Design, synthesis and neuroprotective evaluation of novel tacrine-benzothiazole hybrids as multi-targeted compounds against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2013.05.028

Alzheimer's disease (AD) is a multifactorial disorder with several target proteins contributing to its etiology. In search for multifunctional anti-AD drug candidates, taking into account that the acetylcholinesterase (AChE) and beta-amyloid (Aβ) aggregat

Full text of DOI:10.1016/j.bmc.2013.05.028

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and neuroprotective evaluation of novel
tacrine–benzothiazole hybrids as multi-targeted
compounds against Alzheimer’s disease
Rangappa S. Keri a, Catarina Quintanova a, Sérgio M. Marques a, A. Raquel Esteves b, Sandra M. Cardoso b,c
,
M. Amélia Santos a,
⇑
a Centro de Quimica Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal
b Centro de Neurociencias e Biologia Celular, Universidade de Coimbra, 3030 Coimbra, Portugal
c Faculdade de Medicina, Universidade de Coimbra, 3030 Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Alzheimer’s disease (AD) is a multifactorial disorder with several target proteins contributing to its eti-
ology. In search for multifunctional anti-AD drug candidates, taking into account that the acetylcholines-
terase (AChE) and beta-amyloid (Ab) aggregation are particularly important targets for inhibition, the
tacrine and benzothiazole (BTA) moieties were conjugated with suitable linkers in a novel series of
hybrids. The designed compounds (7a–7e) were synthesized and in vitro as well as in ex vivo evaluated
for their capacity for the inhibition of acetylcholinesterase (AChE) and Ab self-induced aggregation, and
also for the protection of neuronal cells death (SHSY-5Y cells, AD and MCI cybrids). All the tacrine–BTA
hybrids displayed high in vitro activities, namely with IC50 values in the low micromolar to sub-micro-
molar concentration range towards the inhibition of AChE, and high percentages of inhibition of the
self-induced Ab aggregation. Among them, compound 7a, with the shortest linker, presented the best
Received 15 April 2013
Revised 9 May 2013
Accepted 17 May 2013
Available online xxxx
Keywords:
Tacrine
Benzothiazole
Alzheimer’s disease
Acetylcholinesterase inhibitors
Anti-Ab aggregation
Anti-neurodegeneratives
inhibitory activity of AChE (IC50 = 0.34
lM), while the highest activity as anti-Ab42 self-aggregation,
was evidenced for compound 7b (61.3%, at 50
lM. The docking studies demonstrated that all compounds
are able to interact with both catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. Our
results show that compounds 7d and 7e improved cell viability in cells treated with Ab42 peptide. Overall,
these multi-targeted hybrid compounds appear as promising lead compounds for the treatment of Alz-
heimer’s disease.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
involved factors have been identified and found consistent with
its onset. Among them is the impairment of the cholinergic system,
Neurological disorders inflict a heavy burden on healthcare
costs, affecting individuals and their caregivers. Alzheimer’s dis-
ease (AD) is classified as a progressive, neurodegenerative disease
that affects the cholinergic regions of the central nervous system
(CNS) that associates with cognitive function and spatial aware-
ness.1 Data of 2010 reported that it affected approximately 36 mil-
lion people worldwide, being expected to increase up to 114
million by 2050.2 The etiopathogenesis of this multifactorial dis-
ease still remains unknown, although, in the last decades, several
as indicated by the presence of altered cholinergic markers in AD
patients, resulting in a pronounced acetylcholine (ACh) deficiency
which translates into a generalized cognitive decline.3,4 Other hall-
marks of AD patient brains is the misfolding and aggregation of
specific proteins that accumulate intracellularly (e.g., neurofibril-
lary tangles,5,6 containing hyperphosphorylated tau protein) or
extracellularly (e.g., senile plaques,7,8 mostly composed of beta-
amyloid peptide (Ab) aggregates) that interfere with relevant brain
biological functions.
There have been many approaches to potential therapies, but to
date cholinesterase inhibitors (ChEIs) were the first and the only
class of drugs in the market that showed some results in the treat-
ment of AD. Tacrine, donepezil, rivastigmine and galantamine
(Fig. 1) are examples of acetylcholinesterase (AChE) inhibitors that
have been approved by the US Food and Drug Administration
(FDA). These inhibitors have beneficial effects on cognitive, func-
tional and behavioral symptoms of AD.9,10
Abbreviations: AD, Alzheimer’s disease; Ab, beta-amyloid; Ab42, amyloid-b
peptide fragment 1–42; ACh, acetylcholine; AChE, acetylcholinesterase; AChI,
acetylcholine iodide; BTA, benzothiazole; CAS, catalytic active site; DPPH, 2,2-
diphenyl-1-picrylhydrazyl radical; DTNB, 5,50-dithiobis(2-nitrobenzoic acid);
HEPES, 2-[4-(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PAS, peripheral active
site; TRIS, tris(hydroxymethyl)aminomethane.
⇑
Corresponding author. Tel.: +351 218419301; fax: +351 218464455.
E-mail address: masantos@ist.utl.pt (M.A. Santos).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.028
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2
R. S. Keri et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
O
NH2
N
NH2
N
S
NH2
O
N
N
O
Tacrine
Benzothiazole
1. Tacrine
2. Donepezil
O
O
O
CH3
N
O
H
H
N
H3C
N
CH3
S
CH3
CH3
N
linker
N
HO
3. Rivastigmine
4. Galantamine
HN
S
N
Thioflavin-T
Figure 2. Design strategy for the new series of tacrine–benzothiazole hybrids.
N
Figure 1. Chemical structure of reported AChE inhibitors (tacrine, donepezil,
rivastigmine and galantamine), and an Ab-aggregation inhibitor (thioflavin-T).
2. Results and discussion
2.1. Molecular modeling
Among them, tacrine was the first AChE inhibitor approved by
the FDA for the treatment of AD.11,12 Recent studies have demon-
strated that homo- and hetero-dimers can improve the biological
profile of tacrine and even overcome some of its side effects.13 After-
wards, quite a number of tacrine derivatives, some of them as hybrid
potential drugs, have been developed to improve its activity, as
described in a recent review in this topic,14 including tacrine–
8-hydroxyquinoline,15 mercapto-tacrine derivatives,16 tacrine–
fluorobenzoic acid17 and tacrine–multialkoxybenzene hybrids.18
Moreover, based on evidences that reduction of amyloid depos-
its lead to alleviation of the AD’s symptoms,19,20 a considerable
current effort has been directed towards the identification and
the development of potential therapeutic agents able to reduce
the soluble amyloid oligomers burden.21,22 Among the many com-
pounds known for strongly interacting with the Ab peptides, some
include the benzothiazole (BTA) moiety in the main scaffold (e.g.,
the dye thioflavin-T (ThT, Fig. 1)).23 Thus, some BTA derivatives
have been used as Alzheimer’s brain imaging agents,24 while oth-
ers have evidenced capacity for inhibiting the peptide aggregation
and neuro-protective roles.25,26
More recently quite a number of studies have been focused on
the development of therapeutic strategies multitarget drugs to
fight multifactorial neurodegenerative disorders, such as AD. Thus,
one of the most explored design strategies has been focused on the
development of hybrids that combine the ChEs inhibition role, for
the enhancement of the cholinergic neurotransmission, with other
relevant roles (e.g., reduction the Ab fibril self-aggregation, anti-
oxidant and metal depletion).27–30 In this context, the development
of tacrine–BTA hybrid compounds appears as important challenge,
because they could be expected not only to act in the symptomatic
effects but also in the underlying molecular etiology of the dis-
ease.22 Therefore, we have designed a series of novel multifunc-
tional compounds by conjugating a tacrine and a BTA moiety
through different suitable linkers. A selection of these compounds
(Figs. 2 and 3) was synthesized and evaluated for their in vitro
activity as inhibitors of AChE and of self-induced Ab aggregation,
as well as for their antioxidant activity, because oxidative stress
is thought to play a major role in the pathogenesis of AD.31 In order
to evaluate the biological neuroprotective role of these com-
pounds, we determined their effect on the cell viability using dif-
ferent AD cellular models exposed to Ab peptides.
The strategy followed for the design of the new inhibitors con-
sisted of making a pre-selection of several connecting groups
(spacers) between the two main moieties, the tacrine and the
BTA (Fig. 1), and then performing the virtual screening of these
molecules against a target enzyme (AChE) involved in the choliner-
gic loss. The active site of this molecule is found in a deep pocket
were acetylcholine (substrate) can slip inside and be hydrolyzed
into acetic acid and choline, which is no longer a neurotransmitter.
At the base of the pocket there is the catalytic triad, composed of
Ser200, His440 and Glu327. Within the pocket of AChE there is also
the catalytic active site (CAS), which is formed by two important
amino acids, the Phe330 and Trp84. The AChE also contain a
peripheral active site (PAS), which is located at the entrance of
the gorge and is formed by Trp279 and Tyr70 (sequence number-
ing of Torpedo californica AChE, TcAChE).
The docking study was carried out using program GOLD, v.
5.1.32 The crystal structure of TcAChE complexed with an inhibitor
was taken from RCSB Protein Data Bank (PDB, entry 1ODC).33 This
structure was chosen because of the similarity between its inhibi-
tor and the synthesized ligands. The original ligand (N-quinolin-4-
yl-N0-(1,2,3,4-tetrahydroacridin-9-yl)octane-1,8-diamine)
is
formed by tacrine connected through a long carbon chain to an
aminoquinoline moiety, which is structurally very similar to the
BTA moiety. The docking calculations were performed using the
ASP scoring function, since this function has previously proven to
give the best docking predictions for AChE inhibitors.30,34
The docking studies revealed favorable interactions for the new
inhibitors (see Fig. 3), with many similarities in their binding con-
formations. In Fig. 4 are displayed the main docking results. These
show the ligands well inserted into the cavity of the active site
blocking the entrance to the substrate (choline) and water mole-
cules. The tacrine moiety is always found well inserted in the bot-
tom of the gorge of the enzyme, binding to the CAS by
p–p stack
with the aromatic ring of Trp84 and Phe330, overlapping almost
perfectly with the tacrine moiety of the original ligand (see
Fig. S1, Supplementary data). Generally, the spacer seems to be
well accommodated along the hydrophobic cavity. On the other
hand, the BTA moiety was always placed at the entrance of the
gorge, and, except for 7b, it is able to bind to PAS forming aromatic
stacking with Tyr70 and Trp279. For 7b (Fig. 4a) it is the phenyl
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R. S. Keri et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
O
H
N
Y
S
N
H
5
N
N
7(a-e)
Y
Comp.
a
b
c
d
e
-(CH2)3-
7(a-e)
-(CH2)5-
Figure 3. Synthesized tacrine–BTA hybrid compounds.
(a)
(b)
Trp279
Trp279
Tyr70
Tyr70
Tyr121
Tyr121
Phe330
Phe330
Glu327
Trp84
Trp84
His440
Glu327
His440
Figure 4. Docking results for the tacrine–BTA hybrids with AChE: superimposition of 7a (light green) with 7b (pink) (a), and 7d (orange) with 7e (cyan) (b). H-bonds are
represented as solid black lines.
ring of the spacer that forms such aromatic interaction with Tyr70
and Trp279, thus allowing maintaining the strong interactions
with the enzyme. Moreover, a favorable H-bond formed between
the amide NH group of the linker with Tyr121 OH group in the
cases of 7a and 7b, which cannot be formed with 7c, explains the
lower inhibitory capacity of the last compound compared to the
first two (Fig. 4a and Fig. S2). Regarding compounds 7d and 7e
(Fig. 4b), the BTA is in position to performs the same aromatic
interaction with PAS. The possibility of the Tyr121 OH group to
form H-bond with both of these ligands, either through the amide
NH group (7d) or the sulfur atom of the BTA (7e) allows the inhib-
itory activity to increase, with regards to 7c, up to sub-micromolar
values. Considering the vicinity of the NH group of the linker in 7e
with the Tyr121 OH group (Fig. 4b), we may expect that this resi-
due can dynamically alternate the H-bonding between the BTA sul-
fur atom and that NH group. This fact would explain the higher
activity of 7e compared to 7d (see below).
Therefore, we may expect that this new family of hybrid com-
pounds also prevent this type of amyloid fibrillation.
2.2. Chemistry
The tacrine–benzothiazole hybrids (7a–7e) were synthesized as
shown in Scheme 1. The 9-chloro-1,2,3,4-tetrahydroacridine (3)
was prepared from commercially available anthranilic acid, as
previously reported.37,38 Compound 3 was treated with 6-amino-
hexanoic acid, using a catalytic amount of KI and phenol, to give
the 2-(1,2,3,4-tetrahydroacridine-9-ylamino) acid (5). Finally, the
carboxylic group of this compound (5) was condensed with a series
of different amino-benzothiazole derivatives (6) with formation of
amide linkages, using 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride (EDCIꢀHCl) and 4-dimethylaminopyridine
(DMAP), affording the tacrine–BTA hybrid compounds (7).
Globally, these modeling studies foresee the ability of the com-
pounds to interact with both the CAS and PAS, and the features dis-
closed allow explaining the good inhibitory activities observed
with AChE (Table 1). Several studies have pointed out the role of
AChE in the Ab fibrils formation, and that compounds interacting
with PAS may have an inhibitory effect in Ab aggregation.35,36
2.3. Biological activity
2.3.1. AChE inhibition
The enzymatic activities of the newly synthesized compounds
7(a–e) were evaluated by adaptation of the spectroscopic method
described by Ellman.30 The IC50 values for AChE inhibition are
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R. S. Keri et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 1
In vitro activities of the tacrine–BTA derivatives towards AChE inhibition, Ab aggregation and antioxidant activity (DPPH assay)
O
H
N
Y
S
N
H
5
N
N
7(a-e)
a
S. No.
Compd code
Y
AChE inhibitor IC50
(
lM)
Ab aggregation inhibitionb,c
Antioxidant activityd
(lM)
1
2
3
4
5
6
7a
7b
7c
7d
7e
Tacrine
–Ph–
0.34 0.1
0.57 0.1
1.84 0.2
0.96 0.3
0.76 0.4
0.19 0.04
22.3
61.3
29.9
35.0
22.3
n.d.e
32% (1.32 mM)
>1000
>1000
25% (1.22 mM)
>1000
>1000
–PhCH2–
–CH2Ph–
–(CH2)3–
–(CH2)5–
—
a
The values are mean of three independent experiments SD.
Inhibition of self-mediated Ab42 aggregation (in %). The thioflavin-T fluorescence method was used, and the measurements were carried out in the presence of inhibitor
lM).
The values are the mean of two independent measurements in duplicate (SEM < 10%).
EC50 values for the DPPH assay, or the % of quenching for the concentration in brackets.
Not determined.
b
(50
c
d
e
Cl
COOH
O
i
+
H2N
COOH
+
5
NH2
N
(3)
(4)
(2)
R
(1)
ii
O
H
N
HN
COOH
N
H
5
5
iii
N
NH2-R
+
N
7 (a-e)
6 (a-e)
(5)
(6, 7) R =
N
S
N
(a)
(b)
S
N
N
S
n = 3 (d)
5 (e)
(c)
n
S
Scheme 1. Synthesis of the tacrine–BTA hybrids 7(a–e). Reagents and conditions: (i) POCl3, 3 h, 180 °C; (ii) phenol, KI, 4 h, 180 °C; (iii) DCM, EDCIꢀHCl DMAP, 24 h, rt.
summarized in Table 1. As expected, all the tacrine–BTA hybrids
displayed high inhibitory activities against this enzyme, with IC50
values in the sub-micromolar to low micromolar range. Although
the new compounds have less capability to inhibit the enzyme
when compared to the reference drug, tacrine, some of them pre-
sented activities with the same order of magnitude (sub-micromo-
lar). Among these, 7a appears to be the strongest inhibitor, with
linker, as well as its composition and geometry. Analysis of the re-
sults suggests that a five carbon linker seems to provide a suitable
length between the tacrine and the connecting amide bond. On the
other hand, the aromaticity and geometry of the Y spacer near the
BTA group appears to have a relevant role in this enzymatic activ-
ity. In fact, the presence of a simple phenyl group attached to the
BTA moiety, as in 7a, IC50 = 0.34 lM), endowed the highest inhibi-
IC50 value of 0.34
(IC50 = 0.19 M), followed by 7b with 0.51
the weakest inhibitory activity is 7c, with IC50 value of 1.84
structure–activity relationship analysis shows that the inhibitory
potency against AChE is closely related with the length of the
l
M, quite similar to that obtained for tacrine
M. The compound with
M. A
tion capacity. The introduction of a methylene group (and a con-
comitant angle) between the phenyl ring and the BTA, as in 7b,
l
l
l
resulted in small activity reduction (IC50 = 0.51
identical insertion, but between the amide bond and the Ph–BTA
moiety, resulted in considerable activity reduction (7c,
lM), whereas the
a
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R. S. Keri et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
1.84
l
M). On the other hand, when a linear aliphatic Y spacer is
we showed that these new set of hybrid tacrine–BTA compounds
have relevant protective properties that may act as candidate mol-
ecules to prevent or arrest AD pathology.
introduce, there is a retrieval in the inhibitory capacity (7d–e),
which is more pronounced with a longer and more flexible five-
carbon chain (7e, 0.76 lM). These observations are in good agree-
ment with the results obtained from the docking studies (see
above). The fact that the new compounds are weaker inhibitors
than tacrine suggests some associated weakening of their binding
interactions with the enzyme. However, as dual binding-site inhib-
itors, it is expected that the BTA group can bind the peripheral an-
ionic site (PAS) of AChE. According to reported evidences,35 this
feature is expected to reduce the cholinergic-induced aggregation
of the Ab peptide, and thus enhance the overall anti-Ab aggregation
ability of these multifunctional hybrid compounds.
2.3.4. Antioxidant activity
The new compounds were screened for their anti-oxidant activ-
ities (EC50), based on their interaction with the 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH) free radical,44 and the corresponding results
are summarized on Table 1. Analysis of the results clearly shows
that, as expected, none of new compounds exhibits significant
antioxidant activities, as it is shown by the low inhibition percent-
ages at very high concentrations (25–32% at millimolar concentra-
tion). The insignificant radical-capture capacity of the tested
compounds can be explained by the absence of phenolic hydroxyl
groups that are important in the scavenging action of ROS species.
Tacrine moiety is present in all the compounds, and tacrine alone
has no significant antioxidant activity. Since there are no extra-
functional groups in compounds 7a–7e capable of reducing the
DPPH radical, similar results were expected for the new
compounds.
2.3.2. Inhibition of self-mediated Ab42 aggregation
To test the anti-amyloidogenic activity of the new compounds,
our experimental approach relies on in vitro assays with ThT to
monitor and quantify the aggregation of Ab42 synthetic peptide
into fibrils. ThT is a histochemical dye that is known to bind to
the peptide b-sheet conformation, which is the predominant sec-
ondary structure of amyloid-beta fibrils. The presence of fibrils
can be monitored by the fluorescence emission of ThT, with the
absorption peak at 446 nm and the emission peak at 490 nm.39–41
The inhibition studies were carried out upon incubating Ab42
with and without the compounds. The results (summarized in
Table 1) show that all the compounds induced a decrease of
the ThT fluorescence associated with the Ab fibril binding, thus
indicating an inhibition of the Ab42 self-aggregation process. In
particular, all of them presented aggregation inhibition values
above 20% (in a concentration ratio of 1:2.5 [Ab:I]), whereas 7b
presented the best activity (61.3%). This could be explained by
the conformation adopted by this compound in solution, which
may allow the best interaction of the BTA moiety with b-sheet
secondary structure of Ab peptides. Therefore it can be hypothe-
sized that a BTA-moiety composed of a biaryl heterocyclic scaf-
fold is better able to recognize and interact with the abnormal
b-sheet conformation of the Ab peptide, and to endow the inhibi-
tion of the fibrilogenesis.
2.3.5. Pharmacokinetic properties
In order to estimate the potential of the new compounds as
eventual drugs, a few indicators of their pharmacokinetic profiles
were predicted using QikProp program, v. 2.5.45 Parameters such
as the calculated lipo-hydrophilic character (clogP), the ability to
cross the blood–brain barrier (log BB), their ability to be absorbed
through the intestinal tract to the blood (Caco-2 cell permeability),
and the verification of Lipinski’s rule of five, may give an idea of
their druglikeness for being orally used as anti-AD agents.
As observed in Table 2, all the compounds presented clogP (oct-
anol/water) coefficients superior to five, while 7a and 7b are even
greater than 6.5. This fact leads this set of compounds to have a
lipophilic character higher than recommended by Lipinski’s rule.46
The compounds also have molecular weights higher than 500,
which accounts for two violations of this rule, except for com-
pound 7d, which seems to present better results within this set
of compounds.
The high lipophilic character and the low blood–brain barrier
permeability (log BB) indicate that the compounds are not eligible
drug candidates for oral administration, and further improvement
will be necessary to ameliorate compound absorption and entering
into the cells. However, they exhibited very good results in Caco-2
permeability, ranging from ca. 1000 to 2000 nm/s (higher than
500 nm/s is considered good45), indicating that the absorption
through the intestinal tract to the blood is possible.
2.3.3. In vitro neuroprotective activity
To evaluate the potential therapeutic action of tacrine–benzo-
thiazole hybrid compounds, SHSY-5Y cells, AD, MCI (mild cogni-
tive impairment) and control cybrids were treated with Ab42
peptides. In fact, Ab42 treatments in human cell lines constitute
a reliable model for screening potential neuroprotective com-
pounds because they mimic some aspects of neurodegeneration
underlying the AD physiopathology. Moreover, since AD and
MCI cybrids perpetuate the defects found in the brains and
peripheral tissues, it is reasonable to argue that the screening
of new disease-modifying drugs in these ex vivo cellular models
could give valuable information. In addition, the use of MCI cy-
brids, that harbor MCI individuals mitochondria allowed us to
target an early phase of the disease development.42 An early
identification of individuals in the preclinical period that are
not yet demented is imperative, in order to potentiate the effect
of disease-modifying agents.43
Our results (see Fig. 5) evidenced a significant protection of
some compounds, namely 7d and 7e, on Ab42 induced toxicity in
SHSY-5Y cells and in MCI and AD cybrids. However, we did not
see the same for control cybrids where only compound 7a pre-
vented Ab42 toxicity. Our results highlight the importance of mul-
ti-targeted anti-neurodegenative drugs, like tacrine–BTA hybrids
that act as inhibitors of AChE and Ab oligomerization, on the pre-
vention of Ab42 peptides-induced toxicity. By using different cellu-
lar models mimicking AD (AD cybrids) and targeting the
intermediate state between normal aging and AD (MCI cybrids)
3. Conclusions
Taking into account the multifactorial processes involved in
Alzheimer’s disease, the development of drugs that could modulate
multiple targets simultaneously with eventual synergistic effects
seems to be the wisest strategy for improving drug efficacy. In
the present study a set of bi-targeting compounds, based on tacrine
were designed and their protective action, as potential anti-neuro-
degenerative drugs, was investigated. Specifically a novel family of
tacrine–BTA hybrids has been assessed for the AChE inhibition, Ab
self-induced aggregation, antioxidant properties and also protec-
tive roles in cellular models mimicking AD. Our results showed
that four compounds had high cholinesterase inhibitory activities
with IC50 values bellow 1
pound 7a showed good AChE activity (IC50 = 0.34
to the commercial drug tacrine (0.19 M). The length, geometry
lM against AChE. Among them, com-
l
M), similarly
l
and aromaticity of the Y spacer (nearby the BTA moiety) revealed
determinant for the activity. All the synthesized compounds were
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R. S. Keri et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
150
100
50
#
#
*
0
Unt
7a
7b
7c
7d
7e
Tac.
Aβ
7a+Aβ 7b+Aβ 7c+Aβ 7d+Aβ 7e+Aβ Tac.+Aβ
150
#
100
50
***
0
Unt
7a
7b
7c
7d
7e
Tac.
Aβ
7a+Aβ 7b+Aβ 7c+Aβ 7d+Aβ 7e+Aβ Tac.+Aβ
150
###
###
100
50
***
0
Unt
7a
7b
7c
7d
7e
Tac.
Aβ
7a+Aβ 7b+Aβ 7c+Aβ 7d+Aβ 7e+Aβ Tac.+Aβ
150
###
###
100
50
0
***
Unt
7a
7b
7c
7d
7e
Tac.
Aβ
7a+Aβ 7b+Aβ 7c+Aβ 7d+Aβ 7e+Aβ Tac.+Aβ
Figure 5. Effect of tacrine–BTA hybrids on Ab42- induced toxicity in AD cellular models. Cells were treated with Abeta1–42 (1 lM) peptides, for 24 h, in the absence or the
presence of 2.5 lM of compounds 7a–e or tacrine. Evaluation of cell viability was performed by using MTT reduction test. Results are expressed as the percentage of untreated
cells, with the mean SEM derived from 3 different experiments. ⁄p < 0.05; ⁄⁄⁄p < 0.001, significantly different when compared with untreated cells; #p < 0,05; ###p < 0,001,
significantly different when compared with Ab42 treated cells.
capable of inhibiting Ab42 self-aggregation process (above 20% at a
50 M of inhibitor), and 7b showed the best value with 61.3% of
Our cellular studies demonstrated that some of these com-
pounds, in particular 7d and 7e, provided substantial protection
from cell death (SHSY-5Y cells, AD and MCI cybrids) induced by
Ab42 peptides, a reliable model for screening potential neuropro-
tective roles.
Overall, our preliminary findings showed that this new set of
hybrid tacrine–BTA compounds can merge important properties,
such as AChE inhibition and interaction with Ab. Hence, they are
able to act on one of the leading events of AD and on one of AD
l
aggregation inhibition. All the compounds showed low antioxidant
activity (ca. 25–30% for ca. 1 mM towards DPPH). Molecular mod-
eling studies of the AChE-inhibitor interaction allowed the ratio-
nalization of the SAR observed. They confirmed that these hybrid
inhibitors target both the catalytic active site (CAS) and peripheral
anionic site (PAS) of AChE, highlighting their potential to also inhi-
bit the cholinergic-induced Ab aggregation.
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R. S. Keri et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
Table 2
(3–4 times). The aqueous layer was acidified with HCl (concd) up
to pH 2 and then extracted with dichloromethane. The organic
layer was dried over anhydrous Na2SO4 and concentrated in vacuo
to give the pure compound as pale yellow oil (2.73 mg). Yield: 60%;
1H NMR (300 MHz, MeOD) d: 8.37 (d, J = 8.3 Hz, 1H, ArH), 7.97–
7.83 (m, 2H, ArH), 7.60–7.55 (m, 1H, ArH), 3.95 (t, J = 7.3 Hz, 2H,
CH2), 3.02 (s, 2H, CH2), 2.70 (s, 2H, CH2), 2.30 (t, J = 7.20 Hz, 2H,
CH2), 1.96 (br s, 4H, CH2–CH2), 1.85 (t, J = 7.20 Hz, 2H, CH2), 1.62
(t, J = 7.60 Hz, 2H, CH2), 1.51–1.43 (m, 2H, CH2); 13C NMR
(400 MHz, CDCl3) d: 178.7 (C@O), 156.1, 150.3, 138.3, 132.7,
125.8, 125.6, 119.1, 115.7, 111.7 (9 aromatic C), 51.2 (N-CH2 ali-
phatic chain), 48.1, 34.2, 30.6, 28.6, 26.1, 24.4, 22.0, 20.8 (9 ali-
phatic C); m/z: 313 (M+), 314 (M+1)+.
Predicted pharmacokinetic values
a,b
a,c
S. No. Compd
code
Y
clogP
log BB
Caco -2
Violations of
permeabilitya Lipinski’s rule of
(nm/s)
5a
1
2
3
4
5
6
7a
7b
7c
7d
7e
Tacrine
Ph
6.61
ꢃ0.59
ꢃ1.06
ꢃ0.35
ꢃ0.64
ꢃ0.75
0.04
2028
1436
2271
1389
1044
2880
2
2
2
1
2
0
CH2Ph 7.44
PhCH2 5.37
(CH2)3 5.23
(CH2)5 5.14
—
2.53
a
b
c
Predicted values using program QikProp v. 2.5.45
Calculated octanol/water partition coefficient.
Brain/blood partition coefficient.
4.1.4. General procedure for the synthesis of the benzothiazole
derivatives 6(a–e)
pathological hallmark, revealing as potential lead compounds to-
wards AD therapy.
A mixture of 2-aminothiophenol (1 equiv) and different substi-
tuted amino-carboxylic acids (4-aminobenzoic acid, 4-aminophe-
nylacetic acid, 4-(aminomethyl)benzoic acid, 4-aminobutyric acid
and 4-aminohexanoic acid) (1 equiv) in polyphosphoric acid
(15 g) was stirred at 220 °C for 4 h. The resulting brown oil was dis-
solved in 5 M NaOH (50 mL) to obtain basic yellow solution and
this solution was extracted with DCM (2 ꢁ 30 mL). The total organ-
ic phase was then extracted with 0.1 N HCl (2 ꢁ 30 mL) and after-
wards washed DCM (50 mL). The aqueous phase was basified to pH
ꢂ10–12 with 5 M NaOH, and it was extracted with DCM
(2 ꢁ 30 mL). The total organic phase was dried over anhydrous
Na2SO4, and the solvent evaporated. The brown oil obtained was
treated with HCl-saturated methanol until pH ꢂ1, the solution
was evaporated, and the residue was recrystallized from
acetonitrile.
4. Experimental part
4.1. Chemistry
4.1.1. General methods and materials
Analytical grade reagents were purchased from Sigma–Aldrich,
Fluka and Acros and were used as supplied. Solvents were dried
according to standard methods.47 The chemical reactions were
monitored by TLC using alumina plates coated with silica gel 60
F254 (Merck). Column chromatography separations were per-
formed on silica gel Merck 230–400 mesh (Geduran Si 60). The
melting points were measured with a Leica Galen III hot stage
apparatus and are uncorrected. The 1H and 13C NMR spectra were
recorded on Bruker AVANCE III spectrometers at 300 MHz and
400 MHz, respectively. Chemical shifts (d) are reported in ppm
from the standard internal reference tetramethylsilane (TMS).
The following abbreviations are used: s = singlet, d = doublet,
t = triplet, m = multiplet. Mass spectra (ESI-MS) were performed
on a 500 MS LC Ion Trap (Varian Inc., Palo Alto, CA, USA) mass spec-
trometer equipped with an ESI ion source, operated in the positive
ion mode. For the target compounds, the elemental analyses were
performed on a Fisons EA1108 CHNS/O instrument and were with-
in the limit of 0.4%.
4.1.4.1. 4-(Benzo[d]thiazol-2-yl)aniline (6a). 2-Aminothiophe-
nol and 4-aminobenzoic acid afforded the pure title product as a
greenwish solid.49 Yield: 64%; 1H NMR (300 MHz, MeOD) d: 7.80
(d, J = 8.8 Hz, 2H, NC-CH and SC-CH), 7.47 (d, J = 8.9 Hz, 2H, CH-
CHNH2), 7.42 (t, J = 8.9 Hz, 1H, NC-CHCH), 7.30 (t, J = 8.9 Hz, 1H,
SC-CHCH), 6.75 (d, J = 9 Hz, 2H, CH-CHNH2). m/z (ESI) 227.1
(M+H)+.
4.1.4.2. 4-(Benzo[d]thiazol-2-ylmethyl)aniline (6b). 2-Amino-
thiophenol and 4-aminophenylacetic acid afforded the pure title
product as a brown solid.30 Yield: 62%; 1H NMR (300 MHz, MeOD)
d: 7.95 (d, J = 8.9 Hz, 1H, NC-CH), 7.88 (d, J = 9 Hz, 1H, SC-CH), 7.60
(d, J = 8.8 Hz, 2H, CH-CHNH2), 7.48 (t, J = 9 Hz, 2H, NC-CHCH, SC-
CHCH), 7.42 (d, J = 9 Hz, 2H, CHCH-NH2), 4.41 (s, 2H, CH2). m/z
(ESI) 241.1 (M+H)+.
4.1.2. 9-Chloro-1,2,3,4-tetrahydroacridine (3)
To a mixture of anthranilic acid (3.2 g, 23 mmol) and cyclohex-
anone (2.65 mL, 27 mmol) in an ice bath, POCl3 (25 mL) was care-
fully added. The mixture was heated under reflux for 3 h, then
cooled at room temperature, and concentrated to give slurry. The
residue was diluted with EtOAc, neutralized with aqueous K2CO3,
and washed with brine. The organic layer was dried over
anhydrous K2CO3 and concentrated in vacuo, affording a pale
brown solid. It was recrystallized from acetone to give 3 (4.6 g,
91%). Mp 67–69 °C (lit. mp 66–68 °C);48 1H NMR (300 MHz, CDCl3)
d: 8.16 (d, J = 7.5 Hz, 1H, ArH), 7.97 (d, J = 8.3 Hz, 1H, ArH), 7.65 (dd,
J = 9.2, 7.5 Hz, 1H, ArH), 7.52 (t, J = 8.3 Hz, 1H, ArH), 3.10 (t,
J = 6.3 Hz, 2H, CH2), 3.01 (t, J = 4.8 Hz, 2H, CH2), 1.94 (br s, 4H,
CH2–CH2); m/z: 218 (M+), 219 (M+1)+, 220 (M+2)+.
4.1.4.3. (4-(Benzo[d]thiazol-2-yl)phenyl)methanamine (6c). 2-
Aminothiophenol and 4-(aminomethyl)benzoic acid afforded the
pure title product as a yellow solid.30 Yield: 68%; 1H NMR
(300 MHz, MeOD) d: 8.20 (d, J = 8 Hz, 2H, NC-CH and SC-CH), 8.09
(d, J = 8.9 Hz, 2H, HC-CHCH2NH2), 7.60 (d, J = 9 Hz, 2H, HC-
CH2NH2), 7.58 (t, J = 7.8 Hz, 1H, NC-CHCH), 7.51 (t, J = 9 Hz, 1H,
SC-CHCH), 4.24 (s, 2H, CH2). m/z (ESI) 241.1 (M+H)+.
4.1.3. 2-(1,2,3,4-Tetrahydroacridine-9-ylamino)acetic acid (5)
To a mixture of 9-chloro-1,2,3,4-tetrahydroacridine (3.18 g,
1.46 mmol) and 6-amino-hexanoic acid (3.83 g, 2.92 mmol) was
added phenol (1.5 g) and catalytic amount of KI (50 mg). The mix-
ture was heated at 180 °C for 4 h and then cooled to room temper-
ature. The mixture was dissolved in 5% NaOH, washed with diethyl
ether (3–4 times). The collected aqueous layer was adjusted to pH
8 with HCl (concd), then, once again, washed with diethyl ether
4.1.4.4. 3-(Benzo[d]thiazol-2-yl)propan-1-amine (6d). 2-Ami-
nothiophenol and 4-aminobutyric acid afforded the pure title
product as a brown solid. Yield: 69%; 1H NMR (300 MHz, D2O) d:
7.90 (d, J = 7.9 Hz, 1H, NC-CH), 7.83 (d, J = 8 Hz, 1H, SC-CH), 7.53
(t, J = 7.2 Hz, 1H, NC-CHCH), 7.47 (1H, t, J = 7.2 Hz, SC-CHCH),
3.22 (t, J = 7.6 Hz, 2H, H2N(CH2)2CH2), 3.11 (t, J = 7.7 Hz, 2H,
H2NCH2(CH2)2), 2.26–2.04 (m, 2H, H2NCH2CH2CH2). m/z (ESI)
193.0 (M+H)+.
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8
R. S. Keri et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
4.1.4.5. 5-(Benzo[d]thiazol-2-yl)pentan-1-amine (6e). 2-Ami-
nothiophenol and 4-aminohexanoic acid afforded the pure title
product as a brown solid. Yield: 75%; 1H NMR (300 MHz, D2O) d:
7.98 (d, J = 8.0 Hz, 1H, NC-CH), 7.86 (d, J = 8 Hz, 1H, SC-CH), 7.57
(t, J = 7.0 Hz, 1H, NC-CHCH), 7.50 (t, J = 7.2 Hz, 1H, SC-CHCH), 3.19
(t, J = 7.7 Hz, 2H, N(CH2)4CH2), 2.99 (t, J = 7.6 Hz, 2H,
H2NCH2(CH2)4), 1.93–1.83 (m, 2H, H2N(CH2)3CH2CH2), 1.77–1.67
d: 173.6 (C@O), 167.6, 155.1, 154.1, 151.4, 142.3, 139.4, 134.9,
132.3, 131.9, 128.3, 127.5 (2 C). 126.6 (2 C), 125.4, 125.0, 124.5,
123.1, 121.7, 121.0, 116.1, 112.3 (22 aromatic C), 64.0 (N-CH2 ali-
phatic chain), 48.0, 43.0, 35.7, 30.2, 28.9, 25.8, 24.9, 22.05, 20.85
(9 aliphatic C); MS: 535 (M+), 536 (M+1)+. Anal.
(C33H34N4OSꢀ1.8EtOH) C, H, N, S.
(m,
2H,
H2NCH2CH2(CH2)3),
1.52–1.42
(m,
2H,
4.1.5.4.
N-(3-(Benzo[d]thiazol-2-yl)propyl)-6-(1,2,3,4-tetrahy-
H2N(CH2)2CH2(CH2)2). m/z (ESI) 193.0 (M+H)+.
droacridin-9-ylamino)hexanamide (7d). Compounds 5 and 6d
afforded the title compound as colorless crystals. Yield: 35%; mp
94–95 °C; 1H NMR (300 MHz, CDCl3) d: 8.34 (d, J = 8.0 Hz, 1H,
ArH), 8.17 (d, J = 8.6 Hz, 1H, ArH), 7.87 (d, J = 8.0 Hz, 1H, ArH),
7.77 (d, J = 7.2 Hz, 1H, ArH), 7.55 (t, J = 7.3 Hz, 1H, ArH), 7.42–
7.29 (m, 3H, ArH), 6.99 (br s, 1H, NH), 3.86 (t, J = 7.0 Hz, 2H, CH2),
3.39 (t, J = 6.5 Hz, 2H, CH2), 3.18–3.14 (m, 4H, CH2-CH2), 2.61 (t,
J = 6.0 Hz, 2H, CH2), 2.27 (t, J = 7.1 Hz, 2H, CH2), 2.11 (t, J = 7.1 Hz,
2H, CH2), 1.88–1.76 (m, 6H, (CH2)3), 1.70 (t, J = 7.4 Hz, 2H, CH2),
1.46 (t, J = 7.2 Hz, 2H, CH2); 13C NMR (400 MHz, CDCl3), d: 173.1
(C@O), 171.4, 155.0, 153.16, 135.2, 131.9, 126.8 (2 C), 125.1,
125.1, 124.2, 122.9, 121.7 (2 carbons), 116.4, 111.5, 109.2 (16
aromatic C), 63.9 (N-CH2 aliphatic chain), 48.3, 38.9, 35.9, 31.9,
30.5, 29.8, 29.1, 26.0, 24.8, 24.0, 22.1, 21.0 (11 aliphatic C); MS:
487 (M+), 488 (M+1)+. Anal. (C29H34N4OSꢀ2.54H2Oꢀ6.33CH3CN) C,
H, N, S.
4.1.5. General procedure for the synthesis of the tacrine–
benzothiazole compounds (7a–e)
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDCIꢀHCl) (2.5 equiv) and 4-dimethylaminopyridine (DMAP)
(1 equiv) were added to a solution of 5 (1 equiv) and the benzothi-
azole derivative 6 (1 equiv) in DCM (25 mL), and the mixture was
left stirred for 24 h at room temperature under nitrogen. The sol-
vent was removed in vacuo and the residue was purified by col-
umn
chromatography
on
silica
gel
eluted
with
dichloromethane:methanol (6:1) to give the title compounds.
4.1.5.1. N-(4-(Benzo[d]thiazol-2-yl)phenyl)-6-(1,2,3,4-tetrahy-
droacridin-9-ylamino)hexanamide (7a). Compounds 5 and 6a
afforded the title compound as light yellow crystals. Yield: 40%;
mp 154–155 °C; 1H NMR (300 MHz, CDCl3) d: 9.46 (br s, NH),
8.28 (d, J = 8.2 Hz, 1H, ArH), 8.14 (d, J = 8.6 Hz, 1H, ArH), 7.99 (d,
J = 8.2 Hz, 1H, ArH), 7.92–7.85 (m, 4H, ArH), 7.55 (t, J = 7.3 Hz, 1H,
ArH), 7.48–7.44 (m, 1H, ArH), 7.37–7.29 (m, 2H, ArH), 6.76–6.67
(m, 1H, ArH), 3.87 (t, J = 6.9 Hz, 2H, CH2), 3.17 (t, J = 6.0 Hz, 2H,
CH2), 2.57 (t, J = 6.0 Hz, 2H, CH2), 2.51 (t, J = 6.9 Hz, 2H, CH2),
1.91–1.73 (m, 8H (CH2)4), 1.53 (t, J = 6.9 Hz, 2H, CH2); 13C NMR
(400 MHz, CDCl3) d: 174.5 (C@O), 169.4, 157.0, 155.0, 154.4,
147.3, 145.7, 142.9, 135.9, 130.9, 129.8 (2 carbons), 129.1, 127.6,
126.4, 125.8, 125.1, 142.9, 123.5, 122.8, 120.9, 120.1, 115.7 (22 aro-
matic C), 38.2 (N-CH2 aliphatic chain), 37.7, 32.8, 31.7, 27.4, 26.2,
25.7, 23.7, 23.1 (8 aliphatic C); MS 521 (M+), 522 (M+1)+. Anal.
(C32H32N4OSꢀ0.33H2Oꢀ8.65CH3CN) C, H, N, S.
4.1.5.5.
N-(5-(Benzo[d]thiazol-2-yl)pentyl)-6-(1,2,3,4-tetrahy-
droacridin-9-ylamino)hexanamide (7e). Compounds 5 and 6e
afforded the title compound as colorless crystals. Yield: 44%; mp
78–79 °C; 1H NMR (300 MHz, CDCl3) d: 8.36 (d, J = 8.5 Hz, 1H,
ArH), 8.17 (d, J = 8.6 Hz, 1H, ArH), 7.91 (d, J = 8.0 Hz, 1H, ArH),
7.80 (d, J = 7.7 Hz, 1H, ArH), 7.61 (t, J = 7.6 Hz, 1H, ArH),7.45–7.30
(m, 3H, ArH), 6.37 (br s, 1H, NH), 3.86 (t, J = 7.0 Hz, 2H, CH2),
3.28–3.18 (m, 4H, (CH2)2), 3.08 (t, J = 7.5 Hz, 2H, CH2), 2.63 (t,
J = 5.8 Hz, 2H, CH2), 2.24 (t, J = 7.0 Hz, 2H, CH2), 1.90–1.82 (m, 8H,
(CH2)4), 1.70 (t, J = 7.3 Hz, 2H, CH2), 1.58 (t, J = 7.2 Hz, 2H, CH2),
1.49–1.40 (m, 4H, (CH2)2); 13C NMR (400 MHz, CDCl3) d: 173.0
(C@O), 172.0, 155.1, 153.3, 152.1, 135.2, 131.9, 126.0 (2 carbons),
125.0, 124.9, 124.3, 122.5, 121.7 (2 carbons), 116.5, 111.6 (16 aro-
matic C), 64.0 (N-CH2 aliphatic chain), 48.3, 39.3, 35.9, 34.2, 30.4,
29.4, 29.3, 26.5, 25.9, 24.8, 24.1 22.2, 21.0 (13 aliphatic C); MS:
515 (M+), 516 (M+1)+. Anal. (C31H38N4OSꢀ2.02H2Oꢀ2.31CH3CN);
Anal. (C31H38N4OSꢀ2.02 H2Oꢀ2.31CH3CN) C, H, N, S.
4.1.5.2.
N-(4-(Benzo[d]thiazol-2-ylmethyl)phenyl)-6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexanamide (7b). Compounds 5
and 6b afforded the title compound as light yellow crystals. Yield:
42%; mp 104–105 °C; 1H NMR (300 MHz, CDCl3) d: 9.79 (br s, NH),
8.20 (d, J = 8.5 Hz, 1H, ArH), 8.15 (d, J = 8.7 Hz, 1H, ArH), 7.88 (d,
J = 8.1 Hz, 1H, ArH), 7.74–7. 69 (m, 3H, ArH), 7.50 (t, J = 7.6 Hz,
1H, ArH), 7.39–7.35 (m, 1H, ArH), 7.29–7.24 (m, 2H, ArH), 7.15
(d, J = 8.4 Hz, 2H, ArH), 6.62 (br s, NH), 4.28 (s, 2H, CH2-S), 3.81
(s, 2H, CH2), 3.03 (s, 2H, CH2), 2.55–2.50 (m, 4H, CH2-CH2), 1.84
(t, J = 7.0 Hz, 2H, CH2), 1.73 (t, J = 7.0 Hz, 2H, CH2), 1.67 (br s, 4H,
CH2-CH2), 1.42 (t, J = 7.0 Hz, 2H, CH2); 13C NMR (400 MHz, CDCl3)
d: 172.4 (C@O), 171.7, 155.7, 153.2, 151.0, 139.1, 138.4, 135.6,
132.3, 132.2, 129.5 (2 carbons), 126.6, 125.1, 125.0, 124.8, 122.7,
121.7, 120.5, 120.4 (2 carbons), 116.1, 111.5 (22 aromatic C),
64.1 (N-CH2 aliphatic chain), 50.6, 48.0, 40.12, 36.5, 29.9, 28.8,
25.6, 24.7, 22.0, (9 aliphatic C); m/z: 535 (M+), 536 (M+1)+; Anal.
(C33H34N4OSꢀ2.6EtOH) C, H, N, S.
4.2. Acetylcholinesterase inhibition
The determination of the AChE inhibitory capacity of the syn-
thesized compounds was performed by an adaptation of a method
previously described.30 The buffer solution used in this enzymatic
assay was 2-[4-(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid
(HEPES) at 50 mM and pH 8. The Ellman reagent, 5,50-dithiobis-(2-
nitrobenzoic acid) (DTNB, 3 mM) was diluted in a HEPES buffer
solution containing NaCl (50 mM) and MgCl2 (20 mM). Acetylcho-
linesterase stock solution was prepared by dissolving 500 U
(extracted from electrophorus electricus and purchased from
Sigma–Aldrich) in TRIS buffer (50 mM, pH 8) (10 mL). The enzyme
was later diluted with HEPES buffer to give the final AChE concen-
tration conditions, 1.75 U mLꢃ1. A 16 mM aqueous solution of the
enzyme substrate, acetylcholine iodide (AChI), was also prepared.
Stock solutions of the tested compounds were prepared in metha-
nol (1 mg/mL), due to their hydrophobic nature, and five different
concentrations were used in order to obtain AChE inhibition rang-
ing between 20% and 90%.
4.1.5.3. N-(4-(Benzo[d]thiazol-2-yl)benzyl)-6-(1,2,3,4-tetrahy-
droacridin-9-ylamino)hexanamide (7c). Compounds 5 and 6c
afforded the title compound as colorless crystals. Yield: 38%; mp
192–193 °C; 1H NMR (300 MHz, CDCl3) d: 8.20 (d, J = 8.4 Hz, 1H,
ArH), 8.13 (d, J = 8.6 Hz, 1H, ArH), 7.96 (d, J = 8.0 Hz, 1H, ArH),
7.84–7.77 (m, 4H, ArH), 7.53 (t, J = 7.6 Hz, 1H, ArH), 7.44 (t,
J = 7.1 Hz, 1H, ArH), 7.37–7.30 (m, 3H, ArH), 4.45 (s, 2H, CH2-N),
3.71 (t, J = 6.8 Hz, 2H, CH2), 3.03 (t, J = 6.0 Hz, 2H, CH2), 2.51 (t,
J = 5.8 Hz, 2H, CH2), 2.38 (t, J = 7.0 Hz, 2H, CH2), 1.76–1.67 (m, 8H,
(CH2) 4), 1.40 (t, J = 7.1 Hz, 2H, CH2); 13C NMR (400 MHz, CDCl3),
The final assay solution resulted from assembling several solu-
tions: 374 lL of HEPES (50 mM, pH 8), 476 lL of DTNB; a variable
Please cite this article in press as: Keri, R. S.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.05.028

R. S. Keri et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
volume (10, 20, 30, 40, 50
lL) of inhibitor solution, 25
lL of the
4.4. Antioxidant activity
AChE solution and the necessary amount of methanol to attain
0.925 mL of sample mixture in a 1 mL cuvette. The samples were
left to incubate for 15 min. Subsequently, 75 lL of substrate solu-
The antioxidant activity was evaluated by the DPPH method
previously described.44 To a 2.5 mL solution of DPPH (0.002%) in
methanol, four samples of each compound solution were added
with different volumes to obtain different concentrations of the
tested compound. The volume required for the final total volume
(3.5 mL) was attained with methanol. The samples were incubated
for 30 min at room temperature. The absorbance was measured at
517 nm against the corresponding blank (methanol). The antioxi-
dant activity was calculated by Eq. (3),34
tion was added. The initial rate of the enzymatic reaction was mon-
itored by reading the solution absorbance at 405 nm, recorded on a
Perkin-Elmer Lambda 35 UV–Vis spectrophotometer, during the
first 5 min of the reaction time. Assays were also run with a blank
solution containing all the components except the AChE solution
(that was replaced by buffer HEPES), to account for the non-enzy-
matic reactions. The velocities of the reaction were calculated, as
well as the enzyme activity. A control reaction was carried out
using the sample solvent (methanol) in the absence of any tested
compound and it was considered as 100% activity. Each concentra-
tion was analyzed in quintuplicate. The percent inhibition of the
enzyme activity due to the presence of increasing test compound
concentration was calculated by the following Eq. (1),
ADPPH ꢃ Asample
%AA ¼
ð3Þ
ADPPH
in which AA is the antioxidant activity, ADPPH is the absorbance of
the DPPH solution against the blank, Asample is the absorbance of
the sample compound against the blank. The tests were carried
out in triplicate. The compound concentrations providing 50% of
antioxidant activity (EC50) were obtained by plotting the antioxi-
dant activity against the compound concentration.
ꢀ
ꢁ
mI
m0
%I ¼ 100 ꢃ
ꢁ 100
ð1Þ
in which vI is the initial reaction rate in the presence of inhibitor,
and 0 is the initial rate of the control reaction. The inhibition curves
4.5. Human subjects and creation of cybrid cell lines
v
were obtained by plotting the percentage of enzymatic inhibition
versus inhibitor concentration and a calibration curve was drawn
from which the linear regression parameters were obtained. Data
presented are the average of three independent experiments SD.
Subject participation was approved by the Kansas University
Medical Center’s Institutional Review Board. Subjects for this
study were recruited from the University of Kansas Alzheimer’s
Disease Center (KU ADC). Each subject was determined, based
on cognitive testing and by a memory disorders subspecialist cli-
nician, to meet criteria for normal cognition (control status), mild
cognitive impairment (MCI), or sporadic Alzheimer’s disease (AD).
After providing informed consent, sporadic AD (n = 8), MCI (n = 7)
and age-matched control (n = 7) subjects underwent a 10 mL
phlebotomy using tubes containing acid–citrate–dextrose as an
anticoagulant. The age of the AD subject platelet donors was
71.5 9.7 years, the age of the MCI platelet donors was
72.3 6.6, and the age of the control subject platelet donors
was 73.9 7.7.
4.3. Inhibition of self-mediated Ab42 aggregation
Amyloid b-peptide (1–42) (Ab42), was purchased from Aldrich
as a lyophilized powder and stored at ꢃ20 °C. The samples were
treated with 1,1,1,1,1,1-hexafluoropropan-2-ol (HFIP) to avoid
self-aggregation and reserved. HFIP pre-treated Ab42 samples were
re-solubilized with a CH3CN/Na2CO3 (300
(48.3:48.3:4.3, v/v/v) solvent mixture in order to have a stable
stock solution. This Ab42 alkaline solution (500 M) was diluted
in phosphate buffer (0.215 M, pH 8.0) to obtain a 20 M solution.
lM)/NaOH (250 lM)
l
l
Cybrid cell lines were created on an SHSY-5Y cell nuclear back-
ground. To generate the cybrid lines used in these studies, platelets
from human subjects were mixed with SHSY-5Y cells previously
Compounds under study were firstly dissolved in methanol
(1 mg/mL) due to their hydrophobic nature, and then further di-
luted in phosphate buffer to a final concentration of 50 lM.
depleted of endogenous mtDNA (q0 cells). During the overall cy-
To study the Ab42 aggregation inhibition, a reported method,
brid generation procedure several different types of media were
used. For each medium, Dulbecco’s modified Eagle’s medium
(DMEM) medium was obtained from Gibco-Invitrogen, while
non-dialyzed or dialyzed fetal bovine serum (FBS) was obtained
based on the fluorescence emission of thioflavin T (ThT), was fol-
lowed.39,40 Firstly, Ab42 (10
l
L) samples and the tested compounds
L) were diluted with the phosphate buffer to a final concen-
tration of 20 M (Ab) and 50 M (compounds), and then were
(10
l
l
l
from Sigma. SHSY-5Y
supplemented with 10% non-dialyzed FBS, 200
pyruvate, 150 g/mL uridine, and 1% penicillin–streptomycin solu-
q
0 cell growth medium consisted of DMEM
incubated for 24 h at 37 °C, without stirring. As for the control, a
sample of the peptide was incubated under identical conditions
but without the inhibitor. After incubation, the samples were
added to a black, flat-, and clear bottom 96-well plate (BD Falcon)
lg/mL sodium
l
tion. SHSY-5Y cybrid selection medium consisted of DMEM supple-
mented with 10% dialyzed FBS and 1% penicillin–streptomycin
solution. The selection process lasted 6 weeks. After cell line selec-
tion was completed, each line, including SHSY-5Y was continu-
ously maintained in a cybrid growth medium containing DMEM
supplemented with 10% non-dialyzed FBS and 1% penicillin–strep-
tomycin solution for over 2 months prior to biochemical and
molecular assays.
with 180 lL of 1.5 lM ThT in 50 mM glycine–NaOH (pH 8.5). Blank
samples were prepared for each concentration in a similar way, de-
void 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: excitation (446 nm) and
emission (490 nm). The percent inhibition of the self-induced
aggregation due to the presence of the test compound was calcu-
lated on basis of Eq. (2), in which IFi and IF0 correspond to the
fluorescence
4.6. Cell treatments and viability
ꢀ
ꢁ
The tested compounds (7a–7e) were dissolved in DMSO at a
concentration of 10 mM and aliquots were stored at ꢃ20 °C. These
IFi
IF0
I% ¼ 100 ꢃ
ꢁ 100
ð2Þ
compounds were added to the medium at 2.5 lM final concentra-
intensities, in the presence and absence of the test compound,
respectively, minus the fluorescence intensities due to the respec-
tive blanks. The reported values were obtained as the mean SEM
of duplicate of two different experiments.
tion. Before the addition of Ab42, cells were pre-incubated for 3 h
with tacrine–BTA hybrids compounds. The final concentration of
DMSO in culture media did not exceed 0.05% (v/v) and no altera-
tions on cells were observed. For all conditions tested, control
Please cite this article in press as: Keri, R. S.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.05.028

10
R. S. Keri et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
experiments were performed in which the compounds tested or
Ab42 were not added.
Supplementary data
Cell reduction ability as a surrogate of cell viability was mea-
sured by using a quantitative colorimetric assay with MTT accord-
ing to the method of Mosmann (1983).30,50 In brief, cells were
incubated with MTT solution (in sodium medium) for 3 h at
37 °C. The medium was then discarded, the cells were dissolved
with isopropanol/HCl, and thereafter the absorbance at 570 nm
was measured using a Spectramax Plus 384 spectrophotometer
(Molecular Devices). MTT reduction ability was expressed as a per-
centage of the control value obtained for untreated cells.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.028.
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The authors thank the Portuguese NMR Network (IST-UTL Cen-
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support with the project PEst-OE/QUI/UI0100/2013, the postdoc-
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