Synthesis and functional survey of new Tacrine analogs modified with nitroxides or their precursors

Article abstract of DOI:10.1016/j.ejmech.2014.03.026

A series of new Tacrine analogs modified with nitroxides or pre-nitroxides on 9-amino group via methylene or piperazine spacers were synthesized; the nitroxide or its precursors were incorporated into the Tacrine scaffold. The new compounds were tested for their hydroxyl radical and peroxyl radical scavenging ability, acetylcholinesterase inhibitor activity and protection against Aβ-induced cytotoxicity. Based on these assays, we conclude that Tacrine analogs connected to five and six-membered nitroxides via piperazine spacers (9b, 9b/HCl and 12) exhibited the best activity, providing direction for further development of additional candidates with dual functionality (anti Alzheimer's and antioxidant).

Full text of DOI:10.1016/j.ejmech.2014.03.026

European Journal of Medicinal Chemistry 77 (2014) 343e350
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and functional survey of new Tacrine analogs modified with
nitroxides or their precursors
Tamás Kálai a, Robin Altman b, Izumi Maezawa c, Mária Balog a, Christophe Morisseau d,
Jitka Petrlova b, Bruce D. Hammock d, Lee-Way Jin c, James R. Trudell e, John C. Voss b,
,
Kálmán Hideg a
*
a Institute of Organic and Medicinal Chemistry, University of Pécs, H-7624 Pécs, Szigeti St. 12. Pécs, Hungary
b Department of Biochemistry & Molecular Medicine, University of California Davis, Davis, CA 95616, USA
c M.I.N.D. Institute and Department of Pathology and Laboratory Medicine, University of California Davis, Sacramento, CA95817, USA
d Department of Entomology and UC Davis Comprehensive Cancer Center, University of California Davis, Davis, CA 95616, USA
e Department of Anesthesia, Beckman Program for Molecular and Genetic Medicine, Stanford University, Stanford, CA 94305-5117, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new Tacrine analogs modified with nitroxides or pre-nitroxides on 9-amino group via
methylene or piperazine spacers were synthesized; the nitroxide or its precursors were incorporated into
the Tacrine scaffold. The new compounds were tested for their hydroxyl radical and peroxyl radical
Received 7 August 2013
Received in revised form
3 March 2014
Accepted 8 March 2014
Available online 12 March 2014
scavenging ability, acetylcholinesterase inhibitor activity and protection against Ab-induced cytotoxicity.
Based on these assays, we conclude that Tacrine analogs connected to five and six-membered nitroxides
via piperazine spacers (9b, 9b/HCl and 12) exhibited the best activity, providing direction for further
development of additional candidates with dual functionality (anti Alzheimer’s and antioxidant).
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Alzheimer’s disease
Antioxidants
Nitroxides
Spin trapping
Tacrine
1. Introduction
involved in the neurodegenerative cascade [5]. The approach in-
volves the combination of therapeutic agents that act indepen-
Alzheimer’s disease (AD) is an age-related neurodegenerative
process that gradually worsens over time. Many clinical symptoms
are associated with AD including memory loss, disorientation,
language impairment, etc. The etiology of AD has not been eluci-
dently on different etiological targets. This strategy has proven to
be successful in treatment of similarly complex diseases such as
HIV, cancer and hypertension. In the case of AD, a combination of
AChEIs with compounds targeting other pathogenic factors might
offer several benefits, at least in the improvement of clinical
symptoms of this disease. Several innovative strategies have been
published recently, which produced enhanced therapeutic effects
over AChEI monotherapy [6].
In the light of this, Tacrine, an AChEI, has been combined with
carvedilol [7], melatonin [8], and ferulic acid derivatives [9]. We
have recently published the structure of spin-labeled fluorene
(SLF), containing a pyrroline nitroxide group that provides both
dated yet, several factors such as amiloid-b (Ab) deposits [1],
s
ꢀprotein aggregation, oxidative stress [2], and decreased acetyl-
choline levels [3] play significant role in the pathology of disease
[4]. Currently no treatment is available to cure AD and clinical
treatments have only palliative effects. Such treatments include
acetylcholinesterase inhibitors (AChEIs) (Tacrine, donepezil, riva-
stigmine, galantamine) restoring cholinergic deficit, and the NMDA
receptor antagonist (memantine) limiting glutamate excitotoxicity.
In spite of research efforts, drugs that can reverse or halt the pa-
thology of AD are still lacking. Because of the complexity of AD, a
new approach has been proposed for addressing the disease:
simultaneous targeting of the multiple pathological processes
increased cell protection against toxicity of Ab oligomers (AbO) and
a route to directly observe the binding of the fluorene to the A
b
O
assembly by EPR spectroscopy [10]. Among the fluorene de-
rivatives, the pyrroline nitroxide ring-containing derivatives were
found especially useful in counteracting Ab peptide toxicity, as they
posses both antioxidant properties and the ability to disrupt A
species [10].
bO
* Corresponding author.
E-mail address: kalman.hideg@aok.pte.hu (K. Hideg).
http://dx.doi.org/10.1016/j.ejmech.2014.03.026
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

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T. Kálai et al. / European Journal of Medicinal Chemistry 77 (2014) 343e350
Nitroxides are stable free radicals that rapidly cross cell-
membranes, preempt free-radical formation by oxidizing redox-
active metal ions (equation (1), Fig. 1), and function both as intra-
and extracellular SOD mimics (equations (2) and (3)). The reduced
form of the nitroxide, hydroxylamine, also has antioxidant activity;
a proton and electron donor species (equation (4)). The sterically
hindered amine (pre-nitroxide) with ROS scavenging offers a non-
toxic stable nitroxide free radical (equation (5)). Several studies
indicate that modification of cardioprotective agents [11], PARP-
inhibitors [12], or neuroprotective ebselen [13] with nitroxides
has a beneficial influence on their activity, being supplemented by
the nitroxide’s “in status nascent acting” antioxidants and radical
scavengers.
With these concepts in mind, we focused our attention to
combine nitroxide and/or nitroxide precursors with 9-amino-
1,2,3,4-tetrahydroacridine (Tacrine 1b) in the hope that nitroxides
or their precursors (amines and hydroxylamines) may emerge as
building blocks in the search of new dual active compounds to
confront AD, a disease where oxidative stress contributes signifi-
cantly to the pathogenesis. In this paper, we report the synthesis
and biological study of these Tacrineenitroxide hybrids by modi-
fying the amino group of Tacrine or by incorporating the nitroxide
moiety into the Tacrine scaffold via a substitution of the saturated
ring (Scheme 1).
Scheme 1. (a) toluene, piperidine (0.01 equiv.), reflux, 24 h, then work-up, LiAlH4, THF,
2 h, 0 ꢁC / rt., 22e40%.
azide 11 [19] in Cu(I) catalyzed 1,3-dipolar cycloaddition reaction in
DMSO [20] to yield 12, the triazol spacer containing compound.
Alkylation of compound 7 with 2,3,4-trimethoxybenzylchoride
afforded compound 13, which combines an anti-ischemic meta-
bolic agent, trimetazidine [21] and the AChEI Tacrine (Scheme 2).
Reaction of 14 (2-aminobenzonitrile) with 15 (triacetonamine)
in the presence of 2.5 equiv aluminum-chloride in dichloroethane
[14,22] at reflux temperature after work-up gave compound 16b
which could be oxidized with H2O2 in the presence of Na2WO4 to
yield nitroxide 17b. Reaction of 4-oxo-TEMPO with 2-
aminobenzonitrile in the presence of Lewis acid, despite the con-
sumption of starting materials, did not give compound 17b. For the
synthesis of further Tacrine analogs 19b, 20b, 22b and 24b several
diamagnetic ketones 18 [23], 21, 23 were incorporated into the
Tacrine scaffold under the same conditions used in the synthesis of
compound 16b. From these compounds 19b could be oxidized to
20b nitroxide with H2O2 in the presence of Na2WO4. (Scheme 3) To
achieve better solubility both amines and nitroxides were con-
verted to HCl salts by treatment with HCl saturated ethanol [24]
indicated as 1/HCl, 4/HCl, 9/HCl, 16/HCl, 17HCl, 20/HCl, 22/HCl,
24/HCl In the case of nitroxides, this resulted in hydroxylamine
formation.
2. Results and discussion
2.1. Chemistry
In order to alkylate the amino-group of Tacrine (1b) [14] it was
condensed with aldehydes 2 [15] and 3 [16] in toluene in the
presence of piperidine to result in a Schiff-base, which was reduced
with lithium aluminum hydride in THF to offer compounds 4b and
5 (Scheme 1). Treatment of 9-chloro-1,2,3,4-tetrahydroacridine 6
[17] with five equivalents of piperazine in refluxing pentanol
offered compound 7, a key intermediate, which could be alkylated
easily on secondary amine with a paramagnetic allylic bromide 8
[18] to offer compound 9b. Alkylation of 7 with propargyl bromide
gave compound 10 which was conjugated with the paramagnetic
Scheme 2. (a) pentanol, piperazine (5.0 equiv.), 140 ꢁC, 12 h, 58%; (b) 8 (1.1 equiv.),
CHCl3, K2CO3 (1.1 equiv.) reflux, 3 h, 62%; (c) propargyl bromide (1.1 equiv.), CHCl3,
K2CO3 (1.1 equiv.) reflux, 3 h, 78%; (d) DMSO, CuI (0.3 equiv.), 40 ꢁC, 4 h, 62%; (e) 2,3,4-
trimethoxybenzylchloride, CHCl3, K2CO3 (1.1 equiv.) reflux, 3 h, 55%.
Fig. 1. Possible radical scavenging mechanisms and transformations of nitroxides and
pre-nitroxides.

T. Kálai et al. / European Journal of Medicinal Chemistry 77 (2014) 343e350
345
Scheme 3. (a)1,2-dichloroethane, AlCl3 (2.5 equiv.) reflux, 2e3 h, 32e52%; (b) EtOH,
aq. 30% H2O2, Na2WO4$2H2O (0.1 equiv.), rt., 10 h, 22e39%.
Fig. 2. Inhibitory and cell protection activities of Tacrine derivatives. The concentration
of compound resulting in 50% inhibition (IC50) of bovine acetylcholinesterase is rep-
2.2. Cholinesterase inhibitory activity and A
protection
b-induced cytotoxicity
resented by black bars. Bars with arrows represent IC50 values exceeding 40
mM.
Protection against A -induced cytotoxicity in MC65 cells is reported by the concen-
b
tration of compound that provides 50% protection (PC50, red bars). Light red bars
represent compounds that offered no significant protection. Error bars represent the
standard error of the mean from three independent measurements. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
In designing new Tacrine derivatives, nitroxide as an antioxidant
building block was bound to amino functional group with the short
methylene spacers as for compounds 4b, 4/HCl, 5 and compounds
with longer spacers such as compounds 9b, 9HCl and 12. To
demonstrate the importance of the nitroxide moiety or its pre-
cursor diamagnetic compounds 7, 10, 13 also were tested. The other
possibility was the incorporation the pyrroline- or tetrahydropyr-
idine nitroxide ring into the Tacrine scaffold. We synthesized
compounds 16b, 17b, 19b, 20b, 22b and 24b with modified Fried-
lander synthesis. All the new Tacrine analogs were tested on hy-
droxyl radical scavenging activity, peroxyl radical scavenging
activity, acetylcholinesterase (from bovine erythrocyte) inhibitory
into the binding site of acetylcholinesterase. We used a module of
Discovery studio 3.5 (DS3.5, Accelrys Inc., San Diego, CA) to search
for inter-molecular H-bonds as well as
16b fits nicely into the binding site, but generates only one
from N25 to Tyr334 of 2CKM. In contrast, compound 12 provided
one H-bond between Tyr121 on N25. Moreover, it also made four
p-bonds. Fig. 3a shows that
p-bond
p
-
bonds; one from the ring containing C4 to Trp279, one from N36 to
Phe330, and two from N36 to each ring of Trp84. When the total
energies after the final minimization were compared, compound
16b was ꢀ28716.9 versus compound 12 of ꢀ28842.9 kcal/mol; a
difference of 126 kcal/mol. It is likely that the large difference in
activity, and A
scavenging activity and AChEI activity correlation with A
b
-caused cell death inhibitory activity. The ROS
-induced
b
cytotoxicity was investigated to find compounds with best anti-
oxidant and anti Alzheimer’s activity. As in AD, the memory
dysfunction is a consequence of the cholinergic disturbances in the
afflicted areas, AChE inhibitors are used to limit the amount of
acetylcholine in the brain. In consequence, those cells which are
still alive and produce acetylcholine may restore the cholinergic
deficit at synaptic sites. For testing Tacrine analogs it was essential
to determine AChE inhibitory activity of the new derivatives.
Tacrine (1b) and its hydrochloride salt (1/HCl) have only acetyl-
cholinesterase activity and do not exhibit any protection against
A
b
-induced cytotoxicity. Compounds 4b, 4/HCl, 5, 9b, 9/HCl, 10 and
12 exhibit less AChEI activity than compound 1b, but their pro-
tective concentrations against A -induced cytotoxicity are below
20 M. While compounds 13, 16b, 16/HCl, 17b, 17/HCl, 19b, 19/HCl,
b
m
20b, 20/HCl, 22b, 22/HCl, 24b and 24/HCl also exhibit notable
protective activity against the induced cytotoxicity, their AChEI
activity is practically lost (over 40 mM), only compounds 24 and 24/
HCl produced limited AChEI activity. Hence it can be concluded that
beyond compound 13, all the other ineffective analogs contain
heterocyclic (pyrroline or tetrahydropyridine) ring incorporated
into the Tacrine scaffold, e.g., condensed with a quinoline moiety
(Fig. 2.). To understand the difference in AChEI activity of com-
pounds 12, 13 and 16b docking studies were performed.
2.3. Docking of Tacrine derivatives to acetylcholinesterase
A goal of the docking studies was to understand why compound
16b is inactive while compound 12 is active in experimental
studies. Fig. 3a and b show a zoomed-in view of the ligands docked
Fig. 3. a. Zoomed-in view of compound 16b docked into the binding site of acetyl-
cholinesterase. b. Zoomed-in view of compound 12 docked into the binding site of
acetylcholinesterase.

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T. Kálai et al. / European Journal of Medicinal Chemistry 77 (2014) 343e350
interaction energies between compound 12 and 16b accounts for
the lack of activity of compound 16b. The latter molecule provided
only one
p-bond to the receptor whereas compound 12 provided
one H-bond and four
p
-bonds. We did not calculate the more
distributed van der Waals interactions, but they likely also added to
the increased interaction energy of the larger compound 12.
Docking experiments showed that both Tacrine (1b) and compound
13 are bound equally well and the differences in efficacy at the
binding site were not apparent. However, compound 13 structur-
ally more similar to ditacrine (two rigid 1,2,3,4-tetrahydroacridine
unit connected with a heptan-1,7-diamine flexible spacer) and
the binding energy of compound 13 is somehow less than that of
ditacrine (see table in the supplementary information).
2.4. ROS scavenging activity
To study the ROS scavenging activity of the Tacrine analogs, the
signal intensity of the BMPO spin trap [25] was measured by EPR
spectroscopy following induction of either hydroxyl or superoxide
radicals. BMPO produces a distinct EPR line shape, depending on
whether the adduct is formed with the hydroxyl or superoxide
radical (Fig. 4). Fig. 4 illustrates how Tacrine derivatives with
appreciable scavenging activity compete for ROS species (e.g.,
compound 13), thereby producing a reduced BMPO EPR line in-
tensity. This compares to compound 1b, which has no significant
effect on the level of radical detected (Fig. 4). In comparison of the
hydroxyl radical scavenging activity and the protective effect
Fig. 5. Hydroxyl (black) and peroxyl (red) radical scavenging compared to the cell
protection activities of Tacrine derivatives. The bars represent a qualitative determi-
nation of scavenging by measuring the residual amplitude of the third BMPO reso-
nance line in the presence of the indicated compound at
1 mM, with 100%
representing no identifiable scavenging activity. Error bars represent the fractional
error determined from the signal: noise of each experiment. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
provide remarkable ROS scavenging activity and protection against
against Ab-induced cytotoxicity, the results are more or less parallel
Ab
-induced cytotoxicity (Figs. 2 and 5). The exception is the
suggesting that free radical processes have a contribution in the
induced cytotoxicity. Compounds without nitroxide or nitroxide
precursors (1b, 1/HCl, 7, 10) have limited ꢂOH and superoxide
scavenging activity, while compounds 4b, 4/HCl 5, 9b, 9/HCl, 12
diamagnetic trimetazidine derivative (13), compound with
a
notable antioxidant activity [26]. Regarding the anellated de-
rivatives, sterically hindered amines (16b, 16/HCl, 19b, 19/HCl)
Fig. 4. EPR spectra of hydroxyl or superoxide adducts to the BMPO spin trap in the presence of compounds 1b or 13. Final BMPO concentration in the reaction mixture was 10 mM,
and the tacrines were at 1 mM.

T. Kálai et al. / European Journal of Medicinal Chemistry 77 (2014) 343e350
347
Fig. 6. Correlation between ROS scavenging and cell protection activities for Tacrine compounds. Shown for the indicated Tacrines is the index of cell protection (PC50) plotted
according to its percent BMPO spin trap signal. Compounds 1b, 7 and 10 are not included because they do not provide any significant level of cell protection.
provide a lower protective activity compared to nitroxides 17b and
20b and their hydroxylamine salt (17/HCl and 20/HCl). Further-
more, the annulated six membered nitroxide (17b) provides su-
perior ꢂOH scavenging activity compared to the five-membered
annulated nitroxide (20b). While several factors may influence the
thermodynamics and kinetics of these reactions, a general deter-
mining factor is the flexibility of the nitrogen center to planarize
upon oxidation or pyramidalize upon reduction [27]. Thus, mono-
cyclic nitroxides (4b, 5, 9b, 12) readily take part in redox processes,
as does the six-membered nitroxide with the tetrahydroisoquino-
line scaffold (17b). However, in the case of the five-membered rings
condensed into an aromatic system (19, 20), the severely con-
stricted ring impairs reactivity at the nitrogen.
assay (e.g., the presence of lipids and proteins). Thus differences in
compound solubility (in all assays) or uptake (in the cell protection
assay) is likely to produce divergence between the measurements.
Such factors are apparent for the dissimilar performance of the base
and acid species. Compounds 9 and 12 were the most efficient AChE
inhibitors and radical scavengers and exhibited remarkable cell
protection toward A
b
ꢀinduced toxicity, although compounds 4, 5,
17 and 20 also exhibited notable activity (Fig. 6). This dual pro-
tective (AChE inhibitory and antioxidant) profile of compounds 9
and 12 makes these compounds promising leads for developing
disease modifying drugs for the future treatment of AD.
4. Experimental protocols
As expected, diamagnetic derivatives (1b, 7, 10, 13, 16b, 16/HCl,
20b, 20/HCl, 22b, 22/HCl 24b, 24/HCl) lack peroxyl radical scav-
enging activity, while the nitroxide or hydroxylamine containing
compounds (4b, 4HCl 5, 9b, 9HCl, 12, 17b, 17HCl, 20, 20 HCl) are
excellent in this regard. We can also note that paramagnetic Tac-
rines produce better performance in peroxide scavenging than in
4.1. Chemistry
Melting points were determined with a Boetius micro melting
point apparatus and are uncorrected. Elemental analyses (C, H, N, S)
were performed on Fisons EA 1110 CHNS elemental analyzer. Mass
spectra were recorded on a Thermoquest Automass Multi and VG
TRIO-2 instruments and in the EI mode. 1H NMR spectra were
recorded with Varian UNITYINOVA 400 WB spectrometer and
Bruker Avance 3 Ascend 500. Chemical shifts are referenced to
Me4Si. Measurements were run at 298 K probe temperature in
CDCl3 solution. ESR spectra were taken on Miniscope MS 200 in
10ꢀ4 M CHCl3 solution and monoradicals gave triplet line. Flash
column chromatography was performed on Merck Kieselgel 60
(0.040e0.063 mm). Qualitative TLC was carried out on commer-
cially available plates (20 ꢃ 20 ꢃ 0.02 cm) coated with Merck
Kieselgel GF254. Triacetonamine, 1,2,2,6,6-pentamethylpiperidone,
1-methyl piperidone, compound 14, Tacrine and all other chemicals
were purchased from Aldrich, compound 2 [15], 3 [16], 6 [17], 8
[18], 11 [19], 18 [23] were prepared as described earlier.
HOꢂ scavenging (Fig. 5). It is well known that A
b toxicity is related to
its redox properties [28] and contributes to oxidative damage by
inducing lipid peroxidation, which in turn generates additional
cytosolic free radical formation, leading to mitochondrial and
cytoskeletal compromise, depletion of ATP, and ultimate apoptosis.
Our findings that nitroxide or its precursor containing Tacrine an-
alogs (4b, 4/HCl 5, 9, 12, 17b, 17/HCl, 20b, 20/HCl) with free radical
scavenging capabilities (Fig. 1) corroborate the findings that com-
pounds with antioxidant activity (folate, vitamin E, acetyl-L-
carnitine, ferulic acid ethyl ester) provide protection against
A
bꢀinduced toxicity [29,30].
3. Conclusions
A new series of tacrine-nitroxide and nitroxide precursor hybrid
related derivatives were synthesized as dual acetylcholinesterase
inhibitors and antioxidants (radical scavengers). Our synthesis
varied the nitroxide position, tethered to a 9-amino group or
anellated to the Tacrine pyridine ring. The influence of the nitroxide
ring size was also explored. Compounds were tested on hydroxyl
4.1.1. Synthesis of 9-amino substituted Tacrines
In a 250 mL round bottomed flask equipped with a DeaneStark
constant separator which is connected to a reflux condenser, a
solution of Tacrine (1) (1.98 g, 10,0 mmol) and aldehyde 2 or 3
(10.0 mmol) and piperidine 85 mg (1.0 mmol) in toluene (100 mL)
was heated on reflux temperature for 24 h. After cooling the solvent
was evaporated off and the residue was purified by flash column
chromatography to remove starting materials. To the solution of
Schiff-base in dry THF (30 mL) under N2 at 0 ꢁC LiAlH4 (20.0 mmol,
8.3 mL) was added dropwise. After consumption of the Schiff-base
(w2 h) the mixture was poured on mixture of ice and 10% aq. NaOH
radical scavenging, peroxyl radical scavenging, A
death and AChE inhibitory assays. The general correlation between
ROS scavenging capability and protection against A toxicity is
b
ꢀinduced cell
b
illustrated in Fig. 6. The correlation with hydroxyl scavenging ap-
pears a little stronger. It should be noted the solvent and potential
carrier components are significantly different in the cell protection

348
T. Kálai et al. / European Journal of Medicinal Chemistry 77 (2014) 343e350
solution (100 mL) and the mixture was stirred at room temperature
for 30 min. The organic phase was separated, the aqueous phase
was extracted with CH2Cl2 (2 ꢃ 20 mL). The combined organic
phase was dried (MgSO4), filtered and evaporated and the residue
was purified by flash column chromatography (hexaneeEtOAc,
CHCl3eEt2O) to yield the title compounds as yellow crystalline
solid.
2H). 13C NMR (CD3OD)
d
¼ 161.5, 155.1, 148.4, 129.9, 129.2, 128.4,
127.1, 126.4, 125.2, 79.1, 75.4, 53.9, 51.1, 47.8, 34.4, 27.8, 23.9, 23.6.
Ms (EI) m/z (%): 305 (Mþ, 38), 266 (11), 209 (48), 67 (100). Anal
calcd. for: C20H23N3: C, 78.65; H, 7.59; N, 13.76; found: C 78.55, H
7.51, N 13.57.
4.1.3.3. 9-[4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl]-1,2,3,4-
tetrahydroacridine (13) (HO-4392). 127 mg (55%), yellow solid, mp
4.1.1.1. 2,2,5,5-Tetramethyl-3-[(1,2,3,4-tetrahydroacridin-9-yl)amino-
methyl]-2,5-dihydro-1H-pyrrol-1-yloxyl radical (4bþ) (HO-4219).
1.40 g (40%), mp 153e155 ꢁC. Ms (EI) m/z (%): 350 (M , 19), 305 (7),
277 (11), 211 (100), 197 (56). 1H NMR of NOMe derivative (CDCl3)
202e204 ꢁC. 1H NMR (2HCl salt in D2O)
d
¼ 8.14 (d, 1H, J ¼ 8 Hz),
7.90 (s, 2H), 7.73 (t, 1H, J ¼ 8 Hz), 7.28 (d, 1H, J ¼ 8 Hz), 6.95 (d, 1H,
J ¼ 8 Hz), 4.46 (s, 2H) 3.98 (s, 3H), 3.88 (t, 4H, J ¼ 5 Hz), 3.64 (m, 2H),
3.52 (m, 2H), 3.21 (t, 2H, J ¼ 6.0 Hz), 2.90 (t, 2H, J ¼ 6 Hz), 1.95 (m,
d
¼ 7.96e7.93 (m, 2H), 7.58 (t, 1H, J ¼ 8 Hz), 7.38 (t, 1H, J ¼ 8 Hz),
2H), 1.86 (m, 2H). 13C NMR (CD3OD):
148.4, 143.5, 129.8, 129.0, 128.4, 127.2, 127.1, 126.3, 125.3, 124.2,
108.7, 61.8, 61.2, 57.8, 56.6, 55.1, 51.4, 34.4, 27.8, 23.9, 23.6. Ms (EI)
m/z (%): 447 (Mþ, 12), 266 (90), 238 (44), 181 (100). Anal calcd. for:
d
¼ 161.5, 155.2, 154.7, 154.0,
5.65 (s, 1H), 4.02 (s, 1H), 3.73 (s, 2H), 3.65 (s, 3H), 3.10 (br, 2H), 2.74
(br, 2H), 1.95 (br, 4H), 1.29 (s, 6H), 1.26 (s, 6H). Anal calcd. for:
C
22H28N3O: C 75.39, H 8.05, N 11.99, found: C 75.15, H 7.97, N 11.86.
C27H33N3O3: C, 72.46; H, 7.43; N, 9.39; found: C 72.45, H 7.46, N 9.41.
4.1.1.2. 2,2,6,6-Tetramethyl-4-[(1,2,3,4-tetrahydroacridin-9-yl)ami-
nomethyl]-1,2,3,6-tetrahydropyridin-1-yloxyl radical (5) (HO-4637).
800 mg (22%), mp 157e159 ꢁC. Ms (EI) m/z (%): 364 (Mþ, 14), 334
(2), 278 (20), 211 (100), 197 (45). Anal calcd. for: C23H30N3O: C
75.79; H, 8.30; N, 11.53, found: C 75.96, H 8.12, N 11.55.
4.1.4. Modification of the Tacrine analog by a nitroxide with click-
reaction
2,2,6,6-Tetramethyl-4-{4-[4-(1,2,3,4-tetrahydroacridin-9-yl)
piperazin-1-ylmethyl]-1H-1,2,3-triazol-1-yl}piperidin-1-yloxyl
Radical (12) (HO-4564): To a stirred solution of compound 10
(610 mg, 2.0 mmol) and compound 11 (394 mg, 2.0 mmol) in DMSO
(5 mL) CuI (114 mg, 0.6 mmol) was added and the mixture was
stirred under N2 for 4 h at 40 ꢁC. After consumption of the starting
material the mixture was poured onto ice-water mixture (100 mL)
and the precipitated solid was filtered and air-dried. The crude
product was purified further by flash column chromatography
(CHCl3eMeOH) to offer the title compound 657 mg (62%), beige
solid, mp 152e154 ꢁC. Ms (EI) m/z (%): 502 (Mþ, 8), 414(8), 267 (90),
250(100). Anal calcd. for: C29H40N7O C 69.29; H 8.02; N, 19.51,
found: C 69.10, H 7.95, N 19.42.
4.1.2. 9-(Piperazin-1-yl)-1,2,3,4-tetrahydroacridine (7) (HO-4387)
In a sealed tube a stirred mixture of compound 6 (2.18 g,
10.0 mmol) and piperazine (4.30 g, 50.0 mmol) in pentanol (10 mL)
were heated at 140 ꢁC for 12 h. After cooling the mixture was
diluted with water (20 mL) and extracted with CHCl3 (2 ꢃ 20 mL).
The organic phase was dried (MgSO4), the drying agent was filtered
off, solvents were evaporated and the residue was purified by flash
column chromatography (CHCl3eEt2O) to yield the title compound
as an off-white solid 1.54 g, (58%), mp 156e158 ꢁC. Ms (EI) m/z (%):
267 (Mþ, 9), 225 (23), 69(64) 57 (100). 1H NMR of 2HCl salt (D2O)
d
¼ 8.17 (d, 1H), 7.92e7.91 (m, 2H), 7.74e7.71 (m, 1H), 3.87 (t, 4H,
J ¼ 5 Hz), 3.54 (t, 4H, J ¼ 5 Hz), 3.22 (t, 2H, J ¼ 6.0 Hz), 2.92 (t, 2H,
4.1.5. Friedlander reaction for synthesis of 1,2,3,4-tetrahydrobenzo
[b][1,6]naphthyridines and 1H-pyrrolo[3,4-b]quinoline general
procedure (16b, 19b, 22b, 24b)
J ¼ 6 Hz), 1.95 (m, 2H), 1.86 (m, 2H). 13C NMR (CD3OD)
d
¼ 160.9,
157.0, 138.2, 133.2, 127.9, 126.5, 125.5, 124.0, 119.5, 48.3, 43.81, 28.7,
26.2, 21.6, 20.2. Anal calcd. for: C17H21N3: C, 76.37; H, 7.92; N, 15.72
found: C 76.21, H 7.82, N 15.63.
Mixture of cyclic ketone 15 or 18 or 21 or 23 (10.0 mmol) and 2-
aminobenzonitrile (14) (1.18 g, 10.0 mmol) in 1,2-dichloroethane
(30 mL) was stirred at room temperature for 10 min, then anhy-
drous AlCl3 (3.32 g 25.0 mmol) was added in one portion and the
mixture was stirred and refluxed for 2 h. After cooling the mixture
was basified with 10% aq. NaOH (100 mL), the mixture was stirred
at ambient temperature for 30 min. After separation of organic
phase the aqueous phase was extracted with CH2Cl2 (2 ꢃ 20 mL).
The combined organic phase was dried (MgSO4), filtered and
evaporated. The residue was purified by flash chromatography
(hexaneeEtOAc or CHCl3eEt2O) to give the title compounds as
solids in 32e52% yield.
4.1.3. General procedure for alkylation of 9-(piperazin-1-yl)-1,2,3,4-
tetrahydroacridines (9b, 10, 13)
A stirred solution of compound 7 (1.33 g, 5.0 mmol) and com-
pound
8
(5.5 mmol) or propargyl bromide or 2,3,4-
trimethoxybenzylchloride (5.5 mmol) and K2CO3 (760 mg,
5.5 mmol) in CHCl3 (30 mL) was heated at reflux till the con-
sumption of starting material (w3 h). After cooling the inorganic
salt was filtered off, the organic phase washed with water (10 mL),
the organic phase was separated, dried (MgSO4), filtered and
evaporated. The residue was purified by flash column chromatog-
raphy (CHCl3eEt2O) to furnish the title compounds.
4.1.5.1. 1,1,3,3-Tetramethyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyr-
idin-10-amine (16b) (HO-4276). 1.14 g, (45%), yellow solid, mp 141e
4.1.3.1. 2,2,5,5-Tetramethyl-3-[4-(1,2,3,4-tetrahydroacridin-9-yl)
piperazin-1-ylmethyl]-2,5-dihydro-1H-pyrrol-1-yloxyl radical 9b
(HO-4380). 130 mg (62%), yellow solid, mp:138e140 ꢁC. Ms (EI) m/z
(%): 419 (Mþ, 43), 389 (5), 280 (75), 267 (82), 225 (100). Anal calcd.
for: C26H35N4O: C, 74.43; H, 8.41; N, 13.35 found: C 74.29, H 8.33, N
13.26.
143 ꢁC. 1H NMR (CD3OD)
d
¼ 7.96 (d, 1H, J ¼ 8.5 Hz), 7.73 (d, 1H,
J ¼ 8.5 Hz), 7.57 (t, 1H, J ¼ 7 Hz), 7.38 (t, 1H, J ¼ 7 Hz), 2.87 (s, 2H),
1.69 (s, 6H), 1.24 (s 6H). 13C NMR (CD3OD) 157.1, 148.8, 147.13, 130.3,
127.7, 125.2, 122.4, 120.0, 115.7, 53.7, 50.5, 48.1, 29.5, 29.3. Ms (EI) m/
z (%): 255 (Mþ, 2), 240 (100), 223 (34), 43 (62). Anal calcd. for:
C16H21N3 C 75.26, H 8.29, N 16.46, found: C 75.40, H 8.12, N 16.32.
4.1.3.2. 9-[4-(Prop-2-yn-1-yl)piperazin-1-yl]-1,2,3,4-
4.1.5.2. 1,1,3,3-Tetramethyl-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-
tetrahydroacridine (10) (HO-4563). 119 mg (78%) yellow solid, mp.
9-amine (19b) (HO-4278). 1.25 g, (52%), pale yellow solid, mp 152e
94e96 ꢁC. 1H NMR (CD3OD)
d
¼ 8.17 (d, 1H, J ¼ 9 Hz), 7.85 (d, 1H,
154 ꢁC. 1H NMR (D2O)
d
¼ 8.13 (d, 1H, J ¼ 8 Hz), 7.83 (t, 1H, 8 Hz),
J ¼ 8 Hz), 7.60 (t, 1H, J ¼ 7 Hz), 7.46 (t, 1H, J ¼ 8 Hz), 3.44 (d, 2H,
J ¼ 2 Hz), 3.37 (t, 4H, J ¼ 5 Hz), 3.06 (t, 2H, J ¼ 7 Hz), 2.97 (t, 2H,
J ¼ 6.0 Hz), 2.81 (t, 3H, J ¼ 5 Hz), 2.75 (s, 1H), 1.96 (m, 2H), 1.86 (m,
7.76 (d, 1H, J ¼ 8 Hz), 7.58 (t, 1H, J ¼ 8 Hz), 1.92 (s, 6H), 1.89 (s, 6H).
13C NMR (D2O): 154.0, 153.2, 138.8, 134.5, 127.18, 122.7, 119.9, 116.4,
111.1, 68.2, 66.1, 25.71, 24.63. Ms (EI) m/z (%): 241 (Mþ, 2), 226 (100),

T. Kálai et al. / European Journal of Medicinal Chemistry 77 (2014) 343e350
349
211 (25), 43 (18). Anal calcd. for: C15H19N3 C 74.65, H 7.94, N 17.41,
found: C 74.49, H 7.82, N 17.27.
temperature MD. The conformations are then translated into the
binding site. Candidate poses are then created using random rigid-
body rotations followed by simulated annealing. A final minimi-
zation with the CHARMm force field is then used to refine the
ligand poses. We highlighted di-tacrine in the crystal structure of
2CKM and defined a sphere that contained it; approximately 11 A.
This sphere limited the possible positions of the ligands in subse-
quent docking runs. We used 1000 steps of molecular dynamics at
1000 K to produce ligand conformations for docking. After docking,
we selected the ten top ‘Hits’ for refinement using molecular dy-
namics with simulated annealing. For each selected pose, we used
2000 steps of heating to a target temperature of 700 K and then
5000 steps of cooling to a target temperature of 300 K. Then we
optimized each of the resulting poses to a gradient of 0.001 kcal/
mol with DS3.5. We ranked the poses based on two criteria;
CDOCKER energy and CDOCKER interaction energy. The latter value
was the most informative, it is the difference between the total final
energy of the docked complex and the sum of the energies of the
receptor and the ligand. We chose the best complex of compound
16b and compound 12b based on the CDOCKER interaction energy
value. For these two structures we performed an optimization of
the entire receptor plus each ligand with a light restraining force of
10 kcal/(mol ꢃ A).
4.1.5.3. 2-Methyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridin-10-
amine (22b) (HO-4330). 1.00 g (32%), yellow solid, mp 213e215 ꢁC.
1H NMR (CDCl3)
d
¼ 8.55 (d, 1H, J ¼ 8 Hz), 8.32 (bs, 1H), 7.96 (d, 1H),
7.81 (t, 1H, J ¼ 8 Hz), 7.45 (t, 1H, J ¼ 8 Hz), 3.51 (s, 2H), 3.10 (t, 2H,
J ¼ 6 Hz), 2.80 (t, 3H, J ¼ 6 Hz), 2.50 (s, 2H). 13C NMR of 2 HCl salt
(D2O)
d
¼ 151.1, 146.6, 137.5, 133.5, 126.6, 122.2, 119.8, 116.5, 101.1,
66.02, 58.4, 41.1, 30.4, 27.0, 228, 21.7, 20.0. Ms (EI) m/z (%): 313 (Mþ,
71), 212 (100),196 (64),170 (85). Anal calcd. for: C13H15N3 C 73.21, H
7.09, N 19.70, found: C 73.15, H 7.03, N 19.55.
4.1.5.4. 1,1,2,3,3-Pentamethyl-1,2,3,4-tetrahydrobenzo[b][1,6]naph-
thyridin-10-amine (24b) (HO4330). 1.18 g (44%), brownish-yellow
solid, mp 65e67 ꢁC. 1H NMR of 2 HCl salt (D2O)
d
¼ 7.96 (d, 1H,
J ¼ 8 Hz), 7.51 (t, 1H, J ¼ 8 Hz), 7.39 (d, 1H, J ¼ 8 Hz), 7.29 (1H, t,
J ¼ 8 Hz) 2.97 (s, 2H), 2.54 (s, 3H),1.76 (s, 6H),1.17 (s, 6H). 13C NMR of
2 HCl salt (D2O)
d
¼ 154.7, 145.6, 137.4, 134.5, 129.9, 122.5, 119.1,
114.9, 101.0, 49.6, 49.2, 43.1, 24.4. Ms (EI) m/z (%): 269 (Mþ, 4), 254
(100), 223 (39). Anal calcd. for: C17H23N3 C 75.80; H 8.61; N 15.60,
found: C 75.62, H 8.52, N 15.76.
4.1.6. Oxidation of compounds 16b and 19be17b and 20b nitroxide
free radicals
4.3. Biological assays
To a stirred solution of amine 16b or 19b (5.0 mmol) and
Na2WO4$2H2O (164 mg, 0.5 mmol) in EtOH (20 mL) and water 5
(mL) 30% aq. H2O2 (5 mL) was added dropwise at 0 ꢁC. After the
addition the stirring was continued at room temperature and the
course of the reaction was followed by TLC (hexane/EtOAc 2:1) and
once by product (upon prolonged oxidation time pyridine-N-oxyl
also formed) appears (w10 h) the organic solvent was evaporated
and the mixture was extracted with CHCl3 (2 ꢃ 30 mL). The organic
phase was dried (MgSO4) filtered and evaporated to offer the title
nitroxides 17b or 20b as yellow solids.
4.3.1. Antioxidant activity assay
The free radical scavenging activity of the Tacrine compounds
was determined by measuring the adduct levels accumulated by
the spin trap BMPO (5-tert-butoxycarbonyl 5-methyl-1-pyrroline
N-oxide, Applied Bioanalytical Labs, Bradenton, FL). Briefly, a
mixture of horseradish peroxidase (100 ng), hydrogen peroxide
(0.03%), and sodium azide (0.001%) in PBS pH 7.4 was used to
generate superoxide radicals. Hydroxyl radicals were generated by
mixing ferrous ammonium sulfonate (1 mg/mL) and hydrogen
peroxide (0.03%) in PBS. Each of the superoxide or hydroxyl radical
solutions also contained BMPO at a final concentration of 10 mM.
After mixing the solution, an aliquot was immediately removed and
combined with the tacrine (1 mM final concentration), and EPR
measurements were obtained. The superoxide and hydroxyl radi-
cals were measured as BMPOeOOH and BMPOeOH adducts,
respectively. Electron paramagnetic resonance spectroscopy mea-
surements of the BMPO spin traps were obtained at room tem-
perature with a JEOL X-band spectrometer fitted with a loop-gap
resonator. All spectra were recorded at room temperature and ob-
tained by averaging two 2-min scans with a sweep width of 100 G
at a microwave power of 3 mW and modulation amplitude opti-
mized to the natural line width of the spin probe.
4.1.6.1. 10-Amino-1,1,3,3-tetramethyl-3,4-dihydrobenzo[b][1,6]naph-
thyridin-2(1H)-yloxyl radical (17b) (HO-4277). 526þmg (39%), yel-
low solid, mp 179e181 ꢁC. Ms (EI) m/z (%): 270 (M , 21), 240 (18),
225 (100). Anal calcd. for C 71.08, H 7.46, N 15.54 found: 71.19, H
7.31, N 15.40.
4.1.6.2. 9-Amino-1,1,3,3-tetramethyl-1H-pyrrolo[3,4-b]quinolin-
2(3H)-yloxyl radical (20b) (HO-4279).þ281 mg (22%), yellow solid,
mp > 230 ꢁC. Ms (EI) m/z (%): 256 (M , 62), 241 (45), 226 (77), 211
(100). Anal calcd. for C15H18N3O C 71.08, H 7.46, N 15.54 found: C
71.96; H 7.56; N, 15.46.
4.2. Molecular modeling of compounds 12b and 16b
The structures of Tacrine, di-tacrine, compound 12b, and com-
pound 16b were obtained. They were prepared for docking with the
Generate Conformations module of Discovery Studio 3.5 (DS3.5,
Accelrys Inc, San Diego, CA, USA). We considered two X-ray struc-
tures of acetylcholinesterase as the receptor for docking studies; a
receptor with Tacrine docked (PDB ID 1ACJ) and one with di-tacrine
docked (PDB ID 2CKM). We chose the latter structure as di-tacrine
is closer in size and length to the planned ligands; compound 16b
(inactive in our assays) and compound 12b (active in our assays).
The acetylcholinesterase structure 2CKM was prepared for docking
by adding hydrogens, checking for missing atoms, and assigning
the CHARMm force field to all atoms. We used the CDOCKER
module of DS3.5. CDOCKER uses a CHARMm-based molecular dy-
namics (MD) scheme to dock ligands into a receptor binding site.
Random ligand conformations are generated using high-
4.3.2. MTT cytotoxicity assay
The human neuroblastoma cell line (MC65) was used to deter-
mine the ability of the tacrine compounds to protect against A
oligomer-induced toxicity according to previously published pro-
tocols [31,32]. Briefly, the MC65 cell line conditionally expresses the
b
carboxyl-terminal 99 residues of the A
C99) in the absence of the transgene suppressor tetracycline (TC).
is then generated from APP-C99 after cleavage by cellular
secretase. To generate A , tetracycline was removed from the cul-
ture media, and as early as 4 h after TC removal A oligomers began
b precursor protein (APP-
A
b
g-
b
b
to accumulate intracellularly. Tacrine compounds were added
immediately after TC removal and cells were maintained for 3
days without changing the media. Cytotoxicity was determined
using the colorimetric MTT [3-(4,5-dimethylthiazol-2yl)-2,5-
diphenyltetrazoliumbromide] assay.

350
T. Kálai et al. / European Journal of Medicinal Chemistry 77 (2014) 343e350
4-Car-boxamidobenzimidazole-2-ylpyrroline and etetrahydropyridine nitro-
4.3.3. Acetylcholinesterase inhibition assay
xides and their precursors, Journal of Medicinal Chemistry 52 (2009) 1619e
1629.
[13] T. Kálai, G. Mugesh, G. Roy, H. Sies, Z. Berente, K. Hideg, Combining benzo[d]
isoselenazol-3-ones with sterically hindered alicyclic amines and nitroxides:
enhanced activity as glutathione peroxidase mimics, Organic & Biomolecular
Chemistry 3 (2005) 3564e3569.
[14] D. Thomae, G. Kirsch, P. Seck, Synthesis of selenophene analogues of the
tacrine series: comparison of classical route and microwave irradiation,
Synthesis 40 (2008) 1600e1606.
The acetylcholinesterase inhibitory activity of the tacrines was
determined using acetyl-thio-choline and DTNB according to the
method previously published by Wilson and Henderson [33].
Acetylcholinesterase from bovine erythrocytes was used in the
assay at a final concentration of 80 mg/mL, and the buffer used for
the measurements was 100 mM NaPO4 (pH 8.0) containing 0.1 mg/
mL BSA. Results are expressed as means ꢄ standard deviation of at
least three separate experiments.
[15] K. Hideg, H.O. Hankovszky, L. Lex, Gy. Kulcsár, Nitroxyls; VI. Synthesis and
reactions of 3-Hydroxymethyl-2,2,5,5-tetramethyl-2,5-dihydropyrrole-1-oxyl
and 3-formyl derivatives, Synthesis 12 (1980) 911e914.
}
[16] J. Cseko, H.O. Hankovszky, K. Hideg, Synthesis of novel, highly reactive 1-oxyl-
Acknowledgments
2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine derivatives, Canadian Journal
of Chemistry 63 (1985) 940e943.
This work was supported by grants from Hungarian National
Research Fund OTKA K 81123 (KH), National Institutes of Health
[17] P.W. Elsinhorst, C.M.G. Tanarro, M. Gütschow, Novel heterobivalent tacrine
derivatives as cholinesterase inhibitors with notable selectivity toward
butyrylcholinesterase, Journal of Medicinal Chemistry 49 (2006) 7540e7544.
[18] H.O. Hankovszky, K. Hideg, L. Lex, Nitroxyls VII. Synthesis and reactions of
highly reactive 1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydropyrrole-3-ylmethyl
sulfonates, Synthesis 12 (1980) 914e916.
[19] K. Hideg, H.O. Hankovszky, J. Tigyi, Nitroxides, I. Sulfonic esters of 4-hydroxy-
2,2,6,6-tetramethyl-piperidine-1-oxyl and their reaction with nucleophilic
reactants, Acta Chimica Academiae Scientiarum Hungaricae 92 (1977) 85e87.
[20] T. Kálai, W.L. Hubbell, K. Hideg, Click reactions with nitroxides, Synthesis 41
(2009) 1336e1341.
R01
AG029246
(JV),
National
Institutes
of
Health
R01AA02098001A1 (JRT) and National Institutes of Health R01
ES002710 (BDH). The authors thank for Noémi Lazsányi for
elemental analysis and Gergely Gulyás-Fekete (Dept of. Biochem-
istry and Medicinal Chemistry, University of Pécs) for NMR
measurements.
[21] T. Kálai, M. Khan, M. Balog, K.V. Kutala, P. Kuppusamy, K. Hideg, Structure-
activity studies on the protection of trimetazidine derivatives modified with
nitroxides and their precursor from myocardial ischemia-reperfusion injury,
Bioorganic & Medicinal Chemistry 14 (2006) 5510e5516.
[22] C. Rios, J. Ega, J. Marco-Contelles, R. León, A. Samadi, I. Iriepa, I. Moraleda,
E. Gálvez, A.G. Garcia, M.G. López, M. Villaroya, A. Romero, Synthesis inhibi-
tory activity of cholinesterases, and neuroprotective profile of novel 1,8-
naphthyridine derivative, Journal of Medicinal Chemistry 53 (2010) 5129e
5143.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.026.
References
[23] R.M. Dupreye, A. Rassat, P. Rey, Nitroxides. XVIII. Pyrrolidine free radicals,
Bulletin de la Societe Chimque de France (1965) 3643e3644.
[24] M.C. Krishna, W. Degraff, H.O. Hankovszky, P.C. Sár, T. Kálai, J. Jeko, A. Russo,
[1] M. Jucker, L.C. Walker, Pathogenic protein seeding in Alzheimer disease and
other degenerative disorders, Annals of Neurology 70 (2011) 532e540.
[2] R. Sultana, D.A. Butterfield, Role of oxidative stress in the progression of
Alzheimer’s disease, Journal of Alzheimer’s Disease 19 (2010) 341e353.
[3] M.S. Rafii, P.S. Aisen, Recent developments in Alzeimer’s disease therapeutics,
BMC Medicine 7 (2009) 7.
}
J.B. Mitchell, K. Hideg, Studies of structure-activity relationship of nitroxide
free radicals and their precursors as modifiers against oxidative damage,
Journal of Medicinal Chemistry 41 (1998) 3477e3492.
[25] H. Zhao, J. Joseph, H. Zhang, H. Karoui, B. Kalyanaraman, Synthesis and
biochemical applications of a solid cyclic nitrone spin trap: a relatively su-
perior trap for detecting superoxide anions and glutathiyl radicals, Free
Radical Biology & Medicine 31 (2001) 599e606.
[26] C. Guarnieri, C. Muscari, Anti oxy-radical properties of trimetazidine, Research
Communications in Chemical Pathology and Pharmacology 64 (1989) 215e
225.
[27] J.P. Blinco, J.L. Hodgson, B.J. Morrow, J.R. Walker, G.D. Will, M.L. Coote,
S.E. Bottle, Experimental and theoretical studies of the redox potentials of
cyclic nitroxides, Journal of Organic Chemistry 73 (2008) 6763e6771.
[28] V. Kadlcik, C. Sicard-Roselli, T.A. Mattioli, M. Kodicek, C. Houée-Levin, One-
electron oxidation of b-amyloid peptide: sequence modulation of reactivity,
Free Radical Biology & Medicine 37 (2004) 881e891.
[29] S. Dhitavat, D. Ortiz, E. Rogers, E. Rivera, T.B. Shea, Folate, vitamin E and
acetyl-L-carnitine provide synergistyc protection against oxidative stress
resulting from exposure of human neuroblastoma cells to amyloid-beta, Brain
Research 1061 (2005) 114e117.
[30] R. Sultana, A. Ravgna, H. Mohammad-Abdul, V. Calabrese, D.A. Butterfield,
Ferrulic acid ethyl ester protects neurons against amyloid beta-peptide(1-42)-
induced oxidative stress and neurotoxicity: relationship to antioxidant ac-
tivity, Journal of Neurochemistry 92 (2005) 749e758.
[31] I. Maezawa, H.S. Hong, R. Liu, C.Y. Wu, R.H. Cheng, M.P. Kung, H.F. Kung,
K.S. Lam, S. Oddo, F.M. Laferla, L.W. Jin, Congo red and thioflavin-T analogs
detect Abeta oligomers, Journal of Neurochemsitry 104 (2008) 457e468.
[32] J. Petrlova, T. Kálai, I. Maezawa, R. Altman, G. Harishchandra, H.S. Hong,
D.A. Bricarello, A.N. Parikh, G.A. Lorigan, L.W. Jin, K. Hideg, J.C. Voss, The in-
fluence of spin-labeled fluorene compounds on the assembly and toxicity of
[4] H.F. Kung, The b-Amyloid hypothesis in Alzeimer’s disease: seeing is believing,
Medicinal Chemistry Letters 3 (2012) 265e267.
[5] G. Xiong, P.M. Doraiwsamy, Combination drug therapy for Alzheimer’s dis-
ease: what is evidence ebased, and what is not? Geriatrics 60 (2005) 22e26.
[6] M. Dumont, M.F. Beal, Neuroprotective strategies involving ROS in Alzheimer
disease, Free Radical Biology & Medicine 51 (2011) 1014e1026.
[7] M. Rosini, E. Simoni, M. Bartolini, A. Cavalli, L. Ceccarini, N. Pascu,
D.W. McClymont, A. Tarozzi, M.L. Bolognesi, A. Minarin, V. Tumiatti,
V. Andrisano, I.R. Mellor, C. Melchiorre, Inhibition of acetylcholinesterase, b-
amiloid aggregation, and MNDA receptors in Alzheimer’s disease: a promising
direction for the multi-target-directed gold rush, Journal of Medicinal
Chemistry 51 (2008) 4381e4384.
[8] M.I. Rodríguez-Franco, M.I. Fernández-Bachiller, C. Pérez, B. Hernández-
Ledesma, B. Bartolomé, Novel tacrine-melatonine hybrids as dual-actingdrugs
for Alzheimer disease, with improved acetylcholinesterase inhibitory and
antioxidant properties, Journal of Medicinal Chemistry 49 (2009) 459e462.
[9] L. Fanga, B. Krausc, J. Lehmanna, J. Heilmannc, Y. Zhangb, M. Decker, Design
and synthesis of tacrineeferulic acid hybrids as multi-potent anti-Alzheimer
drug candidates, Bioorganic & Medicinal Chemistry Letters 18 (2008) 2905e
2909.
[10] T. Kálai, J. Petrlova, M. Balog, H.H. Aung, J.C. Voss, K. Hideg, Synthesis and
study of 2-amino-7-bromofluorenes modified with nitroxides and their pre-
cursors as dual anti-amyloid and antioxidant active compounds, European
Journal of Medicinal Chemistry 46 (2011) 1348e1355.
[11] I.K. Mohan, M. Kahn, S. Wisel, K. Selvendiran, A. Sridhar, C.A. Carnes,
B. Bognár, T. Kálai, K. Hideg, P. Kuppusamy, Cardioprotection by HO-4038, a
novel verapamil derivative, targeted against ischemia and reperfusion-
mediated acute myocardial infarction, Heart and Circulatory Physiology e
American Journal of Physiology 296 (2009) 140e151.
the ab peptide, PLoS One 7 (2012) e35443.
[33] B.W. Wilson, J.D. Henderson, Determination of cholinesterase in blood and
tissue, Current Protocols in Toxicology 34 (2007) 12.13.1e12.13.16.
}
[12] T. Kálai, M. Balog, A. Szabó, G. Gulyás, J. Jeko, B. Sümegi, K. Hideg, New
poly(ADP-ribose) polymerase-1 inhibitors with antioxidant activity based on