Two novel donepezil-lipoic acid hybrids: Synthesis, anticholinesterase and antioxidant activities and theoretical studies

Article abstract of DOI:10.21577/0103-5053.20170196

Alzheimer disease (AD) is a complex disease related to multiple pathogenic mechanisms. A strategy to develop effective drugs is based on the so-called multi-Target directed ligands (MTDL) by using hybrid compounds. So, in the present study, we have designed and synthesized two hybrids, containing the indanone-piperidine moiety of donepezil, a drug approved for the treatment of AD, and the lipoic acid scaffold, an antioxidant compound endowed with neuroprotective effects. One hybrid was synthesized in four steps with 42percent global yield, and the other hybrid in six steps with 19percent global yield. The latter hybrid displayed moderate inhibitory activity against human acetylcholinesterase (hAChE) and greater activity against human butyrylcholinesterases (hBuChE). The selectivity for hBuChE was further rationalized by theoretical study. Importantly, the second hybrid showed a good antioxidant activity, exhibiting better ability in scavenging 2,2-diphenyl- 1 picrylhydrazyl (DPPH) radicals than lipoic acid.

Full text of DOI:10.21577/0103-5053.20170196

http://dx.doi.org/10.21577/0103-5053.20170196
J. Braz. Chem. Soc., Vol. 29, No. 4, 738-747, 2018.
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Two Novel Donepezil-Lipoic Acid Hybrids: Synthesis, Anticholinesterase and
Antioxidant Activities and Theoretical Studies
Bruna S. Terra,a Pedro H. C. da Silva,a Anna Tramarin,b Lucas L. Franco,a
Elaine F. F. da Cunha,c Fernando Macedo Junior,d Teodorico C. Ramalho,c
Manuela Bartolini,b Maria L. Bolognesib and Ângelo de Fátima*,a
aDepartamento de Química, Universidade Federal de Minas Gerais (UFMG),
Av. Pres. Antônio Carlos, 6627, 31270-901 Belo Horizonte-MG, Brazil
bDepartment of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna,
via Belmeloro, 6, 40126 Bologna, Italy
cDepartamento de Química, Universidade Federal de Lavras (UFLA), Av. Doutor Sylvio Menicucci,
1001, Kennedy, 37200-000 Lavras-MG, Brazil
dDepartamento de Química, Universidade Estadual de Londrina (UEL), Rodovia Celso Garcia Cid,
Pr 445, km 380, 86057-970 Londrina-PR, Brazil
Alzheimer disease (AD) is a complex disease related to multiple pathogenic mechanisms. A
strategy to develop effective drugs is based on the so-called multi-target directed ligands (MTDL) by
using hybrid compounds. So, in the present study, we have designed and synthesized two hybrids,
containing the indanone-piperidine moiety of donepezil, a drug approved for the treatment of
AD, and the lipoic acid scaffold, an antioxidant compound endowed with neuroprotective effects.
One hybrid was synthesized in four steps with 42% global yield, and the other hybrid in six steps
with 19% global yield. The latter hybrid displayed moderate inhibitory activity against human
acetylcholinesterase (hAChE) and greater activity against human butyrylcholinesterases (hBuChE).
The selectivity for hBuChE was further rationalized by theoretical study. Importantly, the second
hybrid showed a good antioxidant activity, exhibiting better ability in scavenging 2,2-diphenyl-
1-picrylhydrazyl (DPPH) radicals than lipoic acid.
Keywords: donepezil-lipoic acid hybrids, Alzheimer disease, multi-target directed ligands
Introduction
neuroinflammatory processes and neuronal loss, mainly
affecting the frontal cortex and hippocampus.4,5 AD is also
characterized by a reduction of acetylcholine (ACh) levels,
which is correlated with the cognitive symptoms.6
The “cholinergic hypothesis”, proposed in 1982
by Bartus et al.,7 postulated that the cognitive decline
experienced by patients withAD resulted from a deficiency
of acetylcholine or cholinergic neurotransmission. In
humans, acetylcholine is degraded in the synaptic cleft by
two main classes of cholinesterase enzymes: acetyl- (AChE)
and butyryl- (BuChE)8 cholinesterases.
Near the amyloid plaques and neurofibrillary tangles,
an extensive oxidative stress has been observed9 which
is a result of an altered balance of formation of reactive
oxygen species (ROS) versus scavenging activity.5,10 The
production of ROS is also related to calcium homeostasis;
the misbalance of calcium influx affects the mitochondrial
Alzheimer disease (AD) is the most common cause
of dementia in aging population. It was estimated that,
in 2010, about 35.6 millions of people suffered from
dementia worldwide, and it is expected that this number
might triplicate in the next 40 years.1 Patients affected by
AD experience progressive cognitive impairment, such as
a decline in short-term memory, loss of speech, language
and motor coordination.2,3
AD is pathologically characterized by an extracellular
deposition of β-amyloid (Aβ) peptide into senile
plaques, intracellular formation of neurofibrillary tangles
(NFTs) containing a hyperphosphorylated form of Tau
protein, oxidative stress, mitochondrial abnormality,
*e-mail: adefatima@qui.ufmg.br

Vol. 29, No. 4, 2018
Terra et al.
739
enzymes and ROS production is a normal part of the
electron transport chain. However, excessive levels
of these species damage proteins, lipids and nucleic
acids.9
and other antioxidant enzymes, (iii) downregulation
of the inflammatory processes, (iv) scavenging of lipid
peroxidation products, (v) redox active transition metal
chelation, (vi) increase of ACh production by activation of
choline acetyltransferase.25 On the basis of such activities,
LA can exert beneficial effects in AD, possibly stabilizing
cognitive functions.26
AD is a complex disease related to multiple pathogenic
mechanisms involving different molecular targets. All the
drugs approved so far are palliative and not curative. A
strategy to develop effective drugs is based on the so-called
multi-target directed ligands (MTDLs)11 approach. This
strategy builds on the development of a single drug that
can simultaneously interact with different targets. The
advantages of this polypharmacological strategy, when
compared with the administration of a combination of
multiple drugs, are the reduction of the risk of drug-drug
interactions and a simplification of the pharmacokinetic and
pharmacodynamic studies. Moreover, the success rate of
the treatment of a complex disease of the elderly, as AD,
should be higher.12
Donepezil (Figure 1), a palliative drug approved in
1996, is indicated for the treatment of mild and moderate
forms ofAD.13 Its structure represents an attractive starting
point for the rational design of new MTDLs that can inhibit
AChE and, at the same time, interact with other targets
involved in AD onset and progression.13,14
Figure 2. Chemical structures of lipoic acid (LA) and of dihydrolipoic
acid (DHLA).
Thus, LA is a good prototype to design new hybrids
to combat AD, and previously developed LA hybrids
maintained the antioxidant activity and showed other
beneficial activities such as inhibition of AChE and
BuChE as well as neuroprotective and anti-inflammatory
activity.27,28
In 2005, Rosini et al.29 reported the synthesis of
lipocrine, an LA-tacrine hybrid, which further inspired the
development of other hybrids featuring an LA fragment
connected with N1-ethyl-N1-(2-methoxy-benzyl)-hexane-
1,6-diamine moiety or with rivastigmine.26
Although there are works involving the hybridization
of the benzyl-piperidine moiety of donepezil with LA, to
our knowledge, there is no report on the hybridization of
the indanone-piperidine moiety with LA. Therefore, in
the present study, following a simple synthetic route, we
have designed and synthesized two hybrids containing
the indanone-piperidine moiety of donepezil and the
LA scaffold with the aim of achieving new MTDLs for
the treatment of AD.11 Here, we report their biological
assessment on human AChE (hAChE), human BuChE
(hBuChE), as well as the evaluation of their antioxidant
activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH)
assay. Finally, docking studies provided further insights
of the binding mode of these novel donepezil-lipoic acid
hybrids with AChE and BuChE.
Figure 1. Chemical structure of donepezil.
Many prototypes for new drugs based on the
hybridization strategy have been developed starting from
donepezil fragments, i.e., indanone-piperidine moiety
or piperidine-benzyl fragment.13 Furthermore, donepezil
hybrids with tacrine,15,16 diaminobenzyl group,17 ferulic
acid,18 coumarin,19 among others13 have been prepared.
Hybrids containing the piperidine-benzyl moiety of
donepezil and lipoic acid (LA) (Figure 2) were described
by the groups of Kim et al.,20 Lee et al.,21 Prezzavento
et al.,22 and Estrada et al.23 The hybrids showed activity
against cholinesterase (ChE) enzymes, antagonism toward
σ1 receptors, β-secretase inhibition and antioxidant activity.
LA is a natural disulfide compound present in almost
all foods from animal and vegetable sources. LA and its
reduced form, dihydrolipoic acid (DHLA) (Figure 2), play
an important role in pathological conditions characterized
by oxidative stress,24,25 such as: (i) scavenger of ROS,
(ii) capacity to increase the level of reduced glutathione
Results and Discussion
Synthesis of hybrid compounds
Two novel donepezil-LA hybrids, differing only by
the linkage between the two units, were designed and
synthesized. In the final hybrid structures, the indanone and
piperidine fragments were preserved and the benzyl group
was replaced by the LA portion (Figure 3).

740
Two Novel Donepezil-Lipoic Acid Hybrids
J. Braz. Chem. Soc.
Figure 3. Design strategy for hybrids 1 and 2.
In the synthesis of hybrid 1, the first step was to
obtain the indanone 4, in high yield, by cyclization of
3-(3,4-dimethoxyphenyl)propanoic acid 3 in the presence of
p-toluenesulfonic acid and phosphorus pentoxide (P2O5).30
The Boc-piperidine-4-carboxaldehyde, obtained via Swern
oxidation of N-Boc-4-piperidinemethanol31,32 was directly
used in aldol condensation with 4 furnishing 5 with 84%
yield33 (Scheme 1).
In the next step, the key intermediate 6 was prepared
in 91% yield by hydrogenation of the aldol adduct 5
with palladium-carbon (Pd-C) as catalyst,34 followed
by removal of the Boc group under acidic aqueous
media.35 Noteworthy, when the hydrogenation reaction
lasted more than 20 min, the deoxygenation product
was observed. The final product 1 was obtained by
coupling the amine 6 and LA using benzotriazol-1-yloxy-
tripyrrolidinophosphonium hexafluorophosphate (PyBOP)
and N,N-diisopropylethylamine (DIPEA) (Scheme 1).36
The synthesis of hybrid 2 featured, as first step, the
protection of 2-bromoethylamine hydrobromide to afford
the tert-butyl (2-bromoethyl)carbamate,37 which was reacted
with 6 under basic conditions to afford the diamine 7 in 39%
yield38 (Scheme 2). Removal of the protection group with
trifluoroacetic acid (TFA) furnished 8 as trifluoroacetate
salt in quantitative yield.39 Specifically, compound 8 is
in the form of mono trifluoroacetate salt as confirmed by
Scheme 1. Synthesis of donepezil-LA hybrid 1. Reagents and conditions: (a) p-toluenesulfonic acid, P2O5, 120 °C, 35 min, 87%; (b) N-Boc-piperidine-
4-carboxaldehyde, NaH, tetrahydrofuran (THF), r.t., 2 h, 84%; (c) H2 (1 atm), Pd-C (10%), THF, r.t., 20 min, then hydrochloric acid (HCl) (3 M), ethyl
acetate (EtOAc), r.t., 3 h, 91%; (d) lipoic acid, DIPEA, PyBOP, dichloromethane (DCM), 0 °C → r.t., 20 h, 63%.

Vol. 29, No. 4, 2018
Terra et al.
741
Scheme 2. Synthesis of donepezil-LA hybrid 2. Reagents and conditions: (a) tert-butyl (2-bromoethyl)carbamate, DIPEA, sodium iodide (NaI), acetonitrile
(MeCN), reflux, 24 h, 39%; (b) TFA, DCM, r.t., 1 h, 100%; (c) lipoic acid, DIPEA, PyBOP, DCM, 0 °C → r.t., 20 h, 74%.
HRMS analysis, which showed the mass-to-charge ratio
(m/z) signals of the mono-protonated amine (m/z C19H28N2O3
[M + H]+ observed: 333.2178; required: 333.2166). Finally,
hybrid 2 was obtained by condensation of LA and 8 in the
same conditions used for the synthesis of 1 (Scheme 2).36
incubated for 30 min in a solution containing the stable
free radical. Figure 4 shows the DPPH radical scavenging
activity of new hybrids and reference compounds,
expressed as percentage of scavenged DPPH radicals. All
tested compounds showed a decrease in the concentration
of DPPH radicals confirming their scavenging ability.
Hybrid 2 was able to scavenge DPPH radicals, showing
a higher activity than LA. The half maximal effective
concentration (EC50), i.e., concentration that causes 50%
decrease in the DPPH radical content, was 300 µM. The
hybrid 1 exhibited lower scavenging activity than hybrid 2,
however, it was similar to that of LA. Scavenging activity of
hybrid 1 and LA was not concentration dependent; similar
results were obtained at all concentrations tested. The
scavenging activity of LA toward DPPH radicals was 27%
at 100 µM, in agreement with data reported in literature.43
hAChE, hBChE and antioxidant assay
Initially, to determine the potential interest of the new
donepezil-LA hybrids for the treatment ofAD, the inhibitory
potency toward hAChE and BuChE from human serum was
assessed by Ellman’s method.40 Results, expressed as half
maximal inhibitory concentration (IC50) values, i.e., the IC50
that reduces the cholinesterase activity by 50%, are listed in
Table 1. In particular, anti-BuChE activity has recently raised
interest because it was shown that with AD progression,
BuChE activity in specific brain regions increases while
AChE activity is greatly reduced.41 Conversely to donepezil
which is anAChE selective inhibitor, hybrid 2 showed to be
a selective BuChE inhibitor. Hybrid 1 was scarcely soluble
in the assay conditions. At the highest tested concentration
(50 mM) hybrid 1 did not significantly inhibit ChE enzymes.
The antioxidant activity was estimated using the
DPPH antioxidant assay.42 For that purpose, different
concentrations (20-640 µM) of the test compounds were
Molecular modeling
To get insights on the binding mode, compounds 1
and 2 were docked in AChE and BuChE enzymes. The
potential binding sites ofAChE and BuChE were calculated
using the built-in cavity detection algorithm from Molegro
program.44,45 The BuChE enzyme has a larger cavity of
482.3 Å3; meanwhile, AChE has a cavity of 363.0 Å3.
Table 1. hAChE and hBuChE activities of hybrids 1, 2 and the reference compound donepezil
Compound
IC50a hAChE SEMb / µM
IC50 hBuChE SEMb / µM
BuChE/AChE
1
2
n.a.c
171 10
0.0203 0.0013
n.a.c
62.9 5.4
7.13 0.19
-
0.37
351
Donepezil
aIC50: Inhibitory concentration; bSEM: standard error of the mean; cn.a.: not active (% inhibition < 10%) at the highest concentration achievable (50 mM)
in the assay conditions.

742
Two Novel Donepezil-Lipoic Acid Hybrids
J. Braz. Chem. Soc.
Figure 4. Antioxidant activity toward DPPH radicals expressed as percentage.
Compound 2 formed a more stable protein-ligand
complex with both ChEs than hybrid 1. It should be kept
in mind that the interaction modes of the ligand with the
active sites were determined as the lowest energy scored
protein-ligand complex used during docking and the
conformers of each compound were mostly associated to
each other. Thus, from the theoretical findings, compound 2
revealed the lowest score energy value. For instance,
differences between compound 2 and hybrid 1 of up to 10.5
and 31.8 kcal mol-1 were obtained for AChE and BuChE,
respectively.
bond formation. In particular, donepezil interacted with
tryptophan Trp86A (amino acid residue of catalytic site,
CAS), tryptophan Trp286A and tyrosine Tyr341A (amino
acid residue of peripheral anion site, PAS) through π-π
interactions; 1 and 2 interacted with tryptophan Trp286A
and tyrosine Tyr341 through π-π interaction. It is observed
that the interaction with the amino acids of CAS is lost,
which explains the lower activity observed of the hybrid 2
in the biological assay. Concerning BuChE, donepezil,
1 and 2 formed hydrogen bond interactions with serotonine
Ser198A; furthermore, donepezil established π-π interaction
with tryptophan Trp231 and the methoxy group of both 1 and
2 interacted with tryptophan Trp231A (Figure 5).
Regarding the interaction with AChE, donepezil, 1 and
2 interacted with phenylalanine Phe295A through hydrogen
Figure 5. Docking models for (a) AChE (1); (b) AChE (2); (c) BuChE (1); (d) BuChE (2).

Vol. 29, No. 4, 2018
Terra et al.
743
It is important to note that the strength of molecular
interactions was lower for compound 1 when compared
to 2, i.e., the addition of a methyl group indicated an
increase of hydrophobic interactions with the residues
of the hydrophobic pocket, resulting in higher binding
affinity with both enzymes. This feature suggests that the
inclusion of a larger linkage group between the two units
can be favorable for the biological activity. Exploring the
fact that in BuChE there is a larger accessible cavity to the
solvent, bulky substituents as well as larger linker chains
will have a greater beneficial impact on selectivity and
interaction with BuChE.
parts per million (ppm) and are referenced to signals
from tetramethylsilane (TMS) or residual solvent signal.
1H NMR data are presented in the following order: chemical
shift in ppm (multiplicity, coupling constant (J) in hertz
(Hz), integration). Melting points (mp) (uncorrected)
were obtained on a Mettler FP 80 HT apparatus. Infrared
spectra were recorded on a PerkinElmer Spectrum One
spectrometer. The high resolution mass spectra were
obtained using a mass spectrometer with an electrospray
ionization source (ESI-MS) model Shimadzu LC-ITTOF.
Synthesis
The activity of hybrid 2, compared with those of benzyl-
piperidine hybrids reported in the literature,20-23 suggest that
both interaction with the CAS and PAS of donepezil are
important for the activity of this drug. When was removed
from the moiety of donepezil, the inhibition of AChE
enzymes was lower.
5,6-Dimethoxy-2,3-dihydro-1H-inden-1-one (4)
In a round bottom flask, P2O5 (6.85 g, 36 mmol) and
toluenesulfonic acid (5.11 g, 36 mmol) was warmed to
120 °C and stirred for 30 min. To the clear homogeneous
solution was added the 3-(3,4-dimethoxyphenyl)propanoic
acid (0.630 g, 3.0 mmol) in one portion and the solution
was stirred at 120 °C for 5 min. Then, ice water was
added to the deep purple solution formed and the resulting
mixture was extracted three times with dichloromethane
(CH2Cl2). The combined organic layers were washed
with saturated solution of sodium bicarbonate (NaHCO3),
dried with magnesium sulfate (MgSO4), filtered and
concentrated under reduced pressure. The resulting brown
solid was purified by chromatography column eluted with
ethylacetate/hexane (1:1, v:v) yielding 4 as a yellow solid
(0.51 g) in 87% yield; mp 118-119 °C (Lit.46 116-118 °C);
IR (attenuated total reflection (ATR)) n / cm-1 3001, 2923,
2853, 1688, 1604, 1590, 1500, 1457, 1440, 1423, 1364,
1309, 1264, 1245, 1211, 1188, 1175, 1156, 1118, 1074,
1038, 985, 962, 847, 816, 780, 710; 1H NMR (400 MHz,
CDCl3) d 2.56-2.59 (m, 2H), 2.96 (t, 2H, 3J = 5.6 Hz), 3.82
(s, 3H), 3.88 (s, 3H), 6.81 (s, 1H), 7.08 (s, 1H); 13C NMR
(100 MHz, CDCl3) d 25.6, 36.6, 56.3, 56.1, 104.3, 107.6,
130.0, 149.5, 150.5, 155.5, 205.7; HRMS (ESI) m/z,
observed: 193.0863 for C11H12O3 [M + H]+; required:
193.0864.
Conclusions
In this work, two donezepil-LA hybrids containing the
indanone-piperidine moiety of donepezil were synthesized.
Hybrid 1, in which the two fragments were connected
directly, was obtained in four steps with 42% global yield,
while hybrid 2, which features a linker between the two
units, was synthesized in six steps, with 19% global yield.
Hybrid 2 proved to be a selective BuChE inhibitor even if
less potent than donepezil and a good antioxidant agent.
In particular, the lower activity can be ascribed to the loss
of the interaction with Trp86A, an amino acid of theAChE
CAS, when the benzyl moiety of donepezil is replaced
by LA. The selectivity of 2 toward hBuChE is explained
by the larger gorge of this enzyme, which can better
accommodate hybrid 2. Finally, and quite interestingly,
hybrid 2 showed better scavenging ability toward DPPH
radicals than LA. The combined anti-ChE and antioxidant
properties exhibited by the hybrid 2 confirm their potential
as anti-AD agents.
Experimental
(E)-tert-Butyl 4-((5,6-dimethoxy-1-oxo-1H-inden-
2(3H)-ylidene)methyl)piperidine-1-carboxylate (5)
General techniques
Under argon atmosphere, a solution of oxalylchloride
(0.2 mL, 2.2 mmol) in anhydrous CH2Cl2 (5 mL) was
cooled to -78 °C and a solution of dimethyl sulfoxide
(DMSO) (0.31 mL, 4.4 mmol) in anhydrous CH2Cl2
(1 mL) was dropwise added. After stirring for 10 min, a
solution of N-Boc-4-piperidinemethanol (0.43 g, 2 mmol)
in anhydrous CH2Cl2 (1 mL) was added. Triethylamine
(1.4 mL, 10.2 mmol) was added after 15 min and the
reaction was allowed to warm up to room temperature. The
All starting materials were obtained from commercially
available sources with high-grade purity and used without
further purification. Proton nuclear magnetic resonance
(1H NMR) and carbon-13 (13C) NMR spectra at 200 and
400 MHz were obtained on a Bruker AVANCE DPX
200 and a Bruker AVANCE DRX 400 spectrometer,
respectively. The chemical shifts (d) are expressed in

744
Two Novel Donepezil-Lipoic Acid Hybrids
J. Braz. Chem. Soc.
reaction was quenched by the addition of water (10 mL)
after 2 h and the mixture extracted four times with CH2Cl2.
The combined organic layers were dried with MgSO4,
filtered and concentrated under reduced pressure. The
crude aldehyde was used in the next step without further
purification.
(m, 4H), 3.20-3.29 (m, 2H), 3.91 (s, 3H), 3.97 (s, 3H), 6.86
(s, 1H), 7.17 (s, 1H); HRMS (ESI) m/z, observed: 290.1761
for C17H23NO3 [M + H]+; required: 290.1756.
2-((1-(5-(1,2-Dithiolan-3-yl)pentanoyl)piperidin-4-yl)methyl)-
5,6-dimethoxy-2,3-dihydro-1H-inden-1-one (1)
To a solution of 5,6-dimethoxy-indanone (4)
(0.19 g, 1 mmol) in anhydrous THF (5 mL) was added
sodium hydride (NaH) (0.05 g, 1.2 mmol, 60% dispersion
in mineral oil). After stirring for 30 min, a solution of
crude aldehyde in THF (1 mL) was added dropwise, and
the resulting mixture was stirred at room temperature
for 2 h. The solvent was concentrated under reduced
pressure, and to the residual product was added water and
extracted three times with CH2Cl2. The combined organic
layers were dried with MgSO4, filtered and concentrated
under reduced pressure. The crude reaction was purified
by chromatography column eluted ethyl acetate/hexane
(1:1, v:v) yielding 5 as oil (0.32 g) in 84% yield; IR (ATR)
n / cm-1 3403, 2926, 2850, 2732, 1682, 1605, 1590, 1500,
1463, 1316, 1265, 1218, 1120, 1040, 1004, 975, 862, 789;
1H NMR (400 MHz, CDCl3) d 1.48 (s, 11H), 1.69-1.72
(m, 2H), 2.44-2.53 (m, 1H), 2.80-2,86 (m, 2H), 3.62 (s, 2H),
3.93 (s, 3H), 3.98 (s, 3H), 4.12-4.15 (m, 2H), 6.62 (d, 1H,
J = 12 Hz), 6.91 (s, 1H), 7.30 (s, 1H); 13C NMR(100 MHz,
CDCl3) d 28.8, 29.8, 31.2, 37.6, 43.6, 56.5, 56.6, 79.9,
105.4, 107.6, 132.1, 136.3, 138.8, 144.7, 149.9, 155.1,
155.8, 192.8; HRMS (ESI) m/z, observed: 410.1931 for
C22H29NO5 [M + Na]+; required: 410.1944.
A solution of lipoic acid (0.06 g, 0.25 mmol) and
PyBOP (0.13 g, 0.25 mmol), in anhydrous CH2Cl2
(4.5 mL), at 0 °C, was stirred for 30 min and then,
cannulated to a flask containing a solution of (6)
(0.1 g, 0.27 mmol), N,N-diisopropylethylamine
(0.25 g, 1.97 mmol), in anhydrous CH2Cl2 (4.5 mL).
The resulting mixture was stirred at room temperature
for 20 h. Then, the reaction was quenched by the
addition of water and the mixture was extracted four
times with CH2Cl2. The combined organic layers
were dried with MgSO4, filtered and concentrated
under reduced pressure. The crude reaction was
purified by chromatography column eluted with
ethyl acetate/methanol (5%) yielding 1 as a beige solid
(0.07 g) in 63% yield; IR (ATR) n / cm-1 2924, 2848,
1691, 1630, 1607, 1591, 1499, 1452, 1437, 1363, 1341,
1312, 1263, 1224, 1213, 1153, 1121, 1072, 1036, 968,
1
861, 840, 792, 765, 730, 700; H NMR (400 MHz,
CDCl3) d 1.23-1.29 (m, 3H), 1.38-1.44 (m, 2H), 1.49-
1.56 (m, 2H), 1.64-1.77 (m, 4H), 1.86-1.95 (m, 5H),
2.44-2.53 (m, 3H), 2.70-2.71 (m, 2H), 3.09-3.22 (m, 3H),
3.28 (dd, 1H, J = 20, 8 Hz), 3.60 (qui, 1H, J = 8 Hz), 3.91
(s, 3H), 3.97 (s, 3H), 6.88 (s, 1H), 7.16 (s, 1H); 13C NMR
(100 MHz, CDCl3) d 25.6 (CH2), 29.2 (CH2), 32.5
(CH2), 33.6 (CH2), 34.4 (CH), 34.7 (CH2), 38.6 (CH2),
38.7 (CH2), 40.5 (CH2), 45.2 (CH), 56.3 (OCH3), 56.4
(OCH3), 56.6 (CH), 104.7 (CAr), 107.6 (CAr), 129.3 (CAr),
148.8(CAr), 149.8 (CAr), 155.9 (CAr), 172.9 (NC=O),
207.3 (C=O); HRMS (ESI) m/z, observed: 500.1859 for
C25H35NO4S2 [M + Na]+; required: 500.1905.
5,6-Dimethoxy-2-(piperidin-4-ylmethyl)-2,3-dihydro-
1H-inden-1-one (6)
To a solution of 5 (0.22 g, 0.57 mmol) in THF (5 mL) the
Pd-C (0.01 g, 10% Pd-C) was added. The reaction mixture
was purged with hydrogen and stirred at room temperature
under hydrogen atmosphere for 20 min. Then, the reaction
was filtered through celite washing with methanol and the
solvent was removed under reduced pressure. The crude
product (0.20 g) was solubilized in ethyl acetate (10 mL)
and HCl 3 M (8 mL) was added. The reaction mixture
was stirred at room temperature for 3 h. After this time,
the solvent was concentrated under reduced pressure
and the residue was dissolved with saturated NaHCO3
solution. The resulting solution was extracted three
times with CH2Cl2. The organic phases were dried over
anhydrous MgSO4 and concentrated under reduced pressure
yielding the desired product (6) in 91% yield; IR (ATR)
n / cm-1 3403, 2926, 2850, 2732, 1682, 1605, 1590, 1500,
1463, 1316, 1265, 1218, 1120, 1040, 1004, 975, 862, 789;
1H NMR (400 MHz, CDCl3) d 1.25-1.39 (m, 3H), 1.70-1.82
(m, 3H), 1.89-1.95 (m, 1H), 2.55 (sl, 2H), 2.69-2.73
tert-Butyl(2-(4-((5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-
2-yl)methyl)piperidin-1-yl)ethyl)carbamate (7)
A solution of compound 6 (0.20 g, 0.61 mmol),
NaI (0.09 g, 0.61 mmol) and N,N-diisopropylethylamine
(0.16 g, 1.22 mmol) in acetonitrile (12 mL) was dropwise
added to a solution of tert-butyl (2-bromoethyl)carbamate
(0.18 g, 0.79 mmol) in acetonitrile (1 mL). Then, the
reaction was heated to reflux temperature and kept under
stirring for 24 h. After that time, a reaction mixture was
concentrated under reduced pressure and the residue was
solubilized in ethyl acetate and washed with solution of
potassium carbonate (K2CO3) 1 M. The organic phase
was extracted with two portions of ethyl acetate. The
organic phase was then dried over anhydrous MgSO4 and

Vol. 29, No. 4, 2018
Terra et al.
745
concentrated under reduced pressure on a rotary evaporator.
The crude reaction was purified by chromatography column
eluted with ethyl acetate/methanol (4:1, v:v) and the desired
product 7 was obtained in 39% (0.10 g); IR (ATR) n / cm-1
3426, 2920, 2852, 1692, 1626, 1468, 1364, 1316, 1256,
1170, 1118, 1042; 1H NMR (400 MHz, CDCl3) d 1.14-1.41
(m, 6H), 1.46 (s, 9H), 1.62-1.81 (m, 4H), 1.88-1.94
(m, 1H), 2.68-2.76 (m, 4H), 3.25 (dd, 1H, J = 20, 8 Hz),
3.91 (s, 3H), 3.96 (s, 3H), 4.08-4.13 (m, 2H), 6.86 (s, 1H),
74% yield; IR (ATR) n / cm-1 3383, 3279, 3071, 2926, 2853,
2641, 2548, 2363, 1739, 1727, 1687, 1606, 1591, 1562,
1548, 1542, 1536, 1500, 1456, 1439, 1365, 1316, 1266,
1223, 1191, 1160, 1121, 1088, 1077, 1066, 1037, 1008,
972, 952, 862, 848, 797, 765, 750; 1H NMR (400 MHz,
CDCl3) d 1.40-1.52 (m, 3H), 1.63-1.76 (m, 7H), 1.83-1.96
(m, 4H), 2.26 (t, 2H, J = 8 Hz), 2.32-2.40 (m, 2H), 2.46
(td, 1H, 2J = 16 Hz, 3J = 8 Hz), 2.67-2.71 (m, 2H), 2.77
(t, 2H, J = 5 Hz), 3.08-3.21 (m, 4H), 3.28 (dd, 1H, J = 20,
8 Hz), 3.50 (q, 2H, J = 5 Hz), 3.58 (qui, 1H, J = 8 Hz),
3.91 (s, 3H), 3.97 (s, 3H), 6.86 (s, 1H), 7.05 (sl, 1H, NH),
7.16 (s, 1H); 13C NMR (100 MHz, CDCl3) d 25.4 (CH2),
29.0 (CH2), 29.8 (CH2), 30.0 (CH2), 32.7 (CH), 33.8 (CH2),
34.7 (CH2), 36.3 (CH2), 38.1 (CH2), 38.6 (CH2), 40.4
(CH2), 44.8 (CH), 53.7 (CH2), 56.3 (CH3), 56.4 (CH3),
56.7 (CH), 57.5 (CH2), 104.6 (CH), 107.6 (CH2), 129.2
(CAr), 148.8 (CAr), 149.8 (CAr), 155.9 (CAr), 173.8 (NC=O),
207.2 (C=O); HRMS (ESI) m/z, observed: 521.2452 for
C27H40N2O4S2 [M + H]+; required: 521.2507.
13
7.17 (s, 1H); C NMR (100 MHz, CDCl3) d 28.7, 32.0,
33.0, 33.6, 34.8, 38.9, 44.2, 45.4, 56.3, 56.4, 79.5, 104.7,
107.6, 129.5, 148.8, 149.8, 155.1, 155.8, 207.7; HRMS
(ESI) m/z, observed: 433.2684 for C24H36N2O5 [M + H]+;
required: 432.2624.
1-(2-Aminoethyl)-4-((5,6-dimethoxy-1-oxo-2,3-dihydro-
1H-inden-2-yl)methyl)piperidin-1-ium 2,2,2-trifluoroacetate
(8)
Trifluoroacetic acid (1.2 mL) was added to a solution
of 7 (0.08 g, 0.18 mmol) in CH2Cl2 (1.2 mL) under ice
bath. The reaction was stirred for 1 h. After this period, the
reaction mixture was concentrated under reduced pressure
and the desired product 8 was obtained in quantitative yield.
IR (ATR) n / cm-1 3422, 2958, 2922, 2872, 2852, 1690,
1610, 1500, 1458, 1318, 1268, 1204, 1128, 1036; 1H NMR
(400 MHz, CD3OD) d 1.35-1.36 (m, 1H), 1.47-1.57 (m,
1H), 1.65-1.74 (m, 2H), 1.92-2.01 (m, 2H), 2.11-2.21 (m,
2H), 2.79-2.83 (m, 2H), 3.18 (sl, 2H), 3.52 (sl, 4H) 3.70
(sl, 2H), 3.92 (s, 3H), 4.00 (s, 3H), 7.12 (s, 1H), 7.21 (s,
1H), 7.94-7.95 (m, 1H, NH); 13C NMR(100 MHz, CD3OD)
d 19.5, 30.9, 33.2, 34.3, 39.0, 46.3, 54.6, 54.9, 56.6, 56.9,
105.5, 109.2, 129.9, 151.3, 151.5, 157.9, 209.9; HRMS
(ESI) m/z, observed: 333.2166 for C19H28N2O3 [M + H]+;
required: 333.2178.
Determination of inhibitory effect on AChE and BuChE
activity
The capacity of compound 2 and donepezil to
inhibit AChE activity was assessed using the Ellman
method.40 Initial rate assays were performed at 37 °C
with a Jasco V-530 double beam spectrophotometer
by following the rate of increase in the absorbance at
412 nm for 3 min. AChE stock solution was prepared
by dissolving human recombinant AChE (E.C.3.1.1.7)
lyophilized powder (Sigma, Italy) in 0.1 M phosphate
buffer (pH = 8.0) containing Triton X-100 (0.1% v:v).
Stock solution of BuChE (E.C. 3.1.1.8) from human
serum (Sigma, Italy) was prepared by dissolving
the lyophilized powder in an aqueous solution of
gelatine (0.1% m:v). The final assay solution consisted of a
0.1 M phosphate buffer pH 8.0, with the addition of 340 mM
5,5’-dithio-bis(2-nitrobenzoic acid), 0.02 unit mL-1 of
human recombinantAChE, or BuChE from human serum
and 550 mM of substrate (acetylthiocholine iodide, ATCh
or butyrylthiocholine iodide, BTCh, respectively). Stock
solutions of 2 were prepared in methanol and diluted
in methanol, while donepezil was solubilized in water
and dilutions were prepared in water. Five different
concentrations of inhibitor were selected in order to obtain
inhibition of the enzymatic activity comprised between
20 and 80%. 50 mL aliquots of increasing concentration
of inhibitor were added to the assay solution and pre
incubated for 20 min at 37 °C with the enzyme before the
addition of the substrate. Assays were carried out with a
blank containing all components except AChE or BuChE
N-(2-(4-((5,6-Dimethoxy-1-oxo-2,3-dihydro-1H-inden-
2-yl)methyl)piperidin-1-yl)ethyl)-5-(1,2-dithiolan-3-yl)
pentanamide (2)
A solution of lipoic acid (0.05 g, 0.22 mmol) and
PyBOP (0.11 g, 0.22 mmol), in anhydrous CH2Cl2
(3.5 mL), at 0 °C, was stirred for 30 min and then
cannulated to a flask containing a solution of 8 (0.11 g, 0.24
mmol), N,N-diisopropylethylamine (0.23 g, 1.76 mmol),
in anhydrous CH2Cl2 (4 mL) and the resulting mixture was
stirred at room temperature for 20 h. Then, the reaction
was quenched by the addition of water and the mixture
was extracted four times with CH2Cl2. The combined
organic layers were dried with MgSO4, filtered and
concentrated under reduced pressure. The crude reaction
was purified by chromatography column eluted with
ethyl acetate/methanol (10%) yielding 2 as oil (0.08 g) in

746
Two Novel Donepezil-Lipoic Acid Hybrids
J. Braz. Chem. Soc.
in order to account for the non-enzymatic reaction. The
reaction rates were compared and the percent inhibition
due to the presence of inhibitor was calculated. Each
concentration was analysed in duplicate, and IC50 values
were determined graphically from log concentration
versus % inhibition curves (GraphPad Prism 4.03
software, GraphPad Software Inc.).47
Supplementary Information
1
Copies of H and 13C NMR spectra of synthesized
compoundsareavailablefreeofchargeathttp://jbcs.sbq.org.br
as PDF file.
Acknowledgments
Scavenging of DPPH radicals
This research was carried out in the framework of
B2AlzD2 Joint lab. Financial support was obtained from
the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), Fundação de Amparo à Pesquisa do
Estado de Minas Gerais (FAPEMIG), Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq)
and from the Italian Ministry of Education, Universities
and Research (MIUR). Dr Christian Bergamini and Prof
Romana Fato from Alma Mater Studiorum University of
Bologna are acknowledged for their contribution to the
characterization of the activity profile of the new hybrids.
The ability of hybrids to scavenge DPPH radical, a
reactive nitrogen species (RNS), was determined according
to Gülçin48 with modifications. The screening was done by
incubating 50 µL of each compound in an ethanolic medium
containing 50 µL of 200 µM DPPH. Final concentrations of
test compounds were between 20-640 µM and the DPPH
was 100 µM. The systems were maintained under stirring
in the dark for 30 min. The absorbance at 517 nm was
recorded. Each concentration was tested in triplicate.
Theoretical calculations
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orientation (encoded as a rotation vector and a rotation
angle), and one angle for each flexible torsion angle in
the ligand.
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Submitted: September 5, 2017
Published online: October 31, 2017