Synthesis, biological evaluation and molecular modeling studies of substitutedN-benzyl-2-phenylethanamines as cholinesterase inhibitors

Article abstract of DOI:10.1039/d0nj00282h

In this work, we report the synthesis of a series of derivatives ofN-benzyl-2-phenylethanamine which is the framework of norbelladine, the natural common precursor of the Amaryllidaceae alkaloids. These compounds were assessed in the inhibition of both AChE and BChE which are the enzymes responsible for the breakdown of acetylcholine and hence they constitute targets in the palliative treatment of Alzheimer's disease. In particular, brominated derivatives exhibited the lowest IC₅₀values against AChE. Interestingly, the presence of iodine in one of the aromatic rings highly increased the inhibition of BChE compared to its analogues, with an IC₅₀value similar to that of galantamine, which is the reference compound currently used in the treatment of AD. A possible mechanism of action for these compounds was determined by molecular modeling studies using combined techniques of docking and molecular dynamics simulations.

Full text of DOI:10.1039/d0nj00282h

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Synthesis, biological evaluation and molecular modeling studies of substituted
N-benzyl-2-phenylethanamine as cholinesterase inhibitors
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Florencia Carmona Vigliancoa, Daniel Zaragoza Pucholb, Oscar Parravicinic, Adriana Garroc,
Ricardo D. Enrizc, Gabriela E. Feresinb, Marcela Kurina-Sanza, Alejandro A. Ordena
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a INTEQUI CONICET, Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis,
Almirante Brown 1455, San Luis, D5700HHW, Argentina
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b Instituto de Biotecnología, CONICET, Facultad de Ingeniería, Universidad Nacional de San Juan. Av.
Libertador General San Martín 1109 (O), CP 5400, San Juan, Argentina
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c IMIBIO-SL CONICET, Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis,
Ejército de los Andes 950, San Luis, D5700HHW, Argentina
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Corresponding author: E-mail aaorden@unsl.edu.ar
Abstract
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In this work we report the synthesis of a series of derivatives of N-benzyl-2-phenylethanamine
which is the framework of norbelladine, the natural common precursor of the Amaryllidaceae
alkaloids. These compounds were assessed in the inhibition of both AChE and BChE which
are the enzymes responsible for the breakdown of acetylcholine and hence they constitute
targets in the palliative treatment of Alzheimer disease. In particular, brominated derivatives
exhibited the lowest IC50 values against AChE. Interestingly, the presence of iodine in one of
the aromatic rings highly increased the inhibition of BChE compared to its analogues, with an
IC50 value similar to that of galantamine, which was the reference compound currently used in
the treatment of AD. A possible mechanism of action for these compounds was determined by
molecular modeling studies using combined techniques of docking and molecular dynamics
simulations.
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Keywords
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Amaryllidaceae alkaloid precursor; AChE and BChE inhibition; norbelladine analogues;
reductive amination; molecular modeling.
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Abbreviations
AD: Alzheimer Disease
ACh: acetylcholine
AChE: Acetylcholinesterase
BChE: Butyrylcholinesterase
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IC50: half maximal inhibitory concentration
Gal: Galantamine
MD: Molecular Dynamics
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1. Introduction
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Alzheimer’s disease (AD) is the most common type of dementia among older adults which is
characterized by chronic neurodegenerative pathology that causes a significant and
progressive functional disability, loss of cognition and altered behavior. Several factors have
been described to play a role in the pathogenesis of AD including a deficit of acetylcholine
(ACh), tau-protein aggregation and extracellular deposits of amyloid plaques. Consequently,
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multiple pharmacological targets can be tackled as a palliative treatment for this disease 1,2
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Cholinesterase inhibitors have been developed as therapeutic agents for AD based on the
cholinergic dysfunction hypothesis which states that low levels of ACh lead to cognitive
impairment and dementia 3. The human brain contains two different cholinesterases:
acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) which
are the enzymes responsible for hydrolyzing the neurotransmitter ACh into choline and
acetate. Currently, only three AChE inhibitors have been approved by the FDA and used for
the palliative treatment of mild to moderate symptoms of AD: galantamine, donepezil and
rivastigmine. AChE has gained great relevance as a target for novel drug discovery because
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of its dual functionality: ACh hydrolysis and amyloid beta peptide aggregation . On the other
hand, BChE has a role in the hydrolysis of ACh but also non-enzymatic functions, such as
being involved in anti-inflammatory pathways and delaying the rate of neurotoxic amyloid-β
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fibril formation
which foster the study of this enzyme as an important target in AD
pharmacotherapy.
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Several studies have been performed in order to discover novel anticholinesterases either as
naturally-occurring compounds or synthetic inhibitors. Plant species and their potentially active
compounds such as terpenes, coumarins, polyphenols and alkaloids have been screened for
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anti-AChE activity being the latter the most potent compounds assessed . Recently, some
molecules have been designed and synthesized based on different scaffolds such as
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chalcone-derivatives 7–9, 1,2,3-triazole-chromenone carboxamides 10, dibenzo-γ-pyrones
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benzofurans 12, spirooxindoles 13, tacrine-ferulic acid and quinoline-ferulic acid hybrids as
multi-target-directed ligands 14,15, among many others 2. Most of them have shown moderate to
significant cholinesterase inhibitory activity. Also, the modification of natural compounds by
xenobiotic biotransformation has been a tool to increase their biological activity 16.
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Norbelladine is an alkaloid-like amine (protoalkaloid) resulting from the condensation of 3,4-
dihydroxybenzaldehyde -protocatechuic aldehyde- (derived from phenylalanine) and tyramine
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(derived from tyrosine). This is the common precursor in the biosynthesis of all Amaryllidaceae
alkaloids which is further regioselectively methylated to give 4’-O-methylnorbelladine that can
undergo three different types of oxidative phenol coupling reactions (para-para’, ortho-para’,
ortho’-para couplings) to give alkaloids such as haemanthamine, lycorine and galantamine.
The latter is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and
summer snowflake (Leucojum aestivum) and has been used in the palliative treatment of
Alzheimer’s disease in the early stages 17,18. The potential health effects of the Amaryllidaceae
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alkaloids have been widely investigated although there are a limited number of studies of
the bioactivities of their precursors. One recent example is the antioxidant and anti-
inflammatory effects of norbelladine via scavenging radicals and inhibiting both COX-1 and 2
enzymes 20.
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Halogens in ligand–target complexes play an important role due to steric aspects that
influence their conformation, allow intermolecular interactions that favorably contribute to the
stability and also increase membrane permeability 21. Although less abundant than fluorine-
containing drugs which are estimated 20 % of all pharmaceuticals, there are interesting
examples of commercially available organobromine drugs, such as the mucolytic drug
bromhexine, the vasodilator nicergoline, the sedative and hypnotic brotizolam and non-
steroidal anti-inflammatory for ophthalmic use bromfenac 22. On the other hand, iodine is
highly used in nuclear medicinal diagnostic techniques and there are a few examples of
iodine-containing organic compounds such as iobenguane, a blocking agent for adrenergic
neurons, I131 iodocholesterol with diagnostic imaging activity, 4'-iodo-4'-deoxydoxorubicin with
antiamyloid activity and 4-iodopropofol, an alkylphenol derivative with anticonvulsant activity 23.
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In the present study, inspired by the common precursor of Amaryllidaceae alkaloids, we
synthesized a series of substituted N-benzyl-2-phenylethanamine based on the norbelladine
structure as a scaffold. Most of the compounds bear a halogen atom on one of the aromatic
rings. These compounds were assessed as cholinesterase inhibitors and a plausible
mechanism of action was explained by molecular modeling studies using combined
techniques such as docking calculations and molecular dynamics simulations. As a result, the
possible stereo-electronic requirements for these ligands were also discussed regarding to
their different affinities.
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2. Results and discussion
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2.1. Synthesis of norbelladine analogues
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N-benzyl-2-phenylethanamine (norbelladine framework) was used as a key unit to design new
analogues with different substitution patterns mainly at the A-ring. 4’-O-methylnorbelladine and
several non-natural analogues were synthesized by condensation of substituted aromatic
aldehydes (1-9) and tyramine (10) or phenylethylamine (11) to form the Schiff base and further
reduced with sodium borohydride. The reductive amination to obtain 4’-O-methylnorbelladine
(12) has been already described in literature with different yields 24–27. Several experiments
were conducted to improve the isolated yields, especially using some Lewis acid catalysts,
bases and dehydrating agent in the reaction mixture as well as different temperatures (data
not shown). The best results were obtained at room temperature in methanol using anhydride
sodium sulfate as a desiccant and triethylamine (TEA) or KOH as a base to increase the
nucleophile strength of the primary amine giving the desired products. Most of the halogenated
norbelladine analogues precipitated during the work-up procedure in the alkaline aqueous
medium at their corresponding isoelectric point and thus were purified by filtration and simple
recrystallization. However, to recover 12 after work-up, it was necessary to perform a partition
with ethyl acetate. As a result, a library of ten synthetic analogues of norbelladine was
obtained in yields ranging from 51-92 % with purity higher than 96 % determined by GC-FID or
GC-MS. The halogenated compounds were synthesized from the corresponding aldehydes as
starting materials.
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12 R1: H R2: OH R3: OCH3 R4: H R5: OH
13 R1: Cl R2: OH R3: OCH3 R4: H R5: OH
14 R1: Br R2: OH R3: OCH3 R4: H R5: OH
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R2: OH R3: OCH3 R4: H R5: OH
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16 R1: H R2: OH R3: OCH3 R4: Br R5: OH
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18 R1: H R2: H
19 R1: H R2: H
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R3: F
R4: H R5: OH
R4: H R5: OH
R4: H R5: OH
R4: H R5: OH
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R3: Cl
R3: Br
R3: OH
21 R1: Br R2: OH R3: OCH3 R4: H R5: H
Fig. 1. Reductive amination for the synthesis of norbelladine analogues
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2.2. Cholinesterase inhibitory activity
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The synthesized compounds were tested for cholinesterase inhibition of both Electrophorus
electricus AChE and equine butyrylcholinesterase BChE according to Ellman’s method 28 with
some modifications 29, and the results were expressed as IC50 values. In order to make the
compounds more water-soluble for the bioassays and avoid the use of co-solvents, the
hydrochloride salts of 12-14 were prepared. No significant differences in the IC50 values for the
inhibition of AChE or BChE were observed for the compounds with free amines compared to
the amine salt (see Table S1).
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Compounds 13, 14, 16 and 21 showed the lowest IC50 values for AChE inhibition. The
introduction of halogenated substituents at the A-ring improved the cholinesterase inhibition
compared to the natural compound 4’-O-methylnorbelladine (12) with the exception of iodine.
Other authors have also discussed how the presence of halogens on thiophene chalcones and
pyrazoline derivatives exerted an increase in the cholinesterase inhibition 7,8. Brominated
compounds on 2’ position 14 and 21 were the most effective inhibitors against AChE exhibiting
IC50 values of 16.79 ± 0.51 and 17.14 ± 3.17 µM, respectively, differing by one order of
magnitude higher than the positive control galantamine (IC50 of Gal: 1.2 ± 0.1 µM). These
studies suggest that the brominated derivatives of 12 show higher cholinesterase inhibition
than their analogues, being good candidates to deepen for this bioactivity. On the other hand,
monosubstituted derivatives (17-20) at the A-ring displayed no inhibition activity towards
AChE. However, with the exception of the fluorinated compound 17, they all showed to some
extent some inhibition of BChE, being the brominated analogue the best inhibitor for this
enzyme with an IC50 value of 23.90 ± 6.26.
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Interestingly, when the OH group at the B-ring was not present (compound 21) the product
exerted a high inhibition of both cholinesterases with IC50 of 17.14 ± 3.17 for AChE and 13.35
± 3.01 for BChE. This latter value was comparable to Gal (IC50 of 15.88 ± 1.6 µM). Also,
compound 15 showed a high selectivity for BChE inhibition showing an IC50 value of 13.34 ±
2.79 µM.
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Table 1
Results of the cholinesterase inhibition with norbelladine analogues.
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Compound
R1
R2
R3
R4
R5
IC50 [µM]
Selectivity
to BChE
AChE
BChE
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H
Cl
Br
I
H
H
H
H
H
Br
OH
OH
OH
OH
OH
H
H
H
H
OH
OCH3
OCH3
OCH3
OCH3
OCH3
F
Cl
Br
OH
OCH3
H
H
H
H
Br
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
69.26±0.52
36.18±4.99
16.79±0.51
173.66±14.78
34.79±4.52
>200
65.48±4.84
62.25±4.64
49.91±3.01
13.34±2.79
30.32±1.81
>200
1.05
0.58
0.34
13.01
1.15
n.d.
n.d.
n.d.
n.d.
1.28
0.08
>200
>200
>200
159.27±28.44
23.90±6.26
52.98±8.58
13.35±3.01
15.88±1.65
21
Gal*
17.14±3.17
1.21±0.06
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AChE and BChE inhibition is expressed as the mean ± SD (n = 3 experiments). Selectivity to BChE:
IC50 for AChE/IC50 for BChE. *Galantamine (Gal) was used as positive control. n.d.: not determined.
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2.3. Molecular modeling studies
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In order to have a better understanding of the experimental results obtained, we carried out a
molecular modeling study for compounds 12-21. It should be noted that calculations were
carried out considering that the amino group is protonated at physiological pH. Results
previously reported by our group regarding to a well-known AChE inhibitor, galantamine, was
also included here for discussion 29,30. This study was performed in three stages. First, we
conducted docking calculations which led us to find the probable modes of interaction between
ligands and the active site of both enzymes. Next, we performed molecular dynamics (MD)
simulations with the aim of analyzing compound behavior over time. In the last stage, using
the trajectories obtained from MD simulations, we calculated free energy of different
complexes and carried out a per-residue analysis in order to identify the AChE and BChE
active site amino acids involved in the intermolecular interactions of the different complexes.
Although biological tests were performed with EeAChE, the crystalline structure of TcAChE
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was used in molecular modeling studies. This is possible since both enzymes are considered
structurally homologous 31. The ligand binding pockets in TcAChE, EeAChE and even AChE in
vertebrates, have almost the same geometry, therefore, they are expected to bind inhibitors in
a very similar manner 31–33
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Considering the experimental results, it should be noticed that most compounds reported here
showed greater inhibitory activity against BChE than that observed against AChE. This might
be due to the difference in the active sites of both enzymes. BChE active site presents a larger
accessible area than the AChE active site since it has a lower number of aromatic residues in
its binding pocket 34. Both enzymes have over 60% of sequence identity and show a similar
response to a number of classical inhibitors since the amino acid sequence at the active site of
both AChE and BChE is well conserved 35. Additionally, the existence of a catalytic triad (Ser,
His and Glu) in the active site is considered important for the catalytic activity of both enzymes
36,37. However, six residues, i.e. Tyr70, Tyr121, Trp279, Phe288, Phe290 and Phe330, with
bulky aromatic side chains present in the AChE active site are substituted by non-aromatic
residues of Asn68, Gln119, Ala277, Val286, Val288 and Ala328 in BChE. This may generate
the appropriate conditions for these compounds to better accommodate in the active site of
BChE.
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4’-O-methylnorbelladine (12) and its halogenated derivatives at C2 (13-15) showed AChE
inhibitory activity, being compound 14 one the most active of all analogues studied here.
Figure 2A shows the main interactions stabilizing 14-AChE complex. These interactions
involve the following enzyme residues: Gln69, Trp84, Gly118, Ser122, Gly123, Ser124,
Phe330 and Tyr334. Our simulations suggest that OH at C3 (A-ring) establishes an H-bond
with the side chain of Gln69 and that OMe at C4 interacts with Ser124 backbone (Figure S1).
The interactions with residues Trp84, Gly118, Ser122 and Phe330 are the same to those
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previously reported for galantamine
(Figure 2B), suggesting that these compounds are
located in the same AChE region. It should be noted that activity increases when H at C2 of
compound 12 is substituted by chlorine and bromine, resulting in compounds 13 and 14,
respectively. It is important to highlight that 14 is about 4 times more active than 12. The
presence of a halogen atom in norbelladine analogues allows intermolecular interactions that
favorably contribute to the stability of the complexes. However, it seems that halogen atoms
also play an important role from the steric point of view; compound 15 with an iodine atom
showed the lowest activity of this series. These results could be related to the AChE active site
size, since compounds with bulky substituents cannot accommodate properly to establish
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favorable interactions compared to the other compounds of the same series (compare
inhibitory effects of compounds 13-15). Figure 3 shows the histogram obtained for compound
15. In this case significant interactions were observed for Asp72, Tyr121 and Trp279, all of
them belonging to the peripheral anionic site or bottleneck region 37. These results suggest
that compound 15 is located out of the AChE active site and establishes a different interaction
pattern in comparison with compounds 13 and 14, which could explain its poor activity.
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Fig. 2. Histograms of interaction energies partitioned with respect to AChE amino acid sequence when
complexed with compound 14 (A), Gal (B) and compound 17 (C). The X-axis denotes the residue
number of AChE and the Y-axis denotes the interaction energy between the compounds and a specific
residue. Negative values and positive values are favorable or unfavorable to binding, respectively.
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Fig. 3. Histograms of interaction energies partitioned with respect to AChE amino acid sequence when
complexed with compound 15. The X-axis denotes the residue number of AChE and the Y-axis denotes
the interaction energy between the compounds and a specific residue. Negative values and positive
values are favorable or unfavorable to binding, respectively.
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Regarding 21, one of the most active compounds with an IC50 value of 17.14 ± 3.17 µM, it is
structurally very similar to 14, being their only difference the lack of OH at B-ring. This
suggests that the presence of this substituent in this position is not an important structural
requirement for inhibition activity.
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On the other hand, monosubstituted derivatives at the A-ring (17-20) showed IC50 values ≥
200 µM for AChE, and therefore, they were considered as inactive. Figure 2C shows the
histogram corresponding to 17, as an example of these inactive compounds. It should be
noted that due to the different pattern of substitutions, the important interactions with Gln69
and Ser122 discussed above are missing for this compound.
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Regarding BChE, Figure 4A shows the histogram obtained for compound 15. The main
interactions stabilizing the complex are Trp110, Glu225, Phe357, and His466 among others.
Similar results were obtained for the rest of the active compounds. Comparing these results
29
with those observed for BChE-Gal complex
(Figure 4B), it is reasonable to assume that
derivatives 12-21 interact with the same region of the enzyme. In all complexes, the
protonated amino group of ligands is oriented towards the carboxyl group of Glu225,
establishing a salt bridge in most cases (Figure S2).
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Fig. 4. Histograms of interaction energies partitioned with respect to BChE amino acid sequence when
complexed with compound 15 (A), Gal (B) and compound 17 (C). The X-axis denotes the residue
number of BChE and the Y-axis denotes the interaction energy between the compounds and a specific
residue. Negative values and positive values are favorable or unfavorable to binding, respectively.
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On the other hand, the inhibitory effect of compounds 13 to 15 increases with the atomic
radius of the halogen in position R1. This might be due to the possibility that the iodine
substituent in 15 establishes a higher number of interactions with neighboring amino acids
than the rest of halogen derivatives tested. Compound 15 with IC50 value lower than
galantamine, establishes strong interactions with Trp110, Glu225 and His466 (Figure 4A). In
this line, the presence of the OH substituent at the B-ring seems to be important for this
compound to show inhibitory activity since an H-bond can be formed with the backbone of
Trp110 (Figure S2). It is important to notice that compound 15 is the most active against BChE
in this series. However, this compound does not display significant inhibitory activity against
AChE. As discussed above, the amino acid sequence in BChE active site allows to
accommodate bulkier ligands if compared to AChE. Figure 5 represents the spatial view of
both cholinesterases active sites when complexed with compound 15. As can be
seen, 15 adopts a different spatial arrangement in each complex. In 15-AChE complex (Figure
5A), the ligand remains close to the surface of the active site interacting with amino acid
residues from the peripheral anionic site and the bottleneck region. In contrast, in the 15-BChE
complex (Figure 5B), the ligand is located deeper in the gorge and can interact with the
catalytic triad and the acyl-binding pocket of the cholinesterase.
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B
A
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Fig. 5. Active sites of Torpedo californica acetylcholinesterase (AChE) (A) and Equus caballus
butyrylcholinesterase (B) when complexed with compound 15 which is represented in ball and stick and
colored in blue. The gorge of each enzyme is depicted by its molecular surface in semi-transparent
gray. The main amino acid residues from both active sites are also shown. The catalytic triad and
oxyanionic subsite residues are in magenta. The acyl-binding pocket amino acids are in orange. The
anionic subsite is colored in yellow. The peripheral anionic site is represented in cyan. The residues
from the bottle neck region are in green.
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The inactive compound 17 (IC50 > 200 µM) showed a different interaction pattern and
interaction energy values in comparison with 15-BChE and 21-BChE. In Figure 4C, it can be
seen that in 17-BChE interactions with Trp110 and His466 are significantly decreased. The
main interactions stabilizing the 17-BChE complex are those with Met109, Val155, Tyr468 and
Glu471.
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An unexpected result was the remarkable inhibitory activity of compound 21 (IC50 = 13.35 ±
3.01 µM) despite the lack of the OH substituent in B-ring. Unlike 14 and 15, in which the OH
group of the phenethyl moiety establishes an H-bond with the backbone of Trp110, 21 adopts
a different conformation that favors the interaction between Ser315 and OH at C3 from A-ring.
Additionally, a better hydrophobic interaction with Trp110 can be observed for this compound
(Figure S3). These results may explain, at least in part, the significant inhibitory activity found
for compound 21.
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3. Conclusions
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The synthesis of a series of N-benzyl-2-phenylethanamine derivatives was optimized and
performed by a simple methodology involving a reductive amination affording the products with
moderate to excellent yields. Most of these compounds exerted significant in vitro inhibition of
AChE and BChE. In particular, brominated norbelladine analogues showed the highest
inhibition values, whereas the presence of iodine showed a high selectivity towards BChE with
a strong IC50 value comparable to galantamine. Moreover, the lack of hydroxyl group on the B-
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ring had an influence on the inhibition of BChE showing a high inhibition with similar values for
both enzymes. All these observations can be explained by molecular modeling studies
considering the size and structural features of the active sites of both enzymes. Due to the
presence of different halogens on one of the aromatic rings, these compounds can adopt
different conformations that allow some of them to accommodate very well in the active site by
establishing the necessary interactions to stabilize the molecular complexes. These results
can be very useful for the design and development of new inhibitors possessing similar
structural characteristics.
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4. Experimental Section
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4.1. General experimental procedures
4.1.1. Chemicals
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3-Hydroxy-4-methoxybenzaldehyde (1), 4-fluorobenzaldehyde (6), 4-chlorobenzaldehyde (7),
4-bromobenzaldehyde (8), 4-hydroxybenzaldehyde (9), tyramine (10), phenylethylamine (11)
were purchased from Sigma–Aldrich, Argentina. Aldehydes 2-5 were prepared by
halogenation reactions. Compounds 12-21 were obtained by reductive amination as described
in Section 4.2.3. Their NMR spectra and GC-FID or GC-MS chromatograms are shown in the
Supplementary Material.
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4.1.2. Analytical methods
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Thin-layer chromatography (TLC) was performed on silica gel 60 F254 plates (Merck) using n-
hexane:ethyl acetate mixtures of different polarity for halogenated aldehydes and
diclorometane:methanol:NH4OH (80:15:5) for norbelladine analogues and visualized by UV
irradiation at 254 nm and further sprayed with acidic anisaldehyde solution. The GC-FID and
GC-MS analyses were performed using a Perkin Elmer Clarus 500 and a Thermo Trace 1300
gas chromatograph coupled to an ITQ900 ion trap mass spectrometer (GC/MS-ITQ Thermo
1
Scientific), respectively. H and 13C NMR spectra were recorded using a Bruker AC-200
spectrometer in CDCl3 or D2O at 200 and 50 MHz or Bruker Avance 400 MHz spectrometer at
400 and 100 MHz, respectively. Melting points were determined with Leitz Wetzlar 553174
(1.25 X) apparatus (Germany).
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4.2. Synthesis
4.2.1. Synthesis of halogenated aldehydes
2-bromo-3-hydroxy-4-methoxybenzaldehyde (2)
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NBS (1.75 g, 9.86 mmol) was dissolved in 60 mL of glacial acetic acid. A solution of 3-
hydroxy-4-methoxybenzaldehyde (1) (1.5 g, 9.86 mmol) in 30 mL of glacial acetic acid, was
added dropwise. The reaction mixture was stirred at room temperature for 4 h. After that the
precipitated solid was filtered and washed with glacial acetic acid and then with water. Finally,
the product was dried under vacuum to obtain 2 as a white solid (1.51 g, 66 % yield). Mp: 200
°C (Mp. Lit. 210 °C 38). 1H NMR (200 MHz, CDCl3) δ 4.01 (s, 3H, CH3), 6.08 (s, 1H, OH), 6.93
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(d, J = 8.6 Hz, 1H, Ar), 7.58 (d, J = 8.6 Hz, 1H, Ar), 10.26 (s, 1H, CHO). C NMR (50 MHz,
CDCl3) δ 56.7, 109.4, 113.0, 122.9, 127.3, 143.3, 151.8, 191.1.
6
7
332
6-bromo-3-hydroxy-4-methoxybenzaldehyde (3)
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9
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To obtain 3 we followed the methodology described by Hazlet and Brotherton
with
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modifications. To a solution of 1 (1 g, 6.6 mmol) in 25 mL of chloroform, a solution of bromine
(1.5 g, 9.5 mmol) in 10 mL of chloroform was added dropwise. The mixture was heated at 60
°C by reflux under argon atmosphere for 1 hour. The reaction was stopped in absence of
starting material and evaporated to dryness. The solid was resuspended in ethyl acetate and
washed with sodium thiosulfate solution (10 %) to eliminate the bromine excess. The organic
phase was washed with H2O and the aqueous phase with chloroform. Organic phases were
dried and evaporated and the crude product was purified by chromatography on silica gel (70–
230 mesh, Sigma-Aldrich) with dichloromethane to afford a pale brown solid (1.1 g, 73 %
yield). Mp: 118-120 °C (Mp. Lit. 112-114°C. 40). H NMR (200 MHz, CDCl3) δ 3.99 (s, 3H,
CH3), 5.63 (s, 1H, OH), 7.06 (s, 1H, Ar), 7.48 (s, 1 H, Ar), 10.18 (s, H, CHO). 13C NMR (100
1
MHz, CDCl3) δ 56.6, 114.6, 115.1, 118.7, 127.4, 145.4, 151.9, 190.8.
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2-chloro-3-hydroxy-4-methoxybenzaldehyde (4)
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To a solution of 3-hydroxy-4-methoxybenzaldehyde (1) (1.5 g, 9.86 mmol) in 30 mL of glacial
acetic acid, was added dropwise a solution of NCS (1.97 g, 14.8 mmol) in 60 mL of glacial
acetic acid. The reaction was carried out for 24 h. The precipitated solid formed was filtered
and washed with glacial acetic acid and water to yield 4. The resulting product was a white
1
solid (582 mg, 48 % yield). Mp: 187-189 °C. H NMR (200 MHz, CDCl3) δ 4.01 (s, 3H, CH3),
5.97 (s, 1H, OH), 6.90 (d, 1H, J = 8.6 Hz, Ar), 7.57 (d, 1H, J = 8.6 Hz, Ar), 10.35 (s, 1H, CHO).
13C NMR (50 MHz, CDCl3) δ 56.7, 108.9, 122.2, 123.2, 126.5, 142.3, 152, 189.1.
353
3-hydroxy-2-iodo-4-methoxybenzaldehyde (5)
41
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The iodination of 3-hydroxy-4-methoxybenzaldehyde was obtained according to literature
with modifications. 3-Hydroxy-4-methoxybenzaldehyde (1.5 g, 9.86 mmol) and NaI (1.73 g,
11.53 mmol) was dissolved in EtOH (30 mL). NaClO (16.4 mL, 9.86 mmol) was added
dropwise to the solution. The precipitate formed was filtered and washed with cold water. The
ocher solid was obtained in 46 % yield. Mp: 170-172 °C. 1H NMR (200 MHz, CDCl3-DMSO-d6)
δ 4.00 (s, 3H, CH3), 6.93 (d, 1H, J = 8.5 Hz, Ar), 7.56 (d, 1H, J = 8.5 Hz, Ar), 10.04 (s, 1H,
CHO). 13C NMR (50 MHz, CDCl3) δ 56.7, 88.2, 110.1, 124.0, 128.8, 145.8, 150.8, 194.9.
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4.2.3. General procedure for the synthesis of N-benzyl-2-phenylethanamines (12-21)
To obtain the norbelladine analogues, we followed the procedure described by Tachy et al.
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with modifications. Aldehydes 1-9 (2.16 mmol) and amines 10-11 (2.16 mmol) were dissolved
in MeOH (12 mL) and anhydride Na2SO4 and triethylamine (TEA) (400 µL) or KOH (100 mg)
were added. The reaction was stirred overnight under inert atmosphere. After that, the imine
formed was reduced with NaBH4 (2.16 mmol) on ice bath until no starting material was
observed by TLC. The solvent was evaporated and the solid was resuspended in water. The
pH of the aqueous phase was adjusted to the corresponding theoretical isoelectric point of the
products. The precipitated product was filtered and dried under vacuum. The solid was
dissolved in hot ethanol and after cooling, the suspension was filtered. The resulting solid was
dried to yield the desired product. To obtain the corresponding hydrochloride, each compound
was dissolved in absolute EtOH and then HCl was added in equimolar relation with the
product. The reaction was stirred for 3 h and after that, the solvent was evaporated and the
residue washed with acetone.
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N-(p-hydroxyphenylethyl)-N-(3-hydroxy-4-methoxy)benzylamine (12) beige solid. 51 % yield.
26
1
Mp: 206-208 °C (Mp. Lit. 208° ). H NMR (200 MHz, D2O) as hydrochloride: δ 2.92 (m, 2H,
CH2), 3.25 (m, 2H, CH2), 3.88 (s, 3H, OCH3), 4.11 (s, 2H, CH2), 6.85-7.18 (m, 7H, Ar). 13C
NMR (50 MHz) 30.5, 47.5, 50.2, 55.7, 112.4, 115.6, 116.5, 122.5, 123.2, 128.0, 130.0, 144.9,
148.2, 154.4.
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N-(p-hydroxyphenylethyl)-N-(2-chloro-3-hydroxy-4-methoxy)benzylamine (13) Light brown
solid. 66 % yield. Mp: 172 °C. 1H NMR (200 MHz, CDCl3) δ 2.72-2.82 (m, 4H, CH2-CH2), 3.82
(s, 2H, CH2), 3.87 (s, 3H, OCH3), 6.69-6.82 (m, 4H, Ar), 7.00 (d, J = 8.4 Hz, 2H, Ar). 13C NMR
(100 MHz, D2O) as hydrochloride: δ 30.5, 47.9, 48.2, 56.2, 110.3, 115.7, 120.7, 121.0, 123.4,
128.0, 130.1, 142.0, 149.5, 154.6.
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N-(p-hydroxyphenylethyl)-N-(2-bromo-3-hydroxy-4-methoxy)benzylamine (14) light brown
1
solid. 77 % yield. Mp: 185-187 °C. H NMR (200 MHz, CDCl3) δ 2.75-2.85 (m, 4H, CH2-CH2),
3.83 (s, 2H, CH2), 3.87 (s, 3H, OCH3), 6.67-6.86 (m, 4H, Ar), 7.02 (d, J = 8.3, 2H, Ar). 13C NMR
(100 MHz, D2O) as hydrochloride: 30.4, 47.8, 50.9, 56.1, 110.7, 111.4, 115.6, 122.4, 123.5,
127.8, 130.0, 143.0, 149.0, 154.4.
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N-(p-hydroxyphenylethyl)-N-(2-iodo-3-hydroxy-4-methoxy)benzylamine (15) Ocher solid 49 %
1
yield. Mp: 202-205 °C (as hydrochloride). H NMR (400 MHz, D2O) as hydrochloride δ 2.93-
3.36 (m, 4H, CH2-CH2), 3.87 (s, 2H, CH2), 4.32 (s, 3H, OCH3), 6.84 (d, J = 8.2 Hz, 2H, Ar),
7.02 (s, 2H, Ar), 7.15 (d, J = 8.2 Hz, 2H, Ar). 13C NMR (100 MHz, D2O) δ 30.6, 48.0, 54.9,
56.2, 89.4, 111.7, 115.8, 123.5, 126.0, 127.9, 130.1, 145.8, 147.8, 154.6.
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400
N-(p-hydroxyphenylethyl)-N-(6-bromo-3-hydroxy-4-methoxy)benzylamine (16) Pale yellow
solid. 56 % yield. Mp: 122-125 °C. 1H NMR (200 MHz, CDCl3) δ 2.75- 2.84 (m, 4H, CH2-CH2),
3.75 (s, 2H, CH2), 3.85 (s, 3H, OCH3), 6.73 (d, J = 8.4 Hz, 2H, Ar), 6.88 (s, 1H, Ar), 6.96 (s,
1H, Ar), 7.05 (d, J = 8.4 Hz, 2H, Ar). 13C NMR (100 MHz, CDCl3) δ 35.4, 50.3, 53.1, 56.2,
112.6, 115.0, 115.3, 116.2, 129.8, 132.1, 144.9, 146.1, 153.8.
401
402
403
404
N-(p-hydroxyphenylethyl)-N-(p-fluor)benzylamine (17) yellow solid. 51 % yield. Mp: 163-165
1
°C. H NMR (400 MHz, D2O) as hydrochloride δ 2.91-3.28 (m, 4H, CH2-CH2), 4.20 (s, 2H,
CH2), 6.87 (d, 2H, Ar), 7.15-7.21 (m, 4H, Ar), 7.42-7.46 (m, 2H, Ar). 13C NMR (200 MHz, D2O)
δ 30.7, 47.9, 50.2, 115.8, 116.0, 116.2, 126.6, 128.2, 130.2, 132.0, 132.1, 154.6, 162.0, 164.4.
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N-(p-hydroxyphenylethyl)-N-(p-chloro)benzylamine (18) pale yellow solid. 55 % yield. Mp: 123-
126 °C. 1H NMR (400 MHz, D2O) as hydrochloride δ 2.91- 3.28 (m, 4H, CH2-CH2) 4.20 (s, 2H,
CH2), 6.87 (d, J = 8.4 Hz, 2H, Ar), 7.16 (d, J = 8.3, 2H, Ar), 7.39 (d, J = 8.4, 2H, Ar), 7.46 (d, J
13
= 8.4, 2H, Ar). C NMR (100 MHz, CDCl3) δ 30.6, 47.9, 50.1, 115.7, 128.1, 129.1, 129.2,
130.1, 131.3, 135.0, 154.5.
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412
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N-(p-hydroxyphenylethyl)-N-(p-bromo)benzylamine (19) white solid. 84 % yield. Mp: 140-143
1
°C. H NMR (200 MHz, CDCl3) δ 2.75-2.84 (m, 4H, CH2-CH2), 3.75 (s, 2H, CH2), 6.73 (d, J =
8.3 Hz, 2H, Ar), 7.04 (d, J = 8.3 Hz, 2H, Ar), 7.15 (d, J = 8.2 Hz, 2H, Ar), 7.42 (d, J = 8.2 Hz,
13
2H, Ar). C NMR (100 MHz, CDCl3) δ 35.2, 50.5, 53.2, 115.6, 121.0, 129.9, 130.1, 131.4,
131.7, 138.8, 154.6.
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416
417
418
419
N-(p-hydroxyphenylethyl)-N-(p-hydroxy)benzylamine (20) white solid. 92 % yield. Mp: 220-222
1
°C (as hydrochloride). H NMR (400 MHz, D2O) as hydrochloride δ 2.87-3.23 (m, 4H, CH2-
CH2), 4.11 (s, 2H, CH2), 6.84 (d, J = 8.5 Hz, 2H, Ar), 6.90 (d, J = 8.5, 2H, Ar), 7.13 (d, J = 8.5
Hz, 2H, Ar), 7.28 (d, J = 8.5 Hz, 2H, Ar). 13C NMR (100 MHz, D2O) δ 30.6, 47.6, 50.3, 115.8,
122.3, 128.2, 130.1, 131.6, 154.5, 156.5.
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N-(phenylethyl)-N-(2-bromo-3-hydroxy-4-methoxy)benzylamine (21) pale yellow solid. 55 %
yield. Mp: 102-105 °C. 1H NMR (400 MHz, CDCl3) 2.85-2.96 (m, 4H, CH2CH2), 3.89 (s, 3H,
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OCH3), 3.91 (s, 2H, CH2), 6.76 (d, J = 8.3 Hz, 1H, Ar), 6.88 (d, J = 8.3 Hz, 1H, Ar), 7.18-7.30
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(m, 5H, Ar). C NMR (100 MHz, CDCl3) δ 35.2, 49.2, 52.4, 56.3, 109.4, 110.3, 121.5, 126.4,
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128.6, 128.7, 138.9, 143.3, 146.6.
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4.3. Microplate assay for AChE and BChE inhibitory activities
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The enzymes AChE from Electrophorus electricus (EeAChE) and BChE from equine serum
(EqBChE) (Sigma-Aldrich) were used. For the AChE and BChE activity assay,
acetylthiocholine iodide and butyrylthiocholine iodide were used as substrates, respectively.
Briefly, 50 µL of AChE or BChE in phosphate-buffered saline (PBS) (8 mM K2HPO4, 2.3 mM
NaH2PO4, 0.15 M NaCl, pH 7.6) and 50 µL of the sample dissolved in the same buffer, were
added to the wells of a microplate. When necessary, the compounds were dissolved in
dimethyl sulfoxide (DMSO) or methanol at a final concentration of 0.02 % and 0.5 %,
respectively The plates were incubated for 30 min at room temperature before the addition of
100 µL of the substrate solution (0.1 M Na2HPO4, 0.5 M DTNB, 0.6 mM ATCI in Millipore
water, pH 7.5). The absorbance was read in a Thermo Scientific Multiskan FC microplate
photometer at 405 nm after 5 min. Enzyme inhibitory activity was calculated as a percentage
compared to an assay using buffer without any inhibitor. The results obtained were analyzed
with the software package Prism (Graph Pad Inc., San Diego, CA, USA). The values were
expressed as half-maximal inhibitory concentration IC50 (µM), and were calculated as means ±
SD of 3 individual determinations. Galantamine (Sigma-Aldrich) was used as a positive
control.
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4.4. Molecular Modeling Studies
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3D models of Torpedo californica AChE (TcAChE) (1DX6)
and Equus caballus BChE
(EqBChE) (UniProtAC Q9N1N9) available at Protein Data Bank (http://www.rcsb.org) were
used for carring out the molecular modeling studies. Water molecules and ligands were
removed from the structures before performing the docking calculations. Receptors and N-
benzyl-2-phenylethanamine derivatives structures were converted from pdb to pdbqt format
using AutoDockTools 1.5.4 43. Gasteiger charges were added for all the compounds and non-
43
polar hydrogen atoms were merged. AutoDockTools
was also used to perform further
graphic manipulations and visualizations required. For docking procedures, Autodock version
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4.0 44 was used. The receptor structure was set as rigid and grid dimension were 60 60 60 for
the X, Y and Z axes, respectively, in the catalytic site of TcAChE and EqBChE with a spacing
resolution of 0.375 Å in both cases. All torsions of the ligand were allowed to rotate during
docking. The number of collected poses was 200. Other parameters were set to default
values. The resulting docked conformations were clustered into families based on the rmsd
between the coordinates of the ligands and were ranked regarding to the binding free energy
of complexes. The structure with lower binding free energy from the cluster with the largest
number of members was chosen as the optimum docking conformation and was used in
subsequent simulations.
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MD simulations for all complexes selected from docking procedures were performed using the
46
Amber16 software package 45. Antechamber software
was used to generate their
47
48
parameters with FF99SB and GAFF force fields. The complex geometries from docking
were soaked in truncated octahedral periodic boxes of explicit water using the TIP3P model 49
with a margin of 10.0 Å in each direction from the solute. Na+ or Cl- ions were placed by Leap
to neutralize the negative and positive charges of AChE and BChE complexes, respectively.
The energy of each system was then minimized with sander module using a steepest-descent
algorithm for 1000 steps. There upon the complexes were equilibrated during 500 ps at
constant volume. The SHAKE algorithm 50 was applied allowing for an integration time step of
2 fs. The systems were heated from 0 to 300 K using Langevin thermostat 51 in order to control
temperature, collision frequency = 1.0 ps-1. Next, three MD simulations were conducted for
each complex at 298 K target temperature. All production was performed under NVT
conditions. The particle mesh Ewald (PME) method 52 was applied using a grid spacing of 1.2
Å, a spline interpolation order of 4, and a real space direct sum cutoff of 8.0 Å. Simulation time
was set to 20 ns, the time step was set to 2.0 fs and coordinates were saved for analysis every
10 ps. Post MD analysis was performed with the program PTRAJ 53. A per-residue interaction
energy decomposition analysis using mm_pbsa program was carried out in order to determine
the residues of AChE and BChE that interact with each ligand. For mm_pbsa methodology 54,
snapshots from the corresponding last 1000 ps of MD trajectories were considered. The
explicit water molecules and counter ions were removed from the snapshots.
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Declaration of Competing Interest
There are no conflicts to declare.
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Acknowledgments
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The manuscript was written through contributions of all authors. Authors thank Lic. Mónica
Ferrari for technical assistance and Dr. Walter Stege for NMR spectra. Funding: This work was
supported by grants from UNSL (PROICO 2-1716), CONICET (PIP 1122015 0100090) and
ANPCyT (PICT 2018-02916, PICT 2014-03425 and PICT 2015-1769), CICITCA UNSJ. F.C.V.
and D.Z.P. are doctoral CONICET fellows; M.K.S, G.E.F., D.E., O.P., A.G. and A.A.O are
members of the Research Career of CONICET.
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Appendix A. Supplementary material
Supplementary data associated with this article can be found in the online version.
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