New Tacrine Hybrids with Natural-Based Cysteine Derivatives as Multitargeted Drugs for Potential Treatment of Alzheimer's Disease

Article abstract of DOI:10.1111/cbdd.12633

Alzheimer's disease (AD) is a devastating age-dependent neurodegenerative disorder. The main hallmarks are impairment of cholinergic system and accumulation in brain of beta-amyloid (Aβ) aggregates, which have been associated with oxidative damage and dyshomeostasis of redox-active biometals. The absence of an efficient treatment that could delay or cure AD has been attributed to the complexity and multifactorial nature of this disease. With this in mind and the recent interest on natural-based drugs, we have explored a set of natural-based hybrid compounds by conjugation of a tacrine moiety with an S-allylcysteine (garlic constituent) or S-propargylcysteine moiety aimed at improving the cholinergic system and neuroprotective capacity. The docking modeling studies allowed the selection of linkers to optimize the bimodal drug interaction with acetylcholinesterase enzyme (AChE) active site. The compounds were evaluated for some representative biological properties, including AChE activity and Aβ aggregation inhibition, as well as for their neuroprotective activity to Aβ- and ROS-induced cellular toxicity.

Full text of DOI:10.1111/cbdd.12633

Chem Biol Drug Des 2016; 87: 101–111
Research Article
New Tacrine Hybrids with Natural-Based Cysteine
Derivatives as Multitargeted Drugs for Potential
Treatment of Alzheimer’s Disease
1
,2
1
Rangappa S. Keri , Catarina Quintanova , S ꢀı lvia
tide fragment 1-42; Ab, beta-amyloid; CAS, catalytic anionic
site; DHE, dihydroethidium; DMEM, Dulbecco’s modified
Eagle’s medium; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical;
1
3
3,4
Chaves , Diana F. Silva , Sandra M. Cardoso
and M. Am eꢀ lia Santos *
1
,
0
DTNB, 5,5 -dithiobis(2-nitrobenzoic acid); HEPES, 2-[4-(2-hy-
1
droxyethyl)-1-piperazine]ethanesulfonic acid; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMM,
N-methylmorpholine; PAS, peripheral anionic active site; RFU,
relative fluorescence units; SAC, S-allylcysteine; SPRC, S-
propargylcysteine; T3P, 2,4,6-tripropyl-1,3,5,2,4,6-trioxat-
riphosphorinane-2,4,6-trioxide; TRIS, tris(hydroxymethyl)amino
methane.
Centro de Qu ꢀı mica Estrutural, Instituto Superior T eꢀ cnico,
Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001
Lisboa, Portugal
2
Centre for Nano and Material Sciences, Jain University,
Jain Global Campus, Bangalore, Karnataka 562112, India
3
CNBC – Centro de Neuroci e^ ncias e Biologia Celular,
Universidade de Coimbra, 3030 Coimbra, Portugal
4
Faculdade de Medicina, Universidade de Coimbra, 3030
Coimbra, Portugal
Received 24 March 2015, revised 3 June 2015 and accepted
for publication 23 July 2015
*
Corresponding author: M. Am eꢀ lia Santos,
masantos@ist.utl.pt
Alzheimer’s disease (AD) is a devastating age-depen-
dent neurodegenerative disorder. The main hallmarks
are impairment of cholinergic system and accumula-
tion in brain of beta-amyloid (Ab) aggregates, which
have been associated with oxidative damage and
dyshomeostasis of redox-active biometals. The
absence of an efficient treatment that could delay or
cure AD has been attributed to the complexity and
multifactorial nature of this disease. With this in mind
and the recent interest on natural-based drugs, we
have explored a set of natural-based hybrid com-
pounds by conjugation of a tacrine moiety with an S-
allylcysteine (garlic constituent) or S-propargylcysteine
moiety aimed at improving the cholinergic system and
neuroprotective capacity. The docking modeling stud-
ies allowed the selection of linkers to optimize the
bimodal drug interaction with acetylcholinesterase
enzyme (AChE) active site. The compounds were eval-
uated for some representative biological properties,
including AChE activity and Ab aggregation inhibition,
as well as for their neuroprotective activity to Ab- and
ROS-induced cellular toxicity. The most promising
results were achieved by compounds 9d for the AChE
inhibition and 9l for the remarkable prevention of
superoxide production and Ab-induced cellular toxic-
ity.
Alzheimer’s disease is a progressive neurodegenerative
age-related disease characterized by dementia, cognitive
impairment, and memory loss and, in advanced stage, it
can lead to incapacitation and finally to death (1). The cur-
rently adopted therapeutic approach has been mostly
focused on increasing the cholinergic neurotransmission,
through acetylcholinesterase inhibitors (AChEI), although
the available inhibitors confer only modest temporary
improvements on cognitive, functional, and behavioral
symptoms of patients with AD (2).
The AD multifactorial nature is believed to be the main rea-
son for the absence of an effective treatment. It has been
recognized that multifaceted diseases may be more suc-
cessfully tackled through
a complex pharmacological
approach than through a single-target strategy. Accord-
ingly, the current drug strategy has been based on the
development of anti-Alzheimer agents with multiple poten-
cies (3,4), such as the inhibition of AChE and amyloid-beta
(Ab) peptide aggregation as well as antioxidant activity.
Moreover, quite an amount of natural-origin products is
being used either as marketed drugs or as bioactive
molecular fragments in the development of hybrid drugs to
combat the above referred major risk factors involved in
Alzheimer’s disease pathogenesis (5–8).
Key words: acetylcholinesterase inhibitors, Alzheimer’s dis-
ease, anti-Ab aggregation, antineurodegeneratives, S-allylcys-
teine, S-propargylcysteine, tacrine
Tacrine was a first drug approved for AD treatment, as
acetylcholinesterase inhibitor (AChEI), for the improvement
of the cholinergic system and the severe memory loss (9).
However, some toxicity limitations led to its withdrawal
Abbreviations: AChE, acetylcholinesterase; AChI, acetylthio-
choline iodide; AD, Alzheimer’s disease; Ab42, amyloid-b pep-
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12633
101

Keri et al.
from the market and substitution by other more moder-
ately active AChEIs, such as rivastigmine, donepezil, and
galantamine, although they only enable palliative treat-
ments. Aimed at overcoming the tacrine side-effects, a
huge amount of researchers have used this drug as inspi-
ration for the development of an enormous variety of poly-
heterocyclic analogs or of tacrine derivatives, as described
in a recent review on this topic (10). Remarkable is also
the number of tacrine hybrids that have been recently
developed through its conjugation with natural-origin prod-
ucts, such as hydroxyquinoline (11), coumarin (12), and
thioflavin derivatives (13), to combine the AChE inhibition
with antioxidant properties, metal-binding capacity, and
inhibition of Ab aggregation, respectively. Continuing our
recent research interest on the development of multifunc-
tional compounds with potential application in AD treat-
ment (13–15), and taking into account the considerable
importance that has been attributed to some natural prod-
ucts with neuroprotective and antioxidant roles (e.g. garlic
derivatives) (6,16), we have decided to develop a novel
series of hybrids conjugating tacrine with S-allylcysteine
effects in neuronal cells, namely against Ab-induced cellu-
lar toxicity and ROS-inducted oxidative stress. These data,
together with the molecular modeling of ligand–enzyme
interaction, allowed also the analysis of the structure/activ-
ity relationship.
Experimental Part
Molecular modeling
The modeling of ligand–protein docking was performed
following a procedure identical to that previously reported
(13). The X-ray crystallographic structure of acetyl-
cholinesterase Torpedo californica AChE (TcAChE) com-
plexed with an inhibitor was taken from RCSB Protein
a
Data Bank (PDB entry 1ODC) to be used as a receptor in
the docking simulations. The original complex structure
b
was treated using a MAESTRO v. 9.3 by removing the origi-
nal ligand, solvent, and co-crystalization molecules and
also by adding hydrogen atoms. The 3D structures of the
ligands were built with Maestro, and their geometry was
(SAC) or with S-propargylcysteine (SPRC) (Figure 1) to
combine the main biological properties of each molecular
moiety (e.g. AChE inhibition and neuroprotective role). The
linkers between these two main molecular moieties (tacrine
and cysteine derivatives) are mainly chosen aimed at
potential dual binding interaction with AChE, namely the
catalytic anionic site (CAS) and the peripheral anionic site
O
R2
HN
HN
S
H
NH2
n
R1
N
(PAS) of this enzyme. As 6-chlorotacrine has been
reported to have higher potency than tacrine as AChE inhi-
bitor (17), that unit was also incorporated in our hybrids.
The selected tacrine–cysteine hybrids (Figure 2) were eval-
uated for some of their most relevant physicochemical and
biological properties, namely the antioxidant capacity, the
inhibitory capacity against AChE and Ab self-aggregation.
They were also assessed for neuroprotective/rescuing
9
9
9
9
(a,b,c) R
1
= H; n = 2,3,4
= Cl;
= H; n = 2,3,4
R
2
=
(Tacrine-SAC)
(d,e,f) R
(g,h,i) R
1
1
R2
=… ……… (Tacrine-SPRC)
1
(j,k,l) R = Cl;
Figure 2: Chemical structure of the tacrine conjugates prepared
and studied.
Figure 1: Design strategy for the new
series of tacrine conjugates.
1
02
Chem Biol Drug Des 2016; 87: 101–111

Tacrine/Cysteine-Conjugated Compounds as Anti-AD Agents
optimized first by making a random conformational search
RCS) of 1000 cycles and then by 2500 optimization steps
phosphate buffer (0.215 M, pH 8.0) to obtain a 40 lM solu-
tion. Due to the hydrophobic nature of the compounds
under study, they were first dissolved in methanol (1 mg/
mL) and then further diluted in phosphate buffer to a final
concentration of 70 lM.
(
with a program GHEMICAL v. 2.0 (18). The ligands were sub-
sequently docked into the AChE structure using GOLD v.
5.1 (19) with the default parameters of GOLD (except ‘allow
early termination’ option) and the ASP scoring function, as
this function had been previously proven to give the best
docking predictions for AChE inhibition (14). The zone of
To study the Ab42 aggregation inhibition, a reported
method, based on the fluorescence emission of thioflavin
T (ThT), was followed (13,20). Firstly, Ab42 (10 lL) samples
and the tested compounds (10 lL) were diluted with the
phosphate buffer to a final concentration of 40 lM (Ab)
and 70 lM (compounds), and then, they were incubated
for 24 h at 37 °C, without stirring. As for the control, a
sample of the peptide was incubated under identical con-
ditions but without the inhibitor. After incubation, the sam-
ples were added to a black, flat-, and clear-bottom 96-
well plate (BD Falcon) with 180 lL of 5 lM ThT in 50 mM
glycine-NaOH (pH 8.5) buffer. Blank samples were pre-
pared for each concentration in a similar way, devoid of
peptide. After 5-min incubation with the dye, the ThT fluo-
rescence was measured using a Spectramax Gemini EM
(Molecular Devices) at the following wavelengths: 446 nm
(excitation) and 490 nm (emission). The percent inhibition
of the self-induced aggregation due to the presence of the
test compound was calculated by the equation I% = 100-
ꢁ
interest was defined as the residues within 10 A from the
original position of the ligand in the crystal structure.
Prediction of some pharmacokinetic proprieties and descrip-
tors was carried out using in silico tools, namely descriptors
c
using QIKPROP v. 2.5 provided by MAESTROb. The following
drug-like properties have been calculated: the lipo-hy-
drophilic character (clog P), blood–brain barrier partition
coefficient (log BB), the ability to be absorbed through the
intestinal tract (Caco-2 cell permeability), and CNS activity.
Acetylcholinesterase activity
The enzymatic activity was measured using an adaptation
of the method previously described (13). The assay solu-
tion contained 374 lL of Hepes buffer (50 mM and pH
8
5
.0), 476 lL of DTNB (3 mM), a variable volume (10–
0 lL) of the stock solution of each compound in metha-
i 0 i 0
(IF/IF 9 100), in which IF and IF corresponded to the
nol (1 mg/mL), 25 lL of AChE stock solution, and the
necessary amount of methanol to attain 0.925 mL of the
sample mixture in a 1-mL cuvette. The samples were left
to incubate for 15 min, and then, 75 lL of acetylthio-
choline iodide (AChI) solution (16 mM) was added. The
reaction was monitored for 5 min at 405 nm. Assays were
run with a blank containing all the components except
AChE, which was replaced by Hepes buffer. The velocities
of the reaction were calculated as well as the enzyme
activity. A control reaction was carried out using the sam-
ple solvent (methanol) in the absence of any tested com-
pound, and it was considered as 100% activity. The
percentage inhibition of the enzyme activity due to the
presence of increasing test compound concentration was
fluorescence intensities, in the presence and absence of
the test compound, respectively, minus the fluorescence
intensities due to the respective blanks. The reported val-
ues were obtained as the mean ꢁ SEM of duplicate of
two different experiments.
Antioxidant activity
The antioxidant activity was evaluated by the DPPH
method previously described (13,21). To a 2.5-mL solution
of DPPH (0.002%) in methanol, four samples of each
compound in solution were added in different volumes to
obtain different concentrations in a 3.5-mL final volume.
The samples were incubated for 30 min at room tempera-
ture. The absorbance was measured at 517 nm against
the corresponding blank (methanol). The antioxidant activ-
ity was calculated by an equation [%AA = (ADPPH–Asample)/
ADPPH 9 100].
calculated by the following equation (%I = 100-(v
9 100), in which v is the initial reaction rate in the pres-
ence of inhibitor and v is the initial rate of the control
i
/
v
o
i
o
reaction. The inhibition curves were obtained by plotting
the percentage of enzymatic inhibition versus inhibitor con-
centration, and a calibration curve was obtained from
which the linear regression parameters were obtained.
The tests were carried out in triplicate. The compound
concations providing 50% of antioxidant activity (EC50
)
were obtained by plotting the antioxidant activity against
the compound concentration.
Inhibition of self-mediated Ab (1–42) aggregation
Amyloid-b peptide (1–42) (Ab ) was purchased from
42
Aldrich as a lyophilized powder and stored at ꢀ20 °C. The
Cells and treatments
The human neuroblastoma SH-SY5Y cells (ATCCCRL-
2266) were grown in 75-cm tissue flasks in DMEM and
Ham’s F12 medium supplemented with 10% non-dialyzed
fetal bovine serum, 100 IU/mL penicillin, and 50 lg/mL
streptomycin and maintained at 37 °C in a humidified
incubator under an atmosphere of 95% air and 5% CO2.
samples were treated with 1,1,1,3,3,3-hexafluoropropan-
3
2
-ol (HFIP) to avoid self-aggregation and reserved. HFIP
pretreated Ab42 samples were re-solubilized with
CH CN/Na CO (300 lM)/NaOH (250 lM) (48.3:48.3:4.3,
a
3
2
3
v/v/v) solvent mixture in order to have a stable stock
solution. This Ab42 alkaline solution (500 lM) was diluted in
Chem Biol Drug Des 2016; 87: 101–111
103

Keri et al.
Cells were plated at a density of 0.1 9 10 cells/mL, in
6
dual binding mode, the adopted strategy consisted of
preselection of several connecting groups (spacers)
between the two main moieties, the tacrine (TAC) and the
S-allylcysteine/S-propargylcysteine (SAC/SPRC) (Figure 1),
and then performing the virtual screening of these mole-
cules against the target enzyme (AChE). The tested spacer
groups included an amide linkage and alkyleneamine
chains with variable length (two, three, or four carbon
atoms) to allow optimization of the spacer between the
two active molecular moieties.
6
2
4-well plates, for toxicity experiments and 0.16 9 10
cells/mL in cover slips for reactive oxygen species determi-
nation. Cells were incubated for 24 h with 1 lM Ab42 pep-
tide added from a stock solution of 276.9 lM prepared in
sterile water, or with 200 lM H O . Compounds 9(a–l) pre-
2
2
pared in DMSO were tested for cellular toxicity in the
.5 lM to 1 mM range of concentrations. Before the addi-
2
tion of Ab42, the cells were preincubated for 2 h with com-
pounds 9(a–l). The final concentration of DMSO in culture
media did not exceed 0.05% (v/v). For all the conditions
tested, control experiments were performed in which the
compounds tested or Ab42 were not added.
In fact, analysis of the AChE structure showed that the
active center of the enzyme was found in the bottom of a
deep and narrow gorge that is formed by two very impor-
tant amino acids, Phe330 and Trp84. The AChE also has
a peripheral anionic site (PAS), which is located at the
entrance of the gorge and also presents two aromatic resi-
dues, Tyr70 and Trp279 (24).
ꢂꢀ
Superoxide anion (O ) formation
2
ꢂꢀ
Superoxide anion (O ) formation was determined using
2
the fluorescence probe dihydroethidium (DHE) (Molecular
Probes, Invitrogen). DHE is permeable to the cell mem-
brane, and in cytoplasm, it is converted to ethidium by
The docking results revealed many similarities between
binding conformations of the new inhibitors, as it can be
seen in Figure 3. Because at physiological pH the com-
pounds should be protonated at the quinoline amino sub-
stituent group, they were modeled in this monoprotonated
form. This Figure shows that the ligands are well accom-
modated in the AChE cavity, and apparently, all the com-
ꢂꢀ
O . Then, ethidium binds to DNA emitting fluorescence
2
2 2
(22). Different H O concentrations (50, 100, 150, 200 lM)
were tested for 24 h to determine superoxide increased
production. The SHSY5Y cells were preincubated for 1 h
with the tacrine hybrids 9(a–l) and then treated with H O2
2
for 24 h (200 lM final concentration). After 24 h, 10 lM
DHE was dissolved in the cell growth medium and the
cells were preincubated during 1 h at 37 °C. Then, cells
were washed once with the Krebs medium and fluores-
cence measurements were taken in the Krebs medium
during 1 h (518-nm excitation; 605-nm emission). The
values were obtained as a slope of RFU absolute values
per 5 min for each condition.
pounds exhibit
a double binding mode with AChE.
Specifically, the tacrine moiety is found well inserted in the
bottom of the enzyme gorge, binding to the CAS by p–p
stacking with Trp84 and Phe330, and almost overlapping
with the tacrine moiety of the original ligand (Figure 3A).
Further analysis of the distance between the carbonyl oxy-
gen of His440 and the protonated quinoline nitrogen atom
of tacrine indicated they can establish a H-bond men-
tioned by Rydberg et al. (25), as the distance between the
quinoline nitrogen atom and carbonyl oxygen from His440
MTT cell proliferation assay
ꢁ
Cell reduction ability as a surrogate of cell viability was
measured by a quantitative colorimetric assay with 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
according to the Mosmann method (23). In viable cells,
cellular dehydrogenases metabolize MTT with the forma-
tion of formazan that absorbs light at 570 nm. Following
the cell treatment protocol, the medium was removed and
for all the compounds was from 2.7 to 3.3 A. Generally,
the spacer and the SAC/SPRC moiety seem to be also
well fitted along the hydrophobic cavity. Moreover, both
the carbonyl oxygen and the NH group of the linker amide
groups show ability for hydrogen bond interactions with
two protein residues, namely tyrosine 121 (Tyr121) and
aspartate 72 (Asp72). A more detailed analysis of the
docking results (Figure 3B) shows that the hybrid models
with 6-chlorotacrine revealed favorable orientations, which
may be due to some electron density reduction on the
tacrine aromatic ring, favoring the p-interaction (26), in
agreement with the expected improvement of the AChE
inhibitory activity. Comparison between the two series of
SAC/SPRC hybrids can be based on the docking of the
corresponding 6-chlorotacrine derivatives (Figure 3C,D).
Although there are some minor changes in the orientation
of the triple/double bonds of the cysteine moieties along
the PAS unit, both of them seem to establish similar p-
stacking interactions with the same residues (Trp 279 and
Tyr70) in the active site. Notably, the ligands with the
shorter linker (n = 2) seem to provide enough length to the
main spacer between the main moieties, therefore
0
.5 mL of MTT (0.5 mg/mL) was added to each well. The
plate was then incubated at 37 °C for 2 h. At the end of
the incubation period, the formazan precipitates were solu-
bilized with 0.5 mL of acidic isopropanol (0.04 M HCl/iso-
propanol). The absorbance was measured at 570 nm
using a Spectramax Plus 384 spectrophotometer (Molecu-
lar Devices). Cell reduction ability was expressed relatively
to control values obtained for untreated cells.
Results and Discussion
Molecular modeling
With the aim of obtaining useful information to design new
inhibitors with optimized binding interactions, ultimately
1
04
Chem Biol Drug Des 2016; 87: 101–111

Tacrine/Cysteine-Conjugated Compounds as Anti-AD Agents
Figure 3: Docking results for the tacrine
hybrids with TCAChE: (A) superimposition of
the original ligand (yellow) and 9a (blue); (B)
superimposition of 9a (blue) and 9d (red); (C)
superimposition of compounds of series
Cl-Tac-allyl: 9d (red), 9e (purple), and 9f
(
green); (D) superimposition of compounds
of series Cl-Tac-propargyl: 9j (ligh blue), 9k
pink), and 9l (orange).
(
enabling bimodal interaction with the both active sites,
while those with the longer linker (n = 4) seem to present
some distortion on the p–p stack of the multiple bonds
with the PAS aromatic residues.
procedures, by nucleophilic aromatic substitution of vari-
ous alkylenodiamines (1,2-diaminoethane, 1,3-diamino-
propane, and 1,4-diaminobutane) at the 9-chloro-position
of tacrine derivatives 3(a, b), under reflux in phenol, in the
presence of a catalytic amount of KI, and were obtained in
60–70% yield (27). The preparation of the L-cysteine-con-
taining a portion of the hybrid compounds involved the
nucleophilic substitution of allyl bromide or propargyl bro-
mide by the thiol group of L-cysteine, according to litera-
ture method, affording the S-allylcysteine (SAC, 6a) or S-
propargylcysteine (SPRC, 6b), respectively. Before conju-
gation of these moieties with the tacrine-based units, the
amino group of (6) was protected by di-tert-butyl dicarbon-
ate (Boc) under basic conditions to afford 7(a, b) in good
yield. The condensation of the cysteine derivatives 7(a, b)
with the different amino-tacrine derivatives 4(a–f) involved
the use of a carboxylic activating agent, 2,4,6-tripropyl-
Overall, these modeling studies predicted a good ability of
these hybrids to target both the CAS and PAS of AChE.
Furthermore, they allowed some rationalization of the
observed differences in the AChE inhibitory activities (see
below, Table 1).
Chemistry
Tacrine–SAC, 9(a–f), and tacrine–SPRC, 9(g–k), hybrids
were obtained following the convergent synthetic approach
depicted in Scheme 1. Details of the synthetic part and
the full characterization of the compounds are described
as supplementary material. The intermediates 3(a, b) were
prepared from anthranilic acid 1a, or 2-amino-4-
chlorobenzoic acid 1b, and cyclohexanone in the pres-
3
1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide, T P, and
NMM, leading to the formation of amide linkages and
affording the prefinal intermediates 8(a–l) (28). As the last
step, the Boc group in 8(a–l) was removed by a standard
method with trifluoroacetic acid (TFA), to give the desired
target hybrid compounds 9(a–l).
ence of POCl , with 90–92% overall yield according to the
3
reported procedure (13). The linker-bearing tacrine inter-
mediates 4(a–f) were prepared as established in previous
Chem Biol Drug Des 2016; 87: 101–111
105

Keri et al.
Table 1: Biological properties of the tacrine–SAC, 9(a–f), and tacrine–SPRC 9(g–l) hybrids, toward AChE inhibition (AChEI), antioxidant
activity (DPPH), and anti-Ab aggregation; calculated water/octanol lipophilicity (cLog P)
AChE
inhibition IC50 (lM)
Antioxidant
Ab aggregation
Inhib (%)
a
b
c
a,d
Compound code
R
1
R
2
n
clog P
EC50 (10 9 lM)
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
H
H
H
Cl
Cl
Cl
H
H
H
Cl
Cl
Cl
–
-CH
»
»
»
»
»
2
CH=CH
2
2
3
4
2
3
4
2
3
4
2
3
4
–
2.829
2.987
3.540
3.394
3.564
3.940
2.603
2.779
2.801
2.887
3.153
3.263
2.53
1.59
0.88
1.20
0.30
0.68
0.51
1.21
1.72
1.21
0.56
0.59
0.97
0.19
65.7
86.5
73.5
55.8
50.5
99.2
23.8
41.7
52.5
56.3
55.1
75.6
>100
8.5
–
–
10.9
14.7
10.3
13.0
–
g
h
i
-CH
»
2
CꢃCH
»
»
»
»
–
j
11.4
12.1
–
k
l
Tac
–
–
The thioflavin T fluorescence method was used, and the measurements were taken in the presence of an inhibitor (80 lM).
a
Predicted values using program QikProc.
The standard deviation is within 10%.
EC50 values for the DPPH assay.
Inhibition of self-mediated Ab (1–42) aggregation (40 lM).
b
c
d
Cl
HN
N
NH2
n
(i)
O
O
R1
N
R2
R1
HN
HN
R
2
H
S
HN
HN
S
H
3
3
a: R = H
1
4
4
a-c: R = H; n = 2, 3, 4
NHBoc
b: R = Cl
1
(iv)
n
NH2
1
d-f: R = Cl; n = 2, 3, 4
(v)
n
1
HS
H
R2
S
R1
N
R1
N
(ii-1,2)
H N
COOH
8a-l
9a-c: R = H; n = 2, 3, 4; R = allyl
1
2
2
H
XHN
9
d-f: R = Cl; n = 2, 3, 4; R = allyl
1
2
COOH
9g-i: R = H; n = 2, 3, 4; R
2
= propargyl
1
5
9j-l: R = Cl; n = 2, 3, 4; R = propargyl
6
a: X =H; R = allyl
1
2
2
6
b: X =H; R = propargyl
2
(iii)
7
7
a: X = Boc; R = allyl
2
b: X = Boc; R = propargyl
2
Scheme 1: General method for the synthesis of the tacrine conjugates 9(a–f). (i) 2 equiv H
bromide, NH OH (2M), rt, 3 h; (iii) di-tert butyl dicarbonate, in tetrahydrofuran and Na CO , rt, 5 h; (iv) 1.5 equiv T
h, (v) THF, CH Cl , rt, 2 h.
2
N(CH
2
)
n
NH
2
, phenol, KI, reflux, 4 h; (ii) allyl
4
2
3
3
P, 2.5 equiv NMM, rt,
3
2
2
AChE inhibition
The AChE inhibitory activity of the newly synthesized com-
pounds 9(a–f) was measured in vitro by Ellman s assay (29)
6-chlorine atom, which, by filling the hydrophobic pocket,
accounts for the enhancement of the AChE inhibitory
potency (30). Although some tacrine hybrids have been
reported with higher AChE inhibitory potency than tacrine,
the present compounds exhibit potencies in the same
range of magnitudes or somewhat lower. However, as their
potency is still in the range of currently used drugs (low
micro- to submicromolar range) and they are endowed with
further functionalities with cooperative antineurodegenera-
tive roles, their interest can be anticipated as found for
other hybrid compounds (10). Among the compounds
investigated, the 6-chlorotacrine hybrids presented high
0
on AChE from Torpedo californica (TcAChE), and their IC50
values are shown in Table 1. Our options for the use of
TcAChE instead of human AChE are due to the fact that
the active site structures are quite conservative and also
the higher price of second one. This set of tacrine hybrids
evidenced high inhibitory potential with IC50 values between
low micromolar (tacrine derivatives) and high nanomolar
range (6-chlorotacrine derivatives). The higher potency
found for the chloro-substituted tacrine [9(d–f) and 9(j–l)],
as compared with non-substituted counterparts, is accord-
ing to our expectations based on a well-known effect of the
inhibitory
activity
in
submicromolar
range
(9d,
IC50 = 0.30 lM) close to that of tacrine (IC50 = 0.19 lM),
1
06
Chem Biol Drug Des 2016; 87: 101–111

Tacrine/Cysteine-Conjugated Compounds as Anti-AD Agents
while the corresponding unsubstituted tacrine analogs are
less potent, ca 0.5–1 order of magnitude.
aggregation is not expected to be relevant because the
chelating capacity of the ligands toward copper appears to
2+
be only moderate, pCu ca 7 (pCu = ꢀlog [Cu ], C
L
/
ꢀ
5
A structure–activity analysis of the inhibitory potency of this
set of hybrids against AChE should be mainly based on
inhibitor–enzyme interactions which should depend on the
type of substituents and the linker length. According to our
expectations and first results from molecular modeling
studies with TAC-SAC hybrids, the spacer with n = 3
C
= 10, C = 10 M) (32).
Cu L
Antioxidant activity
The antioxidant activity of the synthesized tacrine hybrids
9(a–l) was evaluated based on their interaction with 2,2-
diphenyl-1-picrylhydrazyl (DPPH) (15,21). DPPH is a stable
free radical molecule that can accept an electron or hydro-
gen radical and thus be converted into a stable, diamag-
netic molecule. DPPH has an unpaired electron and so it
has a strong absorption band at 517 nm (purple color)
which generally disappears when an antioxidant is present
in the medium. Analysis of the antioxidation results (see
Table 1) shows only moderate activity in the medium-high
micromolar concentration. There is a mild improvement of
the antioxidant activity of these hybrids, as compared with
the measured values for tacrine, SAC, and SPRC (EC50
1000 lM), under the same experimental conditions, which
may be indicative of some synergist effect. The antioxidant
activity measured herein for SAC and SPRC is quite low,
although they have recognized in vitro neuroprotective
activity (33); this may be associated with an increased
antioxidant capacity inside the cells due to enzymatic
deprotection of the sulphhydryl group, similar to the intra-
cellular glutathione, the major antioxidant in nerve cells
(34). Therefore, for the tacrine–organosulfur cysteine
hybrids herein studied, identical neuroprotective activity
inside the cell is also expected, as otherwise recently
reported for other tacrine-mercapto derivatives (35).
(
n = number of carbon atoms of the alkylene chain)
appeared to be better suited for the interaction with both
the CAS and PAS of AChE. However, the reasonable simi-
larity found on the docking interactions for all the range of
n values (2–4) used was the rational for synthesis and
study of the all set of compounds.
Analysis of AChE inhibition results (Table 1) evidences two
groups of values, in the same range of magnitude, which
are mainly determined by the tacrine substitution. Inside
the respective two hybrid groups, the length of the linker
and the cysteine substituent have some but minor effect.
For the 6-chlorotacrine hybrids, the best inhibition was
always obtained for n = 2, independently of the cysteine
substituent group, allyl (9d, IC50 = 0.30 lM) or propargyl
(
9j, IC50 = 0.56 lM). Regarding the non-substituted tacrine
hybrids, the best linker appeared for n = 3 or n = 4,
depending on the respective conjugation with allylcysteine
(
9b,
IC50 = 0.88 lM)
or
propargylcysteine
(9i,
IC50 = 1.21 lM).
After all, the chloro-substituent induces some expected
positive extra-interaction of the hybrids inside the CAS of
AChE, whereas the change from the thioallyl to the thio-
propargyl group on the cysteine unit seems to induce
some perturbation on the interaction with the active upper
gorge region, namely the PAS. Although the new hybrids
are somehow weaker inhibitors than tacrine, the extra
functionalities due to SAC and SPRC groups are expected
to provide other neuroprotective properties and therefore
counterbalance this disadvantage.
2 2
Efficiency in preventing Ab42-and H O -induced
toxicity in human neuroblastoma cells
To evaluate a potential therapeutic action of the com-
pounds 9(a–l), SH-SY5Y cells were treated with Ab42 pep-
tide or H O , which constitute a good in vitro cellular
2
2
model for studying neurodegeneration associated with AD.
The cell proliferation assay makes use of a quantitative
colorimetric method with 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) and accounts for the
viability of the SH-SY5Y cells, with and without treatment.
Dose–response graphs (Figure S1A,B) showed that cell
viability is not significantly affected after a 24-h treatment
with 2.5 lM tacrine hybrids. As oxidative stress has been
described as a key feature of the neurodegenerative pro-
cess in AD, we determined the neuroprotective effect of
Inhibition of Ab42 self-induced aggregation
To evaluate the capacity of the newly synthesized com-
pounds to inhibit Ab42 self-aggregation, an in vitro assay was
performed by monitoring the presence of amyloid fibrils by
thioflavin T (ThT). Amyloid fibrils have a secondary structure
of b-sheet to which ThT is known to bind and its fluores-
cence emission can be monitored, with the absorption peak
at 446 nm and the emission peak at 490 nm (31). The inhibi-
tion studies were carried out upon incubating Ab42 with and
without the compounds at 37 °C, for 24 h. The results
summarized in Table 1 show that the compounds (70 lM)
studied decreased Ab42 (40 lM) self-aggregation in similar
order of magnitudes (9–15%). This can be explained by their
structure, which is not similar to that of ThT and therefore
does not allow an adequate interaction with the b-sheet of
amyloid fibrils. Some effect of the ligands on copper-induced
2 2
the tacrine hybrids in a cellular context. Indeed, H O
(200 mM, 24 h) induced an increase in superoxide radical
production in SHSY-5Y cells (Figure S2). Among the
tacrine hybrids tested 9(a–l), the most effective in prevent-
2 2
ing H O -mediated oxidation were (9a), (9f), (9k), and (9l),
as they significantly reduced superoxide production in cells
exposed to H O2 (Figure 4A,B). Increased levels of ROS
2
can directly damage various macromolecules, through lipid
peroxidation and/or protein oxidation which may have
Chem Biol Drug Des 2016; 87: 101–111
107

Keri et al.
A
A
1
1
0
0
.5
.0
.5
.0
2
2
1
1
5
0
5
0
5
0
**
#
*
#
*
*
**
*
*
***
#
#
B
1
1
.5
.0
B
25
20
15
10
5
0
#
#
##
###
**
##
#
*
**
#
#
#
0.5
0
.0
Figure 5: Effect of tacrine–allylcysteine (A) and tacrine–
propargylcysteine (B) on Ab42 peptide induced toxicity in SH-
SY5Y cells. Cells were treated with Ab42 (1 lM) peptides, for 24 h
in the absence or the presence of tacrine compounds. Evaluation
of cell viability was performed by using MTT reduction test.
Results are expressed as the percentage of SH-SY5Y untreated
cells, with the mean ꢁ S.E.M. derived from three different
experiments. *p < 0.05; **p < 0.01; ***p < 0.001, significantly
different when compared with SH-SY5Y untreated cells;
Figure 4: Effect of tacrine–allylcysteine hybrids (A) and tacrine–
propargylcysteine hybrids (B) on superoxide production in
SH-SY5Y cells challenged with
2
H O
2
following ethidium
fluorescence. Data are expressed as the slope per 5 min obtained
from RFU absolute values and are the mean ꢁ SEM for three
independent experiments. **p < 0.01, significantly different as
#
##
compared to untreated cells;
p < 0.05 and
-treated cells.
p < 0.01
significantly different as compared to H
2 2
O
#
##
###
p < 0.05; p < 0.01;
p < 0.001, significantly different when
compared with Ab42-treated SH-SY5Y cells.
impact on cell survival and contribute to the neurodegen-
erative process in AD.
central nervous system (CNS), a set of physicochemical
and absorptional descriptors were calculated using the
Maestro and QikProp software, which included the lipo-hy-
drophilic character (clog P), blood–brain barrier partition
coefficient (log BB), the ability to be absorbed through the
intestinal tract (Caco-2 cell permeability), and CNS activity
(Table S1). Based on the calculated values of the Lipin-
Ab peptide oligomers, one important histopathological
hallmark in AD, can activate/potentiate the neurodegenera-
tive pathway in AD. Accordingly, Figure 5 shows a signifi-
cant decrease in cell viability after a 24-h treatment with
1
lM Ab42 peptides (about 20% decrease with respect to
the control). Compounds 9d, 9e, 9(g–j), and 9l prevented
cell viability decrease, which indicates that they are able to
overcome Ab42 toxicity. Based on these results obtained
in a cellular context, some of these tacrine hybrids seem
promising to be used in future studies.
0
sky s rule of 5, none of the compounds under study pre-
sented any violation of that rule. All the compounds show
a reasonably good lipophilicity (clogP < 5), although some
of them (9a, 9b, 9i) present the calculated log BB values
close to the limit predicted for a poor distribution in the
brain (< ꢀ1), which may also be related to the prediction
of the corresponding lower penetration to the central ner-
vous system (CNS) (36). The values for the Caco-2 perme-
ability prediction are also inside the normal range for
Blood–brain barrier permeability
To evaluate the ability of the compounds in study to cross
the blood–brain barrier (BBB) and gain access to the
1
08
Chem Biol Drug Des 2016; 87: 101–111

Tacrine/Cysteine-Conjugated Compounds as Anti-AD Agents
drugs. Overall, this set of values can be taken as a broad
indication that most of these compounds permeate the
BBB and access the CNS, but we are aware that perme-
ability is likely more complex due to the enzymes and
efflux transporters that can prevent and control the drug
accessibility.
Serralheiro for the support of the spectrofluorimetric
measurements.
Disclosure of Interest
The authors declare that they have no conflict of interests
concerning this article.
Conclusions
The absence of drugs for effective treatment AD, attribu-
ted to the multifactorial nature of this disease, and the
recent intezcrest on natural-based drugs led us to explore
and make an integrated study of a set of nature-origin
hybrid compounds, combining the tacrine and garlic con-
stituent, namely S-allylcysteine, or the corresponding
S-propargylcysteine derivative to tackle the acetyl-
cholinesterase (AChE) inhibition with neuroprotective roles,
respectively. The docking modeling studies allowed the
selection of linkers to improve the compound accommo-
dation within the AChE active site and the concomitant
enzyme inhibition. The compounds were evaluated for
some of the most relevant biological properties, including
AChE inhibition (AChEI), the neuroprotective effect to Ab-
and ROS-induced toxicity, and also for the antioxidant
activity (DPPH). Among those compounds, the 6-chloro-
tacrine derivatives demonstrated to have better AChEI
activity (submicromolar) than the non-substituted analogs;
the bioassays in ‘neuronal-like’ cells revealed some neuro-
protective activity, although a rationale for the different
behavior is not yet enough clear. The antioxidant activity
determined by the DPPH method was in the micromolar
range. Interestingly, we observed that the compounds that
most efficiently prevented Ab-induced cellular toxicity had
more pronounced antioxidant capacity. Overall, our pre-
liminary explorations of this new set of natural-based
tacrine hybrids revealed neuroprotective activity, namely in
Ab-induced toxicity [compounds 9d, 9e, 9(g–j), and 9l]
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Chem Biol Drug Des 2016; 87: 101–111

Tacrine/Cysteine-Conjugated Compounds as Anti-AD Agents
Supporting Information
Table S1. Predicted pharmacokinetic values.
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Representative graphic of compound dose-re-
sponse citotoxicity in SH-SY5Y cells.
Appendix S1. Experimental details and characterization
Figure S2. Superoxide production in SH-SY5Y cells chal-
for the synthesized compounds.
2 2
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Chem Biol Drug Des 2016; 87: 101–111
111