Exploiting the lipoic acid structure in the search for novel multitarget ligands against Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2011.09.001

Lipoic acid (LA) is a natural antioxidant. Its structure was previously combined with that of the acetylcholinesterase inhibitor tacrine to give lipocrine (1), a lead compound multitargeted against Alzheimer's disease (AD). Herein, we further explore LA as a privileged structure for developing multimodal compounds to investigate AD. First, we studied the effect of LA chirality by evaluating the cholinesterase profile of 1's enantiomers. Then, a new series of LA hybrids was designed and synthesized by combining racemic LA with motifs of other known anticholinesterase agents (rivastigmine and memoquin). This afforded 4, which represents a step forward in the search for balanced anticholinesterase and antioxidant capacities.

Full text of DOI:10.1016/j.ejmech.2011.09.001

European Journal of Medicinal Chemistry 46 (2011) 5435e5442
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Exploiting the lipoic acid structure in the search for novel multitarget ligands
against Alzheimer’s disease
,
a
a
b
a
a
Michela Rosinia , Elena Simoni , Manuela Bartolini , Andrea Tarozzi , Riccardo Matera , Andrea Milelli ,
*
Patrizia Hreliab, Vincenza Andrisanoa, Maria Laura Bolognesia, Carlo Melchiorrea
a Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b Department of Pharmacology, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipoic acid (LA) is a natural antioxidant. Its structure was previously combined with that of the acetyl-
cholinesterase inhibitor tacrine to give lipocrine (1), a lead compound multitargeted against Alzheimer’s
disease (AD). Herein, we further explore LA as a privileged structure for developing multimodal
compounds to investigate AD. First, we studied the effect of LA chirality by evaluating the cholinesterase
profile of 1’s enantiomers. Then, a new series of LA hybrids was designed and synthesized by combining
racemic LA with motifs of other known anticholinesterase agents (rivastigmine and memoquin). This
afforded 4, which represents a step forward in the search for balanced anticholinesterase and antioxidant
capacities.
Received 12 April 2011
Received in revised form
31 August 2011
Accepted 1 September 2011
Available online 7 September 2011
Keywords:
Lipoic acid
Lipocrine’s enantiomers
Alzheimer’s disease
Antioxidants
Ó 2011 Elsevier Masson SAS. All rights reserved.
Acetylcholinesterase
Multitarget compounds
1. Introduction
To date, a variety of antioxidants have been examined as neu-
roprotectants. Animal models and most human epidemiological
Alzheimer’s disease (ADa) is the most common form of irre-
versible dementia, for which effective treatments are urgently
needed. AD is thought to be a complex, multifactorial syndrome
with many related molecular lesions contributing to its pathogen-
esis [1]. Besides the pathological hallmarks of the disease, which
include the accumulation of misfolded protein deposits as amyloid-
studies have supported the assumption of a correlation between
antioxidant intake and cognitive function. However, randomized
clinical trials have generally afforded disappointing results [2,5].
In light of these conflicting data, there are growing calls for
a critical rethinking of the oxidative stress hypothesis. Experi-
mental evidence has recently expanded our knowledge of the role
of oxidative stress, corroborating the functional importance of
oxidative imbalance as a crucial and early event in mediating the
AD pathogenesis [6], and indicating the complexity of the redox
system in vivo as a nodal point [2].
b
(Ab) plaques and neurofibrillary tangles, AD brains exhibit
constant evidence of oxidative damage [2]. In particular, oxidative
injury is observed in the CNS of AD patients in almost every class of
cellular macromolecules, including nucleic acids, proteins, and
lipids. This has been used to support the “oxidative stress
hypothesis” of AD. Positing reactive oxygen species (ROS) as playing
a key role in AD onset and progression, this hypothesis proposes
antioxidants as beneficial therapeutic tools in AD treatment [3,4].
ROS production is due to a variety of different sources including
mitochondrial abnormalities, unbound transition metals, and A
b
peptides themselves [7]. Interestingly, all of these factors are
strictly correlated and establish interconnections with other key
events of AD, such as apoptosis, A
b processing and secretion,
s
phosphorylation, and the disruption of calcium homeostasis [8].
This complex scenario may explain why simple antioxidants did not
provide effective therapies for AD treatment, prompting
researchers to follow new trends in antioxidant development.
In particular, antioxidants presenting additional pharmacolog-
ical effects are currently thought to offer a good chance of
combating complex diseases in which free radicals are significant
Abbreviations: AD, Alzheimer’s disease; Ab, amyloid-b; ROS, reactive oxygen
species; MTDLs, multitarget-directed ligands; LA, lipoic acid; AChE, acetylcholin-
esterase; BuChE, butyrylcholinesterase; PAS, peripheral anionic site.
* Corresponding author. Tel.: þ39 0512099722; fax: þ39 0512099734.
E-mail address: michela.rosini@unibo.it (M. Rosini).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.001

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M. Rosini et al. / European Journal of Medicinal Chemistry 46 (2011) 5435e5442
Table 1
In in vitro models, 1 effectively displayed the expected multiple
biological properties, namely inhibition of AChE activity, inhibition
of AChE-induced Ab aggregation, and ability to protect cells against
ROS (Tables 1 and 2), emerging as a candidate for drug develop-
ment [19].
Inhibition of cholinesterases activity by (R)-1, (S)-1, 2e6 and reference compounds.
Compounds
1b
(S)-1
(R)-1
2
3
4
5
IC50 (M)a ꢀSEM hAChE
IC50 (M)a ꢀSEM hBuChE
(2.53 ꢀ 0.16) ꢁ 10ꢂ10
(4.71 ꢀ 0.17) ꢁ 10ꢂ10
(2.30 ꢀ 0.15) ꢁ 10ꢂ10
(2.66 ꢀ 0.23) ꢁ 10ꢂ9
(1.92 ꢀ 0.25) ꢁ 10ꢂ4
(2.56 ꢀ 0.08) ꢁ 10ꢂ7
(7.41 ꢀ 0.37) ꢁ 10ꢂ5
(2.52 ꢀ 0.17) ꢁ 10ꢂ5
(1.55 ꢀ 0.11) ꢁ 10ꢂ9
(3.03 ꢀ 0.21) ꢁ 10ꢂ6
> 10ꢂ3
(1.08 ꢀ 0.25) ꢁ 10ꢂ8
(1.21 ꢀ 0.03) ꢁ 10ꢂ8
(1.26 ꢀ 0.14) ꢁ 10ꢂ8
(3.06 ꢀ 0.07) ꢁ 10ꢂ8
(2.05 ꢀ 0.02) ꢁ 10ꢂ4
(2.49 ꢀ 0.11) ꢁ 10ꢂ6
(3.98 ꢀ 0.24) ꢁ 10ꢂ7
(8.24 ꢀ 0.65) ꢁ 10ꢂ5
(1.44 ꢀ 0.10) ꢁ 10ꢂ6
(3.01 ꢀ 0.14) ꢁ 10ꢂ7
> 10ꢂ3
In light of this, we wanted to expand the study that afforded 1
and its derivatives by exploiting the LA structure in the search for
novel MTDLs against AD. To this end, we first studied the contri-
bution of LA and tetrahydroacridine portions to the observed
multimodal profile of 1. It has been reported that stereochemistry is
not relevant for the protective effect of LA against oxidative cell
damage [20]. However, to verify whether it affects cholinesterase
inhibition, the two enantiomers of 1, (S)-1 and (R)-1 were synthe-
sized and studied. Moreover, to confirm the hypothesis that the
dithiolane function is indeed responsible for the antioxidant profile
of LA adducts, a thiophene analogue of 1 was synthesized, affording
2 (Fig. 1).
Then, we investigated the role of the 9-amino-6-chloro-1,2,3,4-
tetrahydroacridine fragment, by replacing it with a 2-chloro-4-
amino-6,7-dimethoxy-quinazoline moiety to give 3 (Fig. 1). The
quinazoline function was selected to verify if a chloro-substituted
aromatic group, protonated at physiological pH, was a sufficient
requisite for AChE binding. Then, we focused on designing new LA
adducts in which the antioxidant moiety was conjugated with the
structural features of other classical AChE inhibitors. Thus, in the
present study, we linked LA with the anticholinesterase motifs of
the marketed drug rivastigmine and memoquin, an AD drug
candidate developed in our group [21,22]. This afforded the hybrid
compounds 4e6 (Fig. 1). The selection of the pharmacophoric
features from the abovementioned anticholinesterase inhibitors
deserves comment. We focused on the benzylamino function,
a feature common to both prototypes. This feature has already
proven effective in recognizing the AChE catalytic site for mem-
oquin and its derivatives [21,23] and for a class of polyamine-based
noncovalent inhibitors [24e28]. Thus, combining a N1-ethyl-N1-(2-
methoxy-benzyl)-hexane-1,6-diamine moiety with LA provided 4.
Similarly, although the peculiar mode of inhibition of rivastigmine
is related to its carbamic function, we envisioned that the 3-(1-
(dimethylamino)ethyl)phenol group could also provide a recogni-
tion fragment for the AChE active site. As further support, docking
studies previously showed that this moiety is itself a competitive
AChE inhibitor [29]. Consequently, the 3-(1-(dimethylamino)ethyl)
phenol group should endow 5 and 6 with a binding mode similar to
that of 1 and the other derivatives, i.e. a reversible mixed type
inhibition of AChE, with LA possibly interacting with the PAS of the
enzyme.
6
memoquinc
rivastigmined
LAb
a
Human recombinant AChE and BuChE from human serum were used. IC50
values represent the concentration of inhibitor required to decrease enzyme activity
by 50% and are the mean of two independent measurements, each performed in
triplicate.
b
From ref. [18].
From ref. [21].
From ref. [22]; SEM ¼ Standard Error of the Mean.
c
d
but not exclusive drivers [9]. This concept has been recently
rationalized in the field of neurodegenerative diseases affording the
multitarget-directed ligands (MTDLs) design strategy. This strategy
holds that single molecules, endowed with antioxidant properties
and able to act at different steps in the neurodegenerative process,
can produce additive neuroprotective effects against AD [10,11].
Lipoic acid (LA) is a natural occurring antioxidant. Its antioxidant
activity is attributed to its ability to scavenge free radicals in both
membrane and aqueous domains. It does this by chelating redox-
active transition metals, increasing the levels of reduced gluta-
thione and down-regulating inflammatory processes [12,13]. LA has
also been shown to possess a variety of additional properties which
can interfere with the pathogenic principles of AD [14]. Moreover,
LA is readily absorbed by diet, transported, taken up by cells, and
reduced to the active dihydrolipoic acid in various tissues, including
brain [15]. Therefore, LA could be considered a privileged structure
in designing new MTDLs for the investigation and, conceivably,
treatment of AD [15-17].
In 2005, with this in mind, we developed lipocrine (1), one of
the first multifunctional antioxidants rationally designed to combat
AD [18]. 1 combined, in the same molecule, the structure of LA with
a derivative of tacrine, the first AChE inhibitor approved for AD
treatment. It was also argued that the cyclic moiety of LA could
interact with the peripheral anionic site (PAS) of AChE, which is
associated with the neurotoxic cascade underlying AD through
AChE-induced A
b aggregation. Indeed, kinetic analyses confirmed
that 1 could bind both catalytic and peripheral sites, thus acting as
a mixed type inhibitor [18].
Synthesized compounds were then investigated to assess their
antioxidant, anticholinesterase, and anti-aggregating activities.
Table 2
Effects of compounds on intracellular ROS formation in SH-SY5Y cellsa.
Conc. (
m
M)
Compounds
1
2
3
4
5
6
0
1
5
10
50
82.4 ꢀ 6.6
83.2 ꢀ 7.4
65.0 ꢀ 6.2b
50.0 ꢀ 5.5c
31.0 ꢀ 4.9d
82.4 ꢀ 6.6
81.0 ꢀ 7.9
79.8 ꢀ 3.2
80.2 ꢀ 4.1
Toxe
82.4 ꢀ 6.6
82.0 ꢀ 7.6
73.5 ꢀ 6.5
63.4 ꢀ 5.6b
Toxe
82.4 ꢀ 6.6
80.8 ꢀ 5.9
75.8 ꢀ 5.8
69.6 ꢀ 4.2b
37.6 ꢀ 4.5d
82.4 ꢀ 6.6
82.5 ꢀ 6.5
80.0 ꢀ 5.2
68.8 ꢀ 5.5b
46.4 ꢀ 5.0c
82.4 ꢀ 6.6
80.3 ꢀ 6.2
76.8 ꢀ 6.3
48.0 ꢀ 6.1c
34.4 ꢀ 5.4d
a
The results are expressed as a percentage increase of intracellular ROS induced by exposure to t-BuOOH. Values are reported as mean ꢀ SD of three/four independent
experiments.
b
c
p < 0.05.
p < 0.01.
d
e
p < 0.001 vs untreated at ANOVA with Dunnett post hoc test.
Tox ¼ cytotoxic.

M. Rosini et al. / European Journal of Medicinal Chemistry 46 (2011) 5435e5442
5437
OMe
O
H
N
N
Et
N
Et
N
H
Memoquin
O
OMe
OMe
H
N
O
S
N
Et
S
*
HN
N
N
H
O
S
4
S
NH2
(S)-1
(R)-1
Cl
O
R
N
HN
N
N
H
Tacrine: R = H
S
2
O
S
HN
N
N
H
Cl
HO
S
S
O
S
O
HN
N
H
Lipoic acid (LA)
Cl
Lipocrine (1)
MeO
MeO
S
N
S
N
3
N
Cl
H
N
S
N
MeO
S
O
O
S
5
N
H
O
S
O
N
N
O
6
Rivastigmine
Fig. 1. Design strategy for compounds (R)-1, (S)-1, and 2e6.
2. Chemistry
inhibition profile of 2-6 with respect to prototypes 1, memoquin,
rivastigmine, and LA (Table 1). The obtained data clearly highlight
the role of the different moieties of 1 in AChE inhibition. Indeed, the
Cl-tetrahydroacridine fragment of 1 emerged as a key feature for
AChE binding, as revealed by the dramatic loss of efficacy observed
for the quinazoline analogue 3. Conversely, the replacement of the
dithiolane with a thiophene unit, affording 2, slightly affected AChE
inhibition.
Of compounds 4-6, where the tacrine-like moiety of 1 was
substituted with diverse anticholinesterase motifs, only 4 emerged
as an effective AChE inhibitor, although it was considerably less
potent than the prototype (IC50 ¼ 256 ꢀ 8 nM and 0.253 ꢀ 0.016 nM
for 4 and 1, respectively). No significant anticholinesterase activity
was confirmed for 5 and 6, which bear the 3-(1-(dimethylamino)
ethyl)phenol function of rivastigmine. 4-6 share a benzylamino
group and therefore a cationic head, which is a fundamental feature
for cholinesterase recognition. However, only the o-methox-
ybenzylamino group of 4 was confirmed to effectively anchor the
AChE catalytic site. Conversely, the 3-(1-(dimethylamino)ethyl)
phenol moiety of 5 and 6, lacking the carbamate functionality of
rivastigmine, seemed unable to properly interact with the AChE
binding site.
Concerning the inhibition of BuChE, we note that, in general,
structural modification led to a drop in the inhibitory activity of all
LA-carrying derivatives except 5, which retained the sub-
micromolar potency of the reference compound rivastigmine.
Moreover, although 4 was less active than 1 on hBuChE, it still
showed an inhibitory potency in the micromolar range, with
a selectivity profile similar to that of 1 (BuChE/AChE ¼ 10 and 43 for
4 and 1, respectively). Compound 5 was the only butyryl-selective
derivative in the series. It could be useful in treating the
We have previously reported how a coupling reaction of amines
with LA provides easy access to a variety of derivatives [18,30,31].
Following the same procedure, we here synthesized 2e6 by
coupling intermediates 7 [18], 9, 10 [22], 13 and 16 with LA or 5-
(thiophen-2-yl)pentanoic acid in the presence of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
(Scheme 1). 13 and 16 were obtained through alkylation with tert-
butyl 3-chloropropylcarbamate of 11 and 14, followed by removal
of the BOC group with trifluoroacetic acid.
Concerning 5 and 6, diastereoisomers were not separated
because of their weak AChE inhibitory potency.
(S)-1 and (R)-1 were synthesized according to the procedure
previously reported for 1 [18], starting from 7 and (S)-lipoic acid or
(R)-lipoic acid.
3. Results and discussion
As previously mentioned, the first step of the present study was
to define the role of LA stereochemistry in the cholinesterase
inhibition of 1. Thus, the two enantiomers of 1 ((S)-1 and (R)-1)
were tested on human AChE and BuChE by the method of Ellman
et al. [32]. Due to the lack of activity of racemic LA [18], its enan-
tiomers were not tested. The obtained results clearly show that,
while BuChE did not discriminate between (S)-1 and (R)-1, the
inhibitory potencies of the two enantiomers were slightly different
on AChE. (R)-1 was only twice as potent as (S)-1. Since there is no
biologically relevant difference between racemic 1 and the most
active enantiomer (R)-1, we decided to synthesize and study the LA
adducts 2-6 as racemic compounds. We assessed the cholinesterase

5438
M. Rosini et al. / European Journal of Medicinal Chemistry 46 (2011) 5435e5442
O
HN
N
H
HN
NH2
S
2
Cl
N
Cl
N
7
O
R
N
HN
N
N
S
H
S
MeO
MeO
MeO
S
S
N
N
HO
MeO
: R = Cl
Cl
Cl
O
3
or
8
a
S
9
: R = NH(CH2)3NH2
S
HO
OMe
S
H
N
OMe
O
N
Et
NH2
N
Et
O
EDCI, DMF, 0° C to rt
4
10
Me
S
Me
R
MeO
S
H
N
MeO
N
N
Me
Me
O
11: R = H
5
b
12: R = (CH2)3NHBoc
13: R = (CH2)3NH2
c
O
Me
Me
OR
O
N
H
NMe2
S
S
NMe2
6
14: R = H
15: R = (CH2)3NHBoc
16: R = (CH2)3NH2
b
c
Scheme 1. Reaction conditions: (a) propanediamine, anhydrous THF, rt 12 h; (b) Cl(CH2)3NHBOC, K2CO3, KI, DMF, reflux 24 h; (c) TFA, CH2Cl2, rt 2 h.
moderate/severe form of AD where the BuChE/AChE ratio is deeply
altered by disease progression and levels of AChE are too low for
AChE inhibitors to be effective [33].
30% and 80%, respectively. In particular, the high cytotoxicity
exerted by 2 deserves comment. It was previously reported that 7,
which is a fragment present in both 1 and 2, was highly toxic in the
same assay [18]. A comparison of these data with the toxicity
profiles of compounds 1 and 2, in which the abovementioned
cytotoxic fragment was combined with LA and 5-(thiophen-2-yl)
pentanoic acid, respectively, seems to support the hypothesis of
a protective role for the LA dithiolane moiety.
The activity of 2e6 against intracellular ROS formation was then
assessed in SH-SY5Y cells after treatment with tert-butyl hydro-
peroxide, a compound used to induce oxidative damage [35]. We
used a range of concentrations of tested compounds that did not
Our main goal was to identify a new antioxidant MTDL. To
determine the potential interest of 2e6 for the treatment of AD, we
thus evaluated their antioxidant activity in a cellular context. To
define the suitable concentration range, the cytotoxicity effects of
2e6 were determined by colorimetric MTT [3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay [34] in
human neuronal-like cells, SH-SY5Y. 1 was used as the reference
compound. As reported in Fig. 2, treatment of SH-SY5Y cells with
4e6 (0.1e50
contrast, 50
m
M) did not show, as for 1, modified cell viability. In
mM 3 and 2 produced a decrease of cell viability around
affect cell viability (1e50 mM for 4e6; 1e10 mM for 2 and 3).
Treating SH-SY5Y cells with 4e6 showed a significant dose-
dependent inhibitory effect on ROS formation (Table 2). More-
over, a relevant decrease of ROS production was observed for 3 with
the highest concentration used (10 mM). In contrast, as expected, 2,
devoid of the LA moiety, did not reduce the intracellular ROS
formation in SH-SY5Y cells.
Altogether, the cholinesterase and antioxidant activity data
point to 4 as the lead compound of the present series of antioxidant
MTDLs. In view of its structural similarity with prototypes 1 and
memoquin, which are both effective inhibitors of AChE-induced A
b
aggregation, we checked if the anticholinesterase efficacy of 4 was
accompanied by a concomitant inhibition of the proaggregating
action of AChE. This was assessed through a thioflavin T-based
fluorometric assay [36]. Data showed that 4 is a weak inhibitor of
the
AChE-induced
A
b
aggregation
(%
inhibition
at
Fig. 2. Effects of compounds on cell viability in SH-SY5Y cells.
100 mM ¼ 16.8 ꢀ 2.2), being significantly less potent than the

M. Rosini et al. / European Journal of Medicinal Chemistry 46 (2011) 5435e5442
5439
prototypes (% inhibition at 100
and memoquin, respectively). This finding is consistent with
a lower anticholinesterase efficacy.
m
M ¼ 61.8 ꢀ 0.8 and 87.1 ꢀ 1.7 for 1
was cooled to 0 ꢃC andthentreatedwith 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (EDCI$HCl) (1.2 eq). The mixture
was stirred at 0 ꢃC for further 15 min and then at rt for 2 h in the dark.
Solvent was then removed under vacuum, avoiding heating up the
reaction mixture while the lipoic fragment was present (3e6),
affording an oily residue that was purified by gravity column.
4. Conclusion
In the present investigation, we synthesized and studied a series
of LA adducts, hypothesising that using diverse AChE motifs would
offer a new point of diversity for MTDL development. The small
difference between the inhibitory potencies of (R)- and (S)-1 on
AChE offered the rational basis for the synthesis of the new LA
adducts as racemates. Of these, 4 emerged for its multiple biological
properties, namely, inhibition of AChE activity in the sub-
micromolar range and the ability to protect cells against oxidative
stress. Interestingly, these properties were accompanied by the
absence of a significant cytotoxicity in neuronal-like cells SH-SY5Y
5.1.1.1. Synthesis of N-(3-(6-chloro-1,2,3,4-tetrahydroacridin-9-ylamino)
propyl)-5-(thiophen-2-yl)pentanamide (2). It was synthesized from
N1-(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)propane-1,3-diamine
[18] (7) (0.350 g, 1.21 mmol), and 5-(thiophen-2-yl)pentanoic acid
(0.334 g, 1.82 mmol), and purified by flash chromatography. Elution
with petroleum ether/CH2Cl2/MeOH/aqueous 30% ammonia
1
(6.0:3.5:0.5:0.007) afforded 2 (0.441 g, 80%) as a yellowish wax. H
NMR (CDCl3, 200 MHz) d 8.70 (br t, 1H, exchangeable with D2O), 7.96
(d, J ¼ 8.8 Hz, 1H), 7.88 (d, J ¼ 2.2 Hz,1H), 7.23 (d, J ¼ 1.8 Hz, 1H),
7.09e7.11 (m, 1H), 6.91 (t, J ¼ 3.6 Hz, 1H), 6.76e6.78 (m, 1H), 6.05 (br t,
1H, exchangeable with D2O), 3.48e3.52 (m, 4H), 3.02e3.05 (m, 2H),
2.85 (t, J ¼ 6.6 Hz, 2H), 2.71e2.76 (m, 2H), 2.25 (t, J ¼ 6.6 Hz, 2H),
up to the highest concentration used (50 mM).
With respect to 1, which represents a milestone in the field of
MTDL development for Alzheimer’s disease, 4 is characterized by
a severe decrease in AChE inhibitory potency, followed by a signif-
icant reduction of AChE-induced anti-aggregating efficacy. This
may suggest a reduction in multimodal efficacy of 4, when
compared to 1; however, a more balanced activity profile has been
attained here. In multitarget projects, it is critical to establish an
optimal ratio of the desired activities, in order to maximize efficacy
and safety. In particular, the profile of a lead compound should be
balanced, so that each target is modulated in vitro to an equal or
similar extent in the first instance [37]. However, the in vivo model
proves to be much more complex, with many different factors
contributing to the optimal level of activity for each target. There-
fore, to verify whether or not 4 could be a new lead compound,
in vivo feedback is needed.
1.71e1.90 (m, 10H). 13C NMR (CD3OD, 100 MHz)
d 175.22, 156.59,
150.77, 144.54, 139.05, 138.60, 127.38, 126.26, 125.50, 123.88, 122.52,
117.81, 114.09, 112.26, 45.06, 35.72, 35.39, 31.12, 30.17, 29.01, 28.11,
25.04, 23.60, 21,54, 20.44. MS (ESIþ) : m/z 456 [Mþ1]þ. Anal. calcd for
C25H30ClN3OS: C, 65.84; H, 6.63; N, 9.21. Found: C, 65.61; H, 6.65, N,
9.18.
5.1.1.2. Synthesis of N-(3-(2-chloro-6,7-dimethoxyquinazolin-4-yla-
mino)propyl)-5-(1,2-dithiolan-3-yl)pentanamide (3). It was syn-
thesised from 9 (0.15 g, 0.505 mmol) and LA (0.104 g, 0.505 mmol),
and purified by gravity column chromatography. Elution with
petroleum ether/CH2Cl2/EtOH/aqueous 30% ammonia (5:4.5:0.5:
0.01) afforded 3 (0.110 g, 45%) as a yellowish oil. 1H NMR (CDCl3,
300 MHz)
d
7.46 ( br t, J ¼ 5.8 Hz, 1H, exchangeable with D2O), 7.34
5. Experimental
(s, 1H), 7.14 (s, 1H), 6.22 (t, 1H, exchangeable with D2O), 4.06 (s, 3H),
4.00 (s, 3H), 3.73 (q, J ¼ 4.8 Hz, 2H), 3.58 (m, J ¼ 6.3 Hz, 1H), 3.45 (q,
J ¼ 6.0 Hz, 2H), 3.24e2.91 (m, 2H), 2.53e2.42 (m, 1H), 2.32 (t,
J ¼ 7.5 Hz, 2H), 2.30e1.61 (m, 7H), 1.60e1.47 (m, 2H). 13C-NMR
5.1. Chemistry
Melting points were taken in glass capillary tubes on Buchi SMP-
20 apparatus and are uncorrected.
(CDCl3, 75 MHz) d 174.52, 160.58, 157.02, 155.07, 149.27, 147.93,
107.54, 107.22, 101.08, 56.63, 56.57, 56.47, 40.52, 38.74, 37.34, 36.78,
36.21, 34.86, 29.69, 29.13, 25.75. MS (ESIþ) : m/z 486 [Mþ1]þ. Anal.
calcd for C21H29ClN4O3S2: C, 52.00; H, 6.03; N, 11.55. Found: C,
52.23; H, 6.15, N, 11.67.
Direct infusion ESI-MS spectra were recorded on PerkineElmer
297 and Waters ZQ 4000 apparatus. 1H NMR and 13C-NMR spectra
were recorded on Varian VXR 200, 300 and 400 instruments.
Chemical shifts are reported in parts per millions (ppm) relative to
tetramethylsilane (TMS), and spin multiplicities are given as s
(singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet) or m
(multiplet). The elemental compositions of the compounds agreed
to within ꢀ0.4% of the calculated value. When the elemental
analysis is not included, crude compounds were used in the next
step without further purification. Chromatographic separations
were performed on silica gel columns by flash (Kieselgel 40,
5.1.1.3. Synthesis of 5-(1,2-dithiolan-3-yl)-N-(6-(ethyl(2-methoxybe-
nzyl)amino) hexyl) pentanamide (4). It was synthesized from N1-
ethyl-N1-(2-methoxybenzyl)hexane-1,6-diamine [22] (10) (0.300 g,
1.13 mmol) and LA (0.350 g, 1.70 mmol), and purified by gravity
column chromatography. Elution with a gradient of mobile phase
petroleum ether/toluene/CH2Cl2/EtOH/aqueous 30% ammonia
(7:2:1:1:0.05 to 7:1:1:1:0.05) afforded 4 (0.219 g, 43%) as a waxy
0.040e0.063
mm,
Merck)
or
gravity
(Kieselgel
60,
solid. 1H NMR (CDCl3, 200 MHz)
d 7.48e7.42 (m, 1H), 7.26e7.18 (m,
0.063e0.200 mm, Merck) column chromatography. Reactions were
followed by thin-layer chromatography (TLC) on Merck (0.25 mm)
glass-packed precoated silica gel plates (60 F254), then visualized
in an iodine chamber or with a UV lamp. (S)-lipoic acid and (R)-
lipoic acid were purchased from Waterstone Technology and Sigma
Aldrich, respectively. The term “dried” refers to the use of anhy-
drous sodium sulfate. Compounds were named following IUPAC
rules as applied by Beilstein-Institute AutoNom (version 2.1), a PC
integrated software package for systematic names in organic
chemistry.
1H), 6.99e6.85 (m, 2H), 5.43 (br s,1H, exchangeable with D2O), 3.84
(s, 3H), 3.64e3.53 (m, 1H þ s, 2H), 3.27e3.08 (m, 4H), 2.57e2.43 (m,
5H), 2.17 (t, J ¼ 7.4 Hz, 2H), 2.00e1.82 (m, 1H), 1.73e1.29 (m, 14H),
1.07 (t, J ¼ 7.0 Hz, 3H). 13C-NMR (CDCl3,100 MHz)
d 169.82,
157.78,130.85, 128.76, 126.92, 120.56, 110.41, 56.70, 55.44, 55.34,
53.18, 51.15, 39.91, 39.16, 38.42, 36.42, 31.89, 29.65, 28.89, 28.85,
27.03, 26.66, 25.39, 14.09. MS (ESIþ) : m/z 453 [Mþ1]þ. Anal. calcd
for C24H40N2O2S2: C, 63.67; H, 8.91; N, 6.19. Found: C, 63.79; H, 8.93,
N, 6.17.
5.1.1.4. Synthesis of the diastereomeric mixture of 5-(1,2-dithiolan-3-
yl)- N-(3-((1-(3-methoxyphenyl)ethyl)(methyl)amino) propyl) pen-
tanamide (5). It was synthesised from 13 (0.130 g, 0.59 mmol) and
LA (0.240 g, 1.47 mmol), and purified by gravity column
5.1.1. General procedure for the synthesis of compounds 2e6
A solution of the appropriate amine (1 eq, 0.1 M) and LA (1.5 eq) or
5-(thiophen-2-yl) pentanoic acid (1.5 eq) in dry DMF (5 mL), under N2

5440
M. Rosini et al. / European Journal of Medicinal Chemistry 46 (2011) 5435e5442
chromatography. Elution with petroleum ether/CH2Cl2/EtOH/
residue was dissolved in water, made basic by adding 2 N NaOH and
aqueous 30% ammonia (5.5:3.5:1:0.015) afforded 5 (0.133 g, 55%) as
then extracted with CHCl3 (3 ꢁ 20 mL) affording 13 as a white solid
a waxy solid. 1H NMR (CDCl3, 200 MHz)
d
7.23 (t, J ¼ 7.8 Hz, 1H),
(0.137 g, quantitative yield). 1H NMR (CDCl3, 200 MHz)
d 7.27 (t,
6.90e6.75 (m, 3H), 6.56 (br s, 1H, exchangeable with D2O), 3.79 (s,
3H), 3.58e3.47 (m, 2H), 3.25e3.05 (m, 4H), 2.45e2.37 (m, 3H), 2.20
(s, 3H), 2.03 (t, J ¼ 7.2 Hz, 2H), 1.97e1.82 (m, 1H), 1.73e1.38 (m, 8H),
J ¼ 8.0 Hz,1H), 6.97e6.80 (m, 3H), 3.85 (s, 3H), 3.54 (q, J ¼ 6.6 Hz,1H),
2.74 (t, J ¼ 6.6 Hz, 2H), 2.58e2.30 (m, 2H), 2.25 (s, 3H),1.67e1.55 (m,
2Hþ2H exchangeable with D2O), 1.39 (d, J ¼ 7.0 Hz, 3H).
1.34 (d, J ¼ 6.4 Hz, 3H). 13C-NMR (CDCl3, 100 MHz)
d 172.42 (2C),
159.65 (2C), 129.32 (2C), 120.11 (2C), 113.76 (2C), 112.07 (4C), 64.08
(2C), 56.43 (2C), 55.21 (2C), 52.54 (2C), 40.22 (2C), 38.45 (2C), 37.98
(2C), 36.55 (2C), 34.64 (2C), 29.67 2C), 28.93 (2C), 25.74 (2C), 25.47
(2C), 17.64, 17.60. MS (ESIþ) : m/z 411 [Mþ1]þ. Anal. calcd for
C21H34N2O2S2: C, 61.42; H, 8.35; N, 6.82. Found: C, 61.65; H, 8.36, N,
6.81.
5.1.5. Synthesis of (R,S) 3-(1-(dimethylamino)ethyl)phenol (14)
Compound 14 was synthesised following the procedure
described for the corresponding (R,S)-3-(1-(di-(2H3)methylamino)
ethyl)phenol in Ciszewska et al. [39]. A solution of methylamine
hydrochloride in methanol was slowly additioned with KOH
(3.48 g, 0.62 mol). After 20 min the hydroxyacetophenone (23.73 g,
0.174 mol) was added to the white precipitate formed and the
mixture stirred at room temperature for 15 min. Over the next
30 min, a solution of NaCNBH3 (4.13 g, 0.66 mol) in 50 ml of
methanol was added dropwise to the imine intermediate, the
mixture was stirred at rt for 30 min and further KOH (13.07 g,
0.233 mol) was added. After KOH was completely dissolved, the
mixture was filtered and the filtrate concentrated under vacuum
while keeping the temperature below 30 ꢃC. Elution with a gradient
of mobile phase EtOAc/aqueous 30% ammonia (10:0.07 to10:0.2)
5.1.1.5. Synthesis of the diastereomeric mixture of N-(3-(3-(1-
(dimethylamino)ethyl)phenoxy)propyl)-5-(1,2-dithiolan-3-yl)penta-
namide (6). It was synthesized from 16 (0.150 g, 0.67 mmol) and LA
(0.210 g, 1.02 mmol), and purified by gravity column chromatog-
raphy. Elution with petroleum ether/toluene/CH2Cl2/MeOH/
aqueous 30% ammonia (6:1:1.5:1.5:0.01) afforded 6 (0.080 g, 30%)
as a waxy solid. 1H NMR (CDCl3, 200 MHz)
d
7.27 (t, J ¼ 8.2 Hz, 1H),
6.98e6.78 (m, 3H), 5.99 (br t, 1H), 4.09 (t, J ¼ 6.0 Hz, 2H), 3.62e3.21
(m, 5H), 3.19e3.05 (m, 3H), 2.53e2.40 (m, 1H), 2.32 (s, 6H), 2.22 (t,
J ¼ 7.2 Hz, 2H), 1.99e1.81 (m, 3H), 1.73e1.65 (m, 4H), 1.47 (d,
afforded 14 (65%) as an oil. 1H NMR (CDCl3, 200 MHz)
d 9.08 (br s,
1H, exchangeable with D2O), 7.15 (t, J ¼ 7.6 Hz, 1H), 6.81e6.73 (m,
J ¼ 6.6 Hz, 3H). 13C-NMR (CDCl3, 100 MHz)
d 172.88 (2C), 158.88,
3H), 3.34 (q, J ¼ 10.8 Hz, 1H), 2.25 (s, 6H), 1.42 (d, J ¼ 6.4 Hz, 3H).
158.77, 144.83, 144.25, 129.35 (2C), 120.26, 120.17, 113.56 (2C),
113.38 (2C), 66.24 (2C), 66.16 (2C), 65.91 (2C), 56.37 (2C), 43.04 (2C),
42.91 (2C),40.18 (2C), 38.42, 37.23, 36.42 (2C), 34.58 (2C), 29.159
(2C), 28.95, 28.85, 25.61, 25.39, 19.99, 19.83. MS (ESIþ) : m/z 411
[Mþ1]þ. Anal. calcd for C21H34N2O2S2: C, 61.42; H, 8.35; N, 6.82.
Found: C, 61.62; H, 8.36, N, 6.80.
5.1.6. Synthesis of tert-butyl 3-(3-(1-(dimethylamino)ethyl)
phenoxy)propylcarbamate (15)
A solution of 14 (0.350 g, 2.17 mmol), tert-butyl 3-chloropropy-
lcarbamate (0.420 g, 2.17 mmol) and K2CO3 (0.300 g, 2.17 mmol)
in DMF (10 mL) was stirred under reflux conditions for 24 h.
Evaporation of the solvent afforded a residue which was purified
by gravity column. Elution with CHCl3/MeOH/aqueous 30%
ammonia (9:1:0.02) afforded 15 (0.454 g, 65%) as an oil. 1H NMR
5.1.2. Synthesis of N1-(2-chloro-6,7-dimethoxyquinazolin-4-yl)
propane-1,3-diamine (9)
A solution of the commercial 2,4-dichloro-6,7-dimethoxyquina-
zoline (1.00 g, 3.86 mmol) in anhydrous THF (15 mL) was addi-
tioned with propanediamine (0.572 g,7.72 mmol). The resulting
mixture was stirred at rt overnight. Evaporation of the solvent
afforded a residue which was purified by gravity column. Elution
with CH2Cl2/MeOH/aqueous 30% ammonia (9:1:0.2) afforded 9
(0.840 g, 75%) as a crystalline white solid. Mp ¼ 210ꢃe215 ꢃC (dec.).
(CDCl3, 200 MHz)
d
7.20 (t, J ¼ 8.0 Hz, 1H), 6.79e6.89 (m, 3H), 4.92
(br s, 1H, exchangeable with D2O), 4.02 (t, J ¼ 6.4 Hz, 2H),
3.20e3.33 (m, 3H), 2.20 (s, 6H), 1.93e1.99 (m, 2H), 1.44 (s, 9H),
1.35 (d, J ¼ 6.6 Hz, 3H).
5.1.7. Synthesis of 3-(3-(1-(dimethylamino)ethyl)phenoxy)propan-
1-amine (16)
1H NMR (CDCl3, 200 MHz)
d
8.53 (br s, 1H, exchangeable with D2O)
A solution of 15 (0.200 g, 0.62 mmol) in CH2Cl2 (5 mL) was
additioned with trifluoroacetic acid (1.5 mL). The reaction mixture
was stirred at rt for 2 h and evaporated in vacuum. The obtained
residue was dissolved in water, made basic by adding 2 N NaOH and
then extracted with CHCl3 (3 ꢁ 20 mL). Evaporation of the dried
solvent afforded 16 (0.137 g, quantitative yield). 1H NMR (CDCl3,
7.13 (s, 1H), 6.98 (s, 1H), 3.99 (s, 3H), 3.96 (s, 3H), 3.80 (q, J ¼ 4.4 Hz,
2H), 3.10 (t, J ¼ 5.6 Hz, 2H),1.87 (m, 2H),1.64 (br s, 2H, exchangeable
with D2O).
5.1.3. Synthesis of tert-butyl 3-((1-(3-methoxyphenyl)
ethyl)(methyl)amino)propylcarbamate (12)
200 MHz)
d
7.20 (t, J ¼ 8.0 Hz, 1H), 6.72e6.88 (m, 3H), 4.04
A solution of 1-(3-methoxyphenyl)-N-methylethanamine [38]
(11) (0.320 g, 1.9 mmol), tert-butyl 3-chloropropylcarbamate
(0.370 g, 1.9 mmol), K2CO3 (0.260 g, 1.9 mmol) and a catalytic
amount of KI in DMF (10 mL) was stirred under reflux conditions for
24 h. Evaporation of the solvent afforded a residue which was
purified by gravity column. Elution with CHCl3/MeOH/aqueous 30%
ammonia (9:1:0.005) afforded 12 (0.245 g, 40%) as an oil. 1H NMR
(t, J ¼ 6.2 Hz, 2H), 3.12e3.22 (m, 1H), 2.91 (t, J ¼ 6.6 Hz, 2H), 2.19
(s, 6H), 1.88e1.95 (m, 2H), 1.43 (br s, 2H, exchangeable with D2O),
1.34 (d, J ¼ 6.6 Hz, 3H).
5.1.8. Characterization of S-(5-( [1,2]Dithiolan-3-yl)- N -[3-(6-
chloro-1,2,3,4-tetrahydro-acridin-9-yl)amino]propyl}pentanamide)
((S)-1) and R -(5-( [1,2]Dithiolan-3-yl)- N-[3-(6-chloro-1,2,3,4-
tetrahydro-acridin-9-yl)amino]propyl}pentanamide) ((R)-1)
(CDCl3, 200 MHz)
d
7.20 (t, J ¼ 8.0 Hz, 1H), 6.95e6.74 (m, 3H), 5.38
(br s, 1H, exchangeable with D2O), 3.80 (s, 3H), 3.50 (q, J ¼ 7.0 Hz,
1H), 3.13 (q, J ¼ 6.2 Hz, 2H), 2.52e2.30 (m, 2H), 2.19 (s, 3H),
1.68e1.54 (m, 2H), 1.44 (s, 9H), 1.35 (d, J ¼ 6.6 Hz, 3H).
1H NMR (CD3OD, 400 MHz)
d
8.00 (d, J ¼ 9.2 Hz, 1H), 7.62 (d,
J ¼ 2 Hz, 1H), 7.22 (dd, J ¼ 9.2, 2.4 Hz 1H), 3.48 (t, J ¼ 7.2 Hz, 2H),
3.39e3.35 (m, 1H), 3.19 (t, J ¼ 6.8 Hz, 2H), 3.05e2.91 (m, 2H), 2.85
(br m, 2H), 2.63 (br m, 2H), 2.32e2.24 (m, 1H), 2.07 (t, J ¼ 7.6 Hz,
2H), 1.82e1.79 (m, 4H), 1.76e1.67 (m, 3H), 1.56e1.42 (m, 4H),
5.1.4. SynthesisofN1-(1-(3-methoxyphenyl)ethyl)-N1-methylpropane-
1,3-diamine (13)
1.34e1.24 (m, 2H). 13C-NMR (CD3OD, 100 MHz)
d 174.95, 158.36,
A solution of 12 (0.230 g, 0.62 mmol) in CH2Cl2 (5 mL) was
additioned with trifluoroacetic acid (1.5 mL). The reaction mixture
was stirred at rt for 2 h and evaporated in vacuum. The obtained
152.14, 146.46, 134.42, 125.14, 124.66, 123.88, 117.81, 115.48, 56.11,
45.20, 39.85, 37.90, 36.02, 35.47, 34.26, 32.47, 30.59, 28.45, 25.34,
24.60, 22.46, 21.99. MS (ESIþ) : m/z 478 [Mþ1]þ.

M. Rosini et al. / European Journal of Medicinal Chemistry 46 (2011) 5435e5442
5441
(S)-1:Anal. calcd for C24H32ClN3OS2: C, 60.29; H, 6.75; N, 8.79;
Found C, 60.53; H, 6.78; N, 8.65; (R)-1:Anal. calcd for C24H32ClN3OS2:
C, 60.29; H, 6.75; N, 8.79; found C, 60.41; H, 6.73; N, 8.72.
Circular dichroism spectra of (S)-1 and (R)-1 were reported on
Bertucci et al. [40].
24 h of incubation at 37 ꢃC in 5% CO2, the growth medium was
removed and media containing compounds (0.1e50 M) were
m
added to the cells. After 24 h of incubation, the cells were washed
with phosphate buffered saline (PBS) and then incubated with MTT
(5 mg/mL) in PBS for 4 h. After removal of MTT and further washing,
the formazan crystals were dissolved with isopropanol. The
amount of formazan was measured (405 nm) with a spectropho-
tometer (TECANÒ, Spectra model Classic, Salizburg, Austria). The
cell viability was expressed as percentage of control cells and
calculated by the formula Ft/Fnt ꢁ 100, where Ft ¼ absorbance of
treated neurones and Fnt ¼ absorbance of untreated neurones.
5.2. Biology
5.2.1. Determination of the inhibitory potency on AChE and BuChE
The method of Ellman et al. [32] was followed. Compound 1,
memoquin, rivastigmine and LA were used as reference
compounds. Five different concentrations of each compound were
used to obtain inhibition of AChE or BuChE activity comprising
between 20% and 80%. The assay solution consisted of a 0.1 M
5.2.5. Determination of antioxidant activity
The antioxidant activity of compounds was evaluated by
measuring the formation of intracellular reactive oxygen species
(ROS) evoked by exposure of SH-SY5Y cells to tert-butyl hydro-
peroxide (t-BuOOH), a compound used to induce oxidative stress.
Formation of intracellular ROS was determined using a probe, 20,70-
dichlorodihydrofluorescein diacetate (DCFH-DA), as described by
Wang H. et al. [35]. Briefly, SH-SY5Y cells were seeded in 96-well
microtiter plates at 2 ꢁ 105 cells/well. After 24 h of incubation at
37 ꢃC in 5% CO2, the growth medium was removed and media
phosphate buffer, pH 8.0, with the addition of 340
bis(2-nitrobenzoic acid), 0.02 unit/mL human recombinant AChE or
human serum BuChE (Sigma Chemical), and 550 M substrate
m
M 5,50-dithio-
m
(acetylthiocholine iodide or butyrylthiocholine iodide, respec-
tively). Assay solutions with and without inhibitor were pre-
incubated at 37 ꢃC for 20 min followed by the addition of substrate.
Blank solutions containing all components except AChE or BuChE
were prepared in parallel to account for the non-enzymatic
hydrolysis of the substrate. The reaction rates were compared,
and the percentage of inhibition due to the presence of test
compounds was calculated. Each concentration was analyzed in
triplicate, and IC50 values were determined graphically from log
concentration-inhibition curves.
containing compounds (1e50
24 h of incubation, the cells were washed with PBS and then
incubated with 5
M of DCFH-DA in PBS at 37 ꢃC in 5% CO2 for
mM) were added to the cells. After
m
30 min. After removal of DCFH-DA and further washing, the cells
were incubated with 0.1 mM t-BuOOH in PBS for 30 min. At the end
of incubation, the fluorescence of the cells from each well was
measured (lexc ¼ 485 nm, lem ¼ 535 nm) with a spectrofluorometer
(Wallac VictorÒ Multilabel Counter, Perkin Elmer Inc., Boston, MA).
The results were expressed as percentage increase of intracellular
ROS evoked by exposure to t-BuOOH and calculated by the formula
[(Ft ꢂ Fnt)/Fnt ꢁ 100], where Ft ¼ fluorescence of treated neurones
and Fnt ¼ fluorescence of untreated neurones.
5.2.2. Inhibition of AChE-induced A
b40 aggregation assay
A
b
40 (2 mg mLꢂ1) was dissolved in HFIP and lyophilized.
A 1 mM solution of tested inhibitor was prepared by dissolution in
MeOH.
Aliquots of 2 mL Ab
40 peptide, lyophilized from 2 mg mLꢂ1 HFIP
(1,1,1,3,3,3,-hexafluoro-2-propanol) solution and dissolved in
DMSO, were incubated for 24 h at room temperature in 0.215 M
sodium phosphate buffer (pH 8.0) at a final concentration of
5.2.6. Statistical analysis
230
(final concentration 2.30
in the presence of 2
concentration 100 M) in 0.215 M sodium phosphate buffer pH 8.0
solution were added. Blanks containing A 40 alone, recombinant
hAChE alone, and A 40 plus the tested inhibitor in 0.215 M sodium
phosphate buffer (pH 8.0) were prepared. The final volume of each
vial was 20 L. Each assay was run in duplicate. To quantify amyloid
fibril formation, the thioflavin T fluorescence method was then
applied [36]. The fluorescence intensities due to -sheet confor-
m
M. For co-incubation experiments aliquots (16
M, A /AChE molar ratio 100:1) and AChE
L of the tested inhibitor (final inhibitor
m
L) of hAChE
Data are reported as mean ꢀ SD of at least 3 independent
experiments. Statistical analysis was performed using one-way
ANOVA with Dunnett post hoc test. Differences were considered
significant at p < 0.05. Analyses were performed using PRISM 3
software on a Windows platform.
m
b
m
m
b
b
Acknowledgments
m
This work was supported by grants from the University of
Bologna (Italy). The authors thank Grace Fox for editing and
proofreading the manuscript.
b
mation were monitored for 300 s at lem ¼ 490 nm (lexc ¼ 446 nm).
The percent inhibition of the AChE-induced aggregation due to the
presence of the tested compound was calculated by the following
expression: 100 ꢂ (IFi/IFo ꢁ 100) where IFi and IFo are the fluo-
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