Novel 8-Hydroxyquinoline Derivatives as Multitarget Compounds for the Treatment of Alzheimer′s Disease

Article abstract of DOI:10.1002/cmdc.201600014

We discovered a small series of hit compounds that show multitargeting activities against key targets in Alzheimer′s disease (AD). The compounds were designed by combining the structural features of the anti-AD drug donepezil with clioquinol, which is able to chelate redox-active metals, thus decreasing metal-driven oxidative phenomena and β-amyloid (Aβ)-mediated neurotoxicity. The majority of the new hybrid compounds selectively target human butyrylcholinesterase at micromolar concentrations and effectively inhibit Aβ self-aggregation. In addition, compounds 5-chloro-7-((4-(2-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquinoline (1 b), 7-((4-(2-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquinoline (2 b), and 7-(((1-benzylpiperidin-4-yl)amino)methyl)-5-chloro-8-hydroxyquinoline (3 a) are able to chelate copper(II) and zinc(II) and exert antioxidant activity in vitro. Importantly, in the case of 2 b, the multitarget profile is accompanied by high predicted blood–brain barrier permeability, low cytotoxicity in T67 cells, and acceptable toxicity in HUVEC primary cells.

Full text of DOI:10.1002/cmdc.201600014

DOI: 10.1002/cmdc.201600014
Full Papers
Novel 8-Hydroxyquinoline Derivatives as Multitarget
Compounds for the Treatment of Alzheimer’s Disease
Federica Prati,[a] Christian Bergamini,[a] Romana Fato,[a] Ondrej Soukup,[b] Jan Korabecny,[b]
Vincenza Andrisano,[c] Manuela Bartolini,*[a] and Maria Laura Bolognesi*[a]
We discovered a small series of hit compounds that show mul-
titargeting activities against key targets in Alzheimer’s disease
(AD). The compounds were designed by combining the struc-
tural features of the anti-AD drug donepezil with clioquinol,
which is able to chelate redox-active metals, thus decreasing
metal-driven oxidative phenomena and b-amyloid (Ab)-mediat-
ed neurotoxicity. The majority of the new hybrid compounds
selectively target human butyrylcholinesterase at micromolar
concentrations and effectively inhibit Ab self-aggregation. In
addition, compounds 5-chloro-7-((4-(2-methoxybenzyl)pipera-
zin-1-yl)methyl)-8-hydroxyquinoline (1b), 7-((4-(2-methoxyben-
zyl)piperazin-1-yl)methyl)-8-hydroxyquinoline (2b), and 7-(((1-
benzylpiperidin-4-yl)amino)methyl)-5-chloro-8-hydroxyquino-
line (3a) are able to chelate copper(II) and zinc(II) and exert an-
tioxidant activity in vitro. Importantly, in the case of 2b, the
multitarget profile is accompanied by high predicted blood–
brain barrier permeability, low cytotoxicity in T67 cells, and ac-
ceptable toxicity in HUVEC primary cells.
Introduction
Among neurodegenerative disorders, dementias are responsi-
ble for the greatest burden of disease, with Alzheimer’s disease
(AD) and related disorders affecting more than seven million
people in Europe. Alzheimer’s Disease International (ADI) esti-
mates that there are nearly 44 million people with AD world-
wide (2015). The global cost of AD and dementia is $605 bil-
lion, a value which is equivalent to 1% of the world’s gross do-
mestic product.[1]
gic transmission, and changes in cholinesterase activity in the
cerebral cortex and hippocampus. However, none of the mar-
keted drugs are effective at slowing or stopping progressive
neuronal death and related cognitive impairment.[2] Thus,
a major issue thwarting a significant therapeutic advancement
for AD is the identification of new chemical entities able to
target key pathological processes underpinning the pathogen-
esis and/or progression of AD.
No current therapies are able to effectively target the under-
lying molecular mechanisms of AD. In the last 20 years, the
first-line therapy for the management of AD has been the ad-
ministration of cholinesterase (ChE) inhibitors (i.e., tacrine, do-
nepezil, rivastigmine, and galantamine), on the basis of the
clinically observed cholinergic dysfunction, for example,
marked degeneration of cholinergic neurons, loss of choliner-
AD is a multifactorial pathology characterized by protein ag-
gregation (i.e., extracellular aggregation of the disease-specific
amyloid-beta [Ab] peptide and intracellular aggregation of the
hyperphosphorylated tau protein), as well as oxidative and in-
flammatory processes.[3] Metal dyshomeostasis is also thought
to play an important role in AD,[4,5] as well as in several other
neurodegenerative diseases. In particular, elevated concentra-
tions of copper(II) and zinc(II) have been detected in the neo-
cortex of AD patients and are especially associated with Ab de-
posits.[6] Binding sites for both metal ions have been identified
on Ab oligomers, and they are thought to mediate amyloid
toxicity.[7] In fact, complexes of Ab and metal ions were shown
to promote Ab aggregation[8] and protease resistance, and to
trigger the production of reactive oxygen species (ROS), en-
hancing oxidative stress.[9] On the basis of these observations,
metal chelating therapy is considered an attractive option to
counteract AD progression (reviewed in ref. [10]).
[a] Dr. F. Prati, Dr. C. Bergamini, Prof. R. Fato, Prof. M. Bartolini,
Prof. M. L. Bolognesi
Department of Pharmacy and Biotechnology
Alma Mater Studiorum University of Bologna
Via Belmeloro 6/Via Irnerio 48, 40126 Bologna (Italy)
E-mail: manuela.bartolini3@unibo.it
marialaura.bolognesi@unibo.it
[b] Dr. O. Soukup, Dr. J. Korabecny
Biomedical Research Center, University Hospital Hradec Kralove
Sokolska 581, 500 05 Hradec Kralove (Czech Republic)
[c] Prof. V. Andrisano
Due to the pathological complexity of AD, multifunctional
molecules with multiple complementary biological activities
may offer an important advance over single-target drugs for
effective treatment of this multifactorial disease. In this respect,
the development of multitarget-directed ligands (MTDLs),
namely small molecules able to hit multiple targets responsible
for the underlying neurodegeneration of AD, has been pro-
posed as a promising therapeutic option.[11]
Department for Quality Life Studies
Alma Mater Studiorum University of Bologna
Corso d’Augusto 237, 47921 Rimini (Italy)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600014.
This article is part of a Special Issue on Polypharmacology and
Multitarget Drugs. To view the complete issue, visit:
http://onlinelibrary.wiley.com/doi/10.1002/cmdc.v11.12/issuetoc.
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A possible and effective strategy to generate novel MTDLs
and incorporate activities toward different targets into a single
molecule involves the combination of structural elements de-
rived from selective ligands at each target.[12] This approach
has resulted in numerous potent multifunctional hybrid com-
pounds potentially useful against AD.[13] Among these, Rodrí-
guez-Franco and co-workers[14] reported a series of tacrine-8-
hydroxyquinoline (8HQ) hybrids as novel MTDLs for AD treat-
ment, with cholinergic, antioxidant, and CuII-complexing prop-
erties. Indeed, while tacrine is a well known cholinesterase in-
hibitor, several 8HQ-related compounds have shown neuropro-
tective activity,[15] which correlates with their ability to complex
redox-active metals and to decrease metal-driven oxidative
phenomena and Ab-mediated neurotoxicity. In this context,
the 8HQ derivatives clioquinol (CLQ) and PBT2 (Figure 1) have
zil. In fact, we envisaged that the combination of structural fea-
tures from the ChE inhibitor donepezil with the 8HQ core of
CLQ and PBT2, endowed with metal-chelating, neuroprotec-
tive, and antioxidant properties, should lead to a new class of
MTDLs with a wider spectrum of biological activities and po-
tential disease-modifying properties (Figure 1).
Design
We sought to combine the anti-acetylcholinesterase (AChE) ac-
tivity of donepezil with the metal-chelating properties of the
8HQ scaffold by synthesizing benzylpiperazine/benzylpiperidin-
4-amine-8HQ hybrids 1a–f, 2a–f, and 3a, depicted in
Schemes 1 and 2 below. Donepezil, a marketed drug for AD
treatment, is known to act as AChE inhibitor by establishing
multiple interactions within the enzymatic cavity.[23] In fact, it
orients itself along AChE by spanning the enzyme gorge from
the peripheral anion site (PAS) to the catalytic active site
(CAS).[24] Notably, donepezil is also a micromolar butyrylcholi-
nesterase (BChE) inhibitor.[25]
Both benzylpiperazine and benzylpiperidin-4-amine frag-
ments were employed in the derivatization process to mimic
the 1-benzylpiperidine tail of donepezil. The introduction of an
extra nitrogen on the piperidine ring of donepezil (either
endo- or exocyclic) was mainly related to synthetic issues, con-
sidering the rather more-demanding formation of carbon–
carbon bonds. In fact, the two amino groups of piperazine and
piperidin-4-amine were easily functionalized, with a benzyl
moiety on one side and a 8HQ core on the other, to access
target compounds. In particular, under the optimized reaction
protocol, the secondary amine piperazine was more reactive
than the primary amine piperidin-4-amine and thus was more
readily exploited. The effect of the introduction of electron-do-
nating (OMe) and -withdrawing (Cl, Br) substituents on differ-
ent positions of the benzylic ring of donepezil was also evalu-
ated.
Figure 1. Hybrid compound design strategy.
been investigated for their neuroprotective effect in AD,[16] Par-
kinson’s disease,[17] and Huntington’s[18] disease and have en-
tered clinical trials for AD. Despite good results in AD animal
models and in small clinical trials involving AD patients,[19]
long-term use of CLQ was hampered by adverse side effects.[20]
On the other hand, PBT2 demonstrated good safety, tolera-
bility, and efficacy in patients with early AD in a clinical pha-
se IIa trial.[21] However, in 2014, unsatisfactory results from the
IMAGINE trial made Prana Biotechnology discontinue the fur-
ther development of PBT2 for AD treatment.[22] Indeed, the
IMAGINE trial highlighted that PBT2 was unable to decrease
the number of insoluble amyloid plaques and did not meet
the target endpoints for cognition and function.[22] Despite
clinical trial failures, both CLQ and PBT2 show promising thera-
peutic features as metal–protein attenuation compounds
(MPACs), able to sequester CuII and ZnII from both amyloid
plaques and the synaptic cleft, and act as CuII ionophores to
compensate the AD-related CuII dyshomeostasis.[4]
The 5-chloro-8HQ core of CLQ and PBT2 (compounds in
series 1 and 3), as well as the 8HQ nucleus (compounds in
series 2) were selected for chemical derivatization. These aro-
matic and flat 8HQ cores, in addition to the favorable neuro-
protective properties discussed above, might also mimic the
dimethoxyindanone ring of donepezil, thus possibly retaining
the same p-stacking interactions of the parent compound
within the AChE PAS.
Synthesis
7-((4-Benzylpiperazin-1-yl)methyl)-8-hydroxyquinolines
1a–f
and 2a–f were prepared in a two-step synthetic route, as sum-
marized in Scheme 1. First, piperazine (4a) was treated with
paraformaldehyde and 5-chloro-8HQ (5a) or 8HQ (5b) through
a multicomponent Mannich reaction to provide the corre-
sponding 7-(piperazin-1-ylmethyl)-8HQs 6a and 6b, respective-
ly.
Motivated by these considerations, and as a result of our in-
terest in exploring original framework combinations, we pro-
posed a novel series of hybrids, rationally designed by fusing
the 8HQ scaffold with different benzylpiperidine-like moieties
inspired by the chemical structure of the anti-AD drug donepe-
We optimized an efficient and simple microwave-assisted
synthetic protocol to afford 6a and 6b in good yields (76 and
72%, respectively) and shorter reaction time with respect to re-
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from 9.0 to 63.8% for 5-chloro-8HQ derivatives 1a–f and from
49.2 to 89.1% for 8HQ derivatives 2a–f. Thus, compounds of
both series were BChE-selective inhibitors.
A comparison of the inhibitory activities clearly highlighted
that the inhibitory potency toward BChE was negatively affect-
ed by the presence of a chlorine atom at position 5 of the 8HQ
nucleus, with all derivatives from series 1 being less active
than those from series 2. For derivatives showing greater than
50% inhibition at 40 mm (2a–f and 1b), IC50 values (i.e., the
concentration of inhibitor required to decrease the enzyme ac-
tivity by 50%) were determined. Results are listed in Table 1.
An analysis of the results revealed that BChE inhibition was in-
fluenced by the presence and the position of a substituent on
the benzyl moiety, with the most potent derivatives bearing
a substituent at position 2 (2b, 2e, 2 f). Among the 2-substitut-
ed derivatives, 2b, bearing a methoxy group, showed the
highest potency. Removing either the halogen atom or the
methoxy group from the ortho position significantly decreased
the activity, indicating a favorable effect of a substituent at
such a position. Moving the methoxy group from position 2 to
either position 3 or 4 resulted in derivatives with a statistically
significantly, albeit slightly, lower inhibitory potency. Indeed,
2c (IC50 =47.2 mm) was eightfold less active than 2b (IC50 =
5.71 mm).
These structure–activity relationships (SARs) also apply to
compounds from series 1. In fact, among the 7-((4-benzylpiper-
azin-1-yl)methyl)-5-chloro-8HQ derivatives, 2-methoxy ana-
logue 1b was the only one able to inhibit human BChE
(hBChE) by more than 50% at 40 mm (IC50 =23.3 mm). These
findings are consistent with those obtained in previous studies
on two series of benzyl-substituted polyamine derivatives.[25b,28]
Similarly, it can be speculated that substituents on the benzyl
ring affect the protonation of the basic nitrogen on the pipera-
zine group through inductive and mesomeric effects.
Scheme 1. Synthesis of compounds 1a–f and 2a–f. Reagents and conditions:
a) paraformaldehyde, EtOH, MW 1308C, 40 min; b) DIPEA, DMF, RT, 2–4 h.
ported conventional heating-based procedures.[26] Subsequent-
ly, 6a and 6b were combined with the proper benzylchloride
(7a–f) by classical SN2 nucleophilic substitution to give target
compounds 1a–f and 2a–f in good to excellent yields (47–
97%).
7-(((1-Benzylpiperidin-4-yl)amino)methyl)-5-chloro-8HQ
3a
was synthesized from the commercially available 1-benzylpi-
peridin-4-amine (4b) and 5-chloro-8HQ (5a) in a one-pot reac-
tion, following the previously described Mannich procedure
with minor modifications (Scheme 2).
A comparison of the IC50 values of 1b and 2b confirmed the
detrimental effect of the chlorine atom on the 8HQ nucleus.
Indeed, by simply removing the chlorine atom, the inhibitory
potency increased fourfold (23.3 vs. 5.71 mm). The most active
hBChE inhibitor was 2b.
Scheme 2. Synthesis of compound 3a. Reagents and conditions: a) parafor-
maldehyde, EtOH, MW 708C, 25 min.
Interestingly, 5-chloro-8HQ derivative 3a, in which the piper-
azine nucleus was replaced by a 4-aminopiperidine residue,
showed a completely different activity profile. This structural
modification had a drastically beneficial effect on the inhibitory
activity against hAChE. Indeed, while at 40 mm, piperazine ana-
logue 1a was completely inactive, 3a inhibited hAChE by
54.9% and showed an IC50 value in the micromolar range
(32.0Æ1.9 mm). Compound 3a was therefore the only deriva-
tive endowed with a significant inhibitory activity against
hAChE. This increase in anti-AChE activity was accompanied by
a change in the selectivity profile: while all derivatives within
series 1 and 2 are highly selective hBChE inhibitors, 3a is a non-
selective inhibitor of both cholinesterases (AChE/BChE=1.4).
The higher anti-hAChE activity can be conceivably ascribed
to the presence of an alkylamino functionality endowed with
a higher degree of flexibility. Indeed, previous studies showed
that embedding the amino functionality into a piperidine or pi-
perazine nucleus led to weaker anticholinesterase activity.[29]
Results and Discussion
To determine their potential as MTDLs for the treatment of AD,
1a–f, 2a–f, and 3a were assayed for their inhibitory activity
against human ChE in comparison with CLQ and the drugs do-
nepezil, tacrine, and galantamine. Screening at a single con-
centration ([I]=40 mm) was initially performed to assess inhibi-
tory activity on recombinant human AChE (hAChE) using the
spectrophotometric method of Ellman et al.[27] For the most
soluble compounds, 2a–f, a higher concentration (100 mm)
was also assayed. The lower solubility of compounds 1a–f pre-
vented them from being tested at concentrations higher than
40 mm. As reported in Table 1, derivatives 1a–f and 2a–f were
inactive or were very weak inhibitors of hAChE, with the per-
cent inhibition not exceeding 21% (at 100 mm). On the other
hand, all compounds within series 1 and 2 acted as inhibitors
of BChE from human serum, with inhibition at 40 mm ranging
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Table 1. Inhibitory activity toward hAChE, hBChE, and amyloid self-aggregation by 1a–f, 2a–f, 3a, 4b, 1-benzylpiperazine (1-BP), and reference com-
pounds.
hAChE Inhib. [%][a]
hBChE[a]
Ab42 self-aggregation[c]
Compd
X
R
[I]=40 mm
[I]=100 mm
Inhib. [%]
IC50 [mm][b]
Inhib. [%]
[I]=40 mm
1a
1b
1c
1d
1e
1 f
2a
2b
2c
2d
2e
2 f
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
n.a.
21.9Æ2.7
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
13.7Æ1.4
20.2Æ2.8
14.1Æ2.6
21.0Æ0.2
8.9Æ2.5
15.0Æ3.5
11.7Æ1.1
n.d.
56.3Æ5.3
53.2Æ2.2
55.3Æ5.4
47.6Æ0.6
19.1Æ2.9
41.8Æ7.6
59.4Æ5.4
44.2Æ1.7
42.6Æ0.5
38.1Æ2.3
43.3Æ1.4
54.5Æ11.2
2-OMe
3-OMe
4-OMe
2-Br
2-Cl
H
2-OMe
3-OMe
4-OMe
2-Br
63.8Æ1.2 23.2Æ0.9
9.2Æ0.3
9.0Æ1.6
n.d.
n.d.
n.d.
n.d.
39.2Æ1.4
42.6Æ2.2
53.3Æ1.8 32.8Æ1.7
89.1Æ1.1
5.71Æ0.20
49.2Æ2.6 47.2Æ3.4
67.8Æ0.7 16.3Æ0.7
77.2Æ0.6 11.4Æ0.3
73.0Æ1.0 13.9Æ1.0
n.a.
n.a.
n.a.
H
2-Cl
3a
55.9Æ2.6[d]
74.9Æ3.1[d]
63.5Æ0.4 23.3Æ1.0
65.0Æ2.7
4b
n.a.
n.a.
n.a.
n.a.
n.d.
n.d.
n.d.
n.d.
1-BP
n.a.
n.a.
@90[e]
@90
>90
n.a.
n.s.
@90[e]
@90[f]
>90
clioquinol
donepezil
tacrine
galantamine
curcumin
n.a.
n.a.
34.9Æ3.3
<10
<5[33]
<10
73.7Æ3.2[34]
84.3Æ0.8
7.42Æ0.39[25b]
@90
0.046Æ0.003[33]
65.8Æ2.3 18.8Æ1.2
n.d.
n.d.
n.d.
n.d.
[a] Human recombinant AChE and BChE from human serum were used; Percent inhibition data are the meanÆSEM of two independent experiments each
performed in duplicate. [b] Inhibitor concentration required to decrease enzyme activity by 50%; values are the meanÆSEM of two independent measure-
ments, each performed in duplicate. [c] Inhibition of Ab42 self-aggregation (50 mm) by an equimolar concentration of inhibitor ([I]=50 mm); values are the
meanÆSEM of two independent experiments, each performed in duplicate. [d] IC50hAChE =32.0Æ1.9 mm. [e] IC50hAChE =0.023Æ0.005 mm.[25b] [f] IC50hAChE
=
0.424Æ0.021 mm.[33] n.a.=not active; n.s.=not soluble at the given concentration; n.d.=not determined.
Inhibition of amyloid self-aggregation
Despite their completely different selectivity profiles, 3a and
2b appeared to be the most interesting derivatives in terms of
anticholinesterase activity, with 3a being a balanced inhibitor
of both ChE enzymes, and 2b being a highly selective BChE in-
hibitor with a selectivity ratio of more than two orders of mag-
nitude. It is worth mentioning that the potential value of BChE,
in addition to AChE, as a therapeutic target for AD treatment
has been emphasized by increasing evidence.[30] While AChE
activity is predominant in the healthy human brain, in the AD
brain, AChE activity decreases and BChE activity increases or is
unaltered.[31] Thus, while in the early stages of the disease,
AChE-selective inhibitors are likely to be effective in raising the
cholinergic tone, in moderate to severe forms of AD, balanced
anticholinesterase activity and/or BChE-selective inhibition
should offer higher beneficial effects. Further, it was also dem-
onstrated that administration of BChE inhibitors to rats not
only led to an increase in cognitive function, presumably
through an increase in the concentration of the neurotransmit-
ter acetylcholine (ACh), but also decreased Ab levels.[32]
Soluble Ab aggregates are able to trigger a series of cellular
events leading to cell dysfunction, inflammation, and ultimate-
ly, neuron death. In addition to metal ion-dependent mecha-
nisms,[19] CLQ and hydroxyquinolines could exert a direct anti-
aggregating activity by inhibiting Ab oligomer formation.[35]
Alternatively, formation of ternary complexes with metal
ions and amyloid peptides was also suggested as potential
mechanism of action of 8HQ.[36] Finally, conjugated polymers
containing an 8HQ nucleus were shown to be able to seques-
ter metal ions from Ab protofibril aggregates and diminish
their accumulation.[37] Based on these promising premises, the
inhibitory activity against the spontaneous aggregation of Ab42
was evaluated. The Ab42 peptide was selected from the various
isoforms because it is more hydrophobic and fibrilogenic than
shorter isoforms and is the principal toxic species deposited in
the brain.[38] A single concentration screening assay was carried
out using equimolar concentrations of Ab42 and inhibitor
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(50 mm). Percent inhibition of amyloid fibril formation was de-
termined using a thioflavin T-based fluorimetric assay.[39]
At the selected concentration, both series of derivatives
were able to significantly inhibit Ab42 self-aggregation
(Table 1). For all compounds except 1e, inhibition values were
within a fairly narrow range (38.1–59.4%), indicating that,
unlike ChE inhibition, the anti-aggregating activity was not sig-
nificantly influenced by either the position of the substituent
on the benzyl ring or the presence/absence of the chlorine
atom on the quinoline moiety. Even though a clear correlation
between the inhibitory activity and the type and the position
of the substituent on the benzyl ring could not be drawn, it
seems that an unsubstituted benzyl ring was slightly preferred.
Compounds 1a and 2a were the most active derivatives (al-
though to a limited extent) in series 1 and 2 (inhibition: 56.3
and 59.4%, respectively). Replacing the 1-benzylpiperazine
moiety with a 1-benzylpiperidin-4-amine residue, as in 3a, led
to a significant, albeit slight, increase in the inhibitory potency
(3a vs. 1a). Derivative 3a showed the highest inhibition po-
tency (65%, Table 1), a value quite close to that of the known
anti-aggregating agent curcumin (73.7%).[34] The overlaid time-
course fluorescence spectra of Ab42 recorded after 24 h incuba-
tion in the absence and presence of 3a (as well as that record-
ed in the presence of 2b) are provided in the Supporting Infor-
mation (Figure S1).
Figure 2. a) Overlaid UV/Vis spectra of 2b (25 mm) alone (black) and in the
presence of 50 mm ZnCl2 (red) in phosphate buffer (10 mm, pH 7.4); b) over-
laid difference spectra of mixtures of 2b (25 mm) and increasing concentra-
tions of ZnCl2, ranging from 1.56 to 50 mm.
sulted in significant changes in the absorbance spectrum with
the appearance of a new band centered at 261 nm, with bath-
ochromic shift of ~18 nm from the original band (in the ab-
sence of any metal). An increase in the metal ion concentration
resulted in a higher intensity absorption band at 261 nm as
a result of a higher amount of complex formed (Figure 2b).
The bathochromic shift and trend demonstrated that 2b could
effectively complex metal ion under physiological conditions
(i.e., phosphate buffer, pH 7.4).
Finally, a comparison of the Ab anti-aggregating properties
of the new 8HQ derivatives with those of the reference com-
pound CLQ clearly highlights the better inhibitory activity of
these new compounds. Furthermore, under the same assay
conditions, the anti-AD drugs donepezil, tacrine, and galanta-
mine were not able to significantly decrease amyloid fibril for-
mation.
Similarly, a bathochromic shift was observed when either
CuII or ZnII was added to a solution of 1b or 3a (see Support-
ing Information). The stoichiometry of the 2b–CuII complex
was determined using the molar ratio method. As shown in
Figure 3a, the two straight lines intersected at a mole fraction
of 0.5, indicating a 1:2 stoichiometry of the M–C complex. The
same trend was observed when ZnII was used instead of CuII
(Figure 3b).
Based on their anti-ChE and anti-aggregating profiles, 2b
and 3a were selected for further studies. Indeed, while 3a was
the derivative with the best activity profile toward both tar-
gets, 2b, although slightly less active as an anti-aggregating
agent, was endowed with a better solubility profile. Com-
pound 1b, as the 5-chloro analogue of 2b, was also included
in further studies to obtain information on the role of the
chlorine atom in the multitarget activity profile.
This result is in agreement with the previously reported che-
lating properties of CLQ,[40c] hydroxyquinoline, and selenium-
containing clioquinol derivatives.[41] Furthermore, the change
in the UV spectrum observed for 2b–M complex formation is
similar to that previously reported by Ferrada et al.[40b] for CLQ
in the presence of CuII or ZnII, thus confirming a similar involve-
ment of the 8HQ fragment in metal complex formation.
Derivative 1b, the 5-chloro analogue of 2b, was also able to
complex both metal ions in phosphate buffer (10 mm, pH 7.4).
However, a difference in the ability of this compound to che-
late the two ions was observed. While, similarly to 2b, 1b
could clearly complex CuII with a M/C of 1:2 (data not shown),
it had a lower capacity to complex ZnII ions. Indeed, even if
a bathochromic shift indicated that 1b could chelate ZnII (Fig-
ure S2), no significant change in the UV spectrum was ob-
served at M/C ratios lower than 1, indicating that the complex
was formed only in excess concentrations of ZnII. The stoichi-
ometry of the 1b–ZnII complex could not be determined.
Metal chelating properties of compounds 1b, 2b, and 3a
Based on the idea that metal chelation could decrease metal-
dependent cytotoxic events, providing therapeutically relevant
effects, compounds 1b, 2b, and 3a were evaluated for their
ability to chelate the metal ions CuII and ZnII. The chelating
properties of CLQ and 8HQ, as well as the equilibrium con-
stants for the formation of CuII and ZnII complexes, have been
extensively investigated.[40] However, as introduction of sub-
stituents on the 8HQ nucleus might modulate the chelating
properties, these properties needed to be investigated and
confirmed. Thus, the ability to complex CuII and ZnII was inves-
tigated by UV/Vis difference spectroscopy. Figure 2a shows, as
representative example, the overlaid UV/Vis spectra of 2b in
the absence and presence of ZnCl2 in a metal ion/compound
(M/C) ratio of 2:1. The addition of either CuII or ZnII to 2b re-
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Figure 4. Antioxidant properties of 1b, 2b, and 3a in comparison with
Trolox. Control: colored radical ABTS. + formation after a 3 min reaction in
the presence of metmyoglobin and hydrogen peroxide. The capture of free
oxygen radicals by antioxidants reduces the formation of color species and
the corresponding absorbance (see Experimental Section for details).
methylchroman-2-carboxylic acid), an analogue of vitamin E,
was used as a reference. As shown in Figure 4, all selected
compounds were able to significantly decrease the formation
of free radicals and thus possessed significant antioxidant ac-
tivity. However, while 5-cloro-8HQ derivatives 1b and 3a
showed a slightly lower antioxidant capacity than Trolox (per-
cent inhibition of free radical formation at 27 mm equal to 92.7,
81.6, and 68.4% for Trolox, 1b, and 3a, respectively), the 8HQ
2b was shown to be slightly more potent than Trolox (98.9 vs.
92.7% inhibition), suggesting that the presence of the chloro
substituent is not beneficial for the antioxidant properties of
this class of compounds. The calculated total antioxidant
status (TAS) values were 0.80, 1.63, and 0.66 mm for 1b, 2b,
and 3a, respectively.
Figure 3. Determination of the stoichiometry of a) 2b–CuII complex and
b) 2b–ZnII complex in phosphate buffer (10 mm, pH 7.4) using the molar
ratio method. The final concentration of 2b was 25 mm, and the final con-
centration of copper(II) or zinc(II) ranged from 1.56 to 50 mm. A breakpoint
was observed at M/2b=0.5:1.
Finally, 3a, endowed with the same 5-chloro-8HQ nucleus,
chelated CuII and ZnII (Figure S3a and S3b, respectively) with
a lower affinity than 2b but higher affinity than 1b. The stoi-
chiometry of the 3a–CuII complex was determined as 2:1, anal-
ogous to what was observed for 2b and 1b. Conversely, be-
cause the high concentration of ZnCl2 required to titrate the
tested compound exceeded the solubility of the metal ion and
of the M–C complex, the stoichiometry of the 3a–ZnII complex
could not be determined.
Cytotoxicity
Acute cytotoxicity exerted by 1b, 2b, and 3a in comparison
with CLQ was estimated in a human glioma cell line (T67) and
in primary human umbilical vein endothelial cells (HUVEC)
using a resazurin-based assay.[46] In T67 cells, the cytotoxicity
exerted by the three compounds at concentrations ranging
from 0.5 mm to 50 mm was evaluated first. At the lower concen-
trations tested, compounds 1b, 2b, and CLQ showed similar
toxicity profiles, with none exhibiting significantly toxic at con-
centrations up to 10 mm (cell viability >90%). However, at the
highest tested concentration (50 mm), 1b decreased cell viabili-
ty by 78% (residual cell viability 22%), while cell viability after
treatment with CLQ and 2b remained at 91% and 81%, re-
spectively. On the other hand, and unexpectedly, 3a was
highly cytotoxic even at low concentrations, decreasing cell vi-
ability by 49% at 5 mm and by 85% at 10 mm. At the highest
tested concentration, the cytotoxic effects exerted by 1b and
3a were similar (Figure 5). Thus, among the selected deriva-
tives, 2b was endowed with the best profile, similar to that of
CLQ. Due to the good safety profile at 50 mm, the cytotoxicity
of higher concentrations of 2b (up to 100 mm) was evaluated
to obtain an IC50 value of 65.8Æ1.2 mm (Figure S5).
As a general consideration, the introduction of a chloro-sub-
stituent at position 5 negatively influenced the chelating prop-
erties of the 8HQ moiety in agreement with Ferrada et al.[40b]
who calculated that CLQ has a 17-fold higher affinity for CuII
than for ZnII.
In vitro antioxidant properties
Cumulative evidence suggests that oxidative stress and
damage are early events that precede the appearance of other
pathological hallmarks of AD, such as amyloid plaques and
neurofibrillary tangles.[42] Elevated levels of oxidative stress
products have been detected in the brain, cerebrospinal fluid
(CSF), blood, and urine of AD patients.[43] Thus, drugs that spe-
cifically scavenge oxygen radicals are thought to be potentially
useful for either the prevention or the treatment of AD[44]
when administered in association with other pharmacological
treatments. The antioxidant properties of 1b, 2b, and 3a were
first evaluated in vitro by testing their ability to neutralize free
radicals formed by the reaction of 2,2-azinobis-(3-ethylbenzo-
thiazoline-6-sulfonic acid) (ABTS) with peroxidase (metmyoglo-
bin) and hydrogen peroxide.[45] Trolox (6-hydroxy-2,5,7,8-tetra-
In the primary HUVEC cells, compounds 1b, 2b, and 3a sig-
nificantly decreased cell viability at much lower concentrations
(Figure 6). IC50 values were determined to be 12.8Æ1.2, 21.6Æ
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Figure 5. Effect of CLQ and derivatives 1b, 2b, and 3a on the viability of
T67 glioma cells. Cell viability was determined by a resazurin-based assay
after 24 h of incubation with the compounds at various concentrations (0.5–
50 mm). Data are reported as a percentage of control treated with vehicle
(DMSO) and are the meanÆSD of four independent experiments.
Figure 7. Effect of CLQ and derivatives 1b and 2b on ROS formation in T67
cells. The antioxidant activity was evaluated against ROS induced by expo-
sure to 100 mm TBH for 60 min and detected following 2’,7’-dichlorodihydro-
fluorescein diacetate (DCFDA) oxidation. Experiments were performed in T67
cells treated or not treated with compound (10 mm) for 24 h. Data are ex-
pressed as the meanÆSD. Significance was determined by ANOVA and Dun-
nett post-test between TBH vs. 1b+TBH (*pꢀ0.05).
us to further explore the antioxidant potential of 1b and 2b in
cultured cells. Thus, their activity against oxidative insult was
assayed in human T67 cells, following treatment with tert-butyl
hydroperoxide (TBH). Unexpectedly, at 10 mm, only 1b signifi-
cantly decreased ROS production (15%, Figure 7), whereas the
antioxidant activity of 2b was not significant. However, it
should be noted that, due to cytotoxicity concerns related to
1b, we could not compare the antioxidant activity of 1b and
2b at higher concentrations, such as that used in the in vitro
TAS assay (i.e., 27 mm). In addition, the possibility that the dis-
crepancy between cell-free and cellular-based assays could be
due to a low cellular uptake cannot be ruled out.
Figure 6. Effect of derivatives 1b, 2b, and 3a on HUVEC cell viability. Cell vi-
ability was determined by resazurin-based assay after 24 h of incubation
with each compound at various concentrations (0.5–100 mm). Data are re-
ported as a percentage of control treated with vehicle (DMSO).
Blood–brain barrier penetration
Anti-AD drugs need to be able to cross the blood–brain barrier
(BBB) and reach therapeutic targets in the CNS. Focusing on
this required property, and considering the lipophilic nature of
our compounds, we selected a parallel artificial membrane per-
meability assay (PAMPA) to provide preliminary predictions of
the BBB penetration of compounds 1b, 2b, and 3a. PAMPA-
BBB is a validated method[48] that employs a brain lipid porcine
membrane. Data obtained for the new compounds were corre-
lated to the US Food and Drug Administration (FDA)-approved
AD drugs rivastigmine, donepezil, and tacrine, for which the
CNS availability is known and was previously determined
under the same experimental conditions.[48] Prediction of BBB
penetration of compounds 1b, 2b, and 3a is summarized in
Table 2. It was evident that all tested compounds had a high
potential to be CNS-bioavailable (Pe >4.0 10À6 cmsÀ1), with Pe
values higher than or similar to those determined for the refer-
ence AD drugs.
1.3, and 5.84Æ1.3 mm for 1b, 2b, and 3a, respectively. It is
worth noting that, similar to what was observed in T67 cells,
2b appeared to be the least toxic among the derivatives
tested, while 3a was the most toxic. Although the IC50 value
obtained for 2b in HUVEC cells was about threefold lower
than that obtained in human glioma cells, it must be consid-
ered that primary cell cultures are extremely sensitive to
damage and more susceptible to drug toxicity. As a term of
paragon, the IC50 values of the antipsychotic drugs pimozide
and chlorpromazine were found to be 22Æ2 and 25Æ1 mm,
respectively, which are quite similar to that for 2b.[47]
Cellular antioxidant properties
The good in vitro antioxidant efficacy of 1b and 2b, together
with their very low cytotoxicity in T67 cells at 10 mm, prompted
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cused microwave reactor was used for microwave-assisted reac-
tions. Column chromatography purifications were performed
under flash conditions using Sigma–Aldrich silica gel (grade 9385,
Table 2. Prediction of BBB penetration of test compounds and reference
drugs.
[a]
Compd
Pe [10À6 cmsÀ1
]
Prediction[b]
60 Š, 230–400 mesh). NMR experiments were run on
a Var-
ian VXR400 (400 MHz for 1H; 100 MHz for 13C). 1H and 13C NMR
spectra were acquired at 300 K using deuterated chloroform
(CDCl3) and deuterated methanol (CD3OD) as solvents. Chemical
shifts (d) are reported in parts per million (ppm) relative to the re-
sidual solvent peak as an internal reference, and coupling con-
stants (J) are reported in hertz (Hz). Spin multiplicity is reported as:
s=singlet, brs=broad singlet, d=doublet, t=triplet, q=quartet,
m=multiplet. Mass spectra were recorded on a Waters ZQ 4000
apparatus with electrospray ionization (ESI) in positive mode. All
final compounds showed ꢁ95% purity by elemental analysis.
General procedure for the synthesis of 7-(piperazin-1-ylmethyl)-
8-hydroxyquinolines (6a and 6b): Paraformaldehyde (3.36 mmol)
and hydroxyquinoline 5a or 5b (3.36 mmol) were added to a solu-
tion of piperazine (4a) (20.4 mmol) in dry EtOH (5 mL),. The result-
ing mixture was stirred at room temperature for 10 min and was
subsequently heated at 1308C for 45 min under microwave (MW)
irradiation. After cooling to room temperature, a yellow precipitate
was removed by filtration, and the filtrate was concentrated under
reduced pressure. The residue was re-dissolved in CH2Cl2 (15 mL)
and washed with H2O (2”15 mL) and saturated aqueous NaCl solu-
tion (15 mL). The organic phase was dried over anhydrous Na2SO4,
filtered, and concentrated in vacuo. The crude material was puri-
fied by chromatography, eluting with CH2Cl2/MeOH/NH3 (9:1:0.1
for 6a; 8:2:0.2 for 6b), to afford the title compound (6a or 6b).
1b
2b
3a
13.7Æ1.2
6.9Æ0.4
6.5Æ1.0
7.3Æ0.9
6.6Æ0.5
5.3Æ0.2
CNS(+)
CNS(+)
CNS(+)
CNS(+)
CNS(+)
CNS(+)
donepezil
rivastigmine
tacrine
[a] Values are the meanÆSEM (n=4–8). [b] CNS(+): high BBB permeation
predicted.
To further assess compound safety, any alteration of the in-
tegrity of the phospholipid layer subsequent to compound
passage was excluded using the fluorescent probe lucifer
yellow, which is not able to cross the intact membrane. Lucifer
yellow was added to the donor well, together with the tested
inhibitor, and the fluorescence intensity in the acceptor well
was determined after the incubation time. A negligible fluores-
cence intensity, which was not significantly different from the
control (lucifer yellow without inhibitor), confirmed that the
tested compounds did not alter membrane permeability and
were safe.
Conclusions
5-chloro-7-(piperazin-1-ylmethyl)-8-hydroxyquinoline (6a): Com-
pound 6a was isolated as a yellow powder (yield: 700 mg, 76%):
1H NMR (400 MHz, CDCl3): d=8.91 (dd, J=4.4, 1.6 Hz, 1H), 8.47
(dd, J=8.5, 1.6 Hz, 1H), 7.50 (dd, J=8.5, 4.4 Hz, 1H), 7.34 (s, 1H),
3.86 (s, 2H), 3.01–2.98 (m, 4H), 2.72–2.58 ppm (m, 4H); 13C NMR
(100 MHz, CDCl3): d=152.3, 149.3, 139.7, 132.7, 127.3, 126.0, 121.9,
119.9, 118.0, 60.4, 53.6, 45.8 ppm; MS (ESI+) m/z: 278 [M+H]+.
Several experimental observations support the notion that, in
addition to other triggers, brain metal dyshomeostasis may di-
rectly or indirectly contribute to AD pathogenesis by promot-
ing Ab misfolding, free radical generation, and heightened oxi-
dative damage. Building on this rationale, a new series of
8HQ–donepezil hybrids were designed to exert a carefully se-
lected anti-AD MTDL profile: 1) ChE inhibition, 2) copper(II) and
zinc(II) chelation, 3) ROS scavenging, and 4) anti-aggregating
activity on Ab42. When evaluated in vitro, some of the synthe-
sized compounds displayed a biological profile in compliance
with the underpinning rationale. As another positive point
from a neurodegeneration drug discovery perspective, PAMPA
data predicted that compounds 1b, 2b, and 3a have high pas-
sive BBB permeability. Of note, compound 2b, encompassing
in vitro anti-BChE, anti-aggregating, CuII- and ZnII-complexing,
and antioxidant properties in a single chemical entity, might
be worthy of further investigation.
7-(piperazin-1-ylmethyl)-8-hydroxyquinoline (6b): Compound 6b
was isolated as a yellow powder (yield: 817 mg, 72%): 1H NMR
(400 MHz, CDCl3): d=8.82 (dd, J=4.4, 1.5 Hz, 1H), 8.02 (dd, J=8.5,
1.5 Hz, 1H), 7.31 (dd, J=8.5, 4.4 Hz, 1H), 7.21–7.15 (m, 2H), 3.83 (s,
2H), 2.94–2.92 (m, 4H), 2.57–2.55 ppm (m, 4H); 13C NMR (100 MHz,
CDCl3): d=153.1, 148.9, 139.2, 135.6, 128.4, 127.6, 121.2, 117.6,
117.36, 61.1, 53.7, 45.9 ppm.
General procedure for the synthesis of 7-((4-benzylpiperazin-1-
yl)methyl)-8-hydroxyquinolines (1a–f and 2a–f): DIPEA (36 mL)
and the corresponding benzyl chloride (7a–f, 0.21 mmol) were
added to a solution of 7-(piperazin-1-ylmethyl)-8-hydroxyquinoline
6a or 6b (0.21 mmol) in DMF (1.2 mL), and the reaction mixture
was stirred at room temperature for 2–4 h. The solvent was re-
moved in vacuo, and the resulting residue was re-dissolved in
CH2Cl2 (15 mL) and washed with saturated aqueous NaCl solution
(3”15 mL). The organic layer was dried over anhydrous Na2SO4, fil-
tered, and concentrated under reduced pressure. The crude materi-
al was purified by either trituration with ether or by flash chroma-
tography to afford the title compound (1a–f and 2a–f).
Experimental Section
Chemistry
All commercially available reagents and solvents were purchased
from Sigma–Aldrich, Fluka (Italy), TCI, and Alpha Aesar without fur-
ther purification. Reactions were followed by analytical thin layer
chromatography (TLC), performed on precoated TLC plates
(0.20 mm silica gel 60 with UV254 fluorescent indicator, Merck). De-
veloped plates were air-dried and visualized by exposure to UV
light (l=254 nm and 365 nm). Reactions involving generation or
consumption of amine were visualized using bromocresol green
spray (0.04% in EtOH, made blue by NaOH). A CEM Discover SP fo-
7-((4-benzylpiperazin-1-yl)methyl)-5-chloro-8-hydroxyquinoline
(1a): Purification by flash chromatography (petroleum ether/
CH2Cl2/MeOH/NH3, 3.5:6.0:0.5:0.05) afforded title compound 1a as
1
a yellow powder (yield: 47 mg, 61%): mp: 169.3–170.58C; H NMR
(400 MHz, CDCl3): d=8.91 (dd, J=4.2, 1.6 Hz, 1H), 8.46 (dd, J=8.5,
1.6 Hz, 1H), 7.48 (dd, J=8.5, 4.2 Hz, 1H), 7.34–7.28 (m, 5H), 7.28–
7.23 (m, 1H), 3.86 (s, 2H), 3.53 (s, 2H), 2.79–2.40 ppm (m, 8H);
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13C NMR (100 MHz, CDCl3): d=152.5, 149.3, 139.8, 137.8, 132.7,
129.1, 128.2, 127.2, 127.1, 126.0, 121.9, 119.8, 117.9, 62.8, 60.0, 52.8,
52.7 ppm; MS (ESI+) m/z: 368 [M+H]+; Anal. calcd for
C21H22ClN3O·H2O: C 68.56, H 6.03, N 11.42, found: C 68.47, H 6.10, N
11.58.
1g as a yellow powder (yield: 46 mg, 55%); mp: 144.5–145.28C;
1H NMR (400 MHz, CDCl3): d=8.90 (dd, J=4.1, 1.5 Hz, 1H), 8.45
(dd, J=8.5, 1.5 Hz, 1H), 7.44 (dd, J=8.5, 4.1 Hz, 1H), 7.48–7.42 (m,
2H), 7.35–7.33 (m, 2H), 7.21–7.16 (m, 2H). 3.87 (s, 2H), 3.67 (s, 2H),
2.80–2.65 ppm (m, 8H); 13C NMR (100 MHz, CDCl3): d=152.3, 149.3,
139.7, 135.4, 134.4, 132.7, 130.7, 129.5, 128.2, 127.2, 126.5, 126.0,
121.9, 119.8, 117.9, 59.9, 59.0, 52.7, 52.7 ppm; MS (ESI+) m/z: 402
[M]+; Anal. calcd for C21H21Cl2N3O: C 62.69, H 5.26, N, 10.44, found:
C 62.52, H 5.32, N, 10.61.
5-chloro-7-((4-(2-methoxybenzyl)piperazin-1-yl)methyl)-8-hy-
droxyquinoline (1b): Purification by flash chromatography (petro-
leum ether/CH2Cl2/MeOH/NH3, 3.5:6.0:0.5:0.05) afforded title com-
pound 1b as a yellow powder (yield: 45 mg, 54%); mp: 148.5–
1
149.38C; H NMR (400 MHz, CDCl3): d=8.91 (dd, J=4.1, 1.6 Hz, 1H),
7-((4-benzylpiperazin-1-yl)methyl)-8-hydroxyquinoline (2a): Pu-
rification by flash chromatography (petroleum ether/CH2Cl2/MeOH/
NH3, 3.0:6.0:1.0:0.1) afforded title compound 2a as a yellow
powder (yield: 65 mg, 93%); mp: 147.6–148.18C; 1H NMR
(400 MHz, CDCl3): d=8.84 (dd, J=4.1, 1.6 Hz, 1H), 8.03 (dd, J=8.3,
1.6 Hz, 1H), 7.33 (dd, J=8.3, 4.1 Hz, 1H), 7.30–7.28 (m, 4H), 7.23–
7.22 (m, 1H), 7.20 (d, J=3.2 Hz, 2H), 3.87 (s, 2H), 3.51 (s, 2H), 2.88–
2.35 ppm (m, 8H); 13C NMR (100 MHz, CDCl3): d=153.2, 148.9,
139.2, 137.7, 135.6, 129.1, 128.4, 128.2, 127.6, 127.1, 121.2, 117.6,
117.3, 62.8, 60.4, 52.8, 52.6, 29.7 ppm; MS (ESI+) m/z: 334 [M+H]+;
Anal. calcd for C21H23N3O·0.5H2O: C 73.66, H 7.06, N 12.27, found: C
73.42, H 7.18, N 12.54.
8.46 (dd, J=8.5, 1.6 Hz, 1H), 7.48 (dd, J=8.5, 4.1 Hz, 1H), 7.32–7.33
(m, 2H), 7.27–7.20 (m, 1H), 6.97–6.84 (m, 2H), 3.86 (s, 2H), 3.81 (s,
3H), 3.61 (s, 2H), 2.88–2.40 ppm (m, 8H); 13C NMR (100 MHz,
CDCl3): d=157.8, 152.6, 149.3, 139.8, 132.6, 130.6, 128.2, 127.1,
126.0, 125.6, 121.9, 120.2, 119.7, 117.9, 110.5, 60.2, 55.7, 55.4, 52.8,
52.7 ppm; MS (ESI+) m/z: 398 [M+H]+; Anal. calcd for
C22H24ClN3O2·H2O: C 66.41, H 6.08, N 10.56, found: C 66.58, H 6.01,
N 10.48.
5-chloro-7-((4-(3-methoxybenzyl)piperazin-1-yl)methyl)-8-hy-
droxyquinoline (1c): Purification by flash chromatography (petro-
leum ether/CH2Cl2/MeOH/NH3, 3.5:6.0:0.5:0.05) afforded title com-
pound 1c as a yellow powder (yield: 39 mg, 47%); mp: 145.3–
7-((4-(2-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquino-
line (2b): Purification by flash chromatography (CH2Cl2/MeOH/NH3,
9.5:0.5:0.05) afforded title compound 2b as a yellow powder
1
146.08C; H NMR (400 MHz, CDCl3): d=8.91 (dd, J=4.1, 1.5 Hz, 1H),
8.46 (dd, J=8.5, 1.5 Hz, 1H), 7.48 (dd, J=8.5, 4.1 Hz, 1H), 7.31 (s,
1H), 7.23 (t, J=8.0 Hz, 1H), 6.89 (m, 2H), 6.79 (dd, J=7.2, 2.0 Hz,
1H), 3.86 (s, 2H), 3.80 (s, 3H), 3.51 (s, 2H), 2.93–2.40 ppm (m, 8H);
13C NMR (100 MHz, CDCl3): d=159.6, 152.5, 149.3, 139.8, 139.5,
132.7, 129.2, 127.2, 126.0, 121.9, 121.4, 119.8, 118.0, 114.6, 112.5,
62.7, 60.0, 55.2, 52.8, 52.7 ppm; MS (ESI+) m/z: 398 [M+H]+; Anal.
calcd for C22H24ClN3O2·H2O: C 66.41, H 6.08, N 10.56, found: C
66.34, H 6.13, N 10.45.
1
(yield: 40 mg, 53%); mp: 112.3–113.48C; H NMR (400 MHz, CDCl3):
d=8.85 (dd, J=4.2, 1.7 Hz, 1H), 8.05 (dd, J=8.3, 1.7 Hz, 1H), 7.34
(dd, J=8.3, 4.2 Hz, 1H), 7.31 (dd, J=7.2, 1.6 Hz, 1H), 7.23–7.18 (m,
3H), 6.09 (t, J=7.2 Hz, 1H), 6.84 (d, J=8.0 Hz, 1H), 3.88 (s, 2H),
3.79 (s, 3H), 3.61 (s, 2H), 2.80–2.46 ppm (m, 8H); 13C NMR
(100 MHz, CDCl3): d=157.8, 153.2, 148.9, 139.5, 139.3, 135.6, 130.7,
128.4, 128.3, 127.6, 121.2, 120.3, 117.6, 117.3, 110.5, 60.5, 55.7, 55.4,
52.6 ppm; MS (ESI+) m/z: 364 [M+H]+, 386 [M+Na]+; Anal. calcd
for C22H25N3O2·0.5H2O: C 70.94, H 7.04, N 11.28, found: C 70.73, H
7.18, N 11.41.
5-chloro-7-((4-(4-methoxybenzyl)piperazin-1-yl)methyl)-8-hy-
droxyquinoline (1d): Purification by flash chromatography (petro-
leum ether/CH2Cl2/MeOH/NH3, 3.5:6.0:0.5:0.05) afforded title com-
pound 1d as a yellow powder (yield: 81 mg, 97%); mp: 164.7–
7-((4-(3-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquino-
line (2c): Purification by flash chromatography (CH2Cl2/MeOH/NH3,
9.5:0.5:0.05) afforded title compound 2c as a yellow powder (yield:
68 mg, 89%); mp: 130.0–130.78C; 1H NMR (400 MHz, CDCl3): d=
8.85 (dd, J=4.2, 1.7 Hz, 1H), 8.05 (dd, J=8.3, 1.7 Hz, 1H), 7.34 (dd,
J=8.0, 4.2 Hz, 1H), 7.23–7.18 (m, 3H), 6.89–6.87 (m, 2H), 6.78 (d,
J=8.2 Hz, 1H), 3.86 (s, 2H), 3.79 (s, 3H), 3.50 (s, 2H), 2.67–
2.55 ppm (m, 8H); 13C NMR (100 MHz, CDCl3): d=159.6, 153.2,
148.9, 139.5, 139.3, 135.6, 129.2, 128.4, 127.6, 121.4, 121.2, 117.7,
117.3, 114.5, 112.5, 62.7, 60.5, 55.1, 52.8, 52.7 ppm; MS (ESI+) m/z:
364 [M+H]+, 386 [M+Na]+; Anal. calcd for C22H25N3O2·0.5H2O: C
70.94, H 7.04, N 11.28, found: C 71.12, H 6.89, N 11.52.
1
166.18C; H NMR (400 MHz, CDCl3): d=8.91 (dd, J=4.1, 1.5 Hz, 1H),
8.41 (dd, J=8.5, 1.5 Hz, 1H), 7.44 (dd, J=8.5, 4.1 Hz, 1H), 7.27 (s,
1H), 7.17 (d, J=8.5 Hz, 2H), 6.81 (d, J=8.5 Hz, 2H), 3.81 (s, 2H),
3.75 (s, 3H), 3.43 (s, 2H), 2.82–2.33 ppm (m, 8H); 13C NMR
(100 MHz, CDCl3): d=158.8, 152.4, 149.3, 139.7, 132.6, 130.3, 129.6,
127.2, 126.0, 121.9, 119.8, 117.9, 113.6, 62.1, 60.0, 55.2, 52.6 ppm;
MS (ESI+) m/z: 398 [M+H]+, 420 [M+Na]+; Anal. calcd for
C22H24ClN3O2·H2O: C 66.41, H 6.08, N 10.56, found: C 66.60, H 6.15,
N 10.62.
7-((4-(2-bromobenzyl)piperazin-1-yl)methyl)-5-chloro-8-hydroxy-
quinoline·3HCl (1e): The crude material was dissolved in a mini-
mum amount of saturated methanolic HCl solution, and, after cool-
ing to 08C, diethyl ether was added. The resulting precipitate was
triturated with the diethyl ether and collected by filtration to
afford to title compound 1e as a yellow powder (yield: 76 mg,
7-((4-(4-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquino-
line (2d): Purification by flash chromatography (CH2Cl2/toluene/
MeOH/NH3, 8.0:1.5:0.5:0.05) afforded title compound 2d as
1
a yellow powder (yield: 54 mg, 71%); mp: 149.4–150.68C; H NMR
1
65%); mp: 230.1–230.98C; H NMR (400 MHz, CD3OD): d=9.08 (d,
(400 MHz, CDCl3): d=8.86 (dd, J=4.1, 1.5 Hz, 1H), 8.06 (dd, J=8.3,
1.5 Hz, 1H), 7.35 (dd, J=8.3, 4.1 Hz, 1H), 7.24–7.20 (m, 4H), 6.84 (d,
J=8.6 Hz, 2H), 3.89 (s, 2H), 3.79 (s, 3H), 3.47 (s, 2H), 2.88–
2.35 ppm (m, 8H); 13C NMR (100 MHz, CDCl3): d=158.7, 153.2,
148.9, 139.3, 135.6, 130.3, 129.7, 128.4, 127.5, 121.2, 117.7, 117.3,
113.6, 62.2, 60.5, 55.2, 52.7, 52.6 ppm; MS (ESI+) m/z: 364 [M+H]+;
Anal. calcd for C22H25N3O2·0.5H2O: C 70.94, H 7.04, N 11.28, found:
C 70.75, H 7.13, N 11.02.
J=4.8 Hz, 1H) 8.91 (d, J=8.4 Hz, 1H) 7.97–7.94 (m, 2H), 7.84–7.79
(m, 2H), 7.55 (t, J=8.0 Hz, 1H), 7.45 (t, J=7.2 Hz, 1H), 4.68 (s, 2H),
4.63 (s, 2H), 3.75–3.67 ppm (m, 8H); 13C NMR (100 MHz, CD3OD):
d=152.3, 149.3, 140.0, 137.0, 132.8, 132.7, 130.8, 128.6, 127.4,
127.2, 126.0, 124.8, 122.0, 119.9, 117.6, 61.4, 59.5, 52.6, 52.5 ppm;
MS (ESI+) m/z: 446 [M]+; Anal. calcd for C21H21BrClN3O·3HCl·0.5H2O:
C 44.63, H 4.46, N 7.44, found: C 44.52, H 4.32, N 7.37.
5-chloro-7-((4-(2-chlorobenzyl)piperazin-1-yl)methyl)-8-hydroxy-
quinoline (1 f): Purification by flash chromatography (petroleum
ether/CH2Cl2/MeOH/NH3, 5.0:4.0:0.5:0.05) afforded title compound
7-((4-(2-bromobenzyl)piperazin-1-yl)methyl)-8-hydroxyquinoline
(2e): Purification by flash chromatography (petroleum ether/
CH2Cl2/MeOH/NH3, 3.5:6.0:0.5:0.05) afforded title compound 2e as
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1
a yellow powder (yield: 60 mg, 68%); mp: 117.2–117.98C; H NMR
nents except AChE or BChE to account for non-enzymatic reac-
tions. The reaction rates were compared, and the percent inhibi-
tion due to the presence of tested inhibitor at increasing concen-
trations was calculated. Each concentration was analyzed in dupli-
cate, and IC50 values were determined graphically from log concen-
tration–inhibition curves (GraphPad Prism 4.03 software, GraphPad
Software). Donepezil (Sigma), tacrine (Sigma), and galantamine
(Tocris Bioscience), were used as reference compounds. 4-Amino-1-
benzylpiperidine (4b) (Alfa Aesar) and 1-bezylpiperazine dihydro-
chloride (Sigma) were also tested.
(400 MHz, CDCl3): d=8.86 (dd, J=4.1, 1.5 Hz, 1H), 8.06 (dd, J=8.3,
1.5 Hz, 1H), 7.50 (d, J=7.9 Hz, 1H), 7.40 (d, J=7.5 Hz, 1H), 7.33
(dd, J=8.3, 4.1 Hz, 1H), 7.26–7.20 (m, 3H), 7.07 (t, J=7.6 Hz, 1H),
3.88 (s, 2H), 3.60 (s, 2H), 2.82–2.47 ppm (m, 8H); 13C NMR
(100 MHz, CDCl3): d=153.1, 148.8, 139.2, 137.2, 135.6, 132.8, 130.8,
128.5, 128.4, 127.7, 127.1, 124.7, 121.2, 117.6, 117.4, 61.5, 60.2, 52.8,
52.7 ppm; MS (ESI+) m/z: 413 [M+H]+; Anal. calcd for
C21H22BrN3O: C 61.17, H 5.38, N 10.19, found: C 61.32, H 5.47, N
10.35.
7-((4-(2-chlorobenzyl)piperazin-1-yl)methyl)-8-hydroxyquinoline
(2 f): Purification by flash chromatography (petroleum ether/
CH2Cl2/MeOH/NH3, 4.5:5.0:0.5:0.05) afforded title compound 2 f as
Inhibitory potency on Ab42 self-aggregation: Ab42 samples (Bachem
AG, Switzerland) pretreated with 1,1,1,3,3,3-hexafloro-2-propanol
(HFIP) were solubilized with
a CH3CN/Na2CO3 (0.3 mm)/NaOH
1
a yellow powder (yield: 70 mg, 90%); mp: 121.5–122.38C; H NMR
(250 mm) (48.4:48.4:3.2) mixture to obtain a stable stock solution
([Ab42]=500 mm).[39,49] Experiments were performed by incubating
the peptide in 10 mm phosphate buffer (pH 8.0) containing 10 mm
NaCl at 308C for 24 h (final Ab concentration=50 mm) with and
without inhibitors at 50 mm (Ab/inhibitor=1:1). Blank solutions
containing the tested inhibitors without Ab42 were also prepared
and tested. To quantify amyloid fibril formation, the thioflavin T
fluorescence method was used.[49] After incubation, samples were
diluted to a final volume of 2.0 mL with 50 mm glycine–NaOH
buffer (pH 8.5) containing 1.5 mm thioflavin T. A 300-second time
(400 MHz, CDCl3): d=8.86 (dd, J=4.1, 1.6 Hz, 1H), 8.06 (dd, J=8.3,
1.6 Hz, 1H), 7.42 (dd, J=7.4, 1.8 Hz, 1H), 7.37–7.31 (m, 2H), 7.23–
7.14 (m, 4H), 3.90 (s, 2H), 3.64 (s, 2H), 2.80–2.54 ppm (m, 8H);
13C NMR (100 MHz, CDCl3): d=153.1, 148.9, 139.2, 135.6, 135.5,
134.4, 130.7, 129.5, 128.4, 128.2, 127.7, 126.5, 121.2, 117.5, 117.4,
60.3, 59.0, 52.7, 52.6 ppm; MS (ESI+) m/z: 368 [M+H]+; Anal. calcd
for C21H22ClN3O: C 68.56, H 6.03, N, 11.42, found: C 68.73, H 6.21, N,
11.78.
7-(((1-benzylpiperidin-4-yl)amino)methyl)-5-chloro-8-hydroxyqui-
noline (3a): Paraformaldehyde (0.32 mmol) and 5a (0.39 mmol)
were added to a solution of 4b (0.32 mmol) in dry EtOH (0.5 mL).
The resulting mixture was stirred at room temperature for 10 min
and was subsequently heated at 708C for 25 min under MW irradi-
ation (PW=50 W). After cooling to room temperature, the solvent
was removed under vacuum. The crude material was purified by
chromatography, eluting with CH2Cl2/MeOH/NH3 (8:2:0.2), to afford
title compound 3a as a yellow oil–waxy solid (yield: 35 mg, 28%);
1H NMR (400 MHz, CDCl3): d=8.86 (dd, J=4.1, 1.6 Hz, 1H), 8.06
(dd, J=8.3, 1.6 Hz, 1H), 7.45 (dd, J=8.4, 4.1 Hz, 1H), 7.38 (s, 1H),
7.28–7.21 (m, 5H), 4.12 (s, 2H), 3.51 (s, 2H), 2.87 (d, J=11.6 Hz,
2H), 2.63–2.61 (m, 1H), 2.07–1.95 (m, 4H), 1.59–1.54 ppm (m, 21H);
13C NMR (400 MHz, CDCl3): d=151.8, 148.9, 139.4, 137.5, 132.8,
129.2, 128.2, 127.5, 127.2, 125.8, 122.0, 119.7, 118.8, 62.7, 53.5, 51.8,
47.0, 31.5 ppm; MS (ESI+) m/z: 368 [M+H]+; Anal. calcd for
C22H24ClN3O·H2O: C 69.19, H 6.33, N 11.00, found: C 69.38, H 6.06, N
11.14.
scan of fluorescence intensity was carried out (lexc =446 nm; lem
=
490 nm), and plateau values were averaged after subtracting the
background fluorescence of the thioflavin T solution. The fluores-
cence intensities were compared, and the percent inhibition due
to the presence of the tested inhibitor was calculated.
Determination of metal chelating properties: Complexing studies
were performed in phosphate buffer (10 mm, pH 7.4) at room tem-
perature using a UV/Vis spectrophotometer (HP 8453 Hewlett Pack-
ard). Spectra of the tested compound alone (25 mm) and in the
presence of varying concentrations of CuCl2 or ZnCl2 (from 1.56 to
50 mm), as well as of the corresponding solutions of the metal
alone, were recorded in 1 cm quartz cells (Hellma, Italy) (final
volume=3.0 mL). The difference UV/Vis spectra (related to metal/
compound complex formation) were obtained by subtracting the
spectra of the tested inhibitor alone and of the metal ion alone
from the spectra of the M/C mixtures. The wavelengths of maxi-
mum absorption corresponding to the formation of the metal ion/
compound complexes were determined (l=247 nm for 1b, l=
261 nm for 2b, and l=265 nm for 3a). The stoichiometries of the
inhibitor–CuII and inhibitor–ZnII complexes were then determined
by plotting the change in absorbance (DA) at the selected wave-
length versus the metal ion/compound (M/C) molar ratios (M/C
from 0 to 2.0) (GraphPad Prism 4.03, GraphPad Software). The
break point in the plot corresponds to the molar ratio of the metal
ion in the metal–ligand complex.
Biological methods
Human AChE and BChE inhibition assay: AChE inhibitory activity
was evaluated spectrophotometrically at 378C by Ellman’s
method[27] using a Jasco V-530 double beam spectrophotometer.
The rate of increase in absorbance at 412 nm was followed for
5 min. An AChE stock solution was prepared by dissolving human
recombinant AChE (EC: 3.1.1.7) lyophilized powder (Sigma, Italy) in
0.1m phosphate buffer (pH 8.0) containing Triton X-100 (0.1%). A
stock solution of BChE (EC: 3.1.1.8) from human serum (Sigma,
Italy) was prepared by dissolving the lyophilized powder in an
aqueous solution of gelatin (0.1%). Stock solutions of inhibitors (1
or 2 mm) were prepared in MeOH. The assay solution consisted of
a 0.1m phosphate buffer (pH 8.0), with the addition of 5,5’-dithio-
bis(2-nitrobenzoic acid) (340 mm), human recombinant AChE or
human serum BChE (0.02 UmLÀ1, Sigma), and substrate (550 mm
acetylthiocholine iodide or butyrylthiocholine iodide, respectively).
Fifty microliter aliquots of increasing concentrations of the test
compound were added to the assay solution by pre-incubating for
20 min at 378C with the enzyme, followed by addition of substrate.
Assays were carried out with a blank mixture containing all compo-
Determination of antioxidant activity: The abilities of 1b, 2b, and
3a to neutralize free radicals was assayed using a Total Antioxidant
Status assay kit (Randox Laboratories, UK), using Trolox as a stan-
dard and following the manufacturer’s protocol. This colorimetric
method is based on reactivity of the peroxidase compound met-
myoglobin (6.1 mm, HXFe3+), which, in the presence of hydrogen
peroxide (250 mm) in phosphate buffer (pH 7.4), allows the forma-
tion of oxygen radicals.[45] Moreover, metmyoglobin itself is trans-
formed to ferryl myoglobin (*X-[Fe4+ =O]). Ferryl myoglobin sub-
tracts an electron from a cation (2,2’-azino-di-3-ethylbenzthiazoline
sulfonate [ABTS], 610 mm), transforming back to metmyoglobin
and converting to ABTS in a colored radical, quantifiable at
600 nm.[45] The capture of free oxygen radicals by antioxidants re-
duces the formation of color species and the corresponding ab-
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sorbance. The total amount of oxidant was represented by met-
myoglobin and ABTS incubated at 378C in phosphate buffer
(80 mm, pH 7.4). The absorbance was read at t0 and after 3 min of
incubation. The absorbance intensities in the presence or in the
absence of the test compound (or reference compound Trolox) at
27 mm were compared. The percentage of inhibition was calculated
on the basis of Equation (1):
through the lipid membrane (area=0.28 cm2) to the acceptor well.
The concentration of the drug in both the donor and the acceptor
wells was assessed after 3, 4, 5, and 6 h of incubation in quadrupli-
cate using a Synergy HT UV plate reader (Biotek, USA) at the maxi-
mum absorption wavelength. The concentration of the compound
was calculated from the standard curve and expressed as the per-
meability (Pe), according to Equation (2):[52]
ꢀ
ꢁ
ꢂ
ꢀ
ꢁꢃ
DAblankÀDAtest
½drugŠ
½drugŠ
acceptor
Inhibition ð%Þ ¼
 100
ð1Þ
log Pe ¼ log C Á ln 1 À
DAblankÀDAcontrol
equilibrium
ð2Þ
VD Á VA
where C ¼
ðVD þ VAÞArea Á time
in which DAtest and DAcontrol are the differences in absorbance re-
corded in the presence and absence of the tested compound, re-
spectively. The total antioxidant status (TAS) was calculated follow-
ing the manufacturer’s protocol.
In order to verify the influence of tested compounds on the integ-
rity of the PBL layer, lucifer yellow (Sigma–Aldrich), which is not
able to cross the intact PBL, was used as a fluorescent probe. A so-
lution of lucifer yellow (100 mgmLÀ1 in PBS), with or without the
tested compound, was applied to the donor well. After the incuba-
tion period (3 and 6 h), the fluorescence intensities in the donor
and acceptor wells were measured (lexc =485 nm, lem =535 nm)
and compared.
Cytotoxicity assays. The T67 human glioma cell line was derived by
Lauro et al.[50] from a World Health Organization (WHO) grade III
gemistocytic astrocytoma. T67 cells were cultured in DMEM sup-
plemented with 10% FBS, 100 UImLÀ1 penicillin, 100 mgmLÀ1 strep-
tomycin, and 40 mgmLÀ1 gentamycin, in a 5% CO2 atmosphere at
378C, with saturating humidity. Primary human umbilical vein en-
dothelial cells (HUVEC) were purchased from Gibco-Life Technolo-
gies. Cells were grown in phenol-red-free basal medium M200
(Gibco-Life Technologies) with 10% FBS, 1% glutamine, and
growth factors (LSGS, Gibco-Life Technologies) at 378C in a humidi-
fied atmosphere with 5% CO2.[51] Cytotoxicity of selected com-
pounds was estimated using a resazurin-based assay.[46] T67 cells
were seeded in 24-well plates at 1”105 cells per well, while HUVEC
cells were seeded in gelatin-coated 96-well plates at a density of
7000 cells per well. Experiments were performed after 24 h incuba-
tion at 378C in 5% CO2. After this time, cells were washed and
treated for 24 h with different concentrations of compounds; the
cells were then incubated for 60 min with 100 mm resazurin in cul-
Acknowledgements
This work was supported by the University of Bologna (Italy), the
Italian Ministry for Education, Universities and Research (MIUR),
and University Hospital Hradec Kralove (MH CZ-DRO) (UHHK,
00179906). The authors thank Prof. Silvana Hrelia (University of
Bologna), who kindly provided the HUVEC cell line used in this
study, and Dr. Emanuela Leoncini (University of Bologna) for her
helpful assistance.
ture medium. The fluorescence of each well was measured (lexc
=
580 nm; lem =620 nm) with a spectrofluorimeter (Wallac Victor
multilabel counter, PerkinElmer, USA). Data are reported as the
meanÆSD of at least three independent experiments.
Keywords: Alzheimer’s disease · b-amyloid · antioxidants ·
chelation · cholinesterases · inhibitors
Determination of antioxidant activity in T67 cells: To evaluate the
antioxidant activity of the compounds, T67 cells were seeded in
24-well plates at 1”105 cells per well. After 24 h, cells were
washed and treated for 24 h with 10 mm of CLQ, 1b, and 2b. The
antioxidant activity of the compounds was evaluated after 30 min
incubation with 10 mm fluorescent probe (2’,7’-dichlorofluorescein
diacetate, DCFH-DA) in DMEM by measuring the intracellular ROS
formation evoked by 1 h exposure of T67 cells to 100 mm TBH in
PBS. The increase in fluorescence of the cells from each well was
measured (lexc =485 nm; lem =535 nm) with a spectrofluorimeter
(Wallac Victor multilabel S9 counter, PerkinElmer). Data are report-
ed as the meanÆSD of at least three independent experiments.
PAMPA: In order to predict passive BBB penetration of novel com-
pounds, a modified PAMPA was used, based on a reported proto-
col.[48] The filter membrane of the donor plate was coated with
polar brain lipid (PBL, Avanti, USA) in dodecane (4 mL of
20 mgmLÀ1 PBL in dodecane), and the acceptor well was filled
with 300 mL of PBS pH 7.4 (VD). Each tested compound was dis-
solved first in DMSO and then diluted with PBS (pH 7.4) to reach
a final concentration of 100 mm in the donor well. The concentra-
tion of DMSO in the donor solution did not exceed 0.5% (v/v).
Donor solution (300 mL) was added to the donor wells (VA), and the
donor filter plate was carefully set on the acceptor plate so that
the coated membrane was touching both the donor solution and
the acceptor buffer. Test compound diffused from the donor well
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Published online on February 16, 2016
ChemMedChem 2016, 11, 1284 – 1295
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