Highly potent and selective aryl-1,2,3-triazolyl benzylpiperidine inhibitors toward butyrylcholinesterase in Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2018.12.030

Acetylcholinesterase (AChE) is the key enzyme targeted in Alzheimer's disease (AD) therapy, nevertheless butyrylcholinesterase (BuChE) has been drawing attention due to its role in the disease progression. Thus, we aimed to synthesize novel cholinesterases inhibitors considering structural differences in their peripheral site, exploiting a moiety replacement approach based on the potent and selective hAChE drug donepezil. Hence, two small series of N-benzylpiperidine based compounds have successfully been synthesized as novel potent and selective hBuChE inhibitors. The most promising compounds (9 and 11) were not cytotoxic and their kinetic study accounted for dual binding site mode of interaction, which is in agreement with further docking and molecular dynamics studies. Therefore, this study demonstrates how our strategy enabled the discovery of novel promising and privileged structures. Remarkably, compound 11 proved to be one of the most potent (0.17 nM) and selective (>58,000-fold) hBuChE inhibitor ever reported.

Full text of DOI:10.1016/j.bmc.2018.12.030

Accepted Manuscript
Highly potent and selective aryl-1,2,3-triazolyl benzylpiperidine inhibitors to-
ward butyrylcholinesterase in alzheimer's disease
Peterson de Andrade, Susimaire P. Mantoani, Paulo Sérgio Gonçalves Nunes,
Carlos Roca Magadán, Concepción Pérez, Danilo Jordão Xavier, Elza Tiemi
Sakamoto Hojo, Nuria E. Campillo, Ana Martínez, Ivone Carvalho
PII:
S0968-0896(18)31817-0
https://doi.org/10.1016/j.bmc.2018.12.030
BMC 14679
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 October 2018
18 December 2018
21 December 2018
Please cite this article as: de Andrade, P., Mantoani, S.P., Gonçalves Nunes, P.S., Magadán, C.R., Pérez, C., Xavier,
D.J., Hojo, E.T.S., Campillo, N.E., Martínez, A., Carvalho, I., Highly potent and selective aryl-1,2,3-triazolyl
benzylpiperidine inhibitors toward butyrylcholinesterase in alzheimer's disease, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.12.030
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HIGHLY POTENT AND SELECTIVE ARYL-1,2,3-TRIAZOLYL
BENZYLPIPERIDINE INHIBITORS TOWARD
BUTYRYLCHOLINESTERASE IN ALZHEIMER'S DISEASE
Peterson de Andrade1, Susimaire P. Mantoani1, Paulo Sérgio Gonçalves Nunes1, Carlos
Roca Magadán2, Concepción Pérez3, Danilo Jordão Xavier4, Elza Tiemi Sakamoto
Hojo5, Nuria E. Campillo2, Ana Martínez2 and Ivone Carvalho1*
1School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. Café s/n, 14040-930, Ribeirão
Preto, SP, Brazil
2Centro de Investigaciones Biologicas (CIB-CSIC), Ramiro de Maeztu 9, Madrid-28040, Spain
3Instituto de Química Médica (IQM-CSIC), Juan de la Cierva 3, Madrid-28006, Spain
4Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Av. Bandeirantes 3900, 14049-
900, Ribeirão Preto, SP, Brazil
5Department of Biology, Faculty of Philosophy, Sciences and Letters at Ribeirão Preto, University of São Paulo,
Avenida Bandeirantes 3900, 14040-900, Ribeirão Preto, SP, Brazil
* Corresponding author: phone: +55 16 3315 4709; fax: +55 16 33154879; email: carronal@usp.br
Abstract
Acetylcholinesterase (AChE) is the key enzyme targeted in Alzheimer's disease (AD)
therapy, nevertheless butyrylcholinesterase (BuChE) has been drawing attention due to
its role in the disease progression. Thus, we aimed to synthesize novel cholinesterases
inhibitors considering structural differences in their peripheral site, exploiting a moiety
replacement approach based on the potent and selective hAChE drug donepezil. Hence,
two small series of N-benzylpiperidine based compounds have successfully been
synthesized as novel potent and selective hBuChE inhibitors. The most promising
compounds (9 and 11) were not cytotoxic and their kinetic study accounted for dual
binding site mode of interaction, which is in agreement with further docking and
molecular dynamics studies. Therefore, this study demonstrates how our strategy
enabled the discovery of novel promising and privileged structures. Remarkably,
compound 11 proved to be one of the most potent (0.17 nM) and selective (>58,000-
fold) hBuChE inhibitor ever reported.
Graphical Abstract
Keywords
Alzheimer's disease, Butyrylcholinesterase, Acetylcholinesterase, Click Chemistry,
Inhibitors and Azido amino acids
1

1. Introduction
Known as the most common type of dementia, Alzheimer's disease (AD) is a severe and
progressive neurological disorder characterized by cognitive impairment and
irreversible memory loss. Although AD is not fully understood due to its high
complexity and its origin is also under scientific debate, the presence of extracellular
plaques (accumulation of amyloid-β protein (Aβ)) and neurofibrillary tangles
(hyperphosphorylated tau protein) in the brain may possibly have a causal role on the
cascade leading to AD. Besides these neuropathological hallmarks, inflammation and
oxidative stress processes also contribute to damage of synaptic integrity and
neurodegeneration.1,2 The affected regions by neuronal loss encompass cholinergic
neurons, thus compromising learning and memory processes due to deficiency of the
neurotransmitter acetylcholine (ACh).3
The current treatment is symptomatic and mainly involves restoring of ACh levels
through acetylcholinesterase (AChE, EC 3.1.1.7) inhibition by donepezil, rivastigmine
and galantamine (cholinesterase inhibitors approved by FDA). AChE is the key enzyme
targeted in AD therapy whilst butyrylcholinesterase (BuChE, EC 3.1.1.8), a closely
homologous serine hydrolase encoded by different genes and not predominant in the
central nervous system (CNS), was previously underestimated.4-7 However, it has been
argued that a switch in focus from AChE to BuChE is owing to a more important role
assumed by BuChE in AD advanced stages due to its up-regulation, playing a key
function in the disease maintenance and progression.8,9
Evidences for BuChE involvement in AD and its role as a considerable drug target have
been suggested over the years.10 In fact, selective BuChE inhibition caused raising of
acetylcholine and increased long-term potentiation and learning in rats.11 Likewise, in
vivo BuChE inhibition yielded improvement of memory, cognitive functions and
learning abilities in cholinergic deficit mice model.12 Notably, peripheral cholinergic
side effects were not observed in these studies. Another expressive result refers to a
decreasing of fibrillar Aβ brain plaques (up to 70%) in BuChE knockout mouse, which
suggests that the diminished BuChE activity could prove beneficial in AD.13 Therefore,
it seems reasonable to accept that the discovery of highly potent and selective BuChE
inhibitors may represent a promising therapeutic approach to AD.13-15
2

Although both enzymes are capable of hydrolyzing ACh, they differ in substrate
specificity and sensitivity to inhibitors due to important structural differences in their
active site. The catalytic site of both enzymes is located at the bottom of 20 Å deep
cavity and the peripheral site is located at the entrance of the cavity. hAChE peripheral
site is smaller and narrower (about 300 Å3) because it is constituted by aromatic
residues whilst in hBuChE some of them are replaced by aliphatic residues,
consequently yielding a larger region (about 500 Å3) and favouring bulkier ligands
binding.16-18
In this context, we envisaged the synthesis of novel cholinesterases inhibitors exploiting
a relevant method in the discovery of bioactive molecules, such as "click chemistry"
(Copper-catalyzed Azide-Alkyne Cycloaddition - CuAAC).19 Hence, the target
compounds 1-12 have been designed based on the potent and selective hAChE drug
donepezil (Aricept®) keeping the N-benzylpiperidine moiety and replacing the 5,6-
dimethoxy-1-indanone moiety by other (non)substituted fused (hetero)aromatic
scaffolds, tethered to 1,4-disubstituted 1,2,3-triazole ring (series 1), and by azido amino
acids to afford 1-substituted-4-phenyl-1,2,3-triazoles (series 2) (Figure 1). Thus, we
aimed to evaluate the influence of these replacements upon inhibitory activity and
selectivity towards hAChE and hBuChE considering either a direct coupling (via 1,2,3-
triazole ring) or an indirect coupling (via amide bond) to N-benzylpiperidine moiety.
target compounds
R
N
MeO
O
N
N
N
N
series 1 (1 - 8)
MeO
R = fused aromatic or
heteroaromatic rings
donepezil
N
N
N
( )n
N
H
N
R'HN
series 2 (9 - 12)
O
n = 1 or 4
R' = H or Ac
Figure 1. Design of potential cholinesterase inhibitors exploiting a moiety replacement
approach considering the potent and selective hAChE drug donepezil and a relevant
method in drug discovery ("click chemistry").
3

2. Results and Discussion
2.1 Chemical synthesis
Towards the synthesis of the target compounds 1-8 (series 1), we have proposed a
concise route as shown in Scheme 1.
R
NH
N
N
b
a
N
H2N
N3
N
N
(1 - 8)
4-(aminomethyl)piperidine
R =
13 (32%)
O
O
O
HN
MeO
N
N
H
MeO
O
3 (49%)
1 (80%)
4 (61%)
2 (75%)
HN
N
HN
N
H
H
N
N
MeO
MeO
S
S
N
N
EtO
N
N
8 (61%)*
6 (31%)
7 (70%)*
5 (20%)
Scheme 1. Synthetic route for the synthesis of the target compounds 1-8 (series 1).
Reagents and experimental conditions: a) i. imidazole-1-sulfonyl azide hydrogen
sulfate, CuSO4, NaHCO3, MeOH/H2O; ii. BnCl, K2CO3, acetone; b) terminal alkyne
(RCΞCH), CuSO4, sodium ascorbate, DMF, 100ºC under MW (procedure 1) or terminal
alkyne (RCΞCH), CuSO4, sodium ascorbate, EtOH/H2O/1,10-phenanthroline, room
temperature (*procedure 2).
The key intermediate 13 was synthesized in two steps (32% overall yield) from readily
available 4-(aminomethyl)piperidine by diazo transfer reaction with imidazole-1-
sulfonyl azide hydrogen sulfate (synthesized in 50% yield on a gram scale)20 and CuSO4
in MeOH/H2O mixture at pH 9 followed by benzylation with BnCl in acetone.21
Formation of 1,2,3-triazole-1,4-disubstituted ring from 13 and terminal alkynes readily
available in our lab [commercial (phthalimide 2a and naphthyl 4a) and synthesized
(indanone 1a, indole 3a, quinazolines 5a/6a and mercaptobenzimidazoles 7a/8a -
Supplementary Information)] was initially carried out with CuSO4/sodium ascorbate in
DMF under microwave (MW) heating (condition 1)22 to afford the desired compounds
1-6, mostly in good yields. Due to degradation problems, an alternative condition
4

(EtOH/H2O/1,10-phenanthroline mixture at room temperature)23 was necessary to
obtain compounds 7 and 8 in good yields (70% and 61%, respectively).
Towards the synthesis of the target compounds 9-12 (series 2), two azido amino acid
precursors [Fmoc-L-Ala(N3)-OH (15) and Fmoc-L-Lys(N3)-OH (17)] were efficiently
synthesized in two steps from commercially available Fmoc-L-Asn-OH and Fmoc-L-
Lys(Boc)-OH, respectively (Scheme 2).24,25
NH2
+
N3
NH3
O
a
b
FmocHN
CO2H
FmocHN
CO2H
FmocHN
CO2H
Fmoc-L-Asn-OH
15 (65%)
14 (80%)
+
NH3
NHBoc
N3
a'
b
FmocHN
CO2H
FmocHN
CO2H
FmocHN
CO2H
Fmoc-L-Lys(Boc)-OH
17 (81%)
16 (97%)
Scheme 2. Synthesis of the azido amino acids 15 and 17. Reagents and conditions: a)
[bis(trifluoroacetoxy)iodo]benzene, pyridine, DMF/H2O; a') HCl 4.0 M in dioxane; b)
imidazole-1-sulfonyl azide hydrogen sulfate, CuSO4, NaHCO3, MeOH/H2O.
In a Hofmann rearrangement-based procedure, Fmoc-L-Asn-OH was treated with
[bis(trifluoroacetoxy)iodo]benzene and pyridine in DMF/H2O to afford the primary
amine 14 (80%), which was converted to the corresponding azido compound 15 (65%)
by diazo transfer reaction with imidazole-1-sulfonyl azide hydrogen sulfate and CuSO4
in MeOH/H2O mixture at pH 9.24 Likewise, azido compound 17 was also synthesized
by the same diazo transfer reaction (81%) from 16, which was previously obtained by
Boc-cleavage (97%) of Fmoc-L-Lys(Boc)-OH in acidic condition.25 Besides good
yields, the syntheses were readily scaled up and pure products were obtained without
any further purification for the amide coupling step. Thereafter, we have proposed a
straightforward and efficient route for the target compounds 9-12 (series 2) as shown in
Scheme 3.
5

N
NH
NH
a
b
H2N
H2N
H2N
O
18 (65%)
4-(aminomethyl)piperidine
isonipecotamide
c
N
N
N3
N
d
( )n
N
( )n
N
H
N
H
N
FmocHN
FmocHN
O
O
19 (n = 1) 71%
20 (n = 4) 74%
21 (n = 1) 55%
22 (n = 4) 86%
e
N
N
N
N
N
N
f
( )n
( )n
N
N
H
N
H
N
AcHN
H2N
O
O
9 (n = 1) 61%
10 (n = 4) 81%
11 (n = 1) 60%
12 (n = 4) 85%
Scheme 3. Synthetic route for the synthesis of the target compounds 9-12 (series 2).
Reagents and conditions: a) C6H5CHO, NaBH(OAc)3, DCM, N2(g); b) i. BnBr, K2CO3,
EtOH, 90ºC; ii. LiAlH4, DCM/THF, 70ºC, N2(g); c) azido amino acid 15 or 17, HBTU,
DIPEA, DMF; d) phenylacetylene, CuSO4, sodium ascorbate, t-BuOH/H2O/DCM
(1:1:1); e) morpholine/DCM (1:1); f) Ac2O, pyridine.
After some unsuccessful attempts to synthesize the key intermediate 18 in a more direct
and atom-efficient way
one step from commercial 4-(aminomethyl)piperidine by
reductive amination with NaBH(OAc)3 and benzaldehyde in DCM,26 we have found a
two-step method from isonipecotamide more suitable. Hence, commercially available
isonipecotamide was benzylated with BnBr and K2CO3 in EtOH and the crude product
reduced with LiAlH4 in THF/DCM to afford compound 18 in 65% overall yield.27
Despite the vast number of possibilities for amide bond formation (nature of coupling
reagent, reaction conditions, reagent stoichiometry, solvent, etc), the amide coupling
between the azido amino acids 15/17 and the key intermediate 18 was successfully
achieved after some initial failures due to difficulties to find out the most convenient
condition. Moreover, some by-products formation made the purification also
challenging. Thus, the novel Fmoc-protected azido-building blocks 19 and 20 were
6

synthesized with HBTU as coupling reagent and DIPEA in DMF at room temperature in
good yields (71% and 74%, respectively).28
In our initial efforts to perform the next step with compounds 19 and 20 through
CuAAC reaction (which also present a wide range of procedures), we have tested the
procedure previously described.22 In this case, the formation of 1,2,3-triazole-1,4-
disubstituted ring from commercial phenylacetylene with CuSO4/sodium ascorbate in
DMF under MW heating was unsuccessful due to several by-products formation. In
order to circumvent this problem, a slightly different protocol in another solvent system
(t-BuOH/H2O/DCM 1:1:1) and no heating was tested.29 Thus, the desired products 21
and 22 were obtained in reasonable yields (55% and 86%, respectively). Further Fmoc-
deprotection with morpholine solution in DCM (1:1)30 afforded the target compounds 9
and 10 in acceptable yields (61% and 81%, respectively), but the classical purification
process proved to be difficult because of morpholine contamination and HPLC was
needed. In order to investigate whether a small protecting group at the primary amine
could play an important role affecting the biological outcome, the corresponding
acetylated analogues 11 and 12 were synthesized with Ac2O and pyridine31 in
reasonable yields (60% and 85%, respectively).
2.2 Cholinesterases inhibition assay
The in vitro inhibitory activity of compounds 1-8 (series 1) and 9-12 (series 2) was
determined in both cholinesterases by Ellman’s assay32 and it is summarized in Table 1.
The whole series 1 proved to be only weak hAChE inhibitors (IC50 values ranging from
>10 μM to 3.94 μM). Similarly, hBuChE inhibitory activity was also weak for most
compounds (IC50 values ranging from >10 μM to 1.91 μM), except 7 (IC50 65 nM).
Although some inhibition activity and selectivity have been observed for both enzymes
regarding series 1, these results were not so expressive and do not allow a
comprehensive study of structure-activity relationships. In general, the low inhibitory
activity of this whole series (except compound 7) suggests a lack of flexibility caused
by the direct coupling (via 1,2,3 triazole ring) of benzylpiperidine and the
(non)substituted fused (hetero)aromatic moieties. According to our previous work on
indolylpiperidines,15 the capacity of some molecules to change their conformation or
shape by folding had a great impact in their binding mode towards hBuChE, which
7

resulted in high inhibitory activity. However, contribution of the non-substituted
mercaptobenzimidazole scaffold (7) to the potent and selective hBuChE inhibition is
evident and it indicates that this moiety may have a better fit at hBuChE peripheral site
compared to hAChE. With regard to series 2 (9-12), none of the target compounds
displayed significant inhibitory activity in human recombinant hAChE (IC50 > 10 μM)
compared to donepezil (IC50 5.7 nM). On the other hand, all of them proved to be very
active in human recombinant hBuChE, being the least active (12, IC50 1.02 μM) 9-fold
more potent than donepezil (IC50 9.14 μM) and the most active (11, IC50 0.17 nM)
53,000-fold more potent. A preliminary analysis suggests that short side chain
compounds (9 and 11) are much more potent (nM range inhibition) than the long side
chain ones (10 and 12) (μM range inhibition). Additionally, acetylation of primary
amine from 9 (IC50 9.9 nM) to obtain 11 (IC50 0.17 nM) resulted in considerable
inhibitory activity improvement (58-fold) that surely makes it one of the most potent
and selective (>58,000-fold) hBuChE inhibitor ever reported.10,33
Table 1. Cholinesterase inhibitory results (hAChE and hBuChE) based on Ellman
modified microplate assay: initial concentration of 10 μM to determine inhibition
percentage and subsequent IC50 calculation for compounds 1-8 and 9-12 (donepezil as
control).
hAChE
hBuChE
Structures
Compounds
IC50 ± SEM (μM) IC50 ± SEM (μM)
0.0057±0.0005
4.92±0.71
9.14±0.56
>10
donepezil
1
4.90±0.42
>10
>10
2
3
5.30±0.42
8

>10
7.06±0.18
2.95±0.31
4
5
4.22±0.30
3.94±0.26
>10
6
7.33±0.39
6.90±0.76
0.065±0.002
1.91±0.39
7
8
>10
>10
>10
>10
0.0099±0.0043
9
0.97±0.13
10
11
12
0.00017±0.000021
1.02±0.21
9

Furthermore, the mechanism of hBuChE inhibition for compounds 9 (Figure 2A) and 11
(Figure 2B) was also assessed by means of a kinetic study. This study accounted for
mixed-type inhibition models in both cases, which is in accordance with a likely dual
binding site (catalytic and peripheral sites) mode of interaction.
A
B
Figure 2. Lineweaver-Burk plots for hBuChE inhibition kinetics of compounds 9 (A)
and 11 (B). Reciprocals of enzyme activity vs reciprocals of substrate
(acetylthiocholine) concentration in the presence of inhibitors at different
concentrations.
2.3 Cell viability assays
With the aim to evaluate the cytotoxic effects on human neuroblastoma SH-SY5Y cells
line34 in two recovery periods (24 and 120 hours), the cells were treated with seven
concentrations of the series 2 compounds (9 - 12) based on their hBuChE IC50 to obtain
a concentration-dependent effect on cell viability. After 24 h recovery, none of them
was cytotoxic or significantly inhibited cell growth in the hBuChE inhibitory
concentration, though compounds 9, 10 and 12 showed reduction in cell viability at 39.6
nM, 40 µM and 80 µM, respectively (Figure S1). After 120 h recovery, none of them
was cytotoxic or significantly inhibited cell growth in the expected concentration,
except for the intriguing cell growth inhibition by compound 12 only at 2.5 µM.
Interestingly, the cells were able to recover from the mild cytotoxic effect caused by
compounds 9, 10 and 12 after a longer recovery period (Figure S2).
10

2.4 Docking and molecular dynamic studies
Molecular docking studies34 were performed for a better understanding of the binding
interactions of compounds 9 and 11 in order to rationalize their different potency
towards hBuChE inhibitory activity. In addition, it was also considered some key
information about binding interactions initially provided from a crystal structure (PDB
code 4TPK) of hBuChE in complex with the potent and selective inhibitor (+)23 (IC50
13.4 nM).18 As depicted in Figure 3, the conformational alignment of docked structure 9
(Figure 3A) is very similar to the crystal structure of (+)23 in hBuChE active site
(Figure 3B), once the positively charged nitrogen of piperidine also displays a strong
cation-π interaction with Tyr332 side chain and an ionic interaction with Asp70 (both
from peripheral site). Moreover, 1,2,3-triazole phenyl ring from compound 9 (in
optimum distance from the main chain) fully occupies the acyl-binding pocket (Trp231,
Leu286, Val288 and Phe329), favouring a π-π aromatic interaction (T-stacking) with
Trp231 and Phe329 side chains as well as a π-σ interaction with Leu286 side chain. On
the other hand, docked structure 11 displayed different conformation compared to
compound 9 and this intriguing result was not initially understood.
A
B
OMe
N
N
O
23
Figure 3. Docking model showing proposed interactions of compound 9 with hBuChE
(PDB code 1P0I) (A) and binding interaction of (+)23 crystal structure in the active site
of hBuChE (PDB code 4TPK) (B).
Interestingly, a recent crystal structure (PDB code 5DYW) of hBuChE in complex with
another potent and selective inhibitor (24, IC50 4.9 nM)12 shed light on the outstanding
inhibitory activity of compound 11. As depicted in Figure 4, the conformational
11

alignment of docked structure 11 (predicted pose) (Figure 4A) is very similar to the
crystal structure of 24 in hBuChE active site (Figure 4B) and it allows important
additional interactions, such as π-π aromatic interaction of the benzyl group with
Trp430 and Trp82 side chains (choline-binding pocket) as well as H-bond interaction
between the carbonyl group (acetyl) and Thr284 side chain at the entrance of the gorge.
A
B
OMe
N
N
S
O
O
24
Figure 4. Docking model showing proposed interactions of compound 11 with hBuChE
(PDB code 1P0I) (A) and binding interaction of 24 crystal structure in the active site of
hBuChE (PDB code 5DYW) (B).
Accordingly, the impressive inhibitory activity improvement (58-fold) from compound
9 to 11 may be related to its flipped conformation within the active site (Figure 3A vs
4A). It can be inferred that this conformation has been influenced by the acetyl
protecting group in compound 11, which hides the primary amine and abolishes the
important interaction with Glu197 observed in compound 9, allowing an inverted
conformation of compound 11 towards the entrance of the gorge, where it is stabilized
by H-bond interaction with Thr284 side chain. Indeed, according to the literature
Glu197 in hBuChE can generate a strong electrostatic potential that draws positively
charged ligands into and down the gorge.18,35,36 In addition, the R-enantiomers of
compounds 9 and 11 (not synthesized) were studied in silico to show the key role of
their chirality. The best docking poses in hBuChE for both R-enantiomers showed that
the ligands can interact through a π-π aromatic interaction (T-stacking) with Trp231 and
Phe329 side chains, in a similar way observed for the S-enantiomers. However, the
piperidine ring is reoriented, changing the binding mode in the catalytic gorge and
probably their inhibition activity (Figure S3).
12

Regarding compounds 10 and 12, although they showed a good fit within hBuChE
active site and proved to be 9-fold more potent than donepezil, these long side chain
compounds are more flexible and may compromise the most favourable conformations
toward crucial interactions of 1,2,3-triazole phenyl ring. In this case, even the acetyl
group in compound 12 has not affected its biological activity. The docking studies of R-
enantiomers of compounds 10 and 12 (not synthesized) showed that the chirality is very
important to allow a better binding mode with the target. In both cases, the ligands
flexibility is a key property that allows similar poses for S and R, despite the chirality.
The best binding pose for compound 10 locates the 1,2,3-triazole phenyl ring in the
same place as its S-enantiomer, but the benzylpiperidine group has a different
orientation, which does not allow a good fitting in the cavity. On the other hand,
compound 12 showed a similar binding mode for S and R enantiomers, but different to
the binding pose predicted for compounds 9, 10 and 11, since the acyl group does not
allow the H-bond interaction with Asp70 (Figure S4).
In general, the low hAChE inhibitory activity of compounds 9-12 (IC50 > 10 μM) may
be explained considering that its acyl-binding pocket is comparatively smaller than
hBuChE (more details provided in the supporting information). Therefore, the
replacement of 5,6-dimethoxy-1-indanone moiety (present in donepezil structure) by
amino acids bearing 1,4-disubstituted 1,2,3-triazole phenyl ring abolished the potent
inhibitory activity in hAChE and drastically enhanced inhibition towards hBuChE.
It is also important to highlight that all ligand poses agree to the kinetic studies (mixed-
type inhibition model for compounds 9 and 11), which is in accordance with the dual
binding site (catalytic and peripheral sites) mode of interaction showed in the docking
studies. Additionally, the previously described docking poses were used as basis for
molecular dynamics (MD) simulations34 of 50 ns length to evaluate the stability of the
ligand-protein complexes and how different substituents may affect the binding mode
when the ligands converge into a stable pose.
Hence, the root mean square deviation (RMSD) value of hBuChE backbone atoms was
analysed to estimate the simulation trajectories. By comparing the protein RMSD
without ligand (Figure 5A) and the trajectories for compounds 9 (Figure 5B) and 11
(Figure 5C), it was found they are able to stabilize the protein movement turning it into
13

a more rigid behaviour. As expected, 11 showed higher effect on protein movement
stabilization, which is in agreement with the biological result. On the other hand,
compounds 10 (Figure 5D) and 12 (Figure 5E) do not affect hBuChE behaviour since
they shared the same RMSD profile along the simulation.
Figure 5. RMSD values for the backbone atoms along 50 ns length MD simulation of
hBuChE without ligand (A), hBuChE-compound 9 complex (B), hBuChE-compound 11
complex (C), hBuChE-compound 10 complex (D) and hBuChE-compound 12 complex
(E).
Based on the MD trajectory, ligands have modified their interactions with the target,
converting into a stable pose throughout the simulation. Compound 9 remains stable
along the trajectory, maintaining the 1,2,3-triazole ring close to the catalytic residues
(Ser198 and His438) and the phenyl ring interacting with the acyl-binding pocket
through aromatic interaction (Trp231), but reorienting the benzylpiperidine moiety
towards an ionic interaction (Asp70) and changing the primary amine H-bond from
14

Glu197 to His438 (Figure 6A). On the other hand, compound 11 changes its binding
pose along the trajectory due to a steric hindrance that forces different interactions
within the active site. In this case, the 1,2,3-triazole and phenyl rings still maintain the
same position throughout the trajectory (near to the catalytic residues and the acyl-
binding pocket), but the benzylpiperidine moiety is rearranged to a closer interaction
with Asp70 and Gln71 by two H-bonds. As a result, the benzyl group orients towards
the gorge entrance where its closer interaction with Tyr332 favours the amino acid
movement, causing the gorge entrance closing and reducing the access of endogenous
substrates (Figure 6B). Regarding compounds 10 and 12, since they are much more
flexible molecules due to their long side chain (4 carbons), they become unable to
remain stable in a single pose and fluctuates throughout the simulation.
A
B
Figure 6. Binding mode of compounds 9 (A) and 11 (B) with hBuChE (PDB code
1P0I) at the final 50 ns of the MD simulation.
As MD simulation results suggested different catalytic gorge behaviour when
compounds 9 and 11 interact, a catalytic cavity study variation was carried out as a
further investigation to confirm this finding (more details provided in the supporting
information). Using the TRAPP software,37 the last 5 ns of each trajectory were
analysed (Figure 7A) observing that the cavity volume decreases due to the movement
of both Tyr332 and the acyl-loop when 9 (Figure 7B) and 11 (Figure 7C) interact.
Although the cavity volume decreases in both cases, this effect is more significant for
hBuChE-compound 11 complex, closing the entrance of the gorge. Moreover, it can be
observed compound 9 binding mode tends to occupy and open the side-door (specific
region/channel in the active site formed by six amino acids), while compound 11
15

interacts by moving away from the side-door and keeping those residues in a more rigid
way.
Figure 7. TRAPP results of the catalytic cavity analysis in hBuChE (A), hBuChE-
compound 9 complex (B) and hBuChE-compound 11 complex (C). The regions that
disappear from the gorge along the simulation are shown in blue and the regions that
appear are shown in red. It can be seen that the acyl-loop is moving inwards the cavity
in hBuChE-compound 11 complex (C) and the movement of Tyr332 resulted in the
appearance of a new region at the top of the gorge.
2.5 In silico prediction of pharmacokinetic properties
In the context of designing molecules to target the CNS, some physicochemical
properties such as molecular lipophilicity (LogP), number of hydrogen bond donors and
acceptors (HBD and HBA), molecular weight (MW) and polar surface area (PSA) have
been considered important for optimal brain exposure and may define the attributes of
successful CNS drug candidates.38 In this sense, we have calculated some
16

physicochemical properties using QikProp software v. 2.539 to evaluate the drug-
likeness of compounds 9 and 11 (Supplementary Information - Table S1). Briefly, most
of the calculated parameters are within the acceptable range of effective CNS drugs
(LogP < 5, HBD < 3, HBA < 7, MW < 450).40 More importantly, both compounds can
be orally active due to non-violation of Lipinski’s rule and the predicted human oral
absorption is also promising [9 (65%) and 11 (81%)] compared to donepezil (100%).
The lower values may be explained due to their higher polar surface area, which
decreases their brain/blood partition coefficient compared to donepezil.
3. Conclusions
Two small series of N-benzylpiperidine based compounds have successfully been
designed and synthesized. The proposed synthetic routes were suitable and
straightforward to obtain the target compounds 1-8 (series 1) and 9-12 (series 2), as well
as their corresponding precursors (Fmoc-protected azido amino acids and some terminal
alkynes). Although most assessed compounds from series 1 exhibited low hAChE and
hBuChE inhibitory activity (μM range), compound 7 (IC50 65 nM) was potent and
selective to hBuChE. Regarding series 2, the short side chain 1,2,3-triazolyl amino acids
9 (9.9 nM) and 11 (0.17 nM) were not cytotoxic and displayed highly potent and
selective inhibition towards hBuChE. Additionally, their kinetic studies along with
docking and MD studies were important to rationalize their biological outcome.
Surprisingly, the replacement of 5,6-dimethoxy-1-indanone moiety (present in
donepezil structure) by specific scaffolds tethered via indirect coupling (amide bond) to
N-benzylpiperidine moiety abolished the potent hAChE inhibitory activity and
drastically enhanced inhibition towards hBuChE. Remarkably, compound 11 proved to
be one of the most potent (0.17 nM) and selective (>58,000-fold) hBuChE inhibitor ever
reported. It is important to highlight that the lead compounds 9 and 11 may be very
useful as research tools (i.e. pharmacological probes) to better understand the intriguing
role of hBuChE not only physiologically and in healthy states, but also in the molecular
pathology of severe diseases as Alzheimer. As a follow up work, the crystal structure of
hBuChE in complex with compounds 9 and 11 would evidence the discussed results on
molecular docking and MD simulations. Likewise, the extension of series 2 either by
using different protecting groups at the primary amine of 9 or different (non)substituted
17

fused (hetero)aromatic alkynes would provide new insights of inhibitory activity and
selectivity towards hAChE and hBuChE.
4. Experimental section
4.1 Chemistry
Chemicals were commercially obtained as reagent grade and used without any
purification. Thin layer chromatography (TLC - precoated silica aluminium plates) was
used for reaction monitoring and compounds were visualized by ultraviolet light (UV -
254 nm), ninhydrin staining solution and/or iodine vapour. Reactions performed at room
temperature in this work means a range of temperatures from 20 - 30ºC. CEM Discover
Microwave System was used to perform reactions under microwave irradiation in sealed
tubes. Biotage SP1-B2C flash chromatography system was used for purification using
normal phase cartridges (column 12+S: 21 x 55 mm, 10 g silica flash, 15 mL column
volume;10-20 mL/min flow rate) and semi-preparative HPLC purification was run on a
Shimadzu Prominence using C18 column, eluting with gradient system (0.1% (v/v) TFA
in water and 0.1% (v/v) TFA in methanol). The analytical HPLC was run on a
Shimadzu® Shimpack CLC-ODS(M) (4.6 mm i.d x 25 cm) - 5.0 μM particle diameter
column, eluting with gradient system 10-90% (0.1% (v/v) TFA in water and 0.1% (v/v)
TFA in acetonitrile) and flow rate 1.0 mL/min (column temperature: 40ºC and detector:
1
13
Diode Array Detector). H and C NMR spectra were recorded on a Bruker Advance
spectrometer at 300, 400 or 500 MHz and chemical shifts are expressed in ppm (δ).
Some assignments were aided by COSY (Homonuclear Correlation Spectroscopy) and
HMQC (Heteronuclear Multiple Quantum Correlation) spectra. High resolution mass
spectroscopy (HRMS) was carried out on a Bruker Daltonics MicroOTOF-Q II ESI-Qq-
TOF mass spectrometer using electrospray ionization.
4.1.1 General procedures for CuAAC reaction to obtain compounds 1 to 8
Procedure 1 (compounds 1-6): Sodium ascorbate (0.015 mmol) and CuSO4 (1 mg;
0.006 mmol) (6 µL of 1.0 M aq. sol.) were added to a solution of azido 13 (35 mg, 0.15
mmol) and terminal alkyne 1a - 6a (0.17 mmol) in DMF (1 mL) in a sealed tube. After
15 min at 100ºC (150 W), toluene (2 x 10 mL) was added for solvents removal under
18

vacuum. EtOAc (10 mL) was added to the crude and washed with H2O (2 x 10 mL).
Subsequently, the organic layer was dried over MgSO4, filtered, concentrated and
purified by flash chromatography [column: 12+S; solvent: Hexane/EtOAc; gradient: 80-
100% and 100-100% (v:v); flow: 10 mL/min] to afford 1,4-disubstituted 1,2,3-triazole
compounds in good yields.
Procedure 2 (compounds 7-8): To a solution of CuSO4 (0.01 mmol), 1,10-phenantroline
(0.01 mmol) and sodium ascorbate (0.20 mmol) in EtOH/H2O (2:1) (3 mL), stirred at
room temperature for 5 min, were added azido 13 (51 mg, 0.22 mmol) and terminal
alkyne 7a or 8a (0.20 mmol). The reaction was stirred overnight at room temperature
and after completion the solvents were removed under vacuum. EtOAc (10 mL) was
added to the crude and washed with H2O (2 x 10 mL). The organic layer was dried over
MgSO4, filtered, concentrated and purified by flash chromatography [column: 12+S;
solvent: DCM/CH3OH; gradient: 0-10% and 10-10% (v:v); flow: 9 mL/min] to afford
1,4-disubstituted 1,2,3-triazole compounds in good yields.
4.1.1.1 2-((1-((1-benzylpiperidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-5,6
dimethoxy-2,3-dihydro-1H-inden-1-one (1)
1
Yield: 80% (55 mg; 120 µmol). H NMR (300 MHz, CDCl3) δH (ppm): 7.37-7.25 (5H,
m), 7.23 (1H, s), 7.12 (1H, s), 6.79 (1H, s), 4.11 (2H, dd, J 7.2 Hz, J 4.4 Hz), 3.91 (3H,
s), 3.87 (3H, s), 3.50 (2H, s), 3.32-3.17 (2H, m), 3.15-2.91 (3H, m), 2.86 (2H, dd, J 11.5
13
Hz, J 2.8 Hz), 1.98-1.69 (3H, m), 1.47-1.19 (4H, m). C NMR (75 MHz, CDCl3) δC
(ppm): 206.7, 155.9, 149.6, 144.8, 129.5, 129.3, 128.4, 127.3, 122.5, 107.6, 104.3, 77.6,
63.2, 56.3, 55.7, 53.0, 47.4, 37.0, 31.7, 29.6, 26.8. HRMS (ES+): m/z [M+H]+ calculated
for C27H33N4O3: 461.2553; found: 461.2548.
4.1.1.2 2-((1-((1-benzylpiperidin-4-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)isoindoline-1,3-dione (2)
Yield: 75% (47 mg; 113 µmol). 1H NMR (400 MHz, CDCl3) δH (ppm): 7.85 (2H, dd, J
5.5 Hz, 3.1 Hz), 7.71 (2H, dd, J 5.5 Hz, 3.0 Hz), 7.54 (1H, s), 7.31-7.24 (5H, m), 4.98
(2H, s), 4.17 (2H, d, J 7.2 Hz), 3.50 (2H, s), 2.90 (2H, d, J 11.6 Hz), 2.01-1.86 (3H, m),
1.55 (2H, d, J 12.0 Hz), 1.37 (2H, q, J 11.0 Hz, J 9.7 Hz). 13C NMR (101 MHz, CDCl3)
δC (ppm): 167.8, 142.7, 134.2, 132.2, 129.4, 128.4, 127.3, 123.6, 77.5, 77.2, 63.2, 55.9,
19

53.0, 37.0, 33.2, 29.7. HRMS (ES+): m/z [M+H]+ calculated for C24H26N5O2: 416.2087;
found: 416.2081.
4.1.1.3 N-((1-((1-benzylpiperidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(1H
indol-3-yl)acetamide (3)
1
Yield: 49% (34 mg; 74 µmol). H NMR (400 MHz, CD3OD) δH (ppm): 7.50 (2H, d, J
6.9 Hz), 7.35-7.27 (6H, m), 7.18 (1H, s), 7.05 (1H, dd, J 8.1 Hz, J 1.0 Hz), 6.97 (1H, td,
J 7.5 Hz, J 7.1 Hz, J 1.0 Hz), 4.66 (1H, s), 4.41 (2H, s), 4.14 (2H, d, J 7.1 Hz), 3.68
(2H, s), 3.52 (2H, s), 3.36 (1H, s), 2.87 (2H, d, J 11.9 Hz), 2.01-1.89 (2H, m), 1.74 (1H,
ddp, J 11.3 Hz, J 7.3 Hz, J 3.2 Hz), 1.51-1.39 (2H, m), 1.27 (2H, td, J 12.2 Hz, J 3.2
13
Hz). C NMR (75 MHz, CD3OD) δC (ppm): 173.5, 136.7, 129.5, 127.9, 127.1, 127.0,
123.7, 123.1, 121.2, 118.6, 118.0, 111.0, 107.9, 62.7, 54.9, 52.4, 36.5, 34.4, 32.7, 28.6.
HRMS (ES+): m/z [M+H]+ calculated for C26H31N6O: 443.2559; found: 443.2554.
4.1.1.4 1-benzyl-4-((4-(naphthalen-1-yl)-1H-1,2,3-triazol-1-yl)methyl)piperidine (4)
1
Yield: 61% (35 mg; 92 µmol). H NMR (400 MHz, CDCl3) δH (ppm): 8.41-8.30 (1H,
m), 7.96-7.84 (2H, m), 7.75 (1H, s), 7.71 (1H, dd, J 7.1 Hz, J 1.2 Hz), 7.56-7.48 (3H,
m), 7.38-7.20 (5H, m), 4.33 (2H, d, J 7.2 Hz), 3.52 (2H, s), 2.93 (2H, d, J 11.8 Hz),
2.07-1.93 (3H, m), 1.66 (2H, d, J 12.7 Hz), 1.46 (2H, qd, J 12.3 Hz, J 3.8 Hz).13C NMR
(101 MHz, CDCl3) δC (ppm): 146.8, 137.9, 134.0, 131.2, 129.3, 129.0, 128.6, 128.4,
128.2, 127.4, 127.3, 126.7, 126.1, 125.5, 125.4, 123.2, 63.2, 56.0, 53.0, 37.1, 29.8.
HRMS (ES+): m/z [M+H]+ calculated for C25H27N4: 383.2236; found: 383.2231.
4.1.1.5 N-((1-((1-benzylpiperidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)quinazolin-
4-amine (5)
Yield: 20% (12 mg; 30 µmol). 1H NMR (400 MHz, CDCl3) δH (ppm): 8.68 (1H, s), 7.81
(2H, dd, J 15.1 Hz, J 8.1 Hz), 7.73 (1H, ddd, J 8.3 Hz, J 7.0 Hz, J 1.3 Hz), 7.60 (1H, s),
7.45 (1H, ddd, J 8.2 Hz, J 7.1 Hz, J 1.2 Hz), 7.33-7.21 (5H, m), 6.82 (1H, t, J 5.1 Hz),
4.92 (2H, d, J 5.4 Hz), 4.21 (2H, d, J 7.1 Hz), 3.49 (2H, s), 2.89 (2H, d, J 11.7 Hz),
1.94-1.84 (3H, m), 1.57 (2H, d, J 12.6 Hz), 1.37 (2H, qd, J 12.3 Hz, J 3.8 Hz).13C NMR
(101 MHz, CDCl3) δC (ppm): 159.3, 155.2, 149.5, 144.4, 138.0, 132.9, 129.3, 128.6,
20

128.4, 127.3, 126.3, 123.0, 121.0, 115.2, 63.3, 56.0, 53.0, 37.1, 36.6, 29.8. HRMS
(ES+): m/z [M+H]+ calculated for C24H28N7: 414.2406; found: 414.2401.
4.1.1.6 N-((1-((1-benzylpiperidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-6,7
dimethoxyquinazolin-4-amine (6)
Yield: 31% (22 mg; 47 µmol). 1H NMR (400 MHz, CDCl3) δH (ppm): 8.49 (1H, s), 7.60
(1H, s), 7.26-7.16 (5H, m), 7.13 (1H, s), 7.10 (1H, s), 7.03 (1H, s), 4.83 (2H, d, J 5.2
Hz), 4.14 (2H, d, J 7.1 Hz), 3.91 (3H, s), 3.78 (3H, s), 3.44 (2H, s), 2.84 (2H, d, J 11.7
Hz), 1.88 (2H, dd, J 11.7 Hz, J 2.2 Hz), 1.86-1.77 (1H, m), 1.49 (2H, d, J 11.7 Hz), 1.33
13
(2H, qd, J 12.4, J 3.7Hz). C NMR (101 MHz, CDCl3) δC (ppm): 158.2, 154.6, 153.7,
149.2, 146.3, 145.1, 137.7, 129.4, 128.4, 127.4, 123.4, 109.0, 107.4, 100.3, 63.2, 56.3,
56.0, 52.9, 37.0, 36.3, 29.7. HRMS (ES+): m/z [M+H]+ calculated for C26H32N7O2:
474.2617; found: 474.2612.
4.1.1.7 2-(((1-((1-benzylpiperidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-1H-
benzo[d]imidazole (7)
1
Yield: 70% (59 mg; 140 µmol). H NMR (400 MHz, CDCl3) δH (ppm): 7.52 (1H, s),
7.47 (1H, s), 7.34-7.23 (6H, m), 7.2-7.15 (2H, m), 4.41 (2H, s), 4.17 (2H, d, J 7.1 Hz),
3.49 (2H, s), 2.86 (2H, d, J 11.7 Hz), 1.91 (2H, td, J 11.8 Hz, J 2.3 Hz), 1.87-1.80 (1H,
13
m), 1.48 (2H, d, J 12.6 Hz), 1.31 (2H, qd, J 13.0 Hz, J 12.5 Hz, J 4.0 Hz). C NMR
(101 MHz, CDCl3) δC (ppm): 149.6, 145.0, 137.6, 129.4, 128.4, 127.4, 123.0, 122.5,
63.2, 56.0, 52.9, 36.9, 29.5, 27.0. HRMS (ES+): m/z [M+H]+ calculated for C23H27N6S:
419.2018; found: 419.2011.
4.1.1.8 2-(((1-((1-benzylpiperidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5-
ethoxy-1H-benzo[d]imidazole (8)
1
Yield: 61% (55 mg; 122 µmol). H NMR (400 MHz, CD3OD) δH (ppm): 7.64 (1H, s),
7.38-7.21 (6H, m), 6.92 (1H, s), 6.77 (1H, dd, J 8.8 Hz, J 2.4 Hz), 4.45 (2H, s), 4.16
(2H, d, J 7.1 Hz), 4.00 (2H, q, J 7.0 Hz), 3.52 (2H, s), 2.82 (2H, d, J 11.8 Hz), 2.06-1.83
(2H, m), 1.72 (1H, ddt, J 11.4 Hz, J 7.5 Hz, J 4.1 Hz), 1.38 (3H, t, J 7.0 Hz), 1.35-1.24
13
(2H, m), 1.18 (2H, td, J 12.1 Hz, J 3.2 Hz). C NMR (101 MHz, CD3OD) δC (ppm):
157.2, 145.0, 137.6, 130.9, 129.4, 128.7, 125.1, 113.7, 65.1, 63.9, 56.2, 53.8, 37.8, 37.8,
21

29.7, 28.4, 15.3. HRMS (ES+): m/z [M+H]+ calculated for C25H31N6OS: 463.2280;
found: 463.2274.
4.1.2 General procedure for Fmoc-deprotection to obtain compounds 9 and 10
Compound 21 or 22 (75 µmol) was dissolved in morpholine/DCM (1:1) (1 mL) and
stirred for 2 h at room temperature. After completion, toluene (2 x 10 mL) was added
for solvents removal under vacuum. DCM (15 mL) was added to the crude and washed
with H2O (2 x 10 mL). The organic layer was dried over MgSO4, filtered, concentrated
and the crude residue was purified by flash chromatography [column: 12+S; solvent:
DCM/CH3OH; gradient: 0-20% and 20-20% (v:v); flow: 12 mL/min], as well as HPLC
[column: C18 (Semi-preparative); solvents: H2O (0.1% TFA) and CH3OH (0.1% TFA);
isocratic method: 50% (v:v); flow 10 mL/min; injection: 200 µL (3 mg)], to afford the
desired compounds.
4.1.2.1 (S)-2-amino-N-((1-benzylpiperidin-4-yl)methyl)-3-(4-phenyl-1H-1,2,3-triazol-1-
yl)propanamide (9)
1
Yield: 61% (19 mg; 46 µmol). H NMR (500 MHz, CD3OD) δH (ppm): 8.39 (1H, s),
7.83 (2H, d, J 7.3 Hz), 7.49-7.44 (7H, m), 7.38 (1H, t, J 7.3 Hz), 4.96 (2H, m), 4.48
(1H, t, J 5.8 Hz), 4.22 (2H, s), 3.37 (2H, m), 3.29-3.28 (1H, m), 2.95 (1H, dd, J 7.2 Hz,
J 13.5 Hz), 2.85 (2H, q, J 13.3 Hz), 1.82 (2H, m), 1.71 (1H, br), 1.42-1.32 (2H, m). 13C
NMR (126 MHz, CD3OD) δC (ppm): 167.3, 149.3, 132.3, 131.3, 131.2, 130.4, 130.1,
129.7, 126.7, 123.4, 61.9, 54.1, 53.4, 50.8, 45.2, 35.0, 28.2. HRMS (ES+): m/z [M+H]+
calculated for C24H31N6O: 419.2559; found: 419.2561.
4.1.2.2 (S)-2-amino-N-((1-benzylpiperidin-4-yl)methyl)-6-(4-phenyl-1H-1,2,3-triazol-1-
yl)hexanamide (10)
1
Yield: 81% (28 mg; 61 µmol). H NMR (500 MHz, CD3OD) δH (ppm): 8.34 (1H, s),
7.82 (2H, d, J 7.4 Hz), 7.49-7.44 (7H, m), 7.36 (1H, t, J 7.4 Hz), 4.50 (2H, t, J 6.6 Hz),
4.24 (2H, s), 3.81 (1H, t, J 6.6 Hz), 3.48-3.46 (2H, m), 3.17 (1H, dd, J 6.5 Hz, J 13.6
Hz), 3.05 (1H, dd, J 6.9 Hz, J 13.6 Hz), 2.93 (2H, q, J 12.7 Hz), 2.05-1.98 (2H, m),
1.92-1.85 (4H, m), 1.79-1.71 (1H, m), 1.46-1.35 (4H, m). 13C NMR (126 MHz, CD3OD)
δC (ppm): 170.2, 149.0, 132.3, 131.8, 131.3, 130.4, 130.3, 130.1, 129.5, 126.7, 122.4,
22

61.9, 54.4, 53.4, 50.9, 49.0, 45.0, 35.1, 31.9, 30.6, 28.3, 22.8. HRMS (ES+): m/z [M+H]+
calculated for C27H37N6O: 461.3029; found: 461.3022.
4.1.3 General procedure for acetylation reaction to obtain compounds 11 and 12
Ac2O (400 µL) was added to a solution of compound 21 or 22 (43 µmol) in pyridine
(800 µL) and stirred overnight at room temperature. Afterwards, toluene (2 x 10 mL)
was added for solvents removal under vacuum and the crude was purified by flash
chromatography [column: 12+S; solvent: DCM/CH3OH; gradient: 0-10% and 10-10%
(v:v); flow: 12 mL/min] to afford the desired compounds.
4.1.3.1 (S)-2-acetamido-N-((1-benzylpiperidin-4-yl)methyl)-3-(4-phenyl-1H-1,2,3
triazol-1-yl)propanamide (11)
1
Yield: 60% (12 mg; 26 µmol). H NMR (500 MHz, CD3OD) δH (ppm): 8.29 (1H, s),
7.80 (2H, d, J 7.1 Hz), 7.45-7.34 (8H, m), 4.81 (1H, dd, J 6.6 Hz, J 13.8 Hz), 4.73 (1H,
dd, J 6.8 Hz, J 13.8 Hz), 3.98 (2H, s), 3.20-3.12 (3H, m), 2.97 (1H, dd, J 7.1 Hz, J 13.5
Hz), 2.56 (2H, q, J 10.9 Hz), 1.99 (3H, s), 1.70 (2H, t, J 15.0 Hz), 1.63-1.55 (1H, m),
1.32-1.27 (3H, m). 13C NMR (126 MHz, CD3OD) δC (ppm): 173.4, 170.8, 148.8, 131.8,
131.6, 130.4, 130.1, 130.0, 129.5, 126.7, 123.2, 62.3, 54.9, 53.4, 51.8, 49.0, 45.1, 35.6,
28.6, 22.5. HRMS (ES+): m/z [M+H]+ calculated for C26H33N6O2: 461.2665; found:
461.2654.
4.1.3.2 (S)-2-acetamido-N-((1-benzylpiperidin-4-yl)methyl)-6-(4-phenyl-1H-1,2,3
triazol-1-yl)hexanamide (12)
1
Yield: 85% (18 mg; 36 µmol). H NMR (500 MHz, CD3OD) δH (ppm): 8.32 (1H, s),
7.84-7.80 (2H, m), 7.46-7.42 (7H, m), 7.35 (1H, t, J 7.4 Hz), 4.47 (2H, t, J 6.8 Hz), 4.17
(1H, dd, J 6.1 Hz, J 8.2 Hz), 4.10 (2H, s), 3.10 (1H, dd, J 6.9 Hz, J 13.5 Hz), 3.04 (1H,
dd, J 6.6 Hz, J 13.5 Hz), 2.75 (2H, t, J 12.5 Hz), 1.98 (2H, q, J 7.3 Hz), 1.94 (3H, s),
1.87-1.75 (3H, m), 1.74-1.64 (2H, m), 1.45-1.30 (6H, m). 13C NMR (126 MHz,
CD3OD) δC (ppm): 174.6, 173.4, 148.9, 131.9, 131.8, 130.6, 130.1, 130.0, 129.4, 126.7,
122.2, 55.0, 53.4, 51.1, 44.7, 35.6, 32.2, 30.7, 28.5, 23.8, 22.4. HRMS (ES+): m/z
[M+H]+ calculated for C29H39N6O2: 503.3134; found: 503.3129.
23

4.1.4 General procedure for amide coupling reaction to obtain compounds 19 and 20
HBTU (0.25 g; 0.65 mmol) and DIPEA (0.35 mL; 2.0 mmol) were added to a stirring
solution of azido-amino acid 15 or 17 (0.65 mmol) in DMF (10 mL). After 5 min, the
key intermediate 18 (0.10 g; 0.50 mmol) diluted in DMF (5 mL) was added and the
reaction was stirred overnight at room temperature. DCM (30 mL) was added to the
reaction and washed with 5% HCl aq. sol. (20 mL), sat. NaHCO3 aq. sol. (10 mL) and
brine (10 mL). The organic layer was dried over MgSO4, filtered, concentrated and the
crude was purified by flash chromatography [column: 12+S; solvent: DCM/CH3OH;
gradient: 0-3% and 3-3% (v:v); flow: 8 mL/min] to afford the desired compounds.
4.1.4.1 (9H-fluoren-9-yl)methyl(S)-1-((1-benzylpiperidin-4-yl)methylcarbamoyl)-2-
azidoethylcarbamate (19)
Yield: 71% (194 mg; 0.36 mmol). 1H NMR (500 MHz, CDCl3) δH (ppm): 7.77 (2H, d,
J 7.6 Hz), 7.57 (2H, d, J 7.2 Hz), 7.40 (2H, t, J 7.4 Hz), 7.33-7.22 (7H, m), 6.33 (1H,
m), 5.66 (1H, d, J 6.1 Hz), 4.53-4.40 (2H ,m), 4.29 (1H, s), 4.21 (1H, t, J 6.6 Hz), 3.81
(1H, d, J 9.4 Hz), 3.52 (1H, br), 3.48 (2H, s), 3.16 (2H, t, J 5.9 Hz), 2.87 (2H, d, J 11.4
Hz), 1.92 (2H, t, J 11.2 Hz), 1.62 (2H, d, J 12.3 Hz), 1.49 (1H, s), 1.35-1.20 (2H, m).
13C NMR (126 MHz, CDCl3) δC (ppm): 169.0, 143.7, 141.5, 138.3, 129.3, 128.3, 127.3,
127.2, 125.0, 120.2, 109.7, 67.4, 63.4, 54.3, 53.3, 52.3, 47.3, 45.4, 36.0, 29.9. HRMS
(ES+): m/z [M+H]+ calculated for C31H35N6O3: 539.2771; found: 539.2766.
4.1.4.2 (9H-fluoren-9-yl)methyl(S)-1-((1-benzylpiperidin-4-yl)methylcarbamoyl)-5-
azidopentylcarbamate (20)
Yield: 74% (215 mg; 0.37 mmol). 1H NMR (500 MHz, CDCl3) δH (ppm): 7.76 (2H, m),
7.57 (2H, d, J 7.3 Hz), 7.40 (2H, t, J 7.5 Hz), 7.33-7.21 (7H, m), 6.16-6.05 (1H, m),
5.38 (1H, d, J 7.9 Hz), 4.40 (2H, q, J 10.5 Hz), 4.20 (1H, t, J 6.8 Hz), 4.12-4.06 (1H,
m), 3.48 (2H, s), 3.27 (2H, s), 3.14 (2H, s), 2.86 (2H, d, J 11.3 Hz), 1.96-1.78 (4H, m),
1.65-1.56 (4H, m), 1.53-1.36 (3H, m), 1.32-1.22 (2H, m). 13C NMR (126 MHz, CDCl3)
δC (ppm): 171.5, 156.4, 143.8, 141.5, 138.2, 129.4, 128.3, 127.9, 127.3, 127.2, 125.2,
125.1, 120.2, 67.2, 63.4, 55.1, 53.3, 51.3, 47.3, 45.2, 36.0, 32.2, 29.9, 28.6,22.9. HRMS
(ES+): m/z [M+H]+ calculated for C34H41N6O3: 581.3240; found: 581.3235.
24

4.1.5 General procedure for CuAAC reaction to obtain compounds 21 and 22
Sodium ascorbate (8.40 mg; 42 µmol) and CuSO4 (1.64 mg; 10.25 µmol) were added to
a stirring solution of Fmoc-protected azido-building block 19 or 20 (205 µmol) and
phenylacetylene (35 µL; 307 µmol) in DCM/t-BuOH/H2O (1:1:1) (1.5 mL). The
reaction was stirred overnight at room temperature and after completion, toluene (2 x 10
mL) was added for solvents removal under vacuum. DCM (15 mL) was added to the
crude and washed with H2O (2 x 10 mL). The organic layer was dried over MgSO4,
filtered, concentrated and the crude was purified by flash chromatography [column:
12+S; solvent: DCM/CH3OH; gradient: 0-3%, 3-3% and 5-5% (v:v); flow: 9 mL/min]
to afford the desired compounds.
4.1.5.1 (9H-fluoren-9-yl)methyl(S)-1-((1-benzylpiperidin-4-yl)methylcarbamoyl)-2-(4-
phenyl-1H-1,2,3-triazol-1-yl)ethylcarbamate (21)
1
Yield: 55% (72 mg; 112 µmol). H NMR (500 MHz, CDCl3) δH (ppm): 7.87 (1H, s),
7.79-7.72 (4H, m), 7.56 (2H, d, J 6.8 Hz), 7.38 (4H, t, J 7.6 Hz), 7.34-7.17 (8H, m),
6.58 (1H, s), 6.28 (1H, s), 4.97 (1H, d, J 11.5 Hz), 4.81 (1H, s), 4.65 (1H, d, J 12.3 Hz),
4.44 (2H, q, J 9.9 Hz), 4.19 (1H, t, J 6.7 Hz), 3.31 (2H, s), 3.11 (1H, dd, J 12.6 Hz, J 6.2
Hz), 2.99-2.91 (1H, m), 2.72-2.60 (2H, m), 1.67 (2H, q, J 10.3 Hz), 1.37 (2H, d, J 11.2
Hz), 1.25 (1H, s), 1.14-1.04 (2H, m). 13C NMR (126 MHz, CDCl3) δC (ppm): 168.4,
147.9, 143.7, 143.6, 141.5, 141.4, 130.2, 129.3, 129.0, 128.6, 128.3, 128.1, 128.0,
127.3, 127.1, 125.8, 125.1, 121.8, 120.3, 120.2, 67.7, 63.3, 53.1, 51.3, 47.2, 45.3, 35.9,
29.8. HRMS (ES+): m/z [M+H]+ calculated for C39H41N6O3: 641.3240; found: 641.3234.
4.1.5.2 (9H-fluoren-9-yl)methyl(S)-1-((1-benzylpiperidin-4-yl)methylcarbamoyl)-5-(4-
phenyl-1H-1,2,3-triazol-1-yl)pentylcarbamate (22)
Yield: 86% (120 mg; 176 µmol). 1H NMR (500 MHz, CDCl3) δH (ppm): 7.81 (2H, d, J
7.6 Hz), 7.76-7.71 (3H, m), 7.58-7.51 (2H, m), 7.39 (4H, q, J 7.9 Hz), 7.33-7.20 (8H,
m), 6.24 (1H, s), 5.51 (1H, d, J 7.7 Hz), 4.40-4.32 (4H, m), 4.17 (1H, t, J 6.8 Hz), 4.14-
4.04 (1H, m), 3.44 (2H, s), 3.10 (2H, q, J 6.1 Hz), 2.82 (2H, d, J 11.0 Hz), 2.02-1.94
(2H, m), 1.93-1.82 (4H, m), 1.68 (1H, dd, J 13.4 Hz, J 6.9 Hz), 1.56 (2H, d, J 11.9 Hz),
1.45-1.34 (2H, m), 1.23 (2H, q, J 10.3 Hz). 13C NMR (126 MHz, CDCl3) δC (ppm):
171.4, 148.0, 143.9, 141.5, 138.4, 130.7, 129.3, 129.0, 128.3, 127.9, 127.2, 127.1,
25

125.9, 125.2, 125.1, 120.2, 119.8, 67.2, 63.4, 53.3, 50.0, 47.3, 45.2, 36.0, 31.9, 30.0,
29.8, 22.5. HRMS (ES+): m/z [M+H]+ calculated for C42H47N6O3: 683.3710; found:
683.3710.
4.2 Biological assays
4.2.1 Cholinesterases inhibition assay
Based on Ellman’s method,32 the experiments were performed in 96 well microplate and
the samples were assessed against human recombinant acetylcholinesterase (hAChE)
and human serum butyrylcholinesterase (hBuChE) according to the literature.15
4.2.2 Cell viability assays
Cell viability assay was performed with the XTT (Cell Proliferation Kit II - XTT, Roche
Molecular Biochemicals) colorimetric method using SH-SY5Y cell line derived from
human neuroblastoma according to the literature.34
4.3 Molecular modeling
4.3.1 Docking studies and molecular dynamics
Docking studies and molecular dynamics were carried out using the structure of hAChE
(PDB 4EY7) and hBuChE (PDB 1P0I) according to the literature.34
Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo -
FAPESP (grant 2012/14114-5, 2013/50788-3, 2014/04868-8), Coordenadoria de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq)
Consejo Superior de
Investigaciones Científicas - CSIC (grant i-Link0801) and MINECO (CTQ2015-66313-
R).
26

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