N-Propargylpiperidines with naphthalene-2-carboxamide or naphthalene-2-sulfonamide moieties: Potential multifunctional anti-Alzheimer's agents

Article abstract of DOI:10.1016/j.bmc.2016.11.032

In the brains of patients with Alzheimer's disease, the enzymatic activities of butyrylcholinesterase (BChE) and monoamine oxidase B (MAO-B) are increased. While BChE is a viable therapeutic target for alleviation of symptoms caused by cholinergic hypofunction, MAO-B is a potential therapeutic target for prevention of neurodegeneration in Alzheimer's disease. Starting with piperidine-based selective human (h)BChE inhibitors and propargylamine-based MAO inhibitors, we have designed, synthesized and biochemically evaluated a series of N-propargylpiperidines. All of these compounds inhibited hBChE with good selectivity over the related enzyme, acetylcholinesterase, and crossed the blood-brain barrier in a parallel artificial membrane permeation assay. The crystal structure of one of the inhibitors (compound 3) in complex with hBChE revealed its binding mode. Three compounds (4, 5, 6) showed concomitant inhibition of MAO-B. Additionally, the most potent hBChE inhibitor 7 and dual BChE and MAO-B inhibitor 6 were non-cytotoxic and protected neuronal SH-SY5Y cells from toxic amyloid β-peptide species.

Full text of DOI:10.1016/j.bmc.2016.11.032

Accepted Manuscript
N-Propargylpiperidines with Naphthalene-2-Carboxamide or Naphthalene-2-
Sulfonamide Moieties: Potential Multifunctional Anti-Alzheimer’s Agents
Urban Košak, Damijan Knez, Nicolas Coquelle, Boris Brus, Anja Pišlar, Florian
Nachon, Xavier Brazzolotto, Janko Kos, Jacques-Philippe Colletier, Stanislav
Gobec
PII:
S0968-0896(16)30909-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.032
BMC 13399
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 October 2016
14 November 2016
17 November 2016
Please cite this article as: Košak, U., Knez, D., Coquelle, N., Brus, B., Pišlar, A., Nachon, F., Brazzolotto, X., Kos,
J., Colletier, J-P., Gobec, S., N-Propargylpiperidines with Naphthalene-2-Carboxamide or Naphthalene-2-
Sulfonamide Moieties: Potential Multifunctional Anti-Alzheimer’s Agents, Bioorganic & Medicinal Chemistry
(2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.032
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N-Propargylpiperidines with Naphthalene-2-
Carboxamide or Naphthalene-2-Sulfonamide
Moieties: Potential Multifunctional Anti-
Alzheimer’s Agents
Urban Košak,‡ Damijan Knez,‡ Nicolas Coquelle,ʃ Boris Brus,‡ Anja Pišlar,‡ Florian
Nachon,ø Xavier Brazzolotto,ø Janko Kos,‡ Jacques-Philippe Colletier,ʃ Stanislav Gobec‡,*
‡Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
ʃInstitut de Biologie Structurale, Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France
ø Institut de Recherche Biomédicale des Armées, 91223 Brétigny-sur-Orge, France
1

*Corresponding Author: Stanislav Gobec
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
Tel: +386-1-4769500; Fax: +386-1-4258031; E-mail: stanislav.gobec@ffa.uni-lj.si
Abbreviations
Aβ, amyloid β; ACh, acetycholine; mAChE, murine acetylcholinesterase; AD, Alzheimer’s
disease; BBB, blood-brain barrier; hBChE, human butyrylcholinesterase; ChE(s),
cholinesterase(s); FAD, flavine adenine dinucleotide; hMAO-A, human monoamine oxidase
A; hMAO-B, human monoamine oxidase B; MTDL, multitarget-directed ligand; MTS, (3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt); PAMPA, parallel artificial membrane permeation assay; TFA, 2,2,2-trifluoroacetic
acid.
2

ABSTRACT
In the brains of patients with Alzheimer’s disease, the enzymatic activities of
butyrycholinesterase (BChE) and monoamine oxidase B (MAO-B) are increased. While
BChE is a viable therapeutic target for alleviation of symptoms caused by cholinergic
hypofunction, MAO-B is a potential therapeutic target for prevention of neurodegeneration in
Alzheimer’s disease. Starting with piperidine-based selective human (h)BChE inhibitors and
propargylamine-based MAO inhibitors, we have designed, synthesized and biochemically
evaluated a series of N-propargylpiperidines. All of these compounds inhibited hBChE with
good selectivity over the related enzyme, acetylcholinesterase, and crossed the blood-brain
barrier in a parallel artificial membrane permeation assay. The crystal structure of one of the
inhibitors (compound 3) in complex with hBChE revealed its binding mode. Three
compounds (4, 5, 6) showed concomitant inhibition of MAO-B. Additionally, the most potent
hBChE inhibitor 7 and dual BChE and MAO-B inhibitor 6 were non-cytotoxic and protected
neuronal SH-SY5Y cells from toxic amyloid β-peptide species.
Keywords: multitarget-directed ligands; butyrylcholinesterase inhibitors; MAO-B inhibitors;
Alzheimer’s disease.
3

INTRODUCTION
Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by progressive
deterioration of memory and other cognitive functions. Neurodegeneration and synaptic
dysfunction caused by accumulation of the aggregated amyloid β-peptide (Aβ) and
hyperphosphorylated protein tau, and by oxidative stress [1] severely affect the cholinergic
system [2]. As a result, the levels of the neurotransmitter acetylcholine (ACh) are severely
decreased [3]. This produces memory and cognitive deficits [4], which are distinctive for
patients with AD. Inhibition of ACh hydrolysis in the brain is thus used to restore cognitive
functions and alleviate symptoms of AD.
Two cholinesterases (ChEs) terminate cholinergic neurotransmission through their
catalysis of the hydrolysis of ACh: acetycholinesterase (AChE) and butyrylcholinesterase
(BChE) [5]. Three out of the four approved drugs for the treatment of AD are AChE inhibitors
(Figure 1A, donepezil [6], galantamine [7] and rivastigmine [8]); however, these have
limitations due to undesired AChE inhibition in the peripheral and autonomous nervous
systems that can produce adverse cholinergic side effects. This necessitates limited dosing,
and can result in limited clinical efficacy in advanced stages of the disease [9]. These
limitations can be overcome by selective inhibition of BChE [10]–[12], because as AD
progresses, the enzymatic activity of BChE in the brain increases [10]. This makes BChE a
prospective therapeutic target in AD, with one selective BChE inhibitor (Figure 1B, a
cymserine analog [11]) successfully advanced to Phase I clinical trials.
Monoamine oxidase (MAO) is one of the several proteins that contribute to oxidative
stress in AD [13]. MAO is a flavine adenine dinucleotide (FAD)-containing enzyme that is
located in the outer mitochondrial membrane [14]. In most mammalian tissues, two
isoenzymes are present: MAO-A and MAO-B [13]. In the brains of patients with AD, the
enzymatic activity of MAO-B is increased [13]–[16], which results in increased levels of
4

oxidative deamination reaction products such as hydrogen peroxide, ammonia and aldehydes;
these then contribute to neurodegeneration [13],[15],[16]. Selective inhibition of MAO-B in
the brain might thus be useful for the management of AD [13]–[16]. Accordingly, MAO-B
inhibitors (Figure 1C) are currently in clinical trials for the treatment of AD [13],[14],[16].
MAO inhibitory activity has also been combined with ChE inhibitory activity in
multitargeting compounds (Figure 1D) that use the multitarget-directed ligand (MTDL)
approach. Several multitarget compounds are currently being examined in preclinical and
clinical trials [13],[15],[17][19].
5

Figure 1. Structures of the currently approved ChE inhibitors for the treatment of Alzheimer’s disease
(A), the selective BChE inhibitors (B), the selective MAO-B inhibitors (C), and the multitarget-
directed ligands that use ChE and MAO inhibitory activities (D).
Recently, we described a novel piperidine-3-ylmethanamine–based selective human
(h)BChE inhibitor 1 (Figure 1B) that showed reversible, slow–tight binding inhibition with
6

low nanomolar IC50 [20]. The structure of this inhibitor has already been used to develop an
in-vivo active sulfonamide derivative 2 (Figure 1B)[12]. Additionally, compounds 1 and 2 can
protect neuronal cells from the toxic Aβ species. In the current study, our hypothesis was
1–42
that replacing N-piperidine substituents of our neuroportective selective hBChE inhibitors 1
and 2 with the propargyl moiety of selective irreversible propargylamine MAO-B inhibitors
would produce selective hBChE and MAO-B inhibitors with neuroprotective activity.
We report here the design, synthesis, and biochemical evaluation of a series of N-
propargylpiperidine derivatives, as potent inhibitors of hBChE. The crystal structure of
compound 3 in complex with hBChE reveals its binding mode. Additionally, three of these
compounds (4–6) inhibit MAO-B, which demonstrates the feasibility of dual inhibition of
hBChE and MAO-B using a single compound. Furthermore, two compounds (6, 7) protect
neuronal cells from the toxic Aβ species. Hence, N-propargylpiperidines represent a new
1–42
class of drug candidates for symptomatic treatment of AD.
RESULTS AND DISCUSSION
Design
The crystal structure of compound 1 in complex with hBChE and the chemical structures of
selective irreversible propargylamine MAO-B inhibitors (e.g., selegiline, rasagiline) were
used as the starting point to design the N-propargylpiperidines (Figure 2). It was essential that
this design produced compounds with drug-like properties [21]. Selegiline and rasagiline
contain a propargylamine moiety that has been shown to covalently bind to the N5 atom of the
flavine ring of the FAD cofactor [13],[14]. The piperidine nitrogen in hBChE inhibitor hit
compound 1 was recognized as the most suitable functional group for the simple introduction
of the propargyl moiety [22], and thus for the design of novel piperidine-based
propargylamines.
7

Previous studies on derivatives of hit compound 1 have revealed that the naphthalene-
2-yl moiety is essential for good inhibitory potency, due to its tight binding in the acyl
binding pocket of hBChE [12]. Accordingly, all of the N-propargylpiperidines designed here
retained the naphthalene-2-yl moiety of the parent hit compound 1. Sulfonamides were
designed to explore how the bioisosteric replacement of a carboxamide with a sulfonamide
[23] affects ChE and MAO inhibition. The role of the N-alkyl chain of these inhibitors (Figure
2, R) was studied by its removal or elongation. We removed the N-alkyl chain and designed
compounds with a lower molecular weight and a reduced number of rotatable bonds. This is a
common design strategy in medicinal chemistry that has been used to improve brain exposure
of drug candidates [24]. (CH2)2OMe was kept as the substituent R to directly study the effects
of replacing the 2,3-dihydro-1H-inden-2-yl of hit compound 1 and benzyl group of compound
2 with the propargylamine moiety on the inhibitory potency and selectivity against hBChE,
and Aβ1–42-anti-aggregation activity. To investigate the effects of an additional methylene
group in the N-alkyl chain on ChE inhibition, MAO inhibition and Aβ1–42-anti-aggregation
activity of compounds with the (CH2)3OMe group were also designed. Finally, 1,4-
disubstituted piperidine derivatives were designed to examine the effects of the piperidine
ring disubstitution pattern on ChE and MAO inhibition (Figure 2).
8

Figure 2. Design of the N-propargylpiperidines.
Synthesis
The synthesis of 1,3-disubstituted piperidines without the N-alkyl chain 4 and 8 is presented
in Scheme 1A. The key intermediate, orthogonally protected piperidin-3-ylmethanamine 9
was prepared from commercially available nipecotamide using a straightforward two step
procedure, as previously reported [25]. In the next step, the N-benzyl group of 9 was removed
using cyclohexene in the presence of catalytic amount of Pearlman's catalyst (palladium
hydroxide on carbon) to provide crude secondary amine 10 [26], which was then reacted with
propargyl bromide in the presence of Cs2CO3 in acetone [27], to produce propargyl amine 11.
The tert-butyloxycarbonyl protecting group from compound 11 was removed using HCl
solution in Et2O since using TFA in CH2Cl2 produced an impure desired product. The crude
amine dihydrochloride 12 was then reacted with 2-naphthoyl chloride or naphthalene-2-
sulfonyl chloride, to produce amide 4 or sulfonamide 8, respectively.
9

The synthesis of 1,4-disubstituted piperidines without the N-alkyl chain 5 and 6 is presented
in Scheme 1B. They were prepared from commercially available isonipecotamide using the
same reagents and conditions used to synthesize inhibitors 4 and 8 from nipecotamide, with
the exception that the butyloxycarbonyl protecting group from compound 13 was removed
using TFA in CH2Cl2 which, in this case produced the pure desired product.
Scheme 1. Reagents and conditions: (a) (i) PhCOCl, Et3N, THF, 0 °C to rt, 22-25 h; (ii) LiAlH4,
anhydrous THF, rt to reflux, under argon, 2–3 h, (iii) Boc2O, Et3N, CH2Cl2, 0 °C to rt, 21-24 h, (48–
78% overall yield); (b) cyclohexene, Pd(OH)2/C cat., MeOH, rt, under argon, 20 h (90–91%); (c)
propargly bromide (80 wt. % solution in toluene), Cs2CO3, acetone, rt, 24 h (58–75%); (d) 2 M HCl
10

solution in Et2O, MeOH, 0 °C to rt, under argon, 24 h (99%); (e) 2-naphthoyl chloride, Et3N, CH2Cl2,
0 °C to rt, 24 h (78–91%); (f) naphthalene-2-sulfonyl chloride, Et3N, CH2Cl2, 0 °C to rt, 24 h (81–
82%); (g) TFA, CH2Cl2, rt, 24 h (97%).
The synthesis of 1,3-disubstituted piperidines with the N-alkyl chain 3, 7, 14 and 15 is
presented in Scheme 2A. Commercially available nipecotic acid was first converted into
orthogonally protected piperidin-3-ylmethanamines 20 and 21 using a two-step procedure, as
previously reported [25]. Amine 20 was then converted into amide 3 and sulfonamide 14,
while amine 21 was converted into amide 15 and sulfonamide 7 using the same reagents and
conditions used to synthesize inhibitors 4 and 8 in Scheme 1A.
1,4-Disubstituted piperidines with N-alkyl chain 22–25 were synthesized from commercially
available isonipecotic acid using the same reagents and conditions used to synthesize
inhibitors 3, 7, 14 and 15 (Scheme 2B).
11

Scheme 2. Reagents and conditions: (a) (i) PhCOCl, K2CO3, THF–H2O, 0 °C to rt, 22 h, (ii) 6 M HCl
(aq), 0 °C; (iii) H2N-R, TBTU, Et3N, CH2Cl2, rt, 21–23h; (iv) LiAlH4, anhydrous THF, rt to reflux,
under argon, 2–3 h, (v) Boc2O, Et3N, CH2Cl2, 0 °C to rt, 17–22 h, (56–74% overall yield); (b)
cyclohexene, Pd(OH)2/C cat., MeOH, rt, under argon, 20 h (94–99%); (c) propargly bromide (80 wt.
12

% solution in toluene), Cs2CO3, acetone, rt, 24 h (64–78%); (d) 2 M HCl solution in Et2O, MeOH, 0
°C to rt, under argon, 24 h (93–97%); (e) 2-naphthoyl chloride, Et3N, CH2Cl2, 0 °C to rt, 24 h (92–
96%); (f) naphthalene-2-sulfonyl chloride, Et3N, CH2Cl2, 0 °C to rt, 24 h (79–97%).
In-Vitro Enzyme Inhibition and Structure-Activity Relationships
The inhibitory potencies against hBChE and murine (m)AChE for these N-propargyl
piperidines were determined using the method of Ellman [28], with all of these compounds
showing selectivity toward hBChE over mAChE (Table 1).
The inhibitory potencies against human (h)MAO-A and hMAO-B were determined for
all of the target compounds, using a previously described fluorescence-based Amplex Red
assay [17] with minor modifications. Table 1 reports the structures and inhibitory potencies of
these N-propargyl piperidines. All of these compounds showed moderate selectivity toward
hMAO-B over hMAO-A (Table 1), and three showed IC50 values ranging from 53.9 µM to
72.6 µM against hMAO-B.
13

Table 1. Inhibitory potencies and structures of N-propargyl piperidines, tacrine, clorgyline, and
pargyline.
Selectivity
hMAO-
Bb
IC50
±SEM
(µM)
hBChE mAChE
Selectivity
ratio
hMAO-
Ab
IC50
ratio
IC50hMAO-
A/
IC50hMAO-
B
Compound
number or Pdpa
name
IC50
±SEM
(µM)
IC50
R
X
±SEM IC50mAChE/
(µM)
IC50hBChE
(µM)
23.444
±1.497
3.306
±0.381
8.641
±1.989
54.396
±9.428
4.040
±0.237
4.590
±0.230
1.223
±0.036
0.137
±0.003
0.127
±0.006
2.600
±0.348
0.384
±0.017
0.281
±0.024
0.023
±0.003
not
59.293
±0.002
1,3
H
CO
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>4.27
>30.25
>11.57
>1.84
>24.75
>21.79
>81.77
>729.93
>787.40
>38.46
>260.42
>355.87
5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>1.69
4
1,3 (CH2)2OMe CO
1,3 (CH2)3OMe CO
>100
>100
1
3
1
15
72.611
±1.668
1,4
H
CO
>1.38
5
1,4 (CH2)2OMe CO
1,4 (CH2)3OMe CO
>100
>100
>100
>100
>100
1
22
1
23
1,3
H
SO2
1
8
1,3 (CH2)2OMe SO2
1,3 (CH2)3OMe SO2
1
14
1
7
6
53.904
±4.781
1,4
H
SO2
>1.86
1,4 (CH2)2OMe SO2
1,4 (CH2)3OMe SO2
>100
>100
1
24
1
/
25
0.115
±0.009
not
tested
not
not
tested
0.0034
not
tested
13.568
±0.0003 ±1.157
/
/
/
/
/
/
/
/
/
Tacrine
Clorgyline
Pargyline
/
0.00025
17.27
tested
not
3.368
±0.275
0.195
±0.019
/
tested
tested
apdp = piperidine disubstitution pattern
bdetermined after 15 min preincubation of inhibtor with the enzyme
14

As propargylamines inhibit MAO irreversibly by forming covalent N(5)-flavocyanine
adducts [13],[14] we performed the reversibility test [17] with compound 6 to determine the
mechanism of hMAO-B inhibition. The results of the 100-fold dilution assay showed that this
inhibitor binds irreversibly, maintaining over 70% inhibition after dilution of the preincubated
mixture (Figure 3). The time-dependent mechanism of inhibition was confirmed in the IC50
shift assay, as the IC50 decreased with prolongation of incubation time (Figure 4).
Figure 3. Recovery of hMAO-B activity after 100-fold dilution following 15 min incubation (at 37
°C) of the 100× enzyme concentration with 10-fold the IC50 of compound 6 and pargyline. The control
was carried out in the absence of inhibitor and diluted in the same way. Data are expressed as
percentages of control ±SEM of four independent experiments.
15

Figure 4. The shifted IC50 curves for compound 6 (A) and pargyline (positive control) (B) at various
pre-incubation times indicate time-dependent inhibition of hMAO-B.
The most important findings regarding the structure–activity relationships are
summarized in Figure 5. Replacing the 2,3-dihydro-1H-inden-2-yl and benzyl groups on the
piperidine nitrogen with the propargylamine moiety reduced the inhibitory potency against
hBChE [12],[20]. The N-propargylpiperidine sulfonamides were more potent hBChE
inhibitors compared to their analogous carboxamides. Removal of the N-alkyl chain
[(CH2)nOMe] from the carboxamide and sulfonamide nitrogen reduced the inhibitory potency,
as secondary carboxamides and sulfonamides were significantly weaker inhibitors than their
tertiary counterparts. Elongation of the N-alkyl chain with an additional methylene group did
not affect the inhibitory potency significantly. Analogous to sulfonamide derivatives of hit
compound 1 [12], 1,3 disubstituted N-propargilpiperidines were more potent hBChE
inhibitors than their 1,4 disubstituted counterparts. The most potent hBChE inhibitor of the
series, compound 7 (IC50 = 127 nM) is thus a 1,3 disubstituted piperidine with a sulfonamide
group and (CH2)2OMe chain on the sulfonamide nitrogen. This study of the structure–activity
relationships also showed that the absence of the N-alkyl chain on the carboxamide and
16

sulfonamide nitrogen is imperative for MAO-B inhibition, as compounds bearing the N-alkyl
chain were inactive.
Figure 5. Structure–activity relationships of N-propargylpiperidines.
Crystal Structure of hBChE in Complex with Compound 3
The crystal structure of compound 3 in complex with hBChE was solved at 2.5 Å resolution,
which revealed the molecular basis for the micromolar inhibitory potency of this N-
propargypiperidine. As expected, compound 3 shares a similar binding mode with the parent
hit compound 1, where the naphthalene moiety occupies the acyl-binding pocket and is T-
stacked (i.e., π-π interaction) to Trp231 (Figure 6). Compared to the parent hit compound 1,
the positively charged nitrogen of the piperidine moiety does not interact with the Tyr332 side
chain anymore, as the distance between the cation and the aromatic ring is too large (5.2 vs.
3.5 Å, to the closest aromatic carbon) [20]. These specificities appear to explain the difference
in the inhibitory potencies between these two inhibitors (IC50 (1) = 21.3 nM; IC50 (3) = 3.306
µM). In the complex structure, the (CH2)2OMe side-chain is oriented toward the choline-
binding site and the catalytic His438, but this does not interact directly with any of the amino-
17

acid residues. Finally, the propargyl moiety lies over the backbone atoms of residues Asn68 to
Asp70, and points out of the active site gorge.
Figure 6. Crystal structure of compound 3 bound into the active site of hBChE (PDB code 5LKR).
(A) Alignment of crystal structures of compound 3 (cyan) and hit compound 1 (purple) in their
complexes with hBChE (gray surface). (B) Close-up view of compound 3 (cyan sticks) bound in the
acyl-binding and choline-binding pockets and at the peripheral site of hBChE (green). Important active
site residues are shown as sticks: catalytic residues (Ser198, His438); acyl-binding pocket residues
(Trp231, Leu286, Val288, Phe398); choline-binding site (Trp82); and peripheral site (Asn70, Tyr332).
In-Vitro Blood-Brain Barrier Permeation of Compounds 3–7 and 32–38
As AD is a progressive brain disorder, drugs for its treatment should partition into the brain
by crossing the blood-brain barrier (BBB). On this basis, the brain penetration of N-
propargylpiperidines 4–8, 14, 15 and 22–25 were evaluated using the parallel artificial
membrane permeation assay (PAMPA)–BBB method, which is a low cost and high-
throughput assay that is used to exclude poorly permeable compounds from further testing
(e.g., in-vitro assays on cell lines and in-vivo assays) [30]. Table 2 shows the logarithms of the
18

effective permeabilities (log Pe) of the N-propargylpiperdines and eight reference drugs with
known CNS penetration. For all of the N-propargylpiperdines, the log Pe values are higher
than the limit of uncertain permeability (–5.0), which suggests that these should cross the
BBB by passive diffusion.
Table 2. In-vitro blood-brain barrier permeation of N-propargy piperidines and reference drugs.
Compound name or Log Pea
number
Permeability
prediction
CNS+
CNS±
CNS+
CNS+
CNS+
CNS–
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
Verapamil HCl
–4.1
–5.0
–4.0
–4.6
–4.3
–6.9
–4.1
–4.0
–4.3
–4.0
–4.1
–4.0
–4.0
–3.9
–4.0
–4.3
–4.4
–4.5
–3.9
–3.9
Corticosterone
Progesterone
Lidocaine
Quinidine HCl
Theophylline
Propranolol HCl
Donepezil HCl
3
4
5
6
7
8
14
15
22
23
24
25
a Data are means of two independent experiments
CNS+, log Pe > –5.0, high permeability; CNS–, log Pe ≤ –6.9, low permeability; CNS±, –6.9 < log Pe ≤
–5.0, permeability uncertain
Inhibition of Aβ1–42 Aggregation
Senile (amyloid) plaques that accumulate in the brains of patients with AD [31] are mainly
composed of Aβ1–42 [32]. This 42-amino-acid-long Aβ isoform forms a variety of cytotoxic
structures, which range from monomers to oligomers (i.e., 2-6 peptides) and fibers [1],[33].
The inhibitory activity of these N-propargylpiperidines against Aβ1–42 aggregation was thus
19

evaluated with the thioflavine-T fluorometric assay [34]. Only compound 22 showed Aβ1–42
anti-aggregation effects, with modest 23.4% inhibition. Interestingly, replacement of the 1H-
indene moiety of hit compound 1 (61.7% inhibition) [20] with the propargyl group
(compound 3, no inhibition) diminished the Aβ1–42-anti-aggregation activity (Supplementary
Table S1).
Cytotoxicity and Neuroprotective Effects of Compounds 6 and 7
Hit compound 1 and its most potent sulfonamide derivative (compound 2) are non-cytotoxic
to neuronal cells (SH-SY5Y) and can protect them from the toxic effects of Aβ1–42 [12],[20].
To determine whether N-propargylderivatives share these non-cytotoxic and neuroprotective
properties of the parent compounds, inhibitors 6 (dual hBChE and hMAO-B inhibitor) and 7
(most potent BChE inhibitor) were characterized accordingly.
First, their cytotoxicity profiles were determined using MTS (3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay. At 5
µM, compound 6 was completely non-cytotoxic and had an LD50 of 241.8 µM, a
concentration 90-fold and 4-fold greater than those needed to achieve 50% inhibition of
hBChE and MAO-B, respectively. At 2.5 µM, compound 7 was completely non-cytotoxic and
had an LD50 of 48.13 µM, a concentration 375-fold greater than that needed to achieve 50%
inhibition of hBChE (Figure 7).
20

Figure 7. Concentration-dependent cytotoxicities of compounds 6 and 7. SH-SY5Y cells were
incubated in the presence of increasing concentrations of compounds 6 and 7 (1–100 µM). After 48 h,
cell viability was evaluated using the MTS assay. The control group (DMSO) was considered as 100%
cell viability. Cells were treated in quadruplicate. Data are means ±SD of at least three independent
experiments.
We then determined whether compounds 6 and 7 can protect neuronal cells from toxic
Aβ-species. Therefore, we examined neuronal death induced by Aβ1–42 in the absence and
presence of 5 µM compound 6 and 2.5 µM compound 7. As shown in Figure 8, treatment of
neuronal cells with 5 µM Aβ1–42 caused significant toxicity, whereas compounds 6 and 7
prevented this Aβ1–42-induced cell death. These neuroprotective effects are independent of
Aβ1–42 aggregation, as at 10 µM, compounds 6 and 7 did not inhibit Aβ1–42 aggregation in the
Thioflavin-T (ThT) fluorometric assay (Supplementary Table S1).
21

Figure 8. Neuroprotective effects of compounds 6 and 7 on Aβ1–42-induced cytotoxicity in SH-SY5Y
cells. Cells were treated with 5 µM Aβ1–42 in the absence and presence of 5 µM compound 6 and 2.5
µM compound 7. After 48 h the, neuroprotective effects were evaluated using the MTS assay. The
control group (DMSO) was considered as 100% cell viability. Cells were treated in quadruplicate.
Data are means ±SD of three independent experiments. *P <0.01, **P <0.05
CONCLUSIONS
We have here described the design, synthesis, and biochemical evaluation of 12 N-
propargylpiperidines compounds. All of these compounds selectively inhibit hBChE over
AChE, and the results from the PAMPA-BBB assay suggest that they should cross the BBB.
The crystal structure of compound 3 in complex with hBChE revealed its mode of binding
and also suggested ways to further optimize these N-propargylpiperidines. Three compounds
also selectively inhibited MAO-B over MAO-A (i.e., 4, 5, 6). One of these, the dual BChE
and MAO-B inhibitor 6 (IC50(hBChE) = 2.6 µM; IC50(hMAO-B) = 53.9 µM), and the most
potent hBChE inhibitor 7 (IC50 = 0.127 µM) were non-cytotoxic and protected neuronal cells
from toxic Aβ1–42. Compound 6 confirmed our hypothesis that replacing the N-piperidine
22

substituents of our neuroportective selective hBChE inhibitors 1 and 2 with the propargyl
moiety of selective irreversible propargylamine MAO-B inhibitors produces selective hBChE
and MAO-B inhibitors with neuroprotective activity.
METHODS
General chemistry methods
1H-NMR and 13C-NMR were recorded at 400.130 MHz and 100.613 MHz, respectively, on an
NMR spectrophotometer (Bruker Avance III). The chemical shifts (δ) are reported in parts per
million (ppm) and are referenced to the deuterated solvent used. The coupling constants (J)
are reported in Hz, and the splitting patterns are indicated as: s, singlet; bs, broad singlet; d,
doublet; dd, doublet of doublets; td, triplet of doublets; h, hextet; m, multiplet; t, triplet; bt,
broad triplet; dt, doublet of triplets; tt, triplet of triplets; q, quartet; qd, quartet of doublets.
Infrared (IR) spectra were recorded on a FT-IR spectrometer (System Spectrum BX; Perkin-
Elmer). ATR IR spectra were recorded on a FT-IR spectrometer (Thermo Nicolet Nexus 470
ESP). Mass spectra were recorded on a mass spectrometer (VG-Analytical AutoSpec Q
Micromass). Melting points were determined on a Leica hot-stage microscope and are
uncorrected. Evaporation of the solvents was performed under reduced pressure. Reagents and
solvents were purchased from Acros Organics, Alfa Aesar, Euriso-Top, Fluka, Merck, Sigma-
Aldrich, and TCI Europe, and were used without further purification, unless otherwise stated.
Flash column chromatography was performed on silica gel 60 for column chromatography
(particle size, 230-400 mesh). Analytical thin-layer chromatography was performed on silica
gel aluminum sheets (0.20 mm; 60 F254; Merck), with visualization using ultraviolet light
and/or visualization reagents. Analytical reversed-phase HPLC method A was performed on
an LC system (Dionex Ultimate 3000 Binary Rapid Separation; Thermo Scientific) equipped
with an autosampler, a binary pump system, a photodiode array detector, a thermostated
23

column compartment, and the Chromeleon Chromatography Data System. Analytical
reversed-phase HPLC method B were performed on an LC modular system (Agilent 1100)
equipped with an autosampler, a quarternary pump system, a photodiode array detector, a
thermostated column compartment, a fraction collector compartment, and the ChemStation
data system. The detector on both HPLC systems was set to 210 nm, 254 nm, and 280 nm.
The column used for methods A and B was a C18 analytical column (150 × 4.6 mm, 5 µm;
Zorbax Eclipse Plus; Agilent). An HPLC guard cartridge system was used (octadecyl; 4.0 ×
3.0 mm ID; Security Guard Cartridge C18 CODS; Phenomenex). The HPLC columns were
thermostated at 25 °C.
Method A: The sample solution (10 μL; 0.1 mg/mL in MeCN) was injected and eluted at a
flow rate of 1 mL/min, using a linear gradient of mobile phase A (70% [v/v] 5 mM aqueous
phosphate buffer, pH 8.00, in MeCN) and mobile phase B (30% [v/v] 5 mM aqueous
phosphate buffer, pH 8.00, in MeCN). The gradient for method A (for mobile phase B) was:
0–7 min, 0%–100%; 7–20 min, 100%.
Method B: The sample solution (10 μL; 0.1 mg/mL in MeCN) was injected and eluted at a
flow rate of 1 mL/min, using a linear gradient of mobile phase A (70% [v/v] 5 mM aqueous
phosphate buffer, pH 8.00, in MeCN) and mobile phase B (30% [v/v] 5 mM aqueous
phosphate buffer, pH 8.00, in MeCN). The gradient for method A (for mobile phase B) was:
0–7 min, 0%–100%; 7–15 min, 100%, 15–20 min, 100%–0%.
General synthetic procedures
General procedure for debenzylation (general procedure 1)
24

The benzyl amine (1.0 equiv.) was dissolved in MeOH at room temperature. The solution was
stirred and agitated with a stream of argon for 30 min. Pd(OH)2 on carbon (20 wt.%) (20%
mass of benzyl amine) was added, followed by cyclohexene (10.0 equiv.). The resulting
suspension was refluxed under an atmosphere of argon for 20 h, then filtered under suction
through a pad of Celite, and evaporated to produce the crude secondary amine, which was
used in the next step without further purification.
General procedure for alkylation of piperidine nitrogen with propargly bromide (general
procedure 2)
Piperidine (1.0 equiv.) was dissolved in acetone at room temperature. The solution was stirred
and Cs2CO3 (1.0 equiv.) was added, followed by propargly bromide (80 wt. % solution in
toluene, 1.0 equiv.). The reaction mixture was protected from the light by wrapping the flask
with aluminum foil. After 24 h, the reaction mixture was evaporated, and EtOAc (60 mL) was
added to the residue. The suspension was transferred into a separating funnel and extracted
with water (60 mL) followed by saturated aqueous NaHCO3 solution (60 mL), dried over
anhydrous Na2SO4, and evaporated. The crude product was purified by flash column
chromatography.
General procedure for removal of Boc-protective group with 2 M HCl solution in Et2O
(general procedure 3)
The amine (1.0 equiv.) was dissolved in MeOH at room temperature. The solution was stirred
and agitated with a stream of argon for 15 min, and then cooled to 0 °C. A solution 2 M HCl
in Et2O (10 equiv.) was added drop-wise, then allowed to warm to room temperature, and
stirred for 24 h. The reaction mixture was evaporated, and Et2O (50 mL) was added to the oily
residue obtained. The flask was placed in an ultrasonic bath for 15 min. During this time, the
25

oily residue transformed into a solid. The flask was removed from the ultrasonic bath and the
precipitate was allowed to settle to the bottom of the flask. The supernatant was removed,
Et2O (50 mL) was added, and the flask was placed back in the ultrasonic bath for 1 min. The
flask was removed from the ultrasonic bath and the precipitate was allowed to settle to the
bottom of the flask. The supernatant was removed, Et2O (50 mL) was added again, and this
procedure was repeated two more times. After the final supernatant was removed, the solid
residue was dried at reduced pressure. The crude product was used in the next step without
further purification.
General procedure for removal of Boc-protective group with TFA (general procedure 4)
The amine (1.0 equiv.) was dissolved in DCM at room temperature. The solution was stirred
and TFA (20.0 equiv.) was added drop-wise. After 24 h, the reaction mixture was evaporated
and Et2O (50 mL) was added to the oily residue. The flask was placed in an ultrasonic bath for
15 min. During this time, the oily residue transformed into a solid. The flask was removed
from the ultrasonic bath, and the precipitate was allowed to settle to the bottom of the flask.
The supernatant was removed, Et2O (50 mL) was added, and the flask was placed back in the
ultrasonic bath for 1 min. The flask was removed from the ultrasonic bath, and the precipitate
was allowed to settle to the bottom of the flask. The supernatant was removed, Et2O (50 mL)
was added again, and this procedure was repeated two more times. After the final supernatant
was removed, the solid residue was dried at reduced pressure. The crude product was used in
the next step without further purification.
General procedure for formation of carboxamide bond (general procedure 4) and
sulfonamide bond (general procedure 5)
26

The salt of the amine (1.0 equiv.) was dissolved in DCM and cooled to 0 °C. The reaction
mixture was stirred and trimethylamine (Et3N; 3.0 equiv.) was added drop-wise. After 15 min,
2-naphthoyl chloride (1.0 equiv.) or naphthalene-2-sulfonyl chloride (1.0 equiv.) was added,
and the reaction mixture was allowed to warm up to room temperature, and then stirred for 24
h. The reaction mixture was transferred into a separating funnel, washed with water, followed
by saturated aqueous NaHCO3 solution, dried over anhydrous Na2SO4, and evaporated. The
crude product was purified by flash column chromatography.
All of the final compounds were appropriately characterized and were of high purity (>95%;
analytical HPLC, Supplementary Information).
Characterization of the final compounds
(±)-N-((1-(prop-2-ynyl)piperidin-3-yl)methyl)-2-naphthamide (4)
Rf = 0.49 (DCM/MeOH, 10:1, v/v). mp 112–115 °C. IR (ATR): 3304, 3270, 2929, 2789,
1625, 1534, 1309, 1242, 1115, 897, 779, 734, 689 cm–1. 1H-NMR (400.130 MHz, CDCl3): δ =
1.12–1.21 (1 H, m), 1.59–1.69 (1 H, m), 1.80 (2 H, d, J = 10.0 Hz), 2.04 (1 H, bs), 2.19–2.37
(3 H, m), 2.79 (1 H, d, J = 10.4 Hz), 2.91 (1 H, d, J = 10.9 Hz), 3.35 (2 H, d, J = 2.1 Hz), 3.47
(2 H, t, J = 6.3 Hz), 6.73 (1 H, bs), 7.51–7.58 (2 H, m), 7.82–7.92 (4 H, m), 8.29 (1 H, s). 13C-
NMR (100.613 MHz, CDCl3): δ = 24.43 (CH2), 28.00 (CH), 36.24 (CH2), 43.78 (CH2), 47.23
(CH2), 52.57 (CH2), 56.42 (CH2), 73.12 (CH), 78.81 (C), 123.56 (CH), 126.47 (CH), 127.21
(CH), 127.35 (CH), 127.52 (CH), 128.15 (CH), 128.71 (CH), 131.78 (C), 132.40 (C), 134.44
(C), 167.61 (CO). HRMS (ESI+): m/z calcd for C20H23N2O 307.1810; found 307.1811. HPLC
purity, 99% at 254 nm (method B, tR = 8.27 min).
27

(±)-N-(2-methoxyethyl)-N-((1-(prop-2-ynyl)piperidin-3-yl)methyl)-2-naphthamide (3)
Rf = 0.43 (DCM/MeOH, 10:1, v/v). IR (ATR): 3235, 2929, 2808, 1622, 1476, 1421, 1288,
1194, 1115, 1071, 900, 823, 757 cm–1. 1H-NMR (400.130 MHz, CDCl3): δ = 0.83 (1 H, dd, J1
= 217.1 Hz, J2 = 7.0 Hz), 1.47–2.93 (9 H, m), 3.18–3.54 (7 H, m), 3.51 (2 H, d, J = 28.2 Hz),
3.69–3.78 (2 H, m), 7.43–7.49 (3 H, m), 7.80–7.84 (4 H, m). 13C-NMR (100.613 MHz,
CDCl3): δ = 24.49 (CH2), 24.68 (CH2), 27.57 (CH), 27.93 (CH), 35.00 (CH2), 35.12 (CH2),
44.75 (CH2), 47.07 (CH2), 47.26 (CH2), 47.56 (CH2), 48.03 (CH2), 48.66 (CH2), 52.18 (CH2),
52.70 (CH2), 53.80 (CH2), 55.78 (CH2), 56.47 (CH2), 58.70 (CH3), 70.21 (CH2), 70.44 (CH2),
73.10 (CH), 78.39 (C), 78.86 (C), 124.18 (CH), 126.12 (CH), 126.23 (CH), 126.39 (CH),
126.65 (CH), 127.59 (CH), 128.08 (CH), 128.12 (CH), 132.48 (C), 133.16 (C), 134.02 (C),
172.08 (CO), 172.33 (CO) [35]. HRMS (ESI+): m/z calcd for C23H29N2O2 365.2229; found
365.2221. HPLC purity, 98% at 254 nm (method B, tR = 9.11 min).
(±)-N-(3-methoxypropyl)-N-((1-(prop-2-ynyl)piperidin-3-yl)methyl)-2-naphthamide (15)
Rf = 0.45 (DCM/MeOH, 10:1, v/v). IR (ATR): 3228, 2929, 2805, 1622, 1476, 1423, 1301,
1
1197, 1114, 900, 864, 756 cm–1. H-NMR (400.130 MHz, CDCl3): δ = 0.84 (1 H, dd, J1 =
214.6 Hz, J2 = 8.3 Hz), 1.44–2.26 (9 H, m), 2.65–2.75 (1 H, m), 2.87 (1 H, dd, J1 = 35.6 Hz,
J2 = 7.8 Hz), 3.09–3.38 (8 H, m), 3.49 (2 H, bs), 3.57–3.66 (1 H, m), 7.42 (1 H, d, J = 7.8 Hz),
13
7.46–7.50 (2 H, m), 7.80–7.86 (4 H, m). C-NMR (100.613 MHz, CDCl3): δ = 24.51 (CH2),
24.73 (CH2), 27.38 (CH), 27.62 (CH), 28.04 (CH2), 28.52 (CH2), 34.91 (CH2), 35.15 (CH2),
42.46 (CH2), 46.36 (CH2), 47.10 (CH2), 47.30 (CH2), 47.44 (CH2), 52.20 (CH2), 52.25 (CH2),
52.77 (CH2), 53.00 (CH2), 55.84 (CH2), 56.50 (CH2), 58.27 (CH3), 58.51 (CH3), 69.15 (CH2),
70.23 (CH2), 73.12 (CH), 78.40 (C), 78.85 (C), 123.87 (CH), 124.19 (CH), 125.86 (CH),
126.24 (CH), 126.42 (CH), 126.66 (CH), 127.62 (CH), 128.11 (CH), 132.53 (C), 133.21 (C),
28

134.13 (C), 171.98 (C), 172.09 (C) [35]. HRMS (ESI+): m/z calcd for C24H31N2O2 379.2386;
found 379.2376. HPLC purity, 97% at 254 nm (method B, tR = 9.31 min).
N-((1-(prop-2-ynyl)piperidin-4-yl)methyl)-2-naphthamide (5)
Rf = 0.41 (DCM/MeOH, 10:1, v/v). IR (ATR): 3298, 3264, 2922, 2837, 1638, 1542, 1428,
1
1300, 1147, 1108, 895, 740, 690 cm–1. H-NMR (400.130 MHz, CDCl3): δ = 1.59 (2 H, bs),
1.81 (1 H, bs), 1.90 (2 H, d, J = 12.7 Hz), 2.34 (1 H, s), 2.45 (2 H, bs), 3.05 (2 H, bs), 3.44 (4
13
H, t, J = 6.2 Hz), 6.52 (1 H, bs), 7.52–7.59 (2 H, m), 7.82–7.94 (4 H, m), 8.30 (1 H, s). C-
NMR (100.613 MHz, CDCl3): δ = 29.77 (CH2), 35.44 (CH), 45.35 (CH2), 46.92 (CH2), 51.86
(CH2), 72.91 (CH), 78.82 (C), 123.52 (CH), 126.45 (CH), 127.15 (CH), 127.32 (CH), 127.49
(CH), 128.12 (CH), 128.64 (CH), 131.76 (C), 132.38 (C), 134.44 (C), 167.71 (C). HRMS
(ESI+): m/z calcd for C20H23N2O 307.1810; found 307.1805. HPLC purity, 98% at 254 nm
(method B, tR = 8.08 min).
N-(2-methoxyethyl)-N-((1-(prop-2-ynyl)piperidin-4-yl)methyl)-2-naphthamide (22)
Rf = 0.44 (DCM/MeOH, 10:1, v/v). IR (ATR): 3228, 2924, 1622, 1476, 1422, 1285, 1115,
1
1070, 976, 899, 823, 757 cm–1. H-NMR (400.130 MHz, CDCl3): δ = 0.94–1.03 (1 H, m),
1.51–1.98 (4 H, m), 2.07–2.40 (3 H, m), 2.78 (1 H, d, J = 7.9 Hz), 3.00 (1 H, bs), 3.22–3.42 (7
H, m), 3.50 (2 H, bs), 3.73 (2 H, m), 7.42–7.55 (3 H, m), 7.83–7.86 (4 H, m). 13C-NMR
(100.613 MHz, CDCl3): δ = 29.31 (CH2), 29.87 (CH2), 33.86 (CH), 44.65 (CH2), 46.76
(CH2), 46.90 (CH2), 49.43 (CH2), 50.69 (CH2), 51.63 (CH2), 51.90 (CH2), 55.45 (CH2), 58.71
(CH3), 70.28 (CH2), 70.53 (CH2), 72.92 (CH), 78.58 (C), 78.70 (C), 124.17 (CH), 126.23
(CH), 126.40 (CH), 126.67 (CH), 127.57 (CH), 128.07 (CH), 128.09 (CH), 132.45 (CH),
133.16 (C), 133.83 (C), 134.10 (C), 172.23 (C), 172.32 (C) [35]. HRMS (ESI+): m/z calcd for
29