activity

Article abstract of DOI:10.1007/978-1-4939-7404-7_6

Galantamine (GAL) is a well-known acetylcholinesterase (AChE) inhibitor, and it is widely used for treatment of Alzheimer’s disease. GAL fits well in the catalytic site of AChE, but it is too short to block the peripheral anionic site (PAS) of the enzyme,

Full text of DOI:10.1007/978-1-4939-7404-7_6

Chapter 6
Galantamine Derivatives as Acetylcholinesterase Inhibitors:
Docking, Design, Synthesis, and Inhibitory Activity
Irini Doytchinova, Mariyana Atanasova, Georgi Stavrakov,
Irena Philipova, and Dimitrina Zheleva-Dimitrova
Abstract
Galantamine (GAL) is a well-known acetylcholinesterase (AChE) inhibitor, and it is widely used for
treatment of Alzheimer’s disease. GAL fits well in the catalytic site of AChE, but it is too short to block
the peripheral anionic site (PAS) of the enzyme, where the amyloid beta (Aβ) peptide binds and initiates the
Aβ aggregation. Here, we describe a docking-based technique for designing of GAL derivatives with dual-
site binding fragments – one blocking the catalytic site and another blocking the PAS. The highly scored
compounds are synthesized and tested. Protocols for docking, design, synthesis, and AChE inhibitory test
are given.
Key words Galantamine, Acetylcholinesterase inhibition, Docking, Drug design, Dual-site binding
1 Introduction
The binding site of recombinant human AChE (rhAChE) consists
of several domains [1–5]. The catalytic anionic site (CAS) lies on
˚
the bottom of 20 A deep and narrow binding gorge. It consists of
the catalytic triad: Ser203, Glu334, and His447. The anionic
domain binds the quaternary trimethylammonium choline moiety
of acetylcholine (ACh). Despite its name, it does not contain any
anionic residues but only aromatic ones: Trp86, Tyr130, Tyr337,
and Phe338. They are involved in cation-pi interactions with the
protonated head of ACh. The acyl pocket consists of two bulky
residues, Phe295 and Phe297, and it determines the selective bind-
ing of ACh by preventing the access of larger choline esters. The
oxyanion hole hosts one molecule of structural water and consists
of Gly121, Gly122, and Ala204. The water molecule bridges the
binding between enzyme and substrate by hydrogen-bond net-
working and stabilizes the substrate tetrahedral transition state.
Kunal Roy (ed.), Computational Modeling of Drugs Against Alzheimer’s Disease, Neuromethods,
vol. 132, DOI 10.1007/978-1-4939-7404-7_6, © Springer Science+Business Media LLC 2018
163

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Irini Doytchinova et al.
Finally, the peripheral anionic site (PAS) lies at the entrance to
binding gorge. It is composed of five residues: Tyr72, Asp74,
Tyr124, Trp286, and Tyr341. PAS allosterically modulates catalysis
[6] and is implicated in non-cholinergic functions such as amyloid
deposition [7], cell adhesion, and neurite outgrowth [8, 9]. It was
found that the Aβ peptide binds close to PAS, interacting hydro-
phobically with residues 275–305 [7]. This interaction promotes
the formation of amyloid fibrils characteristic of Alzheimer’s disease
[10, 11]. The blockade of PAS prevents the AChE-induced Aβ
aggregation [7]. This pivotal finding prompted the design of
novel AChEIs with dual binding moieties – one blocking the CAS
at the bottom of the gorge and one blocking the PAS [12–17].
Galantamine (GAL) is an alkaloid initially isolated from the
bulbs and flowers of Galanthus caucasicus, Galanthus woronowii
(Amaryllidaceae), and related genera [18]. Paskov first developed
GAL as an industrial drug under the trade name Nivalin
(Sopharma, Bulgaria) [19]. It has been used for treatment of
myasthenia gravis, myopathy, residual poliomyelitis paralysis syn-
dromes, sensory and motor disorders of CNS, and decurarization
[20–24]. Because of its ability to cross the blood-brain barrier and
to affect the central cholinergic function, in the 1980s, GAL was
investigated for treatment of Alzheimer’s disease (AD) and in 2000
was approved for use in Europe, the United States, and Asia
[25–27].
In addition to its acetylcholinesterase (AChE)-inhibiting abil-
ity, GAL has been identified as an allosteric modulator of nicotinic
acetylcholine receptors (nAChRs) [28–31]. The stimulation of
nAChRs can increase intracellular Ca2+ levels and facilitate nor-
adrenaline release; both effects enhance the cognitive brain func-
tion [32]. Recently, Takata et al. have demonstrated that treatment
of rat microglia with GAL significantly enhanced microglial amy-
loid-β (Aβ) phagocytosis and facilitated Aβ clearance in brains of
rodent AD models [33]. This multiple-target action of GAL makes
it a most valuable drug for AD treatment and prompts the synthesis
of novel GAL derivatives with improved binding to AChE [33–39].
Several series of GAL derivatives with dual-site binding to the
enzyme have been prepared and tested [16, 36, 37, 40–42]. All
of them showed good AChE inhibitory activities.
In the present paper, we summarize our experience in develop-
ing of galantamine derivatives as acetylcholinesterase inhibitors
with dual-site binding [43–45]. We used docking-based design in
a stepwise manner to develop derivatives with binding affinity more
than 1000 times higher than that of GAL.

Galantamine Derivatives as Acetylcholinesterase Inhibitors: Docking. . .
165
2 Materials
GOLD [46] is a docking tool based on a genetic algorithm, and it
has proven successful in virtual screening, lead optimization, and
identification of the correct binding mode of active molecules [47,
48]. GOLD takes into account the flexibility of the ligand as well as
the flexibility of the residues in the binding site. GOLD v. 5.1 was
used in our studies.
2.1 Docking
Software
Fine chemicals: 5-hydroxyindole, 97%; 5-aminoindole, 97%; 4-
hydroxybenzaldehyde, 99%; 1,6-dibromohexane, 98%; 7-
bromoheptanoic acid, 97%; 6-bromohexanoic acid, 98%; 5-
bromovaleric acid, 97%; 4-bromobutyric acid, 98%; aniline,
99.8%; benzylamine, 99%; phenethylamine, 99%; (S)-(þ)-2-phe-
nylglycine methyl ester hydrochloride, 97%; L-phenylalanine
methyl ester hydrochloride, 98%; L-tryptophan methyl ester hydro-
chloride, 98%; galantamine hydrobromide, 95%.
2.2 Reactants for
Synthesis
Reagents: potassium carbonate, 99 þ %; sodium triacetoxybor-
ohydride, 97%; N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride (EDC), 98%; 1-hydroxybenzotriazole hydrate, 99%;
N,N-diisopropylethylamine, for synthesis; 3-chloroperoxybenzoic
acid, 70–75%; ammonia water, 25%, pure for analysis; iron(II)
sulfate heptahydrate, 99 þ %; sodium sulfate, anhydrous, 99 þ %;
sodium bicarbonate, 99 þ %; triethylamine, 99%; hydrochloric acid,
ca. 37% solution in water.
Solvents: 1,2-dichloroethane, extra pure; acetonitrile, 99.9%,
extra dry over molecular sieve; methylene chloride, extra pure;
ꢀ
petroleum ether 40–60 C, extra pure; ethyl acetate, for analysis;
methanol for HPLC.
Electrophorus electricus AChE (Sigma-Aldrich), acetylthiocholine
(ATCl), 5,50-dithio-bis-(2-nitrobenzoic acid) (DTNB), phosphate
buffer (pH 7.6), UV-Vis Spectrophotometer Shimadzu UV-
1203 at 405 nm.
2.3 Reactants for
AChE Inhibition Assay
3 Methods
The GAL derivatives are N-substituted products (Fig. 1). Linkers of
different type and length connect the GAL core to aromatic frag-
ments. The GAL core fits into the catalytic site, while the aromatic
fragment aims to block the PAS (Fig. 2).
3.1 Design
The optimized docking protocol includes the following settings:
1. Protein: X-ray structure of rhAChE in complex with galanta-
3.2 Molecular
Docking Protocol
˚
mine (GAL) (pdb id: 4EY6, R ¼ 2.15 A) [49]. The ligand is
removed.

166
Irini Doytchinova et al.
Fig. 1 N-substituted GAL derivatives
Fig. 2 The GAL core fits into the catalytic site, while the aromatic fragment
blocks the PAS. AChE given as gray surface, inhibitor in cyan, and amyloid beta
peptide in blue
2. Ligands: The structures of the designed molecules are entered
in .mol2 format.
3. Scoring function: GoldScore or ChemPLP.
´
˚
4. Radius of the binding site: 10A.
5. One water molecule (HOH846) is kept within the binding site.
6. Flexible side chains in the binding site: Tyr72, Asp74, Trp86,
Tyr124, Ser125, Trp286, Phe297, Tyr337, Phe338, and
Tyr341.

Galantamine Derivatives as Acetylcholinesterase Inhibitors: Docking. . .
167
The compounds with the highest GoldScores or ChemPLP
scores are selected for synthesis.
Protocol 1: Synthesis of 4-(6-bromohexyloxy)benzaldehyde
3.3 Synthesis
3.3.1 Synthesis of
Bromo-Containing Linkers
of Different Type and
Length Connected to
Aromatic Fragments:
Br-Linker-Ar
1. Load a 100 ml round bottom flask with 4-hydroxybenzaldehyde
(0.5 g, 4.1 mmol), K2CO3 (2.83 g, 20.5 mmol), and CH3CN
(20 ml).
2. Add to the stirred suspension 1,6-dibromohexane (12.3 mmol,
1.86 ml), equip the flask with a condenser, and reflux for 5 hours.
3. Stop the heating, remove the condenser, and concentrate till dry
under reduced pressure.
4. Separate the residue between ethyl acetate and water, extract the
aqueous phase with ethyl acetate, and dry the organic extracts over
Na2SO4. Filter from the solids and concentrate.
5. Purify via flash column chromatography using silica gel as station-
ary phase and a mixture of ethyl acetate/petroleum ether (1:9) as
mobile phase.
Protocol 2: Synthesis of N-(4-(6-bromohexyloxy)benzyl)-1H-indol-5-
amine
1. Load a 50 ml round bottom flask with 1H-indol-5-amine
(0.100 g, 0.757 mmol), 4-(6-bromohexyloxy)benzaldehyde
(0.216 g, 0.757 mmol), and 1,2-dichloroethane (8 ml).
2. Stir the mixture for 30 min at room temperature, add NaBH
(OAc)3 (0.240 g, 1.136 mmol), and continue the stirring for 24 h.
3. Quench the reaction with sat.aq.NaHCO3 (50 ml), extract the
product with CH2Cl2, and dry the organic extracts over Na2SO4.
Filter from the solids and concentrate.
4. Purify via flash column chromatography using silica gel as station-
ary phase and a mixture of petroleum ether/ethyl acetate/triethy-
lamine (2:1:0.5) as mobile phase.

168
Irini Doytchinova et al.
Protocol 3: Synthesis of 5-(6-bromohexyloxy)-1H-indole
1. Load a 50 ml round bottom flask with 1H-indol-5-ol (0.100 g,
0.750 mmol), CH3CN (15 ml), and K2CO3 (0.310 g,
2.250 mmol).
2. Add to the stirred suspension 1,6-dibromohexane (0.220 g,
0.900 mmol) and heat at 60 ꢀC for 24 h.
3. Cool to room temperature, filter through a pad of Celite, and
concentrate till dry under reduced pressure.
4. Purify via flash column chromatography using silica gel as station-
ary phase and a mixture of petroleum ether/ethyl acetate (4:1) as
mobile phase.
Protocol 4: Synthesis of bromo-amide intermediates
aa
Bromo carboxylic acid
Appropriate amine
Product (bromo-amide intermediate)
Yield %
n ¼ 5
n ¼ 6
84
88
n ¼ 4
n ¼ 4
90
67
n ¼ 5
89
(continued)

Galantamine Derivatives as Acetylcholinesterase Inhibitors: Docking. . .
169
Bromo carboxylic acid
Appropriate amine
Product (bromo-amide intermediate)
Yield %
n ¼ 6
94
78
n ¼ 5
n ¼ 6
n ¼ 3
n ¼ 4
n ¼ 5
n ¼ 6
n ¼ 5
80
88
90
86
89
99

170
Irini Doytchinova et al.
1. Load a 50 ml round bottom flask with bromo carboxylic acid
(1.1 mmol), CH2Cl2(20 ml), N-[3-(dimethylamino)propyl]-N-
ethylcarbodiimide (0.210 g, 1.1 mmol), 1-hydroxybenzotriazole
(0.148 g, 1.1 mmol), and the appropriate amine (1 mmol).
2. In the cases of amino acid methyl ester hydrochlorides, add also N,
N-diisopropylethylamine (1.1 mmol).
3. Stir the mixture at room temperature. Follow the reaction develop-
ment by TLC.
4. Concentrate under reduced pressure till dry.
5. Purify via flash column chromatography using silica gel as station-
ary phase and a mixture of dichloromethane/ethyl acetate (20:1)
as mobile phase.
Protocol 5: Synthesis of norgalantamine and its subsequent alkylation
with Br-Linker-Ar:
3.3.2 Synthesis of the
Target Compounds
1. To a stirred suspension of galantamine hydrobromide (0.734 g,
2 mmol) in dichloromethane (10 ml), add 25% aqueous NH4OH
(1 ml) and stir till clear. Separate the organic layer, dry it over
Na2SO4, and concentrate under reduced pressure to isolate free
galantamine base.
2. Dissolve the galantamine (2 mmol) into dichloromethane
(10 ml), add portionwise 3-chloroperoxybenzoic acid (0.542 g,
2.2 mmol), and stir at room temperature for 1 h.
3. Cool the mixture to 0 ꢀC and add methanol (20 ml) followed by
FeSO4.7H2O (0.556 g, 2 mmol). Stir for ½ h at 0 ꢀC and for 3 h
at room temperature.
4. Quench the reaction with 5 N HCl (40 ml), remove the organic
solvents under reduced pressure, and wash the aqueous residue
twice with ether.
ꢀ
5. Cool the aqueous layer to 0 C and basify it with 25% aqueous
NH4OH.
6. Extract with dichloromethane, dry over Na2SO4, and
concentrate.

Galantamine Derivatives as Acetylcholinesterase Inhibitors: Docking. . .
171
7. Purify via flash column chromatography using silica gel as sta-
tionary phase and a mixture of dichloromethane/methanol (5:1)
as mobile phase to isolate norgalantamine.
8. Prepare under argon atmosphere a solution of norgalantamine
(0.200 g, 0.732 mmol) in anhydrous acetonitrile (25 mL).
9. Add the appropriate Br-Linker-Arom (0.951 mmol) and anhy-
drous K2CO3 (0.303 g, 2.2 mmol).
10. Stir at 60 ꢀC for 24 h, cool to room temperature, filter through a
pad of Celite, and remove the solvent under reduced pressure.
11. Purify via flash column chromatography using silica gel as sta-
tionary phase and a mixture of dichloromethane/methanol/
ammonia (20:1:0.05) to isolate the desired product.
Structure elucidation: The structures of the newly synthesized com-
pounds were confirmed by 1D and 2D NMR spectra. Their purity was
proven by elemental analysis or HRMS. The spectral analyses were in
accordance with the assigned structures. Additionally, the compounds
were characterized by melting points and specific rotation for the chiral
compounds.
The AChE activity is assayed as described by Ellman et al. [50] with
some modifications [51]. 50 μL of E. electricus AChE (Sigma-
Aldrich) in buffer phosphate (pH 7.6) and 50 μL of the test
compounds (4–500 μM in methanol) dissolved in 700 μL in the
same buffer are mixed. The mixtures are incubated for 30 min at
room temperature. 100 μL of the substrate solution (0.5 M DTNB,
0.6 mM ATCI in buffer, pH 7.6) is added. The absorbance is read
in Shimadzu spectrophotometer at 405 nm after 3 min. Enzyme
activity is calculated as a percentage compared to an assay using a
buffer without any inhibitor using nonlinear regression. IC50
values are means Æ SD of three individual determinations each
performed in triplicate. GAL is used as a positive control.
3.4 AChE Inhibition
Assay
4 Some Results Illustrating the Docking-Based Design Efficiency
In order to illustrate the efficiency of the proposed docking-based
design of novel dual-site binding GAL derivatives, in Table 1., we
present some results from our in-house database of GAL derivatives
designed to block simultaneously the CAS and PAS [44, 45]. More
than hundred compounds were designed and docked into AChE
and the best scored of them were synthesized and tested.
Most of the compounds were 10–100 times more active than
GAL. However, two of them – compounds 9 and 15 – were more
than 1000 times more active. They are subject to further research.

172
Irini Doytchinova et al.
Table 1
Docking scores and experimental IC50 values of the designed GAL derivatives
Times
IC50 (exp) more active
Docking
scorea
Comp. Linker
Ar
μM
than GAL
1
(CH2)6O
Phenyl-CH2NH-
indolyl
119.15
0.011
95
2
3
4
(CH2)6O
Indolyl
Indolyl
Indolyl
112.32
109.06
113.89
0.012
0.015
0.094
93
72
11
(CH2)5CONH
(CH2)5CONHCH
(COOCH3)CH2
5
(CH2)6CONH
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
100.87
99.82
0.0169
0.0308
0.0308
0.021
63
35
6
(CH2)4CONHCH2
(CH2)4CONH(CH2)2
(CH2)5CONH(CH2)2
(CH2)6CONH(CH2)2
7
101.79
106.09
109.94
107.52
35
8
51
9
0.0008
0.0527
1338
20
10
(CH2)5CONHCH
(COOCH3)
11
12
13
14
15
(CH2)6CONHCH
(COOCH3)
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
111.86
97.53
0.0211
0.0958
0.0264
0.0246
0.0011
1.07
51
11
(CH2)3CONHCH
(COOCH3)CH2
(CH2)4CONHCH
(COOCH3)CH2
104.38
108.24
116.95
74.56
40
(CH2)5CONHCH
(COOCH3)CH2
43
(CH2)6CONHCH
(COOCH3)CH2
1008
1
GAL HBr
Data are taken from Refs. [44, 45]
aGoldScores for compounds 1–4
GAL HBr ChemPLP scores for compounds 5–15
5 Conclusions
Molecular docking is a widely used structure-based method for
virtual screening of huge databases of compounds as well as for
binding prediction of newly designed ligands. It was used solely or
in combination with 2D- and 3D-QSAR or machine learning
methods [52–64]. Here, we described a protocol for molecular
docking of GAL derivatives into the enzyme AChE. The protocol
was optimized stepwise to find the settings (scoring function,

Galantamine Derivatives as Acetylcholinesterase Inhibitors: Docking. . .
173
flexible binding site, radius of the binding site, presence/absence of
a water molecule) correlating best with the binding affinities of
compounds. The optimized protocol was used to predict the affi-
nities of newly designed GAL derivatives with dual-site binding to
AChE. The best scored compounds were synthesized and tested.
All of them are 10–100 times more active than GAL. Even more,
two of them are more than 1000 times more active.
6 Note
The BLAST alignment of AChEs from human, rat, rabbit, and
E. electricus shows that the main residues forming the binding site
are conserved.
Acknowledgments
This work has been supported by the Council on Medical Science at
the Medical University of Sofia, Bulgaria (Grants 2-S/2013, 1-S/
2014, 8-S/2015, and 10-S/2016).
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