First synthesis of racemic trans propargylamino-donepezil, a pleiotrope agent able to both inhibit AChE and MAO-B, with potential interest against Alzheimer’s disease

Article abstract of DOI:10.3390/molecules26010080

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disease towards which pleiotropic approach using Multi-Target Directed Ligands is nowadays recognized as probably convenient. Among the numerous targets which are today validated against AD, acetylcholinesterase (ACh) and Monoamine Oxidase-B (MAO-B) appear as particularly convincing, especially if displayed by a sole agent such as ladostigil, currently in clinical trial in AD. Considering these results, we wanted to take benefit of the structural analogy lying in donepezil (DPZ) and rasagiline, two indane derivatives marketed as AChE and MAO-B inhibitors, respectively, and to propose the synthesis and the preliminary in vitro biological characterization of a structural compromise between these two compounds, we called propargylaminodonepezil (PADPZ). The synthesis of racemic trans PADPZ was achieved and its biological evaluation established its inhibitory activities towards both (h)AChE (IC₅₀ = 0.4 μM) and (h)MAO-B (IC₅₀ = 6.4 μM).

Full text of DOI:10.3390/molecules26010080

molecules
Article
First Synthesis of Racemic Trans Propargylamino-Donepezil, a
Pleiotrope Agent Able to Both Inhibit AChE and MAO-B, with
Potential Interest against Alzheimer’s Disease
Benjamin Guieu 1, Cedric Lecoutey 1, Rémi Legay 1, Audrey Davis 1, Jana Sopkova de Oliveira Santos 1,
Cosimo Damiano Altomare 2, Marco Catto 2 , Christophe Rochais 1,
*
and Patrick Dallemagne 1,
*
1
Normandie Univ, UNICAEN, CERMN, 14000 Caen, France; benjamin.guieu@unicaen.fr (B.G.);
cedric.lecoutey@unicaen.fr (C.L.); remi.legay@unicaen.fr (R.L.); audrey.davis@unicaen.fr (A.D.);
jana.sopkova@unicaen.fr (J.S.d.O.S.)
Department of Pharmacy-Drug Sciences, University of Bari Aldo Moro, 70125 Bari, Italy;
cosimodamiano.altomare@uniba.it (C.D.A.); marco.catto@uniba.it (M.C.)
Correspondence: christophe.rochais@unicaen.fr (C.R.); patrick.dallemagne@unicaen.fr (P.D.);
Tel.: +33-2-31-56-68-13 (C.R. & P.D.)
2
*
Abstract: Alzheimer’s disease (AD) is a multifactorial neurodegenerative disease towards which
pleiotropic approach using Multi-Target Directed Ligands is nowadays recognized as probably
convenient. Among the numerous targets which are today validated against AD, acetylcholinesterase
(ACh) and Monoamine Oxidase-B (MAO-B) appear as particularly convincing, especially if displayed
by a sole agent such as ladostigil, currently in clinical trial in AD. Considering these results, we
wanted to take benefit of the structural analogy lying in donepezil (DPZ) and rasagiline, two indane
derivatives marketed as AChE and MAO-B inhibitors, respectively, and to propose the synthesis and
the preliminary in vitro biological characterization of a structural compromise between these two
compounds, we called propargylaminodonepezil (PADPZ). The synthesis of racemic trans PADPZ
was achieved and its biological evaluation established its inhibitory activities towards both (h)AChE
(IC50 = 0.4 µM) and (h)MAO-B (IC50 = 6.4 µM).
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Guieu, B.; Lecoutey, C.;
Legay, R.; Davis, A.; Sopkova de
Oliveira Santos, J.; Altomare, C.D.;
Catto, M.; Rochais, C.; Dallemagne, P.
First Synthesis of Racemic Trans
Propargylamino-Donepezil, a
Pleiotrope Agent Able to Both
Inhibit AChE and MAO-B, with
Potential Interest against Alzheimer’s
Disease. Molecules 2021, 26, 80.
https://dx.doi.org/10.3390/molecules
26010080
Keywords: Alzheimer’s disease; acetylcholinesterase; monoamine oxidase; donepezil; rasagiline; MTDL
1. Introduction
Among the therapeutic agents getting to clinical trials, more and more examples illus-
trate the concept of Multi-Target Directed Ligands (MTDLs) [1]. Such compounds display
Academic Editors: Diego Muñoz-
Torrero and Beatriz De Pascual-Teresa
Received: 10 December 2020
several activities by interacting with different biological targets that are implied in a given
disease, in order to obtain a synergy of action. MTDLs appear to be particularly interesting
against multifactorial diseases such as Alzheimer’s Disease (AD). As a matter of fact, the
classical pharmacological approach consisting in interacting very selectively with a single
target has shown clinical limitations, failing to restore such complex biological systems.
Thus, the association of drugs has been favored for many years now. Comparatively to this
approach, the advantages of MTDLs, in addition to the synergistic action they theoretically
display, are the absence of drug–drug interactions, and a better patient compliance by
reducing the number of intakes [2].
Accepted: 24 December 2020
Published: 27 December 2020
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional claims
in published maps and institutional
affiliations.
Basically, there are two main types of MTDLs: conjugates, containing two different
active moieties linked by a chemical bond, and molecules that are able to interact indis-
criminately with several therapeutic targets, combining the structural elements needed
for both activities. As an example, our group recently described donecopride, first MTDL
inhibiting acetylcholinesterase (AChE) and activating serotoninergic receptors 5-HT4, and
now in preclinical evaluation [3–5].
Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland. This
article is an open access article distributed
under the terms and conditions of the
Creative Commons Attribution (CC BY)
license (https://creativecommons.org/
licenses/by/4.0/).
Ladostigil (Figure 1) is a novel type of MTDL currently in phase II clinical trials.
It is the first compound able to release an active compound, the hydroxyrasagiline, a
Molecules 2021, 26, 80. https://dx.doi.org/10.3390/molecules26010080
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 80
2 of 17
Monoamine Oxidase B (MAO-B) inhibitor, through the interaction with a first target, the
AChE, which is temporarily inhibited during the process [6].
O
O
O
O
N
O
R
NH
NH
N
(R)-ladostigil
R = H: (R)-rasagiline
donepezil (DPZ)
R = OH: (R)-hydroxyrasagiline
O
O
O
NH
N
propargylaminodonepezil (PADPZ)
Figure 1. Structures of ladostigil, rasagiline, hydroxyrasagiline, donepezil (DPZ) and propargylamin-
odonepezil (PADPZ).
Besides the originality of the mechanism of action of ladostigil, the first results of its
evaluation seem to validate the interest lying in targeting both AChE and MAO-B.
AChE inhibition is the mechanism of action of the main anti-AD drugs, leading to a
symptomatic effect by preventing acetylcholine (ACh) degradation, responsible for AD
symptoms. The efficiency of these drugs, however, decreases with the evolution of the
pathology. Associating the latter with a neuroprotective effect appears theoretically able to
express a disease-modifying effect while maintaining a long-acting symptomatic benefit.
MAO-B is already an identified target for Parkinson’s disease. The enzyme recycles
dopamine in the brain, leading to the formation of hydrogen peroxide, particularly neuro-
toxic. Its inhibition then provides a neuroprotective effect, which is currently evaluated [7].
More recently, MAO-B was also identified as implied in the amyloid cascade, comforting
the therapeutic interest of this target for AD [8–10].
Taking into consideration the interest lying in the association into a sole structure of
activities directed towards both AChE and MAO-B in AD [8], we aimed at conceiving a
new MTDL targeting these enzymes but contrarily to ladostigil in an indifferently manner,
i.e., able to bind to each of the active site of these targets. To achieve such a goal, the
latter have to share sufficient common features liable to be recognized by a same agent. In
support of such allegation, we consider the fact that both donepezil (DPZ), a marketed
AChE inhibitor (AChEI), and rasagiline, a marketed, MAO-B inhibitor (MAO-BI), possess
an indane core, and we designed propargylaminodonepezil (PADPZ) as a perfect structural
compromise between these two drugs. This paper describes our efforts to achieve the first
synthesis of PADPZ and its preliminary biological evaluation towards these two enzymes.
2. Results
2.1. Chemistry
The synthetic strategy we followed for the access to PADPZ (Scheme 1), involved
an aldolisation reaction between a properly N-protected dimethoxyindanone and para-
N benzylpiperidinecarboxal-dehyde. The methylene derivatives would have then to
successively undergo, in various order, a reduction of its alkene moiety, a deprotection of
its amino group and a substitution of the latter by a propargyl group.

Molecules 2021, 26, 80
3 of 17
Scheme 1. Synthetic strategy for the access to PADPZ.
The access to aminodimethoxyindanones (
3
,
5
–7
) involved, in a first step, a Rodionov-
Johnson synthesis of aminodimethoxyphenylpropionic acid (
hyde ( ), followed by a one-pot protection/cyclization reaction, using trifluoroacetic acid
and anhydride, to give the trifluoracetylaminoindanone ( ) in 65% yield, according to a
previous reported sequence (Scheme 2) [ 10]. Hydrolyzing by aqueous hydrochloric
acid yielded the hydrochloric acid salt ( ) in 76% yield. The latter was then converted into
the N-propargyl ( ), -acetyl ( ) and tertbutyloxycarbonyl ( ) derivatives with 38%, 76% and
83% yields, respectively.
At the same time, para N-benzylpiperidine carboxaldehyde (11) was obtained in 45%
overall yield, through the N-benzylation of ethyl 4-piperidinecarboxylate ( ), followed by
) into the alcohol (10) and a final Swern oxidation of the latter
2), starting from veratralde-
1
3
9
,
3
4
5
6
7
8
a reduction of the ester (
(Scheme 3) [11–13].
9
In a first attempt, aldolisation of
3 was undertaken in acidic medium but failed to
give the methylene derivative (12) (Scheme 4). The latter was obtained in alkaline medium,
quasi exclusively under its E form. The latter was attributed through a selective 1D NOE
experiment during which a response in the signal of the proton, located in the para position
of the nitrogen atom of the piperidine ring, was observed upon excitation of H3, accounting
for a close position between the two protons. Further, a significant deshielding of the signal
of the methylene proton was also observed for the E form (6.70 ppm) of 12, due to the
cone-shaped shielding zone of the carbonyl group, versus those of the Z form (6.25 ppm)
observed as a trace.

Molecules 2021, 26, 80
4 of 17
O
O
O
1
a
O
O
O
O
O
b
OH
N
H
F
F
3
NH2
O
O
2
F
O
O
c
O
O
d
f
O
,HCl
O
N
H
NH2
5
4
e
O
O
O
O
O
O
N
O
H
N
H
7
6
O
O
Scheme 2. Synthetic pathways for access to compounds
2
–
7
.
Reagents and conditions: (
) TFA, TFA2O, Rflx, 2 H, 65%; ( ) aq HCl
) TEA, Ac2O, THF, rt, 4 H,
a)
CH2(CO2H)2, CH3CO2NH4, EtOH, Rflx, overnight, 75%; (
b
◦
c
6N, rflx, 30 min, 78%; (
d) BrCH2CCH, K2CO3, CH3CN, 80 C, 5 H, 38%; (
e
76%; (f) Boc2O, TEA, CH2Cl2, rt, 24 H, 83%.
a
O
O
O
O
N
N
H
8
9
b
c
O
HO
N
N
11
10
Scheme 3. Synthesis pathways for access to compounds
9–
11.
Reagents and conditions: (
a)
C6H5CH2Br, TEA, CH2Cl2, rt, overnight, 79%; (
TEA, CH2Cl2, −48 C, 15 min, 60%.
b
) LiAlH4, THF, rflx, 3 H, 89%; (c) (ClCO)2, DMSO,
◦

Molecules 2021, 26, 80
5 of 17
Scheme 4. Synthesis pathway for access to (E)-12. Conditions and reagents: (a) 11, NaOH, EtOH, rt, 5 H, (19%).
All attempts, however, to hydrolyze the trifluoroacetylamino group of 12 in alkaline
medium failed. Such a chemical behavior was already observed with aminoindanones
which, in these conditions, are suitable for an internalization of their double bond likely to
lead to a degradation [14]. This can also explain the low yield observed in the preparation
of 12 (19%).
All attempts aiming at synthesizing the propargyl derivative (13) starting from
5 and
according to a similar manner, failed, probably for the same reasons.
In a similar manner, the N-acetyl and N-Boc methylene derivatives (14) and (15) were
obtained starting from and , exclusively under their E form and in 51% and 46% yield
respectively (Scheme 5). This geometry was attributed by analogy with the structure of
6
7
12. Reduction of the latter succeeded this time, using NaBH4 for 14 and H2 Pd/C for 15
respectively. Only one diastereoisomeric form of the N-acetylcompound 16 was recovered,
explaining the moderate yield observed (35%). Its trans geometry was established by
its X-ray diffractometry study (Figure 2). The two diastereoisomeric forms of the N-boc
derivative 17 were obtained, with a relative proportion of 3 trans for 1 cis and a global yield
of 65%. Attribution of the two forms of 17 was achieved through 1D NOE experiment
which showed, upon excitation of the NH proton, a response observed on the signal of H2
for the trans form and on the signal of the CH2 protons for the cis form, respectively. It was
not possible to separate them. The coupling constant values between H2 and H3 signals
appeared higher in the cis form (6.5 Hz) versus the trans one (3.5 Hz) and confirmed the
major formation of trans-17 by comparison with the 1H-NMR spectrum of trans-16.
All the attempts aiming at deprotecting the N-acetyl group of 16 failed. Compound
18, however, was obtained in 78% yield starting from the N-Boc derivative (17), which was
easily N-deprotected using TFA. Compound 18 was recovered as a mixture of its trans/cis
forms in a 90/10 ratio, from which the trans form was isolated.
,
Finally, the expected PADPZ (19) was successfully obtained under its trans form,
through the N-substitution of trans-18 by a propargyl group.

Molecules 2021, 26, 80
6 of 17
Scheme 5. Synthesis pathways for access to 14
–
19. Conditions and reagents: (
a
)
11, K2CO3, MeOH,
◦
50 C, 2 h 30 min, 51%; (
b)
11, NaOH, EtOH, rt, 46%; (
c) NaBH4, MeOH, rt, 5 min, 35%; (
d) 10%
◦
Pd/C, H2, EtOH, 40 C, 48 H, 65%; (
CH3CN, rt, 24 H, 6%.
e) TFA, CH2Cl2, rt, overnight, 78%; ( ) BrCH2CCH, K2CO3,
f
Figure 2. X-ray structure of trans-16.

Molecules 2021, 26, 80
7 of 17
2.2. In Silico Results
2.2.1. Molecular Modeling Study
The aim of the present molecular modeling work was to check whether PADPZ (19
)
can bind to the active sites of the two targeted proteins and which of the two enantiomers
of its trans form will be the more prone to display the more effective interaction with both
AChE and MAO-B. The initial 3D model of compound 19 was built using the structurally
closed X-ray structure of compound 16, its acetylamino analog. Two enantiomers were
present in the crystal of 19, and therefore, the two enantiomers of trans-19 (R,R and S,S)
were built. The models, further, were protonated on their piperidine nitrogen.
Firstly, the two enantiomers of 19 were docked separately into the (h)AChE active
site and in a comparative manner with donepezil (Figure 3A). In a similar manner as
in our previous studies on (h)AChE [4], a water molecule interacting with protonated
piperidine ring of donepezil was conserved (residue number 931) during docking. The
results suggested different binding affinities for the two enantiomers. The enantiomer R,R
(Figure 3B) took an orientation within the active site of AChE closed to donepezil one,
accounting for a possible inhibitory activity towards the enzyme similar to those of the
reference compound. This enantiomer, indeed, reproduced crucial donepezil interactions:
(i) the charged nitrogen of the piperidine ring was oriented in a position suitable for an
interaction with the water molecule in the proximity of Tyr337 and Tyr341, (ii) the carbonyl
group formed a hydrogen bond with NH of the Phe295 backbone and (iii) both benzene
rings are placed in parallel to the Trp279 and Trp84 indole rings to favor a π-stacking
interaction. An additional interaction is further observed between the piperidine NH of 19
and the OH of Tyr124.
(A)
Figure 3. Donepezil position from the X-ray structure (
(B)
(C)
) and S,S (C) of trans-19
A
) compared to the enantiomer R,R (
B
positioned in (h)AChE binding sites using the docking studies. The compounds and the selected side chains of the binding
site residues are in stick and the protein in ribbon representation. This figure was made with PYMOL (DeLano Scientific,
2002, San Carlo, CA, USA).
On the other hand, the position of the enantiomer S,S of 19 within the AChE groove
did not appear as favorable for an inhibitory interaction (Figure 3C). It lost some crucial in-
teractions through the protonated piperidine nitrogen of 19, the π-stacking with Trp84 and
the hydrogen bond between the carbonyl group of 19 and the NH of Phe295. Contrariwise
this carbonyl group established a hydrogen bond with Tyr124.
To check the ability of the enantiomers of trans-19 to bind to the (h)MAO-B binding
site, a covalent docking was carried into the enzyme. The docked enantiomers were
previously modified, engaging their propargylamino group into a covalent bond with the
FAD co-factor of MAO-B and modifying the bond order in a same manner as observed in
rasagiline-FAD complex (Figure 4A). The covalent docking was then carried out and both
enantiomers were placed into the MAO-B cavity without any steric clash (Figure 4B,C). The

Molecules 2021, 26, 80
8 of 17
interactions established by the two enantiomers with MAO-B are very similar: a hydrogen
bond between their carbonyl group and the Gln206 side chain and another one between
their methoxy groups and a tyrosine residue, Tyr326 for R,R and Tyr188 for S,S. From
these observations, we hypothesized that the two enantiomers should bind to MAO-B with
similar affinities and inhibitory activities.
(A)
Figure 4. Rasagiline position from the X-ray structure (
(B)
) compared to the enantiomer S,S (
(C)
) and R,R (C) of trans-19
A
B
positioned in (h) MAO-B binding sites using the docking studies. The compounds and the selected side chains of the binding
site residues are in stick and the protein in ribbon representation. This figure was made with PYMOL (DeLano Scientific,
2002, San Carlo, CA, USA).
2.2.2. In Silico ADME Parameters
In silico ADME parameters for PADPZ have been calculated using SwissADME
(www.SwissADME.ch). No alert was identified (Table 1).
Table 1. In silico ADME parameters for PADPZ.
Water Solubility
Log S (ESOL)
Solubility
Class
−4.50
−2
−2
1.38 × 10 mg/mL; 3.20 × 10 mol/L
Moderately soluble
Pharmacokinetics
GI absorption
BBB permeant
P-gp substrate
High
Yes
Yes
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Lipinski
No
No
No
Yes
Yes
Yes; 0 violation
2.3. In Vitro Results
Cholinesterases and Monoamine Oxidases Inhibition
The inhibitory activity of trans-PADPZ (19) as well as its acetylamino analogue (16
)
towards (h)AChE and (eq)BuChE was evaluated according to the Ellman test [15]. DPZ
was used as a reference. Results are depicted in Table 2.

Molecules 2021, 26, 80
9 of 17
Table 2. (h)AChE and (eq)BuChE inhibitory activity.
(h)AChE
(eq)BuChE
IC50 (µM)
% Inhibition
IC50 (µM)
(n = 3)
% Inhibition
at 10
Compound
−6
−6
at 10
M
M
(n = 3)
7.4 ± 0.1 1
DPZ
16
98%
4%
0.014 ± 0.0016
-
ND
39%
ND
Trans-PADPZ
(19)
71%
0.442 ± 0.03
74%
4.226 ± 0.375
1
IC50 values are expressed as mean
from Reference [16].
±
standard error of the mean (SEM) of three experiments. Data
The inhibitory activity of trans-PADPZ (19) towards human recombinant MAO-
A and -B was determined as already described [17], measuring the fluorescence of 4-
hydroxyquinoline produced by MAOs in the oxidative deamination of substrate kynu-
ramine (50 µM). Rasagiline and pargiline were used as references, as well as the dimethoxyketo
analogue of rasagiline (5) we synthesized. Results are depicted in Table 3.
Table 3. (h)MAO-B and (h)MAO-A inhibitory activity.
(h)MAO-B
(h)MAO-A
%
% Inhibition
IC50 (µM)
(n = 3)
IC50 (µM)
(n = 3)
Compound
Inhibition
−5
at 10
M
−5
at 10
M
0.014 1
0.7 1
10.9 ± 0.6
ND
Rasagiline
Pargiline
-
-
-
-
2.69 ± 0.48
13.4 ± 3.4
6.43 ± 0.62
5
45%
52%
<10%
13%
Trans-PADPZ (19)
ND
1
IC50 values are expressed as mean
from Reference [18].
±
standard error of the mean (SEM) of three experiments. Data
3. Discussion
The modulation of DPZ and indanone moiety to develop novel AChEI has been largely
explored in recent years [19]. Indeed, the binding site of the enzyme could accommodate
to an important number of chemical structures, and several MTDL have been obtained
starting from an indanone moiety. Indeed, several modulations have generated MTDL
with Aβ-aggregation [20] or BACE1 [21] inhibition. During these SAR, the presence of α-β
insaturated ketone has generally led to a decrease in potency compared to DPZ. If most of
the modulation has been engaged by changing the nature of the piperidine or replacing
the dimethoxy substituent, few examples have been described in introducing substituent
on the indanone moiety. With this aim, we have prepared and focus our attention on
the preparation of the reduced form of PADPZ. Trans-PADPZ (19) showed a noticeable
inhibitory activity towards (h)AChE with an IC50 = 440 nM and a relative selectivity
towards (h)BuChE which was inhibited at a concentration 10-fold greater (IC50 = 4.2 µM).
This AChE inhibitory activity, however, appeared weaker than those of DPZ (IC50 = 14 nM),
but higher than those of the acetamide trans-16 (% inhibition at 10−6 M = 4%) which almost
totally lost its effect.
Tested against (h)MAO-B, trans-PADPZ exhibited again a sound activity with an
IC50 = 6.4 µM, in a same order of magnitude than those of pargiline (IC50 = 2.7 µM), but
weaker with respect to rasagiline (IC50 = 14 nM) used as references. Interestingly, trans-
PADPZ (19) appeared selective towards MAO-B with a weak inhibition of MAO-A (%
inhibition at 10−6 M = 13%). The dimethoxyketo analogue of rasagiline (
5
) displayed also a
modest and selective inhibitory activity towards MAO-B (IC50 = 13.4
µM). According to the
docking study, this inhibition is not influenced by the stereochemistry of the substitution at
the difference of the corresponding rasagiline or ladostigil.

Molecules 2021, 26, 80
10 of 17
Trans-PADPZ (19) appears therefore as a dual AChE/MAO-B with a good selectivity
towards BuChE and MAO-A. These results are consistent with the predictive data issued
from the in silico study, which further gave us an incentive to separate the enantiomers
of Trans-PADPZ, since one of them (R,R) appeared, according to this study, more able to
inhibit AChE than the other one (S,S).
4. Materials and Methods
4.1. Chemistry
4.1.1. General Methods
All chemical reagents and solvents were purchased from commercial sources and used
without further purification. Melting points were determined on a STUART SMP50 melting
point apparatus (Cole-Parmer, Vernon Hills, IL, USA) 1H, 13C and 19F-NMR spectra were
recorded on a BRUKER AVANCE III 400 MHz (Bruker, Billerica, MA, USA) with chemical
shifts expressed in parts per million downfield from TMS as an internal standard and
coupling in Hertz. IR spectra were recorded on a Perkin-Elmer BX FT-IR apparatus (Perkin
Elmer, Wellesley, MA, USA) using KBr pellets. High resolution mass spectra (HRMS) were
obtained by electrospray on a BrukermaXis (Bruker, Billerica, MA, USA). The purities of all
tested compounds were analyzed by LC MS, with the purity all being higher than 95%.
−
Analyses were performed using a Waters Alliance 2695 (Waters corporation, Milford, MA,
USA) as separating module (column XBridge C18 2.5 M/4.6 X 50 mM (Waters corporation,
Milford, MA, USA) using the following gradients: A (95%)/B (5%) to A (5%)/B (95%)
in 4.00 min. This ratio was hold during 1.50 min before return to initial conditions in
0.50 min. Initial conditions were then maintained for 2.00 min (A = H2O, B = CH3CN; each
containing HCOOH: 0.1%). MS were obtained on a SQ detector by positive ESI.
4.1.2. Synthesis of Compounds (2, 3, 5–11)
3-Amino-3-(3,4-dimethoxyphenyl)propanoic acid (2) [12,13]. To a solution of 3,4-dimethoxy
benzaldehyde (1.0 eq., 6 g, 36.1 mmol) in EtOH (200 mL), were added malonic acid (2.0 eq.,
7.52 g, 72.2 mmol) and ammonium acetate (2.0 eq., 5.57 g, 72.2 mmol). The mixture was
stirred to reflux overnight. After cooling to room temperature, the reaction mixture was
filtered in vacuo and washed by hot EtOH to give
235 ◦C; 1H NMR (400 MHz, D2O)
7.16–6.85 (m, 3H), 4.57 (dd, J = 8.2, 6.5 Hz, 1H), 3.84 (s,
3H), 3.82 (s, 3H), 2.87 (dd, J = 16.2, 8.2 Hz, 1H), 2.76 (dd, J = 16.2, 6.5 Hz, 1H); 13C-NMR
2 as a white solid (6.11g, 75%). M.p.:
δ
(101 MHz, D2O)
δ 177.2, 148.5, 148.2, 128.9, 120.0, 111.9, 110.4, 55.6, 55.6, 52.5, 40.3; IR (neat,
cm−1): 3411 (N-H), 2933, 2636 (C-H), 2124 (C=C), 1571, 1549 (C=O), 1398, 1148; LC-MS m/z
[M − H]+ 224.35.
N-(5,6-Dimethoxy-3-oxo-indan-1-yl)-2,2,2-trifluoroacetamide (3) [12,13]. 3-Amino-3-(3,4-
dimethoxy phenyl)propanoic acid (7.60 g, 33.74 mmol) was stirred in trifluoroacetic acid
(3 mL
·
mmol−1), and trifluoroacetic anhydride was added (3 mL mmol−1). The mixture
·
was refluxed for 2 h. After cooling at room temperature, the reaction was stopped by
adding slowly 10 mL of water. The mixture was evaporated in vacuo. The crude was
dissolved in saturated aqueous solution of NaHCO3 (400 mL) and EtOAc (400 mL); then,
the organic layer was dried over MgSO4 and concentrated in vacuo. The compound
obtained as a yellow powder (6.61 g, 65%). M.p: 219 ◦C; 1H NMR (400 MHz, CD3OD)
7.19 (s, 1H), 7.08 (s, 1H), 5.59 (dd, J = 7.5, 3.0 Hz, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 3.15 (dd,
J = 18.8, 7.5 Hz, 1H), 2.57 (dd, J = 18.8, 3.0 Hz, 1H); 13C NMR (100 MHz, CD3OD)
202.2,
156.5, 151.2, 146.6, 146.4, 129.9, 121.1, 106.5, 103.3, 55.4, 55.1, 47.9, 44.2; 19F-NMR (376 MHz,
3 was
δ
δ
CD3OD) δ ) ν max 3303 (N-H), 3095, 2835, 1694 (C=O), 1594, 1269,
-77.23; IR (neat, cm−1
1048, 857; LC-MS m/z [M + H]+ 304.35.
5,6-Dimethoxy-1-oxoindan-3-ylammonium chloride (4) [12,13]. A solution of 3 (6 g, 0.02 mol)
in aqueous HCl 6N (50 mL) was refluxed for 30 min, cooled and filtered. The filtrate was
evaporated to dryness under reduced pressure. The solid residue was recrystallized form
◦
4
(3.8 g, 78%) as colorless crystal. M.p. 208 C; 1H-NMR (400 MHz, DMSO-d6)
Et2O to give
9.00 (br s, 3H), 7.91 (s, 1H), 7.13 (s, 1H), 4.83 (m, 1H), 3.90 (s, 3H), 3.84 (s, 3H), 3.04
δ

Molecules 2021, 26, 80
11 of 17
(dd, J = 18.5, 6.8 Hz, 1H), 2.60 (dd, J = 18.5, 3.4 Hz, 1H); IR (neat, cm−1
)
ν
max 3100–2700
(+NH3), 1680 (C=O), 1325.
5,6-Dimethoxy-3-(prop-2-ynylamino)indan-1-one (
5
). Propargyl bromide (326
µL, 4.3 mmol)
and K2CO3 (560 mg, 4 mmol) were added to a solution of
4
(500 mg, 2 mmol) in acetoni-
trile. The reaction mixture was stirred at 80 ◦C for 5 h. The solvent was removed under
reduced pressure, and the residue was purified by column chromatography on silica using
CH2Cl2/MeOH (100/2) as eluent to give
1H-NMR (400 MHz, CDCl3)
7.15 (s, 1H), 7.08 (s, 1H), 4.53 (dd, J = 6.5, 2.7 Hz, 1H), 3.98 (s,
3H), 3.90 (s, 3H), 3.63–3.45 (m, 2H), 2.97 (dd, J = 18.5, 6.5 Hz, 1H), 2.55 (dd, J = 18.5, 2.8 Hz,
1H), 2.30 (t, J = 2.4 Hz, 1H); 13C NMR (101 MHz, CDCl3)
202.8, 155.6, 150.5, 150.4, 129.9,
106.8, 103.8, 81.9, 72.2, 56.4, 56.2, 55.0, 44.7, 36.7; IR (neat, cm−1
max 3426 (N-H), 2923,
1694 (C=O), 1594, 1500, 1311, 1268; LC-MS m/z [M + H]+ 191.02.
N-(5,6-Dimethoxy-3-oxo-indan-1-yl)acetamide ( ). An aqueous solution of
5
as a brown solid (190 mg, 38%). M.p. 112 ◦C;
δ
δ
)
ν
6
3 (360 mg,
1.18 mmol,) in HCl 6N was refluxed for 24 h. After evaporation, a suspension of the
◦
obtained amino salt in THF was cooled to 0 C, and triethylamine (493
µL, 3.54 mmol)
and acetic anhydride (251 µL, 2.66 mmol) were added. The mixture was stirred at room
temperature for 4 h, then filtered and concentrated under reduced pressure. Purification
1
on silica gel using CH2Cl2/EtOAc (80/20) as eluent provided
NMR (400 MHz, CDCl3)
6
as a white solid (76%). H
δ
7.14 (s, 1H), 7.01 (d, J = 0.7 Hz, 1H), 5.90–5.74 (m, 1H), 5.64
(dddd, J = 8.2, 7.3, 3.0, 0.8 Hz, 1H), 3.98 (s, 3H), 3.91 (s, 3H), 3.20 (dd, J = 19.0, 7.4 Hz, 1H),
2.42 (dd, J = 19.0, 3.0 Hz, 1H), 2.07 (s, 3H).
Tert-Butyl N-(5,6-dimethoxy-3-oxo-indan-1-yl)carbamate (7). Boc2O (671 mg, 3.075 mmol)
and TEA (415 mg, 4.1 mmol) were added to a solution of (500 mg, 2.05 mmol) in CH2Cl2.
4
The reaction mixture was stirred at room temperature for 24 h and then diluted with
water. Extraction with EtOAc, washing of the organic layer with brine and evaporation
to dryness gave a residue. Purification on the latter by column chromatography on silica
1
using CH2Cl2/EtOAC (80/20) as eluent gave
(400 MHz, CDCl3)
3.98 (s, 3H), 3.91 (s, 3H), 3.18 (dd, J = 18.9, 7.3 Hz, 1H), 2.45 (d, J = 18.9 Hz, 1H), 1.49 (s, 9H);
13C-NMR (101 MHz, CDCl3)
202.0, 156.0, 155.8, 150.9, 149.4, 129.8, 106.8, 103.8, 80.3, 56.5,
56.4, 48.7, 45.5, 28.5 (3C);. HRMS/ESI: m/z calcd. [M+Na]+ 330.1317, found 330.1320.
Ethyl 1- benzylpiperidine-4-carboxylate ( ) [14 16]. To a solution of ethyl piperidine-
7
(530 mg, 83%) as a colorless solid. H NMR
δ
7.15 (s, 1H), 7.05 (s, 1H), 5.44–5.20 (m, 1H), 4.84 (d, J = 8.8 Hz, 1H),
δ
9
–
4-carboxylate (3 g, 19.1 mmol, 1.0 eq.) in CH2Cl2 (300 mL,) was added benzyl ◦bromide
(2.72 mL, 22.92 mmol, 1.2 eq.) and triethylamine (3.09 mL, 22.92 mmol, 1.2 eq.), at 0 C. After
1 night at room temperature; the mixture was washed with saturated aqueous solution of
NaHCO3. The organic layer was washed with brine and dried over MgSO4. After filtration
and concentration in vacuo, purification on silica gel using cyclohexane/EtOAc (35:65) as
1
an eluant provided an orange oil (3.75g, 79%). H NMR (400 MHz, CDCl3)
δ
7.37–7.30 (m,
4H), 7.30–7.22 (m, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.51 (s, 2H), 2.88 (dt, J = 12.1, 3.9 Hz, 2H),
2.30 (tt, J = 11.2, 4.1 Hz, 1H), 2.04 (td, J = 11.4, 2.7 Hz, 2H), 1.97–1.86 (m, 2H), 1.86–1.70 (m,
2H), 1.27 (t, J = 7.1 Hz, 3H); 13C-NMR (101 MHz, CDCl3)
δ
175.3, 138.4, 129.1 (2C), 128.2
(2C), 127.0, 63.3, 60.3, 53.0 (2C), 41.3, 28.3 (2C), 14.2; LC-MS m/z [M + H]+ 248.60.
(1-Benzyl-4-piperidyl)methanol (10) [14 16]. To a suspension of LiAlH4 (2.57g, 67.7 mmol,
4eq.) in 40 mL of THF was added dropwise a solution of (4.2 g, 16.9 mmol, 1.0 eq.) in
–
9
40 mL of THF. The mixture was heated to reflux for 3h and was then slowly quenched with
80 mL of ethyl acetate and 25 mL of NaOH 1N solution followed by 30 mL of water to
afford a granular inorganic precipitate. After the night, the solution was filtered through
celite; the solid was washed with EtOAc, and then, the filtrate was concentrated under
reduced pressure to give reduced compound as a primrose yellow oil (3.1g, 89% yield).
1H NMR (CDCl3, 399.8 MHz)
δ 7.35–7.28 (m, 4H), 7.28–7.22 (m, 1H), 3.50 (s, 2H), 3.48
(d, J = 6.5 Hz, 2H), 2.97–2.86 (m, 2H), 2.00–1.92 (m, 3H, H9), 1.75–1.64 (m, 2H), 1.57–1.42
(m, 1H), 1.35–1.21 (m, 2H); 13C-NMR (CDCl3, 100.5 MHz)
127.1, 67.6, 63.5, 53.5 (2C), 38.5, 28.7 (2C).
δ 138.0, 129.5 (2C), 128.2 (2C),

Molecules 2021, 26, 80
12 of 17
1-Benzylpiperidine-4-carbaldehyde (11) [14
30.2 mmol, 2.0 eq.) in CH2Cl2 was cooled down to
2.2 eq.) was added. After 10 min, a solution of the alcohol 9 (3.1 g, 15.1 mmol, 1.0 eq.) in
–
16]. A solution of oxalyl chloride (2.6 mL,
◦
−
48 C, then DMSO (2.36 mL, 33.2 mmol,
CH2Cl2 was added dropwise. After 15 min, Et3N (8.66 mL, 64.2 mmol, 4.2 eq.) was added
and the mixture was brought back to room temperature. Water was then added, and the
organic phase was washed with brine, then dried on MgSO4, filtered and evaporated under
reduced pressure. Purification on alumina gel (CH2Cl2) provided the expected aldehyde
1
10 as a brown oil (60%). H-NMR (400 MHz, CDCl3)
δ
9.60 (d, J = 1.3 Hz, 1H), 7.36–7.14
(m, 5H), 3.45 (s, 2H), 2.77 (dt, J = 11.8, 4.0 Hz, 2H), 2.19 (ttd, J = 10.7, 4.2, 1.3 Hz, 1H), 2.06
(td, J = 11.1, 2.9 Hz, 2H), 1.90–1.81 (m, 2H), 1.64 (dtd, J = 14.2, 10.7, 3.8 Hz, 2H); 13C-NMR
(101 MHz, CDCl3)
δ 204.1, 138.3, 129.1 (2C), 128.2 (2C), 127.1, 63.3, 52.5 (2C), 48.0, 25.5 (2C).
4.1.3. General Procedure for the Aldolization Reaction and Preparation of Compounds
(12, 15)
To a solution of the ketone (1 eq.) in EtOH (500
µL/mmol) were added an aqueous
solution of NaOH 5N (250 L/mmol) and a solution of the aldehyde 10 (1.3 eq.) in EtOH.
µ
The mixture was stirred at room temperature for 5 h and then concentrated under reduced
pressure. Water was added, and the reaction was extracted with EtOAc. The organic layer
was washed with brine and dried over MgSO4 and then filtered and concentrated under
reduced pressure.
N-[(2E)-2-[(1-Benzyl-4-piperidyl)methylene]-5,6-dimethoxy-3-oxo-indan-1-yl]-2,2,2-
trifluoroacetamide (12). The reaction was performed according to the general procedure. The
residue was purified by column chromatography on silica using CH2Cl2/MeOH (90/10)
as eluent, then on alumina using CH2Cl2/EtOAc (80/20)◦ as eluent to provide 12 as an
orange solid (19%) which was separated from 15. M.p. 199 C; 1H-NMR (400 MHz, CDCl3)
δ
7.48–7.27 (m, 4H), 7.14 (s, 1H), 7.00–6.97 (m, 1H), 6.97 (s, 1H), 6.72 (dd, J = 10.8, 1.7 Hz,
1H), 6.17 (d, J = 9.0 Hz, 1H), 3.98 (s, 3H), 3.91 (s, 3H), 3.61 (s, 2H), 2.04 (t, J = 10.4 Hz,
2H), 2.41–2.38 (m, 1H), 2.18–1.55 (m, 6H); 13C-NMR (101 MHz, CDCl3)
190.1, 157.2 (q,
δ
J = 37.7 Hz, CF3), 156.4, 151.2, 144.8, 143.4, 137.8, 135.4, 131.2, 129.3 (2C), 128.4 (2C), 127.3,
115.9 (q, J = 287.7 Hz, CF3), 106.6, 104.3, 63.3, 56.7, 56.1, 52.7, 52.6, 48.5, 37.1, 31.1, 31.0; IR
−1
(neat, cm
) ν max 3435 (N-H), 2922, 2850, 1716 (C=O), 1689, 1648, 1549, 1211, 1182; LC-MS
m/z [M + H]+ 488.81.
Tert-Butyl N-[(2E)-2-[(1-benzyl-4-piperidyl)methylene]-5,6-dimethoxy-3-oxo-indan-1-
yl]carbamate (15). The reaction was performed according to the general procedure. Yellow
1
solid (46%). H-NMR (400 MHz, CDCl3)
δ
7.36–7.27 (m, 5H), 7.23 (s, 1H), 7.06 (s, 1H), 6.70
(dd, J = 10.5, 1.8 Hz, 1H), 5.83 (d, J = 9.9 Hz, 1H), 4.72 (d, J = 9.8 Hz, 1H, NH), 3.98 (s,
3H), 3.93 (s, 3H), 3.55–3.46 (m, 2H), 3.01–2.81 (m, 2H), 2.61–2.46 (m, 1H), 2.17–1.91 (m,
2H), 1.78–1.52 (m, 4H), 1.50 (s, 9H); 13C-NMR (100 MHz, CDCl3)
δ
190.9, 156.1, 155.7,
150.9, 146.1, 143.8, 138.4, 137.2, 131.0, 129.3 (2C), 128.3, 127.1 (2C), 106.9, 104.3, 80.3, 63.6,
56.6, 56.4, 53.0 (2C), 49.7, 36.4, 31.5, 31.4, 28.6 (3C). IR (neat, cm−1
) ν max 3380, 2933, 1701
(C=O), 1500, 1296; LC-MS m/z [M + H]+ 492.83; HRMS/ESI: m/z calcd. [M + H]+ 493.2702,
found 493.2703.
4.1.4. Synthesis of Compounds (14, 16–19)
N-[(2E)-2-[(1-benzyl-4-piperidyl)methylene]-5,6-dimethoxy-3-oxo-indan-1-yl]acetamide (14).
◦
A solution of
6
(200 mg, 0.8 mmol) in MeOH (1 mL) was heated at 50 C. At this temperature,
K2CO3 (774 mg, 5.6 mmol) was added dropwise to 500
µL of water over 30 min. After
20 min, a solution of 11 (196 mg, 0.96 mmol) in MeOH was added. After stirring for
2 h 30 min, the mixture was concentrated under vacuo; then, water was added, and an
extraction was performed using EtOAc. The organic phase was washed with a brine
and dried on MgSO4 and then filtered and concentrated under reduced pressure. After a
column on neutral alumina using CH2Cl2/EtOAc (80:20) as eluent, another purification on
silica gel using EtOAc/MeOH (90/10) as eluent was performed to provide the expected
1
compound as a white solid (176 mg, 51%). H-NMR (400 MHz, CDCl3)
δ 7.40–7.30 (m,
4H), 7.29–7.22 (m, 1H), 7.00 (s, 1H), 6.95 (s, 1H), 6.60 (dd, J = 10.6, 1.8 Hz, 1H), 6.39 (dd,

Molecules 2021, 26, 80
13 of 17
J = 9.4, 3.0 Hz, 1H, NH), 6.14 (dd, J = 9.2, 1.7 Hz, 1H), 3.96 (s, 3H), 3.83 (s, 3H), 3.51 (d, J
= 4.0 Hz, 2H), 2.99–2.90 (m, 2H), 2.46 (dddd, J = 17.9, 10.9, 6.9, 4.1 Hz, 1H), 2.13–1.97 (m,
5H), 1.73–1.53 (m, 4H); 13C-NMR (101 MHz, CDCl3)
δ 191.1, 169.7, 156.1, 150.6, 145.8, 143.7,
138.3, 137.0, 130.8, 129.2 (2C), 128.2 (2C), 127.0, 106.8, 103.8, 63.5, 56.6, 56.0, 53.0, 52.8, 48.0,
36.7, 31.5, 31.2, 23.4.
Trans N-[2-[(1-Benzyl-4-piperidyl)methyl]-5,6-dimethoxy-1-oxo-indan-4-yl]acetamide (16).
To a solution of 14 (230 mg, 0.53 mmol) in MeOH was added in small portions NaBH4
(60 mg, 1.59 mmol). The reaction mixture was concentrated under reduced pressure;
then, water was added, and it was extracted with EtOAc. The organic phase was washed
with a brine and dried over MgSO4 and then filtered and concentrated under reduced
pressure. Purification on neutral alumina gel using dichloromethane MeOH (95/5) as
1
eluent provided the expected product as a colorless solid (80 mg, 35%). H-NMR (400 MHz,
CDCl3) δ 7.34–7.28 (m, 4H), 7.25–7.21 (m, 1H), 7.09 (s, 1H), 6.95 (s, 1H), 5.81 (d, J = 9.1 Hz,
1H, NH), 5.36 (dd, J = 9.1, 3.2 Hz, 1H), 3.95 (s, 3H), 3.89 (s, 3H), 3.48 (s, 2H), 2.87 (t, J = 11.1
Hz, 2H), 2.47 (ddd, J = 9.8, 5.1, 3.3 Hz, 1H), 2.07 (s, 3H), 1.96 (m, 2H), 1.83 (m, 1H), 1.73–1.58
(m, 3H), 1.53 (m, 1H), 1.37–1.25 (m, 2H); 13C NMR (101 MHz, CDCl3)
δ 204.1, 169.8, 156.1,
150.8, 147.9, 138.6, 129.2 (2C), 128.8, 128.2 (2C), 126.9, 106.7, 103.7, 63.4, 56.5, 56.2, 53.7, 53.7,
53.6, 53.5, 37.4, 33.4, 32.9, 31.8, 23.5; LC-MS m/z [M + H]+ 436.96.
Tert-Butyl N-[2-[(1-Benzyl-4-piperidyl)methyl]-5,6-dimethoxy-3-oxo-indan-1-yl] carbamate
(
17). To a stirring solution of 15 (0.41 mmol,1.0 eq.) in EtOH (10 mL) was added 10%
palladium on carbon (0.02 g, 50 mg/mmol). Hydrogen gas was then pumped into the
reaction vessel at 1 atm and left to stir for 48 h to 40 ◦C. The catalyst was then removed
by filtering the reaction mixture through celite. The solvent was then removed by rotary
evaporation to afford the crude product which was purified by column chromatography
to yield the reduced product as a colorless solid (65%) with a mixture of trans/cis (74/26).
Trans 1H-NMR (400 MHz, CDCl3)
δ 7.33–7.28 (m, 4H), 7.26–7.22 (m, 1H), 7.11 (s, 1H), 6.98
(s, 1H), 4.99 (dd, J = 9.9, 3.5 Hz, 1H), 4.82 (d, J = 9.9 Hz, 1H, NH), 3.96 (s, 3H), 3.90 (s, 3H),
3.51 (s, 2H), 2.88 (m, 3H), 2.02 (m, 2H), 1.86 (m, 1H), 1.74–1.54 (m, 4H), 1.48 (s, 9H), 1.33 (m,
2H); 13C NMR (101 MHz, CDCl3)
δ
204.0, 155.9, 155.6, 150.7, 148.0, 138.3, 129.3 (2C), 128.8,
128.1 (2C), 127.0, 106.5, 103.7, 80.1, 63.3, 56.4, 56.2, 55.1, 53.9, 53.6 (2C), 37.2, 33.2, 32.9, 31.6,
28.4 (3C); Cis 1H-NMR (400 MHz, CDCl3)
7.33–7.28 (m, 4H), 7.26–7.22 (m, 1H), 7.12 (s,
δ
1H), 7.00 (s, 1H), 5.42 (dd, J = 10.1, 6.5 Hz, 1H), 4.62 (d, J = 10.1 Hz, 1H, NH), 3.97 (s, 3H),
3.90 (s, 3H), 3.51 (s, 2H), 2.89 (m, 2H), 2.48 (m, 1H), 2.02 (m, 2H), 1.86 (m, 1H), 1.74–1.54
(m, 4H), 1.48 (s, 9H), 1.33 (m, 2H); 13C-NMR (101 MHz, CDCl3)
δ 204.9, 155.8, 155.6,150.7,
147.9, 138.3, 129.3 (2C), 128.7, 128.1 (2C), 127.0, 106.8, 103.7, 80.1, 63.3, 56.5, 56.2, 53.6 (2C),
51.5, 48.3, 33.5, 32.9, 32.5, 32.1, 28.4 (3C); LC-MS m/z [M + H]+ 494.85.
Trans 3-Amino-2-[(1-benzyl-4-piperidyl)methyl]-5,6-dimethoxy-indan-1-one (18). To a so-
lution of 17 (500 mg, 1.01 mmol, 1.0 eq.) in CH2Cl2 was added TFA (2 mL/mmol). The
mixture was stirred at room temperature overnight. After completion, the reaction was
basified with an aqueous solution of NaOH 1N and then extracted with CH2Cl2. The
organic phase was washed with brine, dried over MgSO4, filtered and evaporated under
reduced pressure. Reversed-phase flash chromatography (Acetonitrile/water) provided
1
the expected primary amine as an orange oil (orange oil, 78%). H-NMR (400 MHz, CDCl3)
δ
7.32–7.26 (m, 4H), 7.25–7.20 (m, 1H), 7.11 (s, 1H), 7.04 (s, 1H), 4.06 (d, J = 3.7 Hz, 1H), 3.97
(s, 3H), 3.89 (s, 3H), 3.49 (s, 2H), 2.89 (m, 2H), 2.36 (m, 1H), 1.97 (m, 2H), 1.89–1.76 (m, 2H),
1.69–1.59 (m, 2H), 1.46 (m, 1H), 1.33 (m, 2H). 13C-NMR (100 MHz, CDCl3)
204.9, 155.8,
δ
151.1, 150.3, 138.5, 129.2 (2C), 128.5, 128.1 (2C), 126.9, 106.1, 103.7, 63.4, 57.3, 57.0, 56.4, 56.2,
53.7 (2C), 37.3, 34.0, 33.0, 32.1.
Trans 2-[(1-Benzyl-4-piperidyl)methyl]-5,6-dimethoxy-3-(prop-2-ynylamino)indan-1-one (19).
Propargyl bromide (35 µL, 0.38 mmol) and K2CO3 (42 mg, 0.31 mmol) were added to a
solution of 16 (110 mg, 0.28 mmol) in acetonitrile. The reaction mixture was stirred at
room temperature for 24 h. The solvent was removed under reduced pressure, and the
residue was purified by column chromatography on silica using EtOAc/MeOH/NH4OH
1
(97/3/41) as eluent to give 14 as a colorless solid (7 mg, 6%). H-NMR (400 MHz, CDCl3)
δ

Molecules 2021, 26, 80
14 of 17
7.35–7.27 (m, 4H), 7.26–7.21 (m, 1H), 7.14 (s, 1H), 7.04 (s, 1H), 4.20 (d, J = 2.1 Hz, 1H), 3.99
(s, 3H), 3.91 (s, 3H), 3.62–3.43 (m, 4H), 2.96–2.83 (m, 2H), 2.66–2.57 (m, 1H), 2.31 (t, J = 2.4
Hz, 1H), 1.98 (t, J = 11.5 Hz, 2H), 1.80–1.62 (m, 4H), 1.56–1.48 (m, 1H), 1.47–1.17 (m, 2H);.
13C NMR (100 MHz, CDCl3)
δ 205.6, 155.6, 150.6, 148.8, 138.5, 129.2, 129.2 (2C), 128.1 (2C),
126.9, 106.9, 104.0, 82.0, 72.3, 63.4, 61.7, 56.4, 56.2, 53.8, 53.7, 52.3, 38.5, 36.1, 33.7, 32.7, 32.5;
HRMS/ESI: m/z calcd. [M + H]+ 433.2491, found 433.2507; LC-MS m/z [M + H]+ 432.87.
4.2. X-ray Diffractometry
Single X-ray crystal analysis on compound 16 was carried out on BRUKER D8 Quest
diffractometer (Bruker, Billerica, MA, USA) with a PHOTON II detector (Bruker, Biller-
ica, MA, USA) using 1 µS micro focus X-ray source (Mo Kα, λ = 0.71073 Å). The crystal
structure was solved by direct methods and refined employing full-matrix least-squares
refinement against F2 using SHELX2014 package [22]. All non-hydrogen atoms were re-
fined anisotropically and hydrogen atom positions were determined via difference Fourier
maps and refined with isotropic atomic displacement parameters. The structural data of
compound 16 have been deposited with Cambridge Crystallographic Data Center, the
CCDC (Deposition Number 2052041).
4.3. In Silico Study
The 3D models of compound 19 were built using both enantiomers present in the
solved X-ray structure of similar compound 16. Their protonation states at pH 7.4 were
predicted using standard tools of the ChemAxon Package (http://www.chemaxon.com/)
and the majority microspecies protonated on piperidine nitrogen at this pH were used for
docking studies.
For docking into human AChE, its crystallographic coordinates were retrieved from
X-ray structure of the donepezil/AChE complex (PDB ID 4EY7, a structure refined to 2.35 Å
with an R factor of 17.7%) [23].
The docking of the compound 19 into AChE was carried out with the GOLD program
(v5.7.2) using the default parameters [24,25]. This program applies a genetic algorithm
to explore conformational spaces and ligand binding modes. To evaluate the proposed
ligand positions, the ChemPLP fitness function was used. The binding site in the (h)AChE
model was defined as a 7 Å sphere from the co-crystallized donepezil ligand and a water
molecule interacting with protonated piperidine ring of donepezil was conserved during
the docking (residue number 931).
The starting structure for our docking into the (h)MAO-B was the high-resolution
(2.2 Å) X-ray structure of rasagiline-inhibited human monoamine oxidase B in complex
with 2-(2-benzofuranyl)-2-imidazoline (PDB ID 2XFQ) [26]. The rasagiline and 2-(2-
benzofuranyl)-2-imidazoline were removed before docking. As the compound 19 should
bind covalently to the FAD co-factor through the propargyl moiety in the same way as
rasagaline, the structure of 19 was modified by adding the nitrogen atom on the propargyl
group and modifying its double bonds repartition according to the one observed in the
rasagaline-(h)MAO-B complex. The covalent docking constraint between the nitrogen
atom of FAD group (N5) and added nitrogen on propargyl moiety was applied during
the docking. The binding site in the MaO-B model was defined as a 10 Å sphere from
OH atom of Tyr188, and ChemPLP fitness function was used to evaluate the proposed
ligand positions.
4.4. Biological Evaluation
4.4.1. In Vitro Tests of AChE and BuChE Biological Activity
Inhibitory capacity of compounds on AChE biological activity was evaluated using the
spectrometric method of Ellman [18]. Acetyl- or butyrylthiocholine iodide and 5,5-dithiobis-
(2-nitrobenzoic) acid (DTNB) were purchased from Sigma Aldrich. Lyophilized BuChE
from equine serum (Sigma Aldrich) was dissolved in 0.2 M phosphate buffer pH 7.4 such
as to have enzyme solutions stock with 2.5 units/mL enzyme activity. AChE from human

Molecules 2021, 26, 80
15 of 17
erythrocytes (buffered aqueous solution,
≥
500 units/mg protein (BCA), Sigma Aldrich)
was diluted in 20 mM HEPES buffer pH 8, 0.1% Triton X-100 such as to have enzyme
solution with 0.25 unit/mL enzyme activity. In the procedure, 100 L of 0.3 mM DTNB
dissolved in phosphate buffer pH 7.4 was added to the 96 wells plate followed by 50 L of
L of enzyme (0.05 U final). After 5 min of preincubation
L of 10 mM acetyl- or
µ
µ
test compound solution and 50
at 25 C, the reaction was then initiated by the injection of 50
µ
◦
µ
butyrylthiocholine iodide solution. The hydrolysis of acetyl- or butyrylthiocholine was
monitored by the formation of yellow 5-thio-2-nitrobenzoate anion as the result of the
reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetyl- or
butyrylthiocholine, at a wavelength of 412 nm using a 96-well microplate plate reader
(TECAN Infinite M200, Lyon, France). Test compounds were dissolved in analytical grade
DMSO. Donepezil was used as a reference standard. The rate of absorbance increase at
412 nm was followed every minute for 10 min. Assays were performed in simplicate
during three independent tests, with a blank containing all components except acetyl- or
butyrylthiocholine, in order to account for non-enzymatic reaction. The reaction slopes
were compared, and the percent inhibition due to the presence of test compounds was
calculated by the following expression: 100
in the presence of inhibitor and v0 is the enzyme activity.
First screening of AChE and BuChE activity was carried out at a 10−6 or 10−5
concentration of compounds under study. For the compounds with significant inhibition
50%), IC50 values were determined graphically by plotting the % inhibition versus the
−
(vi/v0
×
100) where vi is the rate calculated
M
(
≥
logarithm of six inhibitor concentrations in the assay solution using the GraphPad Prism
6 software.
4.4.2. In Vitro Tests of MAO-A and MAO-B Inhibitory Activity
Inhibition of human recombinant monoamine oxidases A (250 U/mg) and B (59 U/mg;
microsomes from baculovirus infected insect cells; Sigma Aldrich) was determined as al-
ready described [17], measuring the fluorescence of 4-hydroxyquinoline (exc. 310 nm;
em. 400 nm) produced by MAOs in the oxidative deamination of substrate kynuramine.
Briefly, compounds were tested at 10
µ
M (or in the range 3
×
10−5–10−10 M for determi-
M) in phosphate buffer
nation of IC50) in co-incubation with MAO and kynuramine (50
µ
390 mOsm pH 7.4, at 37 ◦C for 30 min. Assays were performed in triplicate during three
independent tests, in 96-well black polystyrene plates (Greiner Bio-One, Kremsmünster,
Austria) using an Infinite M1000 Pro plate reader (Tecan, Cernusco s.N., Italy). Inhibition
data were obtained as mean
3 independent experiments (for IC50), using GraphPad Prism (version 5.00 for Windows;
GraphPad Software, San Diego, CA, USA).
±
SD (for single concentration points) or mean
±
SEM from
5. Conclusions
In conclusion, we succeeded, within the frame of this work, in the synthesis of the trans
diastereoisomers of PADPZ, a structural compromise between DPZ and rasagiline. PADPZ
appears endowed with both the activities displayed by these reference compounds and
behaves, apparently, in an indifferently manner, as selective AChE and MAO-B inhibitors.
Even if the required level of activities for MTDL are often weaker than those of mono-
targeted ligands, due to the synergistic effect MTDL are supposed to carry on, improving
the activity of trans-PADPZ appears possible through the stereoselective synthesis or
separation of its enantiomers. After the proof of interest lying in this original compound
this preliminary work brought according to us, this second step, as well as the in vivo
evaluation of these enantiomers in animal models of AD, will be quickly undertaken.
Author Contributions: Conceptualization, C.R. and P.D.; methodology, B.G., C.L., C.D.A., M.C., A.D.
and J.S.d.O.S.; molecular modeling, J.S.d.O.S.; validation, M.C., C.R. and P.D.; formal analysis, R.L.;
writing—original draft preparation, C.R. and P.D.; writing—review and editing, C.L., C.R., M.C. and
P.D.; funding acquisition, C.R. and P.D. All authors have read and agreed to the published version of
the manuscript.

Molecules 2021, 26, 80
16 of 17
Funding: This work was supported by funding from the Région Normandie (RIN GreenChem and
ChemImaging) and FEDER through the CERMN’s analytical platform.
Data Availability Statement: Not applicable.
Acknowledgments: The present work was performed thanks to the generous computing resources
provided by the CRIANN (Normandy, France). The authors gratefully acknowledge the European
Community (FEDER) for the molecular modeling software.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
Multi-Target Directed Ligands. Available online: https://www.clinicaltrials.gov (accessed on 10 November 2020).
Cavalli, A.; Bolognesi, M.L.; Mìnarini, A.; Rosini, M.; Tumiatti, V.; Recanatini, M.; Melchiorre, C. Multi-Target-Directed Ligands to
Combat Neurodegenerative Diseases. J. Med. Chem. 2008, 51, 347–372. [CrossRef]
3.
4.
Lecoutey, C.; Hedou, D.; Freret, T.; Giannoni, P.; Gaven, F.; Since, M.; Bouet, V.; Ballandonne, C.; Corvaisier, S.; Malzert Freon, A.;
et al. Design of Donecopride, a Dual Serotonin Subtype 4 Receptor Agonist/Acetylcholinesterase Inhibitor with Potential Interest
for Alzheimer’s Disease Treatment. Proc. Natl. Acad. Sci. USA 2014, 111, E3825–E3830. [CrossRef] [PubMed]
Rochais, C.; Lecoutey, C.; Gaven, F.; Giannoni, P.; Hamidouche, K.; Hedou, D.; Dubost, E.; Genest, D.; Yahiaoui, S.; Freret, T.;
et al. Novel Multitarget-Directed Ligands (MTDLs) with Acetylcholinesterase (AChE) Inhibitory and Serotonergic Subtype 4
Receptor Agonist Activities As Potential Agents against Alzheimer’s Disease: The Design of Donecopride. J. Med. Chem. 2015
,
58, 3172–3187. [CrossRef] [PubMed]
5.
Rochais, C.; Lecoutey, C.; Hamidouche, K.; Giannoni, P.; Gaven, F.; Cem, E.; Mignani, S.; Baranger, K.; Freret, T.; Bockaert, J.; et al.
Donecopride, a Swiss Army Knife with Potential against Alzheimer’s Disease. Br. J. Pharmacol. 2020, 177, 1988–2005. [CrossRef]
[PubMed]
6.
7.
8.
Youdim, M. The Path from Anti Parkinson Drug Selegiline and Rasagiline to Multifunctional Neuroprotective Anti Alzheimer
Drugs Ladostigil and M30. Curr. Alzheimer Res. 2006, 3, 541–550. [CrossRef]
Youdim, M.B.H.; Edmondson, D.; Tipton, K.F. The Therapeutic Potential of Monoamine Oxidase Inhibitors. Nat. Rev. Neurosci.
2006, 7, 295–309. [CrossRef]
Bautista-Aguilera, O.M.; Samadi, A.; Chioua, M.; Nikolic, K.; Filipic, S.; Agbaba, D.; Soriano, E.; de Andrés, L.; Rodríguez-Franco,
M.I.; Alcaro, S.; et al. N-Methyl-N-((1-Methyl-5-(3-(1-(2-Methylbenzyl)Piperidin-4-Yl)Propoxy)-1H-Indol-2-Yl)Methyl)Prop-2-Yn-
1-Amine, a New Cholinesterase and Monoamine Oxidase Dual Inhibitor. J. Med. Chem. 2014, 57, 10455–10463. [CrossRef]
Dallemagne, P.; Rault, S.; Pilo, J.C.; Foloppe, M.P.; Robba, M. One-Pot Cyclization of Alkoxy-3-Aminoindan-1-Ones. Tetrahedron
Lett. 1991, 32, 6327–6328. [CrossRef]
9.
10. Dallemagne, P.; Rault, S.; Pilo, J.C.; Robba, M. A Convenient Route to Indan-1,3-Diones and 3-Hydroxyindan-1-Ones. Bull. Soc.
Chim. Fr. 1993, 130, 121–124.
11. van Greunen, D.G.; Johan van der Westhuizen, C.; Cordier, W.; Nell, M.; Stander, A.; Steenkamp, V.; Panayides, J.-L.; Riley, D.L.
Novel N-Benzylpiperidine Carboxamide Derivatives as Potential Cholinesterase Inhibitors for the Treatment of Alzheimer’s
Disease. Eur. J. Med. Chem. 2019, 179, 680–693. [CrossRef]
12. Diouf, O.; Depreux, P.; Chavatte, P.; Poupaert, J.H. Synthesis and Preliminary Pharmacological Results on New Naphthalene
Derivatives as 5-HT4 Receptor Ligands. Eur. J. Med. Chem. 2000, 35, 699–706. [CrossRef]
13. Punchi Hewage, A.N.D.; Yao, H.; Nammalwar, B.; Gnanasekaran, K.K.; Lovell, S.; Bunce, R.A.; Eshelman, K.; Phaniraj, S.M.; Lee,
M.M.; Peterson, B.R.; et al. Small Molecule Inhibitors of the BfrB–Bfd Interaction Decrease Pseudomonas Aeruginosa Fitness and
Potentiate Fluoroquinolone Activity. J. Am. Chem. Soc. 2019, 141, 8171–8184. [CrossRef] [PubMed]
14. Auvray, P.; Moslemi, S.; Sourdaine, P.; Galopin, S.; Séralini, G.-E.; Enguehard, C.; Dallemagne, P.; Bureau, R.; Sonnet, P.;
Rault, S. Evidence for new non-steroidal human aromatase inhibitors and comparison with equine aromatase inhibition for an
understanding of the mammalian active site. Eur. J. Med. Chem. 1998, 33, 451–462. [CrossRef]
15. Ellman, G.L.; Courtney, K.D.; Andres, V., Jr.; Featherstone, R.M. A new and rapid colorimetric determination of acetyl-
cholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [CrossRef]
16. Wang, L.; Esteban, G.; Ojima, M.; Bautista-Aguilera, O.M.; Inokuchi, T.; Moraleda, I.; Iriepa, I.; Samadi, A.; Youdim, M.B.H.;
Romero, A.; et al. Donepezil + Propargylamine + 8-Hydroxyquinoline Hybrids as New Multifunctional Metal-Chelators, ChE and
MAO Inhibitors for the Potential Treatment of Alzheimer’s Disease. Eur. J. Med. Chem. 2014, 80, 543–561. [CrossRef] [PubMed]
17. Bolognino, I.; Giangregorio, N.; Pisani, L.; de Candia, M.; Purgatorio, R.; Tonazzi, A.; Altomare, C.D.; Cellamare, S.; Catto, M. A
Prospective Repurposing of Dantrolene as a Multitarget Agent for Alzheimer’s Disease. Molecules 2019, 24, 4298. [CrossRef]
[PubMed]
18. Hubálek, F.; Binda, C.; Li, M.; Herzig, Y.; Sterling, J.; Youdim, M.B.H.; Mattevi, A.; Edmondson, D.E. Inactivation of Purified
Human Recombinant Monoamine Oxidases A and B by Rasagiline and Its Analogues. J. Med. Chem. 2004, 47, 1760–1766.
[CrossRef]
19. Patil, S.A.; Patil, R.; Patil, S.A. Recent developments in biological activities of indanones. Eur. J. Med. Chem. 2017, 138, 182–198.
[CrossRef]

Molecules 2021, 26, 80
17 of 17
20. Huang, L.; Miao, H.; Sun, Y.; Meng, F.; Li, X. Discovery of indanone derivatives as multi-target-directed ligands against
Alzheimer’s disease. Eur. J. Med. Chem. 2014, 87, 429–439. [CrossRef]
21. Rampa, A.; Mancini, F.; de Simone, A.; Falchi, F.; Belluti, F.; di Martino, R.M.C.; Gobbi, S.; Andrisano, V.; Tarozzi, A.; Bartolini,
M.; et al. From AChE to BACE1 inhibitors: The role of the amine on the indanone scaffold. Bioorg. Med. Chem. Lett. 2015
25, 2804–2808. [CrossRef]
,
22. Sheldrick, G.M. A short history of SHELX. Acta Cryst. 2008, A64, 112–122. [CrossRef]
23. Cheung, J.; Rudolph, M.J.; Burshteyn, F.; Cassidy, M.S.; Gary, E.N.; Love, J.; Franklin, M.C.; Height, J.J. Structures of human
acetylcholinesterase in complex with pharmacologically important ligands. J. Med. Chem. 2012, 55, 10282–10286. [CrossRef]
24. Jones, G.; Willett, P.; Glen, R.C. Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation.
J. Mol. Biol. 1995, 245, 43–53. [CrossRef]
25. Jones, G.; Willett, P.; Glen, R.C.; Leach, A.R.; Taylor, R. Development and Validation of a Genetic Algorithm for Flexible Docking.
J. Mol. Biol. 1997, 267, 727–748. [CrossRef] [PubMed]
26. Bonivento, D.; Milczek, E.M.; McDonald, G.R.; Binda, C.; Holt, A.; Edmondson, D.E.; Mattevi, A. Potentiation of ligand binding
through cooperative effects in monoamine oxidase B. J. Biol. Chem. 2010, 285, 36849–36856. [CrossRef] [PubMed]